Pesticide Residues in Food - 2016

Pesticide residues in
food — 2016
Joint FAO/WHO Meeting on
Pesticide Residues
EVALUATIONS
2016
Part II — Toxicological
g
Pesticide residues
in food – 2016
Toxicological evaluations
Sponsored jointly by FAO and WHO
Special Session of the Joint Meeting of the

FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Core Assessment Group on Pesticide Residues
Geneva, Switzerland, 9–13 May 2016
The summaries and evaluations contained in this book are, in most cases, based on
unpublished proprietary data submitted for the purpose of the JMPR assessment. A
registration authority should not grant a registration on the basis of an evaluation
unless it has first received authorization for such use from the owner who submitted
the data for JMPR review or has received the data on which the summaries are
based, either from the owner of the data or from a second party that has obtained
permission from the owner of the data for this purpose.
Pesticide residues in food – 2016: toxicological evaluations / Joint Meeting of the FAO Panel of Experts on
Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticide Residues,
Geneva, Switzerland, 9–13 May 2016
ISBN 978-92-4-165532-3
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TABLE OF CONTENTS
Page
List of participants .......................................................................................................................... iv
Abbreviations used.......................................................................................................................... vi
Introduction ...................................................................................................................................... x
Toxicological monographs and monograph addenda ...................................................................... 1

Diazinon* ............................................................................................................................... 2
Glyphosate*.......................................................................................................................... 90
Malathion* ......................................................................................................................... 298
Annex 1. Reports and other documents resulting from previous Joint Meetings of the
FAO Panel of Experts on Pesticide Residues in Food and the Environment and
the WHO Core Assessment Group on Pesticide Residues........................................... 454
* Evaluated within the periodic review programme of the Codex Committee on Pesticide Residues
iii
2016 Special Session of the Joint Meeting of the FAO Panel of Experts on
Pesticide Residues in Food and the Environment
and the WHO Core Assessment Group on Pesticide Residues
Geneva, 8–13 May 2016
List of participants
Professor Alan R. Boobis, Centre for Pharmacology & Therapeutics, Division of Experimental
Medicine, Department of Medicine, Faculty of Medicine, Imperial College London,
Hammersmith Campus, Ducane Road, London W12 0NN, United Kingdom (WHO Chairman)
Ms Marloes Busschers, Assessor of Human Toxicology, Board for the Authorisation of Plant
Protection Products and Biocides, Bennekomseweg 41, 6717 LL Ede, PO Box 2030, 6710 AA
Ede, the Netherlands (WHO Expert)
Dr Carl E. Cerniglia,1 Director, Division of Microbiology, National Center for Toxicological
Research, HFT-250, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079,
United States of America (USA) (WHO Expert)
Dr Sylvaine Cordier,2 Research Director Emeritus, French National Institute of Health and Medical
Research (INSERM U1085), University of Rennes, 2 rue de Tabor, CS 46510, 35065 Rennes,
France (WHO Expert)
Dr David Eastmond, Department of Cell Biology & Neuroscience, 2109 Biological Sciences
Building, University of California, Riverside, CA 92521, USA (WHO Expert)
Professor Dr Andrea Hartwig,3 Karlsruher Institut für Technologie, Institut für Angewandte
Biowissenschaften, Abteilung Lebensmittelchemie und Toxicologie, Adenauerring 20a, Gebäude
50.41 (AVG), Raum 103, Postanschrift: Kaiserstr. 12, 76131 Karlsruhe, Germany (WHO Expert)
Dr Miriam Jacobs, Toxicology Department, Centre for Radiation, Chemical and Environmental
Hazards, Public Health England, Chilton, Oxfordshire, OX11 0RQ, United Kingdom (WHO
Expert)
Dr Virissa Lenters (assisting),1 Division of Environmental Epidemiology, Institute for Risk
Assessment Sciences, Utrecht University, Yalelaan 2, PO Box 80178, Utrecht, the Netherlands
(WHO Expert)
Dr Dugald MacLachlan, Australian Government Department of Agriculture and Water Resources,
GPO Box 858, Canberra, ACT 2601, Australia (FAO Chairman)
Professor Angelo Moretto, Department of Biomedical and Clinical Sciences, University of Milan,
International Centre for Pesticides and Health Risk Prevention (ICPS), ASST Fatebenefratelli
Sacco, Luigi Sacco Hospital, Via GB Grassi 74, 20157 Milano, Italy (Rapporteur)
Dr Matthew Joseph O’Mullane, Director, Chemical Review, Australian Pesticides and Veterinary
Medicines Authority (APVMA), PO Box 6182, Kingston, ACT 2604, Australia (WHO Expert)
Dr Aldert H. Piersma, Professor of Reproductive and Developmental Toxicology, Center for Health
1
Did not attend the meeting.
2
Did not attend the meeting, but her valuable contributions to the methodological setup of the epidemiological
evaluation are gratefully acknowledged.
3
Attended part of the meeting only.
iv
Protection, National Institute for Public Health and the Environment (RIVM), Antonie van
Leeuwenhoeklaan 9, PO Box 1, 3720 BA Bilthoven, the Netherlands (WHO Expert)
Dr Prakashchandra V. Shah, Chief, Chemistry, Inerts and Toxicology Assessment Branch,
Registration Division (MDTS 7505P), Office of Pesticide Programs, United States Environmental
Protection Agency, 1200 Pennsylvania Avenue NW, Washington, DC 20460, USA (WHO
Expert)
Dr Rachel B. Smith (assisting),1 Department of Epidemiology and Biostatistics, School of Public
Health, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom (WHO
Expert)
Dr Raymond Tice, Special Volunteer, Biomolecular Screening Branch, Division of the National
Toxicology Program, National Institute of Environmental Health Sciences, Mail Code K2-17, PO
Box 12233, Research Triangle Park, NC 27709, USA (WHO Expert)
Dr Mireille B. Toledano, Senior Lecturer in Epidemiology, MRC-PHE Centre for Environment and
Health, Department of Epidemiology and Biostatistics, School of Public Health, Faculty of
Medicine, Imperial College London, St Mary’s Campus, Norfolk Place, London W2 1PG, United
Kingdom (WHO Expert)
Dr Midori Yoshida, Commissioner, Food Safety Commission, Cabinet Office, Akasaka Park
Building, 22nd Floor, 5-2-20 Akasaka Minato-ku, Tokyo 107-6122, Japan (WHO Expert)
Dr Jürg Zarn, Federal Food Safety and Veterinary Office (FSVO), Risk Assessment Division,
Schwarzenburgstrasse 155, CH-3003 Bern, Switzerland (WHO Expert)
Secretariat
Mr Enzo Armaroli, Intern, Department of Food Safety and Zoonoses, World Health Organization,
1211 Geneva 27, Switzerland
Dr Richard Brown, Evidence and Policy on Environmental Health, World Health Organization, 1211
Geneva 27, Switzerland
Mr Paul Garwood, Department of Communication, World Health Organization, 1211 Geneva 27,
Switzerland
Dr Kathryn Guyton, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69008
Lyon, France
Ms Marla Sheffer, 1553 Marcoux Drive, Orleans, Ontario, Canada K1E 2K5 (WHO Editor)
Dr Angelika Tritscher, Coordinator, Risk Assessment and Management, Department of Food Safety
and Zoonoses, World Health Organization, 1211 Geneva 27, Switzerland
Dr Philippe Verger, Department of Food Safety and Zoonoses, World Health Organization, 1211
Geneva 27, Switzerland (WHO JMPR Secretary)
Ms Yong Zhen Yang, Plant Production and Protection Division, Food and Agriculture Organization
of the United Nations, Viale delle Terme di Caracalla, 00153 Rome, Italy (FAO JMPR Secretary)
1
Did not attend the meeting.
v
Abbreviations used
AChE acetylcholinesterase
ACP acid phosphatase
ADI acceptable daily intake
AFC antibody-forming cell
AHS Agricultural Health Study
AhR aryl hydrocarbon receptor
ALP alkaline phosphatase
AMPA aminomethylphosphonic acid
aOR adjusted odds ratio
AP apurinic/apyrimidinic
APG alkyl polyglucoside
AR androgen receptor
ARfD acute reference dose
aRR adjusted risk ratio
ASDN androstene-4-ene-3,17-dione
AST aspartate aminotransferase
AUC area under the plasma concentration–time curve
AUCt area under the concentration versus time–curve calculated up to the last detectable
sample
BChE butyrylcholinesterase
Bmax maximum amount of binding
BfR German Bundesinstitut für Risikobewertung
BMD benchmark dose
BMD10 estimated benchmark dose for a 10% inhibition
BoNT botulinum neurotoxin
BUN blood urea nitrogen
bw body weight
CA chromosomal aberrations
CAS Chemical Abstracts Service
CCPR Codex Committee on Pesticide Residues
CEBS Chemical Effects in Biological Systems
cfu colony-forming unit
ChE cholinesterase
CHO Chinese hamster ovary
Ci curie (1 Ci = 3.7 × 1010 becquerel [Bq])
CI confidence interval
Cmax maximum concentration
CYP cytochrome P450
CMC carboxymethylcellulose
CYP cytochromes P450
2,4-D 2,4-dichlorophenoxyacetic acid
DEL yeast deletion (assay)
DEP diethylphosphoric acid
DETP diethylphosphorothioic acid
DMSO dimethyl sulfoxide
DMDTP dimethyl dithiophosphate
DMP dimethyl phosphate
vi
DMTP dimethyl thiophosphate
DNA deoxyribonucleic acid
DPRA direct peptide reactivity assay
DSB double strand break
EDSP Endocrine Disruptor Screening Program
ELISA enzyme-linked immunosorbent assay
ENDO endonuclease
EPSPS 5-enolpyruvylshikimate 3-phosphate synthase
eq equivalent
ER estrogen receptor
ERTA estrogen receptor transcriptional activation
F female
F0 parental generation
F1 first filial generation
F2 second filial generation
F2A second filial generation, first litter
F2B second filial generation, second litter
FAO Food and Agriculture Organization of the United Nations
Fpg formamidopyrimidine-DNA-glycosylase
FSH follicle-stimulating hormone
FSTRA fish short-term reproduction assay
GD guideline
GGT gamma-glutamyltransferase
GIT gastrointestinal tract
GLP good laboratory practice
GSH glutathione
Hb haemoglobin
Hct haematocrit
Hep2 epidermoid cancer
HepG2 hepatocellular carcinoma
HESS Hazard Evaluation Support System
HIC highest ineffective concentration
HPLC high-performance liquid chromatography
HPLC-EC high pressure liquid chromatography-electrochemical¬-electrochemical detection
HPLC/MS-MS high-performance liquid chromatography with mass spectrometry
HPRT hypoxanthine-guanine phosphoribosyltransferase
HTC hepatoma cell
IARC International Agency for Research on Cancer
IC50 median inhibitory concentration
IEDI international estimated daily intake
IL interleukin
IP intraperitoneal
IM isomalathion
IU International Unit
IV intravenous
ISS Istituto Superiore di Sanità
IW-LED intensity-weighted lifetime-exposure days
JMPR Joint FAO/WHO Meeting on Pesticide Residues
Kd dissociation constant
ke/fd killed in extremis or found dead
LABC levator ani plus bulbocavernosus muscle complex
LC50 median lethal concentration
LD50 median lethal dose
LDH lactate dehydrogenase
LEC lowest effective concentration
vii
LED lifetime-exposure days
LH luteinizing hormone
LLNA local lymph node assay
LOAEL lowest-observed-adverse-effect level
M male
MCH mean corpuscular haemoglobin
MCV mean corpuscular volume
MDCA malathion dicarboxylic acid
MIC minimum inhibitory concentration
MMC minimum microbicidal concentration
MMCA malathion monocarboxylic acid
MN micronuclei
MN-PCE micronucleated polychromatic erythrocytes
MOA mode of action
mRNA messenger ribonucleic acid
N sample size
N/A not applicable
NADH nicotinamide adenine dinucleotide (reduced)
NADPH nicotinamide adenine dinucleotide phosphate (reduced)
NB nota bene
NCE normochromatic erythrocyte
ND not determined
NHL non-Hodgkin lymphoma
NI not investigated
no. number
NOAEC no-observed-adverse-effect concentration
NOAEL no-observed-adverse-effect level
NP not provided
NR not reported
N/S not stated
NS not specified
NS not significant
NTE neuropathy target esterase
NTP National Toxicology Program
OASIS Organization for the Advancement of Structured Information Standards
OECD Organisation for Economic Co-operation and Development
8-OHdG 8-hydroxy-2'-deoxyguanosine
OPPTS Office of Prevention, Pesticides & Toxic Substances
OR odds ratio
8-Oxo-dG 8-hydroxy-2'-deoxyguanosine
2-PAM 2-pyridinealdoxime methiodide (in Jenkins, 1988)
2-PAM pyridine-2-aldoxime methochloride (in Frick et al., 1987, from the 1993 JMPR)
PCE polychromatic erythrocyte
PDII primary dermal irritation index
PEG polyethylene glycol
PHA phytohaemagglutinin
PND postnatal day
POE polyoxyethylene ether
POE-APE polyoxyethylene ether phosphates – polyoxyethylene alkyl ether phosphate
POEA polyoxyethyleneamine
POES polyethoxylated tallow amine
PPAR peroxisome proliferator-activated receptor
ppb parts per billion
viii
ppm parts per million
PWG Pathology Working Group
PXR pregnane X receptor
Q quartile
QSAR quantitative structure–activity relationships
ref. reference
RBA relative binding affinity
RfD reference dose
rhCG recombinant human chorionic gonadotrophin
RNA ribonucleic acid
ROS reactive oxygen species
RPCmax maximum level of response
RR risk ratio
rRNA ribosomal ribonucleic acid
RR relative risk
rtER rainbow trout estrogen receptor
S9 9000 × g supernatant fraction from liver homogenate
SCE sister chromatid exchange
SCSA sperm chromatin structure assay
SD standard deviation
SDH succinate dehydrogenase
SDS sodium dodecyl sulfate
SI Stimulus Index
SN2 bimolecular nucleophilic substitution
SSB single strand breaks
StAR steroidogenic acute regulatory protein
T4 thyroxine
TEPP tetraethyl pyrophosphate
TK thymidine kinase
Tmax time to reach the maximum concentration
TAF toxicity adjustment factor
TG test guideline
Tk terminal kill
TLC thin-layer chromatography
TOCP triorthocresyl phosphate
TP testosterone propionate
TPA 12-O-tetradecanoylphorbol-13-acetate
TSH thyroid-stimulating hormone
U enzyme unit
UDS unscheduled DNA synthesis
USDA United States Department of Agriculture
USEPA United States Environmental Protection Agency
UV ultraviolet
VTG vitellogenin
v/v volume per volume
WHO World Health Organization
w/w weight per weight
ix
Introduction
The toxicological monographs contained in this volume were prepared by a WHO Core
Assessment Group on Pesticide Residues that met with the FAO Panel of Experts on Pesticide
Residues in Food and the Environment in a Joint Meeting on Pesticide Residues (JMPR) in Geneva,
Switzerland, on 9–13 May 2016.
The three compounds (diazinon, glyphosate and malathion) were evaluated following the
recommendation of an electronic task force of the WHO Core Assessment Group on Pesticide
Residues that the compounds be re-evaluated due to public health concerns identified by International
Agency for Research on Cancer (IARC) and the availability of a significant number of new studies.
Reports and other documents resulting from previous Joint Meetings on Pesticide Residues are listed
in Annex 1.
The report of the Joint Meeting has been published by the FAO as FAO Plant Production and
Protection Paper 227. That report contains comments on the compounds considered, acceptable daily
intakes and acute reference doses established by the WHO Core Assessment Group. As no residue
data were requested, maximum residue levels previously established by the FAO Panel of Experts for
these compounds remain unchanged and no monographs on residues were prepared.
The toxicological monographs contained in this volume are based on working papers that
were prepared by WHO experts before the 2016 Joint Meeting. A special acknowledgement is made
to those experts and to the experts of the Joint Meeting who reviewed early drafts of these working
papers.
The designations employed and the presentation of the material in this publication do not
imply the expression of any opinion whatsoever on the part of the World Health Organization
concerning the legal status of any country, territory, city or area or of its authorities, or concerning the
delimitation of its frontiers or boundaries. The mention of specific companies or of certain
manufacturers’ products does not imply that they are endorsed or recommended by the World Health
Organization in preference to others of a similar nature that are not mentioned.
Any comments or new information on the biological properties or toxicity of the compounds
included in this volume should be addressed to: Joint WHO Secretary of the Joint FAO/WHO
Meeting on Pesticide Residues, Department of Food Safety and Zoonoses, World Health
Organization, 20 Avenue Appia, 1211 Geneva, Switzerland.
Methodology
Literature search methodology
For each of the 3 compounds under review, the information was collected from 3 sources.
 The individual publications considered by IARC were provided to JMPR.
 The dossiers provided by industry for registration of the compounds in the European
Union, the United States of America and Japan were submitted.
 The JMPR experts performed an update of the literature search done by IARC for
“cancer”, “genotoxicity” and “epidemiological data”.
For the articles related to cancer and cancer-mechanisms, the literature search strategy
involved performing targeted searches on the agents or major metabolites in the following databases:
1) Google Scholar (http://scholar.google.com/);
2) PubMed (http://www.ncbi.nlm.nih.gov/pubmed);
3) WEB OF SCIENCE (https://apps.webofknowledge.com);
x
4) BioOne (http://www.bioone.org/); and
5) ScienceDirect (http://www.sciencedirect.com/).
A keyword searching strategy was employed, using the keywords and the Boolean Operators
(AND, or , and NOT). ([mh] = mesh term; PubMed’s controlled vocabulary, [tiab] = text word to be
searched in the title or abstract
“Comet Assay”[mh] OR “Germ-line-mutation”[mh] OR “Mutagenesis”[mh] OR
“Mutagenicity tests”[mh] OR “Sister-chromatid exchange”[mh] OR “Mutation”[mh] OR
Ames-Assay[tiab] OR Ames-test[tiab] OR Bacterial-Reverse-Mutation-Assay[tiab] OR
Clastogen*[tiab] OR DNA-Repair*[tiab] OR Genetic-toxicology[tiab] OR hyperploid[tiab] OR
micronucleus-test[tiab] OR tetraploid[tiab] OR Chromosome-aberrations[tiab] OR DNA damage[tiab]
OR Mutation[tiab] OR chromosome-translocations[tiab] OR DNA protein crosslinks[tiab] OR DNA-
damag*[tiab] OR DNA-inhibit*[tiab] OR Micronuclei[tiab] OR Micronucleus[tiab] OR
Mutagens[tiab] OR Strand-break*[tiab] OR Unscheduled-DNA-synthes*[tiab] OR chromosomal-
aberration[tiab] OR chromosome-aberration[tiab] OR chromosomal-aberrations[tiab] OR
chromosomal-abnormalit*[tiab] OR chromosome-abnormalit*[tiab] OR genotoxic*[tiab] OR Comet-
assay[tiab] OR Mutagenic[tiab] OR Mutagenicity[tiab] OR mutations[tiab] OR chromosomal-
aberration-test[tiab] OR Sister-chromatid-exchange[tiab]
The search resulted in 157 references for Diazinon–Cancer; 99 for Diazinon–Genotox; 251
for Glyphosate–Cancer; 269 for Glyphosate–Genotox; 227 for Malathion–Cancer; and 182 for
Malathion–Genotox.
For epidemiological literature the search was restricted to identifying articles published after
the three IARC Monographs were published. The search strategy and results are summarized in the
table below.
Number of Hits after screening for

Search terms Search engine hits relevance
(diazinon OR glyphosate OR PubMed (limited to humans; 31 N=2
malathion) AND cancer published in the last 5 years) Koutros et al. (2015); Lerro
Scopus (limited to 2014–2016) 28 et al. (2015)
(diazinon OR glyphosate OR PubMed (limited to humans; 11

malathion) AND (NHL OR published in the last 5 years)
lymphoma OR leukemia OR “lung
cancer” OR “prostate cancer”) Scopus (limited to 2014–2016) 9
Methodology of epidemiological studies

The pre-agreed evaluation process and Tier 1 screening criteria used to evaluate
epidemiological studies on diazinon, glyphosate and malathion are described in “Section 2.2: Methods
for the evaluation of epidemiological evidence for risk assessment” of the JMPR meeting report1.
Evaluation process of epidemiological evidence for risk assessment for glyphosate, malathion and
diazinon
The evaluation process and Tier 1 screening criteria are shown in Fig. 1 below.
1
Pesticide residues in food 2016: Special session of the joint FAO/WHO meeting on pesticide residues May
2016: Report 2016 (http://www.who.int/foodsafety/areas_work/chemical-risks/jmpr/en/)
xi
Fig. 1. Pre-agreed evaluation process and Tier 1 screening criteria
(a) Identification of compound/cancer sites and screening of papers

This assessment was restricted to studies of cancer outcomes. The body of epidemiological
evidence for non-cancer outcomes was not evaluated; numerous studies have assessed risks for
neurodevelopmental, neurodegenerative or reproductive outcomes, among other health outcomes.
Restricting the assessment to non-cancer outcomes was partly driven by feasibility reasons: a
clinically relevant adverse effect size (or an acceptable level of risk) for a non-cancer outcome must
be defined, and the methodologies for hazard identification and characterization based on
observational epidemiological findings of non-carcinogenic adverse effects are less well-established
than those for cancer (see, for example, Clewell & Crump, 2005; Nachman et al., 2011).
The International Agency for Research on Cancer (IARC) monographs on diazinon,
glyphosate and malathion refer to a total of 45 epidemiological studies. Two more recently published
studies evaluated at least one of malathion, diazinon or glyphosate in relation to cancer outcomes
(Lerro et al., 2015; Koutros et al., 2015). An additional study on prostate cancer (Mills & Yang,
2003), which was not included in the IARC monographs, was also identified.
The 45 publications referred to in the IARC monographs and the three publications since
(Mills & Yang, 2003; Lerro et al., 2015; Koutros et al., 2015) covered 48 compound/cancer site
combinations. The current evaluation focuses on the 6 compound/cancer site combinations for which
IARC identified positive associations from the body of epidemiological evidence, that is, those
associations noted in section 6.1 of the monographs, and which underpin IARC’s evaluation of
limited evidence in humans for the carcinogenicity of malathion, diazinon and glyphosate. The
definition for limited evidence of carcinogenicity used by IARC is as follows: “A positive association
has been observed between exposure to the agent and cancer for which a causal interpretation is
xii
considered by the Working Group to be credible, but chance, bias or confounding could not be ruled
out with reasonable confidence” (IARC, 2015). The 6 compound/cancer site combinations are:
A. Malathion / non-Hodgkin lymphoma (NHL)
B. Malathion / prostate cancer
C. Diazinon / NHL
D. Diazinon / leukaemia
E. Diazinon / lung cancer
F. Glyphosate / NHL
When identifying relevant publications it was noted that there were stand-alone analyses for
specific subtypes of NHL (of which there are many). Evaluations of risk for subtypes of NHL were
not undertaken separately as there was insufficient evidence (too few studies or small numbers of
cases); nor were evaluations of risk undertaken for other haematopoietic and lymphoid tumours, as the
positive associations identified by IARC were for total NHL.
There were 26 publications for these 6 compound/cancer site combinations. Seven studies
were excluded from at least one evaluation for a given compound/cancer site during Tier 1 screening,
either because they were not specific to the pesticide in question; because the publication had been
superseded by a later publication on the same cohort and this later publication included longer follow-
up time; or because there was a more complete analysis on the same study population with a greater
number of participants.
(b) Overview of studies included in evaluation

The IARC monograph on malathion (IARC, 2015) provided an overview of the
epidemiological studies which have assessed pesticide exposures and cancer risk. Therefore, only a
brief summary (largely based on the IARC monograph) of the studies contributing to the current
evaluation is provided here for context.
The Agricultural Health Study is a prospective cohort study of pesticide applicators
(predominantly farmers; n ≈ 52 000) and their spouses (n ≈ 32 000) from Iowa and North Carolina,
United States of America, enrolled in 1993–1997. The Study has examined a range of cancer
outcomes and published analyses with longer periods of follow-up (e.g. De Roos et al., 2005; Beane
Freeman et al., 2005; Koutros et al., 2013; Alavanja et al., 2014; Jones et al., 2015; Lerro et al., 2015).
Information on participants’ use of 50 pesticides and other determinants of exposure was gathered
retrospectively via baseline and two follow-up questionnaires. Cumulative lifetime exposure estimates
were calculated. Validation studies have been conducted to assess the reliability and accuracy of
exposure intensity scores (a component of the exposure assessment) (Coble et al., 2005; Hines et al.,
2008; Thomas et al., 2010). The impact of exposure misclassification in this study was to bias risk
estimates towards null (Blair et al., 2011).
The United States Midwest case–control studies are three population-based case–control
studies of cancer conducted in Nebraska (Zahm et al., 1990), Iowa and Minnesota (Brown et al., 1990;
Cantor et al., 1992) and Kansas (Hoar et al., 1986) that have been pooled (748 cases/2236 controls) to
analyse NHL in white males only (Waddell et al., 2001; De Roos et al., 2003; Lee et al., 2004).
Information on participants’ occupational use of pesticides was gathered retrospectively via a
questionnaire. There were some differences in case ascertainment and exposure assessment methods
between the three studies. For 39% of the pooled study population, proxy respondents were used
(Waddell et al., 2001), for whom recall of specific pesticide use could be problematic and subject to
recall bias that may differ for cases and controls. De Roos et al. (2003) used the same study
population as Waddell et al. (2001) to perform an extensive evaluation and adjustment for other
pesticides.
xiii
The Cross-Canada Study of Pesticides and Health (CCSPH) is a population-based case–
control study of haematopoietic cancers in men diagnosed in 1991–1994 across six Canadian
provinces (McDuffie et al., 2001). It includes 517 NHL cases and 1506 controls. A questionnaire was
administered by post, followed by a telephone interview for those that reported pesticide exposure of
10 hours/year or more and for a 15% random sample of the remainder. The study was not restricted to
pesticide exposure experienced by a specific occupational group (McDuffie et al., 2001). Further
analyses stratified by asthma/allergy status – to assess possible effect modification by immune system
modulation – have been conducted (Pahwa et al., 2012). The study has a large sample size and
detailed information of pesticide exposures; however, the proportion exposed to pesticides was low.
The three sets of studies above were deemed as high quality and highly informative by the
IARC Working Group (IARC, 2015).
A number of other case–control studies of pesticide exposure and cancer risk were included in
this evaluation: the Florida Pest Control Worker study (Pesatori et al., 1994); nested case–control
studies within the United Farm Workers of America cohort study (Mills & Yang, 2003; Mills, Yang
& Riordan, 2005); a population-based case–control study of prostate cancer in British Columbia,
Canada (Band et al., 2011); and case–control studies of NHL/haematopoietic cancers from Sweden
(Hardell et al., 2002; Eriksson et al., 2008) and France (Orsi et al., 2009). The IARC Working Group
(IARC, 2015) noted substantial limitations in these studies, either in relation to exposure assessment,
scope for and variation in exposure misclassification, lack of detail in the publication, which hindered
interpretation, lack of specificity due to high correlations between use of different pesticides, and
limited power.
(c) Strengths and limitations of studies included in evaluation

The included studies predominantly examined the occupational pesticide exposures of
farmers and other pesticide applicators, with the vast majority of research being on males only. None
of the studies assessed exposure via food consumption or ambient exposure from agriculture (e.g.
spray drift). The scientific evidence available is therefore limited in its generalizability and the extent
to which it can be translated to general population exposure scenarios and levels that would be
associated with pesticide residues. Nonetheless, these observational epidemiological studies provide
insight into real-world exposure scenarios and allow for observation of the species of interest
(humans) over the long follow-up periods relevant to cancer.
The number of high quality studies is relatively small. Typically the number of exposed cases
in studies is small, particularly when evaluating specific pesticides, which limits study power.
Relatively few studies have assessed exposure quantitatively, meaning the epidemiological
evidence available to inform/establish dose–response relationships is very limited. Exposure
misclassification is a potential issue for all studies. This is expected to be largely non-differential for
cohort studies (i.e. the Agricultural Health Study), resulting in attenuation of risk estimates. All except
one of the studies included are case–control studies, and these may be affected by recall bias, that is,
cases and controls recall past pesticide exposure with differing accuracy, leading to differential
exposure misclassification that can bias risk estimates either towards or away from the null. As a
cohort study, the Agricultural Health Study avoids recall bias.
Given that studies focused on occupational exposures among farmers/pesticide applicators, it
is unlikely that they were exposed to only one specific pesticide, so confounding, possible effect
modification and additive/multiplicative effects due to coexposures are all concerns. However, many
studies were able to adjust risk estimates for other pesticide coexposures, which yields more accurate
risk estimates.
There are some issues in terms of comparing studies and evaluating the consistency of
evidence overall. Results of studies may appear heterogeneous, but usually there are too few studies to
xiv
really assess consistency and heterogeneity. Exposure assessment methods and referent groups vary
between studies.
Finally, changes in disease classifications (particularly that of NHL) or screening/diagnosis
rates (prostate cancer) over time, may limit comparability between studies.
(d) Publication bias

A formal analysis of publication bias was not undertaken because the number of studies (risk
estimates from non-overlapping study populations) available were few and it is advised that funnel
plot tests for asymmetry be used only where there are at least 10 studies to allow sufficient statistical
power to distinguish true asymmetry from chance (Higgins & Green, 2011; Sterne et al., 2011). Other
formal objective statistical tests require a larger number of studies, typically at least 30, to achieve
sufficient statistical power (Lau et al., 2006). As a result, publication bias cannot be fully excluded.
However, given the very considerable resources invested in these types of (large, difficult exposure
assessment) studies, it is unlikely that results would go unpublished.
References
Alavanja MC, Hofmann JN, Lynch CF, Hines CJ, Barry KH, Barker J et al. (2014). Non-Hodgkin lymphoma
risk and insecticide, fungicide and fumigant use in the agricultural health study. PLoS One. 9(10):e109332.
doi:10.1371/journal.pone.0109332.
Band PR, Abanto Z, Bert J, Lang B, Fang R, Gallagher RP et al. (2011). Prostate cancer risk and exposure to
pesticides in British Columbia farmers. Prostate. 71(2):168–83. doi:10.1002/pros.21232.
Beane Freeman LE, Bonner MR, Blair A, Hoppin JA, Sandler DP, Lubin JH et al. (2005). Cancer incidence
among male pesticide applicators in the Agricultural Health Study cohort exposed to diazinon. Am J
Epidemiol. 162:1070–9.
Blair A, Thomas K, Coble J, Sandler DP, Hines CJ, Lynch CF et al. (2011). Impact of pesticide exposure
misclassification on estimates of relative risks in the Agricultural Health Study. Occup Environ Med.
68(7):537–41. doi:10.1136/oem.2010.059469.
Brown LM, Blair A, Gibson R, Everett GD, Cantor KP, Schuman LM et al. (1990). Pesticide exposures and
other agricultural risk factors for leukemia among men in Iowa and Minnesota. Cancer Res. 50(20):6585–
91.
Cantor KP, Blair A, Everett G, Gibson R, Burmeister LF, Brown LM et al. (1992). Pesticides and other
agricultural risk factors for non-Hodgkin’s lymphoma among men in Iowa and Minnesota. Cancer Res.
52(9):2447–55.
Clewell HJ, Crump KS (2005). Quantitative estimates of risk for noncancer endpoints. Risk Anal. 25(2):285–9.
Coble J, Arbuckle T, Lee W, Alavanja M, Dosemeci M (2005). The validation of a pesticide exposure algorithm
using biological monitoring results. J Occup Environ Hyg. 2(3):194–201.
De Roos AJ, Zahm SH, Cantor KP, Weisenburger DD, Holmes FF, Burmeister LF et al. (2003). Integrative
assessment of multiple pesticides as risk factors for non-Hodgkin’s lymphoma among men. Occup Environ
Med. 60:E11. doi:10.1136/oem.60.9.e11.
De Roos AJ, Blair A, Rusiecki JA, Hoppin JA, Svec M, Dosemeci M et al. (2005). Cancer incidence among
glyphosate-exposed pesticide applicators in the Agricultural Health Study. Environ Health Perspect.
113:49–54.
Eriksson M, Hardell L, Carlberg M, Akerman M (2008). Pesticide exposure as risk factor for non-Hodgkin
lymphoma including histopathological subgroup analysis. Int J Cancer. 123(7):1657–63.
Hardell L, Eriksson M, Nordstrom M (2002). Exposure to pesticides as risk factor for non-Hodgkin's lymphoma
and hairy cell leukemia: pooled analysis of two Swedish case-control studies. Leuk Lymphoma.
43(5):1043–9.
xv
Higgins JP, Green S (editors). Cochrane handbook for systematic reviews of interventions version 5.1.0. The
Cochrane Collaboration, 2011.
(http://handbook.cochrane.org/chapter_10/10_4_3_1_recommendations_on_testing_for_funnel_plot_asym
metry.htm, accessed 14 April 2016)
Hines CJ, Deddens JA, Jaycox LB, Andrews RN, Striley CAF, Alavanja MC (2008). Captan exposure and
evaluation of a pesticide exposure algorithm among orchard pesticide applicators in the Agricultural Health
Study. Ann Occup Hyg. 52:153–66.
Hoar SK, Blair A, Holmes FF, Boysen CD, Robel RJ, Hoover R et al. (1986). Agricultural herbicide use and
risk of lymphoma and soft-tissue sarcoma. JAMA. 256(9):1141-7.
IARC (2015). Some organophosphate insecticides and herbicides: diazinon, glyphosate, malathion, parathion,
and tetachlorvinphos. Monograph Volume 112. Lyon (FR): International Agency for Research on Cancer.
Available from: http://monographs.iarc.fr/ENG/Monographs/vol112/
Jones RR, Barone-Adesi F, Koutros S, Lerro CC, Blair A, Lubin J et al. (2015). Incidence of solid tumours
among pesticide applicators exposed to organophosphate insecticide diazinon in the Agricultural Health
Study: an updated analysis. Occup Environ Med. 72:496-503. doi: 10.1136/oemed-2014-102728.
Koutros S, Beane Freeman LE, Lubin JH, Heltshe SL, Andreotti G, Barry KH et al. (2013). Risk of total and
aggressive prostate cancer and pesticide use in the Agricultural Health Study. Am J Epidemiol. 177:59–74.
doi:10.1093/aje/kws225
Koutros S, Silverman DT, Alavanja MC, Andreotti G, Lerro CC, Heltshe S et al. (2015). Occupational exposure
to pesticides and bladder cancer risk. Int J Epidem. pii: dyv195.
Lau J, Ioannidis JP, Terrin N, Schmid CH, Olkin I (2006). The case of the misleading funnel plot. BMJ.
333(7568):597–600.
Lerro CC, Koutros S, Andreotti G, Friesen MC, Alavanja MC, Blair A et al. (2015) Organophosphate
insecticide use and cancer incidence among spouses of pesticide applicators in the Agricultural Health
Study. Occup Environ Med. 72(10):736–44.
McDuffie HH, Pahwa P, McLaughlin JR, Spinelli JJ, Fincham S, Dosman JA et al. (2001). Non-Hodgkin’s
lymphoma and specific pesticide exposures in men: cross-Canada study of pesticides and health. Cancer
Epidemiol Biomarkers Prev. 10(11):1155–63.
Mills PK, Yang R (2003). Prostate cancer risk in California farm workers. J Occup Environ Med. 45(3):249–58.
Mills PK, Yang R, Riordan D (2005). Lymphohematopoietic cancers in the United Farm Workers of America
(UFW), 1988–2001. Cancer Causes Control. 16(7):823–30.
Nachman KE, Fox MA, Sheehan MC, Burke TA, Rodricks JV, Woodruff TJ (2011). Leveraging epidemiology
to improve risk assessment. Open Epidem J. 4:3–29.
Orsi L, Delabre L, Monnereau A, Delval P, Berthou C, Fenaux P et al. (2008). Occupational exposure to
pesticides and lymphoid neoplasms among men: results of a French case-control study. Occup Environ
Med. 66(5): 291–8.
Pahwa M, Harris SA, Hohenadel K, McLaughlin JR, Spinelli JJ, Pahwa P et al. (2012). Pesticide use,
immunologic conditions, and risk of non-Hodgkin lymphoma in Canadian men in six provinces. Int J
Cancer. 131(11):2650–9. doi:10.1002/ijc.27522.
Pesatori AC, Sontag JM, Lubin JH, Consonni D, Blair A (1994). Cohort mortality and nested case-control study
of lung cancer among structural pest control workers in Florida (United States). Cancer Causes Control.
5(4):310–8. doi:10.1007/BF01804981.
Sterne JA, Sutton AJ, Ioannidis JP, Terrin N, Jones DR, Lau J et al. (2011). Recommendations for examining
and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. BMJ. 343: d4002.
doi: http://dx.doi.org/10.1136/bmj.d4002.
Thomas KW, Dosemeci M, Coble JB, Hoppin JA, Sheldon LS, Chapa G et al. (2010). Assessment of a pesticide
exposure intensity algorithm in the Agricultural Health Study. J Expo Sci Environ Epidemiol. 20:559–69.
xvi
Waddell BL, Zahm SH, Baris D, Weisenburger DD, Holmes F, Burmeister LF et al. (2001). Agricultural use of
organophosphate pesticides and the risk of non-Hodgkin’s lymphoma among male farmers (United States).
Cancer Causes Control. 12(6):509–17.
Zahm SH, Weisenburger DD, Babbit PA, Saal RC, Vaught JB, Cantor KP et al. (1990). A case control study of
non-Hodgkin’s lymphoma and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in eastern Nebraska.
Epidemiology. 1:349–56.
xvii
TOXICOLOGICAL MONOGRAPHS
AND MONOGRAPH ADDENDA
DIAZINON
First draft prepared by
Marloes Busschers1, Midori Yoshida2, Mireille B. Toledano3, Rachel B. Smith4, Virissa Lenters5,
Aldert H. Piersma6, Carl Cerniglia7 and Angelo Moretto8
1
Board for the Authorisation of Plant Protection Products and Biocides, Ede, the Netherlands
2
Food Safety Commission, Tokyo, Japan
3
MRC-PHE Centre for Environment and Health, Department of Epidemiology and Biostatistics,
School of Public Health, Faculty of Medicine, Imperial College London, London, United Kingdom
4
Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London,
London, United Kingdom
5
Norwegian Institute of Public Health, Oslo, Norway6
6
Centre for Health Protection, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands
7
Division of Microbiology, US Food and Drug Administration, Jefferson, AR, United States of
America (USA)
8
Department of Biomedical and Clinical Sciences, University of Milan, International Centre for
Pesticides and Health Risk Prevention, Milano, Italy
Explanation ...............................................................................................................................................3
Evaluation for acceptable intake ...............................................................................................................4
1. Biochemical aspects ..........................................................................................................................4
1.1 Absorption, distribution and excretion ........................................................................................4
(a) Oral route ..............................................................................................................................4
(b) Dermal route .........................................................................................................................6
1.2 Biotransformation ........................................................................................................................6
(a) In vitro studies ......................................................................................................................6
(b) In vivo studies.......................................................................................................................7
2. Toxicological studies .........................................................................................................................8
2.1 Acute toxicity .............................................................................................................................8
(a) Dermal irritation ..................................................................................................................9
(b) Ocular irritation ....................................................................................................................9
(c) Dermal sensitization ..........................................................................................................10
2.2 Short-term studies of toxicity ....................................................................................................10
(a) Oral administration .............................................................................................................10
(b) Dermal application .............................................................................................................22
(c) Exposure by inhalation .......................................................................................................24
2.3 Long-term studies of toxicity and carcinogenicity ...................................................................25
2.4 Genotoxicity .............................................................................................................................33
2.5 Reproductive and developmental toxicity ................................................................................43
(a) Multigeneration studies.......................................................................................................43
(b) Developmental toxicity.......................................................................................................44
2.6 Special studies. .........................................................................................................................46
(a) Acute neurotoxicity ............................................................................................................46
(b) Subacute neurotoxicity .......................................................................................................51
(c) Delayed neurotoxicity .........................................................................................................51
(d) Mechanistic studies ............................................................................................................53
(e) Estrogenic or androgenic activities .....................................................................................53
(f) Studies on metabolites or impurities ...................................................................................58
(g) Intestinal microbiota effects ...............................................................................................59
(h) Immunotoxic effects ...........................................................................................................59
6
Formerly at the Division of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht University,
Utrecht, Kingdom of the the Netherlands
DIAZINON 2–88 JMPR 2016

3
3. Observations in humans ..................................................................................................................59

3.1 Studies in volunteers .................................................................................................................59
3.1 Case studies ...............................................................................................................................61
3.3 Epidemiological data .................................................................................................................62
Comments……………………………………………………………………………………..………. .69
Toxicological evaluation ........................................................................................................................75
References ..............................................................................................................................................78
Explanation
Diazinon (Fig. 1) is the common name approved by the International Organization for
Standardization (ISO) for O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate
(International Union of Pure and Applied Chemistry), with the Chemical Abstracts Service (CAS)
number 333-41-5.
Diazinon is a contact organophosphorus insecticide with a wide range of insecticidal activity.
It is effective against adult and juvenile forms of flying insects, crawling insects, acarians and spiders.
Diazoxon, the biologically active metabolite of diazinon, inhibits the activity of cholinesterases.
Diazinon is used mainly as a pesticide in agriculture and as a drug in veterinary medicine.
Thus, the major source of diazinon residues in edible crops is from its use as an agricultural pesticide;
residues in meat, offal and other animal products arise from its use as a veterinary drug containing
active ingredient.
Diazinon has been evaluated by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR)
on several occasions since the first evaluation in 1963. In the most recent evaluation, in 2006, the
Meeting established an acceptable daily intake (ADI) of 0 to 0.005 mg/kg body weight (bw), based on
a no-observed-adverse-effect level (NOAEL) of 0.5 mg/kg bw per day for inhibition of erythrocyte
acetylcholinesterase activity in a 92-day repeated-dose toxicity study in rats. The 2006 Meeting
reaffirmed the acute reference dose (ARfD) of 0.03 mg/kg bw, established by the 2001 JMPR, based
on a NOAEL of 2.5 mg/kg bw observed in a study of acute neurotoxicity in rats.
Diazinon was scheduled within the periodic review programme of the Codex Committee on
Pesticide Residues (CCPR) for 2021. The compound was placed on the agenda by the JMPR
Secretariat following the recommendation of an electronic task force of the World Health
Organization (WHO) Core Assessment Group on Pesticide Residues that it be re-evaluated due to
public health concern identified by the International Agency for Research on Cancer (IARC) and the
availability of a significant number of new studies.
The current Meeting evaluated all previously considered toxicological data in addition to new
published or unpublished toxicological studies and published epidemiological studies on cancer
outcomes. Several study reports evaluated at previous JMPR meetings were not available to the
present Meeting, as they were not submitted in the sponsor’s dossier; for these studies, the evaluations
in this monograph were taken from the 1993 JMPR monograph without further review.
All critical unpublished studies contained statements of compliance with good laboratory
practice (GLP), unless otherwise specified. The studies on human volunteers were conducted in
accordance with the principles expressed in the Declaration of Helsinki or equivalent ethical
standards.

4
Fig. 1. Structure of diazinon
Evaluation for acceptable intake

1. Biochemical aspects
1.1 Absorption, distribution and excretion
The effect of oral and topical administration of diazinon was studied in various animal species
using unlabelled and radiolabelled diazinon. Additional studies were performed in vitro using tissue
slices or cell fractions from different tissues and species to investigate the biotransformation of the
compound.
(a) Oral route

Rats
According to a study by Mücke, Alt & Esser (1970):
After the application of a single oral dose of 0.8 mg/rat (4 mg/kg bw) 14C-labelled diazinon (labels at
2-14C, 4-14C or ethoxy-14C) to four male and two female Wistar rats, radioactivity was eliminated
practically completely during the 168-hour observation period with either label. Excretion of applied
radioactivity amounted to 65–80% in urine, and 16–24% in faeces. The excretion half-time was
estimated to be 12 hours for the pyrimidine-labelled and 7 hours for the ethoxy-labelled compound.
After [applying the] side-chain labelled material, about 6% of the applied dose was eliminated as
14
CO2. The absence of radioactive CO2 in the expired air after application of ring-labelled diazinon
shows that no cleavage of the pyrimidine ring occurred.
After feeding male rats [for] 10 days 0.1 mg/rat per day (0.5 mg/kg bw per day) of 2-14C-diazinon,
2.9% of the applied dose was found in the essential organs 6 hours after the final application, whereas 2
days after cessation of treatment the radioactivity was below the detection limit (< 0.1%). These results
indicate that no accumulation of diazinon or its metabolites occurs in the body (Mücke, Alt & Esser,
1970; study evaluation copied from 1993 JMPR without further evaluation).
In the low-dose group, 93% of the applied radioactivity was excreted in the urine [of] male [rats] and
86% in [the urine of] female [rats] within 24 hours. At the high-dose level, an average of 91% and 58%
in males and females, respectively, was excreted in the first 24 hours. After preconditioning, about 90%
of the 14C-dose was excreted within 24 hours in both sexes. The total amounts eliminated over the
seven-day observation period did not differ between the various dosing regimens. Total urinary
excretion amounted to 96%, and faecal elimination to 3%. These results suggest that complete
absorption occurs following intragastric administration and supports the hypothesis that the small
amount of faecal radioactivity found may be of biliary origin. Tissue concentrations of 14C-diazinon
and metabolites seven days after dosing were mostly less than 0.05 ppm [parts per million] for low-
dose and preconditioned animals. In high-dose animals, residue levels in different organs varied
between 0.1 and 0.4 ppm (red blood cells) with female rats showing consistently slightly higher tissue
residues than males. The 14C in the blood was associated primarily with the cellular fraction, suggesting
that binding occurred (Capps et al., 1989; Craine, 1989; study evaluations copied from the 1993 JMPR
without further evaluation).

5
Guinea-pigs
32
P-Labelled diazinon was administered orally or subcutaneously to male guinea-pigs at a dose level of
45 mg/kg bw. After oral dosing, 80% of the applied radioactivity was excreted in the urine within 48
hours, whereas faecal excretion was 10% mostly eliminated within the first day. After subcutaneous
dosing, urinary excretion was 53%, faecal excretion was minimal. Highest residues were found in the
caecum corresponding to 36% and 5% of the radioactivity administered 16 hours after oral and
subcutaneous dosing, respectively. About 1–2% of the radioactivity was found in the liver after 16
hours. During the 7-day observation period, over 87% of the dose was eliminated in the excreta, mainly
in the urine, indicating that the large amounts found in the caecum after oral treatment ultimately left
the body via the kidneys. The accumulation of radioactivity in the caecum also following subcutaneous
injection indicates that this tissue may play a role in metabolism or elimination of diazinon and/or its
metabolites in guinea-pigs (Kaplanis, Louloudes & Roan, 1962; study evaluation copied from the 1993
JMPR without further evaluation).
Hens
The following studies measured the elimination of 14C-labelled diazinon by hens:
After oral administration of 14C-diazinon to 4 laying hens by capsule for 7 consecutive days at daily
doses of 2.8 mg/animal corresponding to 25 ppm, 79% of the total dose applied was eliminated in the
excreta and 0.1% was found in the tissues at sacrifice, about 24 hours after the final dose. Highest
tissue levels of 0.15 ppm were found in the kidney. Maximum residues in eggs amounted to 0.07 ppm.
The treatment caused some reduction in body weight in most animals but had no influence on the
general health condition (Simoneaux, 1988b; Simoneaux et al., 1988; Burgener & Seim, 1988; study
evaluations copied from the 1993 JMPR without further evaluation).
Dogs
In an intravenous study, 14C-ethoxy-labelled diazinon was injected at a dose level of 0.2 mg/kg bw
[into dogs]. After 24 hours, 58% of the applied radioactivity was recovered in urine (Iverson, Grant &
Lacroix, 1975; study evaluation copied from the 1993 JMPR without further evaluation).
Goats
Two lactating goats were treated daily with 14C-labelled diazinon by capsule for four consecutive days
at a dose of 150 mg/animal (4 mg/kg bw). Urinary excretion amounted to 64% of the applied dose,
whereas faeces contained an average of 10%. Highest residues of 2 ppm 14C were found in kidneys
whereas the levels in the other tissues ranged from 0.2 to 1.2 ppm. Highest levels in milk were 0.5 ppm
(Pickles & Seim 1988; Simoneaux, 1988a,c; Simoneaux et al., 1988).
Cows
According to a study by Robbins, Eddy & Hopkins (1957):
A lactating cow was orally treated by capsule with a dose level of 20 mg/kg bw of 32P-labelled
diazinon. About 74% of the applied dose was excreted in the urine within 36 hours, and 6% in the
faeces. The cumulative percentage of total dose in milk 36 hours after treatment was less than 0.08%
(Robbins, Eddy & Hopkins, 1957; study evaluation copied from 1993 JMPR without further
evaluation).
In summary, diazinon is rapidly and almost completely absorbed and eliminated after oral
application. Excretion occurs mainly via the kidneys. Diazinon or its metabolites do not accumulate in
the body.

6
(b) Dermal route

Rats
According to a study by Ballantine, Marco & Williams (1984):
Groups of 4 rats were dermally treated with dose levels of 1 or 10 mg/kg bw 14C-diazinon dissolved in
tetrahydrofuran. Renal excretion ranged from 70–80% of the applied dose at both dose levels over the
6 days observation period; elimination in faeces was usually less than 10%. Most of the radioactivity
was eliminated within 48 hours after application. After 72 hours, less than 1% of the applied dose was
found on the skin. Highest tissue residues were measured 8 hours after treatment varying between
0.1 and 0.4 ppm in the low-dose animals. Residues in the high-dose animals were correspondingly
higher. These results show that diazinon is easily absorbed through the skin (Ballantine, Marco &
Williams, 1984; study evaluation copied from 1993 JMPR without further evaluation).
Sheep
The following studies assessed the effects of dermal exposure in sheep:
Two sheep were dermally exposed for 3 days to diazinon at a daily dose of 40 mg/kg bw. The sheep
were sacrificed six hours after the final application. Radiolabelled residues were observed in all tissues
at levels ranging from 2.2 to the highest value of 13 ppm in kidney. Because the study was designed to
allow the identification of metabolites in sheep tissue after topical application of 14C-diazinon, no
results were presented with respect to elimination of the compound (Capps et al., 1990; Pickles & Seim
1990; study evaluations copied from 1993 JMPR without further evaluation).
1.2 Biotransformation
Biotransformation of diazinon has been extensively studied in mammalian and avian species.
Considerable breakdown occurs in all the species studied.
(a) In vitro studies

The following study evaluations were copied from the 1993 JMPR without further evaluation:
Diazinon was incubated with liver microsomes from different avian species, rat, guinea-pig, pig, sheep
and cow. Metabolites identified included hydroxydiazinon, isohydroxydiazinon, dehydroxydiazinon,
their oxons and diazoxon. Yields and rates of production of these metabolites varied between the
different species (Machin et al., 1975).
In vitro studies using microsomal preparations from rat liver showed that diazinon was converted
rapidly to water-soluble metabolites (Dahm, 1970). The oxidation of diazinon by the microsomal
enzyme system fortified with NADPH [nicotinamide adenine dinucleotide phosphate (reduced)] or
NADH [nicotinamide adenine dinucleotide (reduced)] occurred through hydroxylation of the ring alkyl
side-chain, desulfuration, and cleavage of the arylphosphate bound. The major metabolic products of
diazinon were hydroxydiazinon, diazoxon and hydroxydiazoxon. Other metabolites identified were 2-
isopropyl-4-methyl-6-hydroxypyrimidine, 2-(2'-hydroxy-2'-propyl)-4-methyl-6-hydroxypyrimidine,
diethyl-phosphorothioic acid and diethylphosphoric acid, which were all produced by the cleavage of
the arylphosphate bound (Shishido et al., 1972).
Diazoxon, the active toxicant, formed by the oxidation of diazinon through desulfuration was degraded
mainly by hydrolysis resulting in the two metabolites diethylphosphoric acid and 2-isopropyl-4-methyl-
6- hydroxypyrimidine (Shishido & Fukami, 1972).
A glutathione conjugate S-(2-isopropyl-4-methyl-6-pyrimidinyl) glutathione was also identified. This
compound was formed by conjugation of reduced glutathione and the pyrimidinyl moiety of diazinon
with the simultaneous cleavage of the phosphate ester bound (Shishido, Usui & Fukami, 1972). In
insects, diazoxon is degraded slowly by the microsomal mixed function oxidase system, while no
diazoxon-hydrolyzing enzyme was found (Shishido & Fukami 1972; Yang, Hodgson & Dautermann,
1971).

7
(b) In vivo studies

Rats
In a metabolic study, Mücke, Alt & Esser (1970) showed that
… rats treated with 14C-labelled diazinon (2-14C, 4-14C or ethoxy-14C labels) by the oral route (4 mg/kg
bw) showed that the parent compound is degraded rapidly yielding the 3 pyrimidinols, 2-isopropyl-6-
methyl-4(1H)-pyrimidinone, 2-(alpha-hydroxyisopropyl)-6-methyl-4(1H)-pyrimidinone and its beta
isomer as main urinary metabolites. A number of polar unidentified substances was also found. In
addition to small amounts of unchanged diazinon the same metabolites were also found in faeces.
Diazoxon as a labile and transient intermediate was absent in the extracts of urine and faeces. The
absence of radiolabelled CO2 indicated that no cleavage of the pyrimidine ring took place. The
intravenous application of these main metabolites revealed that the pyrimidinols are further degraded
yielding some unidentified polar metabolites (Mücke, Alt & Esser, 1970; study evaluation copied from
the 1993 JMPR without further evaluation).
A similar metabolic pattern was found when

…[one group of rats was] treated with single oral doses of 10 or 100 mg/kg bw of 14C-labelled
diazinon. Another group was preconditioned for 14 days with unlabelled diazinon and then dosed with
10 mg/kg bw of 14C-labelled diazinon…. In urine, the three pyrimidinols accounted for 65% of the
applied radioactivity, whereas 15% consisted of polar non-identified metabolites and trace amounts of
diazinon (0.11%), hydroxydiazinon (0.12%) and diazoxon (0.14%) (Capps et al., 1989).
Dogs
Metabolic studies in dogs were performed after oral and intravenous administration of ring-labelled and
ethoxy-labelled diazinon, respectively. After [intravenous administration] application the 14C-ethoxy-
labelled diazinon, diethylphosphoric acid (DEP) and diethylphosphorothioic acid (DETP) were
detected in urine, whereas after the oral dosing with the 14C-ring-labelled diazinon, the pyrimidinols 2-
isopropyl-6-methyl-4(1H)-pyrimidinone and 2-(alpha-hydroxyisopropyl)-6-methyl-4(1H)-
pyrimidinone could be identified. In contrast to the results of an in vitro study (Shishido & Fukami,
1972; study evaluation copied from 1993 JMPR without further evaluation) no evidence was given
from this dog study that the cleavage of the ester bond was glutathione-mediated (Iverson, Grant &
Lacroix, 1975; study evaluation copied from the 1993 JMPR without further evaluation).
Cows, sheep and other mammalian and nonmammalian species

In a metabolic study with cows treated with an oral dose of 20 mg/kg bw 32P-labelled diazinon, DEP
and DETP were found as urinary end-products of diazinon metabolism (Robbins, Eddy & Hopkins,
1957; study evaluation copied from 1993 JMPR without further evaluation).
The pyrimidinols were also identified as the major metabolites in urine and tissues of dermally treated
sheep (Capps et al., 1990; study evaluation copied from 1993 JMPR without further evaluation) and
orally treated goats and hens (Simoneaux 1988c,d; Simoneaux et al., 1988; study evaluations copied
from 1993 JMPR without further evaluation). The respective glucuronides were also identified in these
species (Sachsse & Bathe, 1976; Simoneaux 1988c,d; Simoneaux et al., 1989; Capps et al., 1990; study
evaluations copied from the 1993 JMPR without further evaluation).
In summary, diazinon was found to be readily degraded and the metabolites formed were mainly
eliminated via the kidneys. The main degradative pathway of diazinon in mammals includes the
oxidase/hydrolase-mediated cleavage of the ester bond leading directly and via diazoxon to the
pyrimidinol derivative 2-isopropyl-6-methyl-4(1H)-pyrimidinone, which is further oxidized at the
isopropyl substituent resulting in the hydroxy pyrimidinols either excreted as such or further degraded
to more polar metabolites. Metabolites with an intact pyrimidinyl phosphorus ester bond such as
hydroxydiazinon, diazoxon and its hydroxy derivative are only transient products which are ultimately
cleaved to their corresponding pyrimidine analogs. The uncleaved products were found only in in vitro

8
studies (Hagenbuch & Mücke, 1985; study evaluation copied from the 1993 JMPR without further
evaluation).
[Fig. 2] presents the proposed metabolic pathway of diazinon in mammals.
Fig. 2. Proposed metabolic pathway for diazinon in the rat
2. Toxicological studies
2.1 Acute toxicity
The results of acute toxicity tests on diazinon are summarized in Table 1.

9
Table 1. Summary of acute toxicity studies of diazinon

Species Strain Sex Route Purity (%) Result Reference
Mouse N/S M+F Oral N/S LD50 187 mg/kg bw Bathe (1972a) a
Mouse ICR M+F Oral 96.0 LD50 177 mg/kg bw Ishige et al.
(males) and 178 mg/kg (1986)
bw (females)
Rat N/S M+F Oral 97.1 LD50 422 mg/kg bw Bathe & Gfeller
(1980) a
Rat Sprague M+F Oral 87.9 LD50 1 350 mg/kg bw Kuhn (1989a)
Dawley (males) and 1 160 mg/kg
bw (females)
Rat Sprague M+F Oral 94.6 LD50 1 129 mg/kg bw Dreher (1997)
Dawley (males) and 1 155 mg/kg
bw (females)
Rat N/S M+F Oral 95.7 LD50 300 mg/kg bw Piccirillo (1978) a
Rat N/S M+F Oral 96.1 LD50 1 031 mg/kg bw Schoch & Gfeller
(males) and 870 mg/kg (1985) a
bw (females)
Rat N/S M+F Oral N/S LD50 > 2 150 mg/kg bw Bathe (1972b) a
Rabbit Albino M+F Dermal 94 LD50 > 2 000 mg/kg bw Nissimov (1984a)
Rabbit New M+F Dermal 87.9 LD50 > 2 020 mg/kg bw Kuhn (1989b)
Zealand
White
Rat N/S M+F Inhalation 87.9 LC50 > 2.327 mg/L Holbert (1989) a
Rat Sprague M+F Inhalation 88 LC50 > 5.44 mg/L Holbert (1994)
Dawley
Rat Wistar M+F Inhalation 96.02 LC50 3.1 mg/L Jackson (1987)
Rat Sprague M+F Inhalation 93.0 LC50 > 5.00 mg/L Cummins (1985)
Dawley (males) and 4.873 mg/L
(females)
bw: body weight; LC50: median lethal concentration; F: female; LD50: median lethal dose; M: male; N/S: not stated
a
Study evaluation copied from 1993 JMPR without further evaluation.
(a) Dermal irritation

Diazinon was tested for its potential as an irritant on rabbit skin. No skin irritation was
observed in any of the six animals in one study (Nissimov, 1984b), and very slight to well-defined
erythema and very slight oedema was observed in all six animals in another study; no effects on the
skin could be seen after 14 days (Kuhn, 1989c).
(b) Ocular irritation

Diazinon was evaluated for its potential as an irritant in rabbit eyes. In one study, conjunctival
redness and/or chemosis was observed in four out of six animals 1 hour after instillation. No effects
were seen after 24 hours (Nissimov, 1984c). In another study, conjunctival redness, chemosis and
discharge was observed in all six animals 1 hour after instillation. No effects were seen after 72 hours
(Kuhn, 1989d).

10
(c) Dermal sensitization

In a skin-sensitization study, 10 guinea-pigs were topically treated with undiluted diazinon for
three treatments and with a 10% volume per volume (v/v) solution in ethanol for each of the seven
successive treatments (every 2–3 days). One animal died on day 7. Two weeks after the last induction
treatment, the animals were challenged with a 10% v/v solution in ethanol. No skin reactions were
observed after the challenge treatment (Kuhn, 1989e).
Diazinon caused delayed hypersensitivity after a Magnusson–Kligman maximization test in
Dunkin–Hartley guinea-pigs (Cummins, 1987a).
2.2 Short-term studies of toxicity

Although reported in the summaries of the following studies, inhibition of plasma
cholinesterase was not utilized as a criterion for the NOAEL; inhibition of erythrocyte or brain
acetylcholinesterase activity may be used as an indicator of an adverse effect of anticholinesterase
pesticides. Inhibition of activity greater than 20% compared with that of controls is considered
adverse and toxicologically significant.
(a) Oral administration

Mice
In a 90-day dose range-finding toxicity study, groups of 10 male and 10 female B6C3F1 mice
were fed diazinon (purity 98%) ad libitum in the diet at concentrations of 0, 50, 100, 200, 400, 800,
1600 or 3200 ppm for 13 consecutive weeks. The study was of limited value since only mortality,
body weight and necropsy findings were reported.
Mortality was observed in males and females at 200 ppm (1/10 animals per sex), 1600 ppm
(10/10 animals per sex) and 3200 ppm (10/10 animals per sex). At week 13, body weights were
reduced by more than 10% for males (84% of control value) and females (78% of control value) at
800 ppm. No gross abnormalities were observed in necropsied animals at any dose, and microscopic
examination of the tissues of the animals that survived the highest doses showed no pathological
changes (Angel et al., unknown year; also referenced as NTP, 1979, or NCI, 1979).
Rats
In a 28-day cholinesterase inhibition feeding study, groups of 15 male and 15 female Sprague
Dawley Crl:CD[BR] rats were fed diazinon (purity 88%) in the diet at concentrations of 0, 0.3, 30,
300 or 3000 ppm (equal to 0.02, 2.3, 23 and 213 mg/kg bw per day in males and 0.02, 2.4, 23 and
210 mg/kg bw per day in females, respectively). All the test animals were examined twice daily for
clinical signs and mortality. Individual body weights and feed consumption were recorded weekly.
Five animals per sex per group were terminated at weeks 1, 2 and 4. The cerebellum, cerebral cortex,
hippocampus, cerebrum, striatum and thoracic spinal cord were dissected to assess any differences in
regional sensitivity. Plasma and erythrocyte cholinesterase activities were measured at 0, 1, 2 and
4 weeks after exposure; central nervous system cholinesterase activity was measured at termination
(4 weeks after exposure).
There were no deaths and the predominant treatment-related clinical sign was muscle
fasciculations first observed on day 8 in both sexes (3/15 males and 14/15 females) at 3000 ppm.
Diarrhoea was also observed in three females from day 8. Body-weight gain in males was
significantly reduced (P < 0.01) by 54%, 29%, 26% and 26%, respectively, after each week of
treatment at 3000 ppm. Females at the same dose also had a reduced weekly body-weight gain of
103%, 40%, 45% and 40% (P < 0.01) with all except the gain during week 3 being statistically
significant. Feed consumption at 3000 ppm tended to be lower for females, although this reduction
was not significant (P < 0.01) except during week 1 for males (21%) and females (27%). The mean

11
percentage reductions in cholinesterase activity for each treatment group and averaged over the three
measurements are shown in Table 2.
Table 2. Change in cholinesterase activity in rats exposed to diazinon in the diet for 28 days
Mean per cent change in cholinesterase activity per dose of diazinon
Males Females
0.02 2.3 23 213 0.02 2.4 23 210
mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Exposure bw per bw per bw per bw per bw per bw per bw per bw per
Tissue (weeks) day day day day day day day day
Plasma Week 1 −14* −59** −88** −96** −10 −81** −95** −98**
Week 2 −17 −59** −84** −91** −2 −81** −93** −97**
Week 4 −5 −51** −77** −87** −32 −81** −94** −96**
Erythrocytes Week 1 −1 −39** −89** −94** −9 −38** −86** −94**
Week 2 0 −55** −83** −85** −8 −59** −79** −89**
Week 4 −4 −58** −64** −74** +5 −57** −88** −82**
Cerebellum Week 1 +4 −2 −8* −67** +12 +14 −49** −88**
Week 2 −6 −1 −22** −72** +6 −6 −60** −81**
Week 4 +2 +6 −15* −70** −1 −6 −60** −94**
Cerebral Week 1 −15 −7 −14 −77** +5 −1 −47** −92**
cortex
Week 2 −2 +16 0 −80** +6 +13 −62** −91**
Week 4 +4 +3 −10 −84** +7 −6 −72** −91**
Striatum Week 1 −1 +4 −13 −82** −6 0 −58** −95**
Week 2 −11 −2 −15 −82** −4 +3 −73** −95**
Week 4 +3 +5 −5 −84** −4 −9 −78** −97**
Hippocampus Week 1 +5 +6 +2 −79** +3 +1 −51** −92**
Week 2 +5 0 −14 −79** −5 −2 −72** −94**
Week 4 −5 −9 −9 −84** +11 +2 −71** −94**
Thoracic Week 1 +15 +10 −12 −73** −10 −8 −44** −89**
spinal cord
Week 2 +1 −1 −8 −72** +4 −1 −76** −96**
Week 4 +3 −3 −11 −71** +13 +21 −62** −89**
bw: body weight; *: P ≤ 0.05; **: P ≤ 0.01 (Dunnett t-test)
Results expressed as mean increase (+) or mean decrease (−) in cholinesterase activity relative (%) to the control.
Source: Chang (1994)
Significant and dose-related inhibition of plasma and erythrocyte cholinesterase was evident
in males and females at concentrations equal to and greater than 30 ppm (2.3 mg/kg bw per day) from
week 1. Cholinesterase inhibition in the brain showed little regional variation although females
appeared to be more sensitive with significant dose-related inhibition (P < 0.01) observed in all tested
brain regions from week 1 at 300 ppm (23 mg/kg bw per day), while significance at the same dose
was only apparent in the cerebellum of males. Therefore, in both sexes cholinesterase inhibition in
plasma and erythrocytes was at least an order of magnitude more sensitive to treatment with diazinon
than that observed in regional areas of the brain.
The NOAEL was 0.3 ppm (equal to 0.02 mg/kg bw per day) based on the inhibition of
erythrocyte cholinesterase activity at 30 ppm (equal to 2.3 mg/kg bw per day) (Chang, 1994).

12
In a published study:
Groups of 50 male and 50 female Wistar rats (aged 6 weeks) were given semi-purified diets containing
diazinon (purity, 99.2%) at a concentration of 0 or 2 ppm (equivalent to 0.2 mg/kg bw per day) for 7
days, or, 0 or 25 ppm (equivalent to 2.5 mg/kg bw per day) for 30 days. The diet was prepared before
the start of the studies by mixing diazinon suspended in corn oil with a semisynthetic diet. Food
consumption and body weight were recorded twice weekly and clinical signs were monitored daily.
Blood for measurements of plasma and erythrocyte cholinesterase activity were collected from random
groups of 10 rats at various times (at 3–5 days intervals) except for those at 0 or 25 ppm in the 30-day
study, where the same rats were sampled at each bleed. At days 15 and 30 in the 30-day study, brain
cholinesterase activity was measured in 6 sacrificed rats per group. All cholinesterase activities were
measured using a radiometric method, with tritiated acetylcholine as the substrate.
There were no clinical signs observed at any dose. Treated rats at 2 ppm or 25 ppm had a similar food
consumption and body-weight gain except for female rats at 25 ppm for which mean food consumption
increased by 9% from day 15 onwards. The maximum mean percentage reductions in cholinesterase
activity measured in the two studies are shown in Table 3.
Table 3. Maximum inhibition of cholinesterase activity in rats given diets containing

diazinon
Mean percentage reduction in cholinesterase activity
Dietary Plasma Erythrocyte Brain
concentration Duration
(ppm) (days) Males Females Males Females Males Females
2 7 5 29* [12] 3 ND ND
25 30 52* 76* 44* 85* [3] 6
ND, not determined; ppm: parts per million; *: P ≤ 0.05
Values in square brackets indicate the extent (%) to which the measured activity was greater than that in controls.
Source: Davies & Holub (1980a), copied from the 2006 JMPR without further evaluation
Relative to males, cholinesterase activity in females appeared to be more sensitive to inhibition after
exposure to diet containing diazinon. The NOAEL for this study was 2 ppm (equivalent to 0.2 mg/kg
bw per day) based on a statistically significant (> 20%) inhibition of erythrocyte acetylcholinesterase
activity at the next highest dose of 25 ppm (equivalent to 2.5 mg/kg bw per day) (Davies & Holub,
1980a; study evaluation copied from the 2006 JMPR without further evaluation).
It should be noted that 2 ppm was only fed for 7 days, whereas 25 ppm was fed for 30 days.
In a 90-day dose range-finding toxicity study, groups of 10 male and 10 female F344
(Fischer) rats were fed diets containing 0, 50, 100, 200, 400, 800, 1600 or 3200 ppm diazinon (purity
98%) (equivalent to 0, 5, 10, 20, 40, 80, 160 and 320 mg/kg bw per day) for 13 consecutive weeks.
The study was of limited value since only mortality, body weight and necropsy findings were
reported.
At 3200 ppm, three males and four females died. Body weights were reduced at week 13 at
1600 and 3200 ppm (93% and 78% of control body weight for males, and 85% and 67% for females,
respectively). No gross abnormalities were observed in necropsied animals at any dose, and
microscopic examination of tissues of animals surviving the high doses showed no pathological
changes (NTP, 1979; also referenced as Angel et al., unknown year, or NCI, 1979).

13
In a 3-month toxicity study, groups of 15 male and 15 female Sprague Dawley

Crl:CD[SD]BR rats were fed diets containing diazinon (purity 87.7%) at concentrations of 0, 0.5, 5,
250 or 2500 ppm (equal to 0.03, 0.3, 15 and 168 mg/kg bw per day in males 0.04, 0.4, 19 and 212
mg/kg bw per day in females, respectively). All the animals were observed at least once daily for
clinical signs and mortality. Body weights and feed consumption were recorded at predosing and
weekly thereafter. Ophthalmoscopic, haematological and clinical chemistry observations, including
cholinesterase activity (by colorimetric assay), were conducted during week 13. Organs were weighed
and macroscopic pathology and histopathology conducted. Urine analysis was performed prior to
study termination on weeks 12 and 13.
There were no treatment-related deaths. Potentially treatment-related clinical changes
occurred only at 2500 ppm, with soft faeces (10/15 males and 15/15 females) and a degree of
hypersensitivity to touch and sound intermittently observed throughout treatment in both sexes
(12/15 males and 15/15 females); aggressive behaviour was also noted in three males. Treatment-
related body-weight loss was observed at 2500 ppm; reduced body-weight gain was most evident
(statistically significant, P < 0.01) from day 14 to day 42 in males and day 7 to day 49 in females so
that at the end of treatment males were 6% lighter and females 13% lighter than their controls.
Although no significant changes in water consumption occurred, feed consumption was reduced at
2500 ppm, but only during the first week of treatment in males (17%; P < 0.01) and for the first
2 weeks in females (31% and 13% respectively; P < 0.01).
Ophthalmoscopic examination did not reveal any treatment-related changes. Haematological
assessments showed dose-dependent changes in erythrocytic parameters in females (i.e. erythrocyte
count: 1.3%, 1.8%, 2.8% and 9.5%, respectively; haemoglobin concentration: 1.5%, 2%, 3.6% and
4.1% respectively; erythrocyte volume fraction, 0.018, 0.018, 0.044 and 0.077 respectively), although
only erythrocyte volume fraction at 250 (P < 0.05) and 2500 ppm (P < 0.01) achieved statistical
significance. A corresponding significant (P < 0.01) increase in reticulocytes (3.3-fold) was also
observed in females at 2500 ppm (changes at lower concentrations were not examined). Increased
leukocyte count (P < 0.05) in females at 2500 ppm and eosinophil count (P < 0.05) in males at
0.5 ppm were probably incidental findings as there did not appear to be any dose–response
relationship. Clinical chemistry changes were also characterized by a lack of any dose–response
relationship so that in males the reduced cholesterol (by 18%; P < 0.05), elevated alanine
aminotransferase (by 16%; P < 0.05) at 2500 ppm, and reduced sodium (by 1%; P < 0.05) observed at
5 ppm may not be attributable to treatment. Similarly, in females, reduced alanine aminotransferase
(by 46%: P < 0.01), sodium concentration (1.2%; P < 0.05) and chloride concentration (3.4%;
P < 0.05) and elevated phosphorus concentration (24%; P < 0.01) at 2500 ppm, and reduced alanine
aminotransferase activity at 250 (by 36%; P < 0.05) and 0.5 ppm (by 39%; P < 0.05) may also be
unrelated to treatment. However, reduced cholinesterase activity in erythrocytes, plasma and brain
was attributed to the treatment. These mean percentage reductions in cholinesterase activities are
shown in Table 4.
Cholinesterase activity was significantly inhibited (P < 0.01) in the erythrocytes of females
and the plasma of both males and females at 5 ppm; brain cholinesterase activity was significantly
inhibited once dietary diazinon concentrations reached 250 ppm in females and 2500 ppm in males.
Apart from a significantly increased specific gravity of urine (males: 2.2%; females: 1.6%; P < 0.01
for both) at 2500 ppm that was associated with nonsignificant reductions in urine volume (males:
20%; females: 17%) and water consumption (males: 17%; females: 12%), urine analysis was similar
across treatment groups.

14
Table 4. Change in cholinesterase activity in rats exposed to diazinon in the diet for three months
Mean per cent change in cholinesterase activity (%)
Dietary Plasma Erythrocytes Brain
concentration of
diazinon (ppm) Males Females Males Females Males Females
0.5 +10 −12 +4 −4 +7 +2
5 −26** −78** −4 −17** +5 0
250 −89** −97** −27** −41** −4 −41**
2500 −97** −98** −26** −42** −49** −57**
ppm: parts per million; **: P ≤ 0.01
Results expressed as mean increase (+) or mean decrease (−) in cholinesterase activity relative to the control as a
percentage (%).
Source: Singh, Arthur & McCormick (1988)
Macroscopic inspection of organs at necropsy showed no gross abnormalities, although the

absolute (15%; P < 0.05) and relative-to-body weight (20%; P < 0.01) of the liver was significantly
increased in females at 2500 ppm. The absolute weights of the livers in the males at the same dose
also increased (4%) as did the relative-to-body weight (7%), although neither change was significant.
Although neither males nor females at 2500 ppm had significantly increased liver weights relative to
brain weights, an increase of 6.5% and 12% respectively suggests a physiological adaptation, an
assertion consistent with centrilobular hepatocellular hypertrophy observed in 13 out of 15 females at
2500 ppm (and 3 out of 15 at 250 ppm). The only other significant (P < 0.05) organ-weight change
was the relative increase in weight of the kidneys (12%) in females at 2500 ppm.
In conclusion, rats fed diets containing diazinon at 2500 ppm lost body weight, were
hypersensitive to touch and sound, and excreted soft faeces. Increased liver weight resulting from
hepatocellular hypertrophy was also observed in females at 2500 ppm. The NOAEL was 5 ppm (equal
to 0.3 mg/kg bw per day) based on the inhibition of erythrocyte and brain cholinesterase activity at
250 ppm (equal to 15 mg/kg bw per day) (Singh, Arthur & McCormick, 1988).
In a 3-month study, groups of 15 male and 15 female Sprague Dawley Crl:CD[SD]BR rats
were fed diets containing diazinon (purity 88%) at concentrations of 0, 0.3, 30, 300 or 3000 ppm for
13 weeks (equal to 0.017, 1.7, 17 and 177 mg/kg bw per day in males and 0.019, 1.9, 19 and
196 mg/kg bw per day in females). Feed consumption and body weight were measured weekly and
clinical monitoring performed twice daily. All the rats were palpated weekly (although the
justification for this in a 3-month study was not given). Ophthalmoscopy was performed before and at
the completion of treatment (though not for the satellite group). In each group, five rats were used
exclusively as the source of blood to assess the extent of cholinesterase inhibition (by colorimetric
assay) during weeks 4, 8 and 13. Regional brain cholinesterase activity in these five rats was then
measured at week 13. Neurological tests, namely functional observational battery and figure-eight
maze motor activity, were performed in the presence of white noise for 10 rats in each group 1 week
before treatment and again at week 4, 8 and 13 of treatment. The functional observational battery
comprised observations in the home cage, manipulative measurements, open-field and reflex
responses, neuromuscular tests and physiological functions.
After each functional observational battery, the rats in each group were individually tested in a
figure-eight maze for spontaneous activity (measured by light-beam interruption). After 13 weeks of
treatment, the functional observational battery–tested rats were anaesthetized, terminated by whole-
body perfusion (with glutaraldehyde) and necropsied. The following tissues were
histopathologically examined: brain, spinal cord with ganglia (at each level, i.e. cervical, thoracic,
lumbar and sacral), peripheral nerves (left and right sciatic, fibular, tibial, lateral cutaneous sural),
trigeminal (Gasserian) ganglia, eyes with associated optic nerves, skeletal muscle and any gross

15
lesions detected. Since a preliminary histopathological assessment of sections from the controls and
rats at 3000 ppm showed no significant differences other than for some lesions in the nerve roots of
the sacral spinal cord, this tissue was the only one examined in detail from rats at 0.3 and 300 ppm.
All the rats survived treatment, although clinical signs consistent with organophosphate
toxicity were observed in males (i.e. hypersensitivity to touch and sound) and females (i.e. muscle
fasciculations and tremors) at 3000 ppm. At this concentration, body-weight gain was significantly
(P < 0.01) reduced for the first 6 weeks in males and 12 weeks in females. This reduced weight
gain, associated with a reduction in feed consumption that achieved significance (P < 0.01) during
weeks 1 and 2 in males (average, 15%) and weeks 1, 2 and 4 (average, 14%) in females, resulted in a
generally reduced body weight that achieved significance during weeks 1 to 6 (average, 11%) in
males and weeks 1 to 9 (average, 11%) and 11 in females. Significant changes in body weight
and feed consumption that occurred at other concentrations were considered not treatment-related
because they were transient, lasting no more than 1 week, and had no apparent trend. There were no
treatment-related ophthalmoscopy findings.
Clear treatment-related functional observational battery findings were observed at 3000 ppm.
For males, forelimb and hindlimb grip strength was reduced (average for both, 16%) throughout
treatment (i.e. weeks 4, 8 and 13) but did not achieve significance. These reductions in limb grip
strength, which averaged 25% in females, were significant at weeks 4, 8 and 13 for forelimb grip
strength (P < 0.01) and at week 4 for hindlimb grip strength (P < 0.05). Hindlimb foot splay in
females was also significantly reduced (P < 0.01) by 32% at week 4 and by 26% and 23% (both
not statistically significant) at weeks 8 and 13 respectively. Rectal temperature in females was
significantly reduced (P < 0.01) at week 13, and four had signs of dehydration at week 4. Functional
observational battery changes observed at other concentrations were probably unrelated to treatment
because they did not appear to be dose related. Similarly, figure-eight maze activity in all groups
appeared to be unchanged by treatment.
As shown in Table 5, cholinesterase activity in plasma and erythrocytes was significantly
reduced (P < 0.01) as a function of dose so that, as anticipated, no inhibition was observed at the
lowest tested concentration of 0.3 ppm. Inhibition of plasma cholinesterase activity was generally
more pronounced than that of erythrocyte cholinesterase activity at 30, 300 and 3000 ppm in males
and females, except in males at 30 and 300 ppm. Cholinesterase activity in all regions of the brain
was significantly (P < 0.01) reduced at 300 and 3000 ppm in females and at 3000 ppm in males. The
spurious values observed in the striatum of males at 30 ppm and 300 ppm were attributed to an
inconsistent dissection technique for this small brain region.
Table 5. Change in cholinesterase activity in rats exposed to diazinon in the diet for 13 weeks
Dietary
Cortex
concentration Time
Plasma Erythrocytes Cerebelluma hippocampusa Striatuma
of diazinon point
(ppm) (weeks) M F M F M F M F M F
0.3 4 +5 +12 −16 −19 – – – – – –
8 +2 +3 +11 +7 – – – – – –
13 +5 +5 −11 +9 0 0 +2 +10 −4 −26
Mean 4–13 +4 +7 −5 −1 – – – – – –
30 4 −37* −79* −60** −60** – – – – – –
8 −39* −83* −37* −53* – – – – – –
13 −45* −86* −75* −59* −10 +2 +21 −25* +89 −18
Mean 4–13 −40 −83 −57 −57 – – – – – –
300 4 −72* −91* −71** −84** – – – – – –
8 −78* −94* −86* −81* – – – – – –

16

Dietary
Cortex
concentration Time
Plasma Erythrocytes Cerebelluma hippocampusa Striatuma
of diazinon point
(ppm) (weeks) M F M F M F M F M F
13 −79* −95* −84* −75* −14 −55** −6 −75** +138 −74**
Mean 4–13 −76 −93 −80 −80 – – – – – –
3000 4 −81** −94** −72** −85** – – – – – –
8 −85** −96** −80** −88** – – – – – –
13 −85** −97** −86** −79** −64** −89** −77** −92** −62** −96**
Mean 4–13 −84 −96 −79 −84 – – – – – –
F: female; M: male; ppm: parts per million; *: P ≤ 0.05; **: P ≤ 0.01
Results expressed as mean increase (+) or decrease (−) in cholinesterase activity relative to the control as a percentage (%).
a
Brain cholinesterase activity was measured after termination at week 13.
Source: Pettersen & Morrissey (1994)
No treatment-related findings were observed after gross necropsy, and there were no
histopathological lesions apart from the mild focal degeneration of the sacral spinal-cord nerve root
axons detected in the preliminary screen in two females at 3000 ppm.
In conclusion, rats fed diets containing diazinon at concentrations of 0.3, 30, 300 or 3000 ppm
for 13 weeks lost body weight and had characteristic clinical signs of organophosphate poisoning at
the highest concentration tested (3000 ppm). Functional observational battery studies showed a
reduction in fore- and hindlimb grip strength throughout the treatment in all the rats at 3000 ppm;
with reduced hindlimb foot splay and rectal temperature in females. The NOAEL was 0.3 ppm
(equal to 0.017 mg/kg bw per day) based on significant inhibition of erythrocyte
acetylcholinesterase activity at 30 ppm (equal to 1.7 mg/kg bw per day) (Pettersen & Morrissey,
1994).
Groups of 50 female Wistar rats were fed semi-purified diets containing diazinon (purity
99.2%) at concentrations of 0, 5, 10 or 15 ppm (equivalent to 0, 0.5, 1 and 1.5 mg/kg bw per day,
respectively) for 92 days. Female rats were selected for this study because a previous short-term
study had shown them to be more sensitive than males to inhibition of cholinesterase (Davies &
Holub, 1980a). In order to increase the accuracy for determining a NOAEL based on inhibition of
cholinesterase activity in plasma, erythrocytes and brain, a second and a third study, each with a
reduced duration and concentration of diazinon, were performed. The second study involved groups
of 16 rats (mean body weight, 149 g) fed diets containing diazinon at concentrations of 0, 1, 2, 3 or 4
ppm for 42 days. In the third study, groups of 10 rats (mean body weight, 143 g) were fed diets
containing diazinon at concentrations of 0, 0.1, 0.5, 1 or 2 ppm for 35 days. Food consumption and
body weight were recorded twice weekly and clinical signs were monitored daily.
In the three studies, blood for determining plasma and erythrocyte cholinesterase activity was
collected from groups of 8 to 10 rats at various time points (at intervals of approximately 2–5 days).
Determination of brain cholinesterase activity was confined to the two longer studies in which
satellite groups of six rats were sacrificed at various time points (intervals of 5–14 days).
There were no clinical signs observed at any dose in any of the three studies. Treated rats in
all studies had similar food consumption and body-weight gain except for rats at 0.5 ppm (third study)
where mean body-weight gain was 12% and food consumption 10% less than values for controls. The
maximum mean percentage reductions in cholinesterase activity measured in the three studies are
shown in Table 6.

17
Table 6. Maximum inhibition of cholinesterase activity in rats exposed to diazinon in the diet
Mean per cent reduction in cholinesterase activity (%)
Dietary concentration
of diazinon (ppm) Duration (days) Plasma Erythrocyte Brain
0.1 35 −4 0 –
0.5 35 −16* 0 –
1 35 −28* −16 –
2 35 −42* −12 –
1 42 −33* −8 NS
2 42 −51* −9 NS
3 42 −65* −8 NS
4 42 −61* −4 NS
5 92 −75* −18* −2
10 92 −80* −38* −6
15 92 −85* −55* −2
NS: not significant (approximately equivalent to controls); ppm: parts per million; *: P ≤ 0.05 (Duncan test)
Results expressed as mean decrease (−) in cholinesterase activity relative to the control as a percentage (%).
The apparent significant reduction in cholinesterase activity, due to an unexplained increase in cholinesterase activity in
controls on day 42, was discounted.
Source: Davies & Holub (1980b)
The NOAEL for cholinesterase inhibition in the first study was 5 ppm (equivalent to 0.5
mg/kg bw per day) based on a statistically significant (> 20%) inhibition of erythrocyte
acetylcholinesterase activity at the next higher dose of 10 ppm (equivalent to 1 mg/kg bw per day)
after dosing for 92 days. The NOAEL for females in the second and third studies can be established
at the highest tested doses of 4 ppm (equivalent to 0.4 mg/kg bw per day) and 2 ppm (equivalent to
0.2 mg/kg bw per day) after dosing for 42 and 35 days respectively (Davies & Holub, 1980b).
In another 3-month oral toxicity GLP study conducted in accordance with the test guidelines
of the Ministry of Agriculture, Forestry and Fisheries of Japan, 28 male and 28 female Sprague
Dawley Crl:SD rats were fed diazinon (purity 95.0–95.5%) in the diet at concentrations of 0, 5, 125 or
3000 ppm (equal to 0, 0.3, 7.8 and 198 mg/kg bw per day in males and 0, 0.3, 8.9 and 247 mg/kg bw
per day in females, respectively). Ten male and female rats per group were used to examine subacute
toxicity using common parameters, and six male and female rats per group in both sexes euthanized at
week 2, 4 or 8 to measure erythrocyte cholinesterase activity. The animals in the main groups were
checked weekly; at week 13, grip strength was checked and cholinesterase activities in erythrocyte
and brain measured.
A male in the 125-ppm group died on day 74; the death was not considered treatment related
because other animals in this and higher-dose groups survived until termination. There were no
treatment-related clinical signs. The treatment-related changes are summarized in Table 7. Forelimb
grip strength was weakened at 3000 ppm in females, and motor activities decreased at 3000 ppm in
both sexes. Body weights were significantly (P < 0.01) decreased by about 10% at 3000 ppm in both
sexes during the first 4 weeks for males and 7 weeks for females. These consistent depressions were
considered treatment related. No treatment-related ophthalmological abnormalities were observed.
Urine analysis found lower pH, increased specific gravity and decreased urine volume in both males
and females at 3000 ppm. The female rats at 3000 ppm were found to have slight anaemia.
Statistically nonsignificant increases in alkaline phosphatase and gamma-glutamyltransferase in males
at 3000 ppm were considered to be related to the changes in the liver. The increases in glucose in

18
males at 125 and 3000 ppm and urine nitrogen in females at 3000 ppm were potentially treatment
related although the increases were slight and no corresponding histopathological changes were
observed. Other slight changes in blood chemistry at 3000 ppm were not considered treatment related.
Slight hepatocellular hypertrophy was seen microscopically in males at 3000 ppm. Similarly,
incidences and intensities of hyaline droplets and eosinophils in the kidney proximal tubule
epithelium were increased at 3000 ppm in males only. These changes in males only may have been a
result of accumulation of α2u-globulin, a protein specific to the male rat. However, no additional
histopathological examination was conducted to identify the changes.
Table 7. Summary of treatment-related changes observed in a 90-day oral toxicity study of diazinon
in rats
Treatment-related changes per dose of diazinon
Males Females
Treatment-related
measures 0 ppm 5 ppm 125 ppm 3 000 ppm 0 ppm 5 ppm 125 ppm 3 000 ppm
No. of rats 10 10 9 10 10 10 10 10
Functional observation
Forelimb grip 1 656.23 1 723.07 1 787.04 1 624.67 1 411.85 1 442.76 1 384.93 1 082.07**
strength (g)
Motor activity (count), 1 124.2 1 167.8 922.4 636.9 2 362.2 2 541.5 2 345.5 1 572.1
total of 6 trials
Haematology
Erythrocyte 903.6 881.0 891.2 882.2 855.7 841.8 837.3 825.0*
(104  μL)
Haematocrit (%) 46.21 46.22 44.94 44.70 45.43 45.76 44.60 41.58**
Haemoglobin (g/dL) 15.94 15.81 15.58 15.28 15.72 15.76 15.39 14.45**
Blood chemistry
Aspartate 71.0 75.3 77.8 84.4 64.9 76.6 81.5 72.1
aminotransaminase
(IU/L)
Alkaline phosphatase 266.6 258.4 253.8 376.8 126.3 130.4 150.2 163.4
(IU/L)
Gamma- 0.48 0.78 0.59 1.81 0.88 0.78 0.75 1.34
glutamyltransferase
(IU/L)
Glucose (mg/dL) 159.0 165.7 173.6* 178.1* 149.8 149.5 160.7 144.4
Urinary nitrogen 15.19 13.91 13.66 14.02 14.04 14.10 14.95 16.20*
(mg/dL)
Histopathology
Lung – aggregation, 1 3 2 3 3 2 2 8
macrophage, alveolar
Liver – hypertrophy, 0 0 0 0 0 0 0 7#
hepatocyte,
centrilobular
Kidney – hyaline 3 3 3 9 0 0 0 0
droplet, proximal
tubule epithelium
Kidney – eosinophilic 3 3 3 9 0 0 0 0
body, proximal
tubule epithelium

19
IU: International Unit; no.: number; ppm: parts per million; *: P < 0.01 (Dunnett t-test, one-tailed); #: P < 0.01 (Fisher exact
test)
Source: Sunaga (2010)
Erythrocyte and brain acetylcholinesterase activities are summarized in Table 8. Erythrocyte

acetylcholinesterase activity was decreased consistently by over 20% at 125 and 3000 ppm at each
time point in both sexes. Brain acetylcholinesterase activity was reduced by over 20% at 3000 ppm
for males and at 125 and 3000 ppm for females.
The NOAEL for 90-day oral rat toxicity was 5 ppm (equal to 0.3 mg/kg bw per day) based on
significant inhibition of erythrocyte acetylcholinesterase activity at 125 ppm (equal to 7.8 mg/kg bw
per day) (Sunaga, 2010).
Table 8. Summary of acetylcholinesterase activities in a 90-day oral toxicity study of diazinon in

rats
AChE activity per dose of diazinon
Males Females
Site of AChE
activity / Time 3 000 3 000
point 0 ppm 5 ppm 125 ppm ppm 0 ppm 5 ppm 125 ppm ppm
Erythrocytes
Week 2a 389.3 388.2 225.7# 123.7# 374.0 361.0 224.7# 124.8#
(99.7) (58.0) (31.8) (96.5) (60.1) (33.4)
Week 4a 460.7 452.0 155.2# 108.8# 383.8 364.2 159.5# 105.3#
(98.1) (33.7) (23.6) (94.9) (41.6) (27.4)
Week 8a 330.7 296.2 169.2# 116.2# 317.7 355.7 192.0* 118.2*
(89.6) (51.2) (35.1) (112.0) (60.4) (35.5)
Week 13b 337.1 332.5 175.1# 94.4# 262.2 264.1 124.3# 84.8#
(98.6) (51.9) (16.3) (100.7) (47.4) (32.3)
Brainc 157.5 150.5 147.8# 45.3# 166.6 161.2 115.4# 24.5#
(95.6) (93.8) (28.8) (96.8) (69.3) (14.7)
AChE: acetylcholinesterase; IU: International Unit; ppm: parts per million; *: P < 0.01 (Dunnett t-test, one-tailed);
#: P < 0.01 (Steel test, one-tailed)
Results shown as AChE activity in IU/L with the mean activity relative to control activity as a percentage (%) in
parentheses.
a
Six male and six female rats per group were examined at weeks 2, 4 and 8.
b
Ten, ten and nine male rats at 0, 5, 125 and 3000 ppm, respectively, were examined at week 13. Ten female rats were
examined in each group and at each time point.
Source: Sunaga (2010)
Dogs
In a 90-day toxicity study, groups of four male and four female beagle dogs were fed diets
containing diazinon (purity 87.7%) at concentrations of 0, 0.1, 0.5, 150 or 300 ppm (equal to 0.0034,
0.020, 5.9 and 10.9 mg/kg bw per day in males and 0.0037, 0.021, 5.6 and 11.6 mg/kg bw per day in
females, respectively). Feed consumption and body weight were measured weekly for 2 weeks prior
to treatment and weekly during treatment. Clinical observations were performed daily. Urine was
collected during weeks 5, 9 and 14. Ophthalmoscopic and auditory/physical examinations were
performed before treatment and during weeks 14 and 13, respectively. Haematological and clinical
chemistry, including blood cholinesterase activity, were assessed during treatment weeks 5 and 9 and
then prior to termination (week 13), and brain cholinesterase activity was measured after the terminal

20
kill (week 14). Urine analysis and macroscopic pathology and microscopic pathology were analysed
and organs weighed.
No treatment-related mortalities occurred. The treatment did not adversely affect feed
consumption, organ weights or ophthalmoscopic, haematological, urine analysis or macroscopic
findings. Clinical signs such as emesis or bloody faeces were sporadically observed in males at 300
ppm and in females at 150 ppm. Reduced body-weight gain was observed in females at 150 ppm and
at 300 ppm in both sexes. In males, inhibition of serum cholinesterase activity was 70%, 20% and
15% that of controls at the end of the study at 0.5, 150 and 300 ppm, respectively. Erythrocyte
cholinesterase activity at 75% and 69% of control activity was measured at 150 and 300 ppm, while
brain cholinesterase activity was 69% and 58% of control activity at 150 and 300 ppm, respectively.
In females, a reduction in cholinesterase activity was observed at 150 and 300 ppm: serum and
erythrocyte cholinesterase activities were about 18% and 70% of control activity, respectively, at both
doses, and brain cholinesterase activity was 70% and 55% of control activity at 150 and 300 ppm,
respectively. Other changes in biochemical parameters consisted of a decrease in total protein at 300
ppm in males. The only microscopic alteration that may potentially have been treatment related was
atrophy of the pancreatic acini in one male dog at the highest dose.
The NOAEL was 0.5 ppm (equal to 0.020 mg/kg bw per day) based on greater than 20%
inhibition of erythrocyte and brain cholinesterase activities at a dietary concentration of 150 ppm
(equal to 5.6 mg/kg bw per day) (Barnes, Arthur & Hazelette, 1988).
In a second 90-day oral dog study conducted as a GLP study in accordance with the test
guidelines of the Ministry of Agriculture, Forestry and Fisheries of Japan, four male and four female
beagle dogs (Nasan: Beagle, Japan) per group were administered diazinon (purity 95.0–95.5%) by
gavage in gelatine capsules at 0, 0.3, 3 or 10 mg/kg bw per day for 90 days. (The highest dose was
changed from 15 mg/kg bw per day to 10 mg/kg bw per day on day 8 because of the decreased body
weight and feed consumption in one male and two females; moreover, this male dog was not treated
on days 14 to 28, one of these female dogs was not treated on days 11 to 28, 37 to 50 and 72 to 78 and
the other female was not treated on days 11–28.) All the dogs were monitored daily for clinical signs;
detailed observations and body weight and feed consumption measurement were carried out every
week. Erythrocyte acetylcholinesterase activity was measured at weeks 2, 4, 8 and 13 and brain
activity at week 13. At week 13, all dogs were necropsied and all tissues or organs microscopically
examined.
Treatment-related changes are summarized in Table 9. Vomiting pre- and post-capsule
administration was observed in all dogs at the highest dose, and the frequency increased in three
males and all the females. Other clinical signs included diarrhoea or mucous stool. Body weights and
feed consumption were comparable across all treated groups except in the high-dose dogs that were
temporarily taken off the treatment due to their body weight loss and decreased feed consumptions
(one male and two females). There were no treatment-related changes in ophthalmology or urine
analysis. Although anaemia indicating decreased erythrocytes, haemoglobin or haematocrit was
observed in the dogs that were temporarily removed from treatment at 10 mg/kg bw, other animals in
the treated group showed no treatment-related changes in haematology. Blood biochemistry
parameters were also not affected except in the dogs that were temporarily taken off the treatment at
10 mg/kg bw: total protein was lower in one male and one female, and the one male had increased
aspartate aminotransaminase, alanine aminotransaminase and alkaline phosphatase levels. Erythrocyte
acetylcholinesterase activity was continuously and dose-dependently reduced in both sexes by over
20% at 3 mg/kg bw and above. Brain acetylcholinesterase was also reduced by 20% or more at
3 mg/kg bw and above in both sexes. Microscopically, atrophic change in the pancreas, thymus spleen
or prostate was observed in those dogs temporarily taken off the treatment. These were considered
secondary changes because of the dogs’ decreased body weight at 10 mg/kg bw. Any changes
suggesting primary organ toxicity was not found in the treated group.

21
Table 9. Summary of a 90-day oral toxicity study of diazinon in dogs

No. and incidence per dose of diazinon
Males Females
0.3 10 0.3 10
0 mg/kg mg/kg 3 mg/kg mg/kg 0 mg/kg mg/kg 3 mg/kg mg/kg
bw per bw per bw per bw per bw per bw per bw per bw per
Parameter day day day day a day day day day a
Clinical signsb
Vomiting, after treatment 0/4 0/4 1/4 3/4 0/4 1/4 0/4 4/4
Vomiting, before treatment 1/4 1/4 0/4 4/4 0/4 1/4 0/4 4/4
Diarrhoea 0/4 0/4 1/4 4/4 0/4 0/4 0/4 3/4
Mucous stool 0/4 0/4 1/4 4/4 0/4 1/4 0/4 2/4
c
AChE activity
Erythrocytes
Pretreatment 863.0 736.5 876.3 864.7d 967.3 884.3 970.3 965.5e
(85.3) (101.5) (100.2) (91.4) (100.3) (99.8)
Week 2 1038.8 933.0 554.5* 256.0d 1006.3 895.8 720.0 253.5e
(89.8) (53.4) (24.6) (89.0) (71.5) (25.2)
Week 4 1126.5 944.3 447.0** 118.3d 1053.3 1061.5 609.8# 277.5e
(83.8) (39.7) (16.8) (100.5) (52.9) (21.6)
Week 8 1126.5 890.5 804.5 320.0*,d 949.5 854.8 481.0# 166.0e
(98.6) (90.3) (36.2) (90.0) (50.7) (17.5)
Week 13 1097.3 868.0 318.8** 197.7d 1141.0 1012.8 484.5# 195.5e
(79.1) (29.0) (18.2) (88.8) (50.7) (17.1)
Brain 122.0 146.8 73.0 39.7d 139.8 161.3 85.8 32.5e
(120.3) (59.8) (32.5) (115.4) (61.4) (23.2)
AChE: acetylcholinesterase; bw: body weight; IU: International Unit; no.: number; *: P < 0.05 (Dunnett t-test); #: P < 0.01
(Fisher exact test)
a
The starting high dose of 15 mg/kg bw per day was lowered to 10 mg/kg bw per day on day 8.
b
Results for clinical signs shown as number of dogs affected /number of dogs examined.
c
Results for AChE activity expressed in IU/L and as a percentage (%) relative to the control in parentheses.
d
Three dogs were tested; one male was removed from the calculation of mean value because of a shortened exposure period.
c
Two dogs were tested; two females were removed from the calculation of mean value because of a shortened exposure
period.
Source: Ichido (2010)
The NOAEL for 90-day oral toxicity in dogs was 0.3 mg/kg bw per day based on a greater
than 20% reduction in erythrocyte and brain acetylcholinesterase activity at 3.0 mg/kg bw per day
(Ichido, 2010).
In a 1-year toxicity study, groups of four male and four female pure-bred beagle dogs were
fed diazinon (purity 87.7%) admixed into their normal diet (for approximately 3 hours/day) at
concentrations of 0, 0.1, 0.5, 150 or 300 ppm (equal to 0.0032, 0.015, 4.7 and 7.7 mg/kg bw per day in
males and 0.0037, 0.020, 4.5 and 9.1 mg/kg bw per day in females, respectively) over a period of
52 weeks. Dietary concentration was reduced from 300 to 225 ppm after 14 weeks of treatment (test
day 99) due to a general lack of body-weight gain in this test group. All animals were observed daily
for mortality and clinical signs. Body weight and feed consumption were recorded before treatment
and then weekly for the first 16 weeks and monthly thereafter. Physical and auditory examinations

22
were performed 4 weeks before testing and then during weeks 12, 26, 39 and 51. Ophthalmoscopic
examinations were performed 3 weeks before testing and at week 25 and 53. All the dogs were
terminated and necropsied during week 53. Haematology and biochemistry parameters, including
plasma, erythrocyte and brain cholinesterase activities, and urine analysis were assessed 4 weeks
before treatment and then at weeks 13, 26, (27 & 38, urine analysis only), 39 and 52. At termination,
body weight was measured and the main organs removed and weighed before fixation. All the dogs
including the one that died and another that was terminated in poor condition early in the dosing
period underwent a complete necropsy.
Mortality was not affected by treatment, and haematology, urine analysis, gross pathology
and histopathology assessments showed no treatment-attributable changes. Overt clinical signs of
dehydration and emaciation became evident in one male at 300/225 ppm and the symptoms remained
despite a reduction in the initial dose. Reductions in body-weight gain were observed at 150 ppm and
higher doses in males and at 300/225 ppm in females. However, no clear-cut dose–response
relationship was evident and the differences attained statistical significance relative to the control
group only at certain observation times. Feed consumption was reduced at dietary concentrations of
150 ppm and higher doses, again without a clear dose–response relationship, most probably owing to
reduced palatability of the feed admixtures. Serum cholinesterase activity was reduced at 0.5 ppm and
higher doses in males and at 150 ppm and higher doses in females to about 20% of the control group
activity at 150 and 300/225 ppm in both sexes. Erythrocyte cholinesterase activity in both sexes was
also reduced at 150 and 300/225 ppm to about 70% of the control activity measured. Brain
cholinesterase activity was inhibited to 75% of control activity in females at 150 ppm, and at
300/225 ppm to 65% and 75% of control activity in females and males, respectively.
The NOAEL was 0.5 ppm (equal to 0.015 mg/kg bw per day) based on inhibition of
erythrocyte (males and females) and brain (females only) cholinesterase activity at 150 ppm (equal to
4.5 mg/kg bw per day) (Rudzki, Arthur & McCormick, 1991).
(b) Dermal application

Rats
In a 2-week dose range-finding study, groups of four male and four female Sprague Dawley
Crl:CD[SD] rats were topically treated with diazinon (purity 87.4%) suspended in Epoxol (epoxidized
soybean oil; the control vehicle) at daily doses of 0, 5, 25, 125 or 250 mg/kg bw through a porous
gauze dressing. Clinical conditions, body weights, feed consumption, blood (erythrocyte and plasma)
and brain acetylcholinesterase activities, blood chemistry, brain weight and macropathology were
assessed.
Treatment with diazinon at 125 or 250 mg/kg bw per day resulted in reduced plasma
cholinesterase activity in females (31% and 26% of controls, respectively) and males (68% and 63%
of controls, respectively) on day 1 of treatment. By day 14 of treatment, there were clear reductions of
over 20% in both plasma and erythrocyte enzyme activity in males at doses equal and greater than 25
mg/kg per day and in females at doses equal and greater than 5 mg/kg bw per day, with the degree of
response dose related and generally greater in females. Erythrocyte enzyme activity was 97%, 44%,
27% and 21% of controls in males and 76%, 48%, 19% and 14% of controls in females. Treatment of
males with diazinon at 125 or 250 mg/kg bw per day and of females at doses equal to and greater than
25 mg/kg bw per day resulted in a clear reduction (16–72% of controls) in brain cholinesterase
activity on day 14 of treatment; the degree of response was dose related and higher in females. Males
at 125 and 250 mg/kg bw per day and females at 5 mg/kg bw per day and greater showed a local
reaction to the treatment, namely slight- to well-defined erythema. Males at the higher dose of 250
mg/kg bw per day showed greater mean body-weight loss during the first week of treatment compared
with controls. The treatment had no effect on feed consumption or brain weights; macroscopic
examination did not reveal any treatment-related changes.

23
No NOAEL could be determined; the lowest-observed-adverse-effect level (LOAEL) in this

dose range-finding study was considered to be 5 mg/kg bw per day based on a greater than 20%
inhibition of erythrocyte cholinesterase activity in females (Brennan, 2010).
In a 90-day toxicity study, groups of 10 male and 10 female Sprague Dawley Crl:CD[SD] rats
were topically treated with diazinon (purity 87.4%) suspended in Epoxol (epoxidized soybean oil; the
control vehicle) at dermal doses of 0, 0.3, 1, 3 or 25 (for males) and 0, 0.3, 1, 3 or 10 (for females)
mg/kg bw through a porous gauze dressing. Treatment was daily, for 6 hours for 13 weeks. Five more
males and five more female rats were assigned to each group and similarly treated for 13 weeks,
followed by a 4-week period without treatment to assess recovery. Sensory reactivity, grip strength,
motor activity, body weight, feed consumption, ophthalmology, haematology, blood chemistry,
including erythrocyte and plasma activity, brain acetylcholinesterase activity, urine analysis, organ
weights, macropathology and histopathology were assessed.
There were no treatment-related signs after dose administration or at the routine weekly
physical examinations; sensory reactivity, grip strength and motor activity as well as body weight and
feed consumption were unchanged at the end of the treatment period. Four deaths occurred during the
study but these were all clearly incidental to treatment. There were no treatment-related
ophthalmoscopic findings.
Plasma acetylcholinesterase activity was reduced in females at 1 mg/kg bw per day and
higher doses. In males, plasma acetylcholinesterase activity was only affected at the highest dose,
25 mg/kg bw per day. Recovery was complete, though in the first week of the recovery period a small
reduction in plasma acetylcholinesterase activity remained in those females dosed with 3 or 10 mg/kg
bw per day. Erythrocyte acetylcholinesterase activity was reduced in high-dose males and females,
although there was a wide variation in the magnitude of the response. In males at 25 mg/kg bw per
day, erythrocyte cholinesterase inhibition was 1% to 38% compared with controls; in females at
10 mg/kg bw per day, erythrocyte cholinesterase inhibition was 10% to 44%, reaching statistical
significance at several occasions. After the 13 weeks of treatment, brain acetylcholinesterase activity
was reduced in males at 25 mg/kg/day (10% inhibition) and in females at 3 or 10 mg/kg bw per day
(6% and 11% inhibition, respectively). After 13 weeks of treatment, there was no effect on organ
weights and no treatment-related macroscopic or histopathological findings.
The NOAEL was 3 mg/kg bw per day for males and females, based on greater than 20%
inhibition of erythrocyte cholinesterase activity at 25 mg/kg bw per day for males and 10 mg/kg bw
per day for females (Brennan, 2011).
Rabbits
In a 21-day toxicity study, groups of five male and five female albino HAR:PF/CF(NZW)BR
rabbits were topically treated with diazinon (purity 97.1%) suspended in 50% aqueous polyethylene
glycol 300 (the control vehicle), through a porous gauze dressing, at daily doses of 0, 1, 5 or
100 mg/kg bw diazinon. Treatment was daily, for 6 hours, 5 days per week for 3 weeks. As mortality
in the male rabbits at 100 mg/kg was high (4/5), the highest dose was reduced to 50 mg/kg after 7
study days (5 treatment days). Both eyes of each rabbit were examined prior to dosing and at study
termination. Dermal reactions were graded. Evaluations were conducted once daily immediately prior
to applying the test substance. Each animal was monitored at least twice daily, in the morning and the
afternoon, for appearance, mortality and overt toxicological and/or pharmacological effects.
Haematological and biochemical measurements, including cholinesterase activity, were made prior to
dosing and at termination. Necropsies were performed on every animal, and tissues included all gross
lesions, liver, skin (treated and untreated areas), tissue masses, kidneys and brain harvested.
As a result of four of the male rabbits dying at 100 mg/kg during the first test week (days 3
and 6), the highest dose was reduced to 50 mg/kg at the end of the first week. All the other animals
survived the scheduled experimental period. Anorexia, ataxia, fasciculations, tremors, diarrhoea,

24
hypoactivity, hypotonia and salivation were generally observed in high-dose animals during the first
week. All groups, including vehicle controls, consistently exhibited very slight erythema throughout
the study. Well-defined erythema was occasionally observed at all doses. In addition, dry, flaky areas
of skin were observed among high-dose female rabbits during the second test week. No statistically
significant differences in weekly mean body weights or feed consumption between treated and control
groups were noted. Terminal ocular examination did not reveal treatment-related effects.
Slight, but statistically significant (P < 0.01) decreases in eosinophil and basophil percentages
(male) and platelet counts (females) were observed in the high-dose animals, although the absolute
values of these parameters were within the normal range. Dose-dependent inhibition of serum,
erythrocyte and brain cholinesterase activities were observed in both male and female rabbits.
Statistically significant (P < 0.01) reductions in mean serum cholinesterase occurred only in high-dose
males and females. Serum cholinesterase was reduced by 64.3% in the single surviving male rabbit
and by an average of 56.5% in females. There were no statistically significant reductions in low- or
mid-dose groups. Erythrocyte cholinesterase activity was reduced in high-dose males and females
relative to baselines, but the reduction was statistically significant (P < 0.01) only in high-dose
females. Dose-related reduction in mean brain cholinesterase relative to controls occurred in both
sexes but was statistically significant only in mid-dose (18.1%; P < 0.05) and high-dose (43.3%;
P < 0.01) females. Slight but statistically significant (P < 0.05) increases in serum inorganic
phosphorus concentrations (in males) and serum glucose (in females) occurred in all dose groups.
Similarly, slight but statistically significant (P < 0.05) increases in serum albumin and albumin-to-
globulin ratio and reductions in serum sodium concentrations were observed in high-dose females. In
all instances, the absolute values of these parameters were well within the normal range. Mean organ
weights and organ-weight ratios were comparable in treated and control groups. Macroscopic
examinations did not reveal any gross internal lesions directly associated with the dermal application
of diazinon; however, one high-dose male exhibited slight gastric irritation. Except for minimal
hyperkeratosis of the treated skin among high-dose rabbits (60% of each sex), no histological changes
were observed in the other tissues examined (brain, kidney and liver).
The NOAEL was 5 mg/kg bw based on greater than 20% inhibition of erythrocyte and brain
cholinesterase at 100 mg/kg bw per day (Tai & Katz, 1984). It should be noted that the cholinesterase
measurements were compared to baseline and not to controls.
(c) Exposure by inhalation

In a 21-day toxicity study
… groups of rats (9/sex/group) were exposed to an aerosol of diazinon (purity 97.1%; droplet size < 1
µm 30–40%, 1–7 µm 50%) for 6 hours a day, 5 days per week for three weeks. Only the animals'
snouts were exposed to the aerosol. The mean concentrations were 0, 151, 245 or 559 mg/m 3. The
treatment did not cause changes in the mortality rate, in haematology, macroscopical and
histopathological findings or organ weights that were attributable to the inhalation of diazinon.
Exophthalmos and diarrhoea were observed in animals at all dose levels and tonic-clonic muscle
spasms occurred in the high-dose animals. The symptoms were reversible. Food intake at the highest
dose level was reduced at the beginning of the treatment period. Body-weight gain was reduced in male
rats at 245 and 559 mg/m3, and in female rats at 559 mg/m3. Plasma cholinesterase was inhibited at the
intermediate and high levels, resulting in values corresponding to 56% and 37% of the activity in
control animals, respectively. Erythrocyte cholinesterase was inhibited only at the highest dose level
(34% of the control activity). Brain cholinesterase activity was dose-dependently reduced at all dose
levels resulting in activities of 81, 56 and 37% of the control activity in both sexes at the low-,
medium- and high-dose levels, respectively. The cholinesterase values returned to normal at the end of
the 25-day recovery period.
The NOAEL was [less than] 151 mg/m3 (corresponding to an estimated dose of 55 mg/kg bw/day),
based on lack of significant brain cholinesterase inhibition at the lowest dose level (Zak et al., 1973;
study evaluation copied from the 1993 JMPR without further evaluation).

25
The relevance of the inhibition of brain cholinesterase activity at low doses is unclear as
plasma and erythrocyte cholinesterase inhibition did not occur. Moreover, it is in contrast to the
Hartmann (1990) study described below, which used much lower doses.
In a 21-day toxicity study, groups of 10 male and 10 female rats were exposed by inhalation
(nose only) to measured concentrations of 0 (filtered air), 0 (vehicle control), 0.05, 0.46, 1.57 or
11.6 mg/m3 diazinon (purity 88%) for 6 hours a day, 5 days per week. The mass median aerodynamic
diameter of the aerosol particles ranged from 0.7 to 1.4 µm. All animals were assessed for mortality,
clinical signs, body weight, feed consumption and ophthalmological, haematological and blood
chemistry measured, including plasma and erythrocyte cholinesterase activity. At study termination, a
gross pathology was performed, with brain cholinesterase activity and organ weights assessed and
histopathology determined.
There were no deaths attributable to treatment. Apart from piloerection in most animals at all
doses, no signs of systemic toxicity were observed. No concentration-dependant difference in the
body-weight change or feed consumption could be observed. No treatment-related ophthalmoscopic
effects were recorded. Minimal lower values of erythrocyte parameters (erythrocyte count,
haemoglobin and packed cell volume) were recorded in high-dose females. At the end of the exposure
period, plasma cholinesterase activity was decreased in both males and females at 1.57 and
11.6 mg/m3, and also in females at 0.46 mg/m3, indicating exposure. Greater than 20% inhibition of
erythrocyte cholinesterase activity was observed in both sexes at 11.6 mg/m3; the inhibition in females
exposed to 1.57 mg/m3 diazinon was lower (10%). A statistically significant but not dose-related
decrease value of cholinesterase activity was noted in the brains of females at 0.05 mg/m3,
1.57 mg/m3 and 11.6 mg/m3, with less than 20% inhibition at 0.46 mg/m3, when compared to both the
air and the vehicle control. Minimally lower plasma glucose levels were recorded in males at 1.57 and
11.6 mg/m3. A significantly higher lung-to-body-weight ratio was recorded in females at 0.46 mg/m3
and 1.57 mg/m3, but not in the highest dose group. No dose–response relationship or other deviations
in organ weights and organ-to-body-weight ratios in comparison to the controls could be seen, and no
treatment-related effects in macroscopic pathology or histopathology were recorded.
The NOAEL was 0.46 mg/m3, based on a statistically significant reduction in cholinesterase
activity (> 20%) in the brains of the female group dosed at 1.57 and 11.6 mg/m3. In the absence of
corroborative changes in plasma and erythrocyte cholinesterase activity, the statistically significant
lower value for brain cholinesterase activity noted in females dosed 0.05 mg/m3 was not considered
biologically relevant (Hartmann, 1990).
2.3 Long-term studies of toxicity and carcinogenicity

A study report (NTP, 1979; also referenced as Angel et al., unknown year, or NCI, 1979)
described a bioassay of diazinon for possible carcinogenicity conducted by the National Cancer
Institute in the United States of America. This study is evaluated by test animal species in the
subsections below. Subchronic feeding studies are also described in the reports; these 13-week studies
in rats and mice are described in section 2.2 on short-term studies of toxicity.
Mice
In a pre-GLP carcinogenicity study, groups of 50 male and 50 female B6C3F1 mice were fed
diazinon (purity 98%) admixed in the diet at concentrations of 100 or 200 ppm for 103 consecutive
weeks; control groups of 25 males and 25 females were fed the basal diet only. The mice were
observed for an additional 2 to 3 weeks at the end of treatment. The prepared diets were used within 1
week. All the animals were examined twice a day for signs of toxicity, weighed at 2-week intervals,
palpated for tissue masses at each weighing and subjected to gross and histopathological examination.

26
The study was performed according to National Cancer Institute–established guidelines for
carcinogen bioassays, but deviates from Organisation for Economic Co-operation and Development
(OECD) guidelines 451, 452 and 453 for the following reasons:
 two dose groups rather than the prescribed three were used in the chronic study;
 feed consumption was not measured, so the intake of the test material could not be calculated; and
 haematological or clinical chemistry tests were not conducted.
Since the critical effect of diazinon on cholinesterase activity were not measured, and
histopathological information about non-neoplastic findings were limited, the study was inappropriate
for the evaluation of chronic toxicity. Nevertheless, the study provides useful information on
carcinogenicity.
Compared with the controls, mortality was not increased in any of the dosed groups of mice;
survival was 84% or greater in all dosed and control groups at week 78 (84%, 90% and 98% of
survival in males and 96%, 100% and 98% in females of the control, low- and high-dose groups,
respectively). There was no appreciable effect on mean body weights of either sex, except for the last
20 weeks of the study, when the mean body weights of the dosed females were lower than those of the
controls. Hyperactivity was observed in the dosed groups of mice but was rare in the control groups.
No tumours clearly related to diazinon administration occurred in any of the dosed groups of
mice of either sex (Table 10). Incidence of hepatocellular carcinoma increased to statistically
significant levels in low-dose males; the majority of carcinomas were trabecular carcinomas. The
incidence at the high dose was similar to that in the control group. The incidences of adenoma did not
increase at either dose.
Table 10. Incidence of hepatic tumours in 2-year study of carcinogenicity of diazinon in mice
Incidence of hepatic tumours per dose of diazinon
Control 100 ppm 200 ppm
Morphology Males Females Males Females Males Females
Hepatocellular adenoma 1/21 (5) 1/23 (4) 0 /46 (0) 0/47 (0) 3/48 (6) 1/49 (2)
Hepatocellular carcinoma 4/21 (19) 1/23 (4) 20/46* (43) 0/47 (0) 10/48 (21) 2/49 (4)
Carcinoma and adenoma combined 5/21 (24) 2/23 (9) 20/46 (43) 0/47 (0) 13/48 (27) 3/49 (6)
ppm: parts per million; *: P < 0.05 (Fisher exact test)
Results shown as number of tumour-bearing animals / number of animals examined, with incidence relative to the control as
a percentage (%) in parentheses.
Source: NTP (1979)
A NOAEL for systemic toxicity could not be set due to the deficiencies in study design. No
treatment-related tumours were observed in male or female mice (NTP, 1979; also referenced as
Angel et al., unknown year, or NCI, 1979).
In a 2-year non-GLP carcinogenicity study, male and female B6C3F1 mice (59–61
mice/group for both sexes) were fed diazinon (lot no. P-604; purity unknown,) in the diet at
concentrations of 0, 100, 200 or 300 (for males) or 400 (for females) (equal to 0, 16, 31 and
46 mg/kg bw per day for males and 0, 22, 43 and 86 mg/kg bw per day for females, respectively).
Clinical signs were checked daily and the results of palpating for tumours recorded. Body weight and
feed consumption were measured weekly for the first 13 weeks and every 2 weeks thereafter. Water
intake was measured at 1, 12, 25, 51 and 71 weeks. At 52 and 104 weeks, blood was drawn from the
orbital vein for haematological assessment. Acetylcholinesterase was not measured. Ten mice per
group were allocated for interim necropsy at 52 weeks, and all the survivors were necropsied at

27
termination at 104 weeks. Representative organs were weighed at the interim and terminal kills. All
the mice were histopathologically examined.
There were no treatment-related clinical signs or increased mortality throughout the study
period. At the highest dose, body weights in males were slightly (about 5%) but significantly
(P < 0.05) decreased for the first 41 weeks and in females (about 10%) throughout the study. At the
same dose, feed consumption was slightly decreased in males throughout the study and in females
during the first year, but the changes were not statistically significant at many points. The consistent
changes in body weight and feed consumption at the highest dose were considered treatment related.
No treatment-related haematological changes were observed. The pathological examination,
including organ weights and macroscopic or microscopic examinations, also showed no treatment-
related changes. No increases in incidences or malignancy, or early occurrence of tumours, were
observed in the treated groups compared to the control group. The types of tumour were common in
this strain mouse.
Although this study did not comply with GLP or any test guideline, the major parameters
used were adequate for detecting chronic toxicity and carcinogenicity. The NOAEL for 2-year chronic
toxicity in mice was 200 ppm (equal to 31 mg/kg bw per day) based on the slight decreases in body
weight and feed consumption at 300 ppm (equal to 46 mg/kg per day). No carcinogenicity was
observed in this study. Cholinesterase activity was not measured (Goldsmith, 1983).
Rats
In a pre-GLP carcinogenicity study, groups of 50 male and 50 female F344 (Fischer) rats
were fed diazinon (purity 98%) admixed in the diet at concentrations of 400 or 800 ppm for
103 consecutive weeks; control groups of 25 males and 25 females were fed the basal diet only. The
rats were observed for an additional 1 to 2 weeks after the treatment ended. The prepared diets were
used within 1 week. All animals were observed twice a day for signs of toxicity, weighed at 2-week
intervals, palpated for tissue masses at each weighing, and subjected to gross and histopathological
examination.
The study was performed according to guidelines for carcinogen bioassays established by the
NCI (USA) but this method does not comply with the OECD guidelines:
 two dose groups, rather than the prescribed three, were used in the chronic study;
 feed consumption was not measured, so the achieved intake of test material could be not be
calculated; and
 haematology or clinical chemistry data were not recorded.
Since the critical effect of diazinon on cholinesterase activity were not measured, the study
cannot be used to determine chronic toxicity. However, the study provides useful information on
carcinogenicity.
Mortality was not increased at any of the doses compared to the controls, and survival was
88% or greater in all dosed and control groups of animals at week 78 (96%, 98% and 98% of survival
in males and 92%, 88% and 88% in females in the control, low- and high-dose groups, respectively).
Diazinon did not appreciably affect mean body weights of either sex. Clinical signs observed included
hyperactivity, discoloured urine, bloating, vaginal bleeding and vaginal discharge, tissue masses and
tachypnoea.
There were no treatment-related tumours in any of the dosed groups of rats of either sex
(Tables 11 and 12). Leukaemia or lymphoma occurred in dosed and control groups of rats of each sex
at an incidence higher in low-dose males (25/50 [50%]) than in the controls (5/25 [20%]) or high-dose
males (12/50 [24%]). Malignant lymphoma is rare in F344 rats and is systemically spread from the
lymph node through lymph ducts (Frith, Ward & Chandra, 1993). On the other hand, mononuclear
cell leukaemia (synonium: F344 rat leukaemia or large granular lymphocyte leukaemia) are very
common tumours, occurring at high incidences in ageing F344 rats (Haseman, Arnold & Eustis,

28
1990). The tumour cell of mononuclear cell leukaemia in rats is understood to derive from NK-cells in
the marginal zone of the spleen (Ward, 1983; Losco & Ward, 1984); leukaemia proliferates in the red
pulp of the spleen and systemically spreads out through blood vessels (Frith, Ward & Chandra, 1993).
Mononuclear cell leukaemia in rats has a different profile to that of human NK-cell large granular
lymphocyte leukaemia, although some similarities in histogenesis or pathological features have been
reported (Thomas et al., 2007).
Criteria for diagnosis of mononuclear cell leukaemia in F344 rats were not yet established
when this NTP study (1979) was reported, but the descriptions of leukaemia or lymphoma were
considered to match those of mononuclear cell leukaemia. As a result, the Meeting evaluated the
incidence of leukaemias and lymphomas combined as that of mononuclear cell leukaemia. This
combined incidence was within the historical control data of NTP studies in F344 rats (rate: 33.6%;
range: 10–72% in untreated control male rats in an NTP study from 1977 to 1987; Haseman, Eustis &
Arnold, 1990) and no dose dependency was observed, indicating that the incidence at high dose was
not treatment related.
Endometrial stromal polyps were observed at a higher incidence in the low-dose (8/43; 19%)
and high-dose (11/49; 22%) groups than those in the control group (2/23; 9%). This finding is
common in ageing F344 rats, and both incidences were within the historical control range of NTP
studies from 1977 to 1987 (rate 21.4%; range 8–37%) (Haseman, Eustis & Arnold, 1990).
Table 11. Summary of tumour incidences in rats administered diazinon in the diet
Incidence of tumours per dose of diazinon
Control 400 ppm 800 ppm
Morphology Males Females Males Females Males Females
Haematopoietic system, liver, Peyer
patch
Lymphoma or haematopoietic 5/25 (20) 2/25 (8) 25/50* (50) 6/50 (12) 12/50 (24) 6/50 (12)
system leukaemia
Uterus
Endometrial stromal polyp – 2/23 (9) – 8/43 (19) – 11/49 (22)
–: not evaluated; *: P < 0.05 (Fisher exact test)
Results shown as number of tumour-bearing animals / number of animals examined, with incidence relative to the control
expressed as a percentage (%) in parentheses.
Source: NTP (1979)

29
Table 12. Combined incidence of each leukaemia or lymphoma in the haematopoietic system, liver
and Peyer patch in male rats
Incidence per dose of diazinon
Morphology Control 400 ppm 800 ppm
Haematopoietic system
Leukaemia 5/25 (20) 16/50 (32) 10/50 (20)
Lymphocytic leukaemia 0/25 1/50 (2) 0/50
Monocytic leukaemia 0/25 (0) 6/50 (12) 2/50 (4)
Liver
Leukaemia 0/24 (0) 1/49 (2%) 0/49 (0)
Peyer patch
Malignant lymphoma 0/22 (0) 1/44 (2%) 0/48 (0)
ppm: parts per million; *: P < 0.05 (Fisher exact test)
Results shown as number of tumour-bearing animals / number of animals examined, with incidence relative to the control
expressed as a percentage (%) in parentheses.
Source: NTP (1979)
A NOAEL for systemic toxicity could not be set due to the deficiencies in study design. No
treatment-related tumours were observed in male or female rats (NTP, 1979; also referenced as Angel
et al., unknown year, or NCI, 1979).
In a chronic toxicity study, groups of 30 to 40 male and 30 to 40 female Sprague Dawley

Crl:CD[SD]BR VAF/Plus rats were fed diazinon (purity 87.7%) admixed in the diet at concentrations
of 0, 0.1, 1.5, 125 or 250 ppm (equal to 0, 0.004, 0.06, 5 and 10 mg/kg bw per day for males and 0,
0.005, 0.07, 6 and 12 mg/kg bw per day for females, respectively) for 98/99 weeks. The vehicle-
control group was fed a diet containing 26.5 ppm of epoxidized soybean oil at the same concentration
as that in the highest diazinon dose group.
Up to 10 randomly selected animals per sex from each group were terminated and necropsied
at 1 year, and up to 10 per sex per group from both control groups and the high-dose group were
terminated and necropsied after a 4-week recovery period (1 year plus 4 weeks). The rats were
approximately 6 weeks of age at initiation of treatment, and their body weights ranged from 158.3 to
241.1 grams for males and 126.4 to 216.5 grams for females. Body weight and feed consumption
were measured before starting the treatment and then weekly through weeks 1 to 13 of the dosing
period and monthly thereafter. Haematology and biochemistry assessments were conducted during
weeks 13 to 14, 26 to 27, 51 to 52, 56, 79 to 80 and 97 to 98. All the surviving animals were used to
determine haematological effects at each time point, and 10 animals per sex per group were used to
determine clinical chemistry, including cholinesterase activity. The study was performed in
accordance with GLP and USEPA Guidelines 83-1/83-5 (1982), which are equivalent to OECD
Guideline 453 (1981) and Ministry of Agriculture, Forestry and Fisheries of Japan (1985) guidelines,
and which do not differ significantly from the method prescribed by the European Union (B.33).
However, the USEPA, European Union and OECD guidelines require group sizes of 100 animals (50
males and 50 females) for carcinogenicity studies. The Kirchner, McCormick & Arthur (1991) study
used group sizes of 20 animals per sex, excluding satellite groups, a group size sufficiently large to
determine the chronic toxicity, but not the carcinogenicity, of diazinon.
During week 97, increased mortality (6/20) was observed in males at 0.1 ppm. The major
cause of moribundity or death was senile nephropathy and/or pituitary adenomas, both unrelated to
treatment but associated with senescence in this strain of rats. The study was terminated during weeks
98 to 99 because of the need to have at least five animals per sex per group to provide a meaningful

30
evaluation of cholinesterase activity data. This early termination was not considered to affect the
quality or integrity of the study or any interpretation of the data.
Body-weight gain was increased in males at 0.1 ppm and higher doses and, in some instances,
in females at 125 ppm and higher doses compared with the untreated control rats, but because body-
weight gain in the control group also showed an increase compared with the untreated controls, these
increases in body weight may reflect increased palatability of the feed. In fact, the increases in mean
body-weight gain generally coincided with increases in mean feed consumption in these groups.
Decreases in serum cholinesterase activities were observed at 1.5 ppm and higher doses in
both sexes. A dose-dependent reduction in erythrocyte cholinesterase activity was observed at 125
and 250 ppm, resulting in activities of 80% and 75% of controls in males and females, respectively, at
either dose at the end of the study. Brain cholinesterase activity was also inhibited to 76% and 71% of
the control value in males and females, respectively, at 125 ppm, and to 58% and 52% of the control
value at 250 ppm in males and females, respectively. Similar inhibition was observed after the first
year of the study. A slight reduction of less than 9% was still found after the 4-week recovery period
in erythrocyte and brain cholinesterase activity at 250 ppm (Table 13).
Table 13. Changes in mean erythrocyte and brain cholinesterase activities in rats administered
diazinon in the diet
Erythrocyte Brain
Concentration
of diazinon One year + 4 One year + 4
(ppm) One year weeks recovery Two years One year weeks recovery Two years
Males
0.1 +6 – +7 −4 – −3
1.5 +7 – −5 +1 – −2
125 −16* – −21* −2 – −24*
250 −11* −1 −22* −10 +5 −42*
Females
0.1 +5 – −3 +5 – +1
1.5 0 – −3 +6 – +6
125 −22* – −26* −26* – −29*
250 −20* −7* −25* −40* −9* −48*
–: not evaluated; ppm: parts per million; *: P < 0.05 (two-tailed Student t-test)
Results expressed as mean increase (+) or decrease (−) in cholinesterase activity as a percentage of control cholinesterase
activity.
Source: Kirchner, McCormick & Arthur (1991)
No treatment-related increases in non-neoplastic findings were observed in any diazinon

doses. The incidence of neoplasms in the vehicle- and diazinon-treated groups was comparable to that
seen in the untreated controls.
The NOAEL was 1.5 ppm (equal to 0.06 mg/kg bw per day) based on the inhibition of
erythrocyte and brain cholinesterase activity at dietary concentrations of 125 ppm (equal to 5 mg/kg
bw per day). From the available data, there was no evidence of a tumorigenic response; however, the
group size (N = 20) was too small to allow a conclusion to be reached on carcinogenicity (Kirchner,
McCormick & Arthur, 1991; also referenced as EPA, 1993).

31
In accordance with USEPA Guidelines 83-5 and GLP, chronic toxicity and carcinogenicity of
diazinon was examined in rats with the test compound (purity 96.2–97.02%) admixed in the diet of
male and female F344 rats (75 rats/group in both sexes in the first study, 15 rats/group in both sexes
in the second) at concentrations of 0, 0.1, 1.5 or 22.5 mg/kg bw per day (in the first study) and 0 or
0.025 mg/kg bw per day (in the second study). The second study, started a year after the first, was
conducted to measure erythrocyte cholinesterase activity at 13-week intervals and brain
acetylcholinesterase activity at termination (after 108 weeks of treatment). Mortality and clinical signs
were checked daily. Body weights were measured weekly for 26 weeks after study commencement
and every 2 weeks thereafter. Feed consumption was measured weekly for 13 weeks after study
commencement, and every 13 or 26 weeks thereafter. Water intakes were measured at 1, 12, 25, 51,
77 and 101 weeks. At 12, 25, 51 and 102 weeks, blood was drawn from ocular vein (10 rats/group in
both sexes at each time point) for haematological and blood biochemistry analysis of plasma. Plasma
and erythrocyte acetylcholinesterase activity and plasma butyrylcholinesterase activities were
measured at the same time points as haematology and at 17 and 38 weeks in the first study and 13, 26,
39, 52, 66, 78, 91 and 104 weeks in the second study. Brain acetylcholinesterase and
butyrylcholinesterase were measured at interim kills at 18, 27, 52 (5 rats/group in both sexes) and
104 weeks (6–8 rats/group in both sexes) in the first study. Urine analysis was performed at the same
time points as the haematological analyses. Ophthalmological assessments of 20 rats per group in both
sexes were made at commencement of treatment and at 25, 50, 78 and 102 weeks. At each interim kill
and at termination at 120 weeks, the adrenals, brain, heart, kidneys, lungs, pituitary, spleen,
ovaries/testes and thyroids with parathyroids were weighed. Macroscopic and microscopic
examinations were conducted.
In terms of clinical signs, incidences and frequencies of urogenital wetness or staining and
perianal staining associated with loose stools were increased in females at 22.5 mg/kg bw as was the
incidence and frequency of periorbital staining. These symptoms, which progressed with treatment,
were considered treatment-related cholinergic inhibitory effects. No consistent and dose-dependent
changes were observed in body weight, feed consumption, haematology, blood biochemistry or urine
analysis. Erythrocyte and brain acetylcholinesterase activities are shown in Table 14. Erythrocyte
acetylcholinesterase activities were inhibited by over 20% overall at 1.5 mg/kg bw and higher in a
dose-dependently in both sexes. Brain acetylcholinesterase activity was inhibited by over 20% at
22.5 mg/kg bw in males from 27 week onwards. In females, brain acetylcholinesterase activity was
reduced by over 20% at 22.5 mg/kg bw at all time points and at 1.5 mg/kg bw after 52 weeks.
In the second study, erythrocyte and brain acetylcholinesterase activities at 0.0025 mg/kg bw
were comparable to the control except at 52 weeks when there was a significant inhibitory effect on
erythrocyte acetylcholinesterase activity. The decrease in activity was within 20% of the controls
(86% inhibition), indicating no toxicologically significant effect.

32
Table 14. Summary of erythrocyte and brain AChE activity in rats administered diazinon in the diet
Concentration of AChE per dose of diazinon
Male Female
0 0.1 22.5 0.1 1.5 22.5
mg/kg mg/kg 1.5 mg/kg mg/kg 0 mg/kg mg/kg mg/kg mg/kg
Duration bw per bw per bw per bw per bw per bw per bw per bw per
Parameter (weeks) day day day day day day day day
Erythrocyte 12 592 623 450 247 677 704 496 261
AChE (105) (76)*** (42)*** (104) (72)** (39)***
(IU/L)
17 518 420*** 212*** 130*** 420 420 254*** 136***
(81) (41) (25) (100) (60) (32)
25 598 664* 429* 194*** 619 623 526* 198***
(111) (72) (32) (101) (85) (32)
38 630 680 298*** 158*** 645 601* 313*** 127***
(108) (47) (25) (93) (49) (20)
51 646 778* 273*** 104*** 685 621 370*** 184***
(120) (42) (16) (91) (54) (27)
102 752 740 386*** 235** 594 539 369*** 140***
(98) (51) (31) (91) (62) (24)
Brain AChE 18 7 084 6 939 7 001 6 043* 6 295 6 315 5 815 3 308***
(IU/L) (98) (99) (85) (100) (93) (53)
27 6 146 6 986 6 578 4 446 5 399 4 898 5 669 3 118
(114) (107) (72)* (91) (105) (58)
52 5 390 5 573 5 108 4 446*** 6 371 5 598 4 746 2 243
(103) (95) (75) (88) (74)** (36)***
104 4 896 4 696 4 754 1 384*** 6 371 5 598 5 956* 1 939***
(96) (97) (28) (88) (114) (37)
AChE: acetylcholinesterase; IU: International Unit; *: P < 0.05; **: P < 0.01; ***P: < 0.001
Results shown as concentration of AChE with per cent activity relative to the control (%) in parentheses.
Source: Ashby & Danks (1987)
Absolute and relative thyroid weights were statistically significantly increased at 1.5 and 22.5
mg/kg bw in males at 104 and 120 weeks. The lack of corresponding histopathological change or
ageing periods only (no increases at 27 or 52 weeks) indicated the increases were not treatment
related. Histopathological examinations showed that several treatment-related changes were increased
(Table 15). Continuous stimulating changes, such as ulcer, acanthosis, hyperkeratosis, granulation
tissue or hyperplasia of mucosal epithelium, were increased in the forestomach of males and females
at 22.5 mg/kg bw 2 years after commencing the oral treatment, and these changes in the forestomach
were considered treatment-related. Continuous irritative effects might be involved, because the test
chemical was a slight irritant to the rabbit skin (Brennan, 2010). The incidences of fatty change in
adrenal cortex were increased in males at 1.5 and 22.5 mg/kg bw. As the increases were not dose
dependent or were found in moribund or dead animals only, they were not considered treatment
related. No increased incidence, earlier occurrence or increased malignancy was observed in
neoplastic changes at all treated groups. Their incidences were comparable to those in controls.

33
Table 15. Summary of histopathological changes in rats administered diazinon in the diet
No. of histopathological findings per dose of diazinon
Male Female
0 0.1 1.5 22.5 0 0.1 1.5 22.5
mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
bw bw bw bw bw bw bw bw
Duration per per per per per per per per
Morphology Outcome (weeks) day day day day day day day day
Adrenal
Cortical fatty ke/fd 105–120 3/16 7/18 10/17* 13/21* 11/17 4/17* 5/12 4/21*
vacuolation
Tk 120 9/22 8/17 5/19 6/13 16/22 17/21 19/28 19/23
Forestomach
Ulcer ke/fd 105–120 4/16 5/18 8/17 12/21 3/17 2/17 4/12 9/21
Tk 120 5/22 2/17 6/19 3/13 0/22 1/21 2/28 4/23
Acanthosis ke/fd 105–120 6/16 11/18 8/17 15/21 9/17 4/17 4/12 12/21
Tk 120 7/22 4/17 7/19 4/13 0/22 2/21 3/28 9/23**
Hyperkeratosis ke/fd 105–120 6/16 9/18 8/17 15/21 9/17 4/17 3/12 14/21
Tk 120 7/22 4/17 7/19 4/13 0/22 2/21 3/28 8/23**
Granulation ke/fd 105–120 6/16 11/18 9/17 16/21* 6/17 3/17 5/12 11/21
tissue
Tk 120 6/22 3/17 7/19 5/13 0/22 1/21 3/28 8/23**
Hyperplasia of ke/fd 105–120 0/16 3/18 0/17 7/21* 1/17 0/17 1/12 6/21
epithelium
Tk 120 0/22 0/17 1/19 0/13 0/22 0/21 0/28 1/23
ke/fd: killed in extremis or found dead; no.: number; Tk: terminal kill; *: P < 0.05; **: P < 0.01
Results shown as number of rats with an outcome / number of rats examined.
Source: Ashby & Danks (1987)
The NOAEL for long-term toxicity was 0.1 mg/kg bw per day based on the inhibition of
erythrocyte acetylcholinesterase activity at 1.5 mg/kg bw per day. No treatment-related tumours were
observed in male or female rats (Ashby & Danks, 1987).
2.4 Genotoxicity
A variety of genotoxicity studies were evaluated. These included published scientific studies
as well as unpublished guideline studies. Studies that examined the effects of the oral route of
exposure were considered to be the most relevant for assessing risk from low-level dietary exposure,
whereas those administered by parenteral routes or at near-lethal doses, those that used a mixture
including diazinon, such as formulations, or those that used nonmammalian test species were
considered much less relevant. The studies, presented by the JMPR’s evaluation of quality and
relevance, are presented below. The results of acceptable and relevant studies of genotoxicity,
consisting of unpublished and published studies, are summarized in Table 16. Other studies from open
literature were considered to be less relevant for the evaluation; these are shown in Tables 17 to 18.

Table 16. Results of acceptable studies of diazinon-related genotoxicity (from open literature or submitted by companies)
End-point Test object Concentration Purity (%) Results Comments Reference
In vitro studies
Reverse mutation Salmonella typhimurium 1 000 µg/plate (S9) Pure form from Negative Similar to guideline, toxic at Marshall, Dorough &
TA1535, TA1536, TA1537, stock solution, higher concentrations Swim (1976)
TA1538 standard for residue
analysis
Reverse mutation S. typhimurium TA98, TA100, 10–5 000 µg/plate (+S9), 96.16 Negative Guideline study, GLP Bootman & May
TA1535, T1537, TI1538; 10–1 000 µg/plate (−S9) (1986)
Escherichia coli WP2 uvrA
Reverse mutation S. typhimurium TA98, TA100, 0, 313, 625, 1250, 2 500 and 88 Negative Guideline study, GLP Geleick (1990)
TA1535, T1537; E. coli WP2 5 000 µg/mL (S9)
uvrA
Yeast deletion (DEL) Saccharomyces cerevisiae 0, 1 000, 5 000 and 10 000 NP, obtained from Negative Adequately described, Kirpnick et al. (2005)
assay RS112 µg/mL (S9) Aldrich sufficient details provided
Mammalian cell gene Mouse lymphoma L5178Y 12, 24, 48, 72, 96, 108 and 120 97.2 Negative Guideline study, GLP Dollenmeier (1986)
mutation TK+/- µg/mL (+S9)
6, 12, 24, 36, 48, 54 and 60
µg/mL (−S9)
Chromosome damage Human lymphocytes 5–20 µg/mL (S9) 96.16 Negative Guideline study, GLP Bootman, Hodson-
(chromosomal Walker & Dance
aberrations) (1986)
Chromosome damage Human lymphocytes 0, 175, 200 and 250 µg/mL 96 Negative Guideline study, GLP Allais (2002)
(chromosomal (+S9)
aberrations) 0, 100, 150, 175 and 200 µg/mL
(−S9)
Unscheduled DNA Rat hepatocytes 0, 1.1, 3.3, 10, 30, 60 and 120 88 Negative Guideline study, GLP Hertner & Arni
synthesis µg/mL (1990)
In vivo studies
Chromosome damage Mouse (8Tif: MAGF) bone 0 and 120 mg/kg (oral) sampled 87.5 Negative Guideline study, GLP Ceresa (1988)
(micronucleus formation) marrow erythrocytes 16, 24, 48 h after treatment
0, 30, 60 and 120 mg/kg (oral)
sampled 24 h after treatment
Chromosome damage Mouse (ICR) bone marrow 31.3, 62.5 and125 mg/kg bw per 95.4 Negative Guideline study, GLP Kawamura (2006)

35
End-point Test object Concentration Purity (%) Results Comments Reference

(micronucleus formation) erythrocytes day, on 2 consecutive days
(oral)
Chromosome damage Mouse (albino, NMRI derived) 10.5, 21 and 63 mg/kg bw NP Negative Pre-Guideline study, pre- Hool (1981a)
(micronucleus formation) spermatocyte (oral); 5 treatments over 10 days GLP
Chromosome damage Mouse (albino, NMRI derived) 10.5, 21 and 63 mg/kg bw/day NP Negative Pre-Guideline study, pre- Hool (1981b)
(micronucleus formation) spermatocyte (oral) on 5 consecutive days GLP
Dominant lethal assay Mice (albino, NMRI derived) 0, 15 and 45 mg/kg (oral) Technical grade, Negative Pre-Guideline study, pre- Fritz (1975)
analytical results GLP
not available
DNA damage (SCE) Mouse (ICR, male & female) 10, 50 and 100 mg/kg gavage 88.0 Negative in Industry sponsored GLP USEPA 1992
bone marrow cells males and study; USEPA considered (review of Murli &
females female results unacceptable Haworth, 1990)
due to insufficiently high
dose
bw: body weight; GLP: good laboratory practice; NP: not provided; S9: 9000 × g fraction from Aroclor 1254 or phenobarbital/5,6-benzoflavone–induced pretreated rat liver homogenate;
SCE: sister chromatid exchange; TK: thymidine kinase locus; USEPA: United States Environmental Protection Agency

36
Table 17. Results of less relevant studies or studies deemed inadequate for use in human risk assessment of diazinon
End-point Test object Concentration / dose Purity (%) Results Evaluation (Comments) Reference
In vivo human biomonitoring studies
Chromosome damage Lymphocytes of 0.45 mg/m3 in working Commercial Positive Inadequate and not informative. Kiraly et al. (1979)
(chromosomal workers exposed to place air product Inappropriate statistical analyses. Data
aberrations) pesticides in producing (Basudin E) pooled from all individuals in an exposure
factory group prior to analysis. Mixed, largely
negative, results seen when compared with
factory controls; more positive results seen
when compared with non-factory controls
Chromosome damage Lymphocytes of No data No data Positive Not informative. Exposed to ~48 different De Ferrari et al. (1991)
(chromosomal aberrations workers in the flower pesticides
– structural and industry
numerical)
DNA damage (SCE) Human blood Not determined, Sheep dip Positive Not informative. Confounded by exposure Hatjian et al. (2000)
lymphocytes from non- exposure level did not (Probably to other organophosphorus compounds as
smoking students affect erythrocyte commercial evidenced by the presence of higher levels
AChE activity product) of dimethyl and dimethyl-thiophosphate
metabolites than diazinon-derived diethyl-
and diethyl-thiophosphate metabolites in
urine
In vivo studies in mammals – oral administration
Chromosome damage Rat (Wistar, male) 20 mg/kg bw per day 99 Positive Inadequate. Scoring of MN in rat blood Hariri et al. (2011)
(micronucleus formation) blood erythrocyte for 4 weeks erythrocytes is problematic due to the
ability of the spleen to screen out
micronuclei in erythrocytes. Untreated
animals exhibited unusually high mean MN
frequency of 1%

37
DNA damage (AP sites) Liver and kidney in 2.64 and 5.28 mg/kg NP Positive Inadequate. Compound administered every Tsitsimpikou et al.
female rabbits (New bw per day 2 days for 3 months, followed by 8 months (2013)
Zealand White) without exposure, followed by 1 month of
exposure. Doses associated with organ
pathology and oxidative stress. Concern
about quality and interpretation due to small
sample size (N=2/group), very low
variability in controls, and lack of key
details
In vivo studies in mammals – intraperitoneal administration
Chromosome damage Mouse (strain 615, sex 0.8, 0.4, 0.2 and 0.1 × 98 Positive Inadequate; less relevant route; insufficient Ni et al. (1993)
(micronuclei) NP) LD50 per bw per day data for genotoxicity evaluation including
for 4 days those on dose relationship or positive
controls [in Chinese]
DNA damage (sperm Mouse (CF1, male) 22 and 43 mg/kg bw 60 Positive on day Less relevant route and test used a Sarabia et al. (2009a)
head decondensation) spermatozoa from Exmark 1; negative on formulation. Mice terminated on day 1 and
Laboratory day 32 32 postinjection. Positive response at lowest
(Chile) dose tested (equivalent to 1/3 LD50)
Chromosome damage Mouse (CF1, male) 22 and 43 mg/kg bw 60 Positive Less relevant route, test used a formulation, Sarabia et al. (2009b)
(micronucleus formation) bone marrow cells from Exmerk and extremely high MN frequencies in
Laboratory control (5%) and treated (>25%) animals.
(Chile) Mice terminated on day 1 postinjection.
Positive response at lowest dose tested
(equivalent to 1/3 LD50). Small number of
PCEs scored
Chromosome damage Rat (Wistar, male) 20 mg/kg bw per day NP Positive Less relevant route and single dose. Very Shadboorestan et al.
(micronucleus formation) blood lymphocyte for 30 days large (13.5 times) increase in MN in (2013)
binucleated lymphocytes reported
Chromosome damage Rat (Wistar, male) 20 mg/kg bw per day NP Positive Eliminated from consideration because it Shokrzadeh et al.
(micronucleus formation) blood lymphocyte for 30 days uses the same data as published in (2013)
Shadboorestan et al. (2013)

38
In vitro studies in bacteria and yeast
DNA damage (Rec assay) Bacillus subtilis H17 NP NP Negative (10 Inadequate; no data provided Shirasu et al. (1976)
Rec, M45 Rec mm paper disk
containing 0.02
mL of solution)
Reverse mutation (Ames S. typhimurium TA98, 20 ppm (non-toxic) to ≥ 90 positive (TA98 Inadequate due to lack of experimental Wong et al. (1989)
test) TA102, TA1535, 80 ppm (50% toxic) +S9 only) information and data presentation. Negative
TA1537 control values and variability not shown
No positive controls
Electrochemical study of Calf thymus DNA 0.492, 0.984, 1.476, Analytical Positive for Less relevant as it is a non-validated Kashanian et al. (2008)
interaction with purified 1.968, 2.46, 3.444, reagent grade DNA binding method for assessing DNA binding
DNA 4.428, 5.421 and 6.396 from Merk
 10−5 mol/L in
HEPES
buffer:methanol
(50:50%)
Reverse mutation (Ames S. typhimurium TA98, 0.01 to 1 mmol/L NP, Negative (1 Inadequate; no data provided. Kubo, Urano & Utsumi
test) TA100 (S9) compound mmol/L (2012)
provided by with/without
the Ministry rat liver S9)
of
Environment
of Japan
In vitro studies in mammalian cells
DNA damage (Comet Primary nasal mucosa 0.5, 0.75 and 1.0 99.5 Positive (0.5 No information about quality of cells Tisch et al. (2002)
assay-alkaline) (epithelium from mmol/L (= 152, 228, mmol/L) collected from patients and used for the in
patients by surgery at 304 µg/mL) (−S9) vitro experiments. Data presented only as %
inferior and middle undamaged cells
turbunate)
DNA damage (Comet Primary nasal mucosa 0.05, 0.1, 0.5, 0.75 and 99.2 Positive (dose– No information about quality of cells Tisch, Faulde & Maier
assay –alkaline) cells) 1.0 mmol/L (= 15.2, response) collected from patients and used for the in (2007)
30.4, 152, 228, 304 vitro experiments. Graphical data only
µg/mL) (−S9)

39
DNA damage (sperm Human spermatozoa 0.05, 0.3, 0.5 and 0.7 NP (Chem Weak positive Inadequate. Non-standard test. Figures are Salazar-Arredondo et
chromatin structure assay mmol/L (= 15, 91, Service) (> 500 µmol/L) mislabelled. From the text Fig. 2 presents al. (2008)
(SCSA)) 152, 213 µg/mL) the diazinon results. Positive results also
(−S9) seen for diazoxon at ≥ 300 µmol/L
Chromosome damage Human blood 5, 10, 20 and 30 µg/ NP, obtained Negative Inadequate, no positive control; high and Lopez et al. (1986)
(chromosomal lymphocytes mL (−S9) from Ciba- variable aberration frequencies in negative
aberrations) Geigy control and treated cells
DNA damage (SCE ) LAZ-007 (human 0.02, 0.2, 2 and 20 NP Negative Data on +S9 limited to a single Sobti, Krishan &
lymphoid cell line, B- µg/mL (−S9); 20 (−S9), positive concentration tested only Pfaffenberger (1982)
cell origin) µg/mL (+S9) (+S9)
DNA damage (SCE) Human blood 0.02, 0.2, 2.0 and 20.0 98 or 45 Positive Inadequate. Vehicle-control value falls Hatjian et al. (2000)
lymphocytes µg/mL (−S9) within the middle of the dose–response
curve. Questionable repeated measures and
statistical method used. Significance of
individual treatments not reported.
Formulation more cytotoxic and top two
concentrations could not be scored
Chromosome damage Human blood 750 µmol/L (= 228 NP Positive Inadequate. Questionable, very high Shokrzadeh et al.
(micronuclei in binucleate lymphocytes µg/mL) (−S9) induced MN frequency (>10%); no (2014)
cells) information on source or purity of diazinon;
single concentration; no indication of
cytotoxicity; similarity of the results to
Karamian et al. (2013) in spite of very
different treatment protocols raises
questions
Chromosome damage Human blood 750 umol/L (= 228 NP Positive Inadequate. Questionable very high induced Karamian et al. (2013)
(micronuclei in binucleate lymphocytes µg/mL) (−S9) MN frequency (~10%) occurring after a 1-
cells) hour treatment with diazinon prior to
mitogenic stimulation; single concentration;
no indication of cytotoxicity; similarity of
the results to Shokrzadeh et al. (2014) in
spite of very different treatment protocols
raises questions

40
−12 −10 −8
Chromosome damage MCF-7 cell 10 , 10 and 10 NP, obtained Positive Inadequate. No dose–response with same Ukpebor et al. (2011)
(micronuclei in binucleate mol/L (−S9) from Sigma- induction reported at extremely low
cells) Aldrich concentrations; number of cells with
multiple micronuclei is unusually high
Chromosome damage Human blood 2  10−8, 2  10−7, 97.3 Positive 2- to 4-fold increases seen at all tested Colovic et al. (2010)
(micronuclei in binucleate lymphocytes and skin 2  10−6 and 2  10−5 concentrations; UV-degraded diazinon
cells) fibroblasts mol/L (−S9) showed increased clastogenicity
Chromosome damage Human blood 0.04 and 0.4, 4 µg/mL NP Weak positive Weak ≤ 2-fold increases at all Bianchi-Santamaria et
(micronuclei in binucleate lymphocytes (−S9) (≥ 0.04 ug/mL) concentrations, not concentration related al. (1997)
cells)
Chromosome damage CHO cells NP NP Positive Not informative; the test material was the See, Dunn & San.
(chromosomal organic material isolated from the urine of (1990)
aberrations) orchard workers using various pesticides
Mammalian cell gene Mouse lymphoma cell 0, 6.25, 12.5, 25, 50 NP Positive Very different levels of cytotoxicity and McGregor et al. (1988)
mutation L5178Y TK +/- and 100 µg/mL (−S9 mutation frequencies were seen in the 2
1st trial); 0, 20, 40, 60 trials; no explanation provided. The positive
and 80 µg/mL (−S9 control did not produce an increase in
2nd trial) mutation frequency in the second trial
Micronuclei Cultured primary rat 2, 6, 18 and 54 µg/mL NP Negative Hepatocytes stimulated to proliferate using Frölichsthal & Piatti
hepatocytes from epidermal growth factor [in Italian] (1996)
Sprague Dawley male
rats
Chromosome damage Chinese hamster lung 0.1 mg/mL (0.33 NP Positive (+S9) Diazinon was excessively toxic in the Matsuoka, Hayashi &
(chromosomal cells mmol/L) S9 absence of S9. Inadequate due to inclusion Ishidate (1979)
aberrations) of gaps in the analysis
DNA damage (SCE) V79 Chinese hamster 0, 0.05, 0.1, 0.2 and 99, Wako Negative Adequate, 0.4 µg/mL caused excessive cell Kuroda, Yamaguchi &
cells 0.4 µg/mL Pure cycle delay Endo (1992)
Chemicals
DNA damage (SCE) V79 Chinese hamster 10, 20, 40 and 80 99.2 Ciba- Negative Adequate –S9 study since 80 µg/mL Chen et al. (1981)
cells -S9 (Chen et al., µg/mL Geigy associated with excessive cytotoxicity; Chen, Sirianni & Huang
1981) and +S9 (Chen, Inadequate +S9 as no data on cytotoxicity (1982)
Sirianni & Huang, provided
1982)

41
DNA damage (SCE) CHO cells 0.03, 0.1, 0.3 and 1.0 89, Analytical Negative Adequate Nishio & Uyeki (1981)
mmol/L (= 9, 30, 90, grade
300 µg/mL)
In vivo studies in nonmammalian species
Mutation Drosophila (wing spot 1,3,5,7 and 10 ppm fed 95 Positive Not informative due to use of a Cakir & Sarikaya
test) to larvae nonmammalian species (2005)
Chromosome loss (partial Drosophila (repair 100 ppb feeding to Commercial Negative Not informative as test used a formulation Woodruff, Phillips &
and entire) deficient cross) larvae product in a nonmammalian species Irwin (1983)
DNA damage (comet Freshwater mussels 0.28, 0.55 µg/mL 22.5 Positive at 0.28 Not informative as test used a formulation Conners & Black
assay, alkaline) (Utterbackia imbecillis) µg/mL only in a nonmammalian species (2004)
glochida
DNA damage (SCE) Central mudminnow 0, 5.4  10−11, 5.4  48.72 Positive Not informative as test used a formulation Vigfusson et al. (1983)
(Umbra limi) intestinal 10−10 and 5.4  10−9 in a nonmammalian species
tissue mol/L for 11 days
AP: apurinic/apyrimidinic locus; bw: body weight; CHO: Chinese hamster ovary; LD50: median lethal dose; MN: micronuclei; NP: not provided; PCE: polychromatic erythrocyte; ppb: parts per
billion; ppm: parts per million; S9: 9000 × g fraction from Aroclor 1254 or phenobarbital/5,6-benzoflavone–induced pretreated rat liver homogenate; SCE: sister chromatid exchange; SCSA:
sperm chromatin structure assay; TK: thymidine kinase locus; UV: ultraviolet

42
Table 18. Results of less relevant in vitro genotoxicity studies or those deemed inadequate for use in human risk assessment of diazinon
Metabolite End-point Test object Concentration Purity (%) Results Comments Reference
−5
2-Isopropyl-6- Chromosome Human blood 2  10 mol/L 97.3 Positive Inadequate, dose-related increase Colovic et al.
methyl-4-pyrimidinol damage lymphocyte/skin in MN reported; positive results (2010)
(IMP) (micronuclei in fibroblast also reported for UV-irradiated
binucleate cells) compound but with no negative
control
Diethylthiophosphate DNA damage Blood lymphocytes 1, 10, 25, 50, 100, NP, obtained Positive (1 or 10 Inadequate. Quality issues in data Vega et al. (2009)
(Comet assay) (mitogen-stimulated) 500 µmol/L from Sigma µmol/L in HepG2 or presentation and interpretation.
(alkaline at pH and WRL-68, WRL-68 cell lines pH 12.1 detects single strand
12.1 and >13) HepG2, HeLa cell only at either pH with breaks, pH>13 detects single
lines more migration with strand breaks and alkali labile
pH 12.1 as stated in sites. Interpretation in text does
text) not match data in figure but
increase in DNA migration under
both conditions. Effect
diminished by the addition of a
inhibitor of P450 enzymes
Diazoxon DNA damage Chinese hamster 0.03, 0.1, 0.3, 1.0 97 Positive at the highest Concentration-dependent Nishio & Uyeki
(SCE) ovary cells mmol/L (= 9, 30, concentration tested response but all within the control (1981)
90, 300 µg/mL) only range for other tested chemicals
NP: not provided; MN: micronuclei; SCE: sister chromatid exchange; UV, ultraviolet

2.5 Reproductive and developmental toxicity
(a) Multigeneration studies
In a two-generation study on reproductive toxicity, groups of 30 male and 30 female Sprague
Dawley (CR CD) rats were fed diazinon (purity 94.9%) in the diet at concentrations of 0, 10, 100 or
500 ppm over two generations (F0 and F1). Mean diazinon intakes for the F0 generation during the
premating period were 0, 0.77, 7.48 and 32.85 mg/kg bw per day for males and 0, 0.77, 7.48 and
40.26 mg/kg bw per day for females, respectively. After 10 weeks, the animals were mated (1:1)
within each dose group and allowed to rear the ensuing F1 litters to weaning. Litters were culled to
four male and four female pups, where possible, on day 4 postpartum. The breeding programme was
repeated with the F1 parents selected from the F1 offspring. The test diet was fed continuously
throughout the study. Parental feed consumption and body weights were measured throughout the
study. Reproductive performance, pup survival and developmental parameters were assessed. Gross
necropsy findings and histopathological observations in target organs of parental animals and pups not
selected for mating were recorded.
Treatment-related clinical symptoms consisted of tremors in three high-dose F0 females, and
dystocia followed by death or termination of two high-dose females, both on gestational day 26. In
addition, one intermediate-dose female was found dead on day 24 of gestation after being moribund
and with chromodacryorrhoea and a nasal discharge. The cause of death of this female was unknown
but may have been related to difficulties in delivery. In the F1 generation, four high-dose females
showed tremors during post-mating. Feed consumption was increased in F0 females at 500 ppm,
whereas in the F1 generation, a dose-related reduction of feed consumption was observed at 100 and
500 ppm in males only. Body-weight gain was lower in F0 females at 500 ppm during gestation. In the
F1 generation, reduced body-weight gain was observed at 100 and 500 ppm in males and at 500 ppm
in females.
There were no treatment-related effects on mating behaviour and the reproductive parameters
(including mating, fertility, gestation indices, number of viable pups and number of stillborn pups)
were comparable in the control and treatment groups in the F0 generation and at the low and
intermediate doses in the F1 generation. At 500 ppm, a greater proportion of females in both
generations had a longer gestation. A decrease in the number of pregnancies and viable pups as well
as reduced fertility and mating indices was seen in high-dose F1 females. F1 and F2 litter sizes were
smaller on lactation day 0. A dose-related reduction in survival of F1 pups was observed at 100 ppm
and 500 ppm and in F2 at 500 ppm. Weights of F0 pups were reduced in the 100 and 500 ppm groups,
and in F2 pups at 500 ppm. No treatment-related malformations were found in the pups.
The NOAEL for reproductive effects was 100 ppm (equal to 7.48 mg/kg bw per day for males
and females) based on prolonged gestation duration, decrease in the number of pregnancies, and
reduced fertility and mating indices at 500 ppm (equal to 32.85 mg/kg bw per day for males and
40.26 mg/kg bw per day for females).
The NOAEL for parental effects was 10 ppm (equal to 0.77 mg/kg bw per day for males and
females), based on reduced parental body-weight gain at 100 ppm (equal to 7.48 mg/kg bw per day
for males and females).
The NOAEL for offspring toxicity was 10 ppm (equal to 0.77 mg/kg bw per day for males
and females), based on reduced viability of pups and pup weights at 100 ppm (equal to 7.48 mg/kg bw
per day for males and females) (Giknis, 1989).
In another two-generation study on reproductive toxicity (Weatherholtz, 1982), groups of 13

to 15 male and 26 to 30 female Fischer 344 rats were fed diazinon (purity 97.36%) in the diet at
concentrations of 0, 0.1, 1.0 or 10 mg/kg (equivalent to 0, 0.0067, 0.067 and 0.67 mg/kg bw per day,
assuming concentrations are in mg/kg feed or ppm) over two generations (F 0 and F1). Rationales for
the dose selection or mean diazinon intakes were not provided. After the growth period, the animals
were mated (1:2) within each dose group and allowed to rear the ensuing F1 litters to weaning. Litters

44
were culled to 10 pups, where possible, on day 7 postpartum. The breeding programme was repeated
with the F1 parents selected from the F1 offspring. The test diet was fed continuously throughout the
study. Parental feed consumption and body weights were measured throughout the study.
Reproductive performance, pup survival and developmental parameters were measured. Gross
necropsy findings and histopathological observations in target organs of parental animals and pups not
selected for mating were recorded.
There were no treatment-related effects observed in F0 or F1 parental animals or pups. The
NOAEL for reproductive, parental and offspring effects was 10 ppm (equal to 0.67 mg/kg bw per day
for males and females), the highest dose tested (Weatherholtz, 1982).
(b) Developmental toxicity

Mice
In the following non-GLP and non-guideline study,
[g]roups of mice (6 females/dose) were given oral daily doses of 0, 0.18 or 9 mg/kg bw diazinon
throughout gestation. Treated animals gained less weight during gestation compared to control animals.
Weight gain of pups born to mothers receiving 9 mg/kg bw/day was reduced. Daily testing for
physiological and behavioural development of the pups revealed some evidence of retarded
development among the offspring of high-dose animals (e.g. retardation of eye and ear opening).
Measures of endurance and coordination also gave some evidence of impairment (e.g. increased rod
cling endurance in both groups, reduced rotarod endurance, impaired running performance in a maze
inclined plane test). Impaired reactions were sometimes observed at both dose levels but without a
clear dose-effect relationship. Examination of brain tissue of only 8 of a total number of 132 offspring
at the high-dose level revealed morphological abnormalities in the forebrain. The relationship of these
findings to the observed behavioural changes is unknown (Spyker & Avery, 1977; study evaluation
copied from the 1993 JMPR without further evaluation). The evidence for morphological effects of
diazinon on the developing brain was considered insufficient by an expert (Krinke, 1991; copied from
the 1993 JMPR without further evaluation).
It should be noted that the group size was rather small: six dams per group, with eight pups
per dam (48 pups per dose group).
Rats
In a teratogenicity study in Sprague-Dawley rats at dose levels of 0, 15, 50 or 100 mg/kg bw [per] day
administered orally on days 6 through 15 of gestation, dams at the 100 mg/kg bw/day dose level
showed a marked decrease in food consumption correlating with weight loss at the beginning of the
treatment period. Skeletal assessment showed a slightly higher incidence of incomplete ossification at
different sites in the fetuses at 100 mg/kg bw/day. Visceral examination revealed a dystopia cordis in
association with hypoplasia of lungs in 1/105 fetuses at 100 mg/kg bw/day. This anomaly has been
reported to occur spontaneously in control animals. Because of the single occurrence this anomaly was
not considered to be due to a direct action of diazinon but to be secondary to maternal toxicity (Fritz,
1974; study evaluation copied from the 1993 JMPR without further evaluation).
In another teratogenicity study (Infurna & Arthur, 1985), groups of 27 mated female rats
(Charles River Crl:COBS CD[SD] BR) were administered doses of diazinon (purity 97.4%) by
gavage at 0, 10, 20 or 100 mg/kg bw per day in 0.20% carboxymethyl cellulose containing 0.5%
Tween 80 during gestational days 6 through 15. The study was terminated on day 20 of gestation.
Body weight, feed consumption, mortality and abortion were continuously checked. All the dams
were necropsied, the ovaries examined and corpora lutea counted. Approximately one out of three of
the fetuses from each litter was dissected (organs and glands examined included the brain, eyes, spinal
cord, heart and major blood vessels, nasal passages, trachea, lungs, diaphragm, oral cavity, tongue,
oesophagus, stomach, intestines, liver, pancreas, thymus, spleen, kidneys, ureters, bladder, adrenals,

45
ovaries, uterus or testicles). The skeletons of the remaining two of three fetuses were examined for
abnormalities, size, shape, location and relationship to adjacent ossification centres).
The diazinon treatment had no effect on the mortality of the dams. During the first half of the
dosing period (gestation days 6–10), the high-dose dams lost weight (−11 grams), and body weights
and body-weight gains were significantly reduced throughout gestation (P values between 0.01 and
0.05). Feed consumption was reduced on gestation days 6 to 9 at a dose of 100 mg/kg bw per day. No
compound-related clinical signs and no abnormal gross pathological findings were observed in the
dams. At 100 mg/kg bw per day some reproductive parameters differed from the control values but no
statistical significance was achieved (e.g. increase in number of resorptions and increased per cent
pre- and post-implantation loss, reduction in number of live fetuses). However, at 20 mg/kg bw per
day, the number of resorptions and post-implantation losses were significantly (P < 0.05) reduced.
Male and female fetuses in the high-dose group weighed significantly (approximately 6%) more than
control fetuses. This effect can be attributed to the slightly decreased number of fetuses in the high-
dose group.
On gross observation, one fetus in the intermediate-dose group exhibited exencephaly. In the
high-dose group, 3 of the 262 (1%) fetuses from three litters showed external malformations (single
occurrences of a umbilical hernia, filament tail, sublingual extraneous soft tissue), and there were no
similar effects in the controls. However, umbilical hernia and tail abnormalities are routine
spontaneous malformations in this strain of rat, and these malformations were morphologically
unrelated; as a result, they were considered to be a consequence of the marked maternotoxicity at this
dose level and not due to a teratogenic effect of the test compound. An increased incidence in
rudimentary ribs (T-14) was observed in all treatment groups, attaining statistical significance in the
high-dose group (5% versus 0%). The increased incidence of the skeletal variations observed at the
highest dose level was considered to be related to maternotoxicity at this dose level.
The NOAEL for maternal toxicity was 20 mg/kg bw per day, based on body weight loss on
gestation days 6 to 10, reduced body weight and body-weight gains throughout treatment, and
decreased feed consumption on gestation days 6 to 9 at 100 mg/kg bw per day.
The NOAEL for embryo/fetal toxicity was 20 mg/kg bw per day, based on increased
incidence of rudimentary T14 ribs at 100 mg/kg bw per day (Infurna & Arthur, 1985).
Rabbits
Diazinon (purity 89.2%) was administered to groups of 18 to 22 pregnant rabbits (New
Zealand White) by gavage at 0, 7, 25 or 100 mg/kg bw per day in 0.2% carboxymethyl cellulose or
methyl cellulose from gestation days 6 to 18 (inclusive). The females were monitored daily for
clinical signs. Body weights and feed consumption were recorded at selected intervals during
gestation. On gestation day 30, the rabbits were terminated and all the thoracic and abdominal cavity
organs examined for macroscopic changes. The ovaries, uterus and cervix were weighed and
examined. Corpora lutea were counted and the number and distribution of fetuses and intrauterine
deaths determined. All the fetuses were sexed, weighed and examined for soft tissue and skeletal
malformations and variations. A transverse section of the brain was made, and organ structure was
examined.
An increase in maternal mortality was observed at 100 mg/kg bw per day (9/22, 41% versus
0% in all other groups). Overt clinical signs of maternal toxicity at 100 mg/kg bw per day included
tremors, convulsions, hypoactivity and anorexia. Reduced body-weight gain was found at the highest
dose level. The treatment did not affect the number of corpora lutea, number of implantation sites, live
fetuses per litter or fetal weight. The incidence of visceral and skeletal malformations and variations
showed no treatment-attributable differences between the groups. The study therefore gave no
evidence for an embryotoxic or teratogenic activity of diazinon.
The NOAEL for maternal toxicity was 25 mg/kg bw per day, based on mortality, tremors,
convulsions, hypoactivity, anorexia and reduced body-weight gain observed at 100 mg/kg bw per day.

46
The NOAEL for embryo/fetal toxicity was 100 mg/kg bw per day, the highest dose tested (Harris &
Holson, 1981).
In another developmental toxicity study, diazinon (purity 96.2%) was administered to groups
of 17 to 20 pregnant rabbits (New Zealand White) by gavage at dose levels of 0, 2.5, 10 or 40 mg/kg
bw per day in 5% aqueous solution of gum arabic from gestation days 6 to 18 (inclusive). The females
were monitored daily for clinical signs. Body weights and feed consumption were recorded at selected
intervals during gestation. Cholinesterase activity was not measured. On gestation day 29, the rabbits
were terminated and examined for macroscopic changes. Corpora lutea were counted. The uterus was
opened and examined for the number and distribution of live fetuses and intrauterine deaths. All
fetuses were weighed and examined for external, soft tissue and skeletal variations and
malformations. Clinical signs, body weights and feed consumption were measured regularly and
plasma and erythrocyte cholinesterase measured in blood samples obtained 2 hours after dosing on
day 13, the final day of dosing.
Clinical signs, including unsteadiness, body tremors and abnormal movement and posture
were observed in high-dose females. Feed consumption and body-weight gain were also reduced in
this dose group. Mean litter and fetal weights were lower than control values in high-dose fetuses, the
difference in mean fetal weight being statistically significant (P < 0.05). Litter size and pre- and post-
implantation losses were not affected. There was no obvious adverse effect on incidence of
malformation anomalies or skeletal variants. In the preliminary study, where groups of six non-mated
females were administered 0, 10, 30 and 50 mg/kg bw per day, qualitatively similar but more severe
findings were observed in the high-dose females. In addition, plasma and erythrocyte cholinesterase
activity was dose-dependently inhibited at all dose levels compared to controls (Table 19).
Table 19. Cholinesterase estimation in preliminary study

Mean cholinesterase levels on last day of dosing period (µmol/mL per min)
Dose level of diazinon Blood plasma Erythrocyte
Control 0.70 1.92
10 mg/kg bw per day 0.17 0.59
bw: body weight; min: minute
N = 6 per group.
Source: Edwards & Falconer, 1987
The NOAEL for maternal toxicity was 10 mg/kg bw per day, based on clinical signs,
decreased body weight and reduced feed consumption.
The NOAEL for embryo/fetal toxicity was 10 mg/kg bw per day, based on decreased fetal
weight at 40 mg/kg bw per day.
No NOAEL could be derived in the range-finding study as inhibition of erythrocyte
cholinesterase activity was observed at the lowest dose of 10 mg/kg bw per day (Edwards & Falconer,
1987).
2.6 Special studies

(a) Acute neurotoxicity
In an acute neurotoxicity study, male and female Sprague Dawley rats (10 rats/group) were
administered diazinon by gavage (lot no. 510530; purity 95.4%) at 0, 100, 300 or 500 mg/kg bw. This

47
study was conducted in compliance with GLP and in accordance with Ministry of Agriculture,
Forestry and Fisheries of Japan test guidelines. Cholinesterase activity was not measured.
Four hours after dosing, decreased motor activity, respiratory rate and body temperature as
well as mucous stool or stained fur (perianal, perigenital, perioral, periocular) was observed in one
male at 300 mg/kg bw and in both males and females at 500 mg/kg bw. Motor activity was also
decreased at 300 and 500 mg/kg bw in both sexes. Fore- and hindlimb grip strength was weakened at
500 mg/kg bw. No histopathological changes were found.
The NOAEL was 100 mg/kg bw, based on systemic toxicity and clinical signs of
neurotoxicity observed at 300 or 500 mg/kg bw (Sunaga, 2007a).
Groups of 15 male and 15 female Sprague Dawley rats were administered single doses of
diazinon (purity 88%) by gavage at doses of 0, 2.5, 150, 300 or 600 mg/kg bw. Of the 15 rats, 10 were
used for neurological testing and five for measuring cholinesterase activity. Dose selection was based
on the results of three preliminary studies that assessed median lethal doses (LD50) and determined the
lowest lethal dose to be 750 mg/kg bw and the highest non-lethal dose to be 500 mg/kg bw. As such,
significant neurotoxic effects were expected at the highest selected dose of 600 mg/kg bw. The
selected intermediate doses (150 and 300 mg/kg bw) were anticipated to give intermediate or minimal
effects, while a previous investigation (Glaza, 1993) had found no reduction in erythrocyte and brain
cholinesterase activity at the lowest dose of 2.5 mg/kg bw. Groups of 10 male and 10 female rats were
administered triadimefon (purity 99%) by gavage at 150 mg/kg bw as a positive control for
neurological effects. On the day of treatment, all rats were observed before and after dosing and twice
daily thereafter for general appearance, behaviour, signs of toxicity, morbidity and mortality. Each
week, all the rats were examined in detail, including palpation for tissue masses. Body weights were
estimated before dosing and weekly thereafter, and feed consumption was estimated weekly. A
functional observational battery was performed 1 week before administering the single dose, at the
time of peak effect after dosing (9–11 hours for diazinon, 1 hour for triadimefon) and 7 and 14 days
after dosing with diazinon on the 10 rats intended for neurological testing. Blood samples were
obtained at the estimated time of peak effects and at 14 days for the five rats designated for
cholinesterase testing in each group. Plasma and erythrocyte cholinesterase activity was determined
by colorimetric assay, while brain cholinesterase activity was estimated in whole brain samples at
termination 14 days after administration. The rats used for neurological examination (survivors to
termination and decedents) were necropsied. Sections were made at 10 levels of the brain, cervical,
thoracic, lumbar and sacral spinal cord with ganglia and right and left sciatic nerves, right and left
fibular nerves, right and left tibial nerves and right and left lateral cutaneous sural nerves, and the
Gasserian ganglion. Sections were also taken of skeletal muscle from the right thigh, eyes with the
optic nerve and any gross lesions identified. Only five rats per group in the groups receiving diazinon
at 600 mg/kg bw, the control group and those treated with triadimefon were processed for
histopathological examination.
During the study, two males and one female treated at 600 mg/kg bw died. A single
accidental death occurred in the group of five rats at 300 mg/kg bw intended for cholinesterase
measurement during blood sampling. One control animal was removed from the study because it was
thought to have been wrongly dosed, as it showed signs of cholinergic poisoning and low
cholinesterase activity. The clinical observations included chromodacryorrhoea, reduced activity and
tremors at a dose of 300 mg/kg bw, while the group at 600 mg/kg bw also had chromorhinorrhoea,
diarrhoea and pallor. Significant decreases in body-weight gain were observed in males at doses
greater than or equal to 300 mg/kg bw, while no effects were observed on body weight in females.
Feed consumption was decreased in males at doses greater than or equal to 300 mg/kg bw and in
females at doses greater than or equal to 150 mg/kg bw. In both males and females, effects on
functional observational battery parameters were seen only at the estimated time of peak effect (9–11
hours for diazinon, 1 hour for triadimefon) and not on day 7 or 14 after exposure.
The autonomic parameters affected by diazinon at the time of peak effects in males were
faecal consistency and soiled fur at doses greater than or equal to 150 mg/kg bw; these effects were

48
dose related. Increased salivation, staining of the nose and repeated opening and closing of the mouth
were observed at 300 mg/kg bw, and impaired respiration, lachrymation and staining of the mouth at
600 mg/kg bw. In females, repeated opening and closure of the mouth were observed at doses greater
than or equal to 150 mg/kg bw, and altered faecal consistency, soiled fur and staining of the nose were
observed at doses greater than or equal to 300 mg/kg bw. Impaired respiration and lachrymation were
observed at the highest dose. The neuromuscular parameters affected in males were abnormal gait at
150 mg/kg bw and higher doses; ataxic gait, impaired righting reflex, impaired hindlimb extensor
reflex and decreased hindlimb footsplay at 300 mg/kg bw and higher doses and reduced forelimb grip
strength at 600 mg/kg bw. In the females, ataxic and abnormal gait was observed at 150 mg/kg bw
and higher doses and impaired righting reflex at 300 mg/kg bw and higher doses. Impaired hindlimb
extensor reflex, abnormal hindlimb positioning when held by the tail and reduced forelimb and
hindlimb grip strength were observed at 600 mg/kg bw. Central nervous system excitability
parameters were also affected. In males, tremors were observed in the home cage and open field at in
animals at 300 mg/kg bw and higher, as was twitching or muscle fasciculation. At 600 mg/kg bw, the
arousal level was decreased. Females had tremors in the home cage at the highest dose only and in the
open field at 300 mg/kg bw and higher doses. Twitching in the open field was seen at the highest
dose, and a lowered arousal level at 300 mg/kg bw and higher doses. Touch response was reduced in
females at the highest dose, and the tail-pinch response was reduced in both sexes at this dose.
Reduced body temperature was observed in males at doses greater than or equal to 300 mg/kg bw and
in females at greater than or equal to 150 mg/kg bw. In addition, females at 300 mg/kg bw and higher
doses were dehydrated. Locomotor activity in the figure-eight maze decreased over time in all groups.
At the estimated time of peak effect, the activity of males at doses 300 mg/kg bw and higher and of
females at 150 mg/kg bw and higher was decreased. At 7 and 14 days after dosing, the mean total
activity counts were similar for all groups.
At the time of peak effect after diazinon intake (9–11 hours), plasma cholinesterase activity
was reduced in all the treated groups; however, no differences between groups were observed 14 days
after dosing. Erythrocyte cholinesterase activity was inhibited at 150 mg/kg bw and higher doses in
both males and females at the time of peak effect. At 150, 300 and 600 mg/kg bw, the activity was
18%, 17% and 15% of controls in males and 24%, 23% and 24% of controls in females, respectively.
Partial recovery was seen 14 days after dosing: in males, the activity at 150 mg/kg bw was 91% that
of concurrent controls, while it was 66% and 53% that of concurrent controls at 300 and 600 mg/kg
bw, respectively; in females, the activity was 89%, 57%, 74% and 65% that of concurrent controls at
2.5, 150, 300 and 600 mg/kg bw, respectively. No significant differences in brain cholinesterase
activity were seen in the groups of males. Significantly reduced brain cholinesterase activity (92% of
control value) was seen at termination in females at 150 mg/kg bw; however, as activity in the groups
at the higher doses was comparable to that in the controls, this finding is of no biological significance.
No gross or microscopic treatment-related abnormalities were seen at necropsy. Triadimefon
decreased weight gain and feed consumption during week 1 of the study. At the time of peak effect
after exposure (1 hour), changes in central nervous system excitability parameters (increased
incidence of rearing; increased arousal level) were the only changes seen in the functional
observational battery.
The NOAEL was 2.5 mg/kg bw based on decreased erythrocyte cholinesterase concentrations
and behavioural changes at 150 mg/kg bw (Chow & Richter, 1994).
Groups of albino Crl:CD[BR] VAF/Plus rats were administered diazinon (purity 87.9%) by
gavage. In phase 1 of the study, five males and five females were given diazinon at 100, 250 or 500
mg/kg bw. Subsequently, in order to identify an NOAEL, groups with five females dosed with
diazinon at 25 or 50 mg/kg bw were added. In phase 2 of the study, groups of five males each were
administered diazinon at 0.05, 0.5, 1.0, 10.0, 100 or 500 mg/kg bw by gavage, and groups of five
females received doses of 0, 0.05, 0.12, 0.25, 2.5, 25 or 250 mg/kg bw. In phase 1, clinical
observations were carried out 1, 2, 4 and 8 hours after administration of the test material and daily
thereafter. Body weights were determined before treatment and on days 7 and 14. The rats that died
after day 1 were also weighed; cholinesterase activity in these animals was not measured. In phase 2,

49
clinical observations were made 1, 2 and 4 hours after administration of diazinon, while body weights
were measured before treatment and on day 1. The surviving rats from phase 1 were terminated on
day 14 and subjected to gross necropsy; abnormal tissues were retained for possible histopathological
examination. In phase 2, plasma and erythrocyte cholinesterase activity was measured by colorimetric
assay 24 hours after treatment. The rats were then terminated and subjected to gross necropsy, with
abnormal tissues retained for possible histopathological examination; in addition, the right half of the
brain was removed to determine cholinesterase activity.
One high-dose female died during phase 1 of the study. There was no treatment-related effect
on body weight. Clinical signs were seen in males at 250 and 500 mg/kg bw and in females at 50
mg/kg bw and higher doses; the signs included miosis, hypoactivity, absence of the pain reflex, red-
stained face, yellow-stained urogenital region and soft stools. No pathological changes were observed
that could be attributed to treatment. In phase 2, no deaths occurred. Body-weight loss within 24 hours
of dosing was greater in males at 500 mg/kg bw and in females at 250 mg/kg bw than in concurrent
controls. Clinical signs of toxicity were seen in males at 500 mg/kg bw and in females at 250 mg/kg
bw; these signs included miosis, hypoactivity, absent pain reflex, staggering gait, excessive salivation,
red-stained face and yellow-stained and/or wet urogenital region. The only gross pathological findings
of note were yellow staining of the perineum and red paranasal discharge in males at 500 mg/kg bw
and in females at 250 mg/kg bw.
Plasma cholinesterase activity was reduced in males at 10 mg/kg bw and higher and in
females at 2.5 mg/kg bw and higher. A statistically significant (P ≤ 0.05) and biologically significant
reduction in erythrocyte cholinesterase activity was seen in males at 100 mg/kg bw (to 51% of control
value) and 500 mg/kg bw (to 64% of control value). In females, erythrocyte cholinesterase activity
was statistically and biologically significantly reduced at 25 mg/kg bw (to 65% of control value) and
250 mg/kg bw (55% of concurrent control value). Brain cholinesterase activity was statistically and
biologically significantly reduced in males at 500 mg/kg bw (to 31% of control value) and in females
at 250 mg/kg bw (to 30% of concurrent control). In females at 25 mg/kg bw, brain cholinesterase
activity was 64% that of controls; while this difference was not statistically significant, it is
considered biologically significant.
At necropsy in phase 2, staining of the perineum and red paranasal discharge were seen in rats
of each sex at the highest dose. No treatment-related histological lesions were seen.
The NOAEL was 2.5 mg/kg bw based on the inhibition of brain and erythrocyte
acetylcholinesterase activities in females at 25 mg/kg bw (Glaza, 1993).
One study reported on the time course of acute inhibition of cholinesterase activity. Groups of
15 male and 15 female rats were administered diazinon (purity 88%) at 0, 2.5, 150, 300 or 600 mg/kg
bw. Doses were selected based on the results of a range-finding study (Glaza, 1993) in which whole
brain cholinesterase activity was inhibited 24 hours after dosing in males at 500 mg/kg bw and
females at 250 mg/kg bw by about 70%. Clinical observations were made immediately before blood
sampling from the orbital plexus to determine serum and erythrocyte cholinesterase activity. Five rats
of each sex were terminated at 3, 5 and 9 hours and the remainder at 24 hours to determine brain and
spinal-cord cholinesterase activity. Acetylcholinesterase activity was measured in the cerebellum,
cerebral cortex, striatum and hippocampus and in the thoracic spinal cord using a modification of the
method of Ellman et al. (1961).
Survival was unaffected by treatment. At the highest dose, clinical signs were seen at 3 hours,
with maximum effect at 9 hours in males and some recovery after 24 hours, and maximum effect after
24 hours in females. No significant differences were seen in body weight. Plasma cholinesterase
activity was decreased by more than 20% at the lowest dose at 3 hours and 9 hours, with maximum
reduction at 9 hours in males and females. At 24 hours, the cholinesterase activity at the lowest dose
was decreased by 17% in males and 42% in females compared with that of the controls. Although
erythrocyte cholinesterase activity was significantly decreased in females 9 hours after dosing at
2.5 mg/kg bw, the absence of any inhibition at 3 and 24 hours and at any time in males suggests that

50
this result may be an anomalous finding. In an analysis of cholinesterase activity in regions of the
central nervous system at the lowest dose, cholinesterase activity in males was never less than 80%
that of controls (although it was equal to 80% that of controls in the cerebral cortex at 9 hours). In
females at the lowest dose, no substantial decrease in central nervous system cholinesterase activity
was observed. At the higher doses, significantly decreased activity was observed in all regions and at
all times but was usually greater at 9 hours and 24 hours than at 3 hours (Table 20).
The NOAEL was 2.5 mg/kg bw, based on inhibition of brain and erythrocyte
acetylcholinesterase activities at 150 mg/kg bw. Inhibition was observed beginning at 3 hours post-
dosing, with maximal inhibition at 9 hours post-dosing (Potrepka, 1994).
Table 20. Change in cholinesterase activity in rats administered diazinon

Mean per cent change in cholinesterase activity per dose of diazinon
Male Female
2.5 300 2.5 300
Interval mg/kg 150 mg/kg 600 mg/kg 150 mg/kg 600
Sample (hour) bw mg/kg bw bw mg/kg bw bw mg/kg bw bw mg/kg bw
Plasma 3 −21** −66** −71** −72** −57** −74** −77** −79**
9 −30** −79** −80** −77** −60** −82** −85** −73**
24 −17** −76** −84** −88** −42** −89** −89** −91**
Erythrocyte 3 0 −66** −82** −74** +1 −42** −50** −73**
9 +1 −76** −78** −81** −40** −68** −78** −74**
24 −11 −68** −77** −76** −11 −70** −68** −71**
Cerebellum 3 −1 −51** −76** −80** −7 −54** −48** −66**
9 −6 −59** −78** −84** +2 −65** −79** −77**
24 +3 −45** −60** −80** 0 −68** −74** −81**
Cerebral cortex 3 +16 −31 −67** −75** +4 −34* −35** −56**
9 −20* −62** −82** −85** −5 −63** −75** −78**
24 +23 −45** −60** −80** −1 −73** −77** −85**
Striatum 3 0 −28* −69** −75** +13 −26* −31** −50**
9 +10 −65** −77** −85** +9 −66** −81** −83**
24 +12 −43** −58** −85** −5 −68** −84** −87**
Hippocampus 3 +5 −40** −70** −80** −10 −46** −47** −56**
9 −5 −57** −76** −84** +5 −68** −81** −83**
24 +25 −45** −62** −85** −1 −65** −74** −81**
Thoracic spinal 3 +10 −27 −65** −77** +8 −39** −33* −49**

cord
9 +8 −51** −76** −85** +4 −63** −73** −78**
24 +9 −42** −50** −81** +3 −51** −46** −81**
bw: body weight; *:P ≤ 0.05; **:P ≤ 0.01 (Dunnett t-test)
Results expressed as mean increase (+) or decrease (−) in cholinesterase activity relative to the control (%).
Source: Potrepka (1994)

51
(b) Subacute neurotoxicity

A 90-day repeated neurotoxicity study in rats was conducted in accordance with test
guidelines and GLP. Ten male and female Sprague Dawley rats were fed diazinon in the diet at
concentrations of 0, 25, 125 or 1000 ppm (equal to 0, 1.7, 8.4 or 69.1 mg/kg bw per day in males and
0, 1.8, 9.3 or 82.4 mg/kg bw per day in females) for 90 days. All the rats were checked for mortality
and clinical signs twice a day. Body weights and feed consumption were measured and chemical
intakes calculated on day 1, 4 and 8 and weekly thereafter. Detailed clinical signs and behaviour were
monitored before the treatment and 2, 4, 8 and 13 weeks after starting the treatment. An
ophthalmological examination was conducted before the treatment for all the rats and at week 13 for
the rats at 1000 ppm and the controls. Plasma, erythrocyte and brain acetylcholinesterase activities
were measured at termination. At termination, all the rats were necropsied. Five rats per group in both
sexes were fixed by perfusion of glutaraldehyde and paraformaldehyde solutions under deep
anaesthesia. The fixed tissues and organs in the central and peripheral nervous systems underwent
Mortality was unaffected by the treatment. No treatment-related clinical signs were observed.
Body weight were slightly decreased (within 10% of the control) in females on days 4 to 15, but the
decrease was not considered treatment related because it was slight (<10%), transient and bore no
similarity to the results of the preliminary study. No treatment-related changes were noted in
functional observational battery or neurological, ophthalmological or pathological analyses.
Cholinesterase activities in the plasma, erythrocyte and brain are summarized in Table 21. Erythrocyte
acetycholinesterase activity was inhibited by over 20% at 25 ppm and above in both sexes; brain
acetycholinesterase activities were inhibited by 20% and above at 125 ppm and 1000 ppm.
Table 21. Summary of AChE activity in 90-day neurotoxicity study in rats administered diazinon in
the diet
AChE activity per dose of diazinon
Male Female
1 000 125 1 000
0 ppm 25 ppm 125 ppm ppm 0 ppm 25 ppm ppm ppm
Erythrocyte 544.9 396.0# 223.1## 155.0## 601.4 313.5## 175.0## 152.4##
AChE (IU/L) (73) (41) (28) (52) (29) (25)
Brain AChE 127.4 126.0 117.8 117.8** 125.4 124.0 80.4** 21.6**
(mU/mg) (99) (92) (49) (99) (64) (17)
AChE: acetylcholinesterase; IU: International Unit; ppm: parts per million; U: enzyme unit; #: P ≤ 0.05; ## : P ≤ 0.01
(Mann–Whitney U-test); *: P ≤ 0.05; **: P ≤ 0.01 (Dunnett t-test)
Results shown as concentration in IU/L for erythrocyte AChE and mU/mg for brain AChE, with activity relative to controls
as a percentage (%) in parentheses.
Source: Sunaga (2007b)
The NOAEL for subacute neurotoxicity was not determined based on cholinesterase
inhibitions in the erythrocytes and brain at 25 ppm (equal to 1.7 mg/kg bw per day), the lowest dose
tested (Sunaga, 2007b).
(c) Delayed neurotoxicity

An oral dose of 28 mg/kg bw of diazinon technical (87% purity) was administered to a group
of 18 hens (the target dose was 13 mg/kg bw per day as the approximate LD 50 was doubled due to a
preparation error). The schedule to protect the hens from acute cholinergic effects of diazinon
consisted of a 10 mg/kg bw atropine pretreatment by intramuscular injection and an additional
intramuscular injection of 2-pyridinealdoxime methiodide (2-PAM) at the time of diazinon dosing.

52
Post-treatment consisted of concurrent doses of atropine and 2-PAM 1 and 5 hours after dosing. Since
there were no neurotoxic responses in the test group in the 3 weeks following the treatment, the hens
were treated again with 13 mg/kg bw of diazinon on day 21. One test group hen was found dead on
day 5 after the first dosing and one vehicle control hen was found dead on day 7 after the first dosing,
after exhibiting a slight unsteadiness when walking. After the second dosing on day 21, one test group
hen was found dead 6 hours after the second treatment, and one test group hen exhibited a slight
unsteadiness in walking only on day 41. No neurotoxic signs were apparent during the 3-week
observation period after the second treatment. Histopathological examination showed no lesions in the
brain, spinal cord or peripheral nerves in the diazinon-treated animals, whereas animals from the
positive control (tri-orthocresyl phosphate [TOCP] treatment) showed multiple lesions (axonal
degeneration) consistent with peripheral neuropathy (Jenkins, 1988).
In another delayed neurotoxicity study, groups of 12 hens were administered an oral dose of
0, 10, 30 or 100 mg/kg bw per day of diazinon technical (purity 96.3%) in peanut oil, the high dose
being twice the estimated LD50 of 50 mg/kg bw. TOCP at 500 mg/kg bw was used as a positive
control. To protect the hens against cholinergic toxicity, hens were treated with intramuscular
physostigmine and/or atropine for 24 to 48 hours after the diazinon doze. The hens were observed for
clinical signs and ataxia over 21 days. After perfusion fixation, the peripheral nerves, spinal cord,
brainstem and cerebellum were examined histologically. For biochemical assessment of esterase
activities 24 and 48 hours after administration, satellite animals were dosed accordingly.
One hen at 30 mg/kg bw died on day 1 and was replaced by a reserve animal. In addition, 3 of
10 hens in the 100 mg/kg bw group died within 3 days of administration. These early decedents were
not necropsied because delayed neuropathy requires longer to develop. Dose-related signs of
cholinergic toxicity, including diarrhoea, salivation, vomiting or dyspnoea and reduced activity,
impaired gait and recumbency, lasting for up to 6 days, were observed in the animals dosed 30 and
100 mg/kg bw. Body-weight gains were reduced in 30 and 100 mg/kg bw animals. Severe inhibition
of plasma cholinesterase was observed at all doses. Brain and spinal-cord cholinesterase activity was
reduced in a dose-related manner in hens treated at 30 and 100 mg/kg bw, while those at 10 mg/kg bw
were not affected. Neither erythrocyte activity nor neurotoxic esterase activity in brain and spinal cord
was affected by diazinon. TOCP-treated controls showed signs consistent with organophosphate ester-
induced delayed neuropathy (Classen, 1996).
In a third delayed neurotoxicity study, groups of 12 Sterling Ranger hybrid hens were
administered an oral dose of 20 mg/kg bw of diazinon technical (96.2% purity) in maize oil, this being
the approximate oral median lethal dosage. The hens were treated on day 1 and again 21 days later.
Two other groups of six hens were similarly treated with maize oil (vehicle control) or with TOCP at
700 mg/kg bw (positive control).
The observed marked cholinergic responses and associated motor dysfunction were controlled
using atropine sulfate and pralidoxime therapy. One bird with severe cholinergic signs – body weight
loss and deteriorating condition – was terminated in extremis 3 days after the second administration of
diazinon. The surviving birds generally fully recovered within 2 to 4 days of treatment, and the
majority showed no abnormalities during the remainder of the observation period. One bird showed
reduced activity and slight body weight loss from day 22 onwards and episodes of perianal soiling,
unsteady stance and resting on hocks. Other signs seen in three birds were largely restricted to
transient reduced activity and unsteady stance. The positive control animals showed the expected
signs of delayed neurotoxicity in four of the six animals (disturbed balance, unsteady gait/stance).
Minimal axonal swelling and eosinophilic accumulations within the axons of the upper cervical or
mid-thoracic region of the spinal cord were observed in five of the 12 birds treated with diazinon and
in all the birds treated with TOCP. The lesions were generally less severe but qualitatively similar to
those observed in the upper spinal cord of TOCP-treated birds. Neuromotor changes observed in
diazinon-treated birds (also observed in TOCP-treated birds) largely comprised reduced activity
and/or unsteady stance (four birds) and axonal swelling and eosinophilic accumulation within the

53
axons of the upper cervical or mid-thoracic region of the spinal cord (five birds). However, these was
no intra-animal correlation between the in vivo and histopathological findings. In view of the minimal
nature of both types of change, it was concluded that there was no unequivocal evidence that diazinon
caused acute delayed neurotoxicity in the hen (Cummins, 1987b).
(d) Mechanistic studies

Special study of effects on the pancreas
A study was conducted using a canine model to investigate the induction of pancreatic ductal
hypertension following cholinesterase inhibitor intoxication known to be an important triggering
mechanism in the pathogenesis of acute and chronic pancreatitis. Diazinon was intravenously
administered (25 mg/kg bw) to the pancreatic ampulla of dogs and the tissue cholinesterase activity of
the canine pancreatic sphincters was determined. Two enzymes are responsible for the total
cholinesterase activity of whole blood: a membrane-bound AChE associated with the erythrocyte
membrane, and a soluble enzyme, pseudocholinesterase or butyrylcholinesterase (BChE) in the serum.
In tissues, which also contain the two forms of cholinesterase, AChE is important in the regulation of
neuromuscular activity and parasympathetic ganglion transmission, while the role of BChE is
unknown. The observation of the negative correlation between serum BChE activity and intraductal
pancreatic pressure supports the hypothesis that the pancreatic ductal hypertension which occurs
following cholinesterase inhibitor intoxication is due to a selective reduction in pancreatic BChE
activity (Dressel et al., 1980; study evaluation copied from the 1993 JMPR without further evaluation).
The induction of acute pancreatitis by [organophophates] found in dogs was confirmed in guinea-pigs
but not in cats. These results may reflect species-related differences in the distribution of pancreatic
BChE (Frick et al., 1987; study evaluation copied from the 1993 JMPR without further evaluation).
Special studies on antidotes

Previous reports with respect to the usefulness of PAM [sic] and other oxine reactivators against this
organophosphate have been published (Sanderson & Edson, 1959; Wills, 1959; study evaluations
copied from the 1993 JMPR without further evaluation). A study was undertaken to provide
information on the antidotal effectiveness of pyridine-2-aldoxime methochloride (2-PAM) [sic] against
diazinon poisoning in animals. The administration of atropine (16 mg/ kg bw; [intramuscular]) or 2-
PAM (30 mg/kg bw; [intravenous]) alone, 10 minutes after poisoning rats with doses of 235 mg/kg bw
(corresponding to the approximate oral LD50 or higher) provided little or no protection. Best protection
was achieved when the oxime was given orally in conjunction with [intramuscular] atropine or
followed by a subsequent dose of 2-PAM orally or [intravenously]. Administration of 2-PAM to
diazinon-poisoned rabbits (1600 mg/kg bw) resulted in reactivation of inhibited blood ChE
[cholinesterase] activity concurrent with a decrease in signs of poisoning. Within 2 hours, however, the
animals were again weak and ataxic and blood ChE showed renewed inhibition. The authors suggested
that effective therapy of diazinon intoxication requires repeated doses of oxime to maintain effective
antidote levels in the body (Harris et al., 1969; study evaluation copied from the 1993 JMPR without
further evaluation). In dogs and guinea-pigs, pretreatment with atropine protected the animals against
diazinon-induced pancreatitis (Frick et al., 1987; study evaluations copied from the 1993 JMPR
without further evaluation).
(e) Estrogenic or androgenic activities

Estrogen receptor transcriptional activation
Agonistic activity of diazinon to human estrogen alpha (hER) was investigated using the
transcriptional activation assays using the hERα-HeLa-9903 cell line (HeLa-9903). This assay was
conducted as outlined in the USEPA Office of Prevention, Pesticides & Toxic Substances (OPPTS)
Endocrine Disruptor Screening Program Test Guidelines 890.1300 and in accordance with GLP. A
preliminary study was conducted for cytotoxicity and precipitation to identify a suitable top
concentration of diazinon (purity 98.7%; batch no. 00896074) for use. The final concentrations were
10−11 to 10−3 mol/L. For each concentration, additional replicates were prepared that incorporated the

54
hERα antagonist ICI 182,780. The transcriptional activation assay was also performed for four
reference compounds (17β-estradiol, 17α-estradiol, corticosterone and 17α-methyltestosterone). The
optimum top concentration of diazinon for use in the transcriptional activation assays was 10 −5 mol/L,
based on excessive cytotoxicity (≥ 20% reduction in cell viability) at concentrations greater than or
equal to 10−4 mol/L.
In the assay, diazinon did not result in an increase in luciferase activity (maximum response
< 10%) at any of the viable concentrations tested. Diazinon was not an agonist of human estrogen
receptor alpha (hERα) in the HeLa-9903 model system (Willoughby, 2011a).
Aromatase inhibitory activity

The inhibitory effect of diazinon on aromatase activity were investigated using a human
recombinant aromatase assay using human CYP19 (aromatase) and P450 reductase supersomes. This
study was conducted as outlined in USEPA OPPTS 890.1200 test guideline and in accordance with
GLP. Final concentrations of diazinon (purity 98.7%; batch no. 00896074) tested by the aromatase
assay were 10−10 to 10−3 mol/L. Four independent runs of the aromatase assay were conducted. In
addition, the positive control inhibitor 4-hydroxyandrostenedione was included each time the
aromatase assay was performed.
Increasing concentrations of 4-hydroxyandrostenedione decrease the aromatase activity in a
concentration-dependent manner. In four independent runs of the assay, increasing concentrations of
diazinon resulted in a decrease in aromatase activity (at 10−4 and 10−3 mol/L); however, visual
inspection of diazinon concentrations showed particulate matter and cloudiness at 10−3 mol/L that was
determined to be insoluble test substance. Thus, the top concentration of diazinon suitable for use in
the aromatase assay was established at 10−4 mol/L. According to the data-interpretation procedure
outlined by the USEPA for aromatase inhibition, diazinon was classified as ‘equivocal’. It had a mean
(standard deviation [SD]) value of 63.7% ( 14.3%) control activity at 10−4 mol/L. Diazinon was
determined to be equivocal for aromatase activity while a decrease in aromatase activity was
identified at the highest concentration of diazinon (e.g. 10−3 mol/L). Solubility issues were observed
so this concentration was not included in the data interpretation (Wilga, 2011).
Estrogen receptor binding assay using rat uterine cytosol

The ability of diazinon to interact with the estrogen receptors isolated from the rat uterus was
investigated as outlined in the USEPA OPPTS 890.1250 test guideline and in accordance with GLP.
In a preliminary study, the final concentrations of diazinon (purity 98.7%; batch no. 00896074) tested
in the binding assays were 10−11 to 10−3 mol/L. Three independent runs of the binding assay were
conducted. A complete concentration–response curve for the negative control, octyltriethoxysilane,
and weak positive control, 19-norethindrone, was run each time the binding assay was performed.
The top concentration of diazinon suitable for use in the binding assays was 10 −4 mol/L. No
precipitation was observed at 10−3 mol/L in the first runs so the concentration range was shifted for
the second and third runs. In all three valid independent runs, the mean specific binding was greater
than 93% for the negative control, octyltriethoxysilane. In the first run, the mean specific binding was
greater than 75% at every soluble concentration tested for diazinon. The mean specific binding for
diazinon at 10−3 mol/L was 59.3% of control. Since precipitation was observed at this concentration,
data were not evaluated, resulting in diazinon being classified as ‘non-interacting’. In the second run,
the mean specific binding was greater than 75% at every soluble concentration tested for diazinon,
classifying it as ‘non-interacting’. In the third run, the mean specific binding was 69.8% for 10 -4
mol/L diazinon, classifying it as ‘equivocal’. The mean relative binding affinity (RBA; calculated by
dividing the log of the half maximal inhibitory concentration [LogIC50] of the control/test material by
the LogIC50 of the positive control 17β-estradiol) was 0.6 for 19-norethindrone. There was no RBA to
be calculated for diazinon. Diazinon was classified as ‘non-interacting’ in the first two independent

55
valid runs and as ‘equivocal’ in the third independent valid run, and was therefore considered to be
non-interacting (Willoughby, 2011b).
Androgen receptor binding (rat prostate cytosol)

The ability of diazinon to interact with the androgen receptors isolated from rat prostates was
investigated by a study conducted as outlined in the USEPA OPPTS 890.1150 test guideline and in
accordance with GLP. Concentrations of diazinon (purity 98.7%; batch no. 00896074) for use in the
binding assays were identified at 10−10 to 10−3 mol/L. Three valid independent runs were examined. In
this assay, the classification of a chemical as a binder or non-binder was based on the average results
of three non-concurrent runs, each of which met the performance criteria and, taken together, were
consistent with each other. A run is classified as a ‘non-binder’ if the lowest point on the fitted
response curve within the range of the data was above 75%. A run is classified as ‘equivocal’ if the
average lowest point on the fitted response curves within the range of the data was above 50% but
below 75% (USEPA, 2009d).
The top concentration of diazinon suitable for use in the binding assays was 10−3 mol/L. In the
first run, the mean specific binding was 71.9% at 10−4 mol/L diazinon, classifying it as ‘equivocal’ for
this run. At 10−3 mol/L, the mean specific binding was 44.1%, but precipitation of diazinon was
observed at this concentration so the data were not assessed. In the second run, the mean specific
binding was 41.6% at 10−3 mol/L and 80.5% at 10−4 mol/L diazinon, classifying it as a “binder” for
this run. This resulted in a LogIC50 of −3.6 mol/L and an RBA of 0.4 for diazinon. In addition, two of
the three replicates for 10−10 mol/L diazinon were pulled out of the data analysis as outliers. In the
third valid independent run, the mean specific binding was 36.7% at 10−3 mol/L and 79.0% at 10−4
mol/L diazinon, classifying it as a binder for this run. As diazinon was classified as a binder for only
two of the three runs, the mean RBA could not be calculated, though the mean based on RBA for
those two runs alone was 0.4.
Diazinon was classified as ‘equivocal’ in the first valid independent run and as a ‘binder’ in
the second and third runs (Willoughby, 2012).
The uterotrophic assay

A uterotrophic assay was performed to evaluate the estrogenic effects of diazinon on
ovariectomized rats. This study was conducted as outlined in the USEPA OPPTS 890.1600 test
guideline and OECD 440 test guideline and conducted in accordance with GLP. Ovariectomized
Sprague Dawley rats were allocated to four dose groups. The animals were administered dose levels
of 78 and 250 mg/kg bw per day of diazinon, the vehicle control, or 17α-ethinylestradiol (positive
control) for three consecutive days by gavage. Dose levels of diazinon were selected based the LD 50
and available toxicity data. Following administration of 17α-ethinylestradiol, wet and blotted uterine
weights were significantly increased.
Under the conditions of this uterotrophic assay, which utilized the ovariectomized rat model,
oral administration of diazinon at dose levels of 78 and 250 mg/kg bw per day (maximum tolerated
dose) did not increase uterine weights, indicating an absence of estrogenic effects (Davis & Lea,
2011).
Hershberger bioassay
The Hershberger bioassay was used to screen diazinon for its androgen agonist/antagonist
activity and 5α-reductase inhibition properties. This study was conducted as outlined in the USEPA
OPPTS 890.1400 and OECD 441 test guidelines and in accordance with GLP. Castrated Sprague
Dawley rats were allocated to eight groups: diazinon (purity 98.7%; batch no. 00896074) at 47 or 150
mg/kg bw per day, corn oil (the vehicle control), testosterone propionate at 0.4 mg/kg bw per day as
positive control, co-administration of diazinon at 15, 47 or 150 mg/kg bw per day and co-

56
administration of flutamide at 3.0 mg/kg bw per day. Diazinon and flutamide were administered by
gavage for 10 consecutive days and testosterone propionate was subcutaneously injected. In the
testosterone propionate group, the rats were administered testosterone propionate for 5 days and then
co-administered testosterone propionate and corn oil (the vehicle) for 5 days. Twenty-four hours after
the final treatment, androgen-dependent organs, including the glans penis, ventral prostate, levator ani
plus bulbocavernosus muscle complex, Cowper’s glands and seminal vesicles with coagulating gland
with fluid were weighed.
The rats did not show any severe signs of toxicity during the treatment and their final body
weight at 150 mg/kg bw was approximately 90% of the control. Diazinon treatment at all doses did
not show statistically significant changes in any androgen-dependent organs. However, diazinon co-
administered testosterone propionate at all doses showed statistically significant and dose-dependent
decreases in the weights of these organs though their anti-androgenic activities were weak compared
to those of flutamide. The data on organ weights are summarized in Table 22.
Table 22. Summary of mean organs weights in the Hershberger bioassay in rats administered
diazinon for 10 consecutive days
Body-weight changes and final organ weight
Body-
weight Glans Cowper’s Ventral Seminal
No. of changes penis glands LABC prostate vesicles
Dose group rats (g) (mg) (mg) (mg) (mg) (mg)
Control (corn oil) 8 43.6 40.6 6.4 137.3 15.7 37.0
Diazinon at 47 mg 8 42.6 43.1 5.7 121.7 14.5 30.7
Diazinon at 150 mg 8 15.6* 43.2 4.6 108.7 16.1 35.6
Corn oil + TP 7 66.1 76.4* 49.2* 476.7* 205.6* 647.0*
Diazinon at 15 mg + TP 8 69.4 77.3 35.4 469.0 152.2# 461.1#
Diazinon at 47 mg + TP 8 59.2 68.9 38.0 376.2# 118.2# 409.6#
Diazinon at 150 mg + TP 7 38.2 66.2# 25.8# 265.6# 82.6# 246.3#
Flutamide + TP 7 67.5 61.0§ 15.1§ 262.2§ 46.6§ 91.6§
bw: body weight; LABC: levator ani plus bulbocavernosus muscle complex; no.: number; TP: testosterone propionate;
*: P < 0.05 compared to the vehicle control (t-test); #: P < 0.05 compared to the corn oil + TP group (Dunnett t-test);
§: P < 0.05 compared to the corn oil +TP group (t-test)
Source: Davis (2011)
In the Hershberger bioassay, diazinon did not show any androgen agonist activity; however, it
did show antagonist activity (Davis, 2011).
Steroidogenesis (Human Cell Line – H295R)

The steroidogenesis laboratory proficiency assay to evaluate the ability of diazinon to affect
the testosterone and estradiol/estrone steroidogenic pathway was performed using the H295R human
adrenocortical carcinoma cell line. Forskolin or prochloraz was used as a known hormone inducer or
inhibitor, respectively. This study was conducted in accordance with GLP and as outlined in USEPA
OPPTS 890.1550 test guideline (USEPA, 2009). Diazinon (purity 98.7%; batch no. 00896074) was
tested in the assay at 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 μmol/L. Six independent runs of the
steroidogenesis assay were conducted. Test chemicals and reference chemicals (forskolin and
prochloraz) were tested in replicates of three per plate, while six solvent control wells were analysed
on the quality control plate. The H295R supplemented medium used in the assay at the time of
plating, dosing and harvest contained 10 μmol/L 22R-hydroxycholesterol to increase basal hormone
production. The duration of exposure was 48  2 hours. A quality control plate containing two doses

57
of the reference chemicals forskolin and prochloraz was run each time the assay was performed. Cell
viability was assessed after the approximately 48-hour exposure using the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Testosterone and estradiol levels were
measured using high-performance liquid chromatography with mass spectrometry (HPLC/MS-MS).
The highest concentration of diazinon that could be analysed in the steroidogenesis assay was
10 μmol/L in all three independent runs because of precipitation seen under the microscope at the 100
μmol/L diazinon concentration in two runs (run 1 and run 2) as well as cytotoxicity greater than 20%
at the 100 μmol/L concentration in run 3. The results are summarized in Table 23. A statistically
significant decrease in testosterone was observed after exposure with 10 μmol/L diazinon in all three
runs of the assay. In run 1 of estradiol, statistically significant decreases were observed at the 0.0001,
0.001, 0.01 and 0.1 μmol/L diazinon exposure concentrations, but the decreases were not dose
dependent; an increase in estradiol was observed at 10 μmol/L diazinon. The levels of estradiol at 10
μmol/L in runs 2 and 3 were similar to that in run 1, but not statistically significant.
Table 23. Fold changesa for testosterone or estradiol in steroidogenesis using H295R
Fold changesa
Testosterone Estradiol
Concentration of
diazinon (μmol/L ) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3
0.000 1 1.08 1.11* 1.01* 0.85* 0.99 0.87
0.001 0.98 1.04 1.07* 0.82* 0.91 0.89
0.01 0.97 1.04 1.06* 0.86* 0.97 0.90
0.1 0.99 1.03 1.00 0.86* 0.94 0.94
1 0.93 1.05 1.06 0.98 1.12 1.03
10 0.76* 0.85* 0.92* 1.30* 1.40 1.30
100 N/A N/A N/A N/A N/A N/A
N/A: not analysed because of precipitation and/or cytotoxicity; *: P > 0.05
a
Fold changes over solvent control.
Source: Wagner (2012)
A statistically significant decrease in testosterone in all three runs indicated that the decrease
was induced by the 10 μmol/L diazinon exposure. The decreases in estradiol at multiple
concentrations in run 1 were considered not treatment related as there was no dose relationship or
reproducibility. The increase in estradiol at 10 μmol/L diazinon was seen in run 1 only (Wagner,
2012).
Pubertal development and thyroid function in intact juvenile/peripubertal female and male
rats
An assay was conducted to identify the effects of diazinon on pubertal development and
thyroid function in intact juvenile/peripubertal female and male rats. The studies were conducted as
outlined in USEPA OPPTS 890.1450 (females) or OPPTS 890.1500 (males) test guidelines and
conducted in accordance with GLP. The rats were orally administered diazinon at 50 or 100 mg/kg bw
per day (purity 95.9%; batch no. 00896074) or the vehicle control (corn oil) from postnatal day 22 for
21/22 or 31/32 days in females or males, respectively. The high dose was selected following a review
of previous study reports, including a study that showed no abnormal clinical signs and a decrease in
body-weight gain (males only) following 14 days of administration of 100 mg/kg bw per day diazinon
(Green, 1983). Approximately 2 hours after the final dose, all the animals were examined. While
mean terminal body weights at 100 mg/kg bw per day were 96.2% that of the controls, they were

58
92.7% of mean control body weights on postnatal day 30 (day 8), suggesting the maximum tolerated
dose was approached during the course of the study.
Diazinon treatment did not affect pubertal development of female rats, including the day of
vaginal opening, estrus onset, estrus cyclicity or pituitary, ovarian or uterine weights. At termination,
the rats showed decreases in circulating serum thyroxine concentrations at both dose levels
(3.04  0.73, 2.35  0.58, 2.51  0.68 μg/dL, vehicle control and diazinon at 50 and at 100 mg/kg bw
per day, respectively). However, the decreases were not dose related, and there were no corresponding
changes in thyroid-stimulating hormone, thyroid gland weight or thyroid gland histopathology.
Therefore, the decreases in thyroxine levels were not considered treatment related.
Terminal body weights in male rats at 100 mg/kg bw per day diazinon were 99.4% that of the
vehicle-control body weights; however, on postnatal day 30, after 7 days of dose administration, body
weights were 93.1% that of mean control body weights. Diazinon did not affect pubertal development
including organ weights of androgen-dependent tissues, serum testosterone concentrations or pituitary
weights. After 31/32 days of diazinon administration, there was a decrease in circulating serum
thyroxine concentrations at 50 mg/kg bw per day only. No corresponding changes in thyroid-
stimulating hormone concentrations or thyroid gland weight or histopathology were observed at either
dose levels, and so the decrease was not considered treatment related. In conclusion, administration of
diazinon to intact juvenile/peripubertal female and male rats resulted in no changes in end-points that
suggest an effect on pubertal development (Davis, 2012).
(f) Studies on metabolites or impurities

Several studies with tetraethyl pyrophosphate (TEPP), an impurity no longer permitted in
diazinon, and oxypyrimidine, a metabolite, were submitted (see Table 24).
Table 24. Summary of studies of acute toxicity of diazinon impurities and metabolites
Purity
Test substance Species Strain Sex Route (%) Result Reference
O,O-TEPP Rat Sprague M+F Oral N/S LD50 0.947 mg/kg Kuhn
Dawley bw (M) and 0.658 (1995a)
mg/kg bw (F)
O,S-TEPP Rat Sprague M+F Oral N/S LD50 0.711 mg/kg Kuhn
Dawley bw (M) and 0.460 (1995b)
mg/kg bw (F)
S,S-TEPP Rat Sprague M+F Oral N/S LD50 >10 mg/kg bw Kuhn
Dawley (M) and 3.48 mg/kg (1995c)
bw (F)
oxypyrimidine Rat Sprague M+F Inhalation 93.0 LC50 > 5.32 mg/L Maedgen
Dawley (1987)
bw: body weight; F: female; LC50: median lethal concentration; LD50: median lethal dose; M: male; N/S: not stated;
TEPP: tetraethyl pyrophosphate
In a 5-week toxicity study, groups of 10 male and 10 female Sprague Dawley rats were
administered oxypyrimidine (purity unknown) in aqueous 3% cornstarch with 0.5% Tween 80 by
gavage at a daily dose of 0, 20, 100, 500 or 1000 mg/kg bw per day. All test animals were observed
daily for clinical signs and mortality. Individual body weights and feed consumption were recorded
weekly. Physical and ophthalmoscopic examinations as well as haematological, clinical chemistry and
histopathological (liver only) tests were performed on all the rats. Cholinesterase was not measured.
Body-weight gain in males was significantly and dose-dependantly reduced at all dose levels,
from 89% of control values (not statistically significant) at low doses to 66% of control values at high
doses. Feed consumption was statistically significantly decreased after 7 days in both males and

59
females at 500 or 1000 mg/kg bw per day. Changes in mean serum biochemistries in both males and
females included increase in total bilirubin and calcium at 100 mg/kg bw per day and higher doses;
decreased albumin-to-globulin ratio and chloride and increased alkaline phosphatase, alanine
aminotransferase, total protein, albumin and phosphorus at 500 mg/kg bw per day and higher doses;
and reduction in glucose at 1000 mg/kg bw per day. Absolute and/or relative liver weights were
increased at 500 mg/kg bw per day and higher doses, and relative kidney weight were increased at
1000 mg/kg bw per day. No microscopic findings were observed in the liver.
A NOAEL could not be established, based on reduced body-weight gain in males at 20 mg/kg
bw per day, the lowest dose tested (Spoede-Thompson, Batastini & Arthur, 1990).
(g) Intestinal microbiota effects

An extensive literature search did not show published reports on the potential adverse effects
of diazinon on the intestinal microbiota. Based on the mode of action, diazinon is unlikely to affect
the intestinal microbiota. In addition, no studies were found on the ability of intestinal microbiota to
metabolize diazinon.
(h) Immunotoxic effects

No specific studies on immunotoxicity were submitted. A study in the open literature with
intraperitoneal injection of diazinon in mice (Neishabouri et al., 2004) was not informative. The
submitted repeated-dose toxicity studies do not indicate an immunotoxic potential for diazinon after
oral exposure.
3. Observations in humans
3.1 Studies in volunteers
In a double-blind, placebo-controlled study, healthy, informed male volunteers were given
single, ascending doses of diazinon (purity 97.8%) in corn oil in gelatine capsules. Some volunteers
received a placebo of only corn oil in gelatine capsules. The lowest dose used was 0.03 mg/kg bw,
given initially to one volunteer receiving the test material and one the placebo. In the next phase,
one volunteer received the placebo, six diazinon at 0.03 mg/kg bw and one diazinon at 0.12 mg/kg
bw. In the next phase, one volunteer received the placebo, six diazinon at 0.12 mg/kg bw and one
diazinon at 0.21 mg/kg bw. In the next phase, one volunteer received the placebo, six diazinon at 0.21
mg/kg bw and one diazinon at 0.30 mg/kg bw. In the final phase, three volunteers received the placebo
and seven diazinon at 0.20 mg/kg bw. A complete physical examination, including an
electrocardiogram, was carried out before dosing and 2 days and 15 days after dosing. Vital signs
(respiratory parameters, oral temperature, blood pressure and pulse) were recorded before administering
the test material and 1, 2, 4, 6, 8, 12, 24 and 48 hours and 4, 7 and 14 days afterwards. The volunteers
were asked to report all adverse events. Blood was drawn for clinical chemistry and haematological
examination before dosing and on days 1, 2 and 15 after dosing. Blood was also drawn 1 and 2 days
before and immediately before dosing, and plasma and erythrocyte cholinesterase activity was
determined using a modification of the method of Ellman et al. (1961). Further samples for
determination of cholinesterase activity were taken 1, 2, 4, 6, 8, 12, 24 and 48 hours after dosing and on
days 5, 8 and 15 after dosing. Urine samples were collected 24 hours before dosing and for 0–6, 6–12,
12–24 and 24–48 hours after dosing. Pharmacokinetic and urinary metabolite data collected during
the study according to Protocol Parts B and C were reported separately (reports not submitted to the
JMPR).
The only self-reported adverse event considered to be related to diazinon intake was back
pain in a man at the highest dose. No treatment-related effects on haematological or clinical
chemical parameters were observed, except for changes in cholinesterase activity. Plasma
cholinesterase activity was inhibited by more than 20% at doses greater than 0.12 mg/kg bw. No

60
significant inhibition of erythrocyte cholinesterase activity was seen at any dose; at 0.21 mg/kg bw,
7% inhibition was observed at 4 hours, 4% inhibition at 8 hours and 6–7% inhibition at days 5, 8 and
15. At other times, the erythrocyte cholinesterase activity was greater than or equal to that of the group
given the placebo. Furthermore, data for the man given the highest dose did not suggest any
significant inhibition of erythrocyte cholinesterase activity.
The NOAEL was 0.21 mg/kg bw (the highest dose was ignored as only one volunteer
received it) (Boyeson, 2000; final version reported as Anderson, 2000).
In a preliminary study, four healthy adult male volunteers (age not specified; body weight
79.5, 83.5, 84 and 95 kg) were given gelatine capsules containing either 2.47 mg or 2.85 mg of
technical diazinon (purity 99.5%), depending on whether their body weight was closer to 85 o r
95 kg. The study was conducted in accordance with principles expressed in the Declaration of
Helsinki or equivalent statements prepared for use by national and/or multinational authorities
(Cristie, 2000). The dose was 0.03 mg/kg bw per day for 28, 29 or 31 days, depending on the
participant’s availability. The dose was selected on the basis of the Payot study (Payot, 1966), but
slightly increased because the purity of diazinon had increased with a concomitant reduction in its
acute toxicity. The exclusion criteria included clinically relevant cardiovascular, renal,
haematological or biochemical profiles (including plasma cholinesterase activity) and metabolic or
gastrointestinal disorders likely to influence absorption. Individuals with an abnormal
electrocardiogram or an HIV-positive or hepatitis B-positive diagnosis were also excluded. Blood
was drawn to measure haemoglobin concentrations; erythrocyte, leukocyte and platelet counts; blood
urea nitrogen, glucose, sodium, potassium, chloride, bicarbonate, cholesterol, creatinine, triglyceride
and bilirubin concentrations; and alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase,
creatine kinase and gamma-glutamyltransferase activities before dosing. Blood for estimating
cholinesterase activity was collected on four occasions before dosing (days −28, −27, −7 and −1) and
then 6 hours after dosing. Thereafter, activity was measured on days 1, 2, 3, 8, 13/14, 20, 28, 29 or 30.
Clinical investigations, haematology, clinical chemistry and urine analysis were performed on days 8,
13/14, 20, 28, 29 or 30.
No cholinergic signs were observed. Similarly, there were no treatment-related clinical
changes in haematological or clinical chemical end-points, apart from cholinesterase activity during
the study. Plasma cholinesterase activity was reduced by an average of 48% relative to pretest values
in all four volunteers on day 20 of treatment. A significant reduction in plasma cholinesterase
activity (> 20%) occurred by day 8 of treatment. There was no reduction in erythrocyte
acetylcholinesterase activity at any time during treatment.
Based on the absence of cholinergic signs or inhibition of acetylcholinesterase activity in
erythrocytes, the NOAEL was set at 0.03 mg/kg bw per day (Beilstein, 1998).
The results of the following study by Payot (1966) were reported by the JMPR in 1966,
2001 and 2006. Because of some inaccuracies in reporting the administered doses in the previous
JMPR evaluations, the amended summary from the JMPR 2006 is given below.
Four adult male volunteers (age 30–45 years; body weight, 66, 74, 91 and 95 kg) were given
gelatine capsules containing 0.5 mg of technical diazinon (purity 95.4–95.7%) postprandially. Since
the amount of diazinon in each capsule was fixed and volunteers were given either four or five
capsules depending on whether their body weight was closer to 75 or 100 kg, the actual administered
doses varied slightly at 0.03, 0.027, 0.022/0.027 (alternate day treatment with 4 and 5 capsules) and
0.026 mg/kg bw per day, respectively. To ascertain reversibility of effects, after complete plasma
cholinesterase inhibition in two of the four volunteers was observed after 1 day of treatment, dosing
was suspended on days 5–10 of the 42-day regimen, while the other two (lighter) volunteers who
commenced treatment a month later were treated uninterrupted for 34 days.

61
No plasma cholinesterase activity was measurable on the first 6 days of treatment in two of
the volunteers. As a result, treatment was interrupted for 6 days to enable recovery. Subsequent
treatment at the same dose did not reveal any inhibitory effects on plasma cholinesterase activity. The
fluctuations observed during treatment were similar to those observed during the pretest period. At no
time was erythrocyte acetylcholinesterase activity decreased compared with the pretest values. Other
parameters investigated included haematology and blood chemistry, urine analysis and
symptomatology. No changes were observed that could be attributed to treatment.
The NOAEL was 0.03 mg/kg bw per day on the basis of transitory depression of plasma
cholinesterase activity, the only effect observed at this dose (Payot, 1966).
The results of the following study by Lazanas, Fancher & Calandra (1966) were reported by
the JMPR in 2006 and in earlier monographs; the study evaluation is copied from the 2006 JMPR
without further evaluation.
Three healthy adult male volunteers (Nos. 4, 5 and 6) were given gelatine capsules containing a
mixture of diazinon 50W (50% [weight per weight] wettable powder; purity 49.6%) and corn starch at
a final dose of 0.025 mg/kg bw per day administered as three doses per day, taken before meals at
08:00, 12:00 and 18:00, for 43 days. A concurrent control group of three volunteers (Nos. 1, 2 and 3)
were given gelatine capsules containing corn starch only. In a second dosing regimen, three
different volunteers (Nos. 1, 7 and 8) were treated as above at a dose of 0.020 mg/kg bw per day for
37 days. In the recovery phase, after treatment at 0.025 mg/kg bw per day, volunteers were given
capsules containing corn starch only (placebo) for 101 days; this recovery phase was reduced to
41 days for the group at 0.020 mg/kg bw per day. Plasma and erythrocyte cholinesterase activities in
all eight volunteers were measured on five separate occasions before dosing and then at intervals of
1–5 days throughout treatment and recovery using an electrometric method (∆ pH/h). Body weight
and clinical signs were monitored daily. Haematological parameters, including haemoglobin
concentration, erythrocyte count, total and differential leukocyte count and prothrombin time
were determined at intervals of 4–7 days during treatment and at 4–14 days during recovery, as were
clinical chemistry parameters, i.e. cholinesterase activity (erythrocytes and plasma), blood urea
nitrogen, alkaline phosphatase and alanine aminotransferase, and urine analysis, i.e. pH and
microscopic elements.
No clinical signs or changes in body weight were observed. No significant changes were detected in
any of the haematological, urinary or clinical chemistry parameters measured, except for plasma
cholinesterase activity (average inhibition, approximately 22%). By contrast, mean erythrocyte
cholinesterase activity was not appreciably inhibited (maximum, 2%) at any time during treatment at
0.025 mg/kg bw per day or during the 22 days of recovery.
Treatment at 0.020 mg/kg bw per day resulted in a combined nonsignificant mean plasma
cholinesterase inhibition of 8% and recovery appeared to be complete after 16 days. Similarly,
erythrocyte cholinesterase activity was not significantly affected by treatment at 0.020 mg/kg bw per
day. The NOAEL was 0.025 mg/kg bw per day on the basis of depression of plasma cholinesterase
activity, the only effect observed at this dose (Lazanas, Fancher & Calandra, 1966).
3.2 Case studies

The 1993 JMPR mentioned numerous reports on intentional and accidental intake of diazinon
and other organophosphates. The text below is copied from the 1993 JMPR without further
evaluation.
Numerous reports [describe] the clinical manifestations of organophosphate toxicity and the usefulness
of erythrocyte and serum cholinesterase activity assays for diagnosis as well as the efficacy of
supportive and specific therapies (Kabrawala, Shah & Oza, 1965; Payot, 1966; Banerjee, 1967; Gupta
& Patel, 1968; Zwiener & Ginsburg, 1988).
A case of acute diazinon poisoning complicated by pericarditis and pneumonia followed by recovery
has been reported. The intoxication caused the occurrence of cyanosis, tracheobronchial congestion,
pulmonary oedema and pneumonia. The treatment included the intramuscular injection of atropine
(Banerjee, 1967).

62
Another complication reported following accidental ingestion of diazinon consisted of severe

pancreatitis and a pseudocyst (Dressel et al., 1979). The induction of pancreatitis was reproduced in
dogs treated intravenously with a dose of 5 mg/kg bw diazinon. Pancreatitis may be the result of
hypersecretion and ductal obstruction (Dressel et al., 1980).
A study of 60 poisoning cases with diazinon has been reported. The most common clinical
manifestations were vomiting, giddiness, constricted pupils and signs of bronchoconstriction with
pulmonary congestion. The amount of poison ingested varied from 4 ml to 15 ml with an average of
7.5 ml (% active ingredient not specified in publication). This was ingested by 55 patients with suicidal
intention, whereas in 5 patients the compound was taken accidentally. Atropine injections were
administered repeatedly (up to a total dose of 22.4 g over 42 hours). Five patients died in spite of
intensive atropine therapy initiated more than 8 hours after ingestion. Other cases who had ingested the
same amount of poison as the fatal cases received treatment within three hours of ingestion. These
findings emphasize the importance of early treatment in diazinon poisoning (Gupta & Patel, 1968). In
addition, these results are in agreement with an earlier observation that the combination of diazinon
with cholinesterase occurs in two stages, an early reversible stage and a late irreversible stage (Grob,
1956).
In another study, cases of organophosphate and carbamate poisoning in 37 infants and children were
reported, only 5 of which were associated with diazinon. Miosis, excessive salivation, muscle
weakness, respiratory distress, lethargy and tachycardia were the most common clinical findings.
Erythrocyte cholinesterase activities were determined from 24 patients showing erythrocyte
cholinesterase activities that were less than 50% of the lower limit of the normal range. There were no
differences between the erythrocyte and serum cholinesterase activity in 20 [of the] 24 patients from
whom both tests were obtained, whereas in the remaining 4 cases either the erythrocyte or the serum
activity was decreased. A combined atropine/pralidoxime therapy is recommended in case of
organophosphate toxicity. Although the recommended dose for atropine in infants is 0.01 to
0.02 mg/kg bw, the dose is generally insufficient for treatment of signs and symptoms secondary to
organophosphate poisoning (Zwiener & Ginsburg, 1988).
Neurobehavioural effects of short-term, low-level exposure to diazinon were investigated in 99 pest
control workers. A computer assisted neurobehavioural test battery (including attention, vigilance,
hand-eye coordination, visual perception, verbal ability) was used before and after their work shift. The
diazinon metabolite diethylthiophosphate (DETP) was measured in urine samples collected from
46 diazinon applicators and 56 non-applicators. Post-shift DETP values were 24 and 3 [parts per
billion] for applicators and non-applicators, respectively. The study failed to demonstrate any
behavioural effects of diazinon under the conditions of this study (Maizlish et al., 1987).
3.3 Epidemiological data

Several epidemiological studies on diazinon exposure were available. The review of these
studies focused on the occurrence of three cancer types: non-Hodgkin lymphoma (NHL), leukaemia
and lung cancer, the cancer types that the IARC identified as having positive associations with
diazinon in their recent monograph (IARC, 2015). One prospective cohort study was available, the
Agricultural Health Study (AHS), with a large sample size and detailed exposure assessment. Cohort
studies are considered a powerful design, as recall bias is avoided. All other studies were case–control
studies, usually retrospective, which are more prone to recall and selection biases.
Details of the risk estimates are provided in Table 25.

63
Table 25. Results of Tier 1 details of risk estimates in epidemiological data

Study/
Location Reference Diazinon / NHL Diazinon / Leukaemia Diazinon / Lung cancer
Meta- Schinasi Qualitative exposure only –
analysis & Leon ever- vs never-use of diazinon.
(2014) Meta RR: 1.6 (95% CI: 1.2–
2.2)
Meta-analysis includes
McDuffie et al. (2001);
Waddell et al. (2001); and
Mills, Yang & Riordan
(2005a). Does not include
AHS; N for each meta-analysis
not presented
AHS Lerro et Qualitative (ever/never) Qualitative (ever/never)
al. (2015) Risk estimates – aRR (95% CI) Risk estimates – aRR (95% CI)
Ever-use 0.93 (0.53–1.56) Ever-use 0.92 (0.52–1.64)
N = 18 exposed cases N = 15 exposed cases
AHS Jones et Quantitative exposure – LED
al. (2015) and IW-LED (tertiles – cut-
points given for both LED and
IW-LED)
Risk estimates – RR (95% CI)
No exposure
LED < 20: 1.11 (0.75–1.65)
LED 20.0–38.8: 0.76 (0.44–
1.3)
LED > 38.8: 1.6 (1.11–2.31)
P for trend: 0.02
No exposure
IW-LED <368: 1.09 (0.61–
1.53)
IW-LED 368–1800: 0.99
(0.66–1.52)
IW-LED >1800: 1.41 (0.98–
2.04)
P for trend 0.08
Total N = 22 830, with 283

lung cancer cases
N = 84 exposed cases
AHS Alavanja Quantitative exposure – LED
et al. and IW-LED (tertiles – cut-
(2014) points only given for LED, not
IW-LED)
Ever- vs never-use 1.0 (0.8–
1.3)
No exposure
LED  8.75: 1.1 (0.7–1.6)
LED 8.75–25: 1.0 (0.6–1.8)
LED >25–457.25: 1.2 (0.7–
1.9)
P for trend: 0.52
Median days/year=2.5
Minimum days/year=1
Maximum days/year=7

64
Study/
Minimum days/year=1
Maximum days/year=25
Median days/year=7
Minimum days/year=2
No exposure
IW-LED – Low: 1.1 (0.7–1.8)
IW-LED – Med: 0.9 (0.5–1.5)
IW-LED – High: 1.3 (0.8–2.1)
P for trend: 0.33
Minimum days/year=1
Maximum days/year=29.5
Minimum days/year=1
Maximum days/year=49.5
Median days/year=7
Minimum days/year=2
Total N = 24 211 with 257

incident NHL cases
N = 70 exposed cases (LED
analysis), 69 exposed cases
(IW-LED analysis)
AHS Beane Exclude – Alavanja et al. Quantitative exposure – LEDs Exclude – Jones et al. (2015)
Freeman (2014) has longest follow-up and IW-LEDs (tertiles – cut- has longest follow-up
et al. points only given for LED, not
(2005) IW-LED)
No exposure
LED <20.0: 1.10 (0.32–3.72)
LED 20.0–38.8: 2.62 (0.88–
7.82)
LED >38.8: 3.36 (1.08–10.49)
P for trend: 0.026
Median days/year = 2.5
Minimum = 2.5
Maximum = 7.0
Minimum = 2.5
Maximum = 29.5
Minimum = 2.5
Maximum = 200
No exposure
IW-LED – Tertile 1: 0.99
(0.23–4.24)
(0.94–6.66)
(0.92–9.03)
P for trend: 0.053
Minimum = 2.5
Maximum = 14.5
Minimum = 2.5

65
Study/
Maximum = 29.5
Minimum = 2.5
Maximum = 200
Total N = 23 106, with 32

incident leukaemia cases.
AHS Alavanja Exclude – Jones et al. (2015)
et al. has longest follow-up
(2014)
United De Roos NB. Study population overlaps
States et al. with Waddell et al. (2001) and
Midwest (2003) total N is smaller, but as an
case–control exception this study is not
studies excluded as it provides more
fully adjusted risk estimates for
ever- vs never-use analyses
Qualitative (ever/never)
Risk estimates – aRR (95% CI)
Exposed: 1.9 (1.1–1.3.6), from
a logistic regression model
1.7 (1.0–2.8), from the
hierarchical regression. Both
adjusted for other pesticides
Total N =2 583 (650 NHL

cases, 1 933 controls).
N = 40 and 62 exposed cases
and controls, respectively
United Waddell et NB. Study population overlaps
States al. (2001) with De Roos et al. (2003)
Midwest above. See comment above.
case–control
studies Quantitative exposure – days
of use per year (2 categories –
cut-points are given)
Risk estimates – aOR (95%
CI)
Exposed 1.7 (1.2–2.5)

and controls, respectively
Restricted to direct respondent

farmers:
Exposed: 1.3 (0.8–2.0)
Adjusted for fonofos: 1.2 (0.7–
2.1)
Adjusted for malathion: 1.8
(0.9–3.5)
44/69 exposed cases/controls
Risk estimates – aOR (95%

CI)
Non-farmers: 1.0
<5 days/year: 1.3 (0.5–3.9)
5+ days/year: 2.4 (0.7-8.0)

66
Study/

and controls respectively.
(6/11 for <5 days and 6/6 for
5+ days for cases/controls,
respectively.)
United Zahm et Exclude - not specific to
States al. (1993) diazinon
Midwest
case–control
studies
United Cantor et Exclude – as this study is
States al. (1992) pooled in Waddell et al. (2001)
Midwest and De Roos et al. (2003)
case–control
studies
Qualitative exposure only –
ever-/never-use of diazinon.
Risk estimates – OR (95% CI)

Ever-use of diazinon = 1.5
(0.9–2.5)
Ever-use before 1965 = 2.6
(1.2–5.9)
Total N =1 867 (622 cases,

1 245 controls)
United Brown et Quantitative exposure – days
States al. (1990) of use per year (3 categories –
Midwest cut-points are given).
case–control
studies Risk estimates – OR (95% CI)
Ever-use: 1.2 ( 0.6–2.1)
By days per year of diazinon

handling:
Never farmers (unexposed)
1–4 days: 2.1 ( 0.8–5.6)
5–9 days: 0.5 ( 0.1–2.4)
 10 days – no cases
Total N =1 867 (578 leukaemia

cases, 1245 controls).
N = 17 exposed cases in ever-

/never-use analysis
N = 10 exposed cases in
days/year analysis
Cross- McDuffie Qualitative exposure only –
Canada et al. ever-/never-use of diazinon
Study of (2001)
Pesticides
and Health Risk estimates – OR (95% CI)
Ever-use: 1.69 (0.88–3.24)
Total N = 2 023

67
Study/
517 cases, 1506 controls
(overall). 179 cases, 456
controls (with telephone
interview data, i.e. detailed
pesticide information)
Florida Pest Pesatori et Qualitative (ever/never)
Control al. (1994)
Worker Risk estimates – aOR (95%
Study CI)
Ever-use: 2.0 (0.7–5.5)
deceased controls
Ever-use: 1.3 (0.6–3.1) living
controls
United Farm Mills, Qualitative (high vs low – no Qualitative (high vs low - no
Workers of Yang & cut-points) cut-points)
America Riordan
(2005)
Risk estimates – aOR (95% Risk estimates – aOR (95%
CI) CI)
High vs low: 1.39 (0.76–2.53) High vs low 1.32 (0.65–2.65)
Also reported sex-stratified Also reported sex-stratified
results results
Total 131 cases, 651 controls. Total 131 cases, 651 controls.
N exposed cases/controls not N exposed cases/controls not
reported reported
No. of publications after 6 3 3
exclusions (not counting
meta-analysis)
No. of publications 2 – with different units 2 – with different units 1
eligible for quantitative
risk assessment
Exclusions 3 0 2
AHS: Agricultural Health Study; aOR: adjusted odds ratio; aRR: adjusted risk ratio; CI: confidence interval; IW-LED,
intensity-weighted lifetime-exposure days; LED: lifetime-exposure days; NHL: non-Hodgkin lymphoma; no.: number;
OR: odds ratio; RR: risk ratio
Maximally adjusted risk estimates were extracted by the reviewers.
Diazinon / NHL
There was no significant evidence of a positive association between NHL and diazinon
exposure and no evidence of an exposure–response relationship in the AHS (Alavanja et al., 2014;
Lerro et al., 2015).
In a large pooled case–control study, the unadjusted estimates showed a significant elevated
risk of NHL (relative risk [RR] = 1.7; 95% confidence interval [CI] = 1.2–2.5) associated with ever-
versus never-use of diazinon (Waddell et al., 2001). However, these risks were attenuated and/or no
longer significant when proxy respondents were excluded and analyses were mutually adjusted for
other pesticides (e.g. malathion, fonofos). Although increasing risk across exposure–duration
categories was observed, which was suggestive of a duration–response pattern, confidence intervals
were nonsignificant, wide and overlapping between categories.
Two other studies reported elevated risks of NHL for ever- versus never-use of diazinon
(McDuffie et al., 2001) or high versus low diazinon use (Mills, Yang & Riordan, 2005), but

68
confidence intervals were wide, reflecting uncertainty in the risk estimates, and chance could not be
excluded as an explanation for the findings. Overall, there was no convincing evidence of a positive
association between NHL and exposure to diazinon, as shown in the exposure–response plot below.
Fig. 3. Forest plot showing results of studies of risk of NHL on ever exposure to or high versus low
exposure to diazinon
CI: confidence interval; NHL: non-Hodgkin lymphoma; OR: odds ratio; RR: relative risk
Diazinon / Leukaemia
A significantly increased risk of leukaemia in the highest exposure category (> 38.8 lifetime
days of diazinon exposure; RR = 3.36; 95% CI = 1.08–10.49) and a significant exposure–response
relationship were observed in the AHS. Findings for intensity-weighted lifetime-exposure days
demonstrated a similar pattern, but did not reach significance (Beane Freeman et al., 2005). Two other
studies reported nonsignificantly elevated risks of leukaemia for high versus low diazinon use (Mills,
Yang & Riordan, 2005) and ever- versus never-use of diazinon (Brown et al., 1990), with a
nonsignificant dose–response relationship observed using days of use per year (Brown et al., 1990).
Overall, there is weak evidence of a positive association between leukaemia and exposure to diazinon
from the AHS alone. Note that the number of diazinon-exposed cases was low or not reported in all
three available studies.
Fig. 4. Forest plot showing results of studies of risk of leukaemia on ever exposure to diazinon or
high versus low exposure to diazinon
CI: confidence interval; OR: odds ratio

69
Diazinon / Lung cancer

A significant 60% excess risk of lung cancer in the highest exposure category (> 38.8 lifetime
days of diazinon exposure) and a significant trend across exposure categories were observed in the
AHS. Findings for intensity-weighted lifetime-exposure days demonstrated a similar pattern, but did
not reach significance (Jones et al., 2015). A separate analysis of ever-use versus never-use of
diazinon found no evidence of elevated risk of lung cancer among spouses of farmers or pesticide
applicators; however, there were only 15 exposed cases (Lerro et al., 2015). One other study reported
a nonsignificant elevated risk of lung cancer for ever- versus never-use of diazinon (based on 17
exposed cases) (Pesatori et al., 1994). Overall, there is weak evidence of a positive association
between lung cancer and exposure to diazinon from the AHS cohort study only.
Fig. 5. Forest plot showing results of studies of risk of lung cancer on ever exposure to diazinon
CI: confidence interval; OR: odds ratio; RR: relative risk
Comments
Biochemical aspects
Following oral administration to rats, diazinon was almost completely absorbed and rapidly
eliminated, mainly in the urine. There was no evidence of accumulation (Robbins, Eddy & Hopkins,
1957; Mücke, Alt & Esser, 1970; Burgener & Seim, 1988; Simoneaux, 1988b; Simoneaux et al.,
1989; Capps et al., 1989; Craine, 1989).
Diazinon is metabolized by P450 to diazoxon, the active metabolite. The main degradative
pathway includes the oxidase/hydrolase-mediated cleavage of the ester bond, leading to the
pyrimidinol derivative 2-isopropyl-6-methyl-4(1H)-pyrimidinone, which is further oxidized to more
polar metabolites (Mücke, Alt & Esser, 1970; Iverson, Grant & Lacroix, 1975; Capps et al., 1989).
Toxicological data
The oral LD50 for diazinon in rats ranged from 300 to greater than 2150 mg/kg bw, whereas
the dermal LD50 was greater than 2000 mg/kg bw (Bathe, 1972b, 1980; Piccirillo, 1978; Nissimov,
1984a; Schoch & Gfeller, 1985; Kuhn, 1989a,b; Dreher, 1997). The inhalation median lethal
concentration (LC50) was 3.1 mg/L in rats (Cummins, 1985; Jackson, 1987; Holbert, 1989, 1994).
Diazinon produced mild skin and eye irritation in rabbits (Kuhn, 1989c,d). It caused skin sensitization
in the guinea-pig Magnusson–Kligman maximization test (Cummins, 1987a).
The most sensitive end-point observed in all species given single and repeated doses of
diazinon was inhibition of cholinesterase activity. Brain acetylcholinesterase activity was generally
decreased at doses higher than those that inhibited erythrocyte acetylcholinesterase activity. Clinical

70
signs of cholinergic toxicity occurred at doses causing more than 50% inhibition of brain
acetylcholinesterase activity. Female rats were more sensitive than male rats.
Many repeated-dose toxicity studies are available. In both rats and dogs, no effects other than
those related to cholinesterase inhibition have been observed at the LOAEL; in general, effects
observed at the highest doses can be considered secondary to the cholinergic toxicity. In these studies,
NOAELs ranged from 0.02 to 0.5 mg/kg bw per day, and LOAELs from 1 to 15 mg/kg bw per day,
based on erythrocyte acetylcholinesterase inhibition (i.e. > 20%), with brain acetylcholinesterase
inhibition (i.e. > 10%) generally appearing at the next higher dose and clinical cholinergic signs
appearing at doses above 23 mg/kg bw per day.
In a 28-day acetylcholinesterase inhibition study, rats received diazinon by dietary
administration at a concentration of 0, 0.3, 30, 300 or 3000 parts per million (ppm) (equal to 0, 0.02,
2.3, 23 and 213 mg/kg bw per day for males and 0, 0.02, 2.4, 23 and 210 mg/kg bw per day for
females, respectively). The NOAEL was 0.3 ppm (equal to 0.02 mg/kg bw per day), on the basis of
inhibition of erythrocyte acetylcholinesterase activity at 30 ppm (equal to 2.3 mg/kg bw per day)
(Chang, 1994).
In a short-term toxicity study, rats were fed diazinon at a concentration of 0 or 2 ppm
(equivalent to 0 and 0.2 mg/kg bw per day, respectively) for 7 days or at a concentration of 0 or 25
ppm (equivalent to 0 and 2.5 mg/kg bw per day, respectively) for 30 days. The NOAEL was 2 ppm
(equivalent to 0.2 mg/kg bw per day), based on inhibition of erythrocyte acetylcholinesterase activity
at 25 ppm (equivalent to 2.5 mg/kg bw per day) (Davies & Holub, 1980a).
In a 3-month toxicity study, rats were given diets containing diazinon at a concentration of 0,
0.5, 5, 250 or 2500 ppm (equal to 0, 0.03, 0.3, 15 and 168 mg/kg bw per day for males and 0, 0.04,
0.4, 19 and 212 mg/kg bw per day for females, respectively). The NOAEL was 5 ppm (equal to 0.3
mg/kg bw per day), on the basis of inhibition of erythrocyte and brain acetylcholinesterase activities
at 250 ppm (equal to 15 mg/kg bw per day) (Singh, Arthur & McCormick, 1988).
In a second 3-month toxicity study, rats were fed diets containing diazinon at a concentration
of 0, 0.3, 30, 300 or 3000 ppm (equal to 0, 0.017, 1.7, 17 and 177 mg/kg bw per day for males and 0,
0.019, 1.9, 19 and 196 mg/kg bw per day for females, respectively). The NOAEL was 0.3 ppm (equal
to 0.017 mg/kg bw per day), on the basis of inhibition of erythrocyte acetylcholinesterase activity at
30 ppm (equal to 1.7 mg/kg bw per day) (Pettersen & Morrissey, 1994).
In a third 3-month toxicity study, female rats were fed diets containing diazinon at a
concentration of 0, 5, 10 or 15 ppm (equivalent to 0, 0.5, 1 and 1.5 mg/kg bw per day, respectively)
for 92 days. In the second phase, female rats were fed diets containing diazinon at a concentration of
0, 1, 2, 3 or 4 ppm (equivalent to 0, 0.1, 0.2, 0.3 and 0.4 mg/kg bw per day, respectively) for 42 days.
In the third phase, female rats were fed diets containing diazinon at a concentration of 0, 0.1, 0.5, 1 or
2 ppm (equivalent to 0, 0.01, 0.05, 0.1 and 0.2 mg/kg bw per day, respectively) for 35 days. The
NOAEL in the first phase was 5 ppm (equivalent to 0.5 mg/kg bw per day), based on inhibition of
erythrocyte acetylcholinesterase activity at 10 ppm (equivalent to 1 mg/kg bw per day) after dosing
for 92 days. The NOAEL for females in the second and third phases were the highest tested doses of 4
ppm (equivalent to 0.4 mg/kg bw per day) and 2 ppm (equivalent to 0.2 mg/kg bw per day) after
dosing for 42 and 35 days, respectively (Davies & Holub, 1980b).
In a fourth 3-month toxicity study, rats were fed diets containing diazinon at a concentration
of 0, 5, 125 or 3000 ppm (equal to 0, 0.3, 7.8 and 198 mg/kg bw per day for males and 0, 0.3, 8.9 and
247 mg/kg bw per day for females, respectively). The NOAEL was 5 ppm (equal to 0.3 mg/kg bw per
day), on the basis of inhibition of erythrocyte acetylcholinesterase activity at 125 ppm (equal to 7.8
mg/kg bw per day) (Sunaga, 2010).
In a 90-day repeated-dose neurotoxicity study, rats were dosed in the diet at 0, 25, 125 or
1000 ppm (equal to 0, 1.7, 8.4 and 69.1 mg/kg bw per day for males and 0, 1.8, 9.3 and 82.4 mg/kg
bw per day for females, respectively). A NOAEL could not be identified, as erythrocyte
acetylcholinesterase activity was inhibited at 1.7 mg/kg bw per day, the lowest dose tested (Sunaga,
2007b).

71
In considering the NOAELs and LOAELs identified in the 28-day and 3-month
(neuro)toxicity studies in rats measuring the inhibition of acetylcholinesterase activity, the Meeting
concluded that the extent of acetylcholinesterase inhibition was not dependent on duration of dosing
once steady state had been achieved (within 4 weeks). The overall NOAEL for the 28-day and 3-
month (neuro)toxicity studies in rats was 5 ppm, based on inhibition of erythrocyte
acetylcholinesterase activity at the overall LOAEL of 10 ppm. In studies where feed consumption data
were used to calculate test substance intake, 5 ppm was equal to 0.3 mg/kg bw per day. These
substance intake data are considered to be more accurate than those calculated using a default
conversion factor, in which the NOAEL of 5 ppm is equivalent to 0.5 mg/kg bw per day.
In a 90-day toxicity study, dogs were given diets containing diazinon at a concentration of 0,
0.1, 0.5, 150 or 300 ppm (equal to 0, 0.0034, 0.020, 5.9 and 10.9 mg/kg bw per day for males and 0,
0.0037, 0.021, 5.6 and 11.6 mg/kg bw per day for females, respectively). The NOAEL was 0.5 ppm
(equal to 0.020 mg/kg bw per day), on the basis of inhibition of erythrocyte and brain cholinesterase
activities at a dietary concentration of 150 ppm (equal to 5.6 mg/kg bw per day) (Barnes, Arthur &
Hazelette, 1988).
In a second 90-day toxicity study, dogs were given diazinon at 0, 0.3, 3 or 10 mg/kg bw per
day by gelatine capsule. The NOAEL was 0.3 mg/kg bw per day, on the basis of inhibition of
erythrocyte and brain acetylcholinesterase activities at 3 mg/kg bw per day (Ichido, 2010).
In a 1-year toxicity study in dogs given diazinon in the diet at a concentration of 0, 0.1, 0.5,
150 or 300 ppm (equal to 0, 0.0032, 0.015, 4.7 and 7.7 mg/kg bw per day for males and 0, 0.0037,
0.020, 4.5 and 9.1 mg/kg bw per day for females, respectively), the NOAEL was 0.5 ppm (equal to
0.015 mg/kg bw per day), on the basis of inhibition of erythrocyte (males and females) and brain
(females only) acetylcholinesterase activities at 150 ppm (equal to 4.5 mg/kg bw per day) (Rudzki,
Arthur & McCormick, 1991).
The overall NOAEL for the 90-day and 1-year toxicity studies in dogs was 0.3 mg/kg bw per
day, based on inhibition of erythrocyte and brain acetylcholinesterase activities at 3 mg/kg bw per
day.
In a pre-GLP carcinogenicity study in mice that was considered adequate to evaluate
carcinogenicity but not chronic toxicity, diazinon was administered at a dietary concentration of 0,
100 or 200 ppm (equivalent to 0, 15 and 30 mg/kg bw per day, respectively) over 103 weeks. No
treatment-related tumours were observed (NTP, 1979; also referenced as Angel et al., unknown year,
or NCI, 1979).
In another pre-GLP carcinogenicity study in mice, diazinon was administered at a dietary
concentration of 0, 100, 200, 300 (males) or 400 (females) ppm (equal to 0, 16, 31 and 46 mg/kg bw
per day for males and 0, 22, 43 and 86 mg/kg bw per day for females, respectively) for 104 weeks.
Cholinesterase activity was not measured in this study. The NOAEL for chronic toxicity was 200 ppm
(equal to 31 mg/kg bw per day), based on depression of body weight and lower feed consumption at
300 ppm (equal to 46 mg/kg bw per day). No treatment-related tumours were observed (Goldsmith,
1983).
In a pre-GLP carcinogenicity study in rats that was considered adequate to evaluate
carcinogenicity but not chronic toxicity, diazinon was administered at a dietary concentration of 0,
400 or 800 ppm (equivalent to 0, 20 and 40 mg/kg bw per day, respectively) over 103 weeks. No
treatment-related tumours were observed (NTP, 1979; also referenced as Angel et al., unknown year,
or NCI, 1979).
In a chronic toxicity study, rats received diazinon in the diet at a concentration of 0 (untreated
and vehicle controls), 0.1, 1.5, 125 or 250 ppm (equal to 0, 0.004, 0.06, 5 and 10 mg/kg bw per day
for males and 0, 0.005, 0.07, 6 and 12 mg/kg bw per day for females, respectively) for 98/99 weeks.
The NOAEL was 1.5 ppm (equal to 0.06 mg/kg bw per day) on the basis of inhibition of erythrocyte
and brain acetylcholinesterase activities at 125 ppm (equal to 5 mg/kg bw per day). From the available
data, there was no evidence of a tumorigenic response; however, the group size (N = 20) was too
small to allow a conclusion to be reached on carcinogenicity (Kirchner, McCormick & Arthur, 1991).

72
In a combined chronic toxicity and carcinogenicity study in rats, diazinon was fed in the diet
at concentrations adjusted to achieve target concentrations of 0, 0.025, 0.1, 1.5 or 22.5 mg/kg bw per
day for 104 weeks. The NOAEL for long-term toxicity was 0.1 mg/kg bw per day, based on inhibition
of erythrocyte acetylcholinesterase activity at 1.5 mg/kg bw per day. No treatment-related tumours
were observed (Ashby & Danks, 1987).
The overall NOAEL for chronic toxicity in rats was 0.1 mg/kg bw per day, based on
inhibition of erythrocyte acetylcholinesterase activity at 1.5 mg/kg bw per day.
The Meeting concluded that diazinon is not carcinogenic in mice or rats.
Given the similarity of the sensitivities of mammalian species, an overall NOAEL in all
studies of repeated-dose (neuro)toxicity in rats and dogs could be identified. The overall NOAEL was
0.3 mg/kg bw per day, on the basis of inhibition of acetylcholinesterase activity in erythrocytes at 1
mg/kg bw per day.
In studies submitted by the sponsors, diazinon was tested for genotoxicity in an adequate
range of assays, both in vitro and in vivo. In addition, many studies with diazinon were described in
the published literature, but most of these were considered by the Meeting as inappropriate to evaluate
the genotoxicity of diazinon, as they had major deficiencies in study design or reliability (e.g. lack of
statistical analysis, testing of mixtures of diazinon with other chemicals and similarity between
negative and positive control values). Overall, these studies provided no convincing evidence of
genotoxic effects.
The Meeting concluded that diazinon is unlikely to be genotoxic.
In the multigeneration and developmental toxicity studies, cholinesterase activity was not
measured.
In a two-generation study on reproductive toxicity, rats received diazinon in the diet at a
concentration of 0, 10, 100 or 500 ppm over the course of two generations (F0 and F1). Mean diazinon
intakes for the F0 generation during the premating period were 0, 0.77, 7.48 and 32.85 mg/kg bw per
day for males and 0, 0.77, 7.48 and 40.26 mg/kg bw per day for females, respectively. The NOAEL
for reproductive effects was 100 ppm (equal to 7.48 mg/kg bw per day), based on prolonged gestation
duration, decrease in the number of pregnancies, and reduced fertility and mating indices at 500 ppm
(equal to 32.85 mg/kg bw per day). The NOAEL for parental effects was 10 ppm (equal to 0.77 mg/kg
bw per day), based on reduced parental body-weight gain at 100 ppm (equal to 7.48 mg/kg bw per
day). The NOAEL for offspring toxicity was 10 ppm (equal to 0.77 mg/kg bw per day), based on
reduced viability of pups and pup weights at 100 ppm (equal to 7.48 mg/kg bw per day) (Giknis,
1989).
In another two-generation study on reproductive toxicity, rats received diazinon in the diet at
a concentration of 0, 0.1, 1.0 or 10 mg/kg (equivalent to 0, 0.0067, 0.067 and 0.67 mg/kg bw per day,
assuming concentrations are in mg/kg feed or ppm) over the course of two generations (F 0 and F1). A
rationale for the dose selection was not provided. There were no treatment-related effects observed in
F0 or F1 parental animals or pups. The NOAEL for reproductive, parental and offspring toxicity was
10 ppm (equivalent to 0.67 mg/kg bw per day), the highest dose tested (Weatherholtz, 1982).
In a range of studies on estrogenic and androgenic activities, no estrogenic, androgenic or
anti-androgenic activity was observed at concentrations relevant to human exposure via the diet
(Davis, 2011; Davis & Lea, 2011; Wilga, 2011; Willoughby, 2011a,b, 2012; Wagner, 2012).
Overall NOAELs from the multigeneration studies in rats were identified. The overall
NOAEL for reproductive effects was 100 ppm (equal to 7.48 mg/kg bw per day), based on effects at
500 ppm (equal to 32.85 mg/kg bw per day). The overall NOAEL for parental toxicity was 10 ppm
(equal to 0.77 mg/kg bw per day), based on effects at 100 ppm (equal to 7.48 mg/kg bw per day). The
overall NOAEL for offspring toxicity was 10 ppm (equal to 0.77 mg/kg bw per day), based on effects
at 100 ppm (equal to 7.48 mg/kg bw per day).

73
In a study of developmental toxicity evaluated by the 1993 JMPR, rats were administered
diazinon via gavage at a dose of 0, 15, 50 or 100 mg/kg bw per day. A marked decrease in maternal
feed consumption correlating with weight loss at the beginning of the treatment period and a slightly
higher incidence of incomplete ossification at different sites in the fetuses were observed at 100
mg/kg bw per day. As limited information was available from the previous JMPR monograph, the
Meeting was unable to identify a NOAEL for this study (Fritz, 1974).
In a study of developmental toxicity, rats were administered diazinon via gavage at a dose of
0, 10, 20 or 100 mg/kg bw per day. The NOAEL for maternal toxicity was 20 mg/kg bw per day,
based on body weight loss on gestation days 6 to 10, reduced body weight/body-weight gains
throughout treatment and decreased feed consumption on gestation days 6 to 9 at 100 mg/kg bw per
day. The NOAEL for embryo/fetal toxicity was 20 mg/kg bw per day, based on an increased
incidence of rudimentary 14th ribs at 100 mg/kg bw per day (Infurna & Arthur, 1985).
In a study of developmental toxicity, rabbits were dosed with diazinon via gavage at 0, 7, 25
or 100 mg/kg bw per day. The NOAEL for maternal toxicity was 25 mg/kg bw per day, based on
mortality, tremors, convulsions, hypoactivity, anorexia and reduced body-weight gain observed at 100
mg/kg bw per day. The NOAEL for embryo/fetal toxicity was 100 mg/kg bw per day, the highest dose
tested (Harris & Holson, 1981).
In another developmental toxicity study, diazinon was administered to pregnant rabbits by
gavage at a dose level of 0, 2.5, 10 or 40 mg/kg bw per day. The NOAEL for maternal toxicity was 10
mg/kg bw per day, based on clinical signs, decreased body weight and reduced feed consumption. The
NOAEL for embryo/fetal toxicity was 10 mg/kg bw per day, based on decreased fetal weight at 40
mg/kg bw per day (Edwards & Falconer, 1987).
The overall NOAEL for maternal toxicity in developmental toxicity studies in rabbits was 25
mg/kg bw per day, based on effects at 40 mg/kg bw per day, and the overall NOAEL for embryo/fetal
toxicity was 10 mg/kg bw per day, based on effects at 40 mg/kg bw per day.
The Meeting concluded that diazinon is not teratogenic.
In a limited acute neurotoxicity study in which acetylcholinesterase activity was not

measured, rats were dosed with diazinon at 0, 100, 300 or 500 mg/kg bw by gavage. The NOAEL was
100 mg/kg bw, based on systemic toxicity and clinical signs of neurotoxicity observed at 300 or 500
mg/kg bw (Sunaga, 2007a). In another acute toxicity study, rats were administered a single dose of
diazinon by gavage at 0, 2.5, 150, 300 or 600 mg/kg bw. The NOAEL was 2.5 mg/kg bw, on the basis
of depressed erythrocyte acetylcholinesterase activity and behavioural changes at 150 mg/kg bw
(Chow & Richter, 1994). In a third study, rats were administered a single dose of diazinon by gavage
at 100, 250 or 500 mg/kg bw for males or 0, 0.05, 0.12, 0.25, 2.5, 25 or 250 mg/kg bw for females.
The NOAEL was 2.5 mg/kg bw, on the basis of inhibition of brain and erythrocyte
acetylcholinesterase activities in females at 25 mg/kg bw (Glaza, 1993).
In a study that investigated the time course of acute inhibition of acetylcholinesterase activity,
rats were given a single dose of diazinon by gavage at 0, 2.5, 150, 300 or 600 mg/kg bw, and brain
and blood samples were collected at 3, 9 and 24 hours after dosing. The NOAEL was 2.5 mg/kg bw,
based on inhibition of brain and erythrocyte acetylcholinesterase activities at 150 mg/kg bw.
Inhibition was observed beginning at 3 hours post-dosing, with maximal inhibition at 9 hours post-
dosing (Potrepka, 1994).
The overall NOAEL in all studies of acute toxicity was 2.5 mg/kg bw, on the basis of
inhibition of acetylcholinesterase activity in erythrocytes and in the brain at 25 mg/kg bw in rats of
both sexes.
Three studies were performed on delayed neurotoxicity in the hen. Oral doses of diazinon
technical ranging from 10 to 100 mg/kg bw were administered to hens. Inhibition of cholinesterase
activity was observed from 20 mg/kg bw, but there was no evidence that diazinon caused acute
delayed neurotoxicity in the hen (Cummins, 1987b; Jenkins, 1988; Classen, 1996).

74
No specific studies on immunotoxicity were submitted. A study in the open literature with
intraperitoneal injection of diazinon in mice (Neishabouri et al., 2004) was not informative. The
submitted repeated-dose toxicity studies do not indicate an immunotoxic potential for diazinon after
oral exposure.
Toxicological data on metabolites and/or degradates

No toxicological data were available on any metabolites of diazinon other than diazoxon,
which is the active metabolite of diazinon. However, the Meeting concluded that none of the other
metabolites would be of toxicological concern at the levels present in the diet.
Human data
In a study of acute toxicity in male volunteers given ascending doses of diazinon (seven
volunteers per group given 0.03, 0.12, 0.20 or 0.21 mg/kg bw; one volunteer given 0.30 mg/kg bw),
acetylcholinesterase activity was not inhibited in erythrocytes at 0.21 mg/kg bw, the second highest
dose tested. The highest dose (0.30 mg/kg bw) was not informative, as it was tested in a single
volunteer only. Plasma cholinesterase activity was inhibited by more than 20% at doses above 0.12
mg/kg bw (Boyeson, 2000; final version reported as Anderson, 2000).
Repeated-dose studies in four male volunteers given diazinon for 28–37 days showed that,
although there was some inhibition of plasma cholinesterase activity at the highest tested dose of 0.03
mg/kg bw per day (actual administered doses varied slightly, i.e. 0.03, 0.027, 0.022/0.027 and 0.026
mg/kg bw per day), no inhibition of erythrocyte acetylcholinesterase activity was observed (Payot,
1966; Beilstein, 1998).
Diazinon was evaluated in four male volunteers who received diazinon in capsules at 0.025
mg/kg bw per day for 37–43 days. There were no consistent treatment-related effects on erythrocyte
acetylcholinesterase activity, blood chemistry or urine analysis. No clinical effects were reported. The
NOAEL was 0.025 mg/kg bw per day, the only dose tested (Lazanas, Fancher & Calandra, 1966).
The overall NOAEL from repeated-dose studies in humans was 0.03 mg/kg bw per day.
Several epidemiological studies on cancer outcomes following occupational exposure to
diazinon were available. The review of these studies focused on the occurrence of three cancer types:
NHL, leukaemia and lung cancer (see section 2.2 of the meeting report). One prospective cohort study
was available, the AHS, with a large sample size and detailed exposure assessment. Cohort studies are
considered a powerful design, as recall bias is avoided. All other studies were case–control studies,
usually retrospective, which are more prone to recall and selection biases.
There was no significant evidence of a positive association of NHL with diazinon exposure
and no evidence of an exposure–response relationship in the AHS (Alavanja et al., 2014; Lerro et al.,
2015). In a large pooled case–control study, the unadjusted estimates showed a significant elevated
risk of NHL (RR = 1.7; 95% CI = 1.2–2.5) associated with ever- versus never-use of diazinon
(Waddell et al., 2001). However, these risks were attenuated and/or no longer significant when proxy
respondents were excluded and analyses were mutually adjusted for other pesticides (malathion,
fonofos). Although increasing risk across exposure duration categories was observed, which was
suggestive of a duration–response pattern, confidence intervals were nonsignificant, wide and
overlapping between categories. Two other studies reported elevated risks of NHL for ever- versus
never-use of diazinon (McDuffie et al., 2001) or high versus low diazinon use (Mills, Yang &
Riordan, 2005), but confidence intervals were wide, reflecting uncertainty in the risk estimates, and
chance could not be excluded as an explanation for the findings. Overall, there was no convincing
evidence of a positive association between NHL and exposure to diazinon.
A significantly increased risk of leukaemia in the highest exposure category (> 38.8 lifetime
days of diazinon exposure; RR = 3.36; 95% CI = 1.08–10.49) and a significant exposure–response
relationship were observed in the AHS. Findings for intensity-weighted lifetime-exposure days

75
demonstrated a similar pattern, but did not reach significance (Beane Freeman et al., 2005). Two other
studies reported nonsignificantly elevated risks of leukaemia for high versus low diazinon use (Mills,
Yang & Riordan, 2005) and ever- versus never-use of diazinon (Brown et al., 1990), with a
nonsignificant dose–response relationship observed using days of use per year (Brown et al., 1990).
Overall, there is weak evidence of a positive association between leukaemia and exposure to diazinon
from the AHS only. It is noted that the number of diazinon-exposed cases was low or not reported in
all three available studies.
A significant 60% excess risk of lung cancer in the highest exposure category (> 38.8 lifetime
days of diazinon exposure) and a significant trend across exposure categories were observed in the
AHS. Findings for intensity-weighted lifetime-exposure days demonstrated a similar pattern, but did
not reach significance (Jones et al., 2015). A separate analysis of ever-use of diazinon versus never-
use from the AHS found no evidence of elevated risk of lung cancer among spouses of
farmers/pesticide applicators; however, there were only 15 exposed cases (Lerro et al., 2015). One
other study reported a nonsignificant elevated risk of lung cancer for ever- versus never-use of
diazinon (based on 17 exposed cases) (Pesatori et al., 1994). Overall, there is weak evidence of a
positive association between lung cancer and exposure to diazinon from the AHS cohort study only.
In view of the lack of genotoxicity and the absence of carcinogenicity in mice and rats and
considering the available epidemiological data from occupational exposure, the Meeting concluded
that diazinon is unlikely to pose a carcinogenic risk to humans via exposure from the diet.
The Meeting concluded that the existing database on diazinon was adequate to characterize
the potential hazards to the general population, including fetuses, infants and children.
Toxicological evaluation
The Meeting identified inhibition of acetylcholinesterase activity as the most sensitive end-
point after single or repeated doses of diazinon in all species. After considering all previously
evaluated data and the new studies, the Meeting established an ADI of 0–0.003 mg/kg bw, based on
the overall NOAEL of 0.3 mg/kg bw per day from all repeated-dose toxicity studies, and using a
safety factor of 100. This ADI was supported by the NOAEL of 0.03 mg/kg bw per day, the highest
dose tested, identified in repeated-dose studies that involved a limited number of male volunteers,
with application of a safety factor of 10.
In 2006, the Meeting established an ADI of 0–0.005 mg/kg bw, based on the highest NOAEL
of 0.5 mg/kg bw per day for inhibition of erythrocyte acetylcholinesterase activity at 1 mg/kg bw per
day in a 92-day repeated-dose toxicity study in rats and using a safety factor of 100. In this study, the
dietary concentrations of diazinon were converted to units of milligrams per kilogram body weight
per day using a default conversion factor; the present Meeting considers this less reliable than the
conversion using feed consumption data.
The Meeting reaffirmed the ARfD of 0.03 mg/kg bw established by the 2006 JMPR. This
ARfD was based on the NOAEL of 2.5 mg/kg bw identified in studies of acute (neuro)toxicity in rats,
and using a safety factor of 100. This ARfD was supported by the NOAEL of 0.21 mg/kg bw, the
highest dose tested, identified in the study in which a limited number of male volunteers were given a
single dose of diazinon, with application of a safety factor of 10.
Levels relevant to risk assessment of diazinon

Species Study Effect NOAEL LOAEL
Mouse Two-year study of Toxicity 200 ppm, equal to 31 300 ppm, equal to 46
carcinogenicitya,b mg/kg bw per day mg/kg bw per day
Carcinogenicity 300 ppm, equal to 46 –
mg/kg bw per dayc
Rat Acute (neuro)toxicity Toxicity 2.5 mg/kg bw 25 mg/kg bw
studiesd,e

76

(acetylcholinesterase
inhibition)
Four-week or 3-month Toxicity 5 ppm, equal to 0.3 10 ppm, equivalent to
studies of (neuro)toxicitya,e mg/kg bw per dayf 1 mg/kg bw per day
Two-year studies of toxicity Toxicity 0.1 mg/kg bw per dayf 1.5 mg/kg bw per day
and carcinogenicitya,e
Carcinogenicity 800 ppm, equivalent –
to 40 mg/kg bw per
dayc
Two-generation studies of Reproductive toxicity 100 ppm, equal to 500 ppm, equal to
reproductive toxicitya,b,e 7.48 mg/kg bw per 32.85 mg/kg bw per
day day
Parental toxicity 10 ppm, equal to 0.77 100 ppm, equal to
mg/kg bw per day 7.48 mg/kg bw per
day
Offspring toxicity 10 ppm, equal to 0.77 100 ppm, equal to
mg/kg bw per day 7.48 mg/kg bw per
day
Developmental toxicity Maternal toxicity 20 mg/kg bw per day 100 mg/kg bw per
studyb,d day
Embryo and fetal 20 mg/kg bw per day 100 mg/kg bw per
toxicity day
Rabbit Developmental toxicity Maternal toxicity 25 mg/kg bw per day 40 mg/kg bw per day
studiesb,d,e Embryo and fetal 10 mg/kg bw per day 40 mg/kg bw per day
toxicity
Dog Ninety-day and 1-year Toxicity 0.3 mg/kg bw per dayf 3 mg/kg bw per day
studies of toxicitya,e
Rat, dog Repeat-dose (neuro)toxicity Toxicity 5 ppm, equal to 0.3 10 ppm, equivalent to
studiese mg/kg bw per day 1 mg/kg bw per day
Human Acute toxicity studyd Toxicity 0.21 mg/kg bwc –
Four/five-week studies of Toxicity 0.03 mg/kg bw per –

toxicityd,e dayc
a
Dietary administration.
b
Acetylcholinesterase activity not measured.
c
Highest dose tested.
d
Gavage administration.
e
Two or more studies combined.
f
Included in the overall NOAEL for rats and dogs.
Estimate of acceptable daily intake (ADI)

0–0.003 mg/kg bw
Estimate of acute reference dose (ARfD)

0.03 mg/kg bw
Information that would be useful for the continued evaluation of the compound
Results from epidemiological, occupational health and other such observational studies of
human exposure

77
Critical end-points for setting guidance values for exposure to diazinon

Absorption, distribution, excretion and metabolism in mammals
Rate and extent of oral absorption Nearly complete and rapid (~90% at 10 mg/kg bw within 24 h)
Dermal absorption No data
Distribution Widely distributed at low concentrations
Potential for accumulation No potential for accumulation
Rate and extent of excretion Predominantly in urine (86–93% at 10 mg/kg bw within 24 h)
Metabolism in animals Rapidly degraded to diazoxon and subsequently mainly via
oxidase/hydrolase-mediated cleavage of the ester bond, and further
oxidation at the isopropyl substituent to yield hydroxy pyrimidinols
Toxicologically significant compounds in animals Parent compound and diazoxon
and plants
Acute toxicity
Rat, LD50, oral 300 to > 2 150 mg/kg bw
Rat, LD50, dermal > 2 000 mg/kg bw
Rat, LC50, inhalation 3.1 mg/L
Rabbit, dermal irritation Mildly irritating
Rabbit, ocular irritation Mildly irritating
Guinea-pig, dermal sensitization Sensitizing (Magnusson and Kligman maximization test)
Repeat-dose studies of (neuro)toxicity
Target/critical effect Acetylcholinesterase inhibition
Overall oral NOAEL 0.3 mg/kg bw per day (rat, dog)
Lowest relevant dermal NOAEL 3 mg/kg bw per day (21 days; rat)
Lowest relevant inhalation NOAEC 0.46 mg/m3 (21 days; rat)
Long-term studies of carcinogenicity
Carcinogenicity Not carcinogenic in mice or ratsa
Genotoxicity
No evidence of genotoxicity by the oral routea
Reproductive toxicity
Target/critical effect Mortality, reduced parental body-weight gain, reduced viability of
pups and pup weights, prolonged gestation duration, decrease in
number of pregnancies, and reduced fertility and mating indices
Lowest relevant parental NOAEL 0.77 mg/kg bw per day (rat)
Lowest relevant offspring NOAEL 0.77 mg/kg bw per day (rat)
Lowest relevant reproductive NOAEL 7.48 mg/kg bw per day (rat)
Developmental toxicity
Target/critical effect Clinical signs, reduced maternal body weight and feed consumption,
and reduced fetal weight
Lowest relevant maternal NOAEL 25 mg/kg bw per day (rabbit)
Lowest relevant embryo/fetal NOAEL 10 mg/kg bw per day (rabbit)
Neurotoxicityb
Acute neurotoxicity NOAEL 2.5 mg/kg bw (acetylcholinesterase inhibition; rat)
Developmental neurotoxicity NOAEL No data
Acute delayed neurotoxicity No evidence (hens)
Human data Acetylcholinesterase inhibition:
Acute toxicity NOAEL: 0.21 mg/kg bw, highest dose tested
Subchronic toxicity NOAEL: 0.03 mg/kg bw per day, highest dose
tested (4/5 weeks)
a
Unlikely to pose a carcinogenic risk to humans via exposure from the diet.
b
Ninety-day neurotoxicity study in rats is covered by the overall NOAEL for repeated-dose studies of (neuro)toxicity.

78
Summary
Value Study Safety factor
ADI 0–0.003 mg/kg bw Repeated-dose toxicity studies (rat, dog) 100
ARfD 0.03 mg/kg bw Acute (neuro)toxicity studies (rat) 100
References
risk and insecticide, fungicide and fumigant use in the Agricultural Health Study. PLoS ONE.
9(10):e109332. doi:10.1371/journal.pone.0109332
Allais (2002). Diazol (diazinon) Technical: In vitro mammalian chromosome aberration test in human
lymphocytes. Unpublished report no. R 14220 (MAK 737/014563) from Huntingdon Life Sciences Ltd,
UK. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Angel et al. (unknown): see NTP (1979).
Anderson GD (2000). A randomized, double-blind ascending, acute, oral dose study of diazinon to determine
the no effect level (NOEL) for plasma and red blood cell cholinesterase activity in normal, healthy
volunteers. Unpublished report no. NCP-8373 from Covance Clinical Research Unit Inc., Madison,
Wisconsin, USA. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Ashby R, Danks A (1987). Diazinon: Combined toxicity and oncogenicity study in rats. Unpublished report no.
87/NKL002/378 from Life Science Research Ltd, Eye, Suffolk, UK. Submitted to WHO by Nippon Kayaku
Co. Ltd, Tokyo, Japan.
Ballantine L, Marco GJ, Williams SC (1984). Percutaneous absorption of 2 delta-14C-diazinon in rats.
Unpublished report nos. ABR-84011, 302950 from Ciba-Geigy Corp., Greensboro, NC, USA [cited in
Annex 1, reference 70].
Banerjee D (1967). Pericarditis in acute diazinon poisoning. A case report. Armed Forces Med J India. 23:187–
90 [cited in Annex 1, reference 70].
Barnes TB, Arthur AT, Hazelette JR (1988). Diazinon (MG-8): 90-day oral toxicity study in dogs. Unpublished
report no. 882012 from Ciba-Geigy Corp., Greensboro, NC, USA. Submitted to WHO by ADAMA Asia
Pacific Pte Ltd, Singapore, Singapore.
Bathe R (1972a). Acute oral LD50 of technical diazinon in the mouse. Unpublished report no. Siss 1679 from
Ciba-Geigy Ltd, Sisseln, Switzerland [cited in Annex 1, reference 70].
Bathe R (1972b). Acute dermal LD50 of technical diazinon (G-24480) in the rat. Unpublished report no. Siss
1679 from Ciba-Geigy Ltd, Sisseln, Switzerland[cited in Annex 1, reference 70].
Bathe R, Gfeller W (1980). Acute oral LD50 in the rat of technical G 24480. Unpublished report no. 800478
from Ciba-Geigy Ltd, Sisseln, Switzerland [cited in Annex 1, reference 70].
Epidemiol. 162(11):1070–9.
Beilstein P (1998). Tolerance study in Novartis managers upon repeated oral administration of diazinon.
Unpublished report no. 972019 from Novartis Crop Safety/Human Safety Assessment, CH-4002 Basel,
Switzerland. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Bianchi-Santamaria A, Gobbi M, Cembran M, Arnaboldi A (1997). Human lymphocyte micronucleus genotox-
icity test with mixtures of phytochemicals in environmental concentrations. Mutat Res. 388(1):27–32.
doi:10.1016/S1383-5718(96)00128-3.
Bootman J, May K (1986). Diazinon: assessment of its ability to cause lethal DNA damage in strains of
Escherichia coli. Unpublished report no. 86/NKL041/322 from Life Science Research Ltd, Eye, Suffolk,
UK. Submitted to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.
Bootman J, Hodson-Walker G, Dance C (1986). In vitro assessment of the clastogenic activity of diazinon in
cultured human lymphocytes. Unpublished report no. 86/NKL040/473 from Life Science Research Ltd,
Eye, Suffolk, UK. Submitted to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.

79
Boyeson MG (2000). A randomized, double-blind ascending, acute, oral dose study of diazinon to determine the
no effect level (NOEL) for plasma and red blood cell cholinesterase activity in normal, healthy volunteers –
Part A: Clinical Phase. Unpublished report no. NCP-8373 Part A from Covance Clinical Research Unit
Inc., Madison, Wisconsin, USA.
Brennan CR (2010). Diazinon: 2-week dose range-finding/feasibility study by dermal (semi-occlusive)
administration to rats. Project LEB0012. Huntingdon Life Sciences, Huntingdon, Cambridgeshire, UK.
Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Brennan CR (2011). Diazinon: Toxicity study by dermal (semi occluded) administration to rats for 13 weeks
followed by a 4 week recovery period. Unpublished report project LEB0041 from Huntingdon Life
Sciences, Huntingdon, Cambridgeshire, UK. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Singapore, Singapore.
other agricultural risk factors for leukemia among men in Iowa and Minnesota. Cancer Res. 50(20):6585–
91.
Burgener J, Seim V (1988). Biological report for the metabolism of 14C-diazinon in laying hens dosed at 25
ppm. Unpublished report no. BIOL-88006,302213 from Ciba-Geigy Corp., Vero Beach, FL, USA [cited in
Cakir S, Sarikaya R (2005). Genotoxicity testing of some organophosphate insecticides in the Drosophila
wing spot test. Food Chem Toxicol. 43(3):443–50. doi:l0.1016/j.fct.2004.11.010.
52(9):2447–55.
Capps T, Brown K, Lai K, Tweedy B (1989). Characterization and identification of diazinon metabolites in rats.
Unpublished report no. ABR-88164, 302211 and 302925 from Ciba-Geigy Corp., Greensboro, NC, USA.
Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore [cited in Annex 1, reference
70].
Capps TM, Summer D, Bar HP, Carlin TJ (1990). Characterization and identification of major metabolites in
tissues of sheep treated dermally with 14C-diazinon. Unpublished report no. ABR-90014, 302925 from
Ciba-Geigy Corp., Greensboro, NC, USA [cited in Annex 1, reference 70].
Ceresa C (1988). G 24480: Micronucleus test (mouse). Unpublished report no. 871696 from Ciba-Geigy Ltd,
Basel, Switzerland. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Chang JC (1994). Cholinesterase inhibition in 28 day feeding study in rats. Unpublished report no. F-00186
from Ciba-Geigy Corp., Crop Protection Division, Environmental Health Center, Farmington, CT, USA.
Chen HH, Sirianni SR, Huang CC (1982). Sister chromatid exchanges in Chinese hamster cells treated with
seventeen organophosphorus compounds in the presence of a metabolic activation system. Environ
Mutagen. 4(5):621–4. doi:10.1002/em.2860040513.
Chen HH, Hsueh JL, Sirianni SR, Huang CC (1981). Induction of sister-chromatid exchanges and cell cycle
delay in cultured mammalian cells treated with eight organophosphorus pesticides. Mutat Res.
88(3):307–16. doi:10.1016/0165-1218(81)90042-2.
Chow E, Richter AG (1994). Acute neurotoxity study with DZN diazinon MG87% in rats. Unpublished report
no. F-00175 from Ciba-Geigy Corp., Plant Protection Division, Farmington, Connecticut, USA.
Classen W (1996). Delayed neurotoxicity in hens following acute exposure. Unpublished report no. 952030
(G24480/2411) from Ciba-Geigy Ltd, Switzerland. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Colovic M, Krstic D, Petrovic S, Leskovac A, Jaksic G, Savic J et al. (2010). Toxic effects of diazinon and its
photodegradation products. Toxicol Lett. 193(1):9–18. doi:10.1016/j.toxlet.2009.11.022.
Conners DE, Black MC (2004). Evaluation of lethality and genotoxicity in the freshwater mussel Utterbackia
imbecillis (Bivalvia: Unionidae) exposed singly and in combination to chemicals used in lawn care. Arch
Environ Contam Toxicol. 46(3):362-71. doi:10.1007/s00244-003-3003-z.

80
Craine EM (1989). Study of 14C-diazinon disposition in the rat (biological phase and analytical phase I).
Unpublished report no. WIL-82030,302925 from WIL Research Laboratories Inc., Ashland, OH and Ciba-
Geigy Corp., Greensboro, NC, USA [cited in Annex 1, reference 70].
Cristie B (2000). Doctors revise Declaration of Helsinki. BMJ. 321:913.
Cummins HA (1985). Diazol: Acute inhalation toxicity in the rat. Unpublished report no. 85/MAK051/246 (R
3643) from Life Science Research Ltd, UK. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Cummins HA (1987a). Diazinon: Delayed contact hypersensitivity study in guinea-pigs. Unpublished report no.
86/NKL032/534 from Life Science Research Ltd, Suffolk, UK. Submitted to WHO by Nippon Kayaku,
Tokyo, Japan.
Cummins HA (1987b). Diazinon: Acute delayed neurotoxicity study in the hen. Unpublished report no.
86/NKL039/551 from Life Science Research Ltd, Suffolk, United Kingdom. Submitted to WHO by Nippon
Kayaku Co. Ltd, Tokyo, Japan.
Dahm PA (1970). Some aspects of the metabolism of parathion and diazinon. In: O’Brien RD, Yamamoto I,
editors. Biochemical toxicology of insecticides. New York: Academic Press. 51–63 [cited in Annex 1,
reference 70].
Davis JP (2011). The Hershberger bioassay (OPPTS 890.1400); Diazinon unpublished report no. ILS Study no.
C193-200 from ILS, Inc., Durham, NC, USA. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Davis JP (2012). Pubertal development and thyroid function in intact juvenile/peripubertal female and male rats:
Diazinon. Unpublished report no. ILS Study no. C193-300 from ILS, Inc., Durham, NC, USA. Submitted to
WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Davies DB, Holub BJ (1980a). Comparative subacute toxicity of dietary diazinon in the male and female rat.
Toxicol Appl Pharmacol. 54:359–67.
Davies DB, Holub BJ (1980b). Toxicological evaluation of dietary diazinon in the rat. Arch Environ Contam
Toxicol. 9:637–50.
Davis JP, Lea I (2011). The uterotrophic assay (OPPTS 890.1600); Diazinon. Unpublished report. Study No.
C193-100 (R28280) from ILS, Inc., Durham, NC, USA. Submitted to WHO by ADAMA Asia Pacific Pte
Ltd, Singapore, Singapore.
De Ferrari M, Artuso M, Bonassi S, Bonatti S, Cavalieri Z, Pescatore D et al. (1991). Cytogenetic biomonitoring
of an Italian population exposed to pesticides: chromosome aberration and sister-chromatid exchange
analysis in peripheral blood lymphocytes. Mutat Res. 260(1):105–13. doi:10.1016/0165-1218(91)90086-2.
De Roos AJ, Zahm SH, Cantor KP, Weisenburger DD, Holmes FF, Burmeister LF et al. (2003).
Integrative assessment of multiple pesticides as risk factors for non-Hodgkin’s lymphoma among
men. Occup Environ Med. 60(9):E11. doi:10.1136/oem.60.9.e11.
Dollenmeier P, Müller D (1986). G 24480 techn: L 5178Y/T+/-mouse lymphoma mutagenicity test.
Unpublished report no. 840396 from Ciba-Geigy Ltd, Basel, Switzerland. Submitted to WHO by ADAMA
Asia Pacific Pte Ltd, Singapore, Singapore.
Dreher DM (1997). Acute oral toxicity test in the rat. Unpublished report no. 306/249 (R 9796) from SafePharm
Laboratories Ltd, UK. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Dressel TD, Goodale RL Jr, Arneson MA, Borner JW (1979). Pancreatitis as a complication of
anticholinesterase insecticide intoxication. Ann Surg. 189:199–204 [cited in Annex 1, reference 70].
Dressel TD, Goodale RL, Borner JW, Etani S (1980). A study of the cholinesterase of the canine pancreatic
sphincters and the relationship between reduced butyrylcholinesterase activity and pancreatic ductal
hypertension. Ann Surg. 192:614–9 [cited in Annex 1, reference 70].
Edwards JA, Falconer DM (1987). The effect of diazinon technical on pregnancy of the rabbit. Unpublished
report no. NKU 103/87328 from Huntingdon Research Centre Ltd, Huntingdon, Cambridgeshire, UK.
Submitted to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.
Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961). A new and rapid colorimetric determination
of acetylcholinesterase activity. Biochem Pharmacol. 7: 88–95.

81
EPA (1992). EPA Id# 057801: Diazinon: Review of an in vivo sister chromatid exchange study in mouse bone
marrow. Memorandum. Washington (DC): United States Environmental Protection Agency, Office of
Pesticides and Toxic Substances (www3.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC-
057801_22-Jul-92_103.pdf, accessed 7 December 2015)
Frick TW, Dalo S, O’Leary JF, Runge W, Borner JW, Baraniewski H et al. (1987). Effects of insecticide,
diazinon, on pancreas of dog, cat and guinea-pig. J Environ Pathol Toxicol Oncol. 7(4):1–11 [cited in
Frith CH, Ward JM, Chandra M (1993). The morphology, immunohistochemistry, and incidence of
hematopoietic neoplasms in mice and rats. Toxic Pathol. 21:206–18.
Fritz H (1974). G 24480 Diazinon technical: Reproduction study - rat segment II: (test for teratogenic on
embryotoxic effects). Unpublished report from Ciba-Geigy Ltd, Basel, Switzerland [cited in Annex 1,
reference 70].
Fritz H (1975). G 24480 Diazinon technical: Dominant lethal study mouse. Unpublished report no. 327507 from
Ciba-Geigy Ltd, Basel, Switzerland. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore,
Singapore.
Frölichsthal P, Piatti E (1996). [Evaluation of micronuclei in primary hepatocyte culture in rats treated with
organophosphorus compounds]. Boll Chim Farm. 135(9):541–5. [Italian].
Geleick D, Arni P (1990). Salmonella and Escherichia/liver-microsome test. Unpublished report no. 891346
from Ciba-Geigy Ltd, Basel, Switzerland. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore,
Singapore.
Giknis ML (1989). Diazinon technical: A two-generation reproductive study in albino rats. Unpublished report
no. MIN-852218, 88128 from Ciba-Geigy Corp., Greensboro, NC, USA. Submitted to WHO by ADAMA
Asia Pacific Pte Ltd, Singapore, Singapore.
Glaza SM (1993). Acute oral toxicity study with D.z.n diazinon MG87% in rats. Unpublished report no. HWI
6117-221 from Hazleton Wisconsin Inc., Madison, WI, USA. Submitted to WHO by ADAMA Asia Pacific
Pte Ltd, Singapore, Singapore.
Goldsmith LA (1983). A lifetime carcinogenicity study in mice. Unpublished report no. 21099. Life Science
Research Ltd, Eye, Suffolk, UK. Submitted to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.
Green J (1983). Diazinon (FL-821568): 28-day repeated dose oral toxicity study in albino rats. Final Report:
Lab Project: 41-83. Unpublished study prepared by Ciba-Geigy Corp. MRID 43132204. [cited in Davis,
2012]
Grob D (1956). The manifestations and treatment of poisoning due to nerve gas and other organic phosphate
anticholinesterase compounds. AMA Arch Intern Med. 98:221-39 [cited in Annex 1, reference 70].
Gupta OP, Patel DD (1968). Diazinon poisoning. A study of sixty cases. J Assoc Physicians India. 16:457–63
[cited in Annex 1, reference 70].
Hagenbuch JP, Mücke W (1985). The fate of diazinon in mammals. Biochemistry Product Evaluation 1/85.
Unpublished report from Ciba-Geigy Ltd, Basel, Switzerland [cited in Annex 1, reference 70].
Hariri AT, Moallem SA, Mahmoudi M, Hosseinzadeh H (2011). The effect of crocin and safranal, constituents
of saffron, against subacute effect of diazinon on hematological and genotoxicity indices in rats.
Phytomedicine. 18(6):499–504. doi:10.1016/j.phymed.2010.10.001.
Harris LW, Fleisher JH, Innerebner TA, Cliff W, Sim VM (1969). The effects of atropine-oxime therapy on
cholinesterase activity and the survival of animals poisoned with 0,0-diethyl-0-(2-isopropyl-6-methyl-4-
pyrimidinyl) phosphorothioate. Toxicol Appl Pharmacol. 15:216–24 [cited in Annex 1, reference 70].
Harris SB, Holson JF (1981). A teratology study of diazinon (CAS no. 333-41-5) in New Zealand White rabbits.
Unpublished report no. CGA/SAI 281005 from Science Applications, Inc., La Jolla, CA, USA. Submitted
to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Hartmann HR (1990). G 24480: 21-Day repeated exposure inhalation toxicity in the rat (nose-only exposure).
Unpublished report no. 891205 (G24480/1743) from Ciba-Geigy Ltd, Basel, Switzerland. Submitted to

82
Hertner TH, Arni P (1990). G 24480: Autoradiographic DNA repair test on rat hepatocytes. Unpublished report
no. 891345 from Ciba-Geigy Ltd, Basel, Switzerland. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Haseman JK, Arnold J, Eustis SL (1990). Tumor incidence in Fischer 344 rats: NTP historical data. In:
Boorman GA, Eustis SL, Elwell MR, Montgomery CA Jr, MacKenzie WF, editors. Pathology of the
Fischer rat. San Diego (CA): Academic Press; 555–64.
Hatjian BA, Mutch E, Williams FM, Blain PG, Edwards JW (2000). Cytogenetic response without changes
in peripheral cholinesterase enzymes following exposure to a sheep dip containing diazinon in vivo
and in vitro. Mutat Res. 472(1-2):85–92. doi:10.1016/S1383- 5718(00)00131-5.
Holbert MS (1989). Acute inhalation toxicity study in rats (MG8-FL880045). Study no. 5947-89. Unpublished
report from Stillmeadow Inc., Houston, TX, USA [cited in Annex 1, reference 70].
Holbert MS (1994). Acute inhalation toxicity study in rats. Unpublished report no. 0067-93 from Stillmeadow
Inc., Houston, TX, USA. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Hool G, Müller D (1981a). G 24480: Chromosome studies in male germinal epithelium mouse. Test for
mutagenic effects on spermatocytes. Unpublished report no. 801502 from Ciba-Geigy Ltd, Basel,
Hool G, Müller D (1981b). G 24480: Chromosome studies in male germinal epithelium mouse. Test for
mutagenic effects on spermatogonia. Unpublished report no. 801501 from Ciba-Geigy Ltd, Basel,
IARC (2015). Some organophosphate insecticides and herbicides: diazinon, glyphosate, malathion, parathion,
and tetachlorvinphos. Monograph Volume 112. Lyon (FR): International Agency for Research on Cancer.
Available from: http://monographs.iarc.fr/ENG/Monographs/vol112/
Ichido H (2010). 90-Day repeated dose oral toxicity study of diazinon technical grade (SR09062) in beagle
dogs. Unpublished data. Submitted to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.
Infurna RN, Arthur AT (1985). A teratology study of diazinon technical in Charles River rats. Unpublished
report no. MIN-82296 (G24480/1477) from Ciba-Geigy Corp., Summit, NJ, USA. Submitted to WHO by
Nippon Kayaku Co. Ltd, Tokyo, Japan.
Ishige T, Kubota H, Kurino N, Ohniwa K, Abo Y (1986). Acute oral study in mice. Unpublished data.
Iverson F, Grant DL, Lacroix J (1975). Diazinon metabolism in the dog. Bull Environ Contam Toxicol.
13(5):611–8 [cited in Annex 1, reference 70].
Jackson GC (1987). Diazinon technical: Acute inhalation toxicity in rats 4-hour exposure. Unpublished report
no. NKU 99/861213 from Huntingdon Research Centre Ltd, Huntingdon, Cambridgeshire, UK. Submitted
to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.
Jenkins LJ (1988). Acute delayed neurotoxicity of diazinon mg-8 in domestic fowl. Unpublished report no.
5152-87 from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by Nippon Kayaku Co. Ltd,
Tokyo, Japan.
among pesticide applicators exposed to the organophosphate insecticide diazinon in the Agricultural Health
Study: an updated analysis. Occup Environ Med. 72(7):496-503. doi: 10.1136/oemed-2014-102728.
Kabrawala VN, Shah RM, Oza GG (1965). Diazinon poisoning. (A study of 25 cases). Ind Pract. 18:711–7
Kaplanis JN, Louloudes SJ, Roan CC (1962). The distribution and excretion of P32-labeled diazinon in guinea
pigs. Trans Kans Acad Sci. 65:70–5.
Karamian A, Shokrzadeh M, Ahmadi A (2013). The potential chemoprotective effects of melatonin against
genotoxicity induced by diazinon in human peripheral blood lymphocytes. Toxicol Ind Health. 32(2):360–
6. doi: 10.1177/0748233713500824.
Kashanian S, Gholivand MB, Ahmadi F, Ravan H (2008). Interaction of diazinon with DNA and the protective
role of selenium in DNA damage. DNA Cell Biol. 27(6):325–32. doi:10.1089/dna.2007.0718.

83
Kawamura K (2006). Micronucleus test of diazinon in mice. Unpublished report no. SR06003 from Safety
Research Institute for Chemical Compounds Co. Ltd, Kiyota-ku, Sapporo, Japan. Submitted to WHO by
Nippon Kayaku Co. Ltd, Tokyo, Japan.
Kiraly J, Szentesi I, Ruzicska M, Czeize A (1979). Chromosome studies in workers producing organophosphate
insecticides. Arch Environ Contam Toxicol. 8(3):309–19. doi:10.1007/BF01056247 PMID:507940.
Kirchner FK, McCormick GC, Arthur AT (1991). G 24480 Technical: One/two-year oral toxicity study in rats.
Unpublished report no. 882018 from Ciba-Geigy Corp, Greensboro, NC, USA. Submitted to WHO by
ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Kirpnick Z, Homiski M, Rubitski E, Repnevskaya M, Howlett N, Aubrecht J et al. (2005). Yeast DEL assay
detects clastogens. Mutat Res. 582(1-2):116–34. doi:10.1016/j.mrgentox.2005.01.005.
Krinke GJ (1991). Comment on paper: Neurobehavioral effects of prenatal exposure to the organophosphate
diazinon in mice by Spyker & Avery (1977). J Toxicol Environ Health. 3:989–1002. Letter dated December
17 (1991) prepared by Ciba-Geigy Ltd, Basel, Switzerland [cited in Annex 1, reference 70].
Kubo T, Urano K, Utsumi H (2002). Mutagenicity characteristics of 255 environmental chemicals. J Health Sci.
48(6):545–54. doi:10.1248/jhs.48.545
Kuhn JO (1989a). Diazinon MG 8 FL 880045: Acute oral toxicity study in rats. Unpublished report no. 5942-89
from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Kuhn JO (1989b). Diazinon MG 8 FL 880045: Acute dermal toxicity study in rats. Unpublished report no.
5943-89 from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by ADAMA Asia Pacific Pte
Kuhn JO (1989c). G 24480 tech. (Diazinon MG 8 FL 880045): Primary dermal irritation study in rabbits.
Unpublished report no.5945-89 from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by
ADAMA Asia Pacific Pte Ltd, Singapore, Singapore (including amendment).
Kuhn JO (1989d). G 24480 tech. (Diazinon MG 8 FL 880045): Primary eye irritation study in rabbits.
Unpublished report no. 5944-89 from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by
ADAMA Asia Pacific Pte Ltd, Singapore, Singapore (including amendment).
Kuhn JO (1989e). Diazinon MG 8 FL 880045: Dermal sensitization study in guinea pigs. Unpublished report
no. 5964-89 from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by ADAMA Asia Pacific
Kuhn JO (1995a). OO-TEPP FL 892761: Acute oral toxicity study in rats. Unpublished report study no. 6754-
89-0 (G24480/2402) from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by ADAMA Asia
Kuhn JO (1995b). OS-TEPP FL 892762 and FL 900019: Acute oral toxicity study in rats. Unpublished report
study no. 6755-89-0 (G24480/2404) from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by
Kuhn JO (1995c). SS-TEPP FL 892763 and FL 900020: Acute oral toxicity study in rats. Unpublished report
study no. 6756-89-0 (G24480/2405) from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by
Kuroda K, Yamaguchi Y, Endo G (1992). Mitotic toxicity, sister chromatid exchange, and rec assay of
pesticides. Arch Environ Contam Toxicol. 23(1):13–8. doi:10.1007/BF00225990.
Lazanas JC, Fancher OE, Calandra JC (1966). Report to Geigy Chemicals Corporation. Subacute oral toxicity
study on diazinon 50W – humans. Unpublished report no. IBT D4321 from Industrial Bio-Test
Laboratories, Inc., Northbrook, Illinois, USA [Annex 1, reference 109].
Lerro CC, Koutros S, Andreotti G, Friesen MC, Alavanja MC, Blair A et al. (2015). Organophosphate
Lopez D, Aleixandre C, Merchan M, Carrascal E (1986). In vitro induction of alterations in peripheral blood
lymphocytes by different doses of diazinon. Bull Environ Contam Toxicol. 37(4):517–22.
doi:10.1007/BF01 607798.

84
Losco PE, Ward JM (1984). The early stage of large granular lymphocyte leukemia in the F344 rat. Vet Pathol.
21:286–91.
Machin AF, Rogers H, Cross AJ, Quick MP, Howell LC, Janes NF (1975). Metabolic aspects of the toxicology
of diazinon. I hepatic metabolism in the sheep, cow, pig, guinea-pig, rat, turkey, chicken and duck. Pest
Manag Sci. 6:461–73 [cited in Annex 1, reference 70].
Maedgen JL (1987). Rat acute inhalation toxicity. Oxypyrimidine technical (90.7%) FL-870338. Unpublished
report no. 4686-87 (G24480/2276) from Stillmeadow Inc., Houston, Texas, USA. Submitted to WHO by
Maizlish N, Schenker M, Weisskopf C, Seiber J, Samuels S (1987). A behavioral evaluation of pest control
workers with short-term, low-level exposure to the organophosphate diazinon. Am J Ind Med. 12:153–72
Marshall TC, Dorough HW, Swim HE (1976). Screening of pesticides for mutagenic potential using Salmonella
typhimurium mutants. J Agric Food Chem. 24(3):560–3. doi: 10.1021/jf60205a013.
Matsuoka A, Hayashi M, Ishidate M Jr (1979). Chromosomal aberration tests on 29 chemicals combined
with S9 mix in vitro. Mutat Res. 66(3):277–90. doi:10.1016/0165-1218(79)90089-2
McGregor DB, Brown A, Cattanach P, Edwards I, McBride D, Riach C et al. (1988). Responses of the L5178Y
tk+/tk- mouse lymphoma cell forward mutation assay: III. 72 coded chemicals. Environ Mal Mutagen.
12(1):85–154. doi:10.1002/em.2860120111.
Meyer L (1999). Preliminary summary of a randomized, double-blind ascending, acute, oral dose study of
diazinon to determine the no effect level (NOEL) for plasma and red blood cell cholinesterase activity in
normal, healthy volunteers. Unpublished report no. 8373 from Covance Clinical Research Unit Inc.,
Madison, Wisconsin, USA.
(UFW), 1988–2001. Cancer Causes Control. 16(7):823–30. doi:10.1007/s10552-005-2703-2.
14
Mücke W, Alt KO, Esser HO (1970). Degradation of C-labelled diazinon in the rat. J Agric Food Chem.
18:208–12 [cited in Annex 1, reference 70].
Murli H, Haworth SR (1990b). Mutagenicity test on diazinon MG 8: in vivo sister chromatid exchange assay.
Unpublished report no. HLA-12226-0-458, TX 900093 from Hazleton Laboratories America Inc.,
Kensington, Maryland, USA. Reviewed in EPA (1992).
Neishabouri EZ, Hassan ZM, Azizi E, Ostad SN (2004) Evaluation of immunotoxicity induced by diazinon in
C57bl/6 mice. Toxicology. 2004;196(3):173-9.
NCI, 1979: see NTP (1979).
Ni Z, Li S, Liu Y, Tang Y, Pang D (1993). [Induction of micronucleus by organophosphorus pesticides both in
vivo and in vitro]. Hua Xi Yi Ke Da Xue Xue Bao. 24(1):82–6. [Chinese]
Nishio A, Uyeki EM (1981). Induction of sister chromatid exchanges in Chinese hamster ovary cells by
organophosphate insecticides and their oxygen analogs. J Toxicol Environ Health. 8:939–46. doi:
10.1080/152873981095301 28.
Nissimov S (1984a). “Diazol Tech”: Acute dermal toxicity in rabbits. Unpublished report no. MAK/064/DZL
TECH (R 3633) from Life Science Research Israel Ltd, Israel. Submitted to WHO by ADAMA Asia Pacific
Nissimov S (1984b). “Diazol Tech”: Primary dermal irritation study in rabbits. Unpublished report no.
MAK/066/DZL TECH (R 3635) from Life Science Research Israel Ltd, Israel. Submitted to WHO by
Nissimov S (1984c). “Diazol Tech”: Acute eye irritation study in rabbits. Unpublished report no. MAK/065/DZL
TECH (R 3634) from Life Science Research Israel Ltd, Israel. Submitted to WHO by ADAMA Asia Pacific

85
NTP (1979). Bioassay of diazinon for possible carcinogenicity. Nov. 1976. Carcinogenesis Testing Program,
National Cancer Institute. United States Department of Health, Education and Welfare. DHEW publication
no. (NIH) 79–1392.
Payot PH (1966). Subacute oral toxicity study on diazinon AS - humans. Unpublished report, dated 31 October
1966, from J.R. Geigy, Basel, Switzerland. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Singapore, Singapore [cited in Annex 1, reference 70].
Pesatori AC, Sontag JM, Lubin JH, Consonni D, Blair A (1994). Cohort mortality and nested case–control study
5(4):310–8. doi:10.1007/BF01804981.
Pettersen JC, Morrissey RL (1994). 90-Day subchronic neurotoxicity study with D·Z·N Diazinon MG87% in
rats. Unpublished report no. F-00176 from Ciba-Geigy Corp., Crop Protection Division, Environmental
Health Center, Farmington, Connecticut, USA. Submitted to WHO by ADAMA Asia Pacific Pte Ltd,
Piccirillo VJ (1978). Acute oral toxicity study in rats with diazinon technical. Unpublished report no.483-143
from Hazleton Laboratories America Inc., Kensington, Maryland, USA [cited in Annex 1, reference 70].
Pickles M, Seim V (1988). Biological report for the metabolism of 2-pyrimidinyl-14C-diazinon in a lactating
goat. Unpublished report no. BIOL-88004, 302212 from Ciba-Geigy Corp., Vero Beach, Florida, USA
Pickles M, Seim V (1990). Biological report for the metabolism of 14C-diazinon on sheep. Unpublished report
no. BIOL-89014, 302925 from Ciba-Geigy Corp., Greensboro, NC, USA [cited in Annex 1, reference 70].
Potrepka RF (1994). Acute cholinesterase inhibition time course study with DZN diazinon MG87% in rats.
Unpublished report no. F-00185 from Ciba-Geigy Corp., Farmington, Connecticut, USA. Submitted to
Robbins WE, Eddy GW, Hopkins TL (1957). Metabolism of insecticides, metabolism and excretion of
phosphorus-32-labeled diazinon in a cow. J Agric Food Chem. 5:509–13 [cited in Annex 1, reference 70].
Rudzki MW, Arthur AT, McCormick GC (1991). Diazinon (MG-8): 52-week oral toxicity study in dogs.
Unpublished report no.882014 from Ciba-Geigy Corp., Summit, New Jersey, USA. Submitted to WHO by
Sachsse K, Bathe R (1976). Potentiation study: C9491 (Jodfenphos) versus 2 insecticides, G 24480 (Diazinon),
and C 177 (Dichlorvos) in the rat. Unpublished report. Prepared by Ciba-Geigy Ltd, Basel, Switzerland
Salazar-Arredondo E, de Jesus Solis-Heredia M, Rojas-Garcia E, Hernandez-Ochoa I, Quintanilla-Vega B
(2008). Sperm chromatin alteration and DNA damage by methyl-parathion, chlorpyrifos and diazinon and
their oxon metabolites in human spermatozoa. Reprod Toxicol. 25(4):455–60.
doi:10.1016/j.reprotox.2008.05.055.
Sanderson DM, Edson EF (1959). Oxime therapy in poisoning by six organophosphorus insecticides in the rat. J
Pharm Pharmacol. 11:721-8 [cited in Annex 1, reference 70].
Sarabia L, Maurer I, Bustos-Obregon E (2009a). Melatonin prevents damage elicited by the organophosphorous
pesticide diazinon on the mouse sperm DNA. Ecotoxicol Environ Saf. 72(2):663-8.
doi:10.1016/j.ecoenv.2008.04.023
Sarabia L, Maurer I, Bustos-Obregon E (2009b). Melatonin prevents damage elicited by the organophosphorous
pesticide diazinon on the mouse testis. Ecotoxicol Environ Saf. 72(3):938–42.
doi:10.1016/j.ecoenv.2008.04.022.
Schinasi L, Leon ME (2014). Non-Hodgkin lymphoma and occupational exposure to agricultural pesticide
chemical groups and active ingredients: a systematic review and meta-analysis. Int J Environ Res Public
Health. 11(4):4449–527. doi:10.3390/ijerph110404449.
Schoch M, Gfeller W (1985). G 24480 techn. Acute oral LD50 in the rat. Unpublished report no. 850864 from
Ciba-Geigy Ltd, Basel, Switzerland [cited in Annex 1, reference 70].
See RH, Dunn BP, San RH (1990). Clastogenic activity in urine of workers occupationally exposed to
pesticides. Mutat Res. 241(3):251-9. doi:10.1016/0165-1218(90)90022-T.

86
Shadboorestan A, Shokrzadeh M, Ahangar N, Abdollahi M, Omidi M, Hosseini Payam SS (2013). The

chemoprotective effects of L-carnitine against genotoxicity induced by diazinon in rat blood lymphocyte.
Toxicol Ind Health. 31(12):1334-40. doi:10.1177/0748233713491811.
Shirasu Y, Moriya M, Kato K, Furuhashi A, Kada T (1976). Mutagenicity screening of pesticides in the
microbial system. Mutat Res. 40(1):19–30. doi:10.1016/0165-1218(76)90018-5.
Shishido T, Fukami J (1972). Enzymatic hydrolysis of diazoxon by rat tissue homogenates. Pestic Biochem
Physiol. 2:39–50 [cited in Annex 1, reference 70].
Shishido T, Usui K, Fukami J (1972). Oxidative metabolism of diazinon by microsomes from rat liver and
cockroach fat body. Pestic Biochem Physiol. 2:27–38 [cited in Annex 1, reference 70].
Shishido T, Usui K, Sato M, Fukami J (1972). Enzymatic conjugation of diazinon with glutathione in rat and
American cockroach. Pestic Biochem Physiol. 2:51–63 [cited in Annex 1, reference 70].
Shokrzadeh M, Ahmadi A, Ramezaninejhad S, Shadboorestan A (2014). Hesperidin, a citrus bioflavonoid,
ameliorates genotoxicity induced by diazinon in human blood lymphocytes. Drug Res (Stuttg). 65(2):57–
60. doi: 10.1055/s-0033-1363998.
Shokrzadeh M, Ahangar N, Abdollahi M, Shadboorestan A, Omidi M, Payam SS (2013). Potential
chemoprotective effects of selenium on diazinon-induced DNA damage in rat peripheral blood lymphocyte.
Hum Exp Toxicol. 32(7):759–65. doi:10.1177/0960327112468179.
Simoneaux BJ (1988a). Disposition of 14C-diazinon in goats. Unpublished report no. ABR-88117 from Ciba-
Geigy Corp., Greensboro, NC, USA [cited in Annex 1, reference 70].
Simoneaux BJ (1988b). Disposition of 14C-diazinon in chickens. Unpublished report no. ABR-88116 from by
Ciba-Geigy Corp., Greensboro, NC, USA [cited in Annex 1, reference 70].
Simoneaux BJ (1988c). Characterization of 14C-diazinon metabolites in goats. Unpublished report no. ABR-
88118 from Ciba-Geigy Corp., Greensboro, NC, USA [cited in Annex 1, reference 70].
Simoneaux BJ (1988d). Characterization of 14C-diazinon metabolites in chickens. Unpublished report no. ABR-
88119 from Ciba-Geigy Corp., Greensboro, NC, USA [cited in Annex 1, reference 70].
Simoneaux B, Ballantine L, Brown K, Lai K (1988). Metabolite identification in hens and goats treated with
14
C-diazinon. Unpublished report no. ABR-88135 from Ciba-Geigy Corp., Greensboro, NC, USA [cited in
Simoneaux B, Brown K, Lai K, Summer D (1989). Supplemental report on the nature of residues of diazinon in
hens. Unpublished report no. ABR-89040, 302213 from Ciba-Geigy Corp., Greensboro, NC, USA [cited in
Singh AR, Arthur AT, McCormick GC (1988). Diazinon (MG-8): 13-week oral feeding study in rats.
Unpublished report no. MIN-882011, 88083 from Ciba-Geigy Corp., Summit, New Jersey, USA. Submitted
to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Sobti RC, Krishan A, Pfaffenberger CD (1982). Cytokinetic and cytogenetic effects of some agricultural
chemicals on human lymphoid cells in vitro: organophosphates. Mutat Res. 102(1):89–102.
doi:10.1016/0165-1218(82)90149-5.
Spoede-Thompson S, Batastini GG, Arthur AT (1990). Five-week oral toxicity study in rats. Unpublished report
no. 882289 (G24480/2277) from Ciba-Geigy Corp., USA. Submitted to WHO by ADAMA Asia Pacific Pte
Spyker JM, Avery DL (1977). Neurobehavioral effects of prenatal exposure to the organophosphate diazinon in
mice. J Toxicol Environ Health. 3:989–1002 [cited in Annex 1, reference 70].
Sunaga M (2007a). Oral dose acute neurotoxicity study in rats (SR510530). Unpublished data. Submitted to
WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.
Sunaga M (2007b). 90-Day repeated dose neurotoxicity study of diazinon technical grade (SR05310) in rats.
Unpublished data. Submitted to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.
Sunaga M (2010). 90-Day repeated dose oral toxicity study of diazinon technical grade (SR09060) in rats.
Unpublished data. Submitted to WHO by Nippon Kayaku Co. Ltd, Tokyo, Japan.

87
Tai C, Katz R (1984). Diazinon technical: 21-day dermal toxicity study in rabbits. Unpublished report no.
842007 prepared by Ciba-Geigy Corp. Greensboro, NC, USA. Submitted to WHO by ADAMA Asia
Thomas J, Haseman JK, Goodman JI, Ward JM, Loughran TP Jr, Spencer PJ (2007). A review of large granular
lymphocytic leukemia in Fischer 344 rats as an initial step toward evaluating the implication of the endpoint
to human cancer risk assessment. Toxic Pathol. 99:3–19.
Tisch M, Faulde M, Maier H (2007). [Genotoxic effects of insecticides in current use on mucosal epithelial
cells from human tonsil tissue]. HNO. 55(SOI):Suppl I: El5-22. [German] doi:10.1007/s00106-006-
1481-9.
Tisch M, Schmezer P, Faulde M, Groh A, Maier H (2002). Genotoxicity studies on permethrin, DEET and
diazinon in primary human nasal mucosal cells. Eur Arch Otorhinolaryngol. 259(3):150–3. doi:l0.1007/
s004050100406.
Tsitsimpikou C, Tzatzarakis M, Fragkiadaki P, Kovatsi L, Stivaktakis P, Kalogeraki A et al. (2013).
Histopathological lesions, oxidative stress and genotoxic effects in liver and kidneys following long
term exposure of rabbits to diazinon and propoxur. Toxicology 307:109-14.
doi:10.1016/j.tox.2012.11.002.
Ukpebor J, Llabjani V, Martin FL, Halsall CJ (2011). Sublethal genotoxicity and cell alterations by
organophosphorus pesticides in MCF-7 cells: implications for environmentally relevant concentrations.
Environ Toxicol Chem. 30(3):632–9. doi:10.1002/etc.417.
Vega L, Valverde M, Elizondo G, Leyva JF, Rojas E (2009). Diethylthiophosphate and diethyldithiophosphate
induce genotoxicity in hepatic cell lines when activated by further biotransformation via Cytochrome
P450. Mutat Res. 679(1-2):39–43. doi:10.1016/j. mrgentox.2009.07.009.
Vigfusson NV, Vyse ER, Pernsteiner CA, Dawson RJ (1983). In vivo induction of sister-chromatid exchange
in Umbra limi by the insecticides endrin, chlordane, diazinon and guthion. Mutat Res. 118:61–8.
doi:10.1016/0165-1218(83)90116-7.
Cancer Causes Control. 12(6):509–17. doi:10.1023/A:1011293208949.
Wagner H (2012). Diazinon: Steriodogenesis (human cell line H295R). Unpublished report no. CeeTox study
no. 9141V-100358STER from CeeTox, Inc., Kalamazoo, Michigan, USA. Submitted to WHO by
Ward JM, Reynolds CW (1983). Large granular lymphocyte leukemia. A heterogeneous lymphocytic leukemia
in F344 rats. Am J Pathol. 111(1):1-10.
Weatherholtz WM (1982). Two-generation reproduction study of diazinon (technical) in rats. Unpublished
report no. HLA study no 2132-102 from Hazleton Laboratories America, Inc., Vienna, Virginia, USA.
Wilga MS (2011). Endocrine disruptor screening program: Human recombinant aromatase assay for diazinon.
Unpublished report study no. 9141V-100358AROM from CeeTox, Inc., Kalamazoo, Michigan, USA.
Willoughby JA (2011a). Diazinon: Estrogen receptor transcriptional activation (human cell line (HeLa-9903).
Unpublished report no. CeeTox study no. 9141V-100358ERTA from CeeTox, Inc., Kalamazoo, Michigan,
USA. Submitted to WHO by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Willoughby JA (2011b). Diazinon: Estrogen receptor binding (rat uterine cytosol). Unpublished report no.
CeeTox study no. 9141V-100358ERB from CeeTox, Inc., Kalamazoo, Michigan, USA. Submitted to WHO
by ADAMA Asia Pacific Pte Ltd, Singapore, Singapore.
Willoughby JA (2012). Diazinon: Androgen receptor binding assay using rat prostate cytosol. Unpublished
report no. CeeTox study no. 9141V-100358ARB from CeeTox, Inc., Kalamazoo, Michigan, USA.
Wills JH (1959). Recent studies of organic phosphate poisoning. Fed Proc. 18:1020–5 [cited in Annex 1,
reference 70].

88
Wong PK, Wai CC, Liang E (1989). Comparative study on mutagenicities of organophosphorus insecticides
in Salmonella. Chemosphere. 18:2413–22.
Woodruff RC, Phillips JP, Irwin D (1983). Pesticide-induced complete and partial chromosome loss in screens
with repair-defective females of Drosophila melanogaster. Environ Mutagen. 5(6):835–46.
doi:10.1002/em.2860050608.
Yang RS, Hodgson E, Dautermann WC (1971). Metabolism in vitro of diazinon and diazoxon in rat liver. J
Agric Food Chem. 19:10–3 [cited in Annex 1, reference 70].
Zahm HS, Weisenburger DD, Babbit PA, Saal R, Vaught JB, Cantor KP et al. (1990). A case–control
study of non-Hodgkin’s lymphoma and agricultural factors in Eastern Nebraska. Epidemiology.
1:349–56.
Zahm SH, Weisenburger DD, Saal RC, Vaught JB, Babbitt PA, Blair A (1993). The role of
agricultural pesticide use in the development of non-Hodgkin's lymphoma in women. Arch
Environ Health. 48(5):353-8.
Zak F, Hess R, Luetkemeier H, Sachsse K (1973). 21-Day inhalation study on the rat with technical diazinon.
Unpublished report no. Siss 1679 from Ciba-Geigy, Sisseln, Switzerland [cited in Annex 1, reference 70].
Zwiener RJ, Ginsburg CM (1988). Organophosphate and carbamate poisoning in infants and children.
Pediatrics. 81:121–6 [cited in Annex 1, reference 70].

GLYPHOSATE
P.V. Shah1, Miriam Jacobs2, Carl Cerniglia3, David Eastmond4, Virissa Lenters5, Aldert Piersma6,
Rachel B. Smith7, Mireille B. Toledano8 and Jürg Zarn9
1
Office of Pesticide Programs, Environmental Protection Agency, Washington, DC, United States of
America (USA)
2
Toxicology Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health
England, Chilton, Oxfordshire, England, United Kingdom
3
Division of Microbiology, National Center for Toxicological Research, Food and Drug
Administration, Jefferson, AR, USA
4
Department of Cell Biology & Neuroscience, University of California, Riverside, CA, USA
5
Division of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht University,
Utrecht, the Netherlands
6
Center for Health Protection, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands
7
Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London,
London, England, United Kingdom
8
MRC-PHE Centre for Environment and Health, Department of Epidemiology and Biostatistics,
School of Public Health, Faculty of Medicine, Imperial College London, London, England, United
Kingdom
9
Federal Food Safety and Veterinary Office (FSVO), Risk Assessment Division, Bern, Switzerland
Explanation .............................................................................................................................................90
Evaluation for acceptable intake.............................................................................................................90
1. Biochemical aspects ........................................................................................................................90
1.1 Absorption, distribution and excretion ......................................................................................91
(a) Oral route ............................................................................................................................91
(b) Intraperitoneal route .........................................................................................................102
(c) Intravenous route ..............................................................................................................103
(d) Intramuscular route ...........................................................................................................103
(e) Dermal route .....................................................................................................................103
1.2 Biotransformation ....................................................................................................................105
2. Toxicological studies .....................................................................................................................107
2.1 Acute toxicity .........................................................................................................................107
(a) Oral toxicity ......................................................................................................................111
(b) Acute dermal toxicity .......................................................................................................115
(c) Exposure by inhalation ....................................................................................................118
(d) Dermal irritation ..............................................................................................................121
(e) Ocular irritation ................................................................................................................124
(f) Dermal sensitization .........................................................................................................128
2.2 Short-term studies of toxicity ..................................................................................................133
(a) Oral administration ...........................................................................................................133
(b) Dermal application ...........................................................................................................146
2.3 Long-term studies of toxicity and carcinogenicity .................................................................147
2.4 Genotoxicity ...........................................................................................................................173
(a) In vitro studies ..................................................................................................................174
(b) In vivo studies...................................................................................................................182
(c) Non-traditional tests or tests in phylogenetically distant organisms .................................188
(d) Human biomonitoring studies ..........................................................................................188
(e) Mechanistic considerations ...............................................................................................190
2.5 Reproductive and developmental toxicity ..............................................................................190
(a) Multigeneration studies.....................................................................................................190
(b) Developmental toxicity.....................................................................................................197
2.6 Special studies. .......................................................................................................................205
(a) Neurotoxicity ....................................................................................................................205
MALATHION 297–453 JMPR 2016

90
(b) Immunotoxicity ................................................................................................................206

(c) Effects on the salivary gland .............................................................................................207
(d) Gastrointenstinal tract irritation ........................................................................................210
(e) Endocrine disruption .........................................................................................................210
(f) EDSP studies .....................................................................................................................211
(g) Microbiological effects .....................................................................................................226
2.7 Studies on metabolites: AMPA ..............................................................................................230
2.8 Studies on metabolites: N-Acetyl-glyphosate and N-acetyl-AMPA ......................................233
(a) Biotransfomation of N-acetyl-glyphosate .........................................................................233
(b) Acute toxicity of N-acetyl-glyphosate and N-acetyl-AMPA ............................................234
(c) Subacute toxicity of N-acetyl-glyphosate .........................................................................234
(d) Genotoxicity of N-acetyl-glyphosate and N-acetyl-AMPA ..............................................235
2.9 Studies on other formulation ingredients ...............................................................................235
3. Observations in humans ................................................................................................................244
3.1 Occupational exposure: Biomonitoring studies .......................................................................244
3.2 Occupational exposure: Epidemiological data with specific reference to cancer outcomes....246
Comments……………………………………………………………………………………..……… 252
Toxicological evaluation .....................................................................................................................257
References ............................................................................................................................................260
Appendix 1(a). Results of in vitro genotoxicity with glyphosate in nonmammalian species ...............289
Appendix 1(b). Results of in vivo genotoxicity with glyphosate, Roundup and other formulations in
nonmammalian species .........................................................................................................................290
References to Appendix 1 ....................................................................................................................294
Explanation
Glyphosate is the International Organization for Standardization–approved common name for
N-(phosphonomethyl)glycine (International Union of Pure and Applied Chemistry), with Chemical
Abstracts Service (CAS) number 1071-83-6. It is a broad-spectrum systemic herbicide.
Glyphosate was previously evaluated by the Joint FAO/WHO Meeting on Pesticide Residues
(JMPR) for toxicology in 1986, 1997 (evaluation of the metabolite aminomethylphosphonic acid, or
AMPA), 2004 and 2011 (evaluation of new plant metabolites in genetically modified maize and soya
beans).
Glyphosate was last re-evaluated for toxicology within the periodic review programme of the
Codex Committee on Pesticide Residues (CCPR) in 2004. The compound was reviewed by the
present Meeting following the recommendation of an electronic task force of the World Health
Organization (WHO) Core Assessment Group on Pesticides Residues that it be re-evaluated due to
public health concerns identified by the International Agency for Research on Cancer (IARC) and the
availability of a significant number of new studies.
The current Meeting evaluated all previously considered toxicological data in addition to new
published or unpublished toxicological studies and published epidemiological studies on cancer
outcomes. The evaluation of the biochemical aspects and systemic toxicity of glyphosate was based
on previous JMPR evaluations, updated as necessary with additional information. The particular focus
of the current Meeting was on genotoxicity, carcinogenicity, reproductive and developmental toxicity
and epidemiological studies on cancer outcomes. The scope was restricted to the active ingredient.
All critical unpublished studies contained statements of compliance with good laboratory
practice (GLP), unless otherwise specified. The studies on human volunteers were conducted in
accordance with the principles expressed in the Declaration of Helsinki or equivalent ethical
standards.
GLYPHOSATE 89–296 JMPR 2016

91
The absorption, distribution, metabolism and excretion of glyphosate was studied in rats
following a single oral low dose, a single oral high dose and a single oral low daily dose repeated for
14 days followed by a radioactive dose. In addition, absorption and excretion of glyphosate was
studied via intravenous and intraperitoneal administration in rats and intramuscular administration in
Rhesus monkeys.
Fig. 1 shows the structure of radiolabelled glyphosate
Fig. 1. Structure of glyphosate – 14

C-labelled at the methylene carbon at C1 or C2-glycine carbon
* Denotes position of 14C label.
Fig. 2. Structure of aminomethylphosphonic acid (AMPA)
* Denotes position of 14C label.

(a) Oral route
The excretion and residue levels found by various studies following a single oral dose or
repeated oral administration of glyphosate in rats and rabbits are shown in the Table 1.
Table 1. Total elimination and residues of administered radioactivity after single or repeated oral
administration of 14C-labelled glyphosate
Total tissue and
Total excretion via residual carcass
Dose
urine Total faecal excretion residues
administered /
(%) (%) (%)
No. of doses /
Length of study Species Males Females Males Females Males Females Reference
6.7 mg/kg bw Rat 14–16 35–43 81–85 49–55 0.14–0.65 0.83–1.02 Colvin &
Single dose Millera
(1973a)
120 hours

92
Total tissue and

Dose
administered /
(%) (%) (%)
No. of doses /
10 mg/kg bw Rat 17.9/34.0 12.8/12.5 59.3/60.5 80.3/91.2 ND ND Davies,
Single dose (1996a)
24/48 hours
10 mg/kg bw Rat 13 10.6 88.5 88.7 0.59 0.49 Davies,
Single dose (1996d)
72 hours
10 mg/kg bw Rat 28.6 22.5 62.4 69.4 0.44 0.31 Ridley &
Single dose Mirly,
7 days (1988)
10 mg/kg bw Rat 10.6 10.7 86.6 90.7 0.46 0.41 Daviesb

Repeated dosing (1996c)
72 hours
Repeated dosing Mirlyb
(1988)
7 days
30 mg/kg bw Rat 29.04 30.71 58.84 56.53 0.62 0.64 Powles,
Single dose (1992a)
168 hours
30 mg/kg bw Rat 34.28 34.63 49.64 46.73 0.96 0.83 Powles,
Repeated dosing (1992b)
168 hours
1000 mg/kg bw Rat 16.7 17.5 89.6 84.5 0.52 0.58 Davies
Single dose (1996b)
72 hours
1 000 mg/kg bw Rat 30.55 22.41 53.27 60.37 0.47 0.40 Powles
Single dose (1992b)
168 hours
1 000 mg/kg bw Rat 17.8 14.3 68.9 69.4 0.28 0.24 Ridley &
Single dose Mirly
(1988)
7 days
10 mg/kg bw Rat 22.5 19.4 74.6 84.3 0.33 0.27 McEwenc
Single dose (1995)
168 hours
10 mg/kg bw Rat 30.3 29.5 74.7 74.2 0.31 0.39 McEwenc
Single dose (1995)
168 hours
1 mg/kg bw Rat 18.4 27.2 72.6 62.4 0.8 1.0 Knowles &
Single dose Mookherje
e (1996c)
168 hours
100 mg/kg bw Rat 39.4 43.1 41.2 42.4 0.8 1.0 Knowles &
Single dose Mookherje
e (1996c)
168 hours

93
Total tissue and

Dose
administered /
(%) (%) (%)
No. of doses /
5.7–8.8 mg/kg Rabbit 7–11 ND 80–97 ND 0.1–1.2 ND Colvin &
bw Millera
Single dose (1973c)
120 hours
bw: body weight; ND: not determined; no. number
a
Glyphosate 14C-labelled at the methylene carbon, at the C1-glycine carbon or at the C2-glycine carbon.
b
Groups of male and female rats were given 14 consecutive daily oral doses of 10 mg/kg bw of unlabelled glyphosate
followed by a single oral dose 10 mg/kg bw of [ 14C]glyphosate.
c
Residual activity in carcass only.
The excretion and residue levels found by various studies following single intraperitoneal,
intravenous or intramuscular administration in rats and Rhesus monkeys are shown in Table 2.
Table 2. Residues of administered 14C-labelled glyphosate a after single dose administration

Percentage of administered dose (%)
Total tissue and
Dose / Means of Total excretion via residual carcass
administration / urine Total faecal excretion residues
Length of
observation Species Males Females Males Females Males Females Reference
6.7 mg/kg bw Rat 82–90 ND 6–14 ND <1 ND Colvin &
Intraperitoneal Millera
(1973a)
120 hours
Intravenous Mirly
(1988)
7 days
30 mg/kg bw Rat 85.98 84.18 3.42 1.48 1.35 1.09 Powles
Intravenous (1992b)
168 hours
4 mg Monkey 89.9 ND ND ND ND ND Maibach
Intramuscular (1983)
7 days
bw: body weight; ND: not determined
a
Glyphosate 14C-labelled at the methylene carbon, at the C1-glycine carbon or at the C2-glycine carbon.
Rats
In a pre-GLP study, aqueous solutions of glyphosate 14C-labelled at the methylene carbon, at
the C1-glycine carbon and at the C2-glycine carbon were administered to Wistar rats by gavage. The
radiochemical purity of the labelled materials used were 95% and higher for 14C-methylene
glyphosate, 14C-C1-glycine glyphosate and 14C-C2-glycine glyphosate. For the first series of
experiments, eight male and four female rats were fasted for four hours and then administered, by
gavage, aqueous solutions of [14C]glyphosate at a dose level of 6.7 mg/kg body weight (bw). Two
male rats and one female rat were administered 14C-methylene glyphosate, three male rats and one
female rat were administered 14C-C1-glycine glyphosate, and three male rats and two female rats were
administered 14C-C2-glycine glyphosate. In a second series of experiments, three treatment groups of

94
three male rats each were dosed separately, via intraperitoneal injection, with 14C-methylene
glyphosate (2.33 mg/kg bw), 14C-C1-glycine glyphosate (2.91 mg/kg bw) and 14C-C2-glycine
glyphosate (3.63 mg/kg bw). In a third series of experiments designed to determine the gross
distribution of plant-derived metabolites of glyphosate, aqueous extracts of soybeans grown in
hydroponic solutions of [14C]glyphosate were administered orally to rats. The extracts were obtained
from soybean plants, which had been cultured for 4 weeks in separate hydroponic media containing
the three forms of [14C]glyphosate. Treatment groups composed of three male rats each for each type
of radiolabelled material were dosed separately with the aqueous extracts of the roots of soybeans. A
fourth treatment group of three male rats was also dosed with the aqueous extract of the aerial portion
of soybean plants grown in hydroponic media containing 14C-methylene glyphosate.
Approximately 94–98% of the [14C]glyphosate orally administered to male rats was excreted
in urine and faeces within 48 hours of administration. Approximately 15% of the dose was excreted in
the urine within 120 hours of administration, with most of the remainder excreted in the faeces (81%–
85%). Of the [14C]glyphosate absorbed through the gut, only very small amounts were catabolized.
The percentage of administered radioactivity recovered as expired 14CO2 was 0.5%. Tissue retention
120 hours post-administration was less than 1% of the dose for the three 14C-labelled forms of
glyphosate.
The percentage of radioactivity excreted by female rats after oral administration of
[14C]glyphosate was 82–84% at 48 hours and 91–93% 120 hours. Between 34% and 40% of the
administered radioactivity was excreted in the urine within 120 hours, with most of the remainder
excreted in the faeces (49–55%). The levels of exhaled 14CO2 were also slightly higher for female than
male rats, as were carcass retentions. For female rats, the percentage of administered radioactivity
recovered as expired 14CO2 was 0.72%. Tissue retention at 120 hours was approximately 1% for the
three 14C-labelled forms of glyphosate. For both sexes, the order of retention of radioactivity in tissues
120 days post-administration was similar, although the female tissues contained higher
concentrations. The highest concentrations of radioactivity were found in the liver, kidney and gut,
but in all cases these were 0.20 parts per million (ppm) or less on a fresh-weight basis.
About 74–78% of the dose of [14C]glyphosate administered to male rats via intraperitoneal
injection was excreted in the urine within 12 hours. At 96 hours post-administration, total urinary
excretion ranged from 81–90% of the administered dose. Faecal excretion ranged from 6–14% of the
administered radioactivity within 96 hours and strongly suggests that [14C]glyphosate is also
eliminated via the bile. The percentage of radioactivity recovered as expired 14CO2 was slightly
greater than that following oral administration, but for all three radiolabels was less than 1% of the
administered dose. Tissue retention was also greater in female than in male rats after oral
administration, but in all cases was 1% or less of the administered dose.
When extracts of soybeans grown in hydroponic solutions of [ 14C]glyphosate were orally
administered to male rats, 96–99% of the administered radioactivity was excreted in the faeces and
urine within 120 hours. The exception were the rats dosed with extracts of soybean roots from plants
treated with 14C-C2-glycine glyphosate, for which only 76% of the administered dose was found in
the excreta. The relatively high tissue retention (5.19% and 1.86% of the administered dose) and
14
CO2 expiration (3.67% and 3.49% of the administered dose) by rats administered extracts of roots
from plants treated with 14C-C2-glycine glyphosate and the extracts of the aerial portion of plants
treated with [14C]methylene glyphosate was attributed to the metabolism of natural plant products
since the radioactivity in these extracts was due to 30% and 10% natural products, respectively
(Colvin & Miller, 1973a).
In a pre-GLP study, the accumulation and depletion of glyphosate was investigated by the
daily administration of feed containing 0, 1, 10 and 100 ppm of [14C]glyphosate to Wistar rats (15/sex
per dose) for 14 days, followed by a 10-day depuration period on a control ration. Tissue residues
were measured after 2, 6, 10 and 14 days on dosed feed and 1, 3, 6 and 10 days after withdrawal from
the dosed feed. The excretion of ingested [14C]glyphosate in faeces and urine were determined daily.

95
Body and organ weights indicated that the continuous administration of feed containing 1,
10 and 100 ppm of glyphosate for 14 days had no detrimental effect on the growth or relative organ
size of rats. Of the [14C]glyphosate ingested, 8.3–10.5% of the daily intake was excreted in the urine.
The combined urinary and faecal excretion of radioactivity was approximately equal to the total
intake of [14C]glyphosate after 6 days, indicating that a plateau had been reached. By day 4 of dosing,
radioactivity in the urine plus faeces exceeded 90% of the cumulative intake, and by the end of the 14-
day dosing period the combined excretion of radioactivity was 96, 115 and 93% of the cumulative
intake of the 1, 10 and 100 ppm dosing levels, respectively. Since the amount of radioactivity excreted
was directly proportional to the intake, the elimination kinetic of [ 14C]glyphosate could be described
as a first-order process, precluding the potential of unlimited accumulation. Most tissues reached
maximum [14C]glyphosate residue levels during the dosing period in 10 days or less. There was a
modest cumulative effect in the body as a result of chronic [14C]glyphosate administration, but the
effect was not localized in a single tissue type or organ system. The order of decreasing tissue
propensity for [14C]glyphosate, on a fresh-weight basis, was kidney, spleen, fat, liver, ovaries, heart,
muscle, brain and testes. On a dry-weight base the order was spleen, kidney, ovaries, heart, liver,
testes, fat, brain and muscle. Accumulation of [14C]glyphosate in muscle tissue was very low on either
a fresh- or dry-weight basis, indicating a very low propensity for accumulation. The residues in the
tissues were reversibly bound and began to deplete as soon as the dosed feed was withdrawn (Colvin
& Miller, 1973b).
Seven different test groups of Sprague Dawley (Crl:CD[SD]BR) rats, each with an equal
number of males and females, were dosed with [14C]glyphosate labelled in the methylene position
between the nitrogen and phosphorous atoms (radiochemical purity  98%). Single oral doses (10 and
1000 mg/kg bw) were administered by gastric intubation, and intravenous doses (10 mg/kg bw) were
injected into the lateral tail vein. Another group of five male and five female rats was treated with
unlabelled glyphosate as 14 consecutive oral doses at 10 mg/kg bw per day followed by 14C-labelled
glyphosate as a single oral dose at 10 mg/kg bw. Blood, urine and faeces were sampled at various
time points. At the end the study, the animals were terminated and different tissues as well as the
carcass analysed for radioactivity.
The distribution of radioactivity in the excreta and the tissue samples are summarized in Table 3.
Table 3. Recovery of radioactivity as a percentage of the administered 14C-labelled glyphosate dose

Per cent of administered radioactive dose (%)
Single IV dose Single oral dose Repeated oral dose Single oral dose
10 mg/kg bw 10 mg/kg bw 10 mg/kg bw 1 000 mg/kg bw
Excreta/tissue Males Females Males Females Males Females Males Females
Urine 79.0 74.5 28.6 22.5 30.9 23.1 17.8 14.3
Faeces 4.65 8.30 62.4 69.4 61.0 70.9 68.9 69.4
Organs/tissues 0.09 0.05 0.05 0.02 0.05 0.03 0.04 0.03
Residual carcass 1.18 1.04 0.40 0.29 0.50 0.32 0.25 0.21
Gastrointestinal 0.04 0.04 0.02 0.01 0.01 0.01 0.03 0.04
contents
Cage wash 0.89 1.30 1.30 1.96 0.82 1.96 3.86 8.00
a
Total recovery 86.0 85.3 92.8 94.2 93.3 96.3 90.9 92.1
bw: body weight; IV: intravenous
a
Total recovery is the mean of individual animal data.
Source: Ridley & Mirly (1988)

96
The major route of elimination of an oral dose of 14C-labelled glyphosate at 10 mg/kg body
bw was faeces. After a 7-day elimination period, the faeces contained 62.4% and 69.4% of the
administered dose for males and females, respectively. The majority of the remaining radioactivity,
28.6% of the dose for the males and 22.5% of the dose for females, was excreted in the urine. More of
the administered dose remained in the organs, tissues and residual carcasses of the males than of the
females, although the overall amount of retained radioactivity was very low (< 0.5% of the
administered dose). The tissue with the highest concentrations of radioactivity was bone, with
0.552 ppm and 0.313 ppm found for the males and females, respectively.
For the test group orally dosed at 1000 mg/kg bw, 68.9% and 69.4% of the administered dose
was excreted in the faeces and 17.8% and 14.3% was excreted in the urine of the male and female
rats, respectively. Very low levels (< 0.4%) of the administered dose remained in the gastrointestinal
contents, residual carcasses, organs and tissues 7 days after dosing. The tissues showing more than
1.0 ppm of radioactivity were the liver, kidney, spleen, lung, stomach, small intestines, bone and
residual carcass. Bone retained the greatest amount of radioactivity, 30.6 ppm and 19.7 ppm for the
males and females, respectively.
For the test group treated with 14 daily doses of non-labelled glyphosate at 10 mg/kg prior to
receiving a single oral dose of labelled glyphosate at 10 mg/kg bw, males excreted 61.0% and 30.9%
and females 70.9% and 23.1% of the dose in the faeces and urine, respectively. Very low levels
(< 0.7%) of the administered dose remained in the gastrointestinal contents, residual carcasses, organs
and tissues 7 days after dosing. Again, bone was the tissue with the highest concentration of
radioactivity, containing 0.748 and 0.462 ppm glyphosate equivalents for male and female rats,
respectively.
The half-lives of the α and β elimination phases were 5.9–6.2 hours and 79–106 hours,
respectively, following a single oral dose of 10 mg/kg bw. In the 1000 mg/kg bw dosed group, the α
phase was comparable to 10 mg/kg bw group, but the β phase was found to be 181–337 hours.
Comparison of the area under the curves of plots of radioactivity levels in the blood versus time for
the two groups indicated that the orally administered glyphosate was 30–35% absorbed. These values
are in good agreement with the absorption values of 30–36% found by dividing the per cent urinary
excretion of administered radioactivity for the group dosed orally at 10 mg/kg bw by the per cent
urinary excretion of administered radioactivity from the group dosed intravenously at 10 mg/kg bw.
The results of this study demonstrate that glyphosate is poorly absorbed and rapidly eliminated after a
single oral dose at 10 or 1000 mg/kg bw (Ridley & Mirly, 1988).
In a preliminary study of absorption and distribution, male Sprague Dawley rats were
administered [14C]phosphonomethyl-labelled glyphosate (purity of unlabelled test material = 98.6%;
radiochemical purity = 94.3–97.4%) as a single oral dose at 30 mg/kg bw in 0.9% saline by gavage.
Blood samples were taken from the tail vein of three animals periodically between 0.5 and 48 hours
after dosing. Additional animals were terminated 4, 10 and 24 hours after dosing, and the tissue
distribution of radioactivity was investigated by whole-body autoradiography.
Low levels of radioactivity were detected in plasma. Maximum plasma concentrations (Cmax)
reached within 4 hours were 1.769, 1.137 and 0.705 µg eq/mL. Thereafter, plasma levels decayed
exponentially to non-detectable levels 12 hours post dose. The elimination half-lives were 6.196 hours
and 12.35 hours for two animals. A value could not be obtained for the third animal. The
concentration of radioactivity was highest after 10 hours, with the highest concentrations in bone,
bone marrow, cartilage, parts of the gastrointestinal tract, kidney, urinary tract and nasal mucosa. The
highest concentrations within bone were associated with the epiphyses. Lower concentrations were
found in a number of other tissues. Twenty-four hours after dosing, tissue concentrations of
radioactivity were negligible in all tissues except bone, bone marrow, parts of the gastrointestinal
tract, bladder and kidney cortex (Powles, 1992a).

97
In a study of absorption, distribution and excretion, groups of five male and five female
Sprague Dawley rats were administered [14C]phosphonomethyl-labelled glyphosate (purity of
unlabelled test material = 96.8%; radiochemical purity > 98%) as a single dose of 30 or 1000 mg/kg
bw by gavage in saline or intravenously as a single dose at 30 mg/kg bw. A group of five male and
five female rats was administered unlabelled glyphosate as 14 consecutive oral doses at 30 mg/kg bw
per day followed by [14C]glyphosate as a single oral dose at 30 mg/kg bw. The animals were housed
individually in metabolism cages from which urine, faeces and expired air were collected at regular
intervals. The rats were terminated after 90% of the dose had been eliminated or 7 days after dosing,
whichever was sooner. At necropsy, a blood sample was drawn and selected tissues removed.
Following administration of the single intravenous dose of 30 mg/kg, more than 84% of the
radioactivity was eliminated in urine, mostly within 8 hours. Faecal elimination accounted for less
than 3.5% of the administered radioactivity and only a very small proportion was eliminated in
exhaled air; less than 1.4% remained in tissues and the residual carcass after termination. In contrast,
faeces were the major route of elimination when [14C]glyphosate was administered orally.
Approximately 56–59% of the oral dose of 30 mg/kg was excreted in faeces, mostly within 12–36
hours. Urinary elimination of the oral dose was slower than for the intravenous dose, with 29–31%
eliminated, mostly within 36 hours of dosing. Excretion was unaffected by administering unlabelled
glyphosate for 14 days prior to dosing with [14C]glyphosate, and the routes and rates of excretion of a
high dose of [14C]glyphosate (1000 mg/kg) were essentially identical to that of the low dose. There
was no significant sex difference in the elimination of glyphosate for any dose regimen. Irrespective
of the dose, route or frequency of duration, less than 1.4% of a dose was retained in tissues. The
highest concentration of radioactivity was in bone and lower concentrations were in bone marrow,
kidneys, liver, lungs and the residual carcass (Powles, 1992b).
In a study of absorption, distribution, excretion and metabolism, groups of five male and five
female Sprague Dawley rats were administered [14C]phosphonomethyl-labelled glyphosate (purity of
unlabelled test material 98.9%; radiochemical purity > 98%) as a single dose at 10 or 600 mg/kg bw
by gavage in water. For the excretion study, urine and faeces (5/sex) were collected at selected
intervals for 168 hours. Animals were terminated at 168 hours post dosing and the radioactivity in
blood and selected tissues analysed. For the plasma concentration study, blood samples (total nine per
sex per dose) were drawn at selected intervals up to 168 hours. For the tissue distribution study, 12
rats (six male, six female) were administered single oral doses of either 10 or 600 mg/kg bw per day
by gavage. The animals were divided into two groups of six (three per sex) and terminated by cervical
dislocation 6 and 18 hours (the low-dose study) or 3 and 9 hours (for the high dose) after dosing,
depending on the peak plasma concentrations and half the plasma concentration derived in the
blood/plasma kinetics experiments. Samples of urine and faecal extracts from male and female rats
were pooled and analysed directly by thin-layer chromatography (TLC) or high-performance liquid
chromatography (HPLC).
During the 7-day observation period, up to about 23% and 30% of the radioactivity of the low
dose was excreted in the urine of low- and high-dose animals, respectively. At both doses, about three
quarters of the radioactivity was detected in the faeces within 7 days (75% for males and 84% for
females, 10 mg/kg bw; 75% and 74%, 600 mg/kg bw; Table 4) (McEwen, 1995).
14
Table 4. Radioactivity in rat excreta and tissue over 168 hours after a single dose of C-labelled
glyphosate
Percentage of administered radioactive dose (%)
10 mg/kg bw 600 mg/kg bw
Excretion intervals (h) Males Females Males Females
Urine
0–6 2.63 3.25 11.55 9.08

98
Percentage of administered radioactive dose (%)

Excretion intervals (h) Males Females Males Females
6–24 15.85 12.69 13.85 13.36
24–48 2.82 2.41 2.33 4.40
48–72 0.54 0.44 0.59 1.07
72–96 0.24 0.19 0.30 0.40
96–120 0.15 0.13 0.21 0.24
120–144 0.09 0.07 0.17 0.17
144–168 0.07 0.05 0.13 0.18
Cage wash 0.12 0.14 1.13 0.60
Subtotal (urine plus cage wash) 22.51 19.37 30.26 29.50
Faeces
0–24 60.28 74.59 58.94 46.28
24–48 11.72 7.56 13.41 22.87
48–72 1.18 1.34 1.36 3.83
72–96 0.29 0.36 0.35 0.47
96–120 0.17 0.27 0.36 0.23
120–144 0.35 0.08 0.08 0.12
144–168 0.64 0.10 0.15 0.35
Subtotal faeces 74.63 84.30 74.65 74.15
Residual carcass 0.33 0.27 0.31 0.39
Total 97.47 103.94 105.22 104.04
bw: body weight
Source: McEwen (1995)
After a single dose of 10 mg/kg bw, peak mean concentrations of radioactivity in plasma
occurred at 6 and 2 hours in males (0.22 µg eq/mL) and females (0.28 µg eq/mL) (Table 5). After a
single oral dose of 600 mg/kg bw, peak mean concentrations of radioactivity in plasma occurred at
3 hours in both males (26 µg eq/mL) and females (29 µg eq/mL). The area under the concentration
versus time–curve (AUCt) was calculated at 400 and 355 μg eq/mL*hour in males and females,
respectively. These values were around 120 times higher than the AUCt obtained in the low-dose
group.
Table 5. Pharmacokinetic parameters of total rat plasma radioactivity following single oral doses of
14
C-labelled glyphosate
Measures per administered dose
Parameter Males Females Males Females
Cmax (µg eq/mL) 0.2219 0.2789 25.97 28.84
Tmax (hour) 6.00 2.00 3.00 3.00
AUCt (µg eq/mL*hour) 3.20 3.70 399.90 355.30
AUC (µg eq/mL*hour) 3.80 4.20 419.00 –a

99
Terminal rate constant (per hour) 0.0840 0.0887 0.1174 –a

Terminal half-life (hour) 8.30 7.80 5.90 –a
Absorption rate constant (per hour) 0.2963 0.4239 0.2845 0.4477
AUC: area under the plasma concentration–time curve; AUCt: area under the curve calculated up to the last detectable
sample (calculations done up to 24 hours); bw: body weight; Cmax: maximum concentration; eq: equivalent; Tmax: time to
reach the maximum concentration
a
Could not be calculated accurately as the values were at or close to the limit of reliable measurement.
There was no indication of accumulation of radioactivity in any tissue. Only the

gastrointestinal tract, the stomach, muscles and the kidneys, the organs of excretion contained
concentrations of radioactivity higher than the plasma (Table 6). High levels of radioactivity were
detected in the content of stomach and the gastrointestinal tract. The radioactivity in most tissues had
decreased to around the limit of detection 7 days after dosing.
Table 6. Radioactivity in male and female rat tissue over 168 hours after a single oral dose of 10
mg/kg bw 14C-labelled glyphosate
Proportion of administered dose over time (%)
Malea Femalesa
Tissue 6 hours 18 hours 168 hours 6 hours 18 hours 168 hours
b
Bone 0.12 0.10 0.02 0.10 0.09 0.03
Carcass 2.00 2.69 0.33 1.69 3.03 0.27
Gastrointestinal tract 19.05 10.04 0.01 16.47 5.41 0.01
Gastrointestinal tract contents 31.56 4.89 0.01 34.54 14.30 0.01
Kidneys 0.79 0.36 < 0.01 0.67 0.26 < 0.01
Muscle (skeletal) 0.23 0.13 0.04 0.24 0.11 < 0.03
Stomach 3.47 0.60 0.60 2.56 0.62 < 0.01
Stomach contents 25.16 5.05 0.01 22.90 6.96 0.01
Plasma 0.12 0.03 < 0.01 0.13 0.03 < 0.01
Whole blood 0.20 0.04 < 0.03 0.15 0.05 < 0.03
bw: body weight
Results expressed as mean percentage (%) of applied dose, except bone, which is expressed as percentage (%) of applied
dose/g.
a
N=5
b
n=3
A major component of urine or the [14C]phosphonomethyl-labelled glyphosate-treated

animals was unchanged glyphosate, accounting for 18–27% of both the administered doses. A minor
component, accounting for 0.1–0.3% of the administered dose, was shown to co-chromatograph
(using normal phase TLC and reverse phase HPLC) with aminomethylphosphonic acid.
Unchanged glyphosate was the major component of the faecal extract of the
[14C]phosphonomethyl-labelled glyphosate-treated animals, accounting for 65–78% of both the
administered doses. Two minor metabolites accounted for 0.3–1.6% of the administered dose; one of
these was shown to co-chromatograph with aminomethylphosphonic acid (McEwen, 1995).

100
In a series of experiments that compared the faecal and urinary excretion of 14C-labelled
glyphosate, five male and five female Alpk:APfSD rats were each given a single oral dose of 10
mg/kg bw and 1000 mg [14C]phosphonomethyl-labelled glyphosate (radiochemical purity > 98%) in
deionized water. Excretion was measured over 72 hours, after which the animals were terminated and
the radioactivity in blood and selected tissues including residual carcasses analysed.
Excretion of radioactivity was rapid for both sexes and most of the administered dose was
eliminated, principally in faeces, within 24 hours. Males excreted 13.0% and 88.5% of the lower dose
and 16.7% and 89.6% of the higher dose in urine and faeces, respectively. Females excreted 10.6%
and 88.7% of the lower dose and 17.5% and 84.5% of the higher dose in urine and faeces,
respectively.
At termination, radioactivity in the tissues accounted for only 0.6% and 0.5% of the lower
dose in males and females, respectfully. The highest concentrations were in bone (0.5 and 0.4 µg eq/g
of the lower dose and 50 and 45 µg eq/g of the higher dose for males and females, respectively). All
other tissue concentrations were 0.07 µg/g or less for the lower dose and 7 µg eq/g or less for the
higher dose. No marked sex difference was seen in the tissue distribution of radioactivity (Davies,
1996a,b).
A similar experiment was conducted using five male and five female Alpk:AP fSD rats pre-
treated with 10 mg/kg bw of unlabelled glyphosate (purity 99.2%) for 14 days before being given the
single oral dose of 10 mg/kg bw of [14C]phosphonomethyl-labelled glyphosate (radiochemical purity
> 98%) in deionized water. Once again, excretion was measured over 72 hours. The animals were
then terminated and the radioactivity in blood and selected tissues including residual carcass analysed.
Excretion of radioactivity was rapid in both sexes and most of the administered dose was
eliminated, principally in faeces, within 24 hours. Males excreted 10.6% and 86.6% and females
10.7% and 90.7% of the administered dose in urine and faeces, respectively.
At termination, tissue concentrations of radioactivity accounted for 0.5% of the administered
dose in both sexes. The amount in the tissue and contents of the intestinal tract were 0.12% of the
administered dose in both sexes. The highest concentrations were in bone (0.36 and 0.35 µg eq/g in
males and females, respectively). All other tissue concentrations were 0.07 µg eq/g or lower. No
marked sex difference was seen in the distribution of radioactivity in the tissues. Comparison of these
results with those obtained when [14C]glyphosate was administered without pretreatment shows that
pre-dosing has no significant effect on either the routes or rates of elimination of a single dose of the
radiolabelled test material (Davies, 1996c).
Two male and two female Alpk:APfSD rats were each given a single oral dose of 10 mg/kg
bw [14C]phosphonomethyl-labelled glyphosate (radiochemical purity > 96%) in deionized water.
Excretion was measured throughout the study. At intervals of 24 and 48 hours after dosing, one rat of
each sex was terminated and rapidly frozen for whole-body autoradiography.
Within 24 hours of dosing, male rats excreted 22.3% and 55.5% and female rats 11.9% and
83.8% of the administered dose in the urine and faeces, respectively. Within 48 hours of dosing, the
remaining male rats excreted 34.0% and 60.5% and the female rats 12.5% and 91.2% of the
administered dose in the urine and faeces, respectively.
The whole-body autoradiography showed no marked differences in the distribution of
radioactivity between male and female rats. The high levels of radioactivity in the gastrointestinal
tract were consistent with faeces being the predominant route of elimination; accordingly, these levels
had declined markedly by 48 hours. The greatest intensity of tissue radiolabelling at both 24 and 48
hours was in bone. Some radioactivity was in the kidney after 24 hours but had declined by 48 hours.
No significant levels of radioactivity were apparent in other tissues (Davies, 1996d).

101
In a study of absorption, distribution, excretion and metabolism, groups of five male and five
female Sprague Dawley (Crl:CD BR) rats were administered [ 14C]phosphonomethyl-labelled
glyphosate (two batches of unlabelled test material, purity 95.3% and 96.0%; radiochemical purity
> 99%) as a single gavage dose of 1 or 100 mg/kg bw in water. For the excretion study, urine and
faeces (5/sex per dose) were collected at selected times for 168 hours and samples pooled and
analysed directly by TLC or HPLC. At 168 hours, the animals were terminated and radioactivity in
blood and selected tissues analysed. For the pharmacokinetic study, blood was drawn (5/sex per dose)
at selected intervals up to 72 hours after dosing. For the tissue distribution study, 12 male and 12
female rats were administered a single daily gavage dose of either 10 or 100 mg/kg bw. The treated
animals were divided into four groups (three per sex) and terminated at 4, 12, 24 and 72 hours after
dosing. For the biliary excretion study, seven male and seven female cannulated rats were
administered a single gavage dose of 1 mg/kg bw. Urine, faeces and bile were collected periodically
up to 48 hours after dosing.
Following a single gavage dose of 1 mg/kg bw, the major route of elimination was the faeces
with 72.62% recovered in males and 62.40% in females, mostly within 24 hours of dosing (63.93% in
males and 49.69% in females), suggesting this proportion of the dose was not systemically absorbed.
During the 7-day observation period, 18.44% (male) and 27.15% (female) of radioactivity were
recovered in the urine, representing the systemically absorbed dose. The remainder of the
radioactivity was recovered in the cage wash (6.48% in males and 7.71% in females), cage debris
(0.03% in males and 0.58% in females) and carcass (0.75% in males and 0.98% in females).
Following the single gavage dose of 100 mg/kg bw, elimination of radioactivity in the urine
(39.42% in males and 43.07% in females) was quantitatively more significant than in to the low-dose
group. Faecal elimination accounted for 41.23% in males and 42.37% in females. The remainder of
the radioactivity was recovered in the cage wash (13.85% in males and 11.96% in females), cage
debris (0.98% in male and 0.10% in female) and carcass (0.84% in male and 0.98% in female). Renal
elimination was essentially complete in 48 hours.
In the cannulated rats dosed with 1 mg/kg bw by gavage, the majority of the administered
dose was recovered in faeces (55.33% in male and 60.97% in female) in 48 hours. Renal elimination
accounted for 27.45% in males and 24.21% in females. The remainder of the radioactivity was
recovered in the cage wash (6.57% in male and 6.77% in female), cage debris (0.26% in male and
0.15% in female) and carcass (4.99% in male and 3.82% in female).
The mean terminal elimination half-lives were 10.86 hours and 8.07 hours with corresponding
area under the plasma concentration–time curve (AUC) of 0.319 and 0.340 µg eq/mL*hour in males
and females, respectively (Table 7). As the elimination half-lives could not be calculated for several
high-dose animals, mean AUC0–24 (0.257 and 0.338 µg eq/mL*hour in males and females) were
calculated to compare the results of both groups. Following a single oral dose of 100 mg/kg bw, mean
AUC0–24 were 58.2 and 50.7 μg eq/mL*hour in males and females, respectively.
Table 7. Kinetic parameters in male and female rat plasma after a single oral dose of 14C-labelled
glyphosate
Measures per administered dose
Kinetic parameters Males Females Males Females
Cmax (µg eq/mL) 0.016 0.037 8.909 7.634
Tmax (hour) 3.900 8.000 3.600 4.000
AUC0–24 (µg eq/mL*hour) 0.257 0.338 58.200 50.700
AUC (µg eq/mL*hour) 0.319 0.340 – –

102
Terminal half-life (hour) 10.860 8.065 – –

AUC: area under the plasma concentration–time curve; AUC0–24: area under the plasma concentration–time curve from time
0 to 24 hours; bw: body weight; Cmax: maximum concentration; eq: equivalent; Tmax: time to reach the maximum
concentration
Source: Knowles & Mookherjee (1996)
At 1 mg/kg bw, radioactivity was detected in all tissues 4 hours post dose, indicating rapid
absorption and distribution in the body. Apart from the gastrointestinal tract (and contents) and the
carcass, the kidneys was the only tissue with any notable amounts throughout the observation period.
At 72 hours, post-dose concentrations had decreased or plateaued to less than 2% of the administered
dose in all tissues in both males and females, with the carcass containing most of the remaining
radioactivity. At 100 mg/kg bw, all the tissues were exposed to radiolabelled material 4 hours post
dose. Again, only the gastrointestinal tract, carcass and kidneys contained significant amounts of
radioactivity. After 72 hours, concentrations had decreased or plateaued to less than 2% of the dose in
all tissues in both sex, with the carcass containing most of the remaining radioactivity.
In conclusion, following oral administration of glyphosate at 1 mg/kg bw and 100 mg/kg bw,
the absorption, distribution, metabolism and excretion was independent of dose level and sex.
Metabolism of glyphosate was very low with more than 90% of the administered dose eliminated
unchanged in the urine and faeces. Elimination was essentially completed by 48 hours, and the
majority of the radioactivity was recovered in faeces (Knowles & Mookherjee, 1996).
Rabbits
In a pre-GLP study, glyphosate 14C-labelled at the methylene carbon, at the C1-glycine
carbon and at the C2-glycine carbon was dissolved in isotonic saline and administered by gavage to
male New Zealand White rabbits fasted for 3 hours. In two replicate experiments, three rabbits were
administered 14C-methylene glyphosate, two were administered 14C-C1-glycine glyphosate and two
were administered 14C-C2-glycine glyphosate. All the doses were within a range of 5.7–8.8 mg/kg bw.
Approximately, 80–97% of the oral dose of [14C]glyphosate was excreted in the faeces and 7–
11% in the urine over 120 hours. Less than 1% of the dose was exhaled. Approximately 1.2%, 0.7%
and 0.1% of the dose was retained in the tissues (excluding gastrointestinal tract contents) for 14C-C2-
glycine, 14C-C1-glycine and 14C-methylene glyphosate, respectively. The radioactivity in the tissues
differed between 14C-C2-glycine and 14C-C1-glycine by 4 or 5 times, but the ranking was similar: the
liver had the highest concentrations followed by the kidney, the spleen, the heart, skeletal muscle and
gonads, in that order. Only 14C-C2-glycine radioactivity was incorporated in the fat (Colvin & Miller,
1973c).
(b) Intraperitoneal route

In the previously described Colvin & Miller (1973a) study, three treatment groups each with
three male Wistar rats were dosed via intraperitoneal injection with 14C-methylene glyphosate (2.33
mg/kg bw), 14C-C1-glycine glyphosate (2.91 mg/kg bw) and 14C-C2-glycine glyphosate (3.63 mg/kg
bw). Within 12 hours, 74–78% of the 14C-glyphosate was excreted in the urine. At 96 hours post-
administration, total urinary excretion was 81–90% of the administered radioactivity and faecal
excretion was 6–14% of the administered radioactivity, indicating that [14C]glyphosate is also
eliminated via the bile. The percentage of radioactivity recovered as expired 14CO2 was slightly
greater than that observed following oral administration (Section 1.1 (a)), but for all three radiolabels
was less than 1% of the administered dose. Tissue retention was also greater than after oral
administration, but was in all cases less than or equal or 1% of the administered dose (Colvin &
Miller, 1973a).

103
[14C]glyphosate with a radiochemical purity of 98% was administered by intraperitoneal

injection to nine male and nine female Sprague Dawley rats at a dose level of 1150 mg/kg bw. The
rats were subsequently housed in metabolism cages, and blood samples were collected from three to
six rats at approximately 0.25, 0.50, 1, 2, 4, 6 and 10 hours. At approximately 0.5, 4 and 10 hours
after dosing, three animals of each sex were terminated and the femoral bone marrow isolated. The
plasma and bone marrow samples were analysed for radioactivity by liquid scintillation counting.
Peak levels of radioactivity were observed in plasma and bone marrow about 0.5 hours after
dosing. When expressed as glyphosate acid equivalents, the peak values for bone marrow and plasma
in males and females combined were approximately 340 and 1940 ppm, respectively. The
radioactivity in plasma decreased rapidly but remained more constant in bone marrow over the 10
hours of the experiment. The analysis of the first-order elimination rates indicated a half-life of
elimination from the plasma of approximately 1 hour for both males and females. Elimination from
bone marrow was slower with a half-life of 4.2 hours for females and 7.6 hours for males (Ridley,
1983).
(c) Intravenous route

In a previously described study by Ridley & Mirly (1988) (Section 1.1 (a)), groups of male
and female Sprague Dawley rats (Crl:CD(SD)BR) were injected with a single intravenous dose of 10
mg/kg bw [14C]glyphosate into the lateral tail vein. Urine and faeces were collected at intervals for 7
days, and the animals were terminated and tissues and carcass analysed for radioactivity.
The majority of the dose – 79.0% in males and 74.5% in females – was excreted in the urine.
Faecal excretion was 4.65% and 8.30% of the administered dose, respectively, which suggests that
glyphosate is eliminated via the bile. Very little (< 0.1%) of the administered dose was found in the
tissues and organs. An intravenous dose resulted in significantly higher levels of radioactivity in the
residual carcasses than those found following oral dosing, with the highest concentrations in bone:
1.48 ppm glyphosate equivalents in males and 1.59 ppm glyphosate equivalents in females (Ridley &
Mirly, 1988).
In the previously described absorption, distribution and excretion study by Powles (1992b)
(section 1.1(a)), groups of five male and five female Sprague Dawley rats were administered
[14C]phosphonomethyl-labelled glyphosate (purity of unlabelled test material, 96.8%; radiochemical
purity > 98%) as a single dose of 30 or 1000 mg/kg bw by gavage in saline or intravenously as a
single dose at 30 mg/kg bw.
Following administration of the 30 mg/kg bw intravenous dose, more than 84% of the
radioactivity was eliminated in urine, mostly within 8 hours. Faecal elimination accounted for less
than 3.5% of the administered radioactivity. Only a very small proportion of the radioactivity was
eliminated in exhaled air and less than 1.4% remained in the tissues and the residual carcass after the
animals were terminated. In contrast, faeces were the major route of elimination when [14C]glyphosate
was given orally (Powles, 1992b).
(d) Intramuscular route

In a two-phase excretion study, [14C]glyphosate was mixed with isopropylamine and
unlabelled glyphosate isopropylamine salt and dissolved in water to make a solution of 4 mg
glyphosate/mL. One millilitre of this solution was injected into the thigh muscle of each of four male
Rhesus monkeys. Urine samples were collected at intervals for up to 7 days.
During the 7-day collection period following intramuscular injection, 89.9% of the applied
radioactivity was excreted in urine. The overall urinary elimination half-life was 19.7 hours. There
were two distinct phases to the elimination kinetics, a rapid phase with a half-life of 6.9 hours (over
the first 24 hours) and a slow phase with a half-life of 35.1 hours (Maibach, 1983).

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(e) Dermal route

In vitro
The absorption of glyphosate acid (purity 95.93%) from a dried glyphosate wet cake
preparation through abraded rabbit whole skin was measured in vitro over 24 hours. The dose was
placed on the abraded skin at a nominal rate of 79.8 mg/cm2 (48.3 mg glyphosate acid/cm2),
calculated as equivalent to the 5000 mg/kg bw per day dose administered to rabbits in an in vivo
dermal toxicity study (Johnson, 1982). The diffusion cell was left unoccluded for 6 hours, and the
surface of the skin was then decontaminated with a sponge wash. Physiological saline was used as the
receptor fluid.
The total recovery of the individual cells was 87.3–98.2%, with an overall mean recovery of
93.3% of applied dose. The majority of the applied glyphosate acid (mean 87.9%) was washed off the
skin at 6 hours, with a further 2.38% washed off at 24 hours. A small proportion (0.041%) of the dose
applied was recovered from the epidermis, with 0.243% remaining in the dermis. The mean amount of
glyphosate acid that penetrated abraded rabbit skin into the receptor fluid over the entire 24-hour
experimental period was 1177 μg/cm2, corresponding to 2.42% of the applied dose. The reported total
potentially absorbable amount, represented by the mean absorbed dose together with the mean amount
in the remaining dermis, was 2.66%. The results of this in vitro study indicated that dermal absorption
of glyphosate through abraded rabbit skin is slow (Hadfield, 2012a).
The penetration through human epidermis of glyphosate from a formulation concentrate was
measured in vitro over 24 hours. The glyphosate formulation concentrate, containing a nominal
360 g/L of an isopropylamine salt of glyphosate at a 1:133 weight per volume (w/v) aqueous dilution
was applied to the epidermal membranes at a rate of 10 μL/cm2 and left unoccluded for 8 hours.
Penetration of glyphosate was fastest between 0 and 2 hours after application (0.914 μg/cm²
per hour). The mean penetration rate slowed to 0.074 μg/cm² per hour between 2 and 24 hours. The
mean amount penetrated over the entire 24-hour exposure period was 3.51 μg/cm2, corresponding to
0.096% of the applied dose (Hadfield, 2012b).
The absorption and distribution of glyphosate from a 360 g/L soluble (liquid) concentrate
(MON 79545) through human epidermis was measured in vitro. The doses were applied as the
concentrate formulation (450 g/L of glyphosate) and as 1:15.6 volume per volume (v/v) and 1:188 v/v
(nominally 28.8 and 2.4 g/L of glyphosate) aqueous spray dilutions of the formulation. 14C-
radiolabelled glyphosate was incorporated into the concentrate formulation and dilutions prior to
application. The doses were applied to the epidermal membranes at a rate of 10 μL/cm2 and left
unoccluded for 24 hours.
The mean total amount of absorbed glyphosate in 24 hours was 0.573 μg/cm2 (0.012% of
applied dose) from the 450 g/L concentrate formulation. From the 1:15.6 v/v and 1:188 v/v aqueous
dilutions, the mean total amounts of absorbed glyphosate in 24 hours were 0.379 and 0.021 μg/cm2
(0.129% and 0.082% of applied dose), respectively (Ward, 2010a).
The absorption and distribution of glyphosate from a 360 g/L soluble (liquid) concentrate
(MON 79351) through human epidermis was measured in vitro when doses were applied as the
concentrate formulation (480 g/L of glyphosate) and as 1:16.7 v/v and 1:200 v/v (nominally 28.7 and
2.4 g/L ) aqueous spray dilutions of the formulation. 14C-radiolabelled glyphosate was incorporated
into the concentrate formulation and dilutions prior to application. The doses were applied to the
epidermal membranes at a rate of 10 μL/cm2 and left unoccluded for 24 hours.
The mean total amount of absorbed glyphosate in 24 hours was 0.342 μg/cm2 (0.0070% of
applied dose) from the 480 g/L concentrate formulation. From the 1:16.7 v/v and 1:200 v/v aqueous

105
dilutions of the formulation, the mean total amounts of absorbed glyphosate in 24 hours were 0.0.553
and 0.015 μg/cm2 (0.182% and 0.0488% of applied dose), respectively (Ward, 2010b).
The absorption and distribution of glyphosate from a 360 g/L soluble (liquid) concentrate was
measured in vitro through human epidermis when it was applied as the concentrate formulation
(360 g/L of glyphosate) and a 3:200 v/v aqueous spray strength dilution of the formulation.
14
C-radiolabelled glyphosate was incorporated into the concentrate formulation and dilutions prior to
application. The actual concentrations achieved were 364 g/L and 6.70 g/L of glyphosate for the
concentrate and the spray dilution, respectively. The doses were applied to the epidermal membranes
at a rate of 5 μL/cm2 and left unoccluded for 24 hours.
For the concentrate, the mean rate of absorption in 24 hours was 0.02 µg/cm2 per hour. For
the 3:200 v/v aqueous dilution, the mean rate of absorption in 24 hours was 0.001 µg/cm2 per hour.
For the concentrate, mild skin washing at 6 and 24 hours removed practically all of the applied dose
from the surface of epidermal membrane. For the 3:200 v/v spray dilution skin washing at 6 and 24
hours removed 90.8% and 87.9% of the applied dose, respectively (Davies, 2003).
In vivo
In the dermal penetration phase of the Maibach (1983) study described above (section 1.1
(d)), 25 μL of [14C]glyphosate solution containing 8.9 mg glyphosate was placed on the shaved
abdomens (7.9 cm2 area) of six male Rhesus monkeys. After 24 hours, each abdomen was swabbed
twice with water, twice with acetone and again twice with water to remove any residual glyphosate.
Urine samples were collected periodically for up to 7 days post application.
The washing procedure removed 14.2% of the applied 14C label. A mean total of 1.8% of the
applied dose of [14C]glyphosate was recovered in the urine during the 7-day collection period.
Glyphosate penetrated the monkey skin slowly as only 0.4% of the topically applied dose appeared in
the urine after 24 hours. The urinary elimination half-life for topically applied glyphosate was 59
hours (Maibach 1983).
Seven test groups, each with an equal number (between three and five) of male and female
Sprague Dawley Crl:CD(SD)BR rats, were dosed with N-(phosphono[14C]methyl)glycine glyphosate.
The radiochemical purity was 98% or greater. Single oral doses were administered by gastric
intubation whereas the intravenous doses were injected into the lateral tail vein. Comparison of the
areas under the curves for radioactivity levels in whole blood after oral (mean dose for males: 10.2
mg/kg bw; for females: 10.6 mg/kg bw) and intravenous (mean dose for males: 10.7 mg/kg bw; for
females: 11.0 mg/kg bw) administration of radiolabelled glyphosate indicated that absorption of the
oral dose of glyphosate at the 10 mg/kg bw dose level was 30.4% for males and 35.4% for the
females. Glyphosate was isolated as the predominant radioactive fraction in urine (overall recovery of
81.3%) and faeces (overall recovery of 99.2%), and was positively identified in each case by various
analytical methods. The minimum glyphosate content as a per cent of either urine or faecal extract
contained radioactivity in all of the individual rat excreta samples at 97.46%. HPLC analyses further
indicated that glyphosate in the excreta accounted for 98.50–99.33% of the administered
[14C]glyphosate.
In groups orally treated with a mean dose of 9.41 mg/kg bw for males and 9.28 mg/kg bw for
females and with a mean dose of 10.7 mg/kg bw for males and 10.3 mg/kg bw for females, there was
evidence that glyphosate was metabolized to produce 0.2–0.3% and 0.4% AMPA, respectively. The
remainder of the radioactivity in the excreta was due to low-level impurities in the dosing material or
changes during storage of the excreta samples (Howe, Chott & McClanahan, 1988).

106
Urine and faeces samples from the previously described study by Powles (1992b) (Section 1.1
(c)) were analysed for identification of glyphosate metabolites. Briefly, groups of five male and five
female Sprague Dawley rats were administered [14C]phosphonomethyl-labelled glyphosate (purity of
unlabelled test material: 96.8%; radiochemical purity > 98%) as a single dose of 30 or 1000 mg/kg bw
by gavage in saline or intravenously as a single dose at 30 mg/kg bw. Another group of five male and
five female rats were administered unlabelled glyphosate as 14 consecutive oral doses at 30 mg/kg bw
per day followed by 14C-labelled glyphosate as a single oral dose at 30 mg/kg bw.
The recovery of radioactivity from urine and faecal samples was generally greater than 90%.
For both dose groups only one major region of radioactivity was detected when extracts were
analysed by either liquid chromatography or TLC and this co-chromatographed with a glyphosate
standard. The identity of the major component as glyphosate was confirmed by comparing its Fourier
transform infrared spectroscopy spectrum with a glyphosate standard. Small amounts of other
components were detected but no radiolabelled metabolites were identified (Powles, 1992b).
Urine and faeces samples from the previously described McEwen (1995) study (section 1.1
(a)) were analysed for identification of glyphosate metabolites. Briefly, groups of five female Sprague
Dawley rats were administered [14C]phosphonomethyl-labelled glyphosate (purity of unlabelled test
material: 98.9%; radiochemical purity > 98%) as a single dose at 10 or 600 mg/kg bw by gavage in
water. Urine and faeces were collected for 7 days and analysed for metabolites.
The major urinary component was unchanged glyphosate, accounting for 18–27% of the
administered dose. Only 0.1–0.3% of the administered dose was shown to co-chromatograph, using
normal phase TLC and reverse phase HPLC, to aminomethylphosphonic acid. Faecal extract
contained 65–78% of administered dose as unchanged glyphosate. Two minor metabolites were in
faecal extract, accounting for 0.3–1.6% of the administered dose; one of these two metabolites was
shown to co-chromatograph with aminomethylphosphonic acid (McEwen, 1995).
The biotransformation of 14C-labelled glyphosate was investigated in male and female rats
administered either as a single 10 mg/kg dose or a single 10 mg/kg dose following repeated oral doses
of 10 mg/kg unlabelled glyphosate or as a single 1000 mg/kg bw dose. The metabolites in excreta
from the Davies (1996a,b,c) studies were identified (Section 1.1 (a)). In addition, a single oral dose of
1000 mg/kg of [14C]glyphosate (97.8 radiochemical purity) was administered to male and female
Alpk:APfSD rats fitted with a bile duct cannulae. The structural identification of metabolites isolated
from urine, bile and faeces, collected over 48 hours (biliary study) or 72 hours, was characterized
using various analytical methods.
Biliary excretion of radioactivity over 48 hours was negligible, 0.055% and 0.062% of the
administered dose for male and female rats, respectively. The greater percentage of excreted dose was
in faeces in both male (39.1%) and female rats (30.5%). Urinary excretion accounted for 20.8% of the
administered dose in male rats and 16.3% of the administered dose in female rats. In cannulated rats,
the excreted radioactivity (including cage wash) after 48 hours accounted for 62.5% and 52.0% of the
administered dose in male and female rats, respectively.
The main urinary metabolite was unchanged glyphosate, which accounted for virtually the
entire radioactivity present, with minor amounts of AMPA, which represented less than 1% of the
dose in each study (see Table 8). Solvent extraction of faeces, collected from the various excretion
and tissue distribution studies, resulted in the extraction of 53–79% of the radioactivity present. In
each case the extracts contained a single peak, which corresponded to unchanged glyphosate
(Macpherson, 1996).

107
14
Table 8. Quantification of glyphosate metabolites as percentages of single doses of C-labelled
glyphosate administered orally to rats
Percentage of administered dose (%)
Low-dose study Repeat dose studya High-dose study

10 mg/kg bw 10 mg/kg bw 1000 mg/kg bw
Sample Analyte Male Female Male Female Male Female
Urine Glyphosate 12.7 10.5 10.5 10.5 16.0 16.7
AMPA 0.2 0.1 < 0.1 < 0.1 0.6 0.7
Faeces Glyphosate 74.8 55.2 52.9 72.1 79.3 63.9
Total Glyphosate 87.5 65.7 63.3 82.6 95.3 80.6
AMPA 0.2 0.1 < 0.1 < 0.1 0.6 0.7
AMPA: aminomethylphosphonic acid; bw: body weight
a
Following 14 repeated oral doses of 10 mg/kg bw unlabelled glyphosate.
Source: Macpherson, 1996
Urine and faeces samples from the previously described Knowles & Mookherjee (1996) study
(Section 1.1 (a)) were analysed for identification of glyphosate metabolites. Briefly, five female
Sprague Dawley (Crl:CD BR) rats were administered [14C]phosphonomethyl-labelled glyphosate as a
single dose at 1 or 100 mg/kg bw by gavage in water. For the excretion study, urine and faeces (5/sex
per dose) were collected at selected times for 168 hours.
Metabolite profiles of pooled urine and faecal samples were investigated by HPLC. Only one
major peak was detected in urine and faeces (> 90% of the total activity); this was subsequently
identified as glyphosate. A minor component observed in the radiochromatograms had a similar
retention time to AMPA; however, it could not be positively identified due to very low levels
(Knowles & Mookherjee, 1996).
2.1 Acute toxicity
The results of acute toxicity studies of glyphosate (including skin and eye irritation and
dermal sensitization studies) are summarized in Table 9.
Table 9. Summary of acute toxicity studies with glyphosate
LD50 (mg/kg bw) /
Species Strain Sex Purity (%) Result Reference
Oral
Mouse ICR M+F 96.7 > 10 000 Shirasu & Takahashi
(1975)
Mouse NMRI M+F 98.6 > 2 000 Dideriksen (1991)
Mouse ICR(Crj:CD-1) M+F 95.68 > 5 000 (M) Komura (1995a)
> 5 000 (F)
> 5 000 (combined)
Mouse ICR(Crj:CD-1) M+F 62.34% > 5 000 Enami & Nakamura
glyphosate (1995)
isopropylamine
salt
Rat Sprague Dawley F 96.40 & 96.71 > 5 000 Komura (1995b)

108
LD50 (mg/kg bw) /

Rat HanRcc: WIST F 96.66 > 2 000 Simon (2009a)
Rat CD/Crl:CD(SD) F 97.52 > 2 000 Haferkorn (2009a)
Rat Sprague Dawley F 96.40 & 96.71 > 5 000 You (2009a)
Rat CD/Crl:CD(SD) F 95.23 > 2 000 Haferkorn (2010a)
Rat CD/Crl:CD(SD) F 97.3 > 2 000 Haferkorn (2010b)
Rat Sprague Dawley F 97.23 > 5 000 Merkel (2005a)
derived
Rat Wistar Hannover F 98.05 > 2 000 Do Amaral Guimaraes
(2008a), with addendum
dated 2010
Rat HanRcc: F 95.1 > 2 000 Talvioja (2007a)
WIST(SPF)
Rat Sprague Dawley M+F 97.76 > 5 000 (M) Reagan & Laveglia
> 5 000 (F) (1988a)
> 5 000 (combined)
Rat Wistar M+F 99 5 600 (combined) Heenehan, Rinehart &
Braun (1979)
Rat Sprague Dawley M+F 85.5 > 5 000 Blaszcak (1988a)
Rat Sprague Dawley M+F 98.6 > 5 000 Cuthbert & Jackson
(1989a)
Rat Alpk:APfSD (Wistar M+F 95.6 > 5 000 (male) Doyle (1996a)
derived) > 5 000 (female)
> 5 000 (combined)
Rat HanRcc:WIST(SPF) F 96.1 > 5 000 Arcelin (2007a)
Rat RjHan:WI F 96.3 > 5 000 Tavaszi (2011a)
Rat Wistar M+F 99 5 600 Heenehan (1979a)
Rat Sprague Dawley M+F 62% glyphosate > 5 000 Moore (1999)
derived isopropylamine
salt
Acute dermal
Rat Sprague Dawley M+F Not reported > 2 000 Cuthbert & Jackson
(1989b)
Rat Sprague Dawley M+F 96.40 & 96.71 > 5 050 You (2009b)
Rat SD(Crj:CD) M+F 95.68 > 2 000 Komura (1995c)
Rat HanRcc: WIST(SPF) M+F 96.66 > 2 000 Simon (2009b)
Rat CD/Crl:CD(SD) M+F 97.52 > 2 000 Haferkorn (2009b)
Rat CD/Crl:CD(SD) M+F 95.23 > 2 000 Haferkorn (2010c)
Rat CD/Crl:CD(SD) M+F 96.6 > 2 000 Haferkorn (2010d)
Rat Sprague Dawley M+F 97.23 > 5 000 Merkel (2005b)
Rat Wistar Hannover M+F 98.05 > 2 000 Do Amaral Guimaraes
(2008b)
Rat HanRcc: WIST(SPF) M+F 95.1 > 2 000 Talvioja (2007b)

109
LD50 (mg/kg bw) /

Rat Alpk:APfSD M+F 95.6 > 2 000 Doyle (1996b)
(Wistar derived)
Rat HanRcc: WIST(SPF) M+F 96.1 > 5 000 Arcelin (2007b)
Rat RjHan (WI) Wistar M+F 96.3 > 5 000 Zelenak (2011a)
Rabbit New Zealand White M+F 85.5 > 5 000 Blaszcak (1988b)
Rabbit New Zealand White M+F 97.76 > 5 000 Reagan (1988a)
Rabbit New Zealand White M+F 99 > 5 000 Heenehan (1979b)
Inhalation (nose only)
Rat CD/Crl:CD(SD) M+F 96.6 > 5.18 Haferkorn (2010e)
Rat F344/DuCrj(SPF) M+F 97.56 > 5.48 Koichi (1995)

Rat HsdRcc Han M+F 96.66 > 5.04 Griffiths (2009)
Rat CD/Crl:CD(SD) M+F 97.52 > 5.12 Haferkorn (2009c)
Rat CD/Crl:CD(SD) M+F 95.23 > 5.02 Haferkorn (2010f)
Rat Sprague Dawley M+F 96.40 & 96.71 > 2.24 Carter (2009)
Rat Sprague Dawley M+F 97.23 > 2.04 Merkel (2005c)
Rat Not reported M+F 98.05 > 5.21 Dallago (2008)

Rat HanRcc: WIST(SPF) M+F 95.1 > 3.252 Decker (2007)
Rat Alpk:APfSD (Wistar M+F 95.6 > 4.43 Rattray (1996)
derived)
Rat Wistar RjHan (WI) M+F 96.9 > 5.04 Nagy (2011)
Rat Sprague Dawley M+F 62% glyphosate > 2.08 Wnorowski (1999)
isopropylamine
Rat Hsd:Sprague Dawley M+F 47.2% glyphosate > 5.27 Bonnette (2004)
acid equivalent
Primary dermal irritation
Rabbit New Zealand White M+F 95.1 Non-irritating Talvioja (2007c)
Rabbit Himalayan M 95.23 Non-irritating Leuschner (2009a)
Rabbit New Zealand White F 97.56 Non-irritating Hideo (1995a)
Rabbit Himalayan M 97.52 Non-irritating Leuschner (2009c)
Rabbit Himalayan M 96.6 Non-irritating Leuschner (2010a)
Rabbit New Zealand White M+F 96.71 Non-irritating You (2009c)
Rabbit New Zealand White M 97.23 Non-irritating Merkel (2005d)
Rabbit New Zealand White F 98.05 Non-irritating Canabrava Frossard de
Faria (2008a)
Rabbit New Zealand White M+F 97.76 Non-irritating Reagan & Laveglia
(1988b)
Rabbit New Zealand White M+F 99 Slightly irritating Heenehan (1979c)
Rabbit New Zealand White F 95.6 Non-irritating Doyle (1996c)

110
LD50 (mg/kg bw) /

Rabbit New Zealand White M+F 96.1 Non-irritating Arcelin (2007c)
Rabbit New Zealand White M 96.3 Mildly irritating Zelenak (2011b)
Rabbit New Zealand White M+F 85.5 Slightly irritating Blaszcak (1988c)
Eye irritation
Rabbit New Zealand White M+F 95.1 Mildly irritating Talvioja (2007d)
Rabbit Himalayan M 95.23 Moderately irritating Leuschner (2009b)
Rabbit New Zealand White F 97.56 Severely irritating Hideo (1995b)
None n/a – Not stated pH of a 1% solution in Simon (2009c) a
water was 1.93. Not
tested because pH < 2
indicates corrosive
properties
Rabbit Himalayan M 97.52 Mildly irritating Leuschner (2009d)
Rabbit Himalayan M 96.6 Mildly irritating Leuschner (2010b)
Rabbit New Zealand White M+F 96.40 & 96.71 Moderately irritating You (2009d)
Rabbit New Zealand White M 97.23 Moderately irritating Merkel (2005e)
Rabbit New Zealand White M+F 98.05 Severely irritating Canabrava Frossard de
Faria (2008b)
Rabbit New Zealand White Not 97.76 Severely irritating Reagan & Laveglia
reported (1988c)
Rabbit New Zealand White F 95.6 Mildly irritating Johnson (1997)
Rabbit New Zealand White M+F 96.1 Mildly irritating Arcelin (2007d)
Rabbit New Zealand White M 96.3 Severely irritating Tavaszi (2011b)
Rabbit New Zealand White M+F 85.5 Moderately irritating Blaszcak (1988d)
Rabbit New Zealand White M+F 46.6 Non-irritating Blaszcak (1998e)
Rabbit New Zealand White M+F 57.8% glyphosate Mildly irritating Bonnette (2001)
potassium
(47.13%
glyphosate acid
equivalent)
Rabbit New Zealand White M+F Not reported Non-irritating Branch (1981)
(MON 0139)
Rabbit New Zealand White Not 90.8% Mildly irritating. Busch (1987a)
specified (MON 8722)
Rabbit New Zealand White Not 70.7% Mildly irritating Busch (1987b)
specified (MON 8750)
Rabbit New Zealand White Not 99 Moderately irritating Heenehan (1979d)
specified
Rabbit New Zealand White Not 97.76 Severely irritating Reagan (1988b)
specified
F: female; LD50: median lethal dose; M: male
a
According to Simon (2009c): “A 1% w/w solution of glyphosate technical in purified water was found to have a pH of
1.93. According to Council Regulation (EC) No. 440/2008, B.5. and OECD Guidelines 405, a test item is not required to be
tested if the pH value is less than 2, because it is assumed that the test item has corrosive properties… Therefore, no eye
irritation with glyphosate technical will be performed”

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(a) Oral toxicity

Mice
Groups of 10 ICR mice of each sex were administered a single dose of glyphosate (purity
96.7%) at 1000, 5000 or 10 000 mg/kg bw orally by gavage and were observed for 14 days before
termination.
Decreased locomotor activity was observed in all the mice at doses of 5000 mg/kg bw and
higher. Two of high-dose males and one of the high-dose females died; the others recovered fully
within 2 days. No abnormalities were found during necropsy.
The acute oral median lethal dose (LD50) of glyphosate (96.7%) in mice was greater than
10 000 mg/kg bw (Shirasu & Takahashi, 1975).
Groups of five male and five female Bom:NMRI mice were administered a single dose of
glyphosate (purity 98.6%) at 2000 mg/kg bw by gavage.
All the animals survived until the scheduled termination (day 14). Toxicological signs
included piloerection and sedation in all mice on day 1. No macroscopic abnormalities were observed
at necropsy.
The acute oral LD50 of glyphosate (98.6%) in mice was over 2000 mg/kg bw (Dideriksen,
1991).
Five male and five female ICR(Crj:CD-1) mice were orally dosed with 5000 mg/kg bw
glyphosate (purity 95.68%). The test material was administered as a 25% suspension in 0.5%
carboxymethylcellulose (CMC) sodium solution at 20 mL/kg bw.
Signs of toxicity observed at 1 and/or 3 hours after administration included decreased
spontaneous activity in one female and one male; another male was sedate and had a hunched posture.
One male lost a slight amount of weight on days 0–7 after dosing, but all the mice gained weight over
the 14-day observation period. There were no observed abnormalities at necropsy.
The acute oral LD50 of technical (95.68%) glyphosate in mice was greater than 5000 mg/kg
bw (Komura, 1995a).
Five male and five female ICR(Crj:CD-1) mice were dosed with a formulation (described as a
light viscous solution with a specific gravity of 1.23) containing 62.34% glyphosate isopropylamine
salt. The test material was administered undiluted.
None of the mice died and there were no signs of toxicity. There was a slight retardation in
mean body-weight gain in the males from day 0–7 compared with their controls (5000 mg/kg bw:
32.8–35.1 g; controls: 32.6–37.3 g). No gross pathological abnormalities were observed at gross
necropsy.
The mouse acute oral LD50 of a formulation containing 62.34% glyphosate isopropylamine
salt was greater than 5000 mg/kg bw (Enami & Nakamura, 1995).
Rats
In an acute oral toxicity study, five male and five female Sprague Dawley (Crj:CD) rats were
orally dosed with 5000 mg/kg bw glyphosate (purity 95.68%). The test material was administered as a
25% suspension in 0.5% CMC sodium solution at 20 mL/kg bw.

112
There were no mortalities, but spontaneous motor activity was decreased in five male and
three females, and one male had salivation. All the rats gained weight on days 0–7 and 7–14 after
dosing. No abnormalities were seen at necropsy.
The acute oral LD50 of technical (95.68%) glyphosate in male and female rats was greater
than 5000 mg/kg bw (Komura, 1995b).
Three female albino Sprague Dawley rats were administered 5000 mg/kg bw glyphosate
(purity, 96.40% and 96.71%) by gavage. The test material was mixed with deionized water and
administered as a 40% suspension at 12.5 mL/kg bw.
There were no mortalities. One rat showed slight to moderate signs of salivation, piloerection,
diarrhoea, polyuria and decrease in activity, with recovery by day 8. The other two rats showed no
indications of toxicity. All the rats gained weight days on days 0–7 and 7–14 after dosing. There were
no observed abnormalities at necropsy.
The acute oral LD50 of technical (96.40% and 96.71%) glyphosate in female rats was greater
than 5000 mg/kg bw (You, 2009a).
Two groups of three female HanRcc:WIST rats were orally dosed with 2000 mg/kg bw
technical glyphosate (purity 96.66%). The test material was administered as a 20% suspension in
purified water at a dose volume of 10 mL/kg.
All the rats survived. There were no signs of toxicity. Body-weight gain was normal and no
macroscopic lesions were observed at necropsy.
The acute oral LD50 of technical (96.66%) glyphosate in rats was greater than 2000 mg/kg bw
(Simon, 2009a).
Two groups of three female CD/Crl:CD(SD) rats were orally dosed with 2000 mg/kg bw
technical glyphosate at purities of 97.52%, 95.23% and 97.3%. The test material was administered as
a 20% suspension in 0.8% aqueous hydroxypropylmethylcellulose gel at a dose volume of 10 mL/kg.
All the rats survived. There were no signs of toxicity in the case of any of the test material
purity. Body-weight gain was normal, and no pathological findings were noted at necropsy.
The acute oral LD50 of technical glyphosate (97.52%, 95.23% and 97.3%) in female rats was
greater than 2000 mg/kg bw (Haferkorn, 2009a, 2010a,b).
Three female Sprague Dawley–derived albino rats were orally dosed with 5000 mg/kg bw
technical glyphosate (purity 97.23%). The test material was administered as a 50% w/v suspension in
distilled water (specific gravity: 1.252 g/mL).
All the rats survived. Clinical signs exhibited by all the rats were diarrhoea, anogenital and
facial staining and/or reduced faecal volume, with recovery by day 4. All the rats gained weight on
days 0–7 and 7–14 after dosing. There were no gross abnormalities at necropsy. The acute oral LD50
of technical (97.23%) glyphosate in female rats was greater than 5000 mg/kg bw (Merkel, 2005a).
Two groups of three female Wistar Hannover rats were orally dosed with 2000 mg/kg bw
technical glyphosate (purity 98.05%). The test material was mixed with deionized water to form a
dosing mixture containing 200 mg/mL glyphosate technical.
All the rats survived. There were no signs of toxicity, all gained weight on days 0–7 and 7–14
after dosing, and there were no specific signs at necropsy.

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The acute oral LD50 of technical (98.05%) glyphosate in female rats was greater than
2000 mg/kg bw (Do Amaral Guimaraes, 2008a, with an addendum dated 2010).
Two groups of three female HanRcc:WIST(SPF) rats were administrated 2000 mg/kg bw
technical glyphosate (purity 95.1%) by gavage. The test material was diluted in polyethylene glycol
(PEG 300) to 0.2 g/mL and administered at a dosing volume of 10 mL/kg.
All the rats survived. All showed piloerection at 1–3 or 2–3 hours after dosing. No other
clinical signs were observed. All gained weight on days 1–8 and 8–15 after dosing. There were no
macroscopic signs at necropsy.
The acute oral LD50 of technical (95.1%) glyphosate in female rats was greater than
2000 mg/kg bw (Talvioja, 2007a).
Five male and five female Sprague Dawley rats were orally dosed with 5000 mg/kg bw of
technical glyphosate (purity 97.76%). The test material was administered as a 50% w/v aqueous
suspension.
All the rats survived. All had diarrhoea, with recovery by day 4. In addition, three of the male
and two of the female rats had wet abdomens (“apparent urinary incontinence”) and one male and one
female had hair loss on the abdomen at termination. All gained weight on days 1–8 and 8–15 after
dosing. No internal abnormalities were observed at necropsy.
(Reagan & Laveglia, 1988a).
Groups of five male and five female Wistar albino rats were dosed with 2.5, 3.5 5.0, 7.0 or
9.9 g/kg of technical glyphosate (purity 99%) administered as a 25% solution) in distilled water.
At 2.5 g/kg one of the five males died; at 3.5 g/kg one of the males died; at 5.0 g/kg three
females died; at 7.0 g/kg all the males and three females died; at 9.9 g/kg all the animals died. Signs
of toxicity included ataxia, convulsions, muscle tremors, red nasal discharge, clear oral discharge,
urinary staining of the abdomen, soft stool, piloerection, lethargy and faecal staining of the abdomen.
The rats that died at 2.5 g/kg (day 5) and 3.5 g/kg (day 8) had considerable weight loss. At 7 and 9.9
g/kg, all the deaths occurred on day 1, except for one 9.9 g/kg male, which died on day 12. At
necropsy, the male that died on day 5 after dosing at 2.5 g/kg had urinary and faecal staining of the
abdomen, bright red lungs, stomach containing dark red fluid, upper intestines containing dark grey
fluid, lower intestines distended with air and containing yellow fluid. The male that died on day 8
after dosing at 3.5 g/kg had white lungs. Almost all the surviving rats at 2.5 and 3.5 g/kg had red spots
on the lungs, and mottled or purple livers. Surprisingly, most of the surviving rats at 5.0 g/kg had no
visible abnormalities.
The oral LD50 (combined sexes) of technical glyphosate in rats was calculated to be 5.6 g/kg
(95% confidence limits: 4.9–6.3 g/kg) (Heenehan, Rinehart & Braun, 1979).
Groups of five male and five female fasted CD Sprague Dawley–derived rats were
administered glyphosate (purity 85.5%) as a single dose at 5000 mg/kg bw orally by gavage and
observed for 14 days before termination.
All the animals survived until termination. One of the females exhibited weight loss on day 7
after dosing but gained weight on days 7–14. Toxicological signs included wet rales, faecal staining,
urinary staining and soft stool. Some animals had decreased feed consumption after dosing, which
continued in one animal through day 2. No gross abnormalities were found at necropsy (day 14).
The acute oral LD50 in rats was greater than 5000 mg/kg bw (Blaszcak, 1988a).

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Groups of five male and five female fasted Sprague Dawley rats were administered a single
dose of glyphosate (purity 98.6%) at 5000 mg/kg bw orally by gavage and observed for 14 days
before termination.
All the rats survived until termination. Toxicological signs included piloerection, reduced
activity and ataxia through day 9. No gross abnormalities were found during necropsy.
The acute oral LD50 in rats was greater than 5000 mg/kg bw (Cuthbert & Jackson, 1989a).
Five male and five female Alpk:APfSD (Wistar-derived) rats were dosed at 5000 mg/kg bw
with technical glyphosate (purity 95.6%) administered as a 0.5 g/mL suspension in deionized water.
None of the rats died and there were no signs of toxicity. All gained weight days 1–8 and
8–15 after dosing. At necropsy, two of the males and two of the females had mottled or red areas on
the lungs and one male had red areas on the thymus.
(Doyle, 1996a).
Three female HanRcc:WIST(SPF) rats were dosed at 5000 mg/kg bw with technical
glyphosate (purity 96.1%) administered as a 0.5 g/mL suspension in purified water.
None of the rats died. All had slightly ruffled fur (persisting in one rat through day 3) and all
had hunched posture from 1–5 or 2–5 hours after dosing. All gained weight on days 1–8 and 8–15
after dosing. There were no macroscopic findings at gross necropsy.
The acute oral LD50 of technical (purity 96.1%) glyphosate in female rats was greater than
5000 mg/kg bw (Arcelin, 2007a).
Three female RjHan:WI rats were dosed at 5000 mg/kg bw with technical glyphosate (purity
96.3%), administered as a 0.5 g/mL suspension in 0.5% CMC.
None died and there were no signs of toxicity. All the rats gained weight on days 0–7 and
7–14 after dosing. At necropsy, no abnormalities were noted.
The acute oral LD50 in female rats was greater than 5000 mg/kg bw (Tavaszi, 2011a).
Groups of five male and five female Wistar albino rats were orally dosed with glyphosate
technical (purity 99%) at 2.5, 3.5, 5.0, 7.0 or 9.9 g/kg bw. The test material was administered as a
25% w/v solution in distilled water.
Of the 10 rats in each dose group, one died at 2.5 g/kg, one at 3.5 g/kg, three at 5.0 g/kg, eight
at 7.0 g/kg and all 10 at 9.9 g/kg. Signs of toxicity included ataxia, convulsions, muscle tremors, red
nasal discharge, clear oral discharge, urinary staining of the abdomen, soft stool, piloerection, lethargy
and faecal staining of the abdomen.
The acute oral LD50 in rats was 5.6 g/kg (95% confidence limits: 4.9–6.3 g/kg) (Heenehan,
1979a).
In an acute oral toxicity study five male and five female Sprague Dawley–derived albino rats
were orally dosed with a formulation (described as a clear viscous amber liquid with a specific gravity
of 1.214 g/mL) containing 62% isopropylamine glyphosate.

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There were no deaths. There were no signs of toxicity in the males; four of the females had
anogenital staining, and one of these four had diarrhoea and another, soft faeces. All the rats had fully
recovered by day 3. All the rats gained weight on days 0–7 and 7–14 after dosing. There were no
gross abnormalities at necropsy.
The rat acute oral LD50 of a formulation containing 62% isopropylamine glyphosate was
greater than 5000 mg/kg bw (Moore, 1999).
(b) Acute dermal toxicity

Rats
In an acute dermal toxicity study, five male and five female Sprague Dawley rats were
dermally dosed with 2 000 mg/kg glyphosate technical (purity not reported), moistened with an
unspecified amount of water before application, for 24 hours.
There were no deaths. Clinical signs during exposure consisted of piloerection and reduced
activity. All the rats gained weight on days 0–7 after dosing and all, except a female that lost 30 g,
gained weight on days 7–14 after dosing. No abnormalities were detected at necropsy.
The rat dermal LD50 of technical (purity not reported) glyphosate was greater than 2000
mg/kg bw (Cuthbert & Jackson, 1989b).
Five male and five female Sprague Dawley albino rats were dermally dosed with 5050 mg/kg
glyphosate technical (two analyses: 96.40 and 96.71% purity), for 24 hours. The test material was
moistened with deionized water at 0.284 mL/g test material and placed on the skin.
There were no deaths and no clinical signs. All the rats gained weight on days 0–7 after
dosing; except for one female that lost 3 g of weight, all gained or maintained weight on days 7–14
after dosing. There were no observable abnormalities at necropsy.
The rat dermal LD50 of technical (two analyses: 96.40 and 96.71%) glyphosate was greater
than 5050 mg/kg bw (You, 2009b).
Five male and five female Sprague Dawley (Crj:CD) rats were dermally exposed to technical
glyphosate (purity 95.68%) at a concentration of 2000 mg/kg. Appropriate amounts of finely ground
test material were applied to a shaved 4  5 cm area of skin on each rat. Each site was then covered
with a filter paper moistened with 0.5 mL deionized water. A control group of five male and five
female rats was similarly treated without the test material.
Following a 24-hour exposure, there were no deaths and no clinical signs. All the rats gained
weight on days 0–7 and 7–14 after dosing, and the weight gains were similar in the glyphosate-treated
rats and the controls. There were no abnormalities at necropsy.
The rat dermal LD50 of technical (95.68%) glyphosate was greater than 2000 mg/kg bw
(Komura, 1995c).
In an acute dermal toxicity study, five male and five female HanRcc:WIST(SPF) rats were
dermally exposed to 2000 mg technical glyphosate (purity 96.66%) over a 24-hour exposure. The test
material was formulated in purified water at a concentration of 0.5 g/mL and applied at a volume dose
of 4 mL/kg.
There were no deaths or any clinical signs. There was no dermal irritation in males. Dermal
irritation (slight erythema, scaling, scabs) was seen in four females from day 4, persisting to day 12 at
the latest. All the males gained weight on days 1–8 and 8–15 after dosing. Two females had slight (0.6

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and 1.7 g) weight losses on days 1–8, but all had good weight gains on days 8–15. No macroscopic
findings were observed at necropsy.
The rat dermal LD50 of technical (purity 96.66%) glyphosate was greater than 2000 mg/kg bw
(Simon, 2009b).
In a series of acute dermal toxicity studies using 2000 mg technical glyphosate (purity
97.52%, 95.23% or 96.6%), five male and five female CD/Crl:CD(SD) rats were dermally exposed
over 24-hour periods (Haferkorn, 2009b). In each study, the test material was suspended (0.2 g/mL) in
aqua ad iniectabilia. This suspension was applied to eight layers of gauze, which was placed on a
5  6 cm patch of intact skin site. The gauze was covered with a plastic sheet secured with adhesive
plaster.
There were no deaths. There were no signs of toxicity. All the rats gained weight on days 0–8
and 8–15 after dosing. No skin irritation was observed. No pathological changes were observed at
necropsy.
The rat dermal LD50 of technical glyphosate (purity 97.52%, 95.23% and 96.6%) was greater
than 2000 mg/kg bw (Haferkorn, 2009b, 2010c,d).
Five male and five female Sprague Dawley–derived albino rats were dermally exposed to
5000 mg technical glyphosate (purity 97.23%) for 24 hours. The test material was mixed with distilled
water to form a dry paste (70% w/w mixture in distilled water). An appropriate amount of this paste
was applied to a 2  3 inch (about 5.1  7.6 cm) 4-ply gauze pad which was placed on the skin. The
gauze pad and trunk of the rat were then wrapped with Durapore tape.
There were no deaths and no signs of toxicity. All the rats gained weight on days 0–7 and 7–
14 after dosing. There were no abnormalities at necropsy.
(Merkel, 2005b).
Five male and five female Wistar Hannover rats were dermally exposed to 2000 mg technical
glyphosate (purity 98.05%) for 24 hours. The test material was placed on a porous gauze dressing
moistened with deionized water. The gauze dressing was held on the skin with a non-irritating tape,
and the test site and trunk of the animal covered with adhesive tape.
There were no deaths and no signs of toxicity. All the rats gained weight on days 0–7 after
dosing and on days 7–14, with the exception of two females (one lost 2 g, the other maintained
weight). There were no specific findings at necropsy.
The rat dermal LD50 of technical (980.5 g/kg) glyphosate was greater than 2000 mg/kg bw
(Do Amaral Guimaraes, 2008b).
Five male and five female HanRcc:WIST(SPF) rats were dermally exposed to 2000 mg
technical glyphosate (purity 95.1%) for 24 hours. The test material was diluted in PEG 300 to a
concentration of 0.33 g/mL, and 6 mL/kg of this dilution was applied to intact, shaved skin and
covered with a semi-occlusive dressing that was wrapped around the abdomen and fixed with an
elastic adhesive bandage.
There were no deaths and no clinical signs were observed. All the rats gained weight on days
1–8 and 8–15 after dosing except for one female that maintained weight on days 8–15. There were no
macroscopic findings at necropsy.

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(Talvioja, 2007b).
Five male and five female Alpk:APfSD (Wistar-derived) rats were dermally dosed with 2000
mg technical glyphosate acid (purity 95.6%) for 24 hours. The appropriate amount of test material
was weighed out onto a plastic weighing boat and moistened to a dry paste with 0.6–0.8 mL deionized
water before being applied onto approximately half of a 10  5 cm clipped area of skin. The amount
of test material applied per unit area of exposed skin was about 20.0–21.9 mg/cm2 for males and
16.2–17.3 mg/cm2 for females. The paste was covered by a 4-ply gauze patch (about 7  7 cm) kept in
contact with the skin for 24 hours using an occlusive dressing. The gauze patch was covered by a
patch of plastic film held in place by an adhesive bandage (about 25  7 cm) secured by two pieces of
PVC tape (about 2.5  20 cm).
None of the animals died and there were no significant signs of systemic toxicity. Some rats
showed signs of urinary incontinence, but this is common in dermal toxicity studies because of
bandaging and is not considered toxicologically significant. The skin of all rats was stained cream by
the test material for up to 8 days, but there were practically no signs of skin irritation. One male had
slight erythema on days 2–3 after dosing, and one female had small scabs on days 3–8 after dosing.
All gained weight on days 1–8, and, with the exception of one female that lost 2 g, all gained weight
on days 8–15 after dosing. At necropsy, the only finding was that one female had red mottled lungs,
which was reported as common in rats of this age and strain and not considered treatment related.
The rat acute dermal LD50 of technical (95.6%) glyphosate acid was greater than 2000 mg/kg
bw (Doyle, 1996b).
Five male and five female HanRcc:WIST(SPF) rats were dermally exposed to 5000 mg
technical glyphosate acid (purity 96.1%) for 24 hours. The appropriate amount of test material was
weighed out onto a plastic weighing boat and moistened to a dry paste with 0.5–0.6 mL purified
water. The dry paste was applied evenly on an intact 8 cm2 area of clipped skin which was covered
with tape.
There were no deaths and no clinical signs were observed. All the rats gained weight on days
1–8 and 8–15. There were no macroscopic findings at necropsy.
The rat dermal LD50 of technical (95.6%) glyphosate acid was greater than 5000 mg/kg bw
(Arcelin, 2007b).
Five male and five female Rj:Han (WI) Wistar rats were dermally exposed to 5000 mg
technical glyphosate (purity 96.3%) for 24 hours. Sufficient water to moisten the test material was
used to ensure good contact with the skin. The test material suspension was applied uniformly at the
dermal site. Gauze pads were placed over the site, and these were covered with a hypoallergenic
plaster. The entire trunk of the rat was then wrapped with semi-occlusive plastic wrap for 24 hours.
There were no deaths and no clinical signs were observed. There was no treatment-related
dermal irritation. All the rats gained weight on days 0–7 and 7–14 after dosing. There were no
macroscopic observations at necropsy.
The rat dermal LD50 of technical (96.3%) glyphosate acid was greater than 5000 mg/kg bw
(Zelenak, 2011a).
Rabbits
In an acute dermal toxicity study, five male and five female New Zealand White rabbits were
dermally exposed to 5000 mg/kg bw glyphosate (purity 85.5%) for 24 hours. The test material was

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applied dry to a strip of 8-ply gauze and then moistened with about 15 mL 0.9% saline. The gauze
strip was then placed on the skin.
All the rabbits survived the 14-day observation period, with little or no change in body
weights. No clinical signs were observed. There was no dermal irritation. Nothing remarkable was
observed at gross necropsy.
The rabbit dermal LD50 of glyphosate (85.5%) was greater than 5000 mg/kg bw (Blaszcak,
1988b).
Five male and five female New Zealand White rabbits were dermally exposed to 5000 mg/kg
glyphosate (purity 97.76%) for a 24-hour occluded exposure. The test material was moistened with
0.9% saline (about 1 mL/g of test material). An appropriate amount of this mixture was then applied
to each application site.
One female rabbit died at 14 days, but this death was attributed to mucoid enteropathy and not
to exposure to the test material. Other signs were anorexia, diarrhoea and soft stools. Most rabbits
gained slight amounts of weight in the 14-day observation period. At necropsy, one male rabbit had a
white caseous substance adhering to the lungs but this was not ascribed to exposure to the test
material; otherwise, there was nothing remarkable.
The rabbit dermal LD50 of glyphosate (97.76%) was greater than 5000 mg/kg (Reagan,
1988a).
In an acute dermal toxicity study, two male and two female New Zealand White rabbits were
dermally exposed (on abraded skin) to 5000 mg glyphosate technical (99%)/kg for a 24-hour occluded
exposure. The test material was applied as a 25% w/v solution in physiological saline.
All the rabbits survived. All had a clear nasal discharge, which had cleared by day 6. One
male lost weight over the 14-day observation period. At 24 hours, there was well-defined erythema in
two rabbits and very slight erythema in the two others; two had very slight oedema. At necropsy, there
were no internal or external abnormalities.
The rabbit dermal LD50 of glyphosate technical was greater than 5000 mg/kg (Heenehan,
1979b).
(c) Exposure by inhalation

In an acute inhalation toxicity study, five male and five female CD/Crl:CD(SD) rats were
exposed (nose only) for 4 hours to a mean concentration (HPLC-determined) of 5.18 mg/L
(5.05 mg/L as measured gravimetrically) with glyphosate technical (purity 96.6%).
There were no mortalities. All the rats exhibited tremors and dyspnoea, which remained for 3
hours after exposure (last observation on day 1); these effects were no longer present on test day 2
(the day following exposure). All the rats gained weight on days 0–8 and 8–15 after dosing. There
were no pathological findings at necropsy.
The rat inhalation median lethal concentration (LC50) of glyphosate (purity 96.6%) was
greater than 5.18 mg/L (Haferkorn, 2010e).
In an acute inhalation toxicity study, five male and five female F344/DuCrj(SPF) rats were
exposed (whole body) for 4 hours to a mean concentration (determined analytically) of 5.48 mg/L
glyphosate technical (purity 97.56%).

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There were no deaths. All the rats’ fur in the perioral and periocular regions was wet and
stained red with sticky material, which disappeared by day 4 in males and by day 5 in females. All the
rats gained weight on days 0–7 and 7–14 after dosing. No abnormalities were detected at necropsy.
The rat inhalation LC50 of technical (97.56%) glyphosate was greater than 5.48 mg/L (Koichi,
1995).
In an acute inhalation toxicity study, five male and five female HsdRccHan rats were exposed
(nose only) to a mean concentration (gravimetrically determined) of 5.04 mg/L glyphosate technical
(purity 96.66%).
There were no deaths. All the rats showed an increased respiratory rate, hunched posture,
piloerection and wet fur; these signs were still present 1 hour after exposure but were gone the
following day. All the rats gained weight on days 0–7 after dosing, and all gained or maintained
weight on days 7–14 after dosing. There were no macroscopic observations at necropsy.
The rat inhalation LC50 of technical (96.66%) glyphosate was greater than 5.04 mg/L
(Griffiths, 2009).
In an acute inhalation toxicity study of glyphosate technical (purity 97.52%), five male and
five female CD/Crl:CD(SD) rats were exposed (nose only) to 5.12 mg/L (determined by HPLC).
There were no deaths. All the rats had slight dyspnoea and ataxia which were still present at
1 hour but not at 3 hours. All the rats gained weight on days 0–8 and 8–15 after dosing. There were no
pathological findings at necropsy.
(Haferkorn, 2009c).
In an acute inhalation toxicity study, five male and five female CD/Crl:CD(SD) rats were
exposed (nose only) for 4 hours to a mean concentration (HPLC-determined) of 5.02 mg/L (4.99
mg/L measured gravimetrically) glyphosate technical (purity 95.23% ).
There were no deaths. All rats showed slight ataxia, slight tremors and slight dyspnoea which
were still present in all the animals at 3 hours (last observation on day 1) after exposure; these signs
were no longer present on test day 2 (the day following exposure). All the rats gained weight on days
0–8 and 8–15 after dosing. There were no pathological findings at necropsy.
(Haferkorn, 2010f).
In an acute inhalation toxicity study, five male and five female Sprague Dawley rats were
exposed (nose only) for 4 hours to a mean concentration of 2.24 mg/L (nominal concentration:
7.89 mg/L) glyphosate (two batches: purity 96.40% and 96.71%).
There were no deaths. All the rats showed piloerection and activity decrease from 4.5 hours
after exposure began until day 4. All the rats gained weight on days 0–7 and 7–14 after dosing. There
were no observable abnormalities at necropsy.
The rat inhalation LC50 of glyphosate (two analyses: 96.40% and 96.71%) was greater than
2.24 mg/L (Carter, 2009).
In an acute inhalation toxicity study, five male and five female Sprague Dawley rats were
exposed (nose only) for 4 hours to a gravimetrically determined mean concentration of 2.04 mg/L
(nominal concentration: 8.99 mg/L) glyphosate technical acid (purity 97.23%).

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There were no deaths or signs of toxicity. All the rats gained weight on days 0–7 and 7–14
The rat inhalation LC50 of glyphosate acid technical (97.23%) was greater than 2.04 mg/L
(Merkel, 2005c).
In an acute inhalation toxicity study, five male and five female rats (strain not reported:
“healthy young adults supplied by BIOAGRI’S rearing house”) were exposed (nose only) for 4 hours
to a gravimetrically determined mean concentration of 5.211 mg/L glyphosate acid technical (purity
98.05%).
There were no deaths or signs of toxicity. All the rats gained weight on days 0–7 and 7–14
The rat inhalation LC50 of glyphosate acid technical (purity 98.05%) was greater than 5.211
mg/L (Dallago, 2008).
In an acute inhalation toxicity study, five male and five female HanRcc:WIST(SPF) rats were
exposed (nose only) for 4 hours to a gravimetrically determined concentration of 3.252 mg/L
(nominal: 6.304 mg/L) technical (purity 95.1%) glyphosate.
There were no deaths. Two males had salivation and rales following exposure, and another
male had rales only. Two females had rales. All signs were gone two days after exposure. All gained
weight on days 1–8 and 8–15 after dosing. There were no pathological findings at necropsy.
The rat inhalation LC50 of technical (95.1%) glyphosate was greater than 3.252 mg/L (Decker,
2007).
In an acute inhalation toxicity study, five male and five female Alpk:APfSD (Wistar derived)
rats were exposed (nose only) for 4 hours to a particulate concentration of 4.43 mg/L glyphosate acid
(purity 95.6%); the chemical concentration was 4.27 mg/L. Two males and one female exposed to
4.43 mg/L were found dead and one female was terminated in extremis; these events took place on
days 5, 6 or 9 after dosing. Clinical signs seen in all rats included decreased activity, irregular
breathing, hunched posture and piloerection. Signs observed in some rats included splayed gait,
reduced stability, signs of urinary incontinence, gasping and vocalization. Hunched posture persisted
in some females until day 13 after dosing. All the surviving males and females lost weight on days
1–8, but gained weight days on 8–15 after dosing.
The two males found dead had dark lungs, probably as a result of agonal congestion; the
lungs of the decedent females were normal and the report states that the dark lungs in the males were
probably the result of agonal congestion.
Because of the high mortality at 4.43 mg/L, a second group of five male and five female rats
was exposed to a particulate concentration of 2.47 mg/L glyphosate acid (the chemical concentration
was measured to be 2.43 mg/L). No mortality occurred in this group. Clinical signs seen in all rats
included hunched posture, piloerection and salivation. All the males and four of the females had
abnormal respiratory noise, which was still present in one male on day 15 after dosing. All the rats
gained weight on days 1–8 and 8–15 after dosing. At necropsy one female had dark lungs and another
had a few red spots on the lung. These were probably incidental observations,
The rat inhalation LC50 of glyphosate acid (95.6%) was greater than 4.43 mg/L, although
mortality (in 4/10 rats) occurred at this concentration. No mortality occurred at 2.47 mg/L, although
there were signs of toxicity (Rattray, 1996).

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In an acute inhalation toxicity study, five male and five female Wistar RjHan (WI) rats were
exposed (nose only) for 4 hours to a gravimetrically determined concentration of 5.04 mg/L (nominal:
7.71 mg/L) glyphosate technical (purity 96.9%). The percentage of aerosol that was less than 4 µm
(considered the inhalable portion) was 54.4%.
One male was found dead on day 4. All the rats had laboured and noisy respiration,
respiratory rate increase, gasping, sneezing, decreased activity and looked thin. All the surviving rats
recovered by day 3; the male that died had slight noisy respiration, slight laboured respiration and a
wasted appearance on day 3 (this animal had lost 47 g from day 0–3 after dosing). Specific cause of
death was not determined. All the survivors gained weight on days 0–7 after dosing except for one
male which lost 9 g; all gained weight on days 7–14. At necropsy, the male decedent had dark/red
discolouration of the lungs and thymus. No observations were noted for the surviving rats.
The rat inhalation LC50 of glyphosate technical (96.9%) was greater than 5.04 mg/L, with one
rat dying following exposure to this concentration (Nagy, 2011).
In an acute inhalation toxicity study of NUP5a99 (described as a clear viscous liquid

containing 62% isopropylamine glyphosate and 31% other ingredients), five male and five female
Sprague Dawley–derived albino rats were exposed (whole body) for 4 hours to a gravimetrically
determined concentration of 2.08 mg/L (nominal value: 18.38 mg/L).
There were no deaths. In-chamber clinical observations included ocular and nasal discharge,
hunched posture and hypoactivity, but the rats recovered quickly on removal from the chamber and
the only finding 1 hour post-exposure was test material on the fur. All the rats gained weight on days
0–7 and 7–14 post dosing. There were no gross abnormalities at necropsy.
The inhalation LC50 of NUP5a99 glyphosate MUP (62% isopropylamine glyphosate) was
greater than 2.08 mg/L (Wnorowski, 1999).
In an acute inhalation toxicity study of MON 78623 (47.2% glyphosate acid equivalent;
57.8% potassium salt of glyphosate), two groups of five male and five female Hsd:Sprague Dawley
rats were exposed for 4 hours to either 2.21 or 5.27 mg/L glyphosate equivalent.
There were no deaths at either 2.21 or 5.27 mg/L. At 2.21 mg/L, breathing was congested and
there was dark material around the eyes and/or nose, both of which cleared by day 8 after dosing. At
5.27 mg/L, the rats exhibited congested breathing, with reduced faecal output in two females on day
1. All signs of toxicity had cleared by day 3 after dosing. At 2.21 mg/L, all the rats gained weight on
days 0–7 and 7–14 after dosing. At 5.27 mg/L all the males gained weight on days 0–7 and 7–14 after
dosing, while two females (the ones with reduced faecal output on day 1) lost 2 and 6 g on days 0–7;
another female lost 6 g on days 7–14; otherwise females gained weight on days 0–7 and 7–14 after
dosing. At both 2.21 and 5.27 mg/L, none of the tissues showed any abnormalities at necropsy.
The inhalation LC50 of MON 78623 (47.2% glyphosate acid equivalent; 57.8% potassium salt
of glyphosate) was greater than 5.27 mg/L (Bonnette, 2004).
(d) Dermal irritation

The results of studies of primary dermal irritation with glyphosate are summarized in Table 9.
In a dermal irritation study, three male and three female New Zealand White rabbits were
dermally exposed for 4 hours to 0.5 g glyphosate technical (NUP 05068; purity 95.1%) mixed in
about 0.5 mL purified water and applied to a 4  4 cm gauze patch that was placed on the skin. The
patch was covered with a semi-occlusive dressing that was wrapped around the abdomen and
anchored with tape.

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All irritation scores were zero. The primary dermal irritation index (PDII) was zero. A 4-hour
semi-occluded exposure to glyphosate technical (95.1%) over a skin area of about 16 cm2 (rather than
the usual 6 cm2) resulted in no dermal irritation (Talvioja, 2007c).
In three separate dermal irritation studies, three male Himalayan rabbits per study were
dermally exposed for 4 hours with 1000 or 2000 mg of glyphosate technical (purity 95.23%)
(Leuschner, 2009a), glyphosate technical (purity 97.52%) (Leuschner, 2009c) or glyphosate technical
(purity 96.6%) (Leuschner, 2010a) mixed with 0.5 (for 1000 g) or 1.0 mL (for 2000 g) aqua ad
iniectabilia. This paste (750 mg, containing 500 mg glyphosate) was applied to a 6 cm2 area of skin
on each of the rabbits. The paste was covered with a gauze patch held in place with non-irritating
hypoallergenic) tape.
All irritation scores at 1, 24, 48 and 72 hours after exposure were zero. The PDII was 0.00. A
4-hour dermal exposure to glyphosate technical (purity 95.23%, 97.52% or 96.6%) resulted in no
dermal irritation (Leuschner, 2009a,c, 2010a).
In a dermal irritation study, six female New Zealand White rabbits were dermally exposed for
4 hours to glyphosate technical (HR-001; purity 97.56% ). The test material was finely ground in a
mortar and 0.5 g put on a 2.5  2.5 cm area on each rabbit. A 2.5  2.5 cm gauze patch moistened
with 0.5 mL water was then placed over the test material and held in place with a polyethylene sheet
and non-irritating occlusive tape.
4-hour exposure to HR-001 (97.56% active glyphosate) resulted in no dermal irritation (Hideo,
1995a).
In a dermal irritation study, one male and two female New Zealand White rabbits were
dermally exposed for 4 hours to 500 mg glyphosate technical (purity 96.71%) moistened with 0.2 mL
deionized water. This mixture was applied to each test site and covered with a 2.5  2.5 cm gauze
patch. Each patch was secured in place with a strip of non-irritating adhesive tape. The entire trunk of
each rabbit was loosely wrapped with a semi-permeable orthopaedic stockinette secured at both edges
with strips of tape.
4-hour exposure to glyphosate technical grade (96.71%) resulted in no dermal irritation (You, 2009c).
In a dermal irritation study, three male New Zealand White rabbits were dermally exposed for
4 hours to a 70% w/w mixture of glyphosate acid technical (97.23% active) in distilled water. Some of
this paste (0.71 g) was placed on 1  1 inch (2.54  2.54 cm) 4-ply gauze pads which were applied to
a 6 cm2 area of intact skin on each rabbit. The pad and entire trunk of each rabbit were then wrapped
with semi-occlusive 3-inch Micropore tape.
At 1 hour after exposure, one site scored 1 for erythema using the Draize scoring method; all
other scores were zero. All scores were zero at 24, 48 and 72 hrs. The PDII was 0.08. A 4-hour
exposure to glyphosate acid technical (97.23%) resulted in very slight dermal irritation (Merkel,
2005d).
In a dermal irritation study, three female New Zealand White rabbits were dermally exposed
for 4 hours to glyphosate technical (purity 98.0%). A moistened gauze pad with 0.5 g test material
was placed on a 6 cm2 area of skin and held in place with an adhesive non-irritating tape.

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All irritation scores at 1, 24, 48 and 72 hours after exposure were zero. The PDII was zero. A
4-hour dermal exposure to glyphosate technical (purity 98.05%) resulted in no dermal irritation
(Canabrava Frossard de Faria, 2008a).
dermally exposed for 4 hours to 0.5 g glyphosate (purity 97.76%) moistened with 0.5 mL
physiological saline and applied to two intact test sites per rabbit. The test sites were semi-occluded
with a 1  1 inch (2.54  2.54 cm) gauze patch held in place with Micropore tape.
All irritation scores at 0.5, 24, 48 and 72 hours after exposure were zero. The PDII was 0.00.
A 4-hour dermal exposure to glyphosate (purity 97.76%) resulted in no dermal irritation (Reagan &
Laveglia, 1988b).
dermally treated for 24 hours with 0.5 mL glyphosate technical (purity 99%) as a 25% w/v solution in
distilled water applied to four sites (two intact, two abraded) on each of six albino rabbits.
At 24 hours, one rabbit scored 1 for erythema at an intact site using the Draize scoring
method and 1 for erythema and 1 for oedema at an abraded site. Another rabbit scored 1 for erythema
at an abraded site. All other scores at 24 hours were zero. All scores for irritation at 72 hours after
dosing were zero (Heenehan, 1979c).
In a dermal irritation study, 500 mg of glyphosate acid (purity 95.6%) was moistened with 0.5
mL of distilled water to form a dry paste that was applied to a 2.5  2.5 cm test site on the left flank of
each of six female New Zealand White rabbits. The treated area was covered with an 8-ply 2.5  2.5
cm surgical gauze pad that was secured by two strips of surgical tape. This was covered by
impermeable rubber sheeting that was wrapped once around the trunk of the animal and secured with
adhesive polyethylene tape. Exposure was for 4 hours.
No irritation was observed at 30 minutes to 1 hour or 1, 2 or 3 days after dosing. All irritation
scores were zero. The PDII was 0.00 (Doyle, 1996c).
In a dermal irritation study, 0.5 g of glyphosate technical (96.1% glyphosate acid) was
moistened with about 0.5 mL purified water and placed on a 2.5  2.5 cm 8-ply gauze surgical patch
that was applied to intact skin on the left flank of each of three male and three female New Zealand
White rabbits. Each patch was covered with a semi-permeable dressing that was wrapped around the
abdomen and held in place with tape. Exposure was for 4 hours.
No irritation was observed a 1, 24, 48 or 72 hours after dosing. All irritation scores were zero.
The PDII was 0.00 (Arcelin, 2007c).
In a dermal irritation study, 0.5 g glyphosate technical (purity 96.3%) was dampened with
water, and placed on a 2.5  2.5 cm surgical gauze pad that was kept on the skin of three male New
Zealand White rabbits with hypoallergenic plaster for 4 hours. The entire trunk was wrapped with
plastic wrap held in place with an elastic stocking.
One rabbit had grade 1 erythema at 1 and 24 hours after dosing. All other irritation scores
were zero. The PDII was 0.17 (Zelenak, 2011b).

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In a dermal irritation study, 0.5 g glyphosate wet cake (purity 85.5%) was moistened with 0.5
mL 0.9% saline and applied to the skin of six rabbits (two applications per rabbit). The applications
were covered with 2.5  2.5 cm gauze squares for 4 hours of occluded exposure.
Five of the six rabbits showed grade 1 erythema at one or both sites at 0.5, 24 and/or 48 hours
after dosing. All scores were zero at 72 hours. The PDII was 0.31 (Blaszcak, 1988c).
(e) Ocular irritation

The results of studies of primary eye irritation with glyphosate are summarized in Table 9.
In an eye irritation study, 0.1 g glyphosate technical (purity 95.1%) was instilled into the
conjunctival sac of the left eye of each of three male and three female New Zealand White rabbits.
There was no iridial irritation (all irritation scores were zero). Corneal opacity along with
positive conjunctival irritation (grade 2–3 redness and/or grade 2–3 chemosis) was in all the treated
eyes at 1, 24 and 48 hours after dosing and in 2/3 treated eyes (with grade 2 redness) at 72 hours after
dosing. On day 7 all scores for corneal opacity were zero; three eyes scored 1 (not considered a
positive irritation effect) for conjunctival redness. All scores were zero on days 10 and 14.
Glyphosate technical (purity 95.1%) was considered to have caused significant but reversible
damage to the rabbit eye (Talvioja, 2007d).
In eye irritation studies, 100 mg glyphosate technical (purity 95.23%, 97.52% or 96.6%) were
instilled into the conjunctival sac of the right eye of each of three male Himalayan rabbits for each
strength. An hour after instillation, the eyes were rinsed with 20 mL sodium chloride solution.
At purity 95.23%, corneal opacity (maximum score 1) was in all three eyes at 24, 48 and
72 hours after dosing; in two of the three eyes on day 4 after dosing; and in one of the three eyes on
days 5, 6 and 7 after dosing; by day 8, clearing was complete. The maximum score for iritis was 1,
which was observed in all three eyes at 24 hours, in two eyes at 48 hours, in one eye at 72 hours and
in none of the eyes on day 4 and subsequently. The maximum score for conjunctival redness was 1, as
was the maximum score for chemosis. All scores for conjunctival effects were zero by day 5. A
fluorescein test at 24 hours showed corneal staining of between half and three quarters of the surface
of two eyes, and in one quarter to half of the surface of one eye. A fluorescein test on day 7 showed
corneal staining in one eye (up to one quarter of the surface).
At purity 97.52%, fluorescein testing at 24 hours showed corneal staining in two of the three
eyes. At 24 and 48 hours, two eyes had corneal opacity and one of these still had corneal opacity at 72
hours. All the eyes had completely cleared (all eye irritation scores were zero) by day 4.
At purity 96.6%, all three eyes had corneal opacity at 24, 48 and 72 hours. At 4 days, two
eyes had corneal opacity and one of these also had corneal opacity on day 5. All eyes had completely
cleared (all eye irritation scores were zero) by day 7.
The three reports each concluded that “glyphosate TC was non-irritating to eyes, hence, no
labelling is required” (Leuschner, 2009b, 2009d, 2010b).
In an eye irritation study of HR-001 (purity 97.56%), 0.1 g of the test material was placed in
the conjunctival sac of the left eye of each of 12 female New Zealand White rabbits. Six rabbits
(group A) did not receive an eyewash; three rabbits (group B) had their eyes washed out 30 seconds
after instillation; and three rabbits (group C) had their eyes washed out 2 minutes after instillation.
All six rabbits in group A had corneal opacity through day 4. On day 7, five had corneal
opacity. On day 21, three still had corneal opacity while the remaining three had completely cleared.
In group B, all three rabbits had corneal opacity at 24 and 48 hours, but their eyes had completely

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cleared (all scores were zero) by day 7. In group C, one rabbit was positive for corneal opacity at
24 hours, and none of the rabbits had corneal opacity at 48 hours. One group C rabbit was positive for
conjunctival effects at 72 hours; the other two rabbits had completely cleared (all eye irritation scores
were zero). None of the group C rabbit eyes was positive for irritation on day 4.
The report concluded that the test material had severely irritating potential for the eye mucosa
of rabbits and that irrigation at 30 seconds or 2 minutes after application was effective for reduction of
eye irritation and for recovery (Hideo, 1995b).
In an eye irritation study of glyphosate technical grade (two analyses: 96.40 and 96.71%),
0.1 mL (93.2 mg) was placed into the conjunctival sac of the right eye of each of two male and one
female New Zealand White rabbits.
Of the three eyes, two still had corneal opacity at 24, 48 and 72 hours and at day 4. One eye
had corneal opacity on day 7. All eyes had cleared by day 10.
The test material was rated as “moderately irritating and assigned to [United States
Environmental Protection Agency; USEPA] Toxicity Category II” (You, 2009d).
In an eye irritation study of glyphosate acid technical (purity 97.23%), the test material was
ground to a powder with a mortar and pestle and 0.1 mL (0.06 g) was instilled into the conjunctival
sac of the right eye of three male New Zealand White rabbits. The pH of a 1% solution was reported
as 2.5.
All three eyes were positive for corneal opacity through day 7, and for iritis and conjunctivitis
through day 4 (one eye was also positive for conjunctival redness on day 7). All eyes had cleared (all
irritation scores were zero) by day 10. According to the report,
The Maximum Mean Total Score of Glyphosate Technical is 40.3. Based on the classification system
used the test substance is considered severely irritating to the eye. The classification was raised from
moderately to severely [irritating] because all three animals had scores greater than 10 on day 7 of the
study (Merkel, 2005e).
In an eye irritation study, 0.1 g of glyphosate technical (purity 980.5 g/kg) was instilled in an
eye of each of male and female New Zealand White rabbits. Because of the severity of the effects
only two eyes were tested. The pH of a 1% solution is reported as 2.2.
In one rabbit there was corneal opacity, iritis and conjunctival effects through day 4 with
clearing by day 7. In the other rabbit there was corneal opacity at 1, 24, 48 and 72 hours and at 7, 14
and 21 days after dosing. The eye was also positive for conjunctival irritation on day 14 after dosing
(Canabrava Frossard de Faria, 2008b).
In an eye irritation study, 0.1 g glyphosate (purity 97.76%) was instilled in the conjunctival
sac of one eye of each of six New Zealand White rabbits (sex not reported). The eyes were not
washed out until 24 hours after instillation of the test material.
Corneal opacity and conjunctival irritation with blistering was observed in all the rabbits. One
rabbit (which still had corneal opacity on day 14) was found dead at 20 days after instillation; the
death was considered unrelated to exposure to the test material. Of the five surviving rabbits, three
still had corneal opacity on day 21.
Because the glyphosate (97.76%) was severely irritating to the eye, it was assigned to USEPA
Toxicity Category I for this exposure route (Reagan & Laveglia, 1988c).

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In an eye irritation study of glyphosate acid (purity 95.6%), 100 mg was applied into the
conjunctival sac of one female New Zealand White rabbit. This application caused moderate pain in
this first rabbit so the other five animals were pre-treated with a local anaesthetic. Nevertheless,
“between one quarter and one half of the test material was displaced from the eye of each animal
immediately after dosing”, according to the report (Johnson, 1997).
Corneal, iridial and conjunctival effects were seen in all rabbits for up to 4 days post dosing.
Corneal opacity was seen in five of the six rabbits on day 4, but had cleared in all of them by day 7.
All scores were 0 on day 7 except for one rabbit which had grade 1 (not considered positive for
irritation) conjunctival redness, which had cleared by day 8.
Glyphosate acid (purity 95.6%) was classified as a mild irritant (class 5 on a 1–8 Draize
scoring method) to the rabbit eye (Johnson, 1997).
In an eye irritation study, 0.1 g of glyphosate technical (purity 96.1%) was instilled into the
conjunctival sac of the left eye of each of three New Zealand White rabbits. The pH of the test
material was reported as 2.12.
There was no corneal opacity or iritis. All three rabbit eyes were positive for conjunctival
irritation at 1 hour, and two were positive for these effects at 24, 48 and 72 hours after dosing. All
scores were 0 by day 7.
The report concluded that “…the test item did not induce significant or irreversible damage to
the rabbit eye” (Arcelin, 2007d).
In an eye irritation study, 0.1 g of glyphosate technical (purity 96.3%) was instilled into the
conjunctival sac of the left eye of one male New Zealand White rabbit. The pH of the test material
was reported as 1.99.
An Initial Pain Reaction score of 3 (on a scale of 0–5) was observed. Irritation effects were
scored at 1 and 24 hours after instillation. According to the report (Tavaszi, 2011b):
Conjunctival redness, chemosis and conjunctival discharge, as well as corneal opacity, were observed
in the rabbit at 1 and 24 hours after application. Additionally, corneal erosion, redness of the
conjunctiva with pale areas, pink, clean ocular discharge, oedema of the eyelids, and a few black points
on the conjunctiva and dry surface of the eye were noted at one hour after the treatment. Fluorescein
staining was positive at the 24 hour observation. Based on the symptoms, no further animals were
dosed and the study was terminated after the 24 hour observation…
Glyphosate Technical was classified as corrosive to the eye. (Tavaszi, 2011b).
In an eye irritation study of glyphosate wet cake (purity 85.5%), 0.1 mL (68.9 mg) was
instilled into the lower conjunctival sac of the right eye of six New Zealand White rabbits. The eyes
were not washed out until 24 hours after instillation.
All the rabbits showed positive irritation effects (corneal opacity and/or grade 2 chemosis
and/or redness and/or iritis) at 1–48 hours after dosing, and two rabbits showed positive irritation
effects at 72 hours. None of the eyes was positive for irritation on day 7. The report concluded that
“Glyphosate Wet Cake produced moderate to severe but reversible ocular irritation in all animals…
Five had iritis and corneal opacities” (Blaszcak, 1988d).
In an eye irritation study of MON 77945 (described as an amber liquid, pH 4.59, containing
46.6% glyphosate acid), 0.1 mL was instilled into one eye of each of six rabbits.

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There were no positive irritation effects (one eye scored 1 for conjunctival redness at 1 hour,
all other scores were zero). The report concluded that “under conditions of this study, MON 77945
produced very mild, transient ocular irritation” (Blaszcak, 1998e).
In an eye irritation study of MON 78623 (described as an amber liquid with 57.8% potassium
salt of glyphosate; 47.13% glyphosate acid equivalent), 0.1 mL was instilled into an eye of each of
three rabbits. Two rabbits vocalized following instillation.
There was no corneal opacity. At 1 hour, all eyes scored 1 for iritis, two for conjunctival
redness and two for conjunctival swelling. At 24 hours, one eye scored 1 for iritis. All scores were
zero at 48 hours. The report concluded that, “based on [European Economic Community] labelling
criteria, MON 78623 is classified as a non-irritant to the ocular tissue of the rabbit” (Bonnette, 2001).
In an eye irritation study of MON 0139 (described as an amber liquid, with no information on
pH or the active ingredient) 0.1 mL was instilled into an eye of each of nine rabbits. Six eyes were
unwashed; three were washed out with physiological saline about 20 seconds after instillation.
All irritation scores were zero. No signs of irritation were observed in any rabbit eye (Branch,
1981).
In an eye irritation study of MON 8722 (described as a white powder, 90.8% purity, which
was ground with a mortar and pestle prior to dosing), 0.1 g was instilled into an eye of each of six
rabbits.
There was no corneal opacity or iritis. At 1 hour, conjunctival irritation (grade 2 redness
and/or chemosis) was seen in five of the six eyes. At 24 and 48 hours, some of the eyes scored 1 for
conjunctival redness. At 72 hours, all scores were zero (Busch, 1987a).
In an eye irritation study of MON 8750 (described as a white powder, 70.7% purity, which
was ground with a mortar and pestle prior to dosing), 0.1 g (0.1 mL) was instilled into an eye of each
of six rabbits.
There was no corneal opacity or iritis. At 1 hour, conjunctival irritation (grade 2 redness
and/or chemosis) was seen in five of the six eyes. At 24 hours, one eye scored 1 for conjunctival
redness (not considered a positive irritation effect). At 48 hours all scores were zero (Busch, 1987b).
In an eye irritation study, 0.1 mL of a 25% w/v solution of glyphosate technical (purity 99%),
in distilled water was instilled into the conjunctival sac of an eye of each of nine rabbits. Six eyes
were unwashed, while the other three were washed out for 1 minute with lukewarm water starting
20 seconds after instillation.
One unwashed eye and two washed eyes showed corneal opacity, with clearing by day 4. All
scores were zero by day 7. In this study, glyphosate (purity 99%) was moderately irritating to the eye
(Heenehan, 1979d).
In an eye irritation study, 0.1 g glyphosate (purity 97.76%) was instilled into the conjunctival
sac of one eye of each of six rabbits. Corneal opacity and conjunctival irritation were noted in all
rabbits at 24, 48 and 72 hours and on day 7.
One rabbit was found dead at 20 days; however, the death was considered unrelated to
exposure. On day 21, three of the remaining five rabbits still showed corneal opacity. In this study,
glyphosate (97.76%) was severely irritating to the eye (Reagan, 1988b).

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(f) Dermal sensitization

Results of studies of skin sensitization with glyphosate are shown in Table 10.
Table 10. Results of skin sensitization studies with glyphosate

Species Strain Sex Route Purity (%) Results Reference
Mouse CBA/Ca F LLNA 96.1 Negative Betts (2007)
Mouse CBA/J Rj F LLNA 96.3 Negative Török-Bathó (2011)
Guinea pig Dunkin Hartley F Magnusson– 95.1 Negative Talvioja (2007e)
Kligman
Maximization
Guinea pig Dunkin Hartley F Magnusson– 97.52 Negative Haferkorn (2009d)
Kligman
Maximization
Guinea pig Dunkin Hartley F Magnusson– Two analyses: Negative Haferkorn (2010g)
Kligman 95.23 & 96.4
Maximization
Guinea pig Hartley F Magnusson– 97.56 Negative Hideo (1995c)
Kligman
Maximization
Guinea Pig Hartley M Magnusson– 96.66 Negative Simon (2009d)
Kligman
Maximization
Guinea pig Dunkin Hartley M Magnusson– Two analyses: Negative Haferkorn (2010h)
Kligman 97.52 & 98.8
Maximization
Guinea pig Short-haired M+F Buehler Two analyses: Negative You (2009e)
Hartley albino 96.4 & 95.71
Guinea pig Hartley albino M+F Buehler 97.23 Negative Merkel (2005f)
Guinea pig Hartley M Buehler 98.05 Negative Lima Dallago (2008)
Guinea pig Dunkin Hartley F Magnusson– 95.7 Negative Richeux (2006)
Kligman
Maximization
Guinea pig Albino Crl (HA) F Magnusson– 95.6 Negative Doyle (1996d)
BR Kligman
Maximization
Mouse CBA/Ca F LLNA 96.1 Negative Betts (2007)
Mouse CBA/J Rj F LLNA 96.3 Negative Török-Bathó (2011)
F: female; LLNA: local lymph node assay; M: male
Mouse
In a local lymph node assay, about 25 µL of a 10, 25 or 45% w/v preparation of glyphosate
technical (96.1% glyphosate acid) in dimethyl sulfoxide (DMSO) was applied to the dorsal surface of
each ear of groups of four female CBA/Ca mice. A vehicle control group was similarly treated with
DMSO alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected in the tail vein with about
250 µL of phosphate buffered saline containing 20 µCurie (µCi; 74 × 1010 Bq) [methyl-3H]thymidine.
The mice were terminated after about 5 hours. The drained auricular lymph nodes were removed from

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each animal and, together with the nodes from the other animals in that group, placed in a container of
phosphate buffered saline.
Single cell suspensions were prepared by straining the lymph nodes from a single group
through a 200-mesh stainless steel gauze. The cell suspensions were washed three times by
centrifugation with about 10 mL phosphate buffered saline. Approximately 3 mL of 5% w/v
trichloroacetic acid was added and, after overnight precipitation at 4 °C, the samples were pelleted by
centrifugation and the supernatant was discarded. The cells were resuspended in approximately 1 mL
of trichloroacetic acid, and the suspensions transferred to scintillation vials; 10 mL of scintillant was
added prior to ß-scintillation counting.
The following disintegrations per minute were obtained: 0% (DMSO alone): 3912; 10%:
2394 (Stimulus Index or SI of 0.61 relative to vehicle control); 25%: 3292 (SI: 0.84); 45%: 508 (SI:
1.04). The following disintegrations per minute were obtained from the positive control
(α-hexylcinnamaldehyde in 4 parts acetone and 1 part olive oil): 0%; (vehicle alone): 5939; 5%:
10 111 (SI: 1.70); 10%: 13 747 (SI: 2.31); and 25%: 38 015 (SI: 6.40, positive response > 3).
The study concluded that glyphosate technical material is not a skin sensitizer under these test
conditions (Betts, 2007).
A local lymph node assay of glyphosate technical (96.3%) used groups of four female CBA/J
Rj mice with each mouse topically dosed on the dorsal surface of each ear with 25 µL of 10%, 25% or
50% w/v preparation of glyphosate technical in propylene glycol, propylene glycol alone or 25%
α-hexylcinnamaldehyde in propylene glycol. The procedure was repeated daily for three consecutive
days.
Three days after the third application, all the animals were injected in the tail vein with about
250 µL of phosphate buffered saline containing 20 µCi (74 × 1010 Bq) [methyl-3H]thymidine. The
mice were terminated about 5 hours later. The draining auricular lymph nodes were removed from
each animal and, together with the nodes from the other animals in that group, placed in a Petri dish
containing 1–2 mL phosphate buffered saline.
Single cell suspensions of pooled lymph node cells were prepared and collected in tubes by
gentle mechanical disaggregating of the lymph nodes through a cell strainer. The cell strainer was
washed with phosphate buffered saline. Pooled lymph node cells were pelleted in a centrifuge at about
190 g for 10 minutes at 4 °C. Afterwards, the centrifugation supernatants were discarded. The pellets
were gently resuspended and 10 mL phosphate buffered saline added to the tubes. The washing step
was repeated twice. This was repeated for each group of pooled lymph nodes. After the final washing,
suspensions were centrifuged and most of the supernatant was removed except for a small volume
(< 0.5 mL) above each pellet. Each pellet was resuspended in 3 mL of 5% trichloroacetic acid. After
an 18-hour incubation with 5% trichloroacetic acid at 2–8 °C, the precipitate was recovered by
centrifugation at 190 g for 10 minutes. The supernatants were removed and the pellets resuspended in
1 mL of 5% trichloroacetic acid solution and dispersed using an ultrasonic water-bath. Each
precipitate was transferred to a scintillation vial with 10 mL of scintillation liquid and thoroughly
mixed prior to ß-scintillation counting.
The following disintegrations per minute were obtained after accounting for the background:
0% (propylene glycol alone): 681; 10%: 794 (Stimulus Index or SI of 1.2 relative to vehicle control);
25%: 678 (SI: 1.0); 50%: 683 (SI: 1.0); positive control (α-hexylcinnamaldehyde in propylene
glycol): 25%: 8302 (12.2 SI, positive response > 3).
The study concluded that under the conditions of this local lymph node assay, glyphosate
technical had no skin sensitization potential (i.e. it was a non-sensitizer) (Török-Bathó, 2011).

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Guinea pigs
In a Magnusson–Kligman maximization test with female Dunkin Hartley guinea pigs,
intradermal induction treatments were with a 3% dilution of glyphosate technical (95.1%) in PEG 300
and in an emulsion of Freund’s Complete Adjuvant/physiological saline. Epidermal induction (1 week
after the intradermal induction) was for 48 hours under occlusion with the test material at 50% in PEG
300. Two weeks later, the five control and 10 test guinea pigs were challenged. Patches (3  3 cm) of
filter paper were saturated with about 0.2 mL of the test material at the highest tested non-irritating
concentration of 25% in PEG 300 (applied to the left flank) and about 0.2 mL PEG 300 alone (applied
to the right flank) for 24 hours. The application sites were scored at 24 and 48 hours after exposure
ended.
All challenge irritation scores (for the 10 test and five control animals) were zero. A positive
control assay with α-hexylcinnamaldehyde gave appropriate results. Based on these findings,
glyphosate technical does not have to be classified and labelled as a skin sensitizer (Talvioja, 2007e).
In a Magnusson–Kligman maximization test with female Dunkin Hartley guinea pigs,

intradermal induction treatments were with a 0.01% concentration of glyphosate technical (two
analyses: 95.23% and 96.4%) in aqua ad iniectabilia. The day before topical induction, the
application site was treated with 0.5 mL sodium lauryl sulfate 10% in Vaseline. Topical induction
(1 week after the intradermal induction) was 2 mL of a 50% concentration of glyphosate technical in
aqua ad iniectabilia applied for 48 hours. The challenge, 2 weeks after the intradermal induction, was
2 mL of the test material placed on a filter paper on the left flank of each guinea pig; a filter paper
with 2 mL vehicle was placed on the right flank as a control. The period of exposure was 24 hours,
with scoring at 24 and 48 hours after removal of the filter papers.
All challenge irritation scores (for the 10 test and five control guinea pigs) were zero. A
positive control assay with benzocaine gave the expected results. Glyphosate technical (purity 95.23%
and 96.4%) was determined to be not sensitizing to guinea pigs (Haferkorn, 2010f).
In a Magnusson–Kligman maximization test with female Hartley guinea pigs, a 5%

suspension of glyphosate technical (purity 97.56%) in paraffin oil was intradermally injected. Six
days later, the treatment site was treated with 10% sodium lauryl sulfate in white petrolatum; the
topical induction (the following day) was with 0.4 g of the test material preparation (25% test material
in white petrolatum) on a 2  4 cm piece of filter paper for 48 hours. The challenge application
(2 weeks after the topical induction) was 25% test material in white petrolatum for 24 hours, with
scoring at 24 and 48 hours following the end of this exposure.
None of the 20 induced guinea pigs and none of the 10 negative control guinea pigs showed
any signs of irritation at the application site following challenge. A positive control assay with 2,4-
dinitrochlorobenzene gave appropriate results.
The study concluded that glyphosate technical (purity 97.56%) had no dermal sensitization
potential in guinea pigs (Hideo, 1995c).
In a Magnusson–Kligman maximization test with male Hartley guinea pigs, a 10% w/v
dilution of glyphosate technical (purity 96.66%) in purified water and Freund’s Complete Adjuvant
was intradermally injected. Seven days later the application site was treated with the test material at
50% in purified water (about 0.3 mL applied on a 2  4 cm filter paper) with 48-hour exposure. Two
weeks later, the guinea pigs were treated with the test material at 15% in purified water (about 0.2 mL
applied on a 3  3 cm filter paper) for 24 hours, with scoring at 24 and 48 hours following the end of
this exposure.

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None of the 10 induced guinea pigs and none of the five control guinea pigs showed any signs
of irritation at the application site following challenge. A positive control assay with
α-hexylcinnamaldehyde gave the appropriate results.
Based on the results of this study, there is no sensitization potential of glyphosate technical
(purity 96.66%) in the guinea pig (Simon, 2009d).
In a Magnusson–Kligman maximization test with male Dunkin Hartley guinea pigs,

intradermal inductions were with a 0.5% concentration of glyphosate technical (purity 97.52% and
98.8%) in aqua ad iniectabilia. The day before the topical induction, the application site was treated
with 0.5 mL sodium lauryl sulfate 10% in Vaseline. Topical induction (1 week after the intradermal
induction) was 2 mL of a 50% concentration of glyphosate technical in aqua ad iniectabilia for 48
hours. The challenge was two weeks after the intradermal induction. Filter paper with 2 mL of the test
material was placed on the left flank; as a control, a filter paper with 2 mL of the vehicle was placed
the right flank. The period of exposure was 24 hours, with scoring at 24 and 48 hours after removal of
the filter papers.
and 98.8%) was found to be not sensitizing to guinea pigs (Haferkorn, 2009d).
In a dermal sensitization (Magnusson–Kligman maximization test with male Dunkin Hartley

guinea pigs), intradermal induction was with a 0.5% concentration of glyphosate technical (purity
96.6% and 97.3%) in aqua ad iniectabilia. The day before the topical induction, the application site
was treated with 0.5 mL sodium lauryl sulfate 10% in Vaseline. The topical induction (1 week after
the intradermal induction) was 2 mL of a 50% concentration of the test material in aqua ad
iniectabilia for 48 hours. The challenge was two weeks later: filter paper with 2 mL of the test
material was applied to the left flank for 48 hours, with filter paper with 2 mL of the vehicle applied
to the right flank. The period of exposure was 24 hours, with scoring at 24 and 48 hours after removal
of the filter papers.
and 97.3%) was found to be not sensitizing to guinea pigs (Haferkorn, 2010g,h).
In a Buehler method dermal sensitization study with glyphosate technical (purity 96.40% and
96.71%), 15 male and 15 female short-haired Hartley albino guinea pigs were divided into two
groups: group I (five males and five females) and group II (10 males and 10 females). For each
induction treatment, 400 mg of the test material was placed on a four-ply 2.5  2.5 cm gauze pad and
moistened with 2 mL deionized water. Each gauze pads was secured with non-irritating adhesive tape,
which in turn was covered with a strip of clear polyethylene film. Exposures lasted for at least 6 hours
and took place on days 1, 8 and 15. Group I animals were untreated during this period. After a 2-week
rest period, all animals (groups I and II) were challenged at a previously unexposed site with 400 mg
test material moistened with 2 mL deionized water.
All challenge irritation scores (for the 20 induced and 10 control guinea pigs) were zero. A
positive control assay with α-hexylcinnamaldehyde gave the expected results. Glyphosate technical
(purity 96.40% and 96.71%) did not elicit a sensitizing reaction in guinea pigs (You, 2009e).
In a Buehler method dermal sensitization study, a group of 20 male and 20 female Hartley
albino guinea pigs were exposed once a week to 0.4 g of 70% w/w glyphosate acid technical (purity
97.23%) in distilled water. The mixture was applied to the left side of each test animal using an
occlusive 25 mm Hill Top Chamber, which was secured in place and wrapped with non-allergenic

132
adhesive tape. After each 6-hour exposure, the chambers were removed and any residual test material
gently cleansed off. Twenty-seven days after the first induction dose, 0.4 g of a 70% w/w mixture of
the test material in distilled water was applied to a naive site on the right side of each guinea pig.
These sites were evaluated and scored approximately 24 and 48 hours after the challenge application.
A group of 10 controls was similarly treated with the vehicle alone.
There were no positive irritation scores (defined as > 0.5). A positive control assay with α-
hexylcinnamaldehyde gave the expected results. Glyphosate technical (97.23%) is not considered a
contact sensitizer (Merkel, 2005f).
In a dermal sensitization study (Buehler method), a group of 20 male Hartley guinea pigs
were treated three times with once-a-week 6-hour exposures to 1.0 mL of a 50% w/v solution of
glyphosate technical (purity 98.05%) in a DMSO vehicle. The solution was applied in a cotton lint
patch which covered approximately 6 cm2 of the left flank. A group of 10 control guinea pigs was
similarly treated with 1.0 mL DMSO. Two weeks after the last induction treatment, both induced and
control guinea pigs were exposed for 4 hours to 1.0 mL of a 50% w/v solution of test material in
DMSO on the right flank.
One of the 20 induced guinea pigs had a score of 1 (positive response) at 24 and 48 hours
following challenge. All of the other induced and control animals scored zero.
The study concluded that the epidermal application of glyphosate technical (purity 98.05%)
with DMSO as vehicle does not cause skin sensitization in guinea pigs according to the Buehler test
method (Lima Dallago, 2008).
In a dermal sensitization (Magnusson–Kligman maximization test) study with glyphosate

technical (purity 95.7%), the hair was clipped from an area approximately 4  6 cm on the shoulder
region of each of a group of 20 female Dunkin Hartley guinea pigs on day 0. A row of three injections
(0.1 mL each) was made on each side of the spine. The injections were: a) 1:1 Freund’s Complete
Adjuvant in isotonic sodium chloride; b) a 0.195% (v/v) formulation of the test material in isotonic
sodium chloride; c) a 0.195% (v/v) formulation of the test material in a 1:1 preparation of Freund’s
Complete Adjuvant plus isotonic sodium chloride. On day 6, the scapular region was treated with
10% sodium lauryl sulfate (10% in petroleum jelly). On day 7 the same area used for the intradermal
injections was treated with a 60% w/w mixture of the test material in distilled water, with 48-hour
occluded exposure. The challenge was approximately 2 weeks later. One site was treated with 60%
w/w mixture of the test material in distilled water; a second site was treated with a 30% w/w mixture
of the test material in distilled water. The sites were scored for irritation at 24 and 48 hours following
exposure.
A group of 10 control guinea pigs was similarly treated using the vehicle only during the
induction period.
All 18 induced guinea pigs (2 had died during the study) scored zero at 24 and 48 hours
following challenge, as did all 10 controls. The study reported that glyphosate technical (95.7)
produced a 0% (0/18) sensitization rate and was classified as a non-sensitizer to guinea pig skin under
the conditions of the test (Richeux, 2006).
In a dermal sensitization (Magnusson–Kligman maximization test) with glyphosate acid

(purity 95.6%), a group of albino Crl (HA) BR guinea pigs each had the hair clipped from an area
about 5  5 cm on the scapular region. A row of three injections (0.05–0.1 mL each) was made on
each side of the spine. The injections were: a) 1:1 Freund’s Complete Adjuvant in deionized water; b)
a 0.1% (w/v) preparation of the test material in deionized water; c) a 0.% (w/v) preparation of the test
material in a 1:1 preparation of Freund’s Complete Adjuvant plus deionized water. On day 6 the
application site was clipped and 0.5 mL of a 10% preparation of sodium lauryl sulfate in paraffin wax

133
applied. On day 7 the test area was treated with a topical application of the test material (75% w/v) in
deionized water. The preparation (0.2–0.3 mL) was put on a 4  2 cm piece of filter paper held in
place with surgical tape. The filter paper was covered by a strip of adhesive tape secured using self-
adhesive PVC tape. This occlusive dressing was kept in place for about 2 days. Ten control animals
were similarly treated with deionized water. Challenge (for both the induced animals and their
controls) was at approximately 21 days. An area about 15  15 cm on both flanks of the test and
control animals was clipped free of hair. An occlusive dressing was prepared using two pieces of
approximately 1  1.75 cm filter paper stitched to a piece of rubber sheeting (about 12  5 cm). A
75% w/v preparation of the test material in deionized water (0.05–0.1 mL) was applied to a piece of
filter paper and a 30% w/v preparation in deionized water (0.05–0.1 mL) applied to the second. These
were covered with strips of adhesive bandage (about 25–40 cm  7.5 cm) and secured with a self-
adhesive PVC tape. Exposure was for about 24 hours. The sites were scored for irritation at 24 and 48
hours following the end of exposure.
Exposure to the 75% w/v preparation resulted in mild and scattered redness (score of 1) in
three of the 20 induced and one of the 10 control animals at 24 hours only, with all scores zero at 48
hours. Because the redness was observed at similar incidences in both induced and control guinea pigs
and because it occurred only at 24 hours, it was considered to be due to skin irritation rather than the
test material. All sites exposed to the 30% w/v preparation scored zero at both 24 and 48 hours. A
positive control assay with α-hexylcinnamaldehyde demonstrated the sensitivity of the test system.
The study concluded that glyphosate acid is not a skin sensitizer under the test conditions
(Doyle, 1996d).

(a) Oral administration
Mice
In a 13-week oral toxicity study, groups of 15 male and 15 female CD-1 mice were fed diets
containing glyphosate (purity 98.7%) at dietary concentrations of 0, 5000, 10 000 or 50 000 ppm (0,
944, 1870 and 9710 mg/kg bw per day for males and 0, 1530, 2740 and 14 800 mg/kg bw per day for
the females, respectively).
There was no treatment-related mortality or clinical signs of toxicity, organ-weight change,
macroscopic and histopathological findings. At study termination, body-weight gains of the males and
females at 50 000 ppm were about 24% and 18% lower, respectively, than that of the control animals.
Body-weight gains of both males and females at 5000 ppm and 10 000 ppm were comparable to those
of the controls.
The no-observed-adverse-effect level (NOAEL) in the 13-week toxicity study in mice was
10 000 ppm (equal to 1870 mg/kg bw per day) based on reduced body weights at 50 000 ppm (equal
to 9710 mg/kg bw per day) (Tierney & Rinehart, 1979).
In a 13-week oral toxicity study, groups of 10 male and 10 female CD-1 mice were fed diets
containing glyphosate (purity 99.5%) at a concentration that was adjusted weekly to give doses of
200, 1000 or 4500 mg/kg bw per day. The animals were observed daily for symptoms of ill health and
mortality. Body weights and feed consumption were recorded weekly, and water consumption was
monitored throughout the study. Ophthalmoscopic examinations were performed during week 12.
Blood samples were collected from the orbital sinus for haematology (seven parameters) and from the
dorsal aorta at necropsy for clinical chemistry analysis (16 parameters). However, the small sample
volumes precluded analysis of total protein, albumin and cholesterol. All the animals were terminated
and necropsied, 13 organs were isolated and weighed, and about 35 separate tissues were fixed for
microscopy. All tissues from animals in the highest dose group and in the control group and the

134
kidneys, liver and lungs of animals at the lowest (200 mg/kg) and intermediate (1000 mg/kg) doses
underwent a full histopathological examination.
No treatment-related mortalities, clinical signs, haematological or biochemical findings and
no organ-weight changes were observed. Gross or histopathological examination did not show any
effects of glyphosate administration.
Taking into account the limited range of clinical chemistry parameters evaluated, the NOAEL
in the 13-week toxicity study in mice was 4500 mg/kg bw per day, the highest dose tested in this
study (Perry et al., 1991a).
In a 13-week oral toxicity study, groups of 10 male and 10 female B6C3F1 mice were fed
diets containing glyphosate (purity 99%) at concentrations of 0, 3125, 6250, 12 500, 25 000 or 50 000
ppm (equal to 0, 507, 1065, 2273, 4776 and 10 780 mg/kg bw per day for males and 0, 753, 1411,
2707, 5846 and 11 977 mg/kg bw per day for females). All tissues from the highest-dose and control
animals were examined microscopically. The salivary glands were also examined in all groups
receiving lower doses.
Reduced body-weight gain was observed at 25 000 and 50 000 ppm in both males and
females. There were no differences in feed consumption between control and treated mice. The only
significant gross finding in the study was a “dark” salivary gland in a male at the highest dose; no
other gross abnormalities were observed at necropsy. Histological changes were observed only in the
parotid salivary gland (Table 11). The cytoplasmic alterations consisted of a diffuse increase in the
basophilia of the acinar cells. In more severely affected glands, the cells and acini also appeared to be
enlarged and had fewer ducts. No histological changes were observed in the submandibular and
sublingual glands.
Table 11. Incidence and severity of cytoplasmic alteration of the parotid and submandibular
salivary glands (combined) in mice administered glyphosate for 13 weeks
No. of cases per dietary concentration of glyphosate
0 ppm 3 125 ppm 6 250 ppm 12 500 ppm 25 000 ppm 50 000 ppm
Males 0/10 010 5/10 (1.0) 9/10 (1.6) 10/10 (2.8) 10/10 (4.0)
Females 0/10 0/10 2/10 (1.0) 9/10 (1.3) 10/10 (2.4) 10/10 (3.1)
no.: number; ppm: parts per million
Results presented as number of mice showing cytoplasmic alterations / total number of mice in the group, with average
severity score in parentheses. Severity score is based on a scale of 1 = minimal, 2 = mild, 3 = moderate or 4 = marked.
Source: Chan & Mahler (1992)
The NOAEL in the 13-week toxicity study in mice was 3125 ppm (equal to 507 mg/kg bw per
day) based on parotid salivary gland lesions at 6250 ppm (equal to 1065 mg/kg bw per day) (Chan &
Mahler, 1992).
In a 13-week oral toxicity study, groups of 12 male and 12 female ICR(Crj:CD-1)SPF mice
were administered glyphosate (purity 97.56%) at dietary concentrations of 0, 5000, 10 000 or 50 000
ppm (equal to a mean daily glyphosate intake of 0, 600, 1221 and 6295 mg/kg bw per day for males
and 0, 765, 1486 and 7435 mg/kg bw per day for females).
There were no treatment-related clinical signs, mortality or ophthalmological and
haematological findings. At 50 000 ppm, mean body weights of the males were 91% that of the
controls from week 2 to the end of the treatment; body weights of females were comparable to that of
the controls. Similarly, feed consumption was slightly decreased in males at the highest dose. At

135
50 000 ppm, feed efficiency of males and females was lower than that of the controls at almost all
measuring points during the treatment.
At 50 000 ppm, females showed a significant treatment-related increase in creatine
phosphokinase (P < 0.01). Other statistically significant (P < 0.01) changes in clinical chemistry were
observed in high-dose male and female mice; however, these changes were minor and not associated
with any histological findings and not considered adverse. In all treated groups, males showed a
significant decrease in urinary pH. There were no abnormalities in females of any treated groups.
At 50 000 ppm, males and females showed significant (P < 0.01) increases in both absolute
and relative caecum weights (238% and 263%, respectively, for males, and 187% and 195%,
respectively, for females) (Table 12).
Table 12. Caecum weights of mice administered glyphosate for mice 13 weeks
Absolute and relative weight per dietary concentration of glyphosate
0 ppm 5 000 ppm 10 000 ppm 50 000 ppm
Males
Absolute weight ± SD (mg) a 624 ± 86 609 ± 116 718 ± 177 1 484 ± 359
Relative weight ± SD (%) 1.45 ± 0.19 1.38 ± 0.26 1.61 ± 0.33 3.82 ± 1.15**
Females
Absolute weight ± SD (mg) a 497 ± 96 474 ± 115 604 ± 123 958 ± 163**
Relative weight ± SD (%) 1.43 ± 0.26 1.37 ± 0.30 1.67 ± 0.42 2.79 ± 0.53**
ppm: parts per million; SD: standard deviation; **: P < 0.01
Relative weight expressed as (organ weight / body weight)  100.
a
At 50 000 ppm, both males and females showed significant increases in absolute weights (238% for males and 187%
females).
Source: Kuwahara (1995)
At 50 000 ppm, males and females showed a significant increase in incidence of distension of
the caecum (12/12 males and 10/12 females, in contrast to none in the control group). In addition, at
this dose males showed significant increases in incidence of cystitis (4/12 compared to none in the
control group). There were no significant changes in incidence in females. Although significant
increases in incidence of distension of the caecum were noted for males and females at necropsy,
histopathological examinations failed to reveal any abnormalities in the caecum.
The NOAEL in the 13-week toxicity study in mice was 10 000 ppm (equal to 1221 mg/kg bw
per day) based on the decrease in body weights in males, increase in absolute and relative caecum
weights in both sexes and increased incidence of distension of the caecum in both sexes at 50 000
ppm (equal to 6295 mg/kg bw per day) (Kuwahara, 1995).
Rats
In a 4-week range-finding study of oral toxicity, groups of five male and five female Sprague
Dawley rats were fed diets containing glyphosate (purity 97.7%) at concentrations of 0, 30 000,
40 000 or 50 000 ppm (equivalent to approximately 1500, 2000 and 2500 mg/kg bw per day).
No animals died during the study. The only clinical signs of toxicity were soft stools and/or
diarrhoea, which occurred in both sexes at all doses with diarrhoea being the predominant sign in
animals at the highest dose during the last 3 weeks of the study. Slightly reduced body-weight gains
were noted in both sexes at all the doses, although significant reductions consistently occurred only in
males and females at the highest dose (9.6% and 9.0%, respectively, after 4 weeks). Daily feed

136
consumption was reduced for males at the intermediate and highest dose during the first week of the
study. Feed intake for treated females was comparable to that of controls throughout the study. The
only clinical signs of toxicity were soft stools and/or diarrhoea, which occurred in both sexes at all
doses with diarrhoea being the predominant sign in animals at the highest dose during the last 3 weeks
of the study. Gross and microscopic pathology examinations revealed no treatment-related lesions.
Because of the frequent occurrence of soft stools and/or diarrhoea at all doses, no NOAEL
could be derived from this 4-week dietary toxicity study in rats (Reyna & Thake, 1989).
In a 4-week oral toxicity study, groups of five male and five female Sprague Dawley rats
were fed diets containing glyphosate (purity 99.5%) at a concentration that was adjusted weekly to
give doses of 0, 50, 250, 1000 or 2500 mg/kg bw per day. All the animals were terminated and
necropsied, and the livers, hearts, kidneys, spleens and adrenals of control and highest-dose animals
processed and examined histopathologically. Examination was subsequently extended to include the
kidneys from all females in all the groups.
Soft faeces were noted in three males in the highest-dose group during weeks 3 to 4, but not
in any other group. No treatment-related effects were observed on mortality, clinical signs of toxicity,
body weights, feed and water consumption or haematological parameters. In males, equivocal
increases in plasma alanine transaminase [alanine aminotransferase] and alkaline phosphatase
activities were observed at 250, 1000 or 2500 mg/kg bw. In females, plasma alanine transaminase
activity was significantly increased at the highest dose, as was total bilirubin. In addition, increased
plasma concentrations of phosphate were noted in males at 1000 or 2500 mg/kg bw. There were
neither notable intergroup differences in organ weights nor gross pathological findings. However, an
increase in the incidence of very mild to slight nephrocalcinosis was observed in female rats at 250
mg/kg bw and higher doses (Table 13).
Table 13. Nephrocalcinosis in rats administered glyphosate for 4 weeks

No. per dietary concentration of glyphosate
Males Females
0 50 250 1 000 2 500 0 50 250 1 000 2 500
mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
bw per bw per bw per bw per bw per bw per bw per bw per bw per bw per
day day day day day day day day day day
No. of cases 0 NI NI NI NI 0 0 2 2 4
No. of very
mild/minimal
cases 0 NI NI NI NI 0 0 1 1 2
No. of mild/slight
cases 0 NI NI NI NI 0 0 1 1 2
bw: body weight; NI: not investigated; no.: number
Source: Atkinson et al. (1989)
The NOAEL in the 4-week dietary toxicity study in rats was 50 mg/kg bw per day for slight
nephrocalcinosis in female rats at 250 mg/kg bw per day (Atkinson et al., 1989). This finding was not
confirmed in a separate study by Perry et al., 1991b.
In a 90-day oral toxicity study, groups of 12 male and 12 female Sprague Dawley rats were
fed diets containing glyphosate (purity 95.2%) at concentrations of 0, 1000, 5000 or 20 000 ppm
(calculated mean intakes equal to 0, 63, 317 and 1267 mg/kg bw per day for males and 0, 84, 404 and
1623 mg/kg bw per day for females). Clinical signs, body weight, feed consumption, haematology

137
and clinical chemistry parameters were monitored routinely. Gross examinations were performed for
all groups, and the kidneys, liver and testes weighed after termination. A standard range of tissues
from control and highest-dose animals was microscopically examined as well as the kidneys, livers
and lungs from animals at all doses.
No treatment-related effects were observed at up to the highest dose. However, parotid
salivary glands were not included in the histopathological examination.
The NOAEL in the 90-day dietary toxicity study in rats was 20 000 ppm (equal to 1267
mg/kg bw per day), the highest dose tested (Stout & Johnson, 1987).
In a 13-week oral toxicity study, groups of 10 male and 10 female Sprague Dawley rats were
fed diets containing glyphosate (purity 98.6%) at concentrations that were adjusted weekly to doses of
0, 30, 300 or 1000 mg/kg bw per day. All tissues from control and highest-dose animals, in addition to
the kidneys, liver, lungs and parotid salivary glands of all the test animals, underwent a full
There were no mortalities, clinical signs or changes in body or organ weights, feed and water
consumption, haematological parameters and ophthalmoscopic and macroscopic findings. Females at
the highest dose showed slight but statistically significant increases in concentrations of glucose
(11%; P < 0.05), total protein (9%; P < 0.001), albumin (9%; P < 0.05) and creatinine (8%; P < 0.01)
compared with those in the control group. Urinalysis revealed a reduction in pH in males at the
highest dose.
In contrast to results from a 4-week study in rats conducted at the same testing facility
(Atkinson et al., 1989), the incidence of nephrocalcinosis in this 13-week study was evenly distributed
in dose groups and sexes and was not dose dependant; it is therefore clearly not treatment related.
An increase in the incidence of cellular alterations (deep basophilic staining and enlargement
of cytoplasm) was observed in the parotid salivary glands of both sexes in all treated groups. In
addition, the severity (graded as very mild, mild, moderate, severe and very severe) of these findings
showed a dose-related increase, but only reached statistical significance in males at the highest dose
(Table 14), suggesting these changes are of equivocal toxicological significance.
Table 14. Cytoplasmic alteration of the parotid salivary gland in rats administered glyphosate in the
diet for 13 weeks
Males Females
300 1 000 300 1 000
0 mg/kg 30 mg/kg mg/kg mg/kg 0 mg/kg 30 mg/kg mg/kg mg/kg
day day day day day day day day
Severity a
Very mild 3 7 6 0 2 7 7 1
Mild 0 0 3 2 0 1 2 4
Moderate 0 0 1 3 0 0 0 3
Severe 0 0 0 5* 0 0 0 1
Total incidence 3 7 10** 10** 2 8* 9** 9**
bw: body weight; no.: number; *: P < 0.05; **: P < 0.01
a
Severity graded as very mild, mild, moderate, severe and very severe.
Source: Perry et al. (1991b)

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The NOAEL in this 90-day toxicity study in rats was 300 mg/kg bw per day based on the
more pronounced severity of cellular alterations in the parotid salivary gland at 1000 mg/kg bw per
day (Perry et al., 1991b).
In a 13-week oral toxicity study, groups of 10 male and 10 female F344/N rats were fed diets
containing glyphosate (purity 99%) at concentrations of 0, 3125, 6250, 12 500, 25 000 or 50 000 ppm.
Ten more animals of each sex were included at each dietary concentration for evaluation of
haematological and clinical pathology parameters. The calculated mean intakes were equal to 0, 205,
410, 811, 1678 and 3393 mg/kg bw per day, respectively, for males and 0, 213, 421, 844, 1690 and
3393 mg/kg bw per day, respectively, for females. All tissues from the control and highest-dose
animals were examined microscopically. Salivary glands were also examined for the animals at all
lower doses.
Diarrhoea was seen in males at the highest dose and in all females for the first 50 days of the
study. Weight gain was reduced in males at 50 000 and 25 000 ppm, and the final mean body weight
was approximately 18% and 6% less than that of controls, respectively. Small increases in several
erythrocyte parameters were noted in males at 12 500 ppm and higher doses. These changes were
unremarkable and generally consistent with a mild dehydration. Plasma alkaline phosphatase and
alanine transaminase activities were slightly increased in males at 6250 ppm and greater and in
females at 12 500 ppm and greater. In the absence of histopathological findings in the liver, these
increases are considered not toxicologically significant.
No treatment-related gross abnormalities or organ-weight changes were observed at necropsy.
Histopathological changes were observed only in the parotid and submandibular glands of both male
and female rats. The study authors combined the findings for these two glands (Table 15). The
findings for each gland individually or for individual animals were not reported. No histological
alterations were observed in the sublingual gland. The changes were described as cytoplasmic
alterations and consisted of basophilic changes and hypertrophy of the acinar cells. Considering the
16-fold difference between the lowest dose of 3125 ppm and the highest dose of 50 000 ppm, the
incidence response curve appears to be relatively flat and the degree of change is slight, progressing
from only minimal to moderate, suggesting that any changes are of equivocal toxicological
significance.
Table 15. Cytoplasmic alterations of the parotid and submandibular salivary glands (combined) in
rats administered glyphosate for 13 weeks
Incidence per dietary concentration of glyphosate
0 ppm 3 125 ppm 6 250 ppm 12 500 ppm 25 000 ppm 50 000 ppm
Males 0/10 6/10 (1.0) 10/10 (1.0) 10/10 (1.8) 10/10 (2.7) 10/10 (2.9)
Females 0/10 8/10 (1.0) 10/10 (1.0) 10/10 (2.1) 10/10 (2.4) 10/10 (1.0)
ppm: parts per million
Results presented as number of rats showing cytoplasmic alterations / total number of rats in the group, with average severity
score in parentheses. The severity score is based on a scale of 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.
Source: Chan & Mahler (1992)
The NOAEL in the 13-week dietary toxicity study in rats was 6250 ppm (equal to 410 mg/kg
bw per day) based on the more pronounced cellular alterations in the salivary glands at 12 500 ppm
and above (Chan & Mahler, 1992).
In a 90-day range-finding study, groups of 10 Sprague Dawley rats per sex were administered
daily doses of glyphosate technical (purity 97.5%) at concentrations of 0, 2000, 6000 and 20 000 ppm

139
(equal 0, 125.2, 371.9 and 1262.1 mg/kg bw per day for males and 0, 156.3, 481.2 and 1686.5 mg/kg
bw per day for females) in the diet. Blood was collected pretreatment and at termination to measure
selected haematological and clinical chemistry parameters. At necropsy, selected organs were
weighed. Histopathological examination was conducted on all tissues taken at necropsy.
Diets were homogeneously distributed and stable for at least 10 days. Analytical
concentrations were within 10% of the nominal concentrations. No treatment-related effects was
observed on mortality, body weights, body-weight gains, feed consumption, urine analysis,
haematology and clinical chemistry parameters, ophthalmoscopic examination, organ weights and
macroscopic and microscopic examinations. The only obvious treatment-related clinical observations
was diarrhoea seen in all 10 males and nine females in the 20 000 ppm treatment group.
The NOAEL in the 90-day toxicity study in rats was 6000 ppm (equal to 371.9 mg/kg bw per
day) based on diarrhoea at the lowest-observed-adverse-effect level (LOAEL) of 20 000 ppm (Parker,
1993).
In a 13-week feeding study, groups of 12 Sprague Dawley rats per sex were administered
daily dietary doses of glyphosate (purity 95.3 %) at concentrations of 0, 3000, 10 000 and 30 000 ppm
(equal to 0, 168.4, 569 and 1735 mg/kg bw per day for males and 0, 195.2, 637 and 1892 mg/kg bw
per day for females) in the diet.
There were no treatment-related mortalities or clinical signs of toxicity. At 30 000 ppm, body
weights were slightly lower (by about −5 to −10% in males and −5% in females) than those in the
control. The overall feed consumption by males and females was comparable to the control. No
treatment-related ocular effects or changes in haematological and clinical chemistry parameters were
observed. At 30 000 ppm, urine pH in males and females was significantly lower (P < 0.01) than that
in the control. Urine protein was significantly decreased (P < 0.05) in males and showed a decreasing
trend in females. In addition, females showed a significantly (P < 0.05) higher urine volume, but
males showed a decreasing trend in urine volume compared with the controls. At 10 000 ppm, urine,
pH and protein in males were lower than those in the controls. In females, no statistically significant
changes were observed in any parameter. No statistically significant changes were observed in either
sex at 3000 ppm.
At 30 000 ppm, both sexes showed significant (P < 0.01) increases in absolute and relative
weights of the caecum (with contents). In addition, females in this highest-dose group also showed
significant (P < 0.05) increases in relative weights of the brain and liver. At 10 000 ppm, the absolute
and relative weight of the caecum showed a statistically significant (P < 0.01) increase in males and
increasing trend in females. At 3000 ppm, there were no treatment-related abnormalities in either sex
(Table 16).

140
Table 16. Caecum weights of rats administered glyphosate for 13 weeks

Absolute and relative weight per dietary concentration of glyphosate
0 ppm 3 000 ppm 10 000 ppm 30 000 ppm
Males
Absolute weight ± SD (mg) 2 823 ± 794 3 187 ± 609 3 383 ± 1 081 (11%) 5 854 ± 2 053**
Relative weight ± SD (%) 0.55 ± 0.16 0.62 ± 0.13 0.64 ± 0.20 (11%) 1.22 ± 0.41**
Females
Absolute weight in mg ± SD 2 367 ± 582 2 586 ± 462 3 546 ± 959* 5 268 ± 1 189**
Relative weight ± SD (%) 0.79 ± 0.17 0.84 ± 0.17 1.22 ± 0.32* 1.92 ± 0.41**
ppm: parts per million; SD: standard deviation; *: P < 0.05; **: P < 0.01
Relative weight = (organ weight/body weight)  100.
Results expressed as absolute weight or relative weight and, in parentheses, this weight as a percentage of that of controls for
males only.
Source: Kinoshita (1995)
At 30 000 ppm, 9 of the 12 males and 7 of the 12 females had statistically significantly
distended caeca (P = 0.01). At 10 000 ppm, 3 of the 12 males showed distension of the caecum, but
there were no macroscopic abnormalities in females. At 3000 ppm, there were no macroscopic
abnormalities attributable to the treatment in either sex.
Although histopathological examinations revealed various histological changes in each
treatment group of both sexes, treatment-related changes were not observed. One male at 10 000 ppm
and one female at 30 000 ppm had renal lesions (polycystic kidney) and hepatic lesions (bile ductal
proliferation and cholangiectasis). However, these were considered of a genetic nature and not
treatment related.
The NOAEL in this 90-day toxicity study in rats was 3000 ppm (equal to 168.4 mg/kg bw per
day) based on increased caecum weight at 10 000 ppm and above (Kinoshita, 1995).
In a 90-day oral toxicity study, groups of 12 male and 12 female Alpk:AP Wistar-derived rats
were fed diets containing glyphosate (purity 97.4%) at concentrations of 0, 1000, 5000 or 20 000 ppm
(equal to mean intakes of 0, 81, 414 and 693 mg/kg bw per day for males and 90, 447 and 1821 mg/kg
bw per day for females).
There were no mortalities. A low incidence of diarrhoea and light-coloured faeces was seen in
both sexes at 20 000 ppm in the second week of the study. Males at the highest dose showed
statistically significant reductions in body-weight gain and food utilization efficiency compared with
controls. There was some evidence for a reduction in platelet count in males and females at 5000 and
20 000 ppm. A marginal dose-related increase in prothrombin time was observed in males at all doses.
The differences, however, were small and considered not of haematological significance. Plasma
alkaline phosphatase and alanine transaminase activities were increased in both sexes at 20 000 ppm
and, to a lesser extent, in males at 5000 ppm. In addition, plasma aspartate aminotransferase activity
was increased in females at the highest dose at this early time point, but not at study termination. The
changes in clinical chemistry parameters were small, often lacking a clear dose–response relationship,
and therefore not considered biologically relevant. There were no treatment-related effects on urine
biochemistry and organ weights.
The only notable histopathological finding was a uterine leiomyosarcoma in a female at
5000 ppm. Although these are rare, finding such a tumour in an animal at the intermediate dose was
considered incidental to treatment.
The NOAEL in the 90-day toxicity study in rats was 5000 ppm (equal to 414 mg/kg bw per
day) based on the reduced growth in males at 20 000 ppm (Botham, 1996).

141
In a 90-day feeding study, groups of 10 Sprague Dawley rats per sex were administered daily
doses of glyphosate (purity 95.3%) at concentrations of 0, 1000, 10 000 or 50 000 ppm (equal to 0,
79, 730 and 3706 mg/kg bw per day for males and 0, 90, 844 and 4188 mg/kg bw per day for females)
in the diet.
There were no deaths. Animals of both sexes treated with 50 000 ppm had soft faeces and
diarrhoea throughout the study period from day 4. Both sexes at 50 000 ppm showed a reduction in
body-weight gain over the first 4 weeks of treatment. Body-weight development was unaffected at the
other doses. Both males and females at 50 000 ppm showed a reduction in dietary intake and feed
efficiency over the first 4 weeks of treatment compared with controls. Water consumption, measured
ocular parameters or haematological parameters for either sex were unaffected. Both males and
females at 10 000 or 50 000 ppm showed a statistically significant (P < 0.05 at 10 000 ppm and
P < 0.01 at 50 000 ppm) reduction in plasma calcium concentration and an increase in alkaline
phosphatase compared with controls. A statistically significant (P < 0.05) increase in inorganic
phosphorus and reduction in plasma creatinine were also evident in males and females at 50 000 ppm,
while females at this dose level showed statistically significant (P < 0.01) reductions in total plasma
protein and albumin compared with controls. There were no other treatment-related effects. Both
males and females at 50 000 ppm showed statistically significant increases in relative liver and kidney
weights compared with controls (Table 17).
Table 17. Group mean relative organ-weights of rats administered glyphosate for 90 days
Mean relative organ weight (%)
Dietary Liver Kidney

concentration of
glyphosate (ppm) Male Female Male Female
0 2.974 9 ± 0.2629 2.973 4 ± 0.1558 0.586 1 ± 0.0575 0.651 6 ± 0.0523
1 000 2.886 8 ± 0.2552 2.909 3 ± 0.2146 0.590 1 ± 0.0804 0.6257 ± 0.0375
10 000 2.885 3 ± 0.3758 2.980 1 ± 0.1556 0.607 0 ± 0.0552 0.645 4 ± 0.0532
50 000 3.243 3 ± 0.2452* 3.198 9 ± 0.2098* 0.6963 ± 0.0436** 0.718 0 ± 0.0707*

ppm: parts per million; *: P < 0.05; **: P < 0.001
Results expressed as mean organ-weight as a percentage of mean body-weight, ± standard deviation.
Source: Coles et al. (1996)
At 50 000 ppm all animals had enlarged and fluid-filled caeca while one female had gaseous
distension of the stomach at the final termination. There were no treatment-related macroscopic
abnormalities at 10 000 or 1000 ppm.
Treatment-related changes were observed in the caeca. Atrophy, characterized by flattening of
the intestinal mucosa, was observed in five rats of both sexes at 50 000 ppm (P < 0.05 for male rats)
and for one male and two female rats at 10 000 ppm. The etiology of this change is uncertain and may
represent no more than atrophy of the mucosa resulting from caecal distension. There were no other
treatment-related changes.
The NOAEL in this 90-day toxicity study in rats was 1000 ppm (equal to 79 mg/kg bw per
day) based on the reduced plasma calcium concentration and increased alkaline phosphatase
concentrations at 10 000 ppm (Coles et al., 1996).

142
Dogs
In a 7-day oral toxicity study, one male and one female beagle dog were fed gelatin capsules
containing glyphosate (purity 99.5%) at increasing daily doses of 100, 300 or 1000 mg/kg bw per day.
A second pair of dogs were administered gelatin capsules containing glyphosate at a dose of 1000
mg/kg bw per day for 14 consecutive days.
In the first pair of dogs, no treatment-related clinical signs or effects on body weight, body-
weight gain, feed consumption and haematological parameters were observed. There was a slight
increase in plasma alanine transaminase activity in the male dog, and cholesterol concentrations were
slightly reduced in both the male and female. At termination, there were no treatment-attributable
lesions.
In the second pair of dogs, no treatment-related clinical signs or effects on body weight, body-
weight gain, feed consumption and haematological parameters were observed. Plasma alanine
transaminase activity was slightly increased in the male dog which also had loose faeces throughout
the study. No treatment-attributable lesions were found at termination (Goburdhun & Oshodi, 1989).
Glyphosate technical (purity 94.61%) was continuously fed in the basal diet to groups of four
males and four females beagle dogs for at least 90 days. Dietary concentrations were 0, 1600, 8000
and 40 000 ppm (equal to 0, 39.7, 198 and 1015 mg/kg bw per day for males and 0, 39.8, 201 and
1014 mg/kg bw per day for females).
There were no treatment-related effects on mortality, clinical signs, body weight, feed
consumption, test material intake, ocular changes or macroscopic findings.
Although statistically significant changes in haematology parameters and in some clinical
chemistry parameters were observed in both sexes, these were not dose dependent. At 40 000 ppm,
three females showed a decrease in urine pH at week 13, although these differences were not
statistically significant. Although a statistically significant increase was noted in the relative weight of
the adrenals in females at 1600 ppm, the change was considered incidental due to the lack of dose
dependency. There were no histopathological changes related to the treatment in the treated groups of
either sex. One female in the 40 000 ppm group showed cutaneous histiocytoma which is a
nonspecific lesion in young dogs.
The NOAEL in this 90-day toxicity study in dogs was 40 000 ppm, equal to 1015 mg/kg bw
per day, the highest dose tested (Yoshida, 1996).
Glyphosate acid (purity 99.1%) was administered at doses of 0, 2000, 10 000 or 50 000 ppm
(equal to 0, 68, 323, 1680 mg/kg bw per day for males and 0, 68, 334, 1750 mg/kg bw per day for
females) via the diet for 90 days to one control and three treatment groups each with four male and
four female beagle dogs.
There was neither any mortality nor any treatment-related clinical signs of toxicity. The body-
weight gain of males at the highest dose showed a slight depression throughout the study, but the
differences were not statistically significant. Females at 50 000 ppm showed occasionally statistically
significant slight depressions in body-weight gains throughout the study. No treatment-related
ophthalmological and haematological findings or differences in urine clinical chemistry parameters
and urinary sediment examinations were observed. Changes in clinical chemistry parameters were
small and therefore not considered biologically relevant. Kidney weights of males at 10 000 or 50 000
ppm were slightly but not dose dependently increased. There was also a small increase in liver weight
at these doses, but in male dogs only. No macroscopic or microscopic findings were observed.
The NOAEL in this 90-day toxicity study in dogs was 10 000 ppm (equal to 323 mg/kg bw
per day) based on the decreased body-weight gains in female dogs at 50 000 ppm (Hodge, 1996).

143
In a 90-day feeding study, groups of four beagle dogs per sex were administered glyphosate
technical (purity > 95%) at daily doses of 0, 200, 2000 and 10 000 ppm in the diet (corresponding to
0, 5.3, 53.5 and 252.6 mg/kg bw per day).
All the animals survived until scheduled necropsy. Neither clinical signs of toxicity nor
treatment-related effects on body weights, urine analysis, organ weights, gross pathology or
histopathology were observed.
A significant increase in clotting time and gamma-glutamyltransferase activity was observed
in both sexes at the 45-day interim bleed; however, in the absence of any corresponding changes at
terminal bleed or any histopathological correlate in the liver, this observation is considered to reflect a
systemic error rather than a real effect. Total bilirubin was higher; however, in the absence of a
histopathological correlate on the liver, the effect was not considered adverse.
The NOAEL in this 90-day toxicity study in dogs was 10 000 ppm (equal to 252.6 mg/kg bw
per day), the highest dose tested (Prakash, 1999).
In a 13-week oral toxicity study, groups of four beagle dogs per sex were administered
glyphosate (purity 95.7%) in daily doses of 0, 30, 300 and 1000 mg/kg bw by capsule.
One male and one female at 1000 mg/kg bw per day were euthanized in extremis; one male
that vomited once in week 7 (before dosing) and had liquid faeces frequently in weeks 8 and 9 was
euthanized on day 61. One female was euthanized on day 72; this animal had frequent liquid or soft
faeces from week 4, was seen to vomit in week 10, and was dehydrated from week 9.
No treatment-related clinical signs were noted in the control animals or those at 30 or 300
mg/kg bw per day. The following treatment-related clinical signs were reported in animals at 1000
mg/kg bw per day (excluding those terminated in extremis, which are discussed separately): liquid or
soft faeces on several occasions in all animals; vomiting in two of the three surviving females within
30 minutes or 3–5 hours after treatment; thin appearance in one of the three surviving males and all
the females; dehydration in one of the three males and two of the three females; pale ears and mouth
in one of the three females.
At 30 or 300 mg/kg bw per day, there were no histopathological changes or changes in the
mean body-weight gain. At 1000 mg/kg bw per day, mean body-weight gain in males was slight (+4%
vs +31% in controls) while females lost weight (−7% vs +14% in controls) from day 1. This effect on
body weight was considered treatment-related. Feed consumption was reduced to 25–75% of the
amount given. Neither ophthalmological findings nor treatment-related effects on haematological and
clinical chemistry parameters were observed in any of the treated groups. Urinalysis showed a
decrease in mean specific gravity in one of the three remaining males and all three remaining females
at the highest dose in week 11. Mean absolute and relative prostate weights were reduced by 68% and
56%, respectively, but there were no other treatment-related effects on organ weights. All the
macroscopic changes noted in surviving animals at termination were considered normal variations,
except for the reduced uterus size.
The treatment-related changes in surviving animals at 1000 mg/kg bw per day consisted of
increased number of adipocytes in the sternum of two of the three males and the three females,
prostate atrophy in two of the three males and uterine atrophy in two of the three females.
The NOAEL in this 90-day toxicity study in dogs was 300 mg/kg bw per day for mortality
and decreased body-weight gains at 1000 mg/kg bw per day (Gaou, 2007). This study found very
pronounced toxic effects, results which differ considerably from what was seen in other studies in
dogs or other species.
In a 52-week oral toxicity study, groups of six male and six female beagle dogs were fed
gelatin capsules containing glyphosate (purity 96.13%) at a dose of 0, 20, 100 or 500 mg/kg bw per
day once daily.

144
All the dogs survived. There were no treatment-related effects on body or organ weights or
feed consumption and no clinical signs of toxicity, ocular abnormalities or changes in haematological
or urinary parameters or macroscopic and histological findings.
The NOAEL in this 1-year toxicity study in dogs was 500 mg/kg bw per day, the highest dose
tested (Reyna & Ruecker, 1985).
In a 1-year oral toxicity study, groups of four male and four female beagle dogs were
administered gelatin capsules containing glyphosate (purity 98.6–99.5%) at concentrations of 0, 30,
300 or 1000 mg/kg bw per day once daily for 52 weeks.
There were no mortalities throughout the test period. Changes in faecal consistency
(soft/loose/liquid) were recorded frequently for the highest-dose animals, starting 4 to 6 hours after
dosing; these were also noted on occasion in a few animals at 300 mg/kg bw and were considered to
be treatment related. Feed consumption was maximal or near maximal for all test groups. Mean body-
weight gain showed a non-statistically significant reduction in males at all doses (approximately 83%,
75% and 75% that of the control group for the lowest, intermediate and highest doses, respectively)
and in females at the highest dose (81% that of the control group). Ophthalmoscopic and laboratory
examinations revealed no treatment-related abnormalities. Plasma glyphosate concentrations, which
remained constant throughout the study, suggested that absorption was dose related; mean values
detected were 0.36, 1.82 and 6.08 μg/mL for the lowest, intermediate and highest doses, respectively.
At necropsy, no abnormal gross findings and no significant intergroup organ-weight differences
attributable to treatment with glyphosate were noted. In males, absolute and relative weights of the
liver were slightly but nonsignificantly increased (4%, 8% and 10% above that of the control group
for absolute weights, and 10%, 17% and 19% above that of the control group for relative weights for
the groups for the lowest, intermediate and highest doses, respectively). There were no significant
histopathological findings at any dose.
The NOAEL in this 52-week study in dogs was 300 mg/kg bw per day based on the changes
in faecal consistency (Goburdhun, 1991).
In a 52-week oral toxicity study, groups of four male and four female beagle dogs were fed
diets containing glyphosate (purity 95.6%) at concentrations of 0, 3000, 15 000 or 30 000 ppm (equal
to 0, 91, 440 and 907 mg/kg bw per day for males and 0, 92, 448 and 926 mg/kg bw per day for
females) for 1 year. Selected organs were weighed and specified tissues taken from all groups for
There were no mortalities during the study. There was no effect on feed consumption; only
three dogs left small amounts of feed intermittently during the study. Body weight was slightly
reduced in females at 30 000 ppm, with a maximum reduction of 11% (compared with that of
controls) in week 51. These dogs showed a gradual reduction in growth rate which was consistently
significant from week 23 onwards. A similar change in body-weight gain in females at the lowest
dose, although occasionally reaching statistical significance, was not regarded as treatment related
since a dose–response relationship was lacking. There was no effect on body weight in males at any
dose tested. There were no toxicologically significant effects on any of the haematological and
clinical chemistry parameters measured or any of the clinical chemical parameters measured in urine.
No adverse effects of glyphosate were seen at necropsy, and there were no treatment-related effects
on organ weights. No histopathological changes attributable to administration of glyphosate were
found.
The NOAEL in this 1-tear toxicity study was 15 000 ppm (equal to 448 mg/kg bw per day)
based on the reduced body weights at 30 000 ppm in female dogs (Brammer, 1996).

145
In an 12-month oral toxicity study, groups of four male and four female beagle dogs were
administered glyphosate technical (purity 94.61%) in the diet at concentrations of 0, 1600, 8000 or
50 000 ppm (equal to 0, 34.1, 182 and 1203 mg/kg bw per day for males and 0, 37.1, 184 and 1259
mg/kg bw per day for females, respectively) for 1 year. A detailed histopathological examination was
performed on all sampled tissues of all dogs, except for the femur, larynx, oviducts, tongue, ureter and
vagina.
There were no deaths in any dose groups of either sex. No treatment-related effects were
observed during periodic clinical and eye examinations, in urine analysis, weight change and
macroscopic and histopathological findings. At 50 000 ppm, three of the four males and four of the
four females had loose stools. The animals in the 8000 and 1600 ppm groups did not show any
clinical signs. At the end of the study, mean body weights at 50 000 were reduced by 6% in males and
11% in females compared to the controls, but these reductions were not statistically significant. Feed
consumption was unaffected.
Males showed no significant changes in any haematological parameters. Females at
50 000 ppm had significantly decreased haematocrit, haemoglobin concentrations and erythrocyte
count. However, these changes were small and often lacked a dose–response, and so were not
considered biologically relevant.
Females at 50 000 ppm showed significant changes in clinical chemistry parameters.
However, these changes were within biological variability ranges and therefore not considered
adverse.
The NOAEL in this 12-month toxicity study was 8000 ppm (equal to 182 mg/kg bw per day
based on the loose stools in both sexes and decreased body weights in females at 50 000 ppm
(Nakashima, 1997).
In a 12-month oral toxicity study, groups of four beagle dogs per sex were administered 0, 30,
100 and 300 mg/kg bw per day glyphosate technical (purity 97.5%) daily in gelatin capsules. Dose
formulations were prepared weekly by adding the required amount to the capsules.
No deaths occurred in any group. At the highest dose, all males and females had soft stools,
diarrhoea or mucous faeces and, rarely, bloody stools or faeces visibly containing the test material as
well as vomiting. At 100 mg/kg bw per day, changes were similar to those observed at 300 mg/kg but
at lower frequencies. A histopathological examination of a mid-dose male with bloody faeces
continually from day 346 onward showed an ulcer by intussusception. Changes observed at 30 mg/kg
bw per day were comparable to those observed in untreated animals. A significant decrease in body
weight compared to that of the control group was recorded from week 24 in females at 300 mg/kg
(P < 0.01) and from week 27 in females at 30 mg/kg (P < 0.05) largely continually until the end of the
administration period. There were no treatment-related effects in females at 100 mg/kg. There were
no treatment-related changes in feed consumption, urine analysis, haematology, blood biochemistry,
ophthalmoscopy, organ weights, necropsy or histopathology.
The NOAEL was 30 mg/kg bw per day based on the changes in faecal consistency in male
and female dogs and reduced body weights in females at the LOAEL of 100 mg/kg bw per day group
(Teramoto, 1998).
In a 1-year oral toxicity study, groups of four beagle dogs per sex were administered
glyphosate technical (purity 95.7%) at daily doses of 0, 30, 125 and 500 mg/kg bw per day in gelatin
capsules for 52 consecutive weeks.
No mortalities occurred during treatment. There were no treatment-related effects on clinical
signs, body weight, feed consumptions, haematology and clinical chemistry parameters,
ophthalmoscopic findings, organ weights, macroscopic or microscopic findings.

146
The NOAEL in this 1-year toxicity study in dogs was 500 mg/kg bw per day, the highest dose
tested (Haag, 2008).
(b) Dermal application

Rats
In a 21-day dermal toxicity study, groups of five male and five female Alpk:APfSD rats were
exposed to glyphosate (purity 95.6%) at 0, 250, 500 or 1000 mg/kg bw per day. The test material was
moistened with deionized water and the resultant paste spread on the previously clipped back of each
of the animals on a gauze patch that was covered with occlusive dressing. The application site was
rinsed after 6 hours of exposure. A total of 15 six-hour applications were made over 21 days.
No treatment-related effects were noted on mortality, body or organ weights, body-weight
gains, feed consumption, haematology, clinical chemistry parameters, macroscopic findings and
histopathological findings at any doses.
The systemic toxicity NOAEL in this 21-day dermal toxicity study in rats was 1000 mg/kg
bw per day, the highest dose tested (Pinto, 1996).
Rabbits
In a 21-day GLP-compliant dermal toxicity study, groups of 10 male and 10 female New
Zealand White rabbits were exposed to glyphosate (purity not reported) at 0, 100, 1000 or 5000 mg/kg
bw per day. The test material was moistened with physiological saline and applied onto the skin,
which was then covered with a gauze patch secured with a tape. The material was applied on intact
skin (5/sex per dose) and abraded skin (5/sex per dose) for 6 hours per day, 5 days per week, for 3
weeks. Physiological saline only was applied onto the control group.
There were no deaths and no clear effects on clinical condition. Slight dermal irritation was
noted in both intact and abraded skin at 5000 mg/kg bw per day but not at milder doses or the control.
No treatment-related effects were observed on body weights, body-weight gains, feed consumption,
haematology and clinical chemistry parameters at any doses. At termination, no treatment-related
macroscopic lesions were observed at the application site or in any other tissues or organs from all test
groups. No treatment-related variations in organ-weight or histopathological findings were noted.
The systemic toxicity NOAEL in the 21-day dermal toxicity study in rabbits, was 5000 mg/kg
bw per day, the highest dose tested (Johnson, 1982).
In a 28-day dermal toxicity study, groups of five male and five female New Zealand White
rabbits were exposed to glyphosate (purity 99.6%) at 0, 500, 1000 or 2000 mg/kg bw per day. The test
material was homogenized in water, placed on a gauze pad and then applied to the clipped area of
rabbit skin. The pad was covered with a sheet of polyethylene material secured with tape. The test
material covered approximately 10% of the body surface area.
No treatment-related effects were noted on mortality, body weights, body-weight gains, feed
consumption, haematology, clinical chemistry parameters, macroscopic findings, organ weights and
histopathological findings at any dose. Very slight erythema was observed in 2000 mg/kg bw per day
dose group.
The systemic toxicity NOAEL in the 28-day dermal toxicity study in rabbits was 2000 mg/kg
bw per day, the highest dose tested (Tornai, 1994).

147

Mice
In an unpublished non-GLP carcinogenicity study, glyphosate (purity 99.7%) was
administered in the diet to groups of 50 male and 50 female CD-1 mice per dose at concentrations of
0, 1000, 5000 or 30 000 ppm (equal to 0, 157, 814, 4841 mg/kg bw per day, respectively, for males
and 0, 190, 955, and 5874 mg/kg bw per day, respectively, for females) for 24 months. Cage-side and
detailed clinical observations were conducted and body weight and feed intake monitored throughout
the study. Water consumption was measured during months 12 and 24. Erythrocyte, as well as total
white blood cell counts and differentials, were conducted at months 12, 18 and 24. Tissues and organs
were collected from all mice whether they died during the study or were terminated. Microscopic
analyses were conducted on all collected tissues.
Analysis of treated diets demonstrated that glyphosate homogeneously mixed with rodent diet
remained stable for the 1-week feeding period used in this study. Glyphosate test concentrations
averaged approximately 95% of the target concentrations throughout the study. No treatment-related
physical or behavioural signs of toxicity or mortality were observed. Yellow staining of the anogenital
area, scabbing on the ears, alopecia, excessive lacrimation, displacement of the pupils and ocular
opacities seen in all groups of male and female mice were not dose related; all occurred at low
incidences. Body weights for both males and females at 30 000 ppm were consistently less than the
controls throughout the study. Although the decreases were slight (1%–11%), several were
statistically significant. Other statistically significant decreases were noted in the mid- and low-dose
animals; however, these were sporadic and did not reflect a recognizable dose–response relationship.
Although sporadic statistically significant effects were noted for feed consumption in treated male and
female mice, none were dose or treatment related. Also, no treatment-related effects were observed
for water consumption. No biologically or toxicologically relevant effects were noted on total
erythrocyte or white blood cell counts, haemoglobin, haematocrit or platelet counts. No treatment-
related changes were observed in absolute or relative organ weights. Several statistically significant
changes in organ/body weight ratios were observed, but these were attributed to the statistically
significant decreases in terminal (fasted) body weights rather than to specific organ effects. There
were no dose–response relationships or any correlated gross or microscopic observations in any of the
organs.
No remarkable treatment-related effects were noted at necropsy. Statistically significant
positive trends were observed for central lobular hepatocyte hypertrophy, centrilobular hepatocyte
necrosis (Table 18) and chronic interstitial nephritis in males, and for proximal tubule epithelial
basophilia and hypertrophy in females. Statistically significant increases in the incidence of lesions
were observed for centrilobular hepatocyte necrosis in high-dose males and proximal tubule epithelial
basophilia and hypertrophy in high-dose females. While the incidences and/or dose–response trends
of these individual microscopic kidney lesions were found to be statistically significant, they were
considered part of a spectrum of lesions which, as a whole, constitute spontaneous renal disease.
Table 18. Hepatocellular lesions in mice administered glyphosate for 24 months

Lesion 0 ppm 1 000 ppm 5 000 ppm 30 000 ppm
a
Centrilobular hypertrophy M 9/49 5/50 3/50 17/50
F 0/49 5/50 1/49 1/49
b
Centrilobular necrosis M 0/49 2/50 2/50 10/50 a,b
F: female; M: male; ppm: parts per million
Results presented as number of mice showing hypertrophy or necrosis / number of mice examined.
a
Statistically significant linear trend (P  0.01) using the Cochran–Armitage test.
b
Statistically significant increase compared to control (P  0.01) using the Chi squared test.
Source: Knezevich & Hogan (1983)

148
Neoplastic outcomes were of the type common in mice of this age and strain. Of the tumour
types observed, bronchiolar-alveoli tumours of the lungs, hepatocellular neoplasms and tumours of
the lymphoreticular system, none were dose related and all were seen in all treatment groups (Table
19). Lymphoreticular tumours were more frequently observed in female mice, but the incidences were
low and did not approach statistical significance (nonsignificant trend and pair wise comparison).
With the possible exception of kidney tumours (renal tubular adenomas) in males, all tumour types
were considered spurious and unrelated to treatment (see Table 19).
Table 19. Neoplasia in male and female mice treated with glyphosate for 24 months
Males Females
0 1 000 5 000 30 000 0 1 000 5 000 30 000
Site / Neoplasia ppm a ppm ppm ppm ppm a ppm ppm ppm
Lung
Bronchiolar alveolar adenoma 5/48 9/50 9/50 9/50 10/49 9/50 10/49 1/50
Bronchiolar alveolar 4/48 3/50 2/50 1/50 1/49 3/50 4/49 4/50
adenocarcinoma
Lymphoblastic lymphosarcoma 1/48 4/50 3/50 1/50 – – – –
with leukaemic manifestations
Liver
Hepatocellular adenocarcinoma 5/49 6/50 6/50 4/50 1/49 2/50 1/49 0/49
Hepatocellular carcinoma 0/49 0/50 0/50 2/50 2/49 1/50 0/49 4/49
Lymph node (mediastinal)
Kidney
Renal tubular adenoma 0/49 0/49 1/50 3/50 – – – –
Total lymphoreticular 2/48 6/49 4/50 2/49 5/50 6/48 6/49 10/49
neoplasms (sum of
lymphoblastic lymphosarcoma,
composite lymphosarcoma and
histiocytic sarcoma)
ppm: parts per million; PWG: Pathology Working Group
Results presented as number of neoplasm-bearing animals / number of animals examined.
a
Incidence of effect in controls from the study report prior to PWG re-evaluation.
Source: (Knezevich and Hogan, 1983)
At the request of the USEPA, the Pathology Working Group (PWG) examined all sections of
the kidneys from this study as well as additional renal sections. The PWG evaluation included a renal
tubule adenoma in one control male mouse that was identified during a re-evaluation of the original
renal section. The PWG noted that because differentiation between tubular-cell adenoma and tubular-
cell carcinoma is not always clearly apparent and because both lesions are derived from the same cell
type, it appropriate to combine the incidences for statistical analysis. Statistical analyses performed by
the PWG are presented in Table 20. The PWG concluded that these lesions are not treatment-related
based on the following considerations: 1) renal tubular-cell tumours are spontaneous lesions for which
there is a paucity of historical control data for this mouse stock; 2) there was no statistical significance

149
in a pairwise comparison of treated groups with the controls and there was no evidence of a
significant linear trend; 3) multiple renal tumours were not found in any animal; and 4) treatment-
related nephrotoxic lesions, including pre-neoplastic changes, were not present in male mice in this
study. In addition, there was no increase in non-neoplastic renal tubular lesions in male mice (e.g.
tubular necrosis/regeneration, hyperplasia or hypertrophy). Although the incidence of tubular
adenomas exceeded the testing laboratory’s historical control range (0–3.3%), the increase at the high
dose was not statistically significant compared to the concurrent controls. However, the re-analysis of
the tumour indicated that kidney adenomas and kidney adenoma/carcinoma combined showed
statistically significant positive trend.
Table 20. Results of re-examination of incidence of renal tumours in male mice treated with
glyphosate for 24 months
Incidence of renal tumours per dietary concentration of glyphosate
Tumour type 0 ppm 1 000 ppm 5 000 ppm 30 000 ppm
Adenomas 1/49 (2%) 0/49 (0%) 0/50 (0%) 1/45 (2%)
P = 0.442 2 P = 1.000 0 P = 1.000 00 P = 0.757 6
Carcinomas 0/49 (0%) 0/49 (0%) 1/50 (2%) 2/50 (4%)
P = 0.063 5 P = 1.000 0 P = 0.505 1 P = 0.252 5
Combined 1/49 (2%) 0/49 (0%) 1/50 (2%) 3/50 (6%)
P = 0.064 8 P = 1.000 0 P = 0.757 6 P = 0.316 3
Results presented as the number of tumour-bearing animals / number of animals examined, with the resulting percentage in
parentheses.
P values determined using the Cochran–Armitage test and Fisher Exact test.
Source: Knezevich & Hogan (1983)
The NOAEL for the systemic toxicity in the two-stage carcinogenicity study in mice was
5000 ppm (equal to 814 mg/kg bw per day) based on the slightly reduced body weights, increased
centrilobular hepatocellular necrosis in high-dose males and proximal tubular epithelial basophilia in
high-dose females seen at the systemic LOAEL of 30 000 ppm; equal to 4841 mg/kg bw per day for
males and 5874 mg/kg bw per day for females (Knezevich & Hogan, 1983).
The present Meeting concluded that there is some indication, by trend test but not pairwise
comparison, of induction of kidney adenomas in male mice.
In a 22-month carcinogenicity study, trimethylsulfonium carboxymethylamino-

methylphosphonate (Company code SC-0224; glyphosate trimethylsulfonium; purity 56.17%) was
administered in the diet to groups of 80 ICR(Crl:CD-1)BR mice per sex per dose at concentrations of
100, 1000 or 8000 ppm for 22 months (mean test material intake 11.7, 118 and 991 mg/kg bw per day
for male mice and 16.0, 159 and 1341 mg/kg bw per day for female mice, respectively). One control
group of 60 male and female mice were fed the basal diet only. An additional control group of 80
male and female mice were fed the basal diet plus 1% propylene glycol vehicle. Interim terminations
of different numbers of mice occurred at 6, 12 and 18 months. The number of mice scheduled for the
full 22-month study was 50/sex per dose. Blood samples were drawn from 10 fasted male and female
mice per dose at 6, 12, 18 and 22 months for haematology and clinical chemistry measurements. At
the same time points, brain cholinesterase concentrations from left and right sides of the brains of five
mice/sex per dose were measured; urine analysis for 10 fasted mice/sex per dose was performed; and
ophthalmoscopic examinations of all the mice were conducted. Macroscopic examinations of all the
animals and histopathological examinations of selected tissues from all the animals were conducted.
Selected organs were weighed.

150
The mean body weights of the highest-dose male mice were decreased by 3–11% and that of
the highest-dose female mice were decreased by 4–17% during most of the study. Feed consumption
was also slightly decreased in male and female mice at 8000 ppm. Survival of male mice was not
affected by the treatment and the survival of female mice was apparently increased. There were no
treatment-related effects on clinical signs, urine analysis, haematology and clinical chemistry
parameters or ophthalmoscopic parameters at 6, 12, 18 or 22 months. Similarly, there were no
treatment-related effects on organ weights (absolute or relative to body weight) and palpable masses.
Analysis of the brain, erythrocytes and serum cholinesterase activity did not reveal any
toxicologically significant differences. In female mice, the increased incidence of non-neoplastic
epithelial hyperplasia of the duodenum at 8000 ppm was considered treatment related: the per cent
response of hyperplasia in females was 10, 13, 16, 15 and 24% at 0, 100, 1000 and 8000 ppm,
respectively. Male mice exhibited a treatment-related increased incidence of white matter
degeneration in the lumbar region of the spinal cord at 8000 ppm. Increased white masses in male
mice were 2%, 3%, 4% and 8% at 0, 100, 1000 and 8000 ppm, respectively. There were no treatment-
related neoplastic lesions in male and female mice. In addition, there was no decrease in latency.
The systemic toxicity NOAEL in the 22-month carcinogenicity study in mice was 1000 ppm
(equal to 118 mg/kg bw per day) based on the decreased body weights and feed consumption in both
sexes and increased incidence of white matter degeneration in the lumbar region of the spinal cord in
male mice and epithelial hyperplasia of the duodenum in female mice at 8000 ppm. There were no
treatment-related neoplastic lesions in male and female mice (Pavkov & Turnier, 1987).
Groups of 25 male and 25 female Balb/c inbred albino mice (source not specified; 5–8 weeks
old at the start of treatment) per dose were administered glyphosate technical (batch and purity not
given) for 80 weeks at dietary levels of 0, 75, 150 and 300 ppm. The actual mean daily compound
intake was not calculated.
Survival was not affected by treatment, and there were no overt clinical signs of toxicity.
Body weight in high-dose male animals tended to decrease towards the end of treatment. In females, a
similar trend was obvious from the beginning of the study up to week 21 at the highest and the mid-
dose level; during the last 20 weeks, mean body weight was reduced again but only in females at the
highest dose. Feed consumption was markedly diminished in high-dose males from week 9 onwards
and in high-dose females from week 6. Haematology and clinical chemistry assessments showed no
treatment-related changes after 9 months or after 18 months. Mean organ weights were not affected.
Gross and histopathological examination did not provide evidence of treatment-related lesions. The
incidence of neoplasia was not increased. The total number of tumours was considerably low in all
groups.
The NOAEL for chronic toxicity in the 80-week study in mice was 150 ppm for body weight
and feed consumption changes. When the usual conversion factor of 10 is applied, this value would
correspond to a daily intake of 15 mg/kg bw. A no-observed-effect level could not be established
because a weak effect on body weight in mid-dose females cannot be completely excluded. In
contrast, the study author concluded that toxicological effects did not occur up to the highest dietary
level of 300 ppm although the reduction in body weight and feed consumption was mentioned in the
study report. It should be noticed that body weight and feed intake were not affected at much higher
doses in the other available long-term studies in mice. Thus, it is not likely that these effects were
actually related to treatment (Bhide, 1988).
The draft assessment report concluded that the study is unacceptable for a reliable assessment
of carcinogenicity because the number of animals used was too small. In addition, the highest dose
level of 300 ppm is considered too low. However, the study provides supplementary information
about chronic toxicity.

151
In an 18-month non-GLP carcinogenicity study, glyphosate (purity unknown7) was

administered to groups of 50 male and female CFLP/LATI mice (bred in a facility in Godollo,
Hungary; 26–30 days old at study initiation) at dietary levels of 0, 100 or 300 ppm. The actual daily
intake was not calculated. The administration period was 18 months.
The mortality rate was high in all study groups: only 11, 14 and 23 males and 14, 16 and 14
females survived to the scheduled termination and pathological examination in the control, low- and
high-dose groups. Because clinical signs of toxicity were lacking and the mortality rates were not
dose dependant, a treatment-related effect on survival is not likely. Body weight and feed
consumption were not affected. Gross and histopathological examination did not reveal treatment-
related changes. The overall tumour rate was high in all study groups including the controls.
However, no significant difference in tumour incidence was observed between the groups.
There was no clear evidence of adverse effects of glyphosate administration up to the highest
tested dose of 300 ppm (about 30 mg/kg bw per day), considered the no-observed-effect level in this
study. However, the scientific value of this experiment is limited (Vereczkey & Csanyi, 1982, revised
1992).
The draft assessment report concluded that no conclusion could be reached due to the low
quality of the report. The study is unacceptable as a reliable assessment of carcinogenicity because the
number of animals surviving to scheduled termination and pathological examination was too small. In
addition, the highest dose of 300 ppm was insufficient for evaluating carcinogenicity since no
evidence of toxicity was obtained at that dose level. However, the study can be considered a source of
supplementary information with regard to chronic toxicity.
In an unpublished carcinogenicity study, glyphosate (purity 97.5–100.2%) was administered

to groups of 50 CD-1 mice/sex per dose in the diet at concentrations of 0, 100, 300 or 1000 mg/kg bw
per day for 104 weeks. The dietary concentrations were adjusted weekly for the first 13 weeks and
every 4 weeks thereafter. No interim terminations were conducted. Mortality, body weight, body-
weight gain and feed consumption were monitored throughout the study. White blood cell differential
counts were conducted during weeks 52, 77 and 102. Organs were weighed and tissues collected for
microscopic analyses following pre-terminal deaths or at scheduled termination.
The analytical data indicated that the mixing procedure was adequate and that the variance
between nominal and actual dosage acceptable. There were no unscheduled deaths attributable to the
administration of glyphosate. No treatment-related clinical signs of toxicity or biologically relevant or
toxicologically significant effects on body weight or body-weight gain were observed during the
study. Although statistically significant effects were noted, none were treatment related although test
groups’ responses were typically higher than those in the corresponding control mice. No treatment-
related effects were noted on feed or water consumption. Ophthalmoscopic examinations, urine
analysis and clinical chemistry parameters were not evaluated. Intergroup differences in differential
blood counts in either sex at any of the time points tested were unremarkable. The absolute and
relative to body thymus weights of male mice in the 300 and 1000 mg/kg bw per day groups were
statistically significantly increased, but the increase in thymus weights was slight and lacked a dose–
response. No histological correlates were found. In addition, no increase in absolute or relative
thymus weights were found in female mice. The incidence of lung masses was slightly increased in
high-dose male mice (control: 10/50; low dose: 13/50; mid dose: 12/50; and high dose: 18/50);
however, histopathology failed to reveal adverse lung findings. No increase in lung masses was found
in female mice. The occurrence of mineral deposits in the brain was significantly increased in males at
the highest dose compared with the control group (13/50 vs 4/49). It should be noted that this is a
common finding in this strain of mice at this age.
7
The relevant supplement was not submitted to the Meeting Rapporteur and the manufacturer’s name
was not provided.

152
There were no statistically significant increases in the incidence of any tumours, benign and
malignant, in either sex; however, the number of animals with multiple tumour types was slightly
increased in the high-dose group of both sexes (males: 16/50; females: 11/50) compared to the control
(males: 11/50; females: 6/50). This led to a slight increase in the total number of tumours in the high-
dose group of both sexes (males: 60; females: 43) compared to the control (males: 49; females: 36).
Haemangiosarcoma in the vascular system was evident in 4/50 high-dose males, 2/50 low-
dose females and 1/50 high-dose females compared to 0/50 controls. Of the high-dose mice, one had
tumours in the liver and spleen; one had a tumour in the liver only; one had tumours in the liver,
spleen and prostate; and one had a tumour in the spleen only. The incidence of haemangiosarcoma in
males was positive in Exact trend test and nonsignificant in pairwise comparison (Table 21). In
female mice, incidence of haemangiosarcoma did not achieve statistical significance.
Table 21. Haemangiosarcomas in male mice administered glyphosate for 104 weeks
Measure per dietary dose of glyphosate
0 100 300 1 000
mg/kg bw per day mg/kg bw per day mg/kg bw per day mg/kg bw per day
Haemangiosarcomas 0/47 (0%) 0/46 (0%) 0/50 (0%) 4/45 (9%)
P = 0.002 96** P = 1.000 00 P = 1.000 00 P = 0.053 32
bw: body weight; **: significance of trend (P < 0.01) denoted at control, using Fisher Exact test and Exact Trend test.
Results presented as number of tumour-bearing animals / number of animals examined less those that died before week 52,
with the resulting percentage in parentheses.
Source: Atkinson et al., 1993a
Histiocytic sarcoma in the lymphoreticular/haematopoietic tissue was evident in 2/50 low-

and high-dose males and 3/50 low- and intermediate-dose females and 1/50 high-dose female (none
were evident in the respective controls). Due to a lack of dose relationship and statistical significance,
these changes are not considered treatment related. Other tumours seen were considered typical for
mice of this age and strain.
The NOAEL for systemic toxicity in the 104-week carcinogenicity study in mice was 1000
mg/kg bw per day, the highest dose tested (Atkinson et al., 1993a).
In an 18-month carcinogenicity study, glyphosate (two lots of HR-001, purity 97.56% and
94.61%) was fed in the diet to groups of 50 male and 50 female ICR(Crj:CD-1)(SPF) mice at 0, 1600,
8000 or 40 000 ppm (equal to 0, 165, 838.1 or 4348 mg/kg bw per day for males and 0, 153.2, 786.8
or 4116 mg/kg bw per day for females) for 18 months. During treatment, all animals were observed
for clinical signs and changes in body weight, and feed consumption was measured. At week 21, urine
analysis was carried out on 20 males from all groups. Differential leukocyte counts were determined
in blood smears from 10 males and 10 females from all groups at week 52 and after 78 weeks of
treatment and also in animals terminated in extremis during the treatment, as possible. At final
necropsy after 78 weeks of treatment, organ weights of 10 males and 10 females were analysed to
determine differential leukocyte counts. All animals of both sexes were necropsied and their
histopathology examined.
At 1600 ppm, there were no treatment-related changes in either sex in any parameters. At
8000 ppm, retarded growth was observed in females with statistically significant decreases in weight
at week 6 and weeks 9 to 24. No treatment-related changes were seen in males. At 40 000 ppm, the
incidence of pale skin increased in males. In addition, loose stools were found in all the cages from
week 21 in males and week 20 in females. Retarded growth was persistently observed during
treatment, with statistically significant differences in weight from week 16 to 36 in males and from
week 6 to the end of the treatment in females. These changes were associated with depressed feed

153
consumption and feed efficiency. At necropsy, the increased incidences of distension of the caecum
were noted in males and females in all the animals examined, which were consistent to increases in
absolute and relative weights of the caecum. However, no histopathological abnormalities were
recorded in the caecum. In males, a significant increase was noted for the overall incidence of anal
prolapse that corresponded with erosion/ulcer of the anus.
The incidence of lymphoma was increased in the high-dose males but lacked a clear dose–
response (see Table 22). It was significant by trend test and not by pairwise comparison. In female
mice, the increased incidences of lymphoma were not statistically significant (trend test and pairwise
comparison). The overall incidences of lymphomas observed were well below the historical control
range of 0–18% (Baldrick & Reeve, 2007). Kidney adenomas and carcinomas in male mice were
slightly increased at the high dose of 40 000 ppm. The statistical significance was achieved by the
trend test and not by pairwise comparison. The incidences of kidney tumours in males exceeded the
historical control range. Incidence of haemangiosarcomas was statistically significantly increased in
the mid and high dose according to the trend test but not in a pairwise comparison.
Table 22. Selected neoplastic findings in male and female mice administered glyphosate for 18
months
Neoplastic findings 0 ppm 1 600 ppm 8 000 ppm 40 000 ppm
Males
Lymphoma 2/50 2/50 0/50 6/50
Kidney (adenoma/carcinoma) 0/50 0/50 0/50 2/50
Haemangiosarcoma (various organs) 1/50 0/50 0/50 0/50
Females
Lymphoma 6/50 4/50 8/50 7/50
Kidney (adenoma/carcinoma) 0/50 0/50 0/50 0/50
Haemangiosarcoma (various organs) 0/50 0/50 3/50 5/50
No.: number; ppm: parts per million
Results presented as number of tumour-bearing animals / number of animals examined.
Source: Sugimoto (1997)
Based on these results, the NOAEL was 1600 ppm (153.2 mg/kg bw per day) and the LOAEL
was 8000 ppm (838.1 mg/kg bw per day) for females based upon retarded growth with statistically
significant decreases in weight at week 6 and weeks 9 to 24 (Sugimoto, 1997).
In a 78-week carcinogenicity study, glyphosate (purity 97.5%) was fed to groups of 50 male
and 50 female Crj:CD-1 mice per dose at dietary concentrations of 0, 500, 5000 and 50 000 ppm
(equal to 0, 67.6, 685 and 7470 mg/kg bw per day for males and 0, 93.2, 909 and 8690 mg/kg bw per
day for females) for 78 weeks. Stability, homogeneity and dietary concentrations were evaluated
periodically. Cage-side and detailed clinical observations were conducted and body weight and feed
intake monitored throughout the study. Differential white blood cell counts were performed at week
52, and haematological parameters evaluated at the end of the treatment. Gross pathological
examinations were conducted at termination and on euthanized moribund and pre-terminally dead
mice. Selected organs (brain, liver, both kidneys, both adrenal glands and both testes) were weighed.
The tissue samples from control and high-dose animals and animals that died or were terminated in
extremis were histopathologically examined.

154
Prepared diets were stable at room temperature for 4 months and the test material was
homogeneously distributed in the diet. Analysis of the prepared diet indicated that the measured
concentrations ranged from 80–110% of the nominal concentrations. At 50 000 ppm, all the mice had
loose stools throughout the treatment period, although some showed improvement as treatment
continued. In the same group, nine males and eight females had treatment-related anus prolapse at
week 10 or later. Other clinical signs and incidences were similar in both control and treated groups.
A statistically significant difference in mortality rate in males was noted between the 50 000 ppm
group and the control group at week 26 or later. Mortality in mid- and low-dose males and females at
all doses was unaffected. At 50 000 ppm, body-weight gain significantly decreased or appeared to
decrease throughout the treatment in males and at week 24 or later in females. No effects of treatment
were observed in treated males and females in the mid and low dose at any time compared to controls.
In both males and females at 50 000 ppm, feed consumption decreased compared with controls; the
change was considered treatment related. No treatment-related changes were observed in haematology
parameters. In the females at 50 000 ppm, the relative weights of kidneys (total) significantly
increased. These changes were considered treatment related, though no corresponding
histopathological findings were observed. In addition, decreases in the absolute weights of liver and
right and left kidneys and significant increases in the relative weights of brain, left kidney, left adrenal
gland, and right and left testes in males, and a decrease in the absolute weight of brain in females were
noted at 50 000 ppm. The changes in the adrenal and brain were not considered adverse since they
were not accompanied with histopathological findings. Macroscopic examination revealed luminal
dilation of the large intestine, which may be associated with loose stool, in most of the terminated
males and females at 50 000 ppm. Treatment-related non-neoplastic lesions were found in the kidneys
in males and the rectums in males and females at 50 000 ppm. The renal findings included significant
increases in tubular epithelial cell hypertrophy, tubular dilation, degeneration/necrosis and an
increasing tendency in basophilic tubules proliferation (based on data from all animals). The rectal
findings included significant increases in anus prolapse-associated erosion and luminal dilation (Table
23).
Table 23. Non-neoplastic lesions in mice administered glyphosate for 78 weeks

Male Female
0 500 5 000 50 000 0 500 5 000 50 000
Non-neoplastic lesion ppm ppm ppm ppm ppm ppm ppm ppm
Kidney
Tubular dilation 4/50 7/50 4/50 20**/50 8/50 12/50 5/50 8/50
Tubular epithelial cell hypertrophy 13/50 10/50 13/50 25*/50 13/50 17/50 14/50 13/50
Basophilic tubules 21/50 16/50 17/50 28/50 14/50 14/50 10/50 13/50
Tubular degeneration/necrosis 9/50 6/50 5/50 15/50 5/50 8/50 8/50 7/50
Rectum
Luminal dilation 0/48 0/12 0/7 6*/46 0/44 0/11 0/10 6*/44
Erosion 0/48 0/12 0/7 3/46 0/44 0/11 0/10 6*/44
ppm: parts per million; *: P < 0.05, **: P < 0.01 (Fisher Exact test).
Results presented as number of tumour-bearing animals / number of animals examined.
Source: Takahashi (1999a)
Incidences of lymphomas in female mice were 3/50, 1/50, 4/50 and 6/50 in the control, 500,
5000 and 50 000 ppm dose group, respectively. The increased incidences of lymphoma at high doses
were statistically significant in the trend test but not in a pairwise comparison. Renal cell adenoma
was observed in three males and renal cell carcinoma in one male at 50 000 ppm; renal cell adenoma

155
was also observed in one male at 5000 ppm and none in any of the females (based on data from all
animals). The incidence of other tumour types in glyphosate-treated groups and controls were similar.
These tumours were re-examined by the original study pathologist in 2012 because the
Pesticide Expert Panel, Food Safety Commission of Japan requested more information on historical
control data and association with the non-neoplastic renal findings. The haematoxylin-and-eosin–
stained kidney sections prepared in the original study had faded and could not be evaluated; the
paraffin-embedded blocks of 50 males from each group which had been stored for each observation
period were sectioned and stained by haematoxylin and eosin for microscopic re-examination. The
data from the re-examination and the original data are shown in Table 24.
Table 24. Renal tumours in male mice administered glyphosate for 78 weeks
Dietary No. of cases
concentration of
glyphosate (ppm) Findings Original study Re-examination Incidencea
50 000 Renal cell adenoma 3 1 1/50 (2%)
Renal cell carcinoma 1 1 1/50 (2%)
5 000 Renal cell adenoma 1 1 1/50 (2%)
500 Renal cell adenoma 0 1 1/50 (2%)
a
Results presented as number of tumour-bearing animals / number of animals examined, with the resulting percentage in
parentheses.
Source: Nippon Experimental Medical Research Institute (2012)
Upon re-examination (using Fisher Exact probability test, P > 0.05), the incidence of renal
tumours in each treatment group no longer significantly differed from that in the control group. The
historical control data for the Takahashi (1999a) study were not available, but the historical control
values described in the re-examination document for renal cell carcinoma were 1/725 (0.13%) in
males and 0/725 (0%) in females and for renal cell adenoma were 3/564 (0.53%) in males and 0/564
(0%) in females (Chandra & Frith, 1994; Baldrick & Reeve, 2007). The re-examination report also
provides reference data: 0/55, 0/55, 1/55, 0/55 and 0/55 (0–1.8%) in males and 0/55 for all doses (0%)
in females for renal cell carcinoma; and 0/55, 1/55, 1/55, 1/55, 0/55 (0–1.8%) in males and 0/55, 0/55,
0/55, 0/55, 1/55 (0–1.8%) in females for renal cell adenoma. The results of the re-examination
revealed that the incidence of tubular epithelial cell hypertrophy in each treatment group did not
significantly differ from that in the control group. In addition, the tubular epithelial cell hypertrophy
was localized. These findings indicate no association between the tubular epithelial cell hypertrophy
and the development of renal tumours.
In conclusion, the renal cell tumours observed in this study are not relevant for human risk
assessment because (1) the incidence of renal tumours in males at 50 000 ppm did not significantly
differ from that in the control group up on re-evaluation; (2) none of the females had neoplastic or
non-neoplastic lesions; and (3) the highest dose (50 000 ppm) used in this study far exceeded the limit
dose for mice (7000 ppm) specified by the Organisation for Economic Co-operation and Development
(OECD) and USEPA.
The NOAEL in the 78-week carcinogenicity study in mice was 5000 ppm (equal to
685 mg/kg bw per day) for loose stools, decreased body-weight gain, decreased feed consumption and
increased incidences of rectal and renal non-neoplastic lesions observed in male and female mice at
the LOAEL of 50 000 ppm (equal to 7470 mg/kg bw per day), the highest dose tested (Takahashi,
1999a).
In an 18-month carcinogenicity study, glyphosate (purity > 95%) was fed to groups of
HsdOla:MF1 Swiss Albino mice (50/sex per dose) in the diet at concentrations of 0, 100, 1000 or

156
10 000 ppm (equal to 0, 14.5, 149.7 and 1453 mg/kg bw per day for males and 0, 15.0, 151.2 and
1466.8 mg/kg bw per day for females) for 18 months. The stability, homogeneity and dietary
concentrations were measured periodically. All the prepared diets were stable for 30 days. The test
material was homogenously distributed; mean prepared dietary admixture concentrations were within
10% of the nominal concentration for all diet samples.
A detailed veterinary examination of all mice was conducted before and after grouping and
monthly thereafter. Clinical signs of toxicity, appearance, behaviour and neurological changes and
mortality of all mice were checked daily. Ophthalmological examinations of all mice occurred prior to
the start of treatment and at 6, 12 and 18 months. Mortality, body weight, body-weight gain and feed
consumption were monitored throughout the study. White blood cell differential counts were
conducted at 9 months and at scheduled termination of all surviving animals and those terminated in
extremis. All the animals that died or were terminated in extremis were necropsied immediately or
preserved in 10% buffered neutral formalin until necropsy. All the surviving mice were terminated at
scheduled termination. A gross pathological examination was performed on all mice. Adrenals,
kidneys, liver and gall bladder, ovaries and testes from 10 mice per sex per dose were weighed, and
selected tissues from control and high-dose animals and those animals that died or were terminated in
extremis histopathologically examined.
There were no treatment-related effects on clinical signs, body weights, body-weight gains,
feed consumption, ophthalmoscopic examination or absolute and relative organ weights. The survival
percentage was slightly decreased at the highest dose, but the decrease was not statistically significant
and the mortality at 10 000 ppm remained within the historical control range. There were no
significant treatment-related changes in the white blood cell counts for either sex at 9 or 18 months.
In mice found dead or terminated moribund, cystic glands of the stomach were significantly
increased in high-dose males and for both sexes combined, but did not show dose dependency and
were considered incidental. Observations at lower doses or findings that were not dose dependent
included increased haematopoiesis in femurs of high-dose males and mid- and high-dose combined
sex groups; increased cell debris in tubules of epididymides in mid-dose males; increased incidence of
subcapsular cell hyperplasia in the adrenals of low-dose males; decreased incidence of kidney
nephropathy in mid-dose females; and decreased incidence of lymphocyte infiltration of epididymides
in mid-dose males. At termination, cystic glands of the stomach were significantly increased in low-,
mid- and high-dose males but without a dose–response relationship. Degenerative heart changes were
higher in high-dose males and females, and significantly higher when sexes were combined, but the
incidences were similar to the historical controls and the severity was not dose dependant. In
mandibular lymph nodes, lymphoid hyperplasia was significantly increased in low- and mid-dose
males and when sexes were combined, whereas the incidence was significantly lower in high-dose
females. In addition, extramedullary haematopoiesis was significantly increased in these lymph nodes
at the mid-dose level when sexes were combined. Extramedullary haematopoiesis in the spleen was
significantly increased in females and when the sexes were combined at the low-dose level. In the
absence of any dose relation, these findings, as well as several statistically nonsignificant changes,
were considered incidental.
The number of malignant lymphoma (Table 25) was slightly elevated in the high-dose group
compared to controls. However, this haemolymphoreticular system tumour is one of the most
common, accounting for the highest percentage of spontaneous tumours in mice, and the observed
incidence is considered incidental and not treatment related. A statistically significant increase in
malignant lymphoma was noted in both the male and female high-dose groups. Although malignant
lymphoma are common in mice, accounting for 54.6% of all tumours in this study, that the higher
incidence in the high-dose groups is treatment related cannot be excluded.

157
Table 25. Malignant lymphoma in glyphosate-treated mice

Measure per dietary concentration of glyphosate
Males Females
0 100 1 000 10 000 0 100 1 000 10 000
M F ppm ppm ppm ppm ppm ppm ppm ppm
Dead and
moribund mice
No. examined 75 77 22 20 22 27 16 16 20 20
No. affected 20 49 9 12 13 13 9 10 13 12
a
Incidence (%) 26.7 63.6 41.0 60.0* 59.0* 48.0 56.0 63.0 65.0 60.0
Terminated mice
No. examined 175 173 28 30 28 23 34 34 30 30
No. affected 26 50 1 3 3 6* 9 10 6 13
Incidence (%) a 14.9 28.9 3.6 10.0 10.7 26.1* 26.5 29.4 20.0 43.3*
Mean percentage 14.9 28.8 – – – – – – – –
Range of
percentage 8–24 2–43 – – – – – – – –
All fates
No. examined 250 250 50 50 50 50 50 50 50 50
No. affected 46 99 10 15 16 19* 18 20 19 25
a
Incidence (%) 18.4 39.6 20.0 30.0 32.0 38.0* 36.0 40.0 38.0 50.0*
Mean percentage 18.4 41.6 – – – – – – – –
Range percentage 6–30 14–58 – – – – – – – –
F: females; M: males; –: not examined/not determined; *: significant increase compared with historical controls (no P value
provided)
a
Incidence expressed as number of animals affected as a percentage of the number examined.
Source: Kumar (2001)
The increased incidences of kidney tumours at high doses (0/50, 0/50, 1/50 and 2/50 at 0, 100,
1000 and 10 000 ppm, respectively) were statistically significant in the trend test but not in a pairwise
comparison. No historical control data were available.
The NOAEL for systemic toxicity in the 18-month carcinogenicity study in mice was 1000
ppm (equal to 149.7 mg/kg bw per day) for increased mortality at 10 000 ppm. Glyphosate was not
carcinogenic in mice at doses up to 10 000 ppm, the highest dose tested (Kumar, 2001).
In a carcinogenicity study, glyphosate (purity 95.7%) was fed in the diet to groups of 51 male
and 51 female CD-1 mice per dose at concentrations of 0, 500, 1500 and 5000 ppm (equal to 0, 71.4,
234.2 and 810 mg/kg bw per day for males and 0, 97.9, 299.5 and 1081.2 mg/kg bw per day for
females) for 79 weeks. An additional 12 mice per sex, designated as veterinary controls, were housed
and maintained alongside the treated animals. Ten animals per sex from each group were set aside for
an interim termination (toxicity assessment) at week 39. Stability, homogeneity and dietary
concentrations were evaluated periodically. Cage-side and detailed clinical observations were
conducted, and body weight and feed intake monitored throughout the study. Water consumption was
observed daily. Blood smear samples were collected after 12 months and at termination from all
animals and from mice terminated in extremis. Differential white blood cell counts were performed on
all control and high-dose animals and on the animals terminated in extremis. Gross pathological
examinations were conducted at termination and on moribund and pre-terminally dead mice. Selected

158
organs of 10 mice per sex per dose were weighed. Histopathological examination was performed on
all sampled tissues from control and high-dose animals and on animals that died or were terminated in
extremis.
Analyses indicated that the dose preparations were homogeneous and stable for at least six
weeks and that the mean prepared dietary admixture concentrations were within 5% of the nominal
concentration for all doses except one low-dose sample, which was over 10% of the nominal
concentration.
There were no treatment-related effects on the number of mortalities observed and no
significant differences in mortality rates during the study. No significant treatment-related clinical
observations were reported. Similarly, no treatment-related effects on body weights, body-weight
gains, absolute or relative organ weights, and feed and water consumption were observed. There were
no significant differences in proportion of white blood cell populations of either sex at both 12 and 18
months, no trends in the proportion of palpable masses and no treatment-related macroscopic findings
observed for any of the mice. There appears to be a dose-related increase in malignant lymphomas in
the male mice only (0/51, 1/50, 2/51 and 5/51 at 0, 500, 1500 and 5000 ppm, respectively). The
increased incidences at high doses were statistically significant in the trend test and not in a pairwise
comparison; they are attributed to an unusually low incidence in the controls8 (and presumably also
for the low-dose treated mice). The observed increase appears to be well within the historical range
and thus not biologically significant.
The NOAEL for carcinogenicity and systemic toxicity was 5000 ppm (equal to 810 mg/kg bw
per day) in the 79-week study in mice, the highest dose tested (Wood et al., 2009a).
Roundup Original (glyphosate 41%, polyoxyethyleneamine [POEA] approximately equal to

15%) was evaluated in Swiss mice for tumour promotion via topical administration using a two-stage
cancer model. In this study, a known tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA),
and tumour initiator, 7,12-dimethylbenz[a]anthracene, were used. Proteomic analysis using 2-
dimensional gel electrophoresis and mass spectrometry showed that 22 spots were differentially
expressed (> twofold) on glyphosate, 7,12-dimethylbenz[a]anthracene and TPA application compared
with the untreated control. Among them, nine proteins (translation elongation factor eEF-1 α chain,
carbonic anhydrase III, annexin II, calcyclin, fab fragment anti-VEGF antibody, peroxiredoxin-2,
superoxide dismutase [Cu–Zn], stefin A3 and calgranulin-B) were common and showed similar
expression pattern in glyphosate and TPA-treated mouse skin. The study authors concluded that this
glyphosate formulation has tumour-promoting potential in skin and that its mechanism seemed similar
to that of TPA (George et al., 2010).
Rats
In a non-GLP combined chronic toxicity and carcinogenicity study, groups of Sprague
Dawley rats (50/sex per dose) were fed diets containing glyphosate (purity 98.7%) at concentrations
of 0, 30, 100 or 300 ppm for the first week. Concentrations were subsequently adjusted so actual
doses of 0, 3.05, 10.30 and 31.49 mg/kg bw per day in males and 0, 3.37, 11.22, and 34.02 mg/kg bw
per day in females were maintained for approximately 26 months. The diets were periodically
analysed for stability, homogeneity and dietary concentrations. All the rats were observed twice daily
for mortality and toxic signs. Body weights and feed consumption were determined at pretest, weekly
for 14 weeks and biweekly thereafter. Water consumption was determined for 10 rats/sex per group
for two separate 3-day periods at 18 and 24 months. Blood and urine samples were collected at 4, 8,
12, 18 and 24 months from 10 rats/sex per group. Selected haematological and clinical chemistry
8
The historical control value for lymphomas in CD-1 mice from the testing facility, Harlan laboratory,
in 2000–2010 ranged from 0–32% with a mean of 7.51% (letter from Wood E to Bond A, Regulatory Affairs
Manager, Nufarm UK, Ltd., titled ‘Historical incidences of malignant lymphoma in CD-1 mouse’).

159
parameters were evaluated. Complete necropsies were performed on all rats that died or were
terminated during or at the end of the study. Organ weights were recorded for adrenals, brain, heart,
kidneys, liver, testes/ovaries, pituitary, spleen and thyroid. The tissues were preserved for
histopathology.
There was no significant difference in survival rate between the control and treated groups of
both sexes, and survival was approximately 80–90% through month 20 of the study for all groups. No
treatment-related clinical observations were reported in any of the treated groups. Although
statistically significant differences in mean feed consumption were occasionally noted, these
differences occurred sporadically and were not dose dependant. Water consumption of the treated and
control groups were similar at the 18- and 24-month intervals. During the intermediate months, mean
body weights of the treated animals were slightly lower than that of the controls. Maximum body-
weight reductions for males ranged from 6% in the high-dose group to 2–3% in the low-dose group.
For females these differences were statistically significant only during months 20 and 21 and were not
dose related. From month 24 until study termination the mean body weights of all treated groups were
comparable to the controls. Haematology, blood biochemistry and urine analysis parameters deviated
occasionally and some of them differed significantly from controls, but these differences were not
dose related and not consistent over time or between sexes. No statistically significant differences
were noted in the absolute and relative organ weights of the treated groups compared to the controls.
The few intergroup differences were neither dose related nor consistent. Lesions consisting primarily
of inflammatory and structural changes that are common in rats of this strain in lifetime studies were
similar in incidence and severity to control groups for both sexes. The most frequently observed
changes occurred in the lungs and the kidneys, and were associated with chronic respiratory disease
and chronic progressive nephropathy. Both these sites of lesions (lungs and kidneys) are a common
age-related disease in this strain of rats.
A variety of neoplasms were found in both control and treated animals, the most common
being common spontaneous neoplasms in the pituitary glands and in the mammary glands. Female
rats showed an increased incidence of spleen and liver lymphoma combined (positive trend: 0/50,
0/50, 1/50 and 2/50 at 0, 30, 100 and 300 ppm respectively); however, the pair wise comparison was
nonsignificant. Similarly, pancreatic islet cell tumours were observed in male rats with no clear dose–
response relationship. The incidence of all tumour-bearing animals in the treated groups and the
controls were similar (19–23% combined adenomas and carcinomas for males and 36–42% for
females) and did not exhibit a dose–response relationship.
The pancreatic islet cell tumours were observed in male rats with no clear dose–response
relationship. The Meeting concluded that the pancreatic islet cell adenoma and carcinoma were
incidental for several reasons: the tumours occurred in only one study in males only; other studies that
used appreciably higher doses did not find any excess tumours; there was no dose–response
relationship; and incidences in controls was unusually low; the Meeting also noted that there was a
negative dose–response relationship in females.
Although the incidence of interstitial cell tumours in the testes was increased in the treated
animals (12% at the highest dose at termination), this was not considered relevant to human risk
assessment based on the following weight-of-evidence considerations: 1) a monotonic dose–response
relationship was lacking; 2) pre-neoplastic lesions (i.e. interstitial cell hyperplasia) were absent; 3) the
incidences were within the normal biological variation seen for this tumour type in this strain of rats;
4) the incidences in the concurrent controls (0%) was not representative of the normal background
incidences noted in the historical control animals; and 5) no interstitial cell tumours were seen when
tested at much higher doses in the same strain of rats in an another study of glyphosate (Stout &
Ruecker, 1990); and 6) due to major differences between rodents and humans with respect to
prevalence of different testicular tumour types, hormonal physiology and response and risk factors for
Leydig cell tumours, chemical induction of Leydig cell tumours in rats is generally considered of
limited relevance to humans (Alison, Capen & Prentice, 1994; Clegg et al., 1997; Cook et al., 1999).

160
The NOAEL for systemic toxicity in rats after 26 months of dietary exposure to glyphosate
was 31.5 mg/kg bw per day, the highest dose tested. It was concluded that the glyphosate was not
carcinogenic in rats (Lankas, 1981).
In a 24-month combined chronic toxicity and carcinogenicity study, groups of Sprague

Dawley rats were fed daily dietary doses of 0 (group 0, with 60 rats/sex, was fed basal diet with no
vehicle, and group 1, with 80 rats/sex, was fed the basal diet plus the propylene glycol vehicle), 100
(group 2, with 80 rats/sex), 500 (group 3, with 80 rats/sex) and 1000 (group 4: 90 rats/sex) ppm of
active ingredient (0, 178, 890 and 1779 ppm technical glyphosate trimesium [trimethylsulfonium
carboxymethylamino-methylphosphonate, company code SC-0224]. Average doses for the 2-year
treatment period, based on the nominal concentrations of active ingredient, were 4.2, 21.2 and 41.8
mg/kg bw per day for males and 5.4, 27.0 and 55.7 mg/kg bw per day for females.
Interim terminations of between 10 and 20 rats took place at 6, 12 and 18 months. All the
surviving rats in all groups were terminated at 24 months.
The only indication of toxicity was a significant reduction in growth in both sexes in group 4
(1000 ppm). The test material at the doses tested did not cause dose-related effects involving survival,
histopathological changes or any indications of carcinogenicity. Although various common tumour
types were found in both sexes, the majority were pituitary and mammary gland adenomas and
adrenal pheochromocytomas, which occurred at comparable incidences in the controls.
The NOAEL for systemic toxicity was 500 ppm in rats (equal to 21.2 mg/kg bw per day)
based on the significant reduction in growth at 1000 ppm in both sexes. There was no evidence of
carcinogenicity of glyphosate trimesium in rats in this study (Pavkov & Wyand, 1987).
In a 2-year combined chronic toxicity and carcinogenicity study, groups of Sprague Dawley
rats (60/sex per dose) were fed diets containing glyphosate (purity 96.5%) at dietary concentrations of
0, 2000, 8000 or 20 000 ppm for 24 months (equal to 0, 89, 362 or 940 mg/kg bw per day for males
and 0, 113, 457 or 1183 mg/kg bw per day for females). All animals were observed twice daily for
mortality and moribundity. Detailed observations for clinical signs of toxicity were performed
weekly. Body weights and feed consumption were determined each week for the first 13 weeks and
then every fourth week thereafter. Ophthalmic examinations were performed at pretest and just prior
to termination. Haematology, blood biochemistry and urine analysis tests were conducted on 10
animals per sex per dose at months 6, 12 (the interim termination), 18 and 24 (study termination). Ten
animals per sex per dose were terminated at month 12, and all the survivors at month 24. All animals
were given a complete gross necropsy. Brain, kidneys, liver and testes with epididymides were
weighed. Approximately 40 tissues were preserved and examined microscopically.
Analyses indicated that the neat test material was stable throughout the study, that the
homogeneity of the diet mixtures was adequate, and that average glyphosate concentrations were 95%
of target levels for all dose groups. There were no statistically significant differences in group survival
rates. At the end of the study, the percentages of animals surviving at 0, 2000, 8000, and 20 000 ppm
were 29%, 38%, 34% and 34% for males, respectively, and 44%, 44%, 34% and 36% for females.
Various clinical signs were noted throughout the study, but they were typical of those frequently
observed in chronic studies and appeared to be randomly distributed in all groups. Statistically
significant reductions in body weight were noted in high-dose females from week 7 through
approximately month 20. During this time, absolute body weights gradually decreased to 14% below
the control value. Body-weight gain in high-dose females was also consistently reduced compared to
the controls. At the point of maximum body-weight depression (20 months), cumulative body-weight
gain was 23% less than control. Body-weight gain in all treated male groups was comparable to
controls. No statistically significant decreases in feed consumption in either sex took place at any time
in the study; significant increases were noted frequently in high-dose males.

161
The ophthalmic examination prior to study termination revealed a statistically significant

difference (P < 0.05) in the incidence of cataractous lens changes between control and high-dose
males (0/15 vs 5/20). This incidence (25%) was within the range (0–33%) observed in previous
studies of untreated male CD rats at this laboratory (Monsanto Agricultural Company, St. Louis, MO,
USA). The incidences of cataractous lens changes in low- and mid-dose males, as well as all treated
female groups, were comparable to their respective controls. An examination by an independent
pathologist from Monsanto (Dr Rubin) also showed a statistically significant increase (P < 0.05) in
cataractous lens changes in high-dose male animals (8/19 vs 1/14 for controls) and concluded that a
treatment-related occurrence of lens changes affected high-dose males. Further histopathological re-
evaluation of eyes by Experimental Pathology Laboratories Incorporation revealed cataract and/or
lens fibre degeneration (Table 26). Because the number of rats ophthalmologically examined and
affected at termination was small, the results are difficult to interpret. Nevertheless, the occurrence of
degenerative lens changes appears to be exacerbated by treatment in high-dose males.
Table 26. Cataract and lens fibre degeneration in male rats administered dietary glyphosate for 24
months
Incidence per dietary concentrations of glyphosate
0 ppm 2 000 ppm 8 000 ppm 20 000 ppm
Terminal kill 2/14 3/19 3/17 5/17
All animals 4/60 6/60 5/60 8/60
Results presented as number of rats affected / number of rats examined.
Source: Strout & Ruecker (1990)
While there were various changes in haematology and serum chemistry parameters, these
were not consistently noted at more than one time point; were small and within historical control
ranges; and/or did not occur in a dose-related manner and so were considered either unrelated to
treatment or toxicologically insignificant. There was a statistically significant increase in urine
specific gravity in high-dose males at 6 months and statistically significant reductions in urine pH in
high-dose males at months 6, 18 and 24 months; this may have been due to the excretion of
glyphosate, which is an acid. Statistically significant increases in liver-to-body weight ratio at 12
months and absolute liver weight and liver-to-brain weight ratio at 24 months occurred in males at
20 000 ppm. There were no other statistically significant changes in organ weights. Gross
abnormalities seen at necropsy were not glyphosate related.
Histopathological examination revealed an increase in the number of mid-dose females with
inflammation of the stomach squamous mucosa, the only statistically significant occurrence of non-
neoplastic lesions. Although the incidence (15%) of this lesion in mid-dose females was slightly
outside the laboratory historical control range (0–13.3%), there was no dose-related trend across all
groups of treated females and no significant difference in any male group, leading to the conclusion
that the finding was not treatment related (Table 27).

162
Table 27. Inflammation of the stomach squamous mucosa in rats administered glyphosate for 24
months
Incidence per dietary concentrations of glyphosate
0 ppm 2 000 ppm 8 000 ppm 20 000 ppm

Males 2/58 3/58 5/59 7/59
Females 0/59 3/60 9/60** 6/59
ppm: parts per million; **: P < 0.01 (Fisher Exact test with Bonferroni inequality)
Results presented as number of rats with the inflammation / number of rats examined.
The only statistically significant difference in neoplastic lesions between control and treated
animals was an increase in the number of low-dose males (14%) with pancreatic islet cell adenomas
(Table 28). The historical (1983–1989) control range for this tumour at the testing laboratory was 1.8–
8.5%, but a partial review of reported studies revealed a prevalence of 0–17% in control males with
several values greater than or equal to 8%. The incidences of islet cell adenomas did not follow a clear
dose-related trend in the treated male groups as indicated by the lack of statistical significance in the
Peto trend test, meaning that the distribution of incidences in the four groups was most likely random.
There was also considerable intergroup variability in the numbers of females with this tumour (5/60,
1/60, 4/60 and 0/59 in the control, low-, mid- and high-dose groups, respectively) and no evidence of
dose-related pancreatic damage or pre-neoplastic lesions. The only pancreatic islet cell carcinoma
found in this study occurred in a control male, thus indicating a lack of treatment-induced neoplastic
progression. Taken together, the data support a conclusion that the occurrence of pancreatic islet cell
adenomas in male rats was spontaneous in origin and unrelated to glyphosate administration.
Table 28. Incidence of pancreatic islet cell findings in rats administered glyphosate for 24 months
Finding Sex 0 ppm 2 000 ppm 8 000 ppm 20 000 ppm
Hyperplasia M 2/58 (3%) 0/57 (0%) 4/60 (7%) 2/59 (3%)
F 4/60 (7%) 1/60 (2%) 1/60 (2%) 0/59 (0%)
Adenoma M 1/58 (2%) 8/57** (14%) 5/60 (8%) 7/59*** (12%)
F 5/60 (8%) 1/60 (2%) 4/60 (7%) 0/59 (0%)
Carcinoma M 1/58 (2%) 0/57 (0%) 0/60 (0%) 0/59 (0%)
a
F 0/60 (0%) 0/60 (0%) 0/60 (0%) 0/59 (0%)
Adenoma + carcinoma M 2/58 (3%) 8/57*** (14%) 5/60 (8%) 7/59 (12%)
(combined)
F 5/60 (8%) 1/60 (2%) 4/60 (7%) 0/59 (0%)
ppm: parts per million; **: P < 0.01 (Fisher Exact test with Bonferroni inequality); ***: noted to be statistically significant
but not analysed in the original report
Results presented as number of rats affected / number of rats examined with the resulting percentage in parentheses.
There was a statistically significant trend for hepatocellular adenomas in males only, but a
significant trend was not seen for adenomas and carcinomas combined (P > 0.05) (Table 29). These
tumours were not considered to treatment related since 1) their incidences were within the testing
facility’s historical control range (1–18%); 2) pre-neoplastic lesions (i.e. cell hyperplasia or pre-
neoplastic foci) were absent; and 3) there was no evidence of progression to malignancy (adenoma to
carcinoma).

163
An increased incidence of thyroid C-cell adenomas was observed at 8000 and 20 000 ppm in
both sexes but this did not reach statistical significance compared to the control animals (Table 29).
There was a statistically significant dose trend for C-cell adenomas and adenomas/carcinomas
combined in females. The testing laboratory historical control range for C-cell adenomas was 1.8–
10.6% for males and 3.3–10% for females; the range for C-cell carcinomas was 0–5.2% for males and
0–2.9% for females. These tumours are not considered relevant to human risk assessment because 1)
the increased incidences in males were not statistically significant; 2) there was no evidence of
progression from adenoma to carcinoma; 3) and there were no dose-related increases in the incidence
or severity of pre-neoplastic lesions (hyperplasia); and 4) they occurred in only one study.
Table 29. Thyroid C-cell tumours in male and female rats administered glyphosate for 24 months
Finding Sex 0 ppm 2 000 ppm 8 000 ppm 20 000 ppm
Adenoma M 2/54 (4%) 4/55 (7%) 8/58 (14%) 7/58 (12%)
F 2/57 (4%)* 2/60 (3%) 6/59 (10%) 6/55 (11%)
Carcinoma M 0/54 (0%) 2/55 (4%) 0/58 (0%) 1/58 (2%)
F 0/57 (0%) 0/60 (0%) 1/59 (2%) 0/55 (0%)
Adenoma + carcinoma M 2/54 (4%) 6/55 (11%) 8/58 (14%) 8/58 (14%)
(combined)
F 2/57 (4%)* 2/60 (3%) 7/59 (12%) 6/55 (11%)
F: females; M: males; ppm: parts per million; *: P < 0.05 (Cochran–Armitage Trend Test)
Results presented as number of rats affected / number of animals examined, excluding those that died or were terminated
prior to study week 55, and the resulting percentage in parentheses.
The incidence of benign keratoacanthoma was increased in male rats, but as there was no
dose–response relationship, it was not considered treatment related (Table 30).
Table 30. Skin keratoacanthoma in male rats administered glyphosate for 24 months
Finding 0 ppm 2 000 ppm 8 000 ppm 20 000 ppm
Benign keratoacanthoma 0/36 (0%) 1/31 (3%) 2/33 (6%) 1/32 (3%)
(dead and moribund animals)
(terminal kill)
Results presented as number of rats with skin keratoacanthoma / number of rats assessed, with the resulting percentage in
parentheses.
Lymphoma/lymphosarcoma was observed in multiple tissues in male and female rats;

however, the incidences in treatment groups were lower than in the controls and no dose relationship
was observed.
The NOAEL for toxicity in rats was 8000 ppm (equal to 362 mg/kg bw per day) for decreased
body-weight gains in females and cataractous lens changes in males seen at the LOAEL of 20 000
ppm (Strout & Ruecker, 1990).

164
In a combined 2-year chronic toxicity/carcinogenicity study, glyphosate (two batches, purity

98.9 and 98.7%) was fed in the diet to 85 Sprague Dawley rats/sex per dose for 104 weeks in amounts
adjusted to deliver 0, 10, 100, 300 and 1000 mg/kg bw per day to both sexes throughout the study.
Out of each group of 85 rats, 35 were designated for the toxicity portion of the study while the
remainder was designated for the oncogenicity portion of the study. The animals were inspected twice
daily for signs of toxicity and mortality. All were clinically examined, including palpitation for tissue
masses, prior to the start of the study and weekly thereafter. The animals were weighed and feed
consumption measured weekly during weeks 1–13 and once monthly thereafter. Water consumption
was inspected throughout the treatment period. An ophthalmoscopic examination was carried out on
20 males and 20 females from each dose group in the oncogenicity study before treatment started and
on 20 males and 20 females from the control and high-dose oncogenicity groups at weeks 25 and 51.
In addition, all control and high-dose oncogenicity and toxicity study rats were examined at week 102.
Blood was collected from the retro-orbital sinus of fasted animals for haematology and clinical
chemistry while the animals were under light ether anaesthesia. Samples were obtained from 10
animals/sex per group in the toxicity study at weeks 14, 25, 51, 78 and 102. Urine samples were
obtained from 10 animals/sex per group at weeks 14, 26 and 53 in the oncogenicity study and from 10
animals/sex per group at weeks 14, 25, 51, 78 and 102 in the toxicity study. After 52 weeks, 15 males
and 15 females from each toxicity study group were terminated and necropsied; all the remaining
study animals were terminated and necropsied after 104 weeks. All premature decedents were also
necropsied. Selected organs were weighed from all interim kill animals and 10 males and 10 females
terminated at the end of the oncogenicity study. All collected tissues from all decedents prior to week
52, those terminated at 52 weeks, and the control and high-dose animals terminated at the end of the
study were examined microscopically. Only the salivary glands were examined on the decedents after
52 weeks and the rats from the other dose groups at final termination.
Light-coloured faeces were observed during weeks 16–104 in both sexes at the high dose and
in low-mid and high-mid females; however, this sign was not considered toxicologically significant.
There were no statistically significant differences in survival rates between each group receiving
glyphosate and the control group, in either sex. No treatment-related effect was observed in feed
consumption, water consumption and haematology, ophthalmoscopic examinations and gross
pathology data. High-dose males had statistically lower mean body weight (P < 0.01) by 5–11% from
week 2 until week 104; at termination, mean body weight was 10% lower (−14% weight gain). High-
dose females had statistically lower body weight (P ≤ 0.05) by 5–12% from week 20 through week 80
(with several exceptions); at termination, mean body weight was 8% lower (−11% weight gain).
Statistically significantly increased alkaline phosphatase activities (+46% to +72%) were observed in
high-dose males throughout the study except for week 51 when the mean value was 31% higher than
control. Similarly, elevated alkaline phosphatase activities were observed in females at the high dose
(+34% to +53%) throughout the study and through most of the study at the high-mid dose (by +20%
to +67%, though not always statistically significant). These changes in the alkaline phosphatase
activity are considered of little toxicological significance. Urine analysis data showed reduced pH
(5.5–6) in males at the high dose throughout the study.
The absolute liver weight was statistically significantly decreased in females at 100, 300 and
1000 mg/kg bw per day after 52 weeks, but after correcting for final body weight, the difference was
statistically nonsignificant at all three doses. In males, the absolute liver weight was decreased
significantly at 100, 300 and 1000 mg/kg bw per day after 52 weeks, but after correcting for final
body weight the difference was also not statistically significant. The parotid salivary-gland weight
was increased significantly in males at 100, 300 and 1000 mg/kg bw per day (56–111%) after 52
weeks, but not after 104 weeks; the combined weight of the sublingual and submaxillary salivary
glands was significantly increased by 13% (22% after correcting for body weight) at 1000 mg/kg bw
per day after 52 weeks. In females, the parotid gland was not affected but the sublingual and
submaxillary combined weight was significantly higher by about 15%. The changes in salivary-gland
weights were accompanied by increased incidence of mild to severe parotid salivary gland cell
alterations and slight to moderate mandibular salivary gland cell alterations in both sexes at week 52
and week 104. The lesions were described as cells and/or acini that appeared larger and stained in a
weakly basophilic manner without showing a tendency towards proliferative or degenerative changes

165
over time. In males, the increased incidence and severity of lesions in the parotid gland were
significant (P < 0.01) at all doses at 52 weeks and at high-mid and high doses at 104 weeks. The
increased incidence of lesions in the mandibular gland was significant at high-mid and high doses at
52 weeks and significant (P < 0.001) at all doses at 104 weeks. In females, the increased incidence of
parotid lesions was significant (P = 0.001)at high-mid and high doses at 52 weeks and at all doses at
104 weeks. The increased incidence in the mandibular gland lesions was significant at the high dose at
both 52 and 104 weeks. The incidence and/or severity of kidney nephropathy decreased in males at all
doses at 52 weeks and at the high dose at 104 weeks. Urothelial hyperplasia was significantly
decreased in females from the high-dose group at both the 52-week and 104-week intervals.
Although all groups had neoplastic lesions, none proved to be treatment related when
histopathology data from treated groups were compared to that of controls at 104-week termination.
In conclusion, the liver and the salivary glands were identified as the main target organs of
glyphosate-related toxicity in the long-term study. At 100 mg/kg bw per day, the changes in salivary
glands were only minimal in terms of severity and not considered toxicologically significant. The
NOAEL in the 104-week study was 100 mg/kg bw per day in rats based on the more pronounced
cellular alteration of salivary glands at 300 mg/kg bw per day and greater. There was no treatment-
related increase in tumour incidence at doses up to 1000 mg/kg bw per day (Atkinson et al., 1993b).
In a dietary toxicity study in rats, groups of 24 male and 24 female Alpk:APfSD (Wistar-
derived) rats were fed diets containing glyphosate (purity 95.6%) at concentrations of 0, 2000, 8000
or 20 000 ppm (equal to 0, 141, 560 and 1409 mg/kg bw per day for males and 0, 167, 671 and 1664
mg/kg bw per day for females) for 1 year. Analysis of diets showed that the achieved concentrations,
homogeneity and stability were satisfactory throughout the study. The animals were monitored daily
for mortality and clinical signs. Body weights and feed consumption were measured at weekly
intervals until the end of week 13 and every 4 weeks thereafter until termination, and the rats were
terminated and necropsied. Blood and urine samples were taken for clinical pathology, selected
organs were weighed and specified tissues were taken for subsequent histopathological examination.
None of the pre-terminal deaths during the study could be attributed to the administration of
glyphosate. Apart from a small increase in the number of high-dose male and female animals that
showed wet or dry urinary staining, no treatment-related clinical changes were seen. In addition, there
were no treatment-related ophthalmological findings. Body weights of high-dose animals were lower
than concurrent controls throughout the study; body weights of animals at 8000 ppm were slightly
reduced (but not significantly in males and significantly only from week 46 in females). There was no
effect on body weight in animals on 2000 ppm glyphosate. The changes in body weights in males and
females were not considered biologically significant since the magnitude of change was small (less
than 10%).
Feed consumption was lower and feed utilization was slightly less efficient at 20 000 ppm,
the reductions being most marked at the start of the study. There was a trend for reduced feed intake
for females at 8000 ppm, which correlates with the reduction in body-weight gain at this dose in the
latter stages of the study.
Some statistically significant differences in haematological parameters were seen between
treated and control animals, but the differences were small and inconsistent across the various time
points, and were considered unrelated to the administration of glyphosate. Deviations in some clinical
chemistry parameters, such as reductions in plasma concentrations of cholesterol and triglycerides or
a dose-related increase in plasma alkaline phosphatase activity throughout the study as well as
occasional increases in the activities of plasma aspartate aminotransferase, alanine transaminase and
creatine kinase, were mostly confined to high- and intermediate-dose groups. In the absence of any
histopathological findings these marginal changes are not considered toxicologically significant.
There was no evidence of any effect of glyphosate on urine parameters. At necropsy, there
were no treatment-related gross pathological findings or consistent organ-weight changes. An
increased incidence and severity of focal basophilia of the acinar cells of the parotid salivary gland

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were seen in both sexes at 20 000 ppm. At 8000 ppm, examples of focal parotid basophilia were of
minimal severity and the incidence was slightly above that in the control animals. No other
microscopic findings could be ascribed to administration of glyphosate.
Similar numbers and types of neoplasms were diagnosed in the control group and in the
20 000 ppm group, but the study was too short to be able to reach any conclusions about
carcinogenicity.
The NOAEL for the increased incidence of basophilia of parotid acinar cells in the 1-year
toxicity study in rats was 8000 ppm (equal to 560 mg/kg bw per day) based on the increased incidence
of basophilia of parotid acinar cells at 20 000 ppm (Milburn, 1996).
In a combined chronic toxicity/carcinogenicity study, glyphosate (purity 96.8 and 90.0%, two
batches) was fed in the diet to 50 Wistar rats per sex per dose for up to 2 years at concentrations of 0,
100, 1000 or 10 000 ppm (equal to 0, 6.3, 59.4 and 595.2 mg/kg bw per day for males and 0, 8.6, 88.5
and 886 mg/kg bw per day for females). In addition, one vehicle control (acetone) group with 10 rats
per sex and one high-dose group with 20 rats per sex were included for interim termination at the
twelfth month to study non-neoplastic histopathological changes. Veterinary examinations took place
before and after grouping and at the end of each month of experimental schedule. Individual body
weights were recorded before dosing, at weekly intervals until the end of week 13 and every 4 weeks
thereafter until termination. Feed consumption was recorded once weekly for each cage group from
week 1 to week 13 and subsequently over 1 week in every 4 weeks until termination. Individual blood
samples were collected from 20 rats/sex per group at 3, 6, 12, 18 and 24 months. At scheduled
intervals of 6, 12, 18 and 24 months, blood collected from 10 rats/sex per group underwent clinical
chemistry analysis. Individual urine samples were collected from 10 rats/sex per group at 3, 6, 12, 18
and 24 months. Histopathological examination was carried out on all tissues collected at interim
termination of control and high-dose groups; on all pre-terminally dead and moribund terminated rats
in the low- and mid-dose groups; and on all lesions of the terminated rats from the low- and mid-dose
groups. Selected organs from 10 rats/sex per dose were weighed. The stability of glyphosate was
determined at 2000 and 20 000 ppm which demonstrated that prepared diets were fairly stable for 30
days at room temperature with a degradation of less than 7% of the pure compound. The analysis of
diets indicated that the achieved concentrations were within acceptable range. There were no
treatment-related effects on mortality, clinical observations, body weights, body-weight gains, feed
consumption, urine analysis and haematology. The following significant (P < 0.05) dose-related
changes in blood chemistry parameters were seen at the high dose: decrease in gamma-
glutamyltransferase levels at 12 months in male rats; a decrease in albumin levels at 6 months in
female rats; and increases in alkaline phosphatase levels at 6, 12 and 18 months in female rats. The
increase in alkaline phosphatase in high-dose females were 235, 231, 194, and 249 (U/L) at 6, 12, 18
and 24 months, respectively, while the corresponding control values were 133, 141, 101, 254 for
females at 6, 12, 18 and 24 months, respectively.
Neither treatment-related macroscopic findings nor changes in organ weights or relative organ
weights were observed during the study period. None of the significant microscopic changes or
increased and decreased incidences (in liver, spleen, lymph nodes, adrenals, thymus, gonads, uterus,
mammary gland) showed dose relationships, indicating that they were incidental and not related to the
treatment with the glyphosate. At terminal kill, the incidence of cataracts in males at 0, 100, 1000 and
10 000 ppm was 3/20, 3/20, 1/18 and 6/29, respectively, while in females it was 1/24, 1/26, 5/33 and
4/21, respectively. The historical data on neoplasm incidence for the test species indicates that the
incidences of the various tumours observed are within the normal range. The types of tumours seen
were also comparable to the historical records. No statistically significant intergroup difference
between the control and low-, mid- and high-dose treatment groups was recorded in terms of the
number of rats with neoplasms, number of malignant neoplasms and incidence of metastasis either
sex-wise or for combined sex.

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The NOAEL in this combined chronic toxicity/carcinogenicity study in rats was 10 000 ppm,
the highest dose tested, equal to 595.2 mg/kg bw per day. There was no evidence of carcinogenicity of
glyphosate at doses up to 10 000 ppm in rats at in this study (Suresh, 1996).
In a combined chronic toxicity and carcinogenicity study, groups of 50 Sprague Dawley rats
per sex were fed daily dietary doses of 0, 3000, 15 000 and 25 000 ppm (equal to 0, 180, 920 and
1920 mg/kg bw per day for males and 0, 240, 1130 and 2540 mg/kg bw per day for females)
glyphosate technical for 2 years. In addition, 20 rats/sex per dose were included for interim
termination in week 52 as part of the chronic toxicity study to study non-neoplastic histopathological
changes; the dose levels were the same except the highest dose was 30 000 ppm. Test diets were
prepared weekly by mixing appropriate amounts of the test material with the basal diet. The stability
and homogeneity of the test material in feed was determined in an in-house stability study at all dose
levels before the start of dosing. Analyses for achieved concentrations were performed monthly
during the study period.
No treatment-related clinical signs or deaths were observed in the 52-week chronic toxicity
study. In the 104-week carcinogenicity study, male animals of the high-dose group exhibited slight
but statistically insignificant higher mortalities. No significant toxic signs were observed in treated or
control groups. Significantly reduced body-weight gain that lasted throughout the study was observed
in high-dose males. In all other groups, body-weight gain at termination was comparable to the
control. No treatment-related effects on feed consumption for either sex or any group were noted
during the study. The results show a higher intake for females compared to males for each dose level.
The mean intake in the chronic toxicity study was 0.18, 0.92 and 1.92 g/kg bw per day (males) and
0.24, 1.13 and 2.54 g/kg bw per day (females) for 3000, 15 000 and 30 000 ppm, respectively. The
mean intake in the carcinogenicity study was 0.15, 0.78 and 1.29 g/kg bw per day (males) and 0.21,
1.06 and 1.74 g/kg bw per day (females) for 3000, 15 000 and 25 000 ppm, respectively.
Ophthalmological examinations revealed no abnormalities. Haematological examination
showed no treatment-attributable abnormalities. A significant increase in the alkaline phosphatase
level was only seen at 25 000 ppm in the carcinogenicity study at study termination. Other significant
changes observed in haematological and biochemical parameters were within the range of the
historical control data, indicating that they were of no biological significance. Urine analysis did not
reveal any treatment-attributable abnormalities. No treatment-related macroscopic findings were
observed during the study period.
Significant and dose-dependent effects were found in high-dose males and females in the
chronic toxicity study. In males, weights of kidneys, brain and testes were increased; in females, in
addition to increased weights of kidneys and brain, liver weight was also increased.
Histopathological changes were found at all dose levels including the control, indicating that
these are no treatment-related effects. There were no treatment-related neoplasms observed.
Based on mild effects on body-weight gain and the increased organ weights without
histopathological changes, the NOAEL in rats after chronic exposure to glyphosate technical for 24
months was 15 000 ppm (920 mg/kg bw per day) (Bhide, 1997).
In a 2-year combined chronic toxicity and carcinogenicity study, groups of 50 Sprague

Dawley rats/sex per group were fed daily dietary doses of HR-001 at concentrations of 0, 3000,
10 000 or 30 000 ppm (equal to 0, 104, 354 and 1127 mg/kg bw per day for males and 0, 115, 393 and
1247 mg/kg bw per day for females) for 24 months. In addition, 30 rats per sex per group were
included for interim termination at 26, 52 and 78 weeks.
At 3000 ppm, males exhibited significant increases in incidence of decreased spontaneous
motor activity, bradypnea and soiled fur (predominantly in external genital area and foreleg) and a
significant decrease in incidence of tactile hair loss. Females at 3000 ppm showed significant
increases in incidence of ptosis and tactile hair loss. At 10 000 ppm, the incidence of tactile hair loss

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was significantly decreased in males and significantly increased in females compared to their
respective controls.
At 30 000 ppm, neither sex showed an increase in mortality, although mortality in males was
lower than the control during the last half of the treatment period, with statistical significance most
weeks. In all other groups, mortality was comparable to the control. Males had significant increases in
incidence of bradypnea, palpable masses and soiled fur (at the external genital or perianal region)
compared to controls. Palpable masses in the tail were present in 27 males, a high incidence compared
to 11 for the controls; the incidences of masses in other locations were comparable to the controls.
Males at 30 000 ppm also showed significant decreases in incidence of tactile hair loss, incidence of
wounds and hair loss. In females, a significant increase in incidence of wet fur, mainly in the external
genital area, was observed. In addition, loose stools were observed in all cages from week 24 in males
and week 23 in females until the end of the treatment.
There was an increase in benign keratoacanthoma in males at 24 months that was statistically
significant in trend wise comparison but not in pair wise comparison (Table 31). However, skin
keratoacanthoma is one of the most common spontaneous benign neoplasms in male Sprague Dawley
rats (Chandra, Riley & Johnson, 1992). Adenomas of the kidney were observed in four males in the
30 000 ppm grouped compared to zero in the controls. The background incidence of this tumour in
this strain of rat is reported to be 0.7% (0–2.9%), and the incidence of the tumour in the 30 000 ppm
group was only slightly higher than this background incidence. Because there was no statistically
significant difference in incidence between the control and the 30 000 ppm group, the slightly higher
incidence was not considered due to the treatment with glyphosate.
Table 31. Skin keratoacanthoma in male rats administered HR-001 for 24 months
Incidence per dietary concentration of HR-001
Finding 0 ppm 3 000 ppm 10 000 ppm 30 000 ppm
(dead and moribund animals)
(terminal kill)
Results presented as number of male rats with skin keratoacanthoma / number assessed, with resulting percentage in
parentheses.
Source: Enomoto (1997)
The NOAEL for chronic toxicity was 3000 ppm (104 mg/kg bw per day) and the LOAEL
10 000 ppm (354 mg/kg bw per day) based on an increase in ptosis and of tactile hair loss in female
rats in 24-month study. There was an increased incidence of multiple clinical signs at 30 000 ppm
(Enomoto, 1997).
In a combined chronic toxicity and carcinogenicity study, groups of Fischer F344/DuCrlCrlj

rats (50/sex per dose) were fed diets containing glyphosate (purity 97.5%) at concentrations of 0, 500,
4000 or 32 000 ppm (equal to 0, 25, 201 and 1750 mg/kg bw per day for males and 0, 29.7, 239 and
2000 mg/kg bw per day for females) for 104 weeks. An interim termination was conducted on 14 rats
per sex per dose after one year. Achieved concentration was assessed regularly and the stability and
homogeneity of glyphosate in diet determined. Clinical observations (including ophthalmoscopy),
body weights, feed consumption, haematology and clinical biochemistry (blood and urine) were
measured throughout the study. A functional observational battery, including motor activity, was
conducted in week 52 in animals allocated to the chronic toxicity assessment of the study. At the end
of the scheduled period the animals were terminated and necropsied. Blood samples were taken for

169
clinical pathology, selected organs weighed and specified tissues prepared for subsequent
Prepared diets were stable at room temperature for 4 months and the test material was
homogeneously distributed in the diet. Analysis of the prepared diet indicated that the measured
concentrations ranged from 80–110% of the nominal concentrations. All males and females at 32 000
ppm had diarrhoea or soft stools from immediately after the start of administration and throughout the
administration period. Mortality was not affected. Statistically significantly reduced body weights
were observed throughout the study in high-dose males (beginning week 1) and females (beginning
week 2). Feed consumption in all dosed group decreased or increased (no statistical significance) at
various intervals. The only treatment-related effects observed in urine analysis were increased urinary
proteins in three high-dose females at week 104. These changes were thought to be related to the
histological changes in the kidney. There were no remarkable changes in females at any other dose or
other examination time or in males at any dose. Males and females at 32 000 ppm showed statistically
significant decreases or tendencies towards decreases in erythrocyte count, haematocrit and
haemoglobin concentration in weeks 26, 52 and 78, and males in this group also showed significant
increases in platelet count and leukocyte count in week 52 and a significant increase in platelet count
in week 78. At 4000 ppm, females showed a significant decrease in erythrocyte count in week 26
(94% of the control value) and males showed significant decreases in erythrocyte count (96% of the
control value) and haematocrit (95% of the control value) in week 52. In males and females at 500
ppm, there were no significant differences compared to the controls at 0 ppm in any examination
parameter. The historical control values for haematological parameters from the performing
laboratory were not available, however, and the historical control data for Fisher Inbred Strain F344/
DuCrlCrlj were used to compare with study results. Throughout the study, except at week 104, the
control group had higher erythrocyte counts and haematocrit values than the range reported in the
literature for this strain of rats. This suggests that erythrocyte and haematology values for the control
groups of the TAC study were unusually high, and that statistically significant decreases in test groups
may not be toxicologically significant or relevant. Males and females at 32 000 ppm showed a
tendency towards a decrease in albumin at each examination time, and the values were statistically
significant in males and females in week 26 and in males in week 78 compared to controls. In
addition, males in this group showed significant increases in gamma-glutamyltransferase, alkaline
phosphatase and total bilirubin in week 52. Otherwise the following changes were not observed
continuously or at 32 000 ppm and were therefore considered unrelated to administration of the test
material: significant decreases in creatinine, alanine transaminase [serum glutamic pyruvic
transaminase] and total bilirubin in males or females in week 26 at 32 000 ppm and significant
increases in creatinine, total protein and albumin in females at 500 ppm. Ophthalmoscopic
examination indicated treatment-related opacity in one high-dose female at week 104 but was
considered incidental. At 32 000 ppm, a statistically significant increase in relative kidney weights
was observed in males after the scheduled termination in week 79 and in males and females at the
scheduled termination in weeks 105–106. Otherwise, the following changes were recorded, but were
thought to be due to suppressed body-weight gain as there were no corresponding abnormalities in
histopathological examination: significant increases in the relative weights of the brain and liver in
males at the week 79 and week 105–106 scheduled terminations and females in the week 105–106
scheduled termination; a significant decrease in the absolute weight of the adrenal in high-dose males
in the week 105–106 scheduled termination; and a significant decrease in the absolute weight of the
brain in mid-high (4000 ppm) males in the week 79 scheduled termination. High-dose males and
females showed an increase in luminal dilatation of the large intestine at necropsy at the week 79
termination, but there were no histological changes. Thymic involution increased in all females at
32 000 and 500 ppm. However, these effects were thought to be incidental since they are age-related
changes.
Histopathological examination showed an increase in glomerulosclerosis in females at 4000
ppm and 32 000 ppm during the scheduled necropsy at week 105–106 and increases in eosinophilic
granule/hyaline droplets in the tubular epithelium in the kidney in females at the week 79 necropsy
and in males and females at the week 105–106 scheduled necropsy. Monsanto and TAC co-sponsored
the PWG to re-evaluate the microscopic kidney findings, specifically glomerulosclerosis, chronic

170
nephropathy and hyaline droplet renal tubule degeneration in female rats. The PWG concluded
(Hardisty, 2013) that the kidneys of male and female rats did not confirm the study pathologist's
reported conclusions that the incidence of glomerulosclerosis and the presence of eosinophilic
granules/hyaline droplets of renal tubule epithelium were treatment related. The PWG found no
histological evidence of renal toxicity in the sections of kidneys examined. The only frequently
observed finding in the kidneys of male and female rats was chronic progressive nephropathy which,
however, was similar in incidence and severity in control and treated groups. No treatment-related
tumours were observed.
In conclusion, the NOAEL for chronic toxicity of glyphosate in rats was 4000 ppm (equal to
201 mg/kg bw per day) based on the decrease in body weights, transient haematological effects,
diarrhoea, urine parameters, clinical chemistry effects, increased kidney weight relative weight seen at
32 000 ppm, the highest dose tested, in this 104-week study. Glyphosate was not carcinogenic in rats
at doses up to 32 000 ppm (Takahashi, 1999b).
In a combined chronic toxicity/carcinogenicity study, glyphosate (purity 97.6%) was fed to

64 Alpk:APfSD Wistar-derived rats per sex per dose in the diet for up to 2 years at concentrations of
0, 2000, 6000 or 20 000 ppm (equal to 0, 121, 361 and 1214 mg/kg bw per day for males and 0, 145,
437 and 1498 mg/kg bw per day for females). An interim termination was conducted on 12 rats per
sex per dose after one year. Achieved concentration was assessed regularly and the stability and
homogeneity of glyphosate in the diet determined. Clinical observations (including ophthalmoscopy),
body weights, feed consumption, haematology and clinical biochemistry (blood and urine) were
conducted throughout the study. A functional observational battery, including motor activity, was
conducted in week 52 in animals allocated to the chronic toxicity assessment part of the study. At the
end of the scheduled study period, the animals were terminated and necropsied. Cardiac blood
samples were taken for clinical pathology, selected organs weighed and specified tissues taken for
subsequent histopathological examination.
The mean achieved concentrations of glyphosate in each dietary preparation were within 10%
of the nominal concentration, and the overall mean concentrations were within 1% of nominal. The
diets were homogenously distributed and prepared diets were stable at room temperature for 45 days.
Survival in control, low- and mid-dose males approached 25% by week 104 of the study (criteria for
termination of the study) although survival in the high-dose group was significantly better. Survival in
the females was similar across all groups and better than in the lower-dose males. Treatment-related
increase in the incidence of red-brown staining of tray papers (particularly in males) and isolated
observations of red/brown coloured urine were noted in three males and one female at 20 000 ppm.
The body weights of the high-dose rats were statistically significantly lower than controls throughout
the study; however, these differences were not considered toxicologically relevant since maximum
decreased in body weights were approximately 5% and 8% for males and females, respectively. Feed
consumption and feed utilization were statistically significantly lower in high-dose males and females.
Ophthalmoscopic examination did not reveal any treatment-related effects, and no treatment-related
observations were noted in the functional observational battery, grip strength measurements, motor
activity, landing foot splay measurements and time to tail flick. Haematological parameters were not
affected by the treatment. Statistically significant increases in alkaline phosphatase activity occurred
at all doses in both sexes up to week 79. There was evidence at one or more time points of increases
in plasma alanine transaminase and aspartate aminotransferase activities and total bilirubin, but
statistical significance was reached only at 6000 and 20 000 ppm. In the absence of any
histopathological findings these marginal changes are not considered toxicologically significant.
Plasma triglycerides and cholesterol were consistently decreased for all or part of the study in males at
20 000 ppm. Plasma creatinine values were lower in all treated female groups at week 27 and in
females at 6000 and 20 000 ppm at week 14, but in the absence of any effects later in the study, this is
considered not toxicologically significant. Urinary pH was lower than that of controls in high-dose
males throughout the study. An increase in the incidence and severity of blood/red blood cells was
seen in males and, to a lesser extent, in females at 20 000 ppm. There were no consistent, dose-related
effects on organ weights indicative of a toxicologically significant effect of glyphosate.

171
Macroscopic findings consisting of a minor increase in incidence of enlarged kidneys, single

masses in the liver, firmness of the prostate and a reduction in the incidence of reduced testes were
seen in males at 6000 and 20 000 ppm. A minor increase in the incidence but not the severity of
proliferative cholangitis in the liver was observed at interim and terminal kills in high-dose males.
Moreover, an increased incidence of hepatitis and periodontal inflammation was observed in high-
dose males. There were a number of changes in the kidneys of high-dose males and females, notably
renal papillary necrosis, with or without papillary mineralization, and transitional cell hyperplasia; the
incidence was greater in males than females. These findings are considered treatment related but are
consistent with ingesting high doses of an acidic material, which may also have caused the
microscopically observed prostatitis and periodontal inflammation. The decrease in the incidence of
tubular degeneration of the testis in high-dose males is considered of no consequence (Table 32). The
incidence of prostatitis was higher than the control groups in all treated males but it was within
historical background levels in all treated groups; however, as the control value in this study was low,
the relationship to treatment at the high-dose level cannot be entirely dismissed.
Table 32. Selected microscopic findings in rats administered glyphosate for 2 years
Males Females
2 000 6 000 20 000 2 000 6 000 20 000
Organ / Finding 0 ppm ppm ppm ppm 0 ppm ppm ppm ppm
Liver: Proliferative cholangitis 56 57 55 64 55 58 59 61
Liver: Hepatitis 8 6 9 13 6 7 4 6
Kidney: Papillary necrosis 0 1 0 14 0 1 2 5
Kidney: Transitional cell hyperplasia 2 3 0 5 3 1 0 1
Prostate: Prostatitis 13 22 23 37 – – – –
Testis: Unilateral tubular degeneration 18 13 18 5
Periodontal inflammation 25 27 23 42 18 24 32 28
no. number; ppm: parts per million
Results presented as number of rats with the finding. N = 64 for male and for female rats.
Source: Brammer (2001)
In contrast to a previously described 1-year feeding study in rats (Milburn, 1996),

microscopic changes were seen in the liver and kidneys of high-dose rats but not the salivary glands,
even though the study was conducted on the same strain of the rats and in the same laboratory.
The incidence of hepatocellular adenomas in male rats at the high dose increased compared to
the controls (0/52 at 0 ppm, 2/52 [4%] at 2000 ppm, 0/52 [0%] at 6000 ppm and 5/52 [10%] at
20 000). However, this increase was considered incidental rather than treatment related, for the
following reasons: 1) the absence of a dose–response relationship; 2) the lack of progression to
malignancy; 3) no evidence of pre-neoplastic lesions; 4) the incidences were within the range (0–
11.5%) of historical controls for this strain (Wistar) of rats in 26 studies conducted between 1984 and
2003 at the testing laboratory; and 5) the 0% incidence in the concurrent controls is lower than the
average background incidence for liver adenomas in male Wistar rats, which distorts the comparison.
In conclusion, the NOAEL for chronic toxicity of glyphosate in rats was 6000 ppm (equal to
361 mg/kg bw per day) based on kidney, prostate and liver toxicity seen at 20 000 ppm (equal to
1214 mg/kg bw per day) in this 2-year study. There was no evidence of carcinogenicity in rats at
glyphosate doses up to 20 000 ppm (Brammer, 2001).

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In a combined chronic toxicity/carcinogenicity study, glyphosate (purity 95.7%) was fed to

Han Crl:WI (GLx/BRL/HAN) IGS BR Wistar rats (51/sex per dose) in the diet for up to 104 weeks at
concentrations of 0, 1500, 5000 or 15 000 ppm (equal to mean achieved doses of 0, 95.0, 316.9 and
1229.7 mg/kg bw per day). To ensure that a dose of 1000 mg/kg bw per day overall was received, the
highest dose was progressively increased to 24 000 ppm. In addition, three satellite groups with
15 rats per sex each were included for interim termination at the twelfth month to study non-
neoplastic histopathological changes. A satellite control group with 12 rats per sex served as
veterinary control; these animals were to be used for investigations should any health problems have
developed with the study animals. As no such problems occurred, observations of these animals have
not been included in the report.
The prepared diets were stable for at least 6 weeks and their achieved dietary concentrations
were within acceptable ranges.
Clinical signs, functional observations, body-weight changes and feed and water consumption
were monitored throughout the study. Clinical chemistry and haematological examinations were
performed on 10 animals per sex from the satellite and main groups at 3, 6 and 12 months. More
haematological and clinical chemistry investigations were performed on 20 animals per sex from the
main groups at 18 and 24 months. Urine analysis of 10 animals per sex from satellite groups at 3, 6
and 12 months and from main groups at 18 and 24 months was conducted. All survivors at study
termination (main groups: 104 weeks; satellite groups: 52 weeks) were necropsied as were all pre-
terminal decedents or those terminated in extremis. Selected organs of 10 animals/sex per group
terminated at the end of the study and all the animals from satellite groups were weighed.
Histopathological examination was initially carried out on all tissues collected from control and high-
dose groups; all pre-terminally dead and moribund euthanized rats and on all lesions and palpable
masses of the terminated rats from the low- and mid-dose groups. Since there were no indications of
treatment-related bone marrow changes, examination was subsequently extended to the remaining
treatment groups.
No significant treatment-related effects were observed on mortality, clinical signs,
behavioural assessments, functional performance tests (motor activity, grip strength values), sensory
reactivity, body weights, body-weight gains, feed consumption, water consumption, palpable masses,
ophthalmoscopic examinations, haematology, clinical chemistry, urine analysis, organ weights and
macroscopic findings.
Adipose infiltration of bone marrow was seen in the majority of animals examined, with both
sexes being more or less equally affected in terms of incidence and severity. However, generally
greater effects were seen in male rats at 15 000 ppm and this attained statistical significance for
terminal kill animals, indicating the possibility of myeloid hypoplasia as a consequence of treatment.
However, given the normal variability of this condition and the effect of other pathological conditions
upon marrow cellularity in ageing rats, the effect – although not altogether convincing – cannot be
dismissed as a similar effect was not seen in male rats in the remaining treatment groups. A higher
incidence of higher grades of severity of adipose infiltration was seen in premature decedents of both
sexes at 5000 ppm and females only at 1500 ppm. However, the variable duration of exposure and
significant background pathology for pre-terminal decedents further negates this as an effect of
treatment upon marrow cellularity for female rats.
At the highest dose, differences in the site of mineral deposition in the kidneys were
significant compared with controls. Pelvic mineralization was commonly seen in both sexes and was
more prevalent in female rats; however, corticomedullary mineralization was seen in female rats only.
Nephrocalcinosis in rats is generally considered to be related to diet and hormonal status. There was a
lower incidence of pelvic/papillary deposition and an increase in the corticomedullary deposition. At
the same time the incidence of renal pelvic hyperplasia was reduced in in both sexes as a consequence
of the decreased mineral deposition. The effects on pelvic and corticomedullary mineralization as well
as hyperplasia of the pelvic/papillary epithelium were confined to high-dose animals and there was no
indication of a similar effect at any other treatment level for either sex.

173
Treatment did not affect the development of neoplasia in any organ or tissue or the overall
frequency of benign or malignant tumours.
In conclusion, the NOAEL in rats after chronic exposure to glyphosate technical for 24
months was 15 000 ppm (equal to mean achieved dose level of 1229.7 mg/kg bw per day), the highest
dose tested. Glyphosate was not carcinogenic in rats at doses up to and including 15 000 ppm, the
highest dose tested (Wood et al., 2009b).
In a published drinking water study, ammonium salt of glyphosate (13.85% solution) was
administered to groups of 85 male and 85 female Wistar–RIZ rats in drinking water at concentrations
of 0, 300, 900 or 2700 mg/L for 2 years. Examination of peripheral blood parameters and bone
marrow smears did not reveal any harmful effects. In addition, there was no treatment-related effects
on the blood or urine biochemical parameters evaluated. The study authors concluded that glyphosate
has no effect on neoplastic pathogenesis (Chruscielska et al., 2000a). The study report lacks detailed
information on the formulated product or detailed description of the methodology, histopathological
examination and tumour description.
In a published study, the health effects of a Roundup-tolerant NK603 genetically modified

maize (from 11% in the diet), cultivated with or without Roundup application and Roundup alone
(from 0.1 parts per billion [ppb] of the full pesticide containing glyphosate and adjuvants) in drinking
water, were evaluated for 2 years in groups of 10 male and 10 female rats/dose. This study was used
to evaluate the long-term toxicity and was not a carcinogenicity evaluation. The test material is a
formulated product and the study report lacked details of the results (Séralini et al., 2014).
2.4 Genotoxicity
Glyphosate and its formulation products have been extensively tested for genotoxic effects
using a variety of end-points in a wide range of organisms. These tests have ranged from standard,
validated tests in bacteria and mammalian model organisms to less common and non-validated tests in
phylogenetically distant species such as plants, earthworms, clams, frogs, tropical fish and caimans. In
these studies, the test materials were administered through a variety of routes including parenteral
routes used for specialized studies but considered largely irrelevant for assessing risks resulting from
low-level dietary exposures. The reviewed studies for glyphosate are briefly summarized in the text
and tables below (genotoxicity studies on AMPA, N-acetyl-glyphosate, N-acetyl-AMPA and other
formulation ingredients are in Section 2.7, 2.8 and 2.9). Summary tables of studies conducted in non-
traditional or phylogenetically distant organisms are shown in Appendix 1. In addition, a number of
studies were conducted of humans exposed occupationally or environmentally to glyphosate and/or its
formulation products. Many of these involved co-exposures to many different pesticides and were
considered uninformative; however, the few studies that considered glyphosate the major agent are
summarized and briefly discussed below.
A much smaller number of studies have been conducted on the glyphosate metabolite,
AMPA, as well as the plant metabolites, N-acetyl-glyphosate and N-acetyl-AMPA. The results are
shown in Tables 33, 34 and 35. The in vivo studies (Table 35) investigated the ability of these
metabolites to induce micronuclei in the bone marrow erythrocytes of mice and have largely been
negative although a modest positive response was reported by Manas (2009b) when AMPA was
administered in male mice by intraperitoneal injection. Studies by other investigators using the more
relevant oral route of administration did not show an increase in micronuclei in either male or female
mice.
In the in vitro studies, increases in mutation in bacteria were not seen for AMPA or the
acetylated metabolites. Both positive and negative results were reported in studies of chromosome
aberrations and DNA damage for AMPA. AMPA was negative in two studies of unscheduled DNA

174
synthesis in isolated rat hepatocytes. Studies of chromosome aberrations and gene mutation in
mammalian cells using the acetylated metabolites were negative.

Bacteria
Glyphosate or Roundup was used in approximately 40 studies of mutagenicity in bacteria.
Most were conducted with and without metabolic activation (using S9, 9000 × g supernatant fraction
from induced male rat liver homogenate). The actual number of tests performed was well over 150 as
multiple tester strains with and without S9 were used in most studies. Glyphosate or Roundup was
found to be negative for genotoxic effects in almost all of these; weak positive results were reported in
only one or two studies. Glyphosate was also reported to be negative in three assays measuring DNA
repair (rec) in Bacillus subtilis and positive in one SOS-chromotest assay in Escherichia coli. Several
studies reported that glyphosate could enhance DNA strand breaks or interfere with DNA strand break
repair in cyanobacteria following exposure to ultraviolet-B radiation.
In the case of AMPA or the acetylated metabolites, no increases in mutation in bacteria were
seen in the in vitro studies (Table 33).
Table 33. Summary of in vitro genotoxicity studies with glyphosate, glyphosate formulations,
AMPA or their metabolites in bacteria
GLP Results
(Yes/
End-point Test object Concentration Purity No) −S9 +S9 Reference
Point Salmonella 0.1–1 000 Glyphosate No Negative Negative Kier (1978)
mutations typhimurium µg/plate (98.4%)
TA98, 100, 1535,
1537
Point S. typhimurium 0.005–50 Glyphosate Yes Negative Negative Majeska
mutations TA98, 100, 1535, µL/plate trimesium (1982)
1537, 1538 SC-0224
(19.2%)
Point S. typhimurium 10– 5 000 Glyphosate No Negative Negative Li & Long
mutations TA98, 100, 1535, µg/plate (98%) (1988)
1537, 1538; E. coli
WP2 uvrA
Point S. typhimurium 1.6–5 000 Glyphosate Yes Negative Negative Callander
mutations TA98, 100, 1535, µg/plate trimesium (1988a)
1537, 1538; E. coli ICIA 0224
WP2 uvrA
Point S. typhimurium 313–5 000 AK-01 Yes Negative Negative Yanagimoto
mutations TA98, 100, 1535, µg/plate Technical (1991)
1537; E. coli WP2 (glyphosate
uvrA acid)
(96.4%)
Point S. typhimurium 160–5000 Glyphosate Yes Negative Negative Jensen
mutations TA98, 100, 1535 µg/plate (98.6%) (1991a)
and 1537
Point S. typhimurium 33–10 000 Glyphosate No Negative Negative Chan &

mutations TA97, 98, 100, µg/plate (98.6%) Mahler
1535 (1992)
Point S. typhimurium 50–5 000 Rodeo Yes Negative Negative Kier et al.
mutations strains TA98, 100, µg/plate (40% (1992)
1535, 1537 glyphosate)

175
GLP Results
(Yes/
Point S. typhimurium 100–5 000 Glyphosate Yes Negative Negative Callander
mutations TA98, 100, 1535, µg/plate trimesium (1993)
1537, 1538; E. coli TMSC
WP2, WP2 uvrA (95%)
Point S. typhimurium 180–1 440 Roundup No Weak Weak Rank et al.

mutations TA98, TA100 µg/plate positive / positive / (1993)
equivocal equivocal
Point S. typhimurium 156–5 000 HR-001 Yes Negative Negative Akanuma
mutations TA98, 100, 1535, µg/plate (95.7%) (1995a)
1537
Point S. typhimurium 50–5 000 Glyphosate Yes Negative Negative Thompson
mutations strains TA98, 100, µg/plate (95.3%) (1996)
1535, 1537; E. coli
WP2 uvrA
Point S. typhimurium 100–5 000 Glyphosate Yes Negative Negative Callander
mutations TA98, 100, 1535, µg/plate (95.6%) (1996)
1537; E. coli
WP2,WP2 uvrA
Point S. typhimurium 1–5 000 Glifos (360 No Negative Negative Vargas (1996)
mutations TA97a, 98, 100, µg/plate g/L
1535 glyphosate)
Point S. typhimurium 0.025–0.3 Glyphosate No Negative Negative Chruscielska

mutations TA97a, 98, 100, µg/plate formulation et al. (2000b)
102 Perzocyd
10, soluble
liquid
concentrate
Point S. typhimurium 10–5000 Glyphosate Yes Negative Negative Schreib
mutations TA98, 100, 102, µg/plate technical (2012)
1535, 1537 (97%)
Point S. typhimurium 648–5000 Glyphosate Yes Negative Negative Riberri do Val

1535, 1537 Helm
(98%)
Point S. typhimurium 3–5000 Glyphosate Yes Negative Negative Sokolowski
1537; E. coli WP2
uvrA
mutations TA98, 100, 1535, µg/plate (97.7%) (2007b)
1537; E. coli WP2
uvrA
mutations TA98, 100, 1535, µg/plate (95%) (2007c)
1537; E. coli
WP2 uvrA
Point S. typhimurium 1–1000 Glyphosate Yes Negative Negative Miyaji (2008)
mutations TA97a, 98, 100, µg/plate TC (98%)
102, 1535
Point S. typhimurium 31.6–3160 Glyphosate Yes Negative Negative Flügge
mutations TA98, 100, 102, µg/plate TC (97.5%) (2009a)
1535, 1537

176
GLP Results
(Yes/
1537; E. coli WP2,
WP2 uvrA
Point S. typhimurium 31.6–5000 Glyphosate Yes Negative Negative Donath
mutations TA98, 100, 102, µg/plate (> 96%) (2010)
1535, 1537
Point S. typhimurium 31.6–3160 Glyphosate Yes Negative Negative Flügge (2010)

mutations TA98, 100, 102, µg/plate TC (95.2%)
1535, 1537
Point S. typhimurium 31.6–5000 Glyphosate Yes Negative Negative Schreib
mutations A98, 100, 1535, µg/plate (96%) (2010)
1537; E. coli WP2
uvrA
mutations TA98, 100, 1535, µg/plate (> 95%) (2010)
1537; E. coli WP2 spiked with
uvrA glyphosine
(0.63%)
Point S. typhimurium 31.6–5000 Glyphosate Yes Negative Negative Wallner
mutations TA98, 100, 102, µg/plate (> 95.8%) (2010)
1535, 1537
Point S. typhimurium 10–2000 Glyphosate Yes Negative Negative Donath
mutations TA98, 100, 102, µg/plate (> 95.4%) (2011a)
1535, 1537
mutations TA98, 100, 1535, µg/plate (98.8%) (2011b)
1537; E. coli WP2
uvrA
mutations TA98, 100, 1535 µg/plate (97.8%) (2011c)
1537; E. coli WP2
uvrA
Point S. typhimurium 1.5–5000 Glyphosate Yes Negative Negative Thompson

1537; E. coli WP2
uvrA
Point S. typhimurium 10–5000 Glyphosate Yes Negative Negative Schreib
1537; E. coli WP2 (94.1%)
uvrA
DNA damage B. subtilis Rec 20–2 000 Glyphosate No Negative Negative Li & Long
assay H17 and µg/disk (98%) (1988)
M45
DNA damage B. subtilis Rec 15–240 µg/disc AK-01 Yes Negative Negative Yanagimoto
assay H17 and Technical (1992b)
M45 (glyphosate
acid)
(96.4%)
DNA damage B. subtilis Rec 7.5–240 Glyphosate Yes Negative Negative Akanuma
assay H17 and µg/disk (95.7%) (1995b)
M45

177
GLP Results
(Yes/
DNA damage E. coli SOS 0.1–0.25 µg Roundup No Positive N/A Raipulis et al.
chromotest (2009)
Enhanced Cyanobacteria 10 µmol/L Glyphosate No Positive Negative Wang et al.
UV-induced (Scytonema (2012)
DNA strand javanicum)
breaks
Delayed UV– Cyanobacteria 10 µmol/L Glyphosate No Positive N/A Chen et al.
B-induced (Anabaena sp.) (2012)
DNA strand
break repair
Delayed UV- Cyanobacteria 10 µmol/L Glyphosate No Positive N/A Chen et al.
B-induced (Microcystis (2012)
DNA strand viridis)
break repair
DNA damage Acellular prophage 75 mmol/L Glyphosate No Negative N/A Lueken et al.
superhelical PM2 (98.4%) (2004)
DNA
AMPA
Point S. typhimurium 200–5 000 AMPA Yes Negative Negative Akanuma
1537; E. coli WP2
uvrA
Point S. typhimurium 1.6–5 000 AMPA Yes Negative Negative Callander
mutations TA98, 100, 1535, µg/plate (> 99%) (1988b)
1537, 1538; E. coli
WP2 uvrA
Point S. typhimurium 310–5 000 AMPA Yes Negative Negative Jensen
1537
N-Acetyl-AMPA
Point S. typhimurium 50–5 000 N-acetyl- Yes Negative Negative Wagner &
mutations TA98, 100, 1535, µg/plate AMPA Klug (2007)
1537; E. coli WP2 (76%; IN-
uvrA EY252)
N-Acetyl-glyphosate
Point S. typhimurium 100–5 000 N-acetyl- Yes Negative Negative Mecchi
mutations TA98, 100, 1535, µg/plate glyphosate (2004)
1537; E. coli WP2 sodium salt
uvrA (84.3%)
AMPA, aminomethylphosphonic acid; GLP: good laboratory practice; N/A: not applicable; S9: 9000 × g supernatant
fraction from induced male rat liver homogenate; −S9: without metabolic activation; +S9: with metabolic activation; UV:
ultraviolet
Mammalian cells
Glyphosate and its formulation products were tested for various types of genetic damage in
mammalian cells in vitro (Table 34). The results are summarized as follows. Of the four in vitro
studies of gene mutation in mammalian cells induced by glyphosate or its formulation products, no
increases were reported. In contrast, nine of 10 studies investigating DNA strand breaks induced by
glyphosate or Roundup in mammalian cells reported positive results, 4 of 11 studies of chromosome
aberrations reported positive results. For two of these (Lioi et al., 1998a,b), the effects were seen at
much lower concentrations than the other studies reporting negative results. Two studies reported

178
negative results for polyploidy. One study of the glyphosate formulation product Herbazed (Amer et
al., 2006) reported an induction of chromosome aberrations in mouse splenocytes in vitro (see further
discussion of Herbazed below). Five of eight studies of micronuclei were positive, two were negative
and one was equivocal; three of the positive studies required S9 whereas two did not. Of the eight
studies of sister chromatid exchanges induced in peripheral blood lymphocytes, seven were positive;
four were in human peripheral blood lymphocytes, two were in bovine peripheral blood lymphocytes,
and one was in mouse splenocytes. Both in vitro studies of unscheduled DNA synthesis in rat
hepatocytes were negative.
AMPA was negative in two studies of unscheduled DNA synthesis in isolated rat hepatocytes
(Bakke, 1991; Nesslany, 2002). Studies of chromosome aberrations and gene mutation in mammalian
cells using the acetylated metabolites were negative.
Table 34. Summary of in vitro genotoxicity studies with glyphosate, AMPA, metabolites of AMPA
and formulants in mammalian cells
GLP Results
(Yes/
Glyphosate
Gene CHO cells 2–25 mg/mL Glyphosate No Negative Negative Li & Long
mutation (98%) (1988)
(HPRT)
Gene Mouse 0.094–5 Glyphosate Yes Negative Negative Cross (1988)
mutation (TK) lymphoma mg/mL trimesium
cells (L5178Y ICIA 0224
TK±) (57.6%)
Gene Mouse 0.52–5 mg/mL Glyphosate Yes Negative Negative Jensen
mutation (TK) lymphoma (98.6%) (1991b)
cells (L5178Y
TK±)
Gene Mouse 44–1 500 Glyphosate Yes Negative Negative Clay (1996)
mutation (TK) lymphoma µg/mL (95.6%)
cells (L5178Y
TK±)
Chromosomal Mouse 0.1–50 mmol/L Herbazed No Positive N/A Amer et al.
aberrations splenocytes (glyphosate, (2006)
84%)
Chromosomal CHO cells 4–10 µL/mL Glyphosate Yes Negative Negative Majeska
aberrations trimesium (1985)
SC-0224
(55.6%)
Chromosomal Chinese 37.5–1 200 AK-01 Yes Negative Positive Yanagimoto
aberrations hamster cells µg/mL Technical (1992a)
(CHL/IU) (glyphosate
acid) (96.4%)
Chromosomal Chinese 62.5–1 000 HR-001 Yes Negative Negative Matsumoto
aberrations hamster lung µg/mL (95.7%) (1995)
cells
Chromosomal Human 33–562 µg/mL Glyfosaat Yes Negative Negative Van de Waart
aberrations peripheral (1995)
blood
lymphocytes
Chromosomal Chinese 39–1250 Glyphosate Yes Negative Negative Wright (1996)
aberrations hamster lung µg/mL (technical
cells grade; 95.3%)

179
GLP Results
(Yes/
Chromosomal Bovine 17–170 Glyphosate No Positive N/A Lioi et al.
aberrations lymphocytes µmol/L (1998a)
Chromosomal Human 100–1250 Glyphosate Yes Negative Negative Fox (1998)
aberrations peripheral µg/mL (95.6%)
blood
lymphocytes
Chromosomal Human 5–51 µmol/L Glyphosate No Positive N/A Lioi et al.
aberrations peripheral (≤ 98%) (1998b)
blood
lymphocytes
Chromosomal Human 100–4 000 TMS Yes Equivocal Equivocal Griffiths &
aberrations peripheral µg/mL Chloride Mackay
blood (95%) (1993)
lymphocytes [Glyphosate
trimesium]
Chromosomal Human 0.2–6 mmol/L Glyphosate No Negative N/A Manas et al.
aberrations peripheral (analytical (2009a)
blood grade; 96%)
lymphocytes
Micronucleus CHO K1 cells 5–100 µg/mL Glyphosate No Negative Positive Roustan et al.
(2014)
Micronucleus Bovine 28–560 Glyphosate No Equivocal N/A Piesova
lymphocytes µmol/L isopropylami (2004)
ne salt
mixture
(62%)
Micronucleus Bovine 28–560 µg/mL Glyphosate No Equivocal Negative Piesova
lymphocytes isopropylami (2005)
ne salt
mixture
(62%)
Micronucleus Bovine 28–1 120 Glyphosate No Negative N/A Sivikova et al.
lymphocytes µmol/L isopropylami (2006)
ne salt
mixture
(62%)
Micronucleus Human 0.5–580 Glyphosate No Negative Positive Mladinic et al.
peripheral µg/mL (technical (2009)
blood grade; 98%)
lymphocytes
Micronucleus Human 10–20 mg/L Glyphosate No Positive N/A Koller et al.
epithelial (95%) (2012)
cancer cell line
TR146
Micronucleus Human 10–20 mg/L Roundup No Positive N/A Koller et al.
epithelial (2012)
cancer cell line
TR146
Micronucleus CHO K1 cells 5–100 µg/mL Glyphosate No Negative Positive Roustan et al.
(2014)
DNA strand Human 75 mmol/L Glyphosate No Negative N/A Lueken et al.
breaks fibroblast cell (98.4%) alone; (2004)
(Comet assay) line GM5757 positive in
presence
of H2O2

180
GLP Results
(Yes/
DNA strand Human 4.5–6.5 nmol/L Glyphosate No Positive N/A Lopez et al.
breaks fibrosarcoma (technical (2005)
(Comet assay) cell line grade)
HT1080
DNA strand Human 4.5–6.5 nmol/L Glyphosate No Positive N/A Lopez et al.
breaks fibroblast cell (technical (2005)
(Comet assay) line GM38 grade)
DNA strand Human liver 1–10 ppm Roundup No Positive N/A Gasnier et al.
breaks HepG2 cell (R400 ) (2009)
(Comet assay) line
DNA strand Human Hep2 3–7.5 mmol/L Glyphosate No Positive N/A Manas et al.
breaks cell line (analytical (2009a)
(Comet assay) grade; 96%)
DNA strand Human 0.5–580 Glyphosate No Positive Positive Mladinic et al.
breaks peripheral µg/mL (technical (2009)
(Comet assay) blood grade; 98%)
lymphocytes
DNA strand Human 10–2 000 mg/L Glyphosate No Positive N/A Koller et al.
breaks epithelial (95%) (2012)
(Comet assay) cancer cell line
TR146
DNA strand Human 10–2 000 mg/L Roundup No Positive N/A Koller et al.
breaks epithelial (2012)
(Comet assay) cancer cell line
TR146
DNA strand Human 0.000 7–0.7 Glyphosate No Positive N/A Alvarez-Moya
breaks peripheral mmol/L isopropylami et al. (2014)
(Comet assay) blood ne (96%)
lymphocytes
DNA strand Mouse 60–180 mg/L Glyphosate Positive N/A Ming et al.
breaks spermatogonia (2014)
Sister Mouse 0.1–50 mmol/L Herbazed No Positive N/A Amer et al.
chromatid splenocytes (glyphosate, (2006)
exchange 84%)
Sister CHO cells 4–10 µL/mL Glyphosate Yes Negative Negative Majeska
chromatid trimesium (1985)
exchange SC-0224
(55.6%)
Sister Bovine 28–1 120 Glyphosate No Positive N/A Sivikova et al.
chromatid lymphocytes µmol/L isopropylami (2006)
exchange ne salt
mixture
(62%)
Sister Bovine 17–170 Glyphosate No Positive N/A Lioi et al.
chromatid lymphocytes µmol/L (1998a)
exchange
Sister Human 0.25–25 Roundup No Positive N/A Vigfusson &
chromatid peripheral mg/mL Vyse (1980)
exchange blood
lymphocytes
Sister Human 0.33–6 µg/mL Glyphosate No Positive N/A Bolognesi et
chromatid peripheral (analytical al. (1997a)
exchange blood grade; 99.9%)
lymphocytes

181
GLP Results
(Yes/
Sister Human 0.1–0.33 Roundup No Positive N/A Bolognesi et
chromatid peripheral µg/mL (30.4% al. (1997a)
exchange blood glyphosate)
lymphocytes
Sister Human 5–51 µmol/L Glyphosate No Positive N/A Lioi et al.
chromatid peripheral (≥ 98%) (1998b)
exchange blood
lymphocytes
Unscheduled Rat 0.000 012 5– Glyphosate No Negative N/A Li & Long
DNA hepatocytes 0.125 mg/mL (98%) (1988)
synthesis
Unscheduled Rat 0.2–111.7 Glyphosate Yes Negative N/A Rossberger
DNA hepatocytes mmol/L (≥ 98%) (1994)
synthesis
AMPA
Gene Mouse 0.31–5.0 99.2% Yes Negative Negative Jensen
mutation lymphoma mg/mL (1993b)
cells (L5871Y)
Chromosomal Human 0.9–1.8 99% No Weak N/A Manas et al.
aberrations peripheral mmol/L positive (2009b)
lymphocytes
Micronucleus CHO K1 cells 0.005–0.1 µg/L AMPA N/S Positive Positive Roustan et al.
(purity (2014)
unspecified)
Micronucleus CHO K1 cells 5–100 Glyphosate + N/S Negative Negative Roustan et al.
AMPA (2014)
DNA strand Human Hep2 2.5–7.5 99% No Positive N/A Manas et al.
breaks cell line mmol/L (2009b)
(Comet assay)
Unscheduled Rat 5–2 500 94.4% N/S Negative N/A Bakke (1991)
DNA hepatocytes µg/mL
synthesis
Unscheduled Rat 0.078–10 99.9% N/S Negative N/A Neslany
DNA hepatocytes mmol/L (2002)
synthesis
N-Acetyl-AMPA
Chromosomal Human 191–1 530 76%; IN- Yes Negative Negative Gudi & Rao
aberrations peripheral µg/mL EY252 (2007)
blood
lymphocytes
Gene CHO cells 100–1 531 72%; IN- Yes Negative Negative Glatt (2007)
mutation µg/mL (active EY252
(HPRT) ingredient,
adjusted for
purity)
N-Acetyl-glyphosate
Gene CHO cells 250–2 091 N-acetyl- Yes Negative Negative Glatt (2006)
mutation µg/mL (active glyphosate
(HPRT) ingredient, sodium salt
adjusted for (63%)
purity)

182
GLP Results
(Yes/
Chromosomal CHO cells 960–2 800 N-acetyl- Yes Negative N/A Murli (2004)
aberrations µg/mL glyphosate
sodium salt
(84.3%)
AMPA: aminomethylphosphonic acid; CHO: Chinese hamster ovary; GLP: good laboratory practice; HepG2: hepatocellular
carcinoma; Hep2: epidermoid cancer; HPRT: hypoxanthine-guanine phosphoribosyltransferase; N/A: not applicable; N/S:
not stated; ppm: parts per million; S9: 9000 × g supernatant fraction from male rat liver homogenate; −S9: without metabolic
activation; +S9: with metabolic activation; TK: thymidine kinase
(b) In vivo studies

Mammalian studies
Oral route
Thirty-three in vivo genotoxicity studies assessed the effect of orally administered glyphosate
or its formulation products on rodents (29 in mice and four in rats). The end-points investigated
included chromosomal alterations, micronuclei, sister chromatid exchanges, unscheduled DNA
synthesis and dominant lethal mutations (Table 35). Fourteen of the studies were conducted using
glyphosate (≥ 90% pure) with the remainder involving formulation products or less pure forms of
glyphosate. The results were negative for 29 of the 33 studies. The majority of the studies were of
good or acceptable quality, and included sponsored GLP studies conducted in compliance with OECD
Guideline 474.
The four positive studies are briefly described here. A twofold statistically significant increase
in micronucleus frequency was reported by Suresh (1993a) in female (but not male) mice treated with
two 5000 mg/kg doses of glyphosate. (The JMPR committee noted that this dose exceeds the limit
dose of 2000 mg/kg recommended by the OECD [2014] and the International Conference on
Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [2011]. The
micronucleus frequencies in the concurrent control were also higher than normal, and historical
control frequencies for the lab were not provided. In addition, a study published the following year by
the same group using the same doses of glyphosate did not see an increase in glyphosate-induced
chromosome aberrations.] The three other positive studies were described in one article, a study
published by Amer et al. (2006). In this article, positive results in both bone marrow cells and
spermatocytes were reported after the administration of seven or more doses of a glyphosate
formulation product called Herbazed (other positive results from that study are presented below). In
contrast, in a repeated-dose study conducted by the United States National Toxicology Program
(Chan & Mahler, 1992), increases in micronuclei were not seen in bone marrow erythrocytes of male
and female mice administered glyphosate in the diet for 13 weeks. In another repeated-dose study,
increases in chromosome aberrations were not seen in rat bone marrow cells harvested after 5 days of
treatment with glyphosate trimesium (Matheson, 1982). Amer et al. (2006) also reported an increase
in sister chromatid exchanges in mouse bone marrow cells after a single Herbazed dose.
Intraperitoneal injection
The JMPR committee concluded that genotoxic effects in animals treated with glyphosate or
its formulation products by intraperitoneal injection were of limited value in assessing risks due to
low-level dietary exposure. The following description of results is presented for completeness.
Twenty-one studies of micronuclei and chromosomal alterations were performed in the bone
marrow cells of rodents administered glyphosate or its formulation products by intraperitoneal
injection. Positive results were reported in approximately one third of the studies and
negative/equivocal results for the remaining two thirds. The positive studies were reported in articles
by four groups (Bolognesi et al., 1997; Prasad et al., 2009, Manas et al., 2009a; Rodrigues et al.,

183
2011) and involved the administration of both glyphosate and its formulation products. The Rodrigues
et al. (2011) and Prasad et al. (2009) studies reported increases in micronuclei at doses (≥ 0.75 mg/kg
bw and ≥ 25 mg/kg bw of Roundup, respectively) that were considerably lower than those reported as
negative by other investigators (e.g. Jensen, 1991c [5000 mg/kg bw] and Kier, Flowers & Huffman,
1992 [850–3400 mg/kg bw]). When positive results were seen and when a direct comparison could be
made, the formulation product was more potent than glyphosate itself (Bolognesi et al., 1997).
Positive results in mouse spermatocytes were also reported with administration of 50 mg/kg bw of the
glyphosate formulation product Herbazed for 5 days or more (but not 1 or 3 days) (Amer et al., 2006).
Increases in DNA strand breaks in the liver and kidney of mice were reported for both
glyphosate and Roundup by Bolognesi et al. (1997). Heydens et al. (2008) conducted a follow-up
study using the same Roundup formulation and reported that significant toxicity occurred in the liver
and kidney when dosing was by intraperitoneal injection. They postulated that the DNA damage
reported by Bolognesi et al. (1997) was likely a secondary effect of toxicity.
Bolognesi and colleagues (Peluso et al., 1998) also reported an increase in DNA adducts in
mouse liver and kidney by the sensitive but nonspecific 32P-postlabelling method following
intraperitoneal administration of Roundup, but not glyphosate. They attributed the adducts to an
unknown component of the herbicide mixture. This same group of investigators reported that
intraperitoneal administration of glyphosate and Roundup resulted in an increase in 8-hydroxy-2'-
deoxyguanosine (8-OHdG) DNA adducts in the liver (glyphosate) and kidney (Roundup). A follow-
up study on Roundup by Heydens et al. (2008) was unable to replicate the 8-OHdG adduct results.
Table 35. Summary of in vivo genotoxicity studies with glyphosate, glyphosate formulation
products and AMPA and their metabolites in mammalian species
GLP
(Yes/
End-point Test object Concentration Purity No) Results Reference
Glyphosate
Oral administration
Dominant Mouse fetuses 200–2 000 mg/kg Glyphosate Yes Negative Rodwell (1980)
lethal test and (98.7%)
resorptions
Chromosomal Mouse bone 50–5 000 mg/kg Glyphosate Yes Negative in Suresh (1994)
aberrations marrow cells on 2 days (96.8%) males and
females
Chromosomal Mouse bone 1 080 mg/kg bw Roundup (> 90% No Negative in Dimitrov et al.
aberrations marrow cells purity) males (2006)
Chromosomal Mouse bone 50 and 100 mg/kg Herbazed No Positive in Amer et al.
aberrations marrow cells bw (daily up to (glyphosate, 84%) males (2006)
21 days)
Chromosomal Mouse 50 and 100 mg/kg Herbazed No Positive in Amer et al.

aberrations spermatocytes bw (daily up to (glyphosate, 84%) males (2006)
21 days)
Chromosomal Rat bone 21–188 mg/kg Glyphosate No Negative in Majeska (1982b)

aberrations marrow cells trimesium SC- males at all
0224 (58.5%) time points up
to 5 days of
exposure
Micronucleus Mouse bone 400–1 100 mg/kg Glyphosate Yes Negative in Majeska (1986)
marrow trimesium SC- males and
erythrocytes 0224 (55.3%) females

184
GLP
(Yes/
Micronucleus Mouse bone 3–50 mg/kg in Glyphosate No Negative in Chan & Mahler
marrow the diet (98.6%) males and (1992)
erythrocytes females
Micronucleus Mouse bone 50–5 000 mg/kg Glyphosate Yes Negative for Suresh (1993a)
marrow bw; administered (96.8%) males; weak
erythrocytes twice positive /
equivocal for
females at
highest dose
Micronucleus Mouse bone 5 000 mg/kg bw Glyphosate Yes Negative in Fox & Mackay
marrow (95.6%) males and (1996)
Micronucleus Mouse bone 2 000 mg/kg bw Glyphosate Yes Negative in Jones (1999)
marrow potassium salt males
erythrocytes (49% glyphosate
acid by analysis)
[indicated 59.3%
in text]
Micronucleus Mouse bone 500–2 000 mg/kg MON 78634 Yes Negative in Erexson (2003)
marrow bw (65.2% males
erythrocytes glyphosate)
Micronucleus Mouse bone 500–2 000 mg/kg AK-01 Technical Yes Negative in Inoue (2004)
marrow bw (99.1%) males
erythrocytes
Micronucleus Mouse bone 500–2 000 mg/kg Glyphosate Yes Negative in Honarvar (2005)
marrow bw technical (97.73%) males and
Micronucleus Mouse bone 1 080 mg/kg bw Roundup (> 90% No Negative in Dimitrov et al.
marrow purity) males (2006)
erythrocytes
Micronucleus Mouse bone 8–30 mg/kg bw Glyphosate Yes Negative / Zoriki Hosomi
marrow technical Helm equivocal in (2007)
erythrocytes (≥ 95%) males
Micronucleus Mouse bone 500–2 000 mg/kg Glyphosate Yes Negative in Honarvar (2008)
marrow bw (99.1%) males
erythrocytes
Micronucleus Mouse bone 500–2 000 mg/kg MON 79864 Yes Negative in Xu (2008a)
Micronucleus Mouse bone 500–2 000 mg/kg MON 76171 Yes Negative in Xu (2008b)
Micronucleus Mouse bone 500–2 000 mg/kg MON 76313 Yes Negative in Xu (2008c)
Micronucleus Mouse bone 2 000 mg/kg bw Glyphosate Yes Negative in Negro Silva
marrow (A17035A) (280 males (2009)
erythrocytes g/L)
Micronucleus Mouse bone 500–2 000 mg/kg MON 79991 Yes Negative in Xu (2009a)
Micronucleus Mouse bone 500–2 000 mg/kg MON 76138 Yes Negative in Xu (2009b)

185
GLP
(Yes/
Micronucleus Mouse bone 500–2 000 mg/kg MON 78910 Yes Negative in Xu (2010)
marrow bw (30.3% males [amended
erythrocytes glyphosate) version of
Erexson (2006)]
Micronucleus Mouse bone 500–2 000 mg/kg TROP M Yes Negative in Flügge (2010)
marrow bw (Glyphosate 480) males and
erythrocytes (358.4 g/L females
glyphosate acid;
483.6 g/L
glyphosate
isopropylamine
salt)
Micronucleus Mouse bone 2 000 mg/kg bw Glyphosate soluble Yes Negative in Negro Silva
marrow liquid concentrate males (2011)
erythrocytes (A13013Z) (500
g/L)
Micronucleus Mouse bone 500–2 000 mg/kg MON 78239 Yes Negative in Xu (2011)
marrow bw (36.6% males [amended
erythrocytes glyphosate) version of
Erexson (2003)]
Micronucleus Mouse bone 2 000 mg/kg bw Glyphosate Yes Negative in Roth (2012)
marrow (96.3%) males
erythrocytes
Micronucleus Mouse bone 2 000 mg/kg bw Glyphosate TGAI Yes Negative in Patel (2012)
marrow (98.9%) males
erythrocytes
Micronucleus Rat bone 500–2 000 mg/kg Glyphosate Yes Negative in Flügge (2009b)
marrow bw technical grade males and
erythrocytes (98.8%) females
Micronucleus Rat bone 500–2 000 mg/kg Glyphosate 75.5 Yes Negative in Flügge (2010)
marrow bw DF (69.1% males and
erythrocytes glyphosate) females
Unscheduled Rat liver 150–600 mg/kg Glyphosate Yes Negative in Kennelly (1990)
DNA hepatocytes bw trimesium males
synthesis ICIA0224 (57.6%)
Sister Mouse bone 50–200 mg/kg bw Herbazed No Positive in Amer et al.
chromatid marrow cells (glyphosate, 84%) males (2006)
exchange
Intraperitoneal administration
Chromosomal Rat bone 1 000 mg/kg bw Glyphosate (98%) No Negative in Li & Long
aberrations marrow cells males and (1988)
females
Chromosomal Mouse bone 50 mg/kg bw Herbazed No Positive in Amer et al.
aberrations marrow cells (daily up to 5 (glyphosate, 84%) males (2006)
days)
Chromosomal Mouse 50 mg/kg bw Herbazed No Positive in Amer et al.
aberrations spermatocytes (daily up to 5 (glyphosate, 84%) males (2006)
days)
Chromosomal Mouse bone 25 and 50 mg/kg Roundup (> 41%) No Positive in Prasad et al.
aberrations marrow cells bw males (2009)
Micronucleus Mouse bone 5 000 mg/kg bw Glyphosate Yes Negative in Jensen (1991c)
marrow (98.6%) males and

186
GLP
(Yes/
Micronucleus Mouse bone 850–3 400 mg/kg Rodeo formulation Yes Negative in Kier, Flowers &
marrow bw (40%) males and Huffman (1992)
Micronucleus Mouse bone 100–200 mg/kg Glyphosate No Negative in Rank et al.
marrow bw isopropylamine combined (1993)
erythrocytes salt males and
females
Micronucleus Mouse bone 133 and 200 Roundup (480 g/L) No Negative in Rank et al.
marrow mg/kg bw as combined (1993)
erythrocytes glyphosate males and
isopropylamine females
salt
Micronucleus Mouse bone 68–206 mg/kg bw Glifos (360 g/L No Negative in Zaccaria (1996)
marrow glyphosate) males and
Micronucleus Mouse bone 300 mg/kg bw Glyphosate No Positive in Bolognesi et al.
marrow (analytical grade; males (1997)
erythrocytes 99.9%)
Micronucleus Mouse bone 450 mg/kg bw; Roundup (30.4%) No Positive in Bolognesi et al.
marrow 135 mg/kg as males (1997)
erythrocytes glyphosate
Micronucleus Mouse bone 188–563 mg/kg Glyphosate Yes Negative in Carvalho
marrow bw technical Nufarm combined Marques (1999)
erythrocytes (95%) males and
females
Micronucleus Mouse bone 300 mg/kg bw Glyphosate No Negative in Chruscielska et
marrow technical grade males al. (2000b)
erythrocytes
Micronucleus Mouse bone 90 mg/kg bw Glyphosate No Negative in Chruscielska et
marrow formulation males al. (2000b)
erythrocytes Perzocyd 10
soluble liquid
concentrate
Micronucleus Mouse bone 50–200 mg/kg bw Glyphosate No Negative (sex Nascimento &
marrow (Roundup 69) not specified) Grisolia (2000)
erythrocytes
Micronucleus Mouse bone 1 008–3 024 Glifosato IPA Yes Negative in Gava (2000)
marrow mg/kg bw Technico Nufar; males and
erythrocytes glyphosate females
isopropylamine
salt (613 g/kg salt
equivalent)
Micronucleus Mouse bone 50–200 mg/kg bw Roundup (480 g/L) No Negative in Grisolia (2002)
marrow combined
erythrocytes males and
females
Micronucleus Mouse bone 150–600 mg/kg Glyphosate Yes Negative/equiv Durward (2006)
marrow bw technical grade ocal in males
erythrocytes (95.7%)
Micronucleus Mouse bone 15.6–62.5 mg/kg Glyphosate Yes Negative in Costa (2008)
marrow bw technical grade males and
erythrocytes (98%) females
Micronucleus Mouse bone 25 and 50 mg/kg Roundup (> 41%) No Positive in Prasad et al.
marrow bw males (2009)
erythrocytes

187
GLP
(Yes/
Micronucleus Mouse bone 100–400 mg/kg Glyphosate No Positive in Manas et al.
marrow bw (analytical grade; combined (2009a)
erythrocytes 96%) males and
females
Micronucleus Mouse bone 0.148–1.28 Roundup No Positive (sex Rodrigues et al.
marrow mg/kg bw not specified) (2011)
erythrocytes
DNA strand Liver and 300 mg/kg bw Glyphosate No Positive in Bolognesi et al.
breaks kidney of (analytical grade; males (1997)
mice 99.9%)
DNA strand Liver and 900 mg/kg bw; Roundup (30.4%) No Positive in Bolognesi et al.
breaks kidney of 270 mg/kg bw as males (1997)
mice glyphosate
DNA adducts Liver and 130 and 270 Glyphosate No Negative in Peluso et al.
32
by P- kidney of mg/kg isopropylammoniu combined (1998)
postlabelling mice m salt males and
females
DNA adducts Liver and 400–600 mg/kg Roundup (30.4%) No Positive in Peluso et al.
by 32P- kidney of combined (1998)
postlabelling mice males and
females
Oxidative Liver and 300 mg/kg bw Glyphosate No Positive in Bolognesi et al.
DNA adducts kidney of (analytical grade; males (1997)
(8-OHdG) mice 99.9%)
Oxidative Liver and 900 mg/kg bw; Roundup (30.4%) No Positive in Bolognesi et al.
DNA adducts kidney of 270 mg/kg bw as males (1997)
(8-OHdG) mice glyphosate
Oxidative Liver and 600 and 900 Glyphosate No Negative in Heydens et al.
DNA adducts kidney of mg/kg bw formulation males (2008)
(8-OHdG) mice (30.4%)
AMPA
Micronucleus Mouse bone 100–1 000 mg/kg AMPA (94.4%) Yes Negative in Kier & Stegeman
marrow bw males and (1993)
erythrocytes IP females
Micronucleus Mouse bone 5 000 mg/kg bw AMPA (99.2%) Yes Negative in Jensen (1993c)
marrow oral route males and
Micronucleus Mouse bone 200–400 mg/kg AMPA (99%) No Positive Manas et al.
marrow bw (2009b)
erythrocytes IP
N-acetyl-AMPA
Micronucleus Mouse bone 500–2 000 mg/kg N-acetyl-AMPA Yes Negative in Donner (2007)
marrow bw (active (72%; IN-EY252) males and
erythrocytes ingredient, females
adjusted for
purity)
oral route

188
GLP
(Yes/
N-Acetyl-glyphosate
Micronucleus Mouse bone 500–2 000 mg/kg N-acetyl- Yes Negative in Donner (2006)
marrow bw (active glyphosate (63%; males and
erythrocytes ingredient, IN-MCX20) females
adjusted for
purity)
oral route
Other related chemicals
Chromosomal Mouse bone 10 and 100 mg/kg Series of α- No Positive Naydenova et al.
aberrations marrow cells bw aminophosphonic (2007)
acids
AMPA: aminomethylphosphonic acid; bw: body weight; GLP: Good laboratory practice; IP: intraperitoneal; N/S: not stated;
8-OHdG: 8-hydroxy-2'-deoxyguanosine
(c) Non-traditional tests or tests in phylogenetically distant organisms

The results of genotoxicity studies in phylogenetically distant organisms or using non-
traditional and generally non-validated assays are presented in Appendix 1. Studies were performed
both in vitro and in vivo with most of the tests measuring DNA strand breakage or micronucleus
formation. Approximately two thirds of these studies reported positive results. Mixed positive and
negative results were seen in mutation studies in Drosophila. The reason for the differences in
response between these species and those seen in mammals orally administered glyphosate is not
known. Surfactants and other components of the glyphosate formulation products have been reported
to be toxic to fish and other species, and this may contribute to the observed differences in test results
(Howe et al., 2004; Guilherme et al., 2012a; Navarro & Martinez, 2014). For example, the surfactant
polyoxyethylene amine, a common component in glyphosate formulations, was shown to induce
several indices of toxicity in the neotropical fish Prochilodus lineatus at all of the doses tested
(Navarro & Martinez, 2014).
(d) Human biomonitoring studies

The association between exposure to glyphosate and increase in micronucleus frequencies in
peripheral blood lymphocytes, as well as the persistence of any effects over time, was evaluated over
several months in individuals living in three areas of Colombia where glyphosate formulations were
aerially sprayed over illicit and legal crops (Bolognesi et al., 2009). Significant increases in
micronucleus frequencies were observed several days after spraying, but these increases did not
correlate with glyphosate spray rates. Over time, the induced micronucleus frequencies decreased
among the people in one area, remained the same among those in another, and increased among those
in the third. In addition, in all three communities, the micronucleus frequencies of individuals who
reported being directly exposed to glyphosate did not differ from those who reported no glyphosate
exposure.
The JMPR committee reviewed the studies and considered the results to be inconclusive or
equivocal. It noted that the micronucleus frequencies in the reference population were unusually low
and that the frequencies within the glyphosate-exposed communities fall well within the normal range
for non-exposed individuals (Bonassi et al., 2001). The results were considered to be inadequate to
reach a conclusion on the potential chromosome-damaging properties of glyphosate in humans.
Another study used the Comet assay to determine the frequency of DNA strand breakage in
the peripheral blood lymphocytes of individuals living in an Ecuadorian community within 3
kilometres of where glyphosate was sprayed. The frequency of DNA strand breakage was reported to
be significantly higher than that of individuals living in a community 80 kilometres away where

189
glyphosate was not used (Paz-y-Mino et al., 2007). The samples were collected from exposed
individuals 2 weeks to 2 months after the spraying had occurred. In reviewing the study, the JMPR
committee noted that the study had some major deficiencies; the blood samples of the two groups
were collected and processed at different times, a key consideration for an assay that is highly prone
to technical artefacts during sample preparation. In addition, the two populations were located at
considerable distance from each other, the background frequencies of DNA breakage in these
communities was not known, and the median DNA migration values were identical for 20 of the 21
subjects in the control population, a result that was considered to be highly unusual.
The JMPR committee concluded that the study was inconclusive as problems with study
design severely limit the conclusions that can be drawn.
In a follow-up study by the same authors, the frequency of structural chromosomal
aberrations in peripheral blood lymphocytes was measured in the study population that two years
previously had been exposed to glyphosate; the frequencies were found to be normal (Paz-y-Mino,
2011). The study results were considered to be negative but minimally informative as many types of
chromosome alterations do not persist for extended periods of time.
In another study, the levels of 8-OHdG, a lesion formed from oxidative damage to DNA,
were measured in the peripheral blood lymphocytes of workers spraying glyphosate (Koureas et al.,
2014). A modestly elevated but statistically nonsignificant increase was reported.
Summaries of these biomonitoring studies are shown in Table 36.
Table 36. Summary of human biomonitoring studies

GLP
(Yes/
Structural Human Aerial spraying, Glyphosate- No Negative Paz-y-Mino et
chromosomal peripheral Ecuadorian region containing mixture al. (2011)
aberrations blood cells bordering
Colombia
Micronucleus Human Aerial spraying, Herbicide mixtures No Equivocal/inc Bolognesi et al.
peripheral Narino, Colombia containing onclusive (2009)
blood glyphosate and
lymphocytes adjuvant
Micronucleus Human Aerial spraying, Herbicide mixtures No Equivocal / Bolognesi et al.
peripheral Putumayo, containing inconclusive (2009)
blood Colombia glyphosate and
lymphocytes adjuvant
Micronucleus Human Aerial spraying, Roundup 47 No Equivocal / Bolognesi et al.
peripheral Valle del Cuaca, inconclusive (2009)
blood Colombia
lymphocytes
DNA strand Human Aerial spraying, Roundup Ultra No Equivocal/ Paz-y-Mino et
breaks/Comet peripheral Ecuadorian region (44%) inconclusive al. (2007)
blood cells bordering
Colombia
DNA adducts Human Pesticide Glyphosate No Negative Koureas et al.
(8-OHdG) peripheral applicators (2014)
blood cells
8-OHdG: 8-hydroxy-2'-deoxyguanosine

190
(e) Mechanistic considerations

Neither glyphosate nor its metabolites possess the chemical structural motifs commonly
associated with mutagenesis or carcinogenesis (Ashby et al., 1989; Kier and Kirkland, 2013).
However, one study investigating the effects of a series of α-aminophosphonic acids with structural
similarities to glyphosate, reported moderate clastogenic activity in the mouse bone marrow
chromosome aberration test when administered by intraperitoneal injection (Naydenova et al., 2007).
In contrast, glyphosate bioassay results in 620 assays screening biological activity including
cytotoxicity are reported in PubChem (accessed 20 April 2016). Positive results were seen only in 21
of the 620 assay reports, the majority of which appear to be closely related to glyphosate’s herbicidal
mechanism of action in plants. The few other positives involved protein-ligand binding and inhibition
of the metabolic enzyme CYP71B1. These results indicate that, at the concentrations tested and at the
end-points examined, glyphosate had few off-target molecular or cellular effects.
Summary:
The overall weight of evidence indicates that administration of glyphosate and its formulation
products at doses as high as 2000 mg/kg bw by the oral route, the route most relevant to human
dietary exposure, was not associated with an increase in chromosome alterations or other types of
genetic damage. The majority of the in vivo studies were conducted in rodents, a model considered
physiologically relevant for assessing genotoxic risks to humans. The genotoxic effects reported to
occur in vitro or in phylogenetically distant organisms have not been observed in vivo in appropriately
treated mammalian models.
When administered by intraperitoneal injection, mixed, largely negative, results have been
reported in studies of chromosomal damage of glyphosate, its formulation products and metabolites.
Mixed, and somewhat contradictory, results have been reported in the few studies (all conducted by
intraperitoneal injection) that have investigated DNA adducts induced by glyphosate or Roundup.
Results obtained by this route of administration are considered to have limited relevance when
estimating risks from human dietary exposure.
The positive results reported by Amer et al. (2006) using both oral and intraperitoneal routes
of administration appear anomalous, and may have been due to impurities or other components within
the Herbazed formulation product.
Biomonitoring studies of DNA and chromosomal alterations in humans conducted in five to
six communities by several investigators found equivocal associations between glyphosate exposure
and genetic damage.

(a) Multigeneration studies
In a non-GLP three-generation reproduction study, glyphosate (purity 100%) was fed in the
diet to 12 male and 24 female CD rats at concentrations of 0, 3, 10 or 30 mg/kg bw per day starting 63
days prior to mating. Each male was mated with two females. The first litters (F 1A, F2A, and F3A) from
each mating were raised to weaning and then terminated. Second matings (F1B and F2B) were selected
to become parents for subsequent generations or to undergo complete gross necropsy (F 3B). Tissues
were also evaluated microscopically (10/sex/group) from the control and high-dose parental animals
for all generations and F3B offspring.
Analytical results demonstrated that glyphosate was stable and homogenously distributed in
the diet. Analysis of various batches showed an average of 98.0 (± 6.8)% of the target concentration.
No treatment-related adverse effects were observed on mortality, clinical signs, body weights, feed
consumption, feed efficiency, organ weights or histopathological changes for parental animals of
either generation. No adverse effects were observed for mating performance, pregnancy rate or

191
duration of pregnancy in either generation. Litter size and viability were not affected by treatment. No
adverse effects were noted for offspring body weights or development.
No adverse effects were noted in the study. The NOAEL for parental, reproductive and
offspring toxicity was 30 mg/kg bw per day, the highest dose tested (Schroeder & Hogan, 1981).
In a two-generation reproduction study, glyphosate (purity 97.67%) was administered to

Sprague Dawley rats (30/sex per dose) in the diet at concentrations of 0, 2000, 10 000 or 30 000 ppm
(equal to 0, 132, 666 and 1983 mg/kg bw per day for males and 0, 160, 777 and 2322 mg/kg bw per
day for females). After approximately 11 weeks of treatment, pairs of animals within each dose group
were mated on a 1:1 basis to produce the F1 litters. At weaning, 30 of these F1 generation rats
(referred to as F1A in study report) per sex per dose were similarly exposed (approximately 14 weeks)
and mated twice to produce F2A and F2B generations. On day 4 postpartum, litters were standardized
(four males and four females when possible). Offspring not selected for mating, F2A and F2B pups, and
adult females which had littered were terminated on or after day 21 of lactation. Adult males were
terminated after mating. Organs were retained from all parental animals and one pup per sex per litter
from F2A and F2B. Tissues from control and high-dose animals were examined microscopically.
The stability and homogeneity of glyphosate in the diet were acceptable. Analytical
concentrations were, on the average, 95–96.7% of target levels. No treatment-related adverse effects
were observed on mortality, feed consumption, organ weights or histopathological changes for
parental animals of either generation. The incidence of soft stools was increased for high-dose adult
animals in both generations (Table 37). Reduced body weights were noted in parental animals of both
generations at termination: body weights were approximately 8–10% lower than controls for the F0
generation and 10–13% lower than controls in the F1 generation (Table 38).
No adverse effects were observed for mating performance, pregnancy rate or duration of
pregnancy in either generation. Compared to the controls, there was a slight reduction in average litter
size for F0 dams in the highest dose group; an even smaller difference was noted after the first F l
mating. However, the slight reduction in average litter size was not statistically significant. The F la
adults were re-mated to produce the F2b generation. There was no dose-related decrease in litter size in
this second mating. Since the reductions in litter size were neither statistically significant nor
consistently observed in all generations, the relationship to treatment could not be conclusively
established. Therefore, it was concluded that litter size and viability were not affected by treatment.
No adverse effects were noted for offspring body weights or development. Statistically
significant differences in pup body weights compared to controls were observed at mid and high dose,
but these differences were small and within biologically variability.
Table 37. Soft stools in two successive generations of rats administered glyphosate
0 ppm 2 000 ppm 10 000 ppm 30 000 ppm
F0 – males
No. of animals 0 0 0 30/30
No. of occurrences 0 0 0 457
F0 – females
No. of occurrences 0 0 0 116
F1 – males
No. of animals 0 0 1/30 30/30
No. of occurrences 0 0 1/30 698
F1 – females

192

0 ppm 2 000 ppm 10 000 ppm 30 000 ppm
Number of occurrences 0 0 0 537
ppm: parts per million; F0: parental generation; F1: first filial generation; No.: number
Results presented as number of animals with soft stools / number of animals examined.
Source: Reyna (1990)
Table 38. Terminal body weights in two successive generations of parental rats administered
glyphosate
Weight per dietary concentration of glyphosate
0 2 000 ppm 10 000 ppm 30 000 ppm
F0
Males 549.6 ± 46.8 550.2 ± 80.7 540.0 ± 58.1 503.5 ± 45.7 (↓8%)
Females 296.3 ± 23.6 290.6 ± 19.5 290.7 ± 25.4 265.9 ± 15.4 (↓10%)
F1
Males 625.0 ± 53.1 632.1 ± 74.6 591.0 ± 70.1 543.4 ± 58.1 (↓13%)
Females 316.2 ± 37.4 313.7 ± 30.5 312.4 ± 26.7 284.7 ± 18.4 (↓10%)
ppm: parts per million; F0: parental generation; F1: first filial generation; no.: number; ↓: decrease
Results presented as mean weight in grams ± standard deviations, with per cent change relative to controls in parentheses for
the high-dose group only.
Source: Reyna (1990)
The NOAEL for parental toxicity was 10 000 ppm (equal to 666 mg/kg bw per day) based on
decreased body weights and increased incidence of soft stools in rats at 30 000 ppm. As there were no
effects on reproductive parameters or offspring measurements, the NOAEL for reproductive and
offspring toxicity was 30 000 ppm (equal to 1983 mg/kg bw per day (Reyna, 1990).
In a two-generation reproduction study, groups of 28 male and 28 female Crl:CD(SD)BR

VAF/Plus rats (aged 6 weeks at the start of treatment) were fed diets containing glyphosate technical
(purity 99.2%) at concentrations of 0, 1000, 3000 or 10 000 ppm (equal to 0, 66.4, 196.8 and 668.1
mg/kg bw per day for males and 0, 75.3, 226.0 and 752.3 mg/kg bw per day for females) for 70 days
before their first mating and until termination. Each generation was mated twice, changing partners
for the second mating and avoiding sister/brother matings throughout. On postnatal day 4, litters were
standardized (four males and four females, when possible). The remaining pups and those not selected
for mating were terminated and underwent gross pathological examinations. Treatment was continued
for parental animals until day 21 of weaning of the second litter when animals were terminated for
organ weighing, gross pathological examination and histopathological examination. Initial
histopathological examinations were performed in the control and highest dose groups. Other dose
groups were analysed when an effect was seen in a tissue at the highest dose.
No treatment-related adverse effects on mortality, clinical signs, body weights, feed
consumption, feed efficiency or organ weights were observed for parental animals of either
generation. No adverse effects were observed for mating performance, pregnancy rate or duration of
pregnancy in either generation. Litter size and viability were not affected by treatment. No adverse
effects were noted for offspring body weights or development.
Treatment-related histopathological changes were found in the parotid salivary gland of both
sexes and submaxillary salivary gland of females in both generations (Table 39). The changes were

193
described as hypertrophy of acinar cells with prominent granular cytoplasm (minimal severity).
Increased incidence of the effects was observed at the highest dose tested.
Table 39. Cellular alterations in salivary glands of two successive generations of rats administered
glyphosate
Males Females
Site of cellular 1 000 3 000 10 000 1 000 3 000 10 000
alteration 0 ppm ppm ppm ppm 0 ppm ppm ppm ppm
F0
Parotid gland 2/27 2/28 3/28 12/26 0/28 2/27 5/28 17/28
Submaxillary gland 0/27 – – 0/26 0/28 1/27 4/28 14/28
F1
Parotid gland 1/24 0/24 4/23 11/23 0/24 0 4/24 9/23
Submaxillary gland 0/24 – – 0 0/24 0 0/24 3/23
ppm: parts per million; F0: parental generation; F1: first filial generation; –: not examined.
Initial histopathological examinations were performed in the control and highest dose groups. Other dose groups were
analysed when an effect was seen at the highest dose.
Results presented as number of animals with hypertrophy of acinar cells with prominent granular cytoplasm / number of
animals examined.
Source: Brooker et al. (1992)
The NOAEL for parental toxicity was 3000 ppm (equal to 196.8 mg/kg bw per day, based on
increased incidence of histopathological effects observed in the parotid (males and females) and
submaxillary (females only) salivary glands in both generations of rats at 10 000 ppm (equal to 668.1
mg/kg bw per day). As there were no effects on reproductive parameters or offspring measurements,
the NOAEL for reproductive and offspring toxicity of glyphosate in rats is 10 000 ppm (equal to
668.1 mg/kg bw per day) (Brooker et al., 1992).
In a two-generation reproduction study, glyphosate (purity 96.8%) was administered to Wistar

(30 rats/sex per dose) in the diet at concentrations of 0, 100, 1000 or 10 000 ppm (equivalent to 0, 6.6,
66.0 and 660 mg/kg bw per day) for two successive generations with one litter per generation. The
mean daily intake of glyphosate was not reported for all dietary levels; however, the low dose of 100
ppm corresponds to an average of 7.7 mg/kg bw per day according to the original study report. After
10 weeks of treatment, animals were paired within each dose group on a 1:1 basis to produce the F1
litters. On day 4 postpartum, litters were standardized (four males and four females, if possible). At
weaning, 30 males and 30 females from each dose group were selected to produce the F 1 generation;
these rats were dosed for at least 10 weeks and paired within their dose group to produce F2 litters. All
parental animals, non-selected pups from F1 and all pups from F2 were necropsied. Only parental
tissue was collected.
No treatment-related adverse effects were observed on mortality, clinical signs, body weights,
feed consumption, feed efficiency, organ weights or histopathological changes for parental animals of
duration of pregnancy in either generation. Litter size and viability were not affected by treatment. No
adverse effects were noted for offspring body weights or development.
As no adverse effects were noted in the study, the NOAEL for parental, reproductive and
offspring toxicity in rats was 10 000 ppm (equivalent to 660 mg/kg bw per day), the highest dose
tested (Suresh, 1993b).

194
In a two-generation reproduction study, glyphosate (purity 94.61%) was administered to 24

Crl:CD(SD) rats/sex per dose at concentrations of 0, 1200, 6000 and 30 000 ppm (equal to 0, 83.6,
417 and 2150 mg/kg bw per day for males and 0, 96.9, 485 and 2532 mg/kg bw per day for females)
for two successive generations with one litter per generation. After 10 weeks of treatment, animals
were paired within each dose group on a 1:1 basis to produce the F1 litters. On day 4 postpartum,
litters were standardized (four males and four females, if possible). At weaning, 24 males and 24
females from each dose group were selected to produce the F1 generation. Unselected offspring were
terminated and underwent gross necropsy. The offspring selected for the F1 generation were dosed for
at least 10 weeks and paired within dose group to produce F2 litters. At weaning, parental animals and
their offspring were terminated and examined macroscopically. Organs were taken from all parental
animals for weights and histopathological examination. For offspring, the same organs were taken
from one animal per sex per litter at random. The overall calculated mean daily intake of glyphosate
was 0, 84, 417 and 2150 mg/kg bw per day for F0 males; 0, 97, 485 and 2532 mg/kg bw per day for F0
females; 0, 92, 458 and 2411 mg/kg bw per day for F1 males; and 0, 105, 530 and 2760 mg/kg bw per
day for F1 females.
There were no treatment-related adverse effects on mortality, body weights, feed
consumption, feed efficiency or histopathological changes for parental animals of either generation.
The incidence of loose stools was increased for high-dose parental animals in both generations (Table
40). In addition, the incidences of caecum distension were increased in high-dose parental animals in
both generations (Table 41). Although increases in liver and kidney weights were noted in the high-
dose group, these changes were not considered adverse given the magnitude of the change and/or lack
of corresponding histopathological changes in these organs.
Table 40. Loose stools in two generations of rats administered glyphosate

Pre-mating Mating/gestation Lactation/post-weaning
1 200 6 000 30 000 1 200 6 000 30 000 0 1 200 6 000 30 000
0 ppm ppm ppm ppm 0 ppm ppm ppm ppm ppm ppm ppm ppm
F0
M 0/24 0/24 0/24 3/24 0/23 0/24 0/24 2/24 N/A N/A N/A N/A
F 0/24 0/24 0/24 1/24 0/24 0/24 0/24 0/24 0/24 0/24 0/24 6/24
F1
M 0/24 0/24 0/24 13/24 0/23 0/24 0/23 0/24 N/A N/A N/A N/A
F 0/24 0/24 0/24 4/24 0/23 0/23 0/21 0/19 0/23 0/23 0/21 2/19
F: female; F0: parental generation; F1: first filial generation; M: male; N/A: not applicable; ppm: parts per million
Results presented as number of animals with loose stools / number of animals examined.
Source: Takahashi (1997)

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Table 41. Incidence of caecum distension in three generations of rats administered glyphosate
0 ppm 1 200 ppm 6 000 ppm 30 000 ppm
F0
Males 0/24 0/24 0/24 21/24
Females 0/24 0/24 0/24 24/24
F1
Males 0/24 0/24 0/24 19/24
Females 0/24 0/24 0/24 17/24
Pups 0/136 0/141 0/143 89/141
F2
Pups 0/182 0/183 0/164 111/149
ppm: parts per million; F0: parental generation; F1: first filial generation; F2: second filial generation
Results presented as number of animals with caecum distension / number of animals examined.
No adverse effects were observed for mating performance, pregnancy rate or duration of
pregnancy in either generation. Litter size and viability were not affected by treatment. Body weights
of offspring at high doses were decreased in both generations, starting typically on postnatal day 14
(Table 42). Gross pathological examinations found an increased incidence of caecum distension in
high-dose offspring of both generations.
Table 42. Mean body weights of two generations of offspring of rats administered glyphosate
Mean body weights per dietary concentration of glyphosate
F1 pups – male F2 pups – male
1 200 6 000 1 200
PND 0 ppm ppm ppm 30 000 ppm 0 ppm ppm 6 000 ppm 30 000 ppm
0 6.7 ± 0.6 6.8 ± 0.5 6.7 ± 0.4 7.2 ± 0.7* 7.0 ± 0.5 6.9 ± 0.6 7.3 ± 0.7 7.1 ± 0.5
4 11.6 ± 1.2 11.6 ± 1.2 11.7 ± 1.0 11.6 ± 1.2 12.0 ± 1.2 12.1 ± 1.5 12.5 ± 1.5 12.5 ± 1.3
7 19.5 ± 1.7 19.1 ± 2.0 19.5 ± 1.6 19.3 ± 1.2 19.8 ± 1.5 20.0 ± 1.9 20.4 ± 2.2 20.6 ± 1.7
14 39.5 ± 3.2 39.4 ± 2.6 39.3 ± 2.6 36.6 ± 2.6** 40.1 ± 3.0 39.0 ± 2.8 38.7 ± 2.9 39.1 ± 2.8
21 63.9 ± 4.4 63.8 ± 4.1 62.4 ± 3.7 55.1 ± 3.5*** 58.6 ± 5.1 59.4 ± 4.4 58.3 ± 4.3 53.1 ± 4.4**
F1 pups – female F2 pups – female
0 6.3 ± 0.6 6.4 ± 0.5 6.4 ± 0.5 6.8 ± 0.6* 6.6 ± 0.5 6.6 ± 0.7 6.8 ± 0.6 6.8 ± 0.6
4 11.1 ± 1.2 11.2 ± 1.1 11.3 ± 0.9 11.3 ± 1.2 11.6 ± 1.2 11.5 ± 1.6 12.0 ± 1.5 12.1 ± 1.1
7 18.6 ± 1.8 18.4 ± 1.9 18.8 ± 1.5 18.3 ± 1.6 18.9 ± 2.0 19.1 ± 2.1 19.6 ± 2.2 19.9 ± 1.4
14 38.4 ± 3.6 37.9 ± 2.6 38.2 ± 2.2 35.4 ± 2.6** 38.7 ± 3.5 38.0 ± 2.2 37.5 ± 2.9 38.1 ± 2.9
21 61.0 ± 4.8 60.6 ± 3.9 59.8 ± 3.1 53.2 ± 4.0*** 56.4 ± 5.5 57.1 ± 4.4 56.2 ± 4.5 51.8 ± 4.2*
F1: first filial generation; F2: second filial generation; PND: postnatal day; ppm: parts per million; *: P ≤ 0.05; **: P ≤ 0.01;
***: P ≤ 0.001
Results presented are mean weights in grams ± standard deviation. Statistics from study report.

196
The NOAEL for parental toxicity was 6000 ppm (equal to 417 mg/kg bw per day) based on
increased incidence of loose stools and caecum distension in both generations at 30 000 ppm (equal to
2150 mg/kg bw per day). As there were no effects on reproductive parameters the NOAEL for
reproductive toxicity was 30 000 ppm (equal to 2150 mg/kg bw per day). The NOAEL for offspring
toxicity was 6000 ppm (equal to 417 mg/kg bw per day) based on decreased pup body weights and
increased incidence of caecum distension in both generations at 30 000 ppm (equal to 2150 mg/kg bw
per day) (Takahashi, 1997).
In a two-generation reproduction study, groups of 26 male and female Wistar-derived

Alpk:APfSD rats (aged 5–6 weeks at the start of treatment) were fed diets containing glyphosate
technical (purity 97.6%) at concentrations of 0, 1000, 3000 or 10 000 ppm (equal to 0, 99.4, 292.6 and
984.7 mg/kg bw per day for males and 0, 104.4, 322.8 and 1054.3 mg/kg bw per day for females) for
10 weeks before their first mating and until termination. Each generation was mated twice avoiding
sister/brother matings throughout. Males were terminated after completion of mating and females on
or soon after day 29 of lactation, after which their organs were weighed and gross pathological and
histopathological examinations conducted. The offspring not selected for mating were also terminated
on day 29 postpartum, with one pup/sex per litter used for organ-weight determination and two pups
/sex per litter given macroscopic examinations. All the remaining pups were terminated with no
further examination.
feed consumption, feed efficiency, organ weights or histopathological changes for parental animals of
duration of pregnancy in either generation. Litter size and viability were not affected by treatment.
The body weights of F1A pups were lower compared to the control group from day 8 onwards,
but a similar effect was not seen in the F2A pups. There was no treatment-related effect on total litter
weight (Table 43).
Table 43. Mean body weights of two successive generations of offspring of rats administered
glyphosate
Mean body weights per dietary concentration of glyphosate (g)
Males Females
10 000 10 000
PND 0 ppm 1 000 ppm 3 000 ppm ppm 0 ppm 1 000 ppm 3 000 ppm ppm
F1A
1 5.8 6.1 6.0 6.1 5.4 5.8 5.6 5.7
5 9.2 9.1 8.9 8.5 9.0 8.5 8.4 8.1**
8 13.8 13.4 13.2 12.6* 13.3 12.8 12.4 12.1**
15 26.8 26.1 25.8 24.6* 26.1 25.2 24.5 23.8*
22 43.4 42.4 41.4 39.2* 41.9 40.3 39.4 37.7*
29 81.7 79.5 79.6 74.6* 77.1 74.0 74.1 69.9**
F2A
1 6.3 6.3 6.3 6.2 6.1 5.9 5.9 5.8
5 9.7 9.9 9.3 9.5 9.3 9.6 9.1 9.1
8 14.3 14.7 13.8 14.2 13.8 14.2 13.4 13.7
15 27.4 28.3 26.4 27.5 26.7 27.5 25.8 26.5
22 44.5 46.2 43.1 44.9 42.7 44.8 41.8 42.9

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Mean body weights per dietary concentration of glyphosate (g)

Males Females
10 000 10 000
PND 0 ppm 1 000 ppm 3 000 ppm ppm 0 ppm 1 000 ppm 3 000 ppm ppm
29 83.0 86.0 80.6 82.8 77.7 80.6 75.6 77.4
F1A: first filial generation, first litter; F2A: second filial generation, second litter; PND: postnatal day; ppm: parts per million;
*: P = 0.05 (Student t-test, 2 sided); **: P = 0.01 (Student t-test, 2 sided)
Source: Moxon (2000)
As no adverse effects were noted in the study, the NOAEL for parental and reproductive
toxicity was 10 000 ppm (equal to 984.7 mg/kg bw per day), the highest dose tested. The NOAEL for
offspring toxicity was 3000 ppm (equal to 292.6 mg/kg bw per day) based on reduced pup weights in
the F1A generation seen at 10 000 ppm; equal to 984.7 mg/kg bw per day (Moxon, 2000).
In a two-generation reproduction study, glyphosate (purity 95.7%) was administered in the

diet to 28 Crl:CD(SD) IGS BR rats per sex per dose at 0, 1500, 5000 or 15 000 ppm (equal to 0, 104,
351 and 1063 mg/kg bw per day in males and 0, 126, 423 and 1273 mg/kg bw per day in females) for
two successive generations with one litter per generation. After 10 weeks of treatment, animals were
paired within each dose group on a 1:1 basis to produce the F1 litters. At weaning, 24 males and 24
females from each dose group were selected to produce the F2 generation. Surviving adult females and
males and unselected offspring were terminated on day 21 postpartum. All adult animals and
offspring underwent macroscopic examinations and parental organs were weighed. A small subset of
organs were taken from one male and one female offspring from the F0 and F1 pairings (where
available). Tissues from control and high-dose F0 and F1 animals underwent histopathological
examination. As there were indications of changes in the adrenal glands of F1 animals, microscopic
examination was extended to include all dose groups.
feed or feed efficiency, organ weights or histopathological changes in parental animals of either
generation. No adverse effects were observed on mating performance, pregnancy rate or duration of
pregnancy in either generation. Litter size, viability and offspring body weights were not affected by
treatment. Complete preputial separation was delayed by 2.9 days in high-dose F1 male pups (2.9
days) and body weights were increased by 10% at attainment. There were no treatment-related effects
on the age or weight at attainment of vaginal opening.
As there were no effects for parental animals or on reproductive parameters, the NOAEL for
parental and reproductive toxicity was 15 000 ppm (equal to 1063 mg/kg bw per day), the highest
dose tested. The NOAEL for offspring was 5000 ppm (equal to 351 mg/kg bw per day), based on
delayed age and increased weight at attainment of preputial separation at 15 000 ppm (equal to 1063
mg/kg bw per day) (Dhinsa, 2007).

Rats
In a pre-GLP developmental toxicity study, glyphosate (purity 98.7%) suspended in 0.5%
aqueous Methocel was administered to 25 copulated CD female rats per dose by oral gavage at
concentrations of 0, 300, 1000 or 3500 mg/kg bw per day from gestation day 6 through 19. On
gestation day 20, the dams were terminated, pregnancy status determined and numbers of corpora
lutea, implantations and live fetuses recorded. All live fetuses were weighed, sexed and examined for
external, visceral and skeletal abnormalities.
Soft stools, diarrhoea, red nasal discharge, reduced activity and rales (abnormal respiratory
noise) were noted in the highest dose group. By gestation day 17, six rats in this group had died. A

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reduced mean body-weight gain due to a loss in mean maternal weight over the first three days of
treatment was noted in the high-dose group. No significant differences between the 300 and 1000
mg/kg bw per day dosage groups and the control group were observed in terms of the mean number of
viable fetuses, implantations, post-implantation losses, corpora lutea or mean fetal body weight. The
mean number of total implantations, viable fetuses and mean fetal body weight were significantly
decreased in the 3500 mg/kg bw per day dosage group compared to controls. In addition, the dams in
the high-dose group had a significant increase in early resorptions, causing a slight increase in post-
implantation losses.
At 3500 mg/kg bw per day, the number of litters with malformations was identical to that of
the control group, but the number of fetuses with malformations was increased. However, since the
number and type of malformations observed were similar to those observed in historical control data,
it was concluded that they were not treatment related. There were an increased number of fetuses with
unossified sternebrae in the high-dose group; although treatment related, this is considered a
developmental variation rather than a teratogenic malformation. No malformations were observed in
the 300 and 1000 mg/kg bw per day dosage groups.
The NOAEL for maternal toxicity was 1000 mg/kg bw per day based on mortality, soft stools
and reduced body-weight gain at 3500 mg/kg bw per day. The NOAEL for developmental toxicity
was 1000 mg/kg bw per day based on the decreased mean number of total implantations, viable
fetuses, mean fetal body weight, increased early resorptions and increased number of fetuses with
unossified sternebrae at 3500 mg/kg bw per day (Tasker, Rodwell & Jessup, 1980a).
In a developmental toxicity study, glyphosate (purity 98.6%) suspended in a 1.0% aqueous

solution of methylcellulose was administered to 25 mated Crl:CD(SD)BR VAF/Plus female rats per
dose by oral gavage at concentrations of 0, 300, 1000 or 3500 mg/kg bw per day from gestation days
6 through 15. On gestation day 20, the dams were terminated, pregnancy status determined and
numbers of corpora lutea, implantations and live fetuses recorded. All live fetuses were weighed,
sexed and examined for external, visceral and skeletal abnormalities.
At the highest dose, clinical abnormalities included salivation, loose stools and rales. The
latter was also observed in two animals at the intermediate dose on one occasion. There were two
maternal mortalities at the highest dose following signs of respiratory distress. Body-weight gain was
markedly reduced at the highest dose (by 16–81% of control values, gestation days 6–20) and
marginally reduced at the intermediate dose (by 86–97% of control values, gestation days 6–20). Feed
consumption was slightly decreased at the highest dose during the dosing period (75–94% of control
values, gestation days 6–15), but was comparable with controls thereafter. Water intake was increased
at the highest dose (139–205% of control values, gestation days 6–15). No treatment-related changes
were observed at any dose at necropsy.
A total of 23, 23, 25 and 22 dams had live young on day 20 in the control group and at 300,
1000 and 3500 mg/kg bw per day, respectively. Treatment had no significant effect on embryonic
losses, litter size or sex ratio, but the litter weights were reduced at the highest dose (90% of control
values) and mean fetal weights were statistically significantly reduced at the highest dose (94% of
control values; P < 0.01). The occurrence of malformations was not significantly increased by
treatment. However, the incidence of rib distortion (wavy ribs) was markedly higher at the highest
dose and slightly higher at the intermediate dose; the incidences based on fetuses were 1, 0, 3 and 28
and on litters were 1, 0, 2 and 11 at 0, 300, 1000 and 3500 mg/kg bw per day, respectively. In
addition, reduced ossification was seen slightly more frequently at the highest and intermediate doses.
The percentage of fetuses showing skeletal anomalies (variations) was significantly increased at the
two higher doses, but the percentage of fetuses affected at the intermediate dose exceeded the
historical background range (21.9–27.2%) only slightly (Table 44).

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Table 44. Skeletal anomalies in fetuses and litters of rats administered glyphosate
0 300 mg/kg bw per 1000 mg/kg bw per 3500 mg/kg bw per
mg/kg bw per day day day day
Fetal anomoliesa 19/155 36/143 46/166 55/142
b
Litter anomolies 11/23 16/23 19/25 19/22
Fetal skeletal 11.7 22.6 28.4* 35.7**
variations (%)c
Historical range 21.9–27.2
bw: body weight; *: P < 0.05; **: P < 0.01
Kruskal–Wallis H-statistic followed, if significant, by intergroup comparison with control (distribution-free Williams’ test).
a
Results presented as number of fetuses with skeletal anomalies / total number of fetuses.
b
Results presented as number of litters with skeletal anomalies / total number of litters.
c
Results expressed as number of fetuses with skeletal variations (with malformed fetuses excluded) as a percentage of the
total number of fetuses examined.
Source: Brooker et al. (1991a)
The NOAEL for maternal toxicity was 300 mg/kg per day based on clinical signs and reduced
body-weight gain at 1000 mg/kg bw per day and higher. The NOAEL for developmental toxicity was
300 mg/kg per day based on an increased incidence of delayed ossification and an increased incidence
of fetuses with skeletal anomalies at 1000 mg/kg bw per day and higher (Brooker et al., 1991a).

solution of sodium carboxymethylcellulose was administered to 24 copulated Crj:CD(SD) female
rats/dose by oral gavage at concentrations of 0, 30, 300 or 1000 mg/kg bw per day from gestation day
There were no treatment-related changes in mortality, body weight, feed consumption or
macroscopic findings in dams. An increased incidence of slightly loose stools was observed during
the dosing period in 20 of the 22 pregnant females at 1000 mg/kg bw per day. Of these 20 animals, 9
still displayed the effect on the day after the last dosing.
There were no effects on number, growth or survival of fetuses. Any external, visceral or
skeletal abnormalities were considered secondary to maternal toxicity; furthermore, the effects were
also seen in the control group, incidences of the effects were low and/or there was no dose–response
relationship for the effect.
The NOAEL for maternal toxicity was 300 mg/kg bw per day based on the increased
incidence of slightly loose stools observed in dams at 1000 mg/kg bw per day. As there were no
developmental effects, the NOAEL for developmental toxicity was 1000 mg/kg bw per day
(Hatakenaka, 1995).
In a developmental toxicity study, glyphosate acid (purity 95.6%) in deionized water was
administered to 24 time-mated female Alpk:APfSD (Wistar-derived) rats/dose by oral gavage at 0,
250, 500 or 1000 mg/kg bw per day from gestation day 7 through 16. On gestation day 22, the dams
were terminated, pregnancy status determined and numbers of corpora lutea, implantations and live
fetuses recorded. All the fetuses were weighed, sexed and examined for external, visceral and skeletal
abnormalities.

200
One control animal was terminated on day 7 due to incorrect dosing. There were no
treatment-related changes in clinical observations, body weight, feed consumption or macroscopic
findings for dams.
There were no effects on number, growth or survival of fetuses and no treatment-related
external, visceral or skeletal abnormalities.
As there were no maternal or developmental effects, the NOAEL for maternal and
developmental toxicity was 1000 mg/kg bw per day (Moxon, 1996a).
Rabbits
Methocel solution was administered to 16 Dutch Belted female rabbits per dose by oral gavage at
concentrations of 0, 75, 175 or 350 mg/kg bw per day from gestation day 6 through 27. On gestation
day 28, the dams were terminated, pregnancy status determined and numbers of corpora lutea,
implantations and live fetuses recorded. All fetuses were weighed, sexed and examined for external,
visceral and skeletal abnormalities. This study was conducted prior to GLP.
Incidence of mortality was increased in the high-dose group. The number of spontaneous
deaths in the control, low-, mid- and high-dose groups was 0/16, 1/16, 2/16 and 10/17, respectively. A
slight increase in the incidence of soft stools and diarrhoea was noted in the medium–high-dose group
(individual data not reported). At 350 mg/kg bw per day, soft stool and/or diarrhoea were observed in
each animal at least once during treatment. An increased incidence of nasal discharge was also noted
in the high-dose group (individual data not reported). There were no treatment-related changes in
body weight or macroscopic findings for dams.
Due to the increased mortality at the high dose, the number of animals (6 pregnant females)
available for evaluation of developmental effects was insufficient. The numbers of pregnant dams
were also low for the other doses (12, 15 and 11 in the control, low- and mid-dose groups,
respectively). limiting the evaluation of developmental effects in this study. The available data for the
control, low- and mid-dose groups indicate no treatment-related adverse effects on the number,
growth or survival of fetuses. Any external, visceral or skeletal abnormalities were not considered
treatment related.
The NOAEL for maternal toxicity was 175 mg/kg bw per day based on increased incidence of
clinical signs (soft stools and diarrhoea) and mortality at 350 mg/kg bw per day in rabbits. Individual
data were not provided for the clinical signs at 175 mg/kg bw per day, and the increase in incidence
was only slight at this dose. Due to the low number of pregnant dams, developmental effects could not
be evaluated; however, the available data indicate no evidence of developmental effects (Tasker,
Rodwell & Jessup, 1980b).
In a developmental toxicity study, glyphosate (purity 95%) suspended in a 0.1% aqueous gum
acacia solution was administered to 15 New Zealand White female rabbits per dose by oral gavage at
concentrations of 0, 125, 250 and 500 mg/kg bw per day, respectively, from gestation day 6 through
18. On gestation day 29, the dams were terminated, pregnancy status determined and numbers of
corpora lutea, implantations and live fetuses recorded. All live fetuses were weighed, sexed and
examined for external, visceral and skeletal abnormalities.
There were no treatment-related adverse changes in mortality, feed consumption or
macroscopic findings for dams. Two abortions were noted in the high-dose group. A slight decrease in
body-weight gain was also noted at 500 mg/kg bw per day.
There were no treatment-related adverse effects on the number, growth or survival of fetuses.
The mean number of viable implants per litter was lower at the high dose than the other treatment
groups and controls and accordingly the mean number of non-viable implants per litter was higher

201
than the other treatments groups; however, when taking into account the variability for these
measurements, the changes were not considered adverse.
Incidences of external, visceral or skeletal variations/malformations in fetuses in the low- and
mid-dose groups did not differ from those of the control group (Table 45). At 500 mg/kg bw per day,
incidences of variations/malformations were higher than in the control group, but in many cases the
increase was minimal or similar to the 125 and 250 mg/kg bw per day dose groups when evaluated on
a litter basis. These increases in incidences of variations/malformation were observed in the presence
of severe maternal toxicity. The occurrences of a variety of low-incidence fetal effects
(malformations) were slightly increased at higher dose levels. These increases are considered
secondary to maternal toxicity.
Table 45. Malformations and variations in fetuses and litters of rabbit administered glyphosate
0 mg/kg bw 125 mg/kg 250 mg/kg bw 500 mg/kg
Malformations / variations per day bw per day per day bw per day
Number of litters examined 13 14 14 12
Number of fetuses examined 109 113 120 78
Malformations
Tail abnormal 1 (1) 1 (1) 2 (2) 3 (2)
Low-set ears 0 (0) 1 (1) 1 (1) 2 (1)
Ventricular septal defect 0 (0) 1 (1) 1 (1) 2 (2)
Postcaval lung lobe absent 0 (0) 1 (1) 2 (2) 4 (3)
Kidney(s) absent 1 (1) 2 (2) 2 (2) 6 (4)
Rudimentary rib (no. 14) 1 (1) 0 (0) 2 (2) 5 (2)
Variations
Tail blunt tipped 1 (1) 0 (0) 3 (2) 5 (4)
Irregular rugae on palate 0 (0) 2 (1) 3 (2) 2 (2)
Lateral ventricles of cerebrum dilated 0 (0) 2 (2) 2 (2) 6 (4)
Right ventricle smaller than normal 1 (1) 3 (2) 3 (2) 5 (3)
Globular heart 2 (2) 0 (0) 3 (2) 5 (4)
Incomplete separation of lung lobes 1 (1) 2 (1) 2 (1) 4 (2)
Parietal fetal atelectasis 0 (0) 1 (1) 1 (1) 1 (1)
Liver irregular shape 0 (0) 2 (1) 2 (2) 6 (4)
Kidney(s) globular shape 0 (0) 0 (0) 2 (1) 5 (3)
Cervical central 1–3 and/or 4 bilobed 1 (1) 0 (0) 1 (1) 2 (2)
Anterior arch of the atlas poorly ossified 2 (1) 2 (1) 1 (1) 4 (2)
Anterior arch of the atlas split 0 (0) 0 (0) 2 (1) 3 (1)
Extrathoracic centrum and arch 1 (1) 3 (2) 2 (1) 5 (3)
Thoracic centrum only one ossification centre 1 (1) 0 (0) 1 (1) 3 (2)
Thoracic centra fused 2 (1) 1 (1) 1 (1) 2 (1)
Extra ribs on thoracic centra and arch 13 bilateral 1 (1) 0 (0) 3 (2) 5 (4)
Sternebra – 6 poorly ossified 2 (1) 1 (1) 2 (1) 4 (2)
Sternebra(e) split 2 (1) 2 (1) 1 (1) 5 (3)
Sternebra(e) unossified 3 (2) 1 (1) 3 (2) 6 (4)

202

0 mg/kg bw 125 mg/kg 250 mg/kg bw 500 mg/kg
Malformations / variations per day bw per day per day bw per day
Number of litters examined 13 14 14 12
Number of fetuses examined 109 113 120 78
Pubis, poorly ossified 3 (2) 2 (2) 3 (1) 4 (3)
Some ossification in knee area 1 (1) 3 (2) 2 (1) 2 (2)
Skull bones poorly ossified 1 (1) 3 (2) 2 (1) 2 (2)
Frontal, hole in bone 0 (0) 1 (1) 2 (2) 2 (2)
Reduced number of caudal segments 1 (1) 2 (2) 1 (1) 3 (2)
bw: body weight
Results presented as number of fetuses with malformations and variations and, in parentheses, the number of litters with
malformations and variations.
Source: Bhide & Patil (1989)
The NOAEL for maternal toxicity was 250 mg/kg bw per day based on abortions observed at
500 mg/kg bw per day in rabbits. The NOAEL for developmental toxicity was 250 mg/kg bw per day
based on increased incidence of variations/malformations observed at 500 mg/kg bw per day in
rabbits. It should be noted that individual data, uterine weights, maternal necropsy results and
statistical analyses were not provided for this study; therefore, the NOAEL and LOAEL values are
based on the available data (Bhide & Patil, 1989).
In a developmental toxicity study, glyphosate acid (purity 98.6%) suspended in a 1% aqueous

methylcellulose solution was administered to 19, 19, 16 or 20 New Zealand White rabbits per dose by
oral gavage at concentrations of 0, 50, 150 or 450 mg/kg bw per day, respectively, from gestation day
There were no treatment-related adverse changes in body weight, feed consumption or
macroscopic findings for dams. One high-dose animal was found dead on day 20 following signs of
abortion on day 19 and soft/liquid faeces, a reduction in feed intake and body-weight loss from the
start of treatment. The incidence of soft/liquid faeces was increased at the high dose (13/20 animals).
At termination, 18, 12, 15 and 13 pregnant females were available for evaluation in the control, low,
mid and high doses, so evaluation of developmental effects is limited at the low and high doses.
Embryo/fetal death and post-implantation loss were increased in all treatment groups; however, there
was no dose–response and the values were within or slightly above the historical control range.
Any external, visceral or skeletal abnormalities were not considered treatment related. There
was a slightly increased incidence of cardiac malformation (interventricular septal defect) at the high
dose (4/13 pregnant animals); however, it was barely outside of the historical control range from
studies conducted during the same period, and the number of litters to evaluate this dose was reduced.
Furthermore, this effect was considered secondary to the maternal toxicity observed at 450 mg/kg bw
per day.
The NOAEL for maternal toxicity was 150 mg/kg bw per day based on clinical signs
(soft/liquid faeces) at 450 mg/kg bw per day in rabbits. The NOAEL for developmental toxicity was
150 mg/kg bw per day based on the post-implantation loss, late embryonic death and an increase in
cardiac malformations at 450 mg/kg bw per day (Brooker et al., 1991b).

203
In a developmental toxicity study, glyphosate acid (purity 96.8%) suspended in a 0.5%

aqueous CMC solution was administered to 26, 17, 16 and 16 presumed-mated New Zealand White
rabbits per dose by oral gavage at concentrations of 0, 20, 100 or 500 mg/kg bw per day, respectively,
from gestation day 6 through 18. On gestation day 28, the dams were terminated, pregnancy status
determined and numbers of corpora lutea, implantations and live fetuses recorded. All the fetuses
were weighed, sexed and examined for external, visceral and skeletal abnormalities.
There were no treatment-related adverse changes in body weight, feed consumption or
macroscopic findings for dams. There were two, zero, four and eight deaths in the control, low-, mid-
and high-dose groups, respectively; the deaths in the control group were definitively attributed to
gavage error. An increased incidence of soft stool/liquid faeces was observed at the high dose
(12/15 animals). Other clinical signs at the high dose included rales, weakness, dyspnoea and ocular
discharge; however, the incidence of these effects was low and some effects may indicate gavage
error. At necropsy, various findings were noted in the lungs and trachea in mid- and high-dose
animals, which also suggests possible gavage errors and/or issues with animal husbandry.
However, the number of pregnant females available for evaluation in the control and the low-, mid-
and high-dose groups was 20, 13, 12 and 6, respectively, limiting the study of developmental effects.
Total litter loss was recorded for one female in the high-dose group. Any external, visceral or skeletal
abnormalities were not considered treatment related. Major visceral malformations primarily affected
the heart, but occurred in single incidences and/or showed no dose–response relationship except for
the dilated heart; however, interpreting the dose–response relationship is difficult given the limited
number of litters available, especially at the high dose. In addition, this effect was considered
secondary to the maternal toxicity observed at 500 mg/kg bw per day.
Based on the uncertainties regarding gavage errors and mortalities across doses in this study
and the reduced number of pregnant females, the study is considered unacceptable (Suresh, 1993c).

solution of sodium carboxymethylcellulose was administered to 18 artificially inseminated Japanese
white rabbits (Kbl:JW) per dose by oral gavage at concentrations of 0, 10, 100 or 300 mg/kg bw per
day from gestation day 6 through 18. On gestation day 27, the dams were terminated, pregnancy
status determined and numbers of corpora lutea, implantations and live fetuses recorded. All live
fetuses were weighed, sexed and examined for external, visceral and skeletal abnormalities.
There were no treatment-related changes in body weight, feed consumption or macroscopic
findings for dams. One dam died on gestation 20 without showing any clinical signs, and the cause of
death was undetermined. An increased incidence of loose stools was observed during the dosing
period in four of the 17 remaining pregnant females In the high-dose group; two continued to display
this effect during the post-dosing period and one aborted on gestation day 26.
There were no effects on number, growth or survival of fetuses. All observations of external
or visceral malformations were sporadic in nature and not considered treatment related. Skeletal
malformations and variations were also not considered treatment-related since these effects were also
seen in the control group, incidences of the effects were low and/or there was no dose–response
relationship for the effect.
The NOAEL for maternal toxicity was 100 mg/kg bw per day based on the increased
incidence of loose stools observed in dams at 300 mg/kg bw per day. There were no developmental
effects; therefore, the NOAEL for developmental toxicity is 300 mg/kg bw per day (Hojo, 1995).
In a developmental toxicity study, glyphosate (purity 95.3%) suspended in a 1% CMC was

administered to 18 mated New Zealand White female rabbits per dose by oral gavage at
concentrations of 0, 50, 200 or 400 mg/kg bw per day from gestation day 7 through 19. On gestation
day 29, the dams were terminated, pregnancy status determined and numbers of corpora lutea,

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implantations and live fetuses recorded. All fetuses were weighed, sexed and examined for external,
visceral and skeletal abnormalities.
There were no treatment-related changes in body weight, feed consumption or macroscopic
findings for dams. One high-dose female was found dead prior to dosing on day 19 and another was
terminated in extremis on day 20; one death also occurred in the control group and in the mid-dose
group. An increased incidence of diarrhoea was observed at the high dose in 10 of the 16 surviving
pregnant females. All other clinical observations were isolated or a dose–response relationship was
not observed.
The increases in late fetal deaths and post-implantation loss noted at the high doses were not
considered adverse once the variability in the measurements were taken into consideration. In
addition, the increase can mainly be attributed to one animal with nine late-death fetuses. No
treatment-related external, visceral or skeletal abnormalities were observed.
diarrhoea in dams at 400 mg/kg bw per day. As there were no developmental effects, the NOAEL for
developmental toxicity was 400 mg/kg bw per day (Coles & Doleman, 1996).
In a developmental toxicity study, glyphosate acid (purity 95.6%) in deionized water was
given to 20 time-mated New Zealand White female rabbits per dose by oral gavage at concentrations
of 0, 100, 175 or 300 mg/kg bw per day from gestation day 8 through 20. On gestation day 30, the
dams were terminated, pregnancy status determined and numbers of corpora lutea, implantations and
live fetuses recorded. All fetuses were weighed, sexed and examined for external, visceral and skeletal
abnormalities.
There were no treatment-related adverse changes in mortality, body weight, feed consumption
or macroscopic findings for dams. There was a significant increase in the incidence of either diarrhoea
or decreased faecal output at the mid and high doses (no statistical significance was provided) (Table
46). The incidence of staining in the genital area was also increased at the high dose.
Table 46. Clinical signs in pregnant rabbits administered glyphosate by gavage

100 mg/kg bw per 175 mg/kg bw per 300 mg/kg bw per
Clinical sign 0 mg/kg bw per day day day day
Few faeces in tray 3 3 9 9
Signs of diarrhoea 4 5 11 19
Staining in genital area 2 2 3 11
bw: body weight; no. number
Source: Moxon (1996b)
Although mean fetal weight was reduced at the high dose, this was not considered adverse once the
variability in the measurements was taken into account. In addition, the decrease could be attributed to
two litters with lower weights. Any external, visceral or skeletal abnormalities were not considered
treatment related.
clinical signs (decreased faecal output or signs of diarrhoea) in rabbits at 175 mg/kg bw per day. As
there were no developmental effects, the NOAEL for developmental toxicity was 300 mg/kg bw per
day (Moxon, 1996b).

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2.6 Special studies

(a) Neurotoxicity
Cell cultures
In a non-guideline experiment, a cell culture model was used to determine if chronic exposure
to organophosphate pesticides can alter the sensitivity of nerve cells to subsequent acute exposure to
organophosphates or other compounds. NB2a neuroblastoma cells were grown in the presence of
diazinon at a concentration of 25μmol/L for 8 weeks. The organophosphate was then withdrawn and
the cells were induced to differentiate in the presence of various other pesticides, including glyphosate
(purity > 99%). The resulting outgrowth of neurite-like structures was measured by light microscopy
and quantitative image analysis and the median inhibitory concentration (IC50) for each
organophosphate or formulation calculated. The IC50 values in cells pre-exposed to diazinon were
compared with the equivalent values in cells not pre-exposed to diazinon. The IC50 for inhibition of
neurite outgrowth by acute application of diazinon, pyrethrum, glyphosate or a commercial
formulation of glyphosate was decreased by between 20% and 90% after pretreatment with diazinon.
According to the study authors, the data support the view that long-term exposure to an
organophosphate may reduce the threshold for toxicity of some environmental agents (Axelrad,
Howard & McLean, 2003).
Rats
In an acute neurotoxicity study, groups of fasted (24 hours), approximately 42-day-old
Alpk:APfSD rats (10/sex per dose) were given a single oral dose of glyphosate (purity 95.6%) in
deionized water at concentrations of 0, 500, 1000 or 2000 mg/kg bw. They were then observed for 2
weeks. Neurobehavioural assessment (functional observational battery and motor activity testing) was
performed in all animals in week −1 (pre-dosing), on day 1 (approximately 6 hours after dosing), day
8 and day 15. At study termination, five animals/sex per dose were euthanized and perfused. Of the
perfused animals, the control and highest dose groups were used for neuropathological examinations
with brain and peripheral nervous system tissues undergoing histopathological evaluation.
Administration of a single dose of glyphosate produced treatment-related clinical signs of
general toxicity at 2000 mg/kg bw. On day 1, approximately 6 hours after dosing, three high-dose
females were observed with decreased activity, subdued behaviour, hunched posture and/or
hypothermia. Diarrhoea was also seen in another female at this dose. Full recovery was established by
day 2. These clinical signs do not reflect signs of neurotoxicity and were mostly likely associated with
the excessively high dose of glyphosate. No treatment-related effects were observed on mortality,
body weight or brain weight. Similarly, neuropathological and histopathological examinations showed
no treatment-related effects, and functional observational battery and motor activity tests revealed no
treatment-related effects. Although overall motor activity at 2000 mg/kg bw for both sexes on day 1
was lower than that of controls, these differences were not statistically significant or dose dependent.
The NOAEL for neurotoxicity in rats was 2000 mg/kg bw. The NOAEL for systemic toxicity
was 1000 mg/kg bw based on clinical signs of general toxicity (decreased activity, subdued
behaviour, hunched posture, hypothermia and diarrhoea) and lethality at 2000 mg/kg bw. The LOAEL
for systemic toxicity in rats was 1000 mg/kg bw (Horner, 1996a).
In a subacute neurotoxicity study, glyphosate (purity 95.6%) was administered to 12

Alpk:APfSD rats per sex per group in the diet at concentrations of 0, 2000, 8000 or 20 000 ppm
(equal to 0, 155.5, 617.1 and 1546.5 mg/kg bw per day for males and 0, 166.3, 672.1 and 1630.6
mg/kg bw per day for females) for 13 weeks. Neurobehavioural assessment (functional observational
battery and motor activity testing) was performed in all animals at weeks −1, 1, 5, 9 and 14. At study
termination, six animals/sex per group were euthanized and perfused. Of these, the control and highest

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dose groups were used for neuropathological examinations and brain and peripheral nervous system
tissues histopathologically evaluated.
Overall mean body weight (92.8% of the controls; P < 0.05) and feed utilization (P < 0.01)
were reduced in high-dose males with no treatment-related effect on feed consumption. Group mean
body-weight was also lower than the controls in males at 8000 ppm from weeks 6–14 (not statistically
significantly). No treatment-related effects on mortality, clinical signs or brain weight were observed.
Functional observational battery and locomotor activity testing revealed no treatment-related effects.
Neuropathological and histopathological examinations of the peripheral and nervous system did not
yield any treatment-related effects from glyphosate administration.
The NOAEL for neurotoxicity in rats was 20 000 ppm, equal to 1547 mg/kg bw per day. The
NOAEL for systemic toxicity was 20 000 ppm, equal to 1546.5 mg/kg bw per day (Horner, 1996b).
Hens
In an acute delayed neurotoxicity study, 20 hens (hybrid brown laying strain – Lohmann
Brown) were given a single oral dose of glyphosate (purity 95.6%) of 2000 mg/kg bw. In addition, 12
negative control hens were dosed with distilled water and 12 positive control hens with 1000 mg/kg
bw of triorthocresyl phosphate (TOCP). This was followed by an observation period of 21/22 days.
The hens were examined for any clinical signs twice daily and for ataxia daily, and weighed weekly.
Brain acetylcholinesterase, brain neuropathy target esterase (NTE) and lumbar spine NTE
measurements were made on three hens, 48 hours after dosing. At the end of the observation period,
six hens from each treatment group were selected for termination and macroscopic and
histopathological examination. After perfusion through the heart with fixative, the selected tissues
were processed and examined histopathologically.
No treatment-related mortality was observed in the study. There was no evidence of clinical
ataxia in any of the negative controls or in any of the hens dosed with glyphosate. Of the 12 hens
dosed with TOCP (positive controls), five developed clinical ataxia, starting between days 11 and 21.
There was no effect on body weights for hens dosed with glyphosate, but TOCP-dosed hens showed
an overall weight loss. Acetylcholinesterase was reduced by 6% in glyphosate-treated hens and 19%
in TOCP-treated hens. There was no effect on NTE levels in brain or spinal cord for the glyphosate-
treated hens, but compared to the negative controls, brain NTE levels were reduced by 84% and spinal
cord NTE levels by 78% in the positive controls. No macroscopic abnormalities were seen in any of
the hens examined. Histopathological examination revealed no evidence of acute delayed
neurotoxicity or any other treatment-related changes in glyphosate-treated hens. Hens dosed with
TOCP showed significant axonal degeneration in spinal cord, peripheral nerve and cerebellum,
demonstrating the validity of the test system.
In conclusion, oral administration of a single dose of 2000 mg/kg bw of glyphosate produced
no clinical signs of delayed neurotoxicity, no significant reduction in acetyl cholinesterase and no
histopathological findings in hens. The NOAEL for acute delayed neurotoxicity of glyphosate in hens
was 2000 mg/kg bw (Johnson, 1996).
(b) Immunotoxicity
In an unpublished immunotoxicity study, glyphosate (purity 85.2%) was administered to
female B6C3F1/Crl mice (10/dose) in the diet at dose levels of 0, 500, 1500 or 5000 ppm (equal to 0,
150.1, 449.1 and 1447.5 mg/kg bw per day, respectively) for 28 days. The positive control group (10
females) was administered 50 mg/kg bw per day of cyclophosphamide (10 mL/kg at a concentration
of 5 mg/mL) by intraperitoneal injection from study days 24–27. On day 24, all the animals in all the
groups received a single intravenous dose of 7.5  107 sheep red blood cells (SRBC) in 0.2 mL of
Earle’s Balanced Salt Solution with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. At
termination, the spleen and thymus were removed and weighed. The T-cell–dependent antibody
response to SRBC was measured with antibody-forming cell (AFC) assay.

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There were no pre-terminal deaths, no treatment-related clinical signs and no treatment-

related effects on feed and water consumption, mean body weights, organ weights and macroscopic
findings in all treated groups. The body weights of the positive control group treated with
cyclophosphamide did not differ significantly from those of the vehicle control group, but the absolute
and relative spleen and thymus weights decreased statistically significantly (P < 0.01).
The systemic NOAEL was 5000 ppm (equal to 1448 mg/kg bw per day), the highest dose
tested.
No statistically significant differences were observed in anti-SRBC antibody-forming cell
responses for specific activity (AFC/106 spleen cells) and total spleen activity (AFC/spleen) in treated
groups compared to the vehicle control group. The positive control group had a statistically significant
(P < 0.05) decrease in spleen cell numbers, mean specific activity and mean total spleen activity. This
confirmed the ability of the test system to detect immunosuppressive effects and confirmed the
validity of the study design. Natural killer cell activity was not evaluated in this study.
The NOAEL for immunotoxicity was 5000 ppm (equal to 1448 mg/kg bw per day), the
highest dose tested (Haas, 2012).
In a published study, female CD-1 mice were exposed to Tordon 202C (2,4-
dichlorophenoxyacetic acid [2,4-D] and picloram) or Roundup in drinking water for 26 days at
concentrations from 0–0.42% or 0–1.05%, respectively. Glyphosate isopropylammonium salt was
administered in distilled drinking water at concentrations of 0%, 0.35%, 0.70% or 1.05%
(approximately equal to 335, 670 and 1000 mg/kg bw). The mice were inoculated with SRBC to
produce a T-lymphocyte macrophage-dependent antibody response on day 21 of the herbicide
exposure period. Roundup exposure did not alter weight gain or water consumption. Antibody
production was also unaffected by Roundup dosing, suggesting that Roundup is unlikely to cause
immune dysfunction under normal conditions of application (Blakley, 1997).
The role of glyphosate in developing asthma and rhinitis among farmers was evaluated in a
published study. The aim of this study was to explore the mechanisms of glyphosate-induced
pulmonary pathology by utilizing murine models and real environmental samples. C57BL/6,
TLR4_/_, and IL-13_/_ mice inhaled extracts of glyphosate-rich air samples collected on farms during
spraying of herbicides or inhaled different doses of glyphosate and ovalbumin. The cellular response,
humoral response and lung function of exposed mice were evaluated. Inhalation exposure to
glyphosate-rich air samples as well as glyphosate alone increased eosinophil and neutrophil counts,
mast cell degranulation and production of the cytokines interleukin-33 (IL-33), thymic stromal
lymphopoietin, interleukin-13 (IL-13) and interleukin-5 (IL-5). In contrast, in vivo systemic
interleukin-4 (IL-4) production was not increased. Co-administration of ovalbumin with glyphosate
did not substantially change the inflammatory immune response. However, deficiency in IL-13
resulted in diminished inflammatory response, but did not have a significant effect on airway
resistance upon methacholine challenge after 7 or 21 days of glyphosate exposure. Glyphosate-rich
farm air samples as well as glyphosate alone were found to induce pulmonary IL-13–dependent
inflammation and promote Th2-type cytokines, but not IL-4 for glyphosate alone (Kumar et al.,
2014).
(c) Effects on the salivary gland

Groups of 24 male Alpk:APfSD (Wistar-derived; AP), Sprague Dawley (Charles River; CD)
and Fischer 344 (F344) rats were fed diets containing 0 or 20 000 ppm glyphosate acid for 28
consecutive days. Eight animals from each group were terminated of day 29, and the remaining rats
retained without treatment for an additional 4 (eight rats/group) or 13 weeks (eight rats/group).

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Dietary exposure to 20 000 ppm glyphosate acid resulted in significant reductions in body
weight and minor reductions in feed consumption in AP and CD rats, but not in F344 rats. Salivary
gland weight was unaffected in the CD rat but was increased in both AP and F344 rats at the end of
the 4-week dietary exposure period. Microscopic examination of the salivary glands showed that the
most pronounced effected occurred in the F344 strain, where there was diffuse cytoplasmic basophilia
and enlargement of the parotid acinar cells. Similar but slight effects involving small foci of cells
occurred in the AP and CD strains.
After four weeks on the control diet, the salivary glands of the F344 strain had significantly
recovered, while AP and CD rats were indistinguishable from their corresponding controls.
After 13 weeks on the control diet, slightly more glyphosate-treated F344 rats showed minor
focal changes in the salivary glands compared to their controls, and group mean salivary-gland
weights were increased slightly (Allen, 1996).
In a study of the mechanism of induction of salivary gland lesions performed by the National
Toxicology Program, two groups of four male F344/N rats were fed diets containing glyphosate
(purity 99%) at a concentration of 50 000 ppm (the highest dose used in a short-term study on
toxicity) and given a continuous subcutaneous infusion of propanolol (a β-blocker; 1.2 mg/kg bw per
day) or a vehicle (water). Three additional groups of four male rats were fed a control diet and given a
continuous subcutaneous infusion of isoproterenol (a β-adrenergic agonist; 1.0 mg/kg bw per day),
isoproterenol plus propanolol, or a vehicle (water). After 14 days of treatment, the animals were
terminated, and the parotid and submandibular/sublingual glands were removed, weighed and
processed for electron and light microscopy.
All the rats survived to the end of the study. Rats subcutaneously infused with isoproterenol
were hypoactive and had increased respiratory rates on day 1, but behaved normally by the following
day. While there was no effect on feed consumption in any group, there was a significant decrease in
body-weight gains in the groups fed glyphosate (6.3 g and 6.0 g, compared with 16.0 g in controls).
Both glyphosate and isoproterenol produced increased salivary-gland weights, with the parotid gland
being more affected (280% or 154% of weights in the control group for glyphosate or isoproterenol,
respectively). When both compounds were given along with propanolol, parotid weights were 194%
of those of the controls for glyphosate but only 109% of those of the controls for isoproterenol. In the
parotid and in the submandibular gland, increased weights were associated with cytoplasmic changes
of acinar cells (basophilic change, fine vacuolation, swelling, loss of the normal positive periodic
acid–Schiff reactivity of the secretory granules). The study authors concluded that the salivary gland
effects induced by glyphosate were mediated through an adrenergic mechanism (Chan & Mahler,
1992).
The hypothesis that glyphosate produced the changes to the salivary gland via β-adrenergic
activity was questioned in a recent review paper (Williams, Kroes & Munro, 2000). The authors
emphasized that if glyphosate was a β-agonist, it would stimulate β-receptors in other effector organs
and produce a characteristic set of cardiocirculatory effects, such as increased heart rate and cardiac
output as well as decreased blood pressure and peripheral resistance; none of these effects were noted
in other toxicological studies. Similarly, it is known that isoproterenol and other β-agonists cause
myocardial necrosis and enlargement of heart ventricles after prolonged treatment. Glyphosate did not
produce any effects in heart tissue, even after long-term exposure at very high doses, further
supporting for the argument that glyphosate does not act as a β-agonist. The authors concluded that
glyphosate has no significant β-adrenergic activity and could not produce salivary-gland changes via
β-agonist activity. They proposed a number of other potential mechanisms for salivary gland
alteration, including non-chemical modes of action. For example, salivary gland secretion has been
shown to be affected by the texture and moistness of feed, and salivary gland enlargement has been
caused by malnutrition. Glyphosate could be acting by just such a non-chemical mechanism. Because
glyphosate is a strong organic acid, dietary administration at relatively high concentrations may cause

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mild oral irritation leading to increased salivary gland size and flow. In the long-term exposure studies
with glyphosate, there were several changes to the salivary glands. These changes were most
pronounced in the parotid gland, responsible for secreting serous fluid in response to stimuli such as
acidic materials; absent in the sublingual gland that releases mucous fluid in response to other stimuli;
and observed to an intermediate degree in the submandibular gland that contains a mixture of mucous
and serous secreting cells. This pattern of observations was considered consistent with the hypothesis
that the changes are a biological response to the acidic nature of glyphosate. These alterations are not
known to represent any pathological condition and were not considered toxicologically significant or
adverse (Williams, Kroes & Munro, 2000).
A 2-month exploratory study evaluated the effects of a low pH diet on the parotid salivary
glands of rats. Five groups, each with 10 male Crl:CD (SD) rats, were dosed for 56 consecutive days.
Group 4 animals were fed a low pH diet containing 14 000 ppm citric acid, and group 5 a high pH diet
with 21 400 ppm trisodium citrate dihydrate and a citrate ion concentrate equivalent to group 4’s.
Group 2, the controls, were fed the basal diet. Group 3 were administered citric acid in deionized
water by gavage at 791–1316 mg/kg bw per day, with the dosing calculated to maintain citric acid
dose levels approximately equal to group 4’s. Group 1 were gavaged with deionized water.
Treatment-related effects consisted of statistically significant higher parotid salivary-gland
weights in group 4, compared to the group 2 controls. The higher parotid salivary-gland weights seen
in groups 3 (gavaged citric acid) and 5 (fed a trisodium citrate dihydrate diet) were not statistically
significant.
The report states that with the absence of microscopic findings such as cytotoxicity and
hyperplasia, the observed effects are likely adaptive responses to the low pH diet causing local
irritation in the oral cavity rather than adverse effects (Haas, 2010).
A 4-month study examined the effects of glyphosate acid on the salivary glands of different
rat strains (AP, CD and F344) after feeding diets containing 20 000 ppm glyphosate acid to male rats
for 28 consecutive days and monitoring recovery over 4 or 13 weeks.
Differences in terms of systemic toxicity (changes in body weight and feed consumption)
were minor, but marked differences were seen in the severity of effect on the parotid salivary gland.
Significant reductions in body weight with minor reductions in feed consumption were seen in AP and
CD rats but not in F344 rats. In contrast, salivary-gland weight was unaffected in CD rats but was
increased in both AP and F344 rats. Microscopic examination showed the most pronounced effect to
be in F344 rats where cytoplasmic basophilia were diffuse and parotid acinar cells enlarged. Similar
but lesser effects involving only small foci of cells occurred in the AP and CD strains.
Complete recovery was seen in AP and CD rats following the 4-week recovery period.
Although significant recovery of salivary-gland change was observed in F344 rats, it may not have
been complete after a 13-week recovery period (Wood, 1996).
In an in vivo study, five male and five female Sprague Dawley (CD) rats were dosed with
glyphosate technical (purity 95.3%) at a dose level of 5000 mg/kg bw with similar sized control
groups receiving the vehicle only. Approximately 1 hour after dosing, control and treated animals
were examined for either haematological, electrocardiographic or behavioural/functional changes.
There were no differences in response between treated and control animals.
Ex vivo studies evaluated the effect of saturated solutions of glyphosate technical on isolated
guinea pig ileum and isolated rat gastrocnemius muscle. Glyphosate technical caused a contractile
response in isolated guinea pig ileum similar to that seen with acetylcholine; the effect was negated
when the ileum was pre-incubated with atropine sulfate (Wood, 1996).

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(d) Gastrointestinal tract irritation

In a study comparing the irritant effects on the stomach and ileum of a glyphosate formulation
containing isopropylamine salt (41%) and surfactant (15%) with hydrochloric acid, a Teflon-coated
catheter was inserted into intestinal duct of beagle dogs to administer each ration of the test solutions.
Each sample was left in the stomach and intestine for 30 minutes and then the tissues were washed
with physiological saline and examined. Based on the histopathological findings, the study concluded
that the mucosal damage in the stomach and intestine caused by glyphosate formulation was mild,
equivalent to that caused by 0.25 eq/L hydrochloric acid. The intestine was more severely damaged
than the stomach in every case (Mizuyama, 1987).
(e) Endocrine disruption

For the USA pesticide regulatory risk assessment, the USEPA Endocrine Disruptor Screening
Program (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate
the potential of chemicals to interact with the estrogen-, androgen- or thyroid-signalling pathways.
This interaction includes agonism and antagonism at estrogen and androgen receptors as well as at the
hypothalamic–pituitary–gonadal and hypothalamic–pituitary–thyroid axes, and altered
steroidogenesis. In determining whether glyphosate interacts with estrogen-, androgen- or thyroid-
signalling hormone pathways, the number and type of effects induced, the magnitude of responses and
the pattern of responses observed across studies, taxa and sexes were considered. In addition, the
conditions under which effects occur were considered, and in particular, whether endocrine-related
responses occurred at doses that also resulted in systemic or overt toxicity.
This evaluation re-examines the data evaluated by the EDSP Tier 1 Assay Weight-of-
Evidence Review Committee of the Office of Pesticide Programs as well as the Office of Science
Coordination and Policy weight-of-evidence analysis of the potential interaction of glyphosate with
the estrogen, androgen or thyroid hormone pathways, conducted on September 17, 2014, and concurs
with the overarching conclusions.
For the estrogen pathway, there was no evidence of potential interaction of glyphosate with
the estrogen pathway in the EDSP Tier 1 in vitro assays (i.e. estrogen-receptor binding assay,
estrogen-receptor transactivation assay, aromatase and steroidogenesis assays). While glyphosate has
been reported to show estrogen-receptor agonism in vitro with estrogen-dependent human breast
cancer cells (Thongprakaisang et al., 2013), there were confounding issues with this study, and other
in vitro estrogen receptor studies with glyphosate have not demonstrated an interaction (e.g. Kojima et
al., 2004).
In addition, glyphosate was negative in the Tier 1 in vivo mammalian assays (i.e. uterotrophic
or female pubertal assays). In the fish short-term reproduction assay (FSTRA), the non-treatment-
responsive decrease (only significant at mid-treatment) in vitellogenin (VTG) was seen in isolation in
the absence of any treatment-related effects in the other estrogen-related end-points such as gonado-
somatic index, gonadal staging, fecundity and fertilization. In addition, there was no notable gonadal
histopathology. In the open literature, glyphosate did not increase plasma VTG in juvenile rainbow
trout (Xie et al., 2005). There were no treatment-related effects on female reproductive parameters in
the existing glyphosate Part 158 US Toxicological Data Requirement mammalian or wildlife studies
(only decreases in offspring body weight were reported in one avian reproduction study). Therefore,
there is no convincing evidence of a potential interaction with the estrogen pathway for glyphosate.
Tier 1 in vitro assays showed no evidence of glyphosate interacting with the androgen
pathway via androgen-receptor binding, and glyphosate was negative in an androgen-receptor
transactivation assay (Kojima et al., 2004; Kojima, Takeuchi & Nagai, 2010). However, evidence for
the aromatase and steroidogenesis assays is conflicting: these were negative for glyphosate alone in
the USEPA evaluation and a murine in vitro model (Forgacs et al., 2012), but positive for the
coformulants in another laboratory (Benachour et al., 2007; Defarge et al., 2016), with mechanistic
underpinning via both the regulatory steroidogenic acute regulatory protein (StAR) and the P450scc
cleavage enzyme first shown by Walsh et al. (2000).

211
The in vivo Tier 1 FSTRA and mammalian assays (i.e. Hershberger) and male pubertal assays
were negative in the absence of overt toxicity. The only treatment-related effects observed in the Part
158 mammalian studies in the absence of overt toxicity were decreases in sperm count in the
subchronic rat study (1678 mg/kg bw per day) and a delay in preputial separation at 1234 mg/kg bw
per day in the post-1998 two-generation reproduction study in rats (the EDSP Tier 2 study). Both
effects were observed at a dose that was above the limit dose (1000 mg/kg bw per day) for those
studies. No androgen-related effects were seen in the wildlife Part 158 studies (decreases in offspring
body weight observed in one avian reproduction study).
For the thyroid pathway, there was no convincing evidence of potential interaction of
glyphosate. There were no treatment-related effects on thyroid hormones (thyroxine [T4] and thyroid-
stimulating hormone [TSH]), thyroid weights or thyroid histopathology in the male pubertal assay in
the absence of overt toxicity; nor were there any thyroid-related effects observed in the female
pubertal assay. In the amphibian metamorphosis assay, there were no developmental effects or
alterations in thyroid histopathology. No thyroid-related effects were noted in any of the Part 158
studies.
There is little information about any endocrine-mediated effects of glyphosate, for example,
in relation to retinoids, vitamin D receptors, metabolic syndrome, obesogens, glucocorticoids, etc.,
which is a major data gap. In nonmammalian models, two endocrine-relevant pathways have been
reported: retinoic-acid dysfunction was observed in tadpoles exposed to glyphosate formulation,
whereas inhibition of cortisol response in fish by selected pesticides was notable in an academic (non-
industry funded) report because glyphosate did not present a stress response inhibition, unlike most of
the other test pesticides (Koakoski et al., 2014). Mechanistic information on the induction of receptors
such as aryl hydrocarbon receptor (Takeuchi et al., 2008; Kojima, Takeuchi & Nagai, 2010),
peroxisome proliferator-activated receptors (Vainio et al., 1983; Takeuchi et al., 2008; Kojima,
Takeuchi & Nagai, 2010) and pregnane X receptor (PXR) (Kojima, Takeuchi & Nagai, 2010) are all
negative. While glyphosate was not included in the recent Toxcast screens due to solubility issues,
some of the coformulants were, with positive results noted for FD&C Blue No. 1 in some of the
endocrine end-points.
Adverse endocrine effects due to glyphosate poisoning in humans have not been reported by
poison centres (Bradberry, Proudfoot & Vale, 2004; Kamijo, Takai & Sakamoto,. 2016).
(f) EDSP studies

In vitro assays
Androgen-receptor binding
In an in vitro androgen-receptor competitive binding assay, the binding of a single
concentration (1 nmol/L) of [3H]-R1881 (reference androgen) in the presence of increasing
concentrations (10−10 to 10−3 mol/L) of glyphosate (purity 95.93%) was measured. Sprague Dawley rat
ventral prostate cytosol was the source of the androgen receptor for the study. Low-salt TEGD buffer
(which consists of tris hydrochloride or tris base, ethylenediaminetetraacetic acid, glycerol and
dithiothreitol) was used as the vehicle. Altogether three runs were performed, each including
dexamethasone as a weak positive control and R1881 as the ligand reference standard.
The saturation binding curves showed a dissociation constant (Kd) for [3H]-R1881 of 0.613 (±
0.041) nmol/L and an estimated maximum amount of binding (Bmax) of 0.817 (± 0.049) fmol per 100
µg protein for the batch of prostate cytosol used in the study. In the competitive binding runs, the
estimated mean log IC50 for R1881 (strong positive control) was 9.0 mol/L and for the weak positive
control (dexamethasone) was −4.6 mol/L; the mean relative binding affinity for the weak positive
control, dexamethasone, was 0.004%. At glyphosate concentrations of 10−10 to 10−3 mol/L, specific
binding of [3H]-R1881 was 92.4–101.3% with the exception of one concentration (10−9 mol/L) in run
1, which had an average binding of 66.5%. Review of the data indicated that this value was a result of
a single replicate with a specific binding of 7.5%. Excluding this value yielded a mean specific

212
binding of 96.0%, which concurs with the other runs. Since the specific binding was greater than 75%
at all concentrations of glyphosate in all runs, no IC50 or relative binding affinity values were
estimated. Based on the results from the three runs, glyphosate does not competitively bind to the
androgen receptor (Willoughby, 2012a).
Estrogen-receptor binding
In an estrogen-receptor binding assay, the binding of a single concentration of [3H]-17β-
estradiol (1 nmol/L) in the presence of increasing concentrations (10−10 to 10−3 mol/L) of glyphosate
(purity 95.93%) was measured. TEGD buffer was used as the solvent vehicle for glyphosate. A total
of three runs was performed, each including 19-norethindrone as a weak positive control,
octyltriethoxysilane as a negative control and 17β-estradiol as the natural ligand reference chemical.
The Kd for [3H]-17β-estradiol was 0.331 (± 0.061) nmol/L and the estimated Bmax was 74.55
(± 3.03) fmol per 100 µg protein for the prepared rat uterine cytosol. The Kd for each run was within
the expected range of 0.03–1.5 nmol/L. In the competitive binding experiment, the estimated mean
log IC50 for 17β-estradiol was −9.0 mol/L and for 19-norethindrone was −5.5 mol/L. The mean
relative binding affinity was 0.032% for 19-norethindrone, compared to the natural ligand. Glyphosate
was tested over a concentration range (10−10 to 10−3 mol/L) that fully defined the top of the curve.
Across all runs, the lowest average per cent radiolabelled estradiol binding in the presence of
glyphosate was greater than 81% (i.e. showed less than 25% displacement) at concentrations up to
10−3 mol/L. Based on the results from the three runs, glyphosate does not competitively bind to the
estrogen receptor (Willoughby, 2012b).
Estrogen receptor transcriptional activation

In an estrogen receptor transcriptional activation (ERTA) assay, hERα-HeLa-9903 cells
cultured in vitro were exposed to glyphosate (purity 85.14%) at logarithmically increasing
concentrations from 10−10 to 10−3 mol/L in cell culture media for 24 hours in three independent runs.
The experiments were performed using 96-well plates, and each glyphosate concentration was tested
in six wells/plate in each run. The solvent vehicle was the culture media for glyphosate and DMSO
(0.1%) for the reference chemicals. Cells were exposed to the test agent for 24 (± 2) hours to induce
reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen
receptor was measured using a luciferase assay.
Glyphosate was tested up to the limit dose, with no precipitation or cytotoxicity observed at any tested
concentration. At concentrations up to 10−3 mol/L, the relative transcriptional activation of glyphosate
was less than or equal to 2.4%. Glyphosate was only able to reach a maximum of 0.8–2.4% of the
positive control, 1 nmol/L 17β-estradiol, when tested up to the highest concentration. Because the
RPCmax (maximum level of response induced by a test chemical, expressed as a percentage of the
response induced by the positive control) was less than the PC10 (concentration of a test chemical at
which the response is 10% of the response induced by the positive control in both assay runs),
glyphosate was considered negative for estrogen receptor transcriptional activation in this test system
(Willoughby, 2012c).
Aromatase
Glyphosate (purity 95.93%) was evaluated for its potential to inhibit aromatase activity by
incubating with human recombinant aromatase and tritiated androstenedione ([1β-3H(N)]-androstene-
4-ene-3,17-dione; [3H]ASDN) at log concentrations of 10−10 to 10−3 mol/L glyphosate. The solvent
vehicle was 0.1 mol/L phosphate buffer for glyphosate, ethanol for ASDN and DMSO for
4-hydroxyandrostenedione (4-OH ASDN), with a final assay volume of less than or equal to 1%
DMSO. Aromatase activity was determined by measuring the amount of tritiated water produced at

213
the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified
using liquid scintillation counting. Each run included a full activity control, a background activity
control, a positive control series (10−10 to 10−5 mol/L) with a known inhibitor (4-OH ASDN) and the
test chemical series (10−10 to 10−3 mol/L) with three repetitions per concentration.
Aromatase activity in the full activity controls was 0.676 (± 0.072) nmol∙mg-protein−1∙min−1.
The response of each full activity control within a run was between 90% and 110% of the average full
activity. Activity in the background controls ranged from 0.23% to 0.38% and averaged 0.30% of the
full activity control. For the positive control substance (4-OH ASDN), the estimated log IC50 averaged
−7.29 mol/L and the Hill slope was −0.96. For glyphosate, aromatase activity averaged
0.673 (± 0.066) nmol∙mg-protein−1∙min−1 at the lowest tested concentration of 10−10 mol/L and
0.741 (± 0.100) nmol∙mg-protein−1∙min−1 at the highest tested concentration of 10−3 mol/L. The
average aromatase activity was greater than or equal to 99.67% of the control at all tested glyphosate
concentrations for all runs. The results indicate that glyphosate does not inhibit aromatase activity
(Wilga, 2012).
Steroidogenesis
The purpose of this study was to validate the use of a standardized steroidogenesis assay as
detailed in OECD Guideline for the Testing of Chemicals: Draft Proposal for a New Guideline 4XX –
The H295R Steroidogenesis Assay. In this validation study, 28 chemicals were selected as a screen
for potential effects of endocrine-disrupting chemicals on the production of testosterone and 17β-
estradiol. These chemicals were selected based on their known or suspected endocrine activity, or lack
thereof, and included inhibitors and inducers of different potencies as well as positive and negative
controls. In this steroidogenesis assay, H295R cells cultured in vitro in 24-well plates were incubated
with glyphosate (purity and lot no. not provided) at seven concentrations between 0.0001 and 100
μmol/L for 48 hours in triplicate for three independent experiments. A quality control plate was run
concurrently with each independent run of a test chemical plate to demonstrate that the assay
responded properly to positive control agents at two concentrations; positive controls included the
known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.
Testosterone and 17β-estradiol levels were measured using radioimmunoassays or enzyme-linked
immunosorbent assay (ELISA); responses of the quality control plates measured by these assays were
confirmed by liquid chromatography–mass spectrometry. In this validation study, the laboratories
demonstrated that glyphosate does not affect testosterone or estradiol levels via this assay (Hecker et
al., 2011).
In vivo assay
Hershberger assay
To screen for potential anti-androgenic activity, glyphosate in 0.5% methylcellulose (w/v)
was administered daily via oral gavage to groups of six 54- or 55-day old, castrated male Sprague
Dawley rats at concentrations of 0 (vehicle), 100, 300 or 1000 mg/kg bw per day with a daily dose of
reference androgen testosterone propionate at 0.2 mg/kg bw per day by subcutaneous injection. The
anti-androgenic positive control group consisted of six castrated rats exposed to 0.2 mg/kg bw per day
testosterone propionate by subcutaneous injection and 3 mg/kg bw per day flutamide via oral gavage.
Testosterone propionate alone was used as the anti-androgenic negative control. For both components
of the assay, body weights were determined daily. The animals were dosed for 10 consecutive days
and terminated approximately 24 hours after the final dose. At necropsy, the five androgen-dependent
tissues were collected and weighed.
In the androgen-agonist assay, there were no treatment-related effects on body weights,
overall body-weight gains or the weights of accessory sex organs for any glyphosate dose group.
Animals in the positive testosterone propionate control group had increased (P < 0.01) accessory sex
organ weights as follows: 437% in seminal vesicles; 728% in the ventral prostate; 200% in levator

214
ani-bulbocavernosus; 361% in the Cowper gland; and 45% in the glans penis. The performance
criteria indicated that this assay was performing as expected.
In the anti-androgen assay, there were no treatment-related effects on body weights, overall
body-weight gains or the weights of accessory sex organs for any glyphosate dose group. Animals
dosed with testosterone propionate plus flutamide (positive control) had decreased (P < 0.01)
accessory sex organ weights as follows: 76% in seminal vesicles; 80% in ventral prostate; 63% in the
levator ani-bulbocavernosus; 70% in the Cowper glands; and 29% in glans penis. The performance
criteria indicated that this assay was performing as expected.
Statistically significant changes were not seen in two or more of the five androgen sensitive
tissue weights. Glyphosate was negative for androgenicity and anti-androgenicity in the Hershberger
assay (Stump, 2012a).
Uterotrophic assay
In a uterotrophic assay conducted to screen for potential estrogenic activity, glyphosate
(purity 85.14%) in 0.5% methylcellulose (w/v) was administered daily via oral gavage to groups of
six ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300 or 1000 (limit
dose) mg/kg bw per day on postnatal days 66/67 to 68/69. The positive control group was treated with
a daily dose of 17α-ethynyl estradiol at 3 μg/kg per day by oral gavage. Body weights were
determined daily. All the animals were terminated and necropsied approximately 24 hours after the
final dose was administered on postnatal day 69/70 to determine wet and blotted uterine weights.
All the animals survived until scheduled termination and no treatment-related clinical findings
were observed in glyphosate-dosed animals. Body weights, body-weight gains and uterine weights in
the glyphosate groups were comparable to the vehicle control. As expected, absolute wet and blotted
uterus weights were increased by 758% and 256%, respectively, in the positive control (17α-ethynyl
estradiol) group.
The conclusion reached was that glyphosate was negative in the uterotrophic assay (Stump,
2012b).
Male pubertal assay

In a male pubertal assay, 15 Crl:CD(SD) male rats per dose group were treated daily via oral
gavage (5 mL/kg) with glyphosate (purity 95.93%) in 0.5% methylcellulose at 0, 100, 300 or 1000
mg/kg bw per day (limit dose) from postnatal day 23–53. The animals were examined for preputial
separation daily beginning on postnatal day 30, and age and weight at day of attainment were
recorded. Following termination on postnatal day 53, blood was taken for total thyroxine,
testosterone, TSH and clinical chemistry analysis. The hormones were analysed by radioimmunoassay
or chemiluminescence.
Treatment-related clinical findings were limited to rales approximately 4 hours post dosing in
9/15 rats at 300 mg/kg bw per day and 14/15 rats at 1000 mg/kg bw per day. This finding persisted in
the daily examinations in seven high-dose males throughout the study. On postnatal day 53, final body
weights in the 300 and 1000 mg/kg bw per day groups were decreased (P < 0.05) by 7–10%. A
treatment-related delay in the mean age of attainment of complete preputial separation was noted at
1000 mg/kg bw per day (48.0 days) compared to controls (45.9 days). However, it was determined
that this delay at this dose was a result of the treatment-related decrease in body weight, rather than a
direct anti-androgenic effect. No treatment-related effects on organ weights were observed at any
dose. No treatment-related effects on T4, TSH or testosterone levels were observed at any dose. At
1000 mg/kg bw per day, there was a slight increase in the number of animals with thyroid colloid area
grade 4 (five treated vs one control) and grade 5 (one treated vs zero controls). There were no
treatment-related effects on follicular cell height at any dose compared to controls; nor were there any
treatment-related findings in the testes, epididymides or kidneys.

215
In conclusion, glyphosate did not affect maturation and did not produce any thyroid toxicity at
doses up to 1000 mg/kg bw per day (Stump, 2012c).
Female pubertal assay

In a female pubertal assay, 15 Crl:CD(SD) Sprague Dawley rats/dose group were treated
daily via oral gavage with glyphosate (purity 95.93%) in 0.5% methylcellulose at doses of 0, 100, 300
or 1000 mg/kg bw per day (limit dose) from postnatal day 22–42. The animals were examined daily
for vaginal opening beginning on postnatal day 22, and age and weight at day of attainment were
recorded. Following termination on postnatal day 42, blood was collected for clinical chemistry
analyses, including electrochemiluminescent immunoassay (to analyse total thyroxine) and a magnetic
[125I]rTSH gamma counter immunoassay (to analyse TSH).
One animal in the control group was terminated in extremis on postnatal day 27 due to
impairment of the right forelimb (due to possible mechanical injury). There were no treatment-related
differences in age of attainment of vaginal opening, body weights at vaginal opening, final body
weights or body-weight gains in the treated groups relative to controls. One control female and one at
300 mg/kg bw per day failed to attain vaginal opening. There were no statistically significant
differences in mean age at first vaginal estrus, mean cycle length or per cent cycling. The cycle status
at necropsy was similar across all groups. Serum total thyroxine and TSH concentrations were not
affected by treatment, and no adverse treatment-related effects on any clinical chemistry parameter
were observed at any dose. There were no treatment-related microscopic findings in the thyroid,
ovaries, uterus or kidneys at any dose.
In conclusion, glyphosate did delay the maturation and no treatment-related effects were seen
in thyroid toxicity (Stump, 2012d).
Additional literature reports

The published literature was reviewed and is included with the EDSP data, in the summary
Table 47.
Estrogen pathway
With in vitro studies of estrogen receptor activation, Thongprakaisang et al. (2013) reported
estrogen receptor agonism by glyphosate at concentrations from 10−12 to 10−6 mol/L in estrogen-
dependent human breast cancer cells, but did not test the estrogen receptor α antagonism as
recommended by the test developers (Evans, Gray & Wilson, 2012). In contrast, other studies
reported negative results in reporter gene–transfected Chinese hamster ovary (CHO) cells (Kojima et
al., 2004; Kojima, Takeuchi & Nagai, 2010) or that glyphosate formulations reduced the transcription
of estrogen receptor α and estrogen receptor β in HepG2 cells transiently transfected with the reporter
gene ERE, but the glyphosate parent did not (Gasnier et al., 2009).
In an in vivo rainbow trout VTG assay, glyphosate did not increase plasma VTG in juvenile
rainbow trout, and plasma VTG levels in glyphosate plus surfactant–treated trout were only
marginally greater than the controls, with no trend and no significance (Xie et al., 2005).
In conclusion, there is no convincing evidence of a potential interaction with the estrogen

pathway for glyphosate. The one positive in vitro study has not been reproduced by another
laboratory.

216
Androgen pathway
The Séralini laboratory conducted several androgen pathway–related assays in equine testes
utilizing principally non-validated in vitro assays as well as ex vivo assays. These suggested effects of
glyphosate for anti-androgenicity and inhibition of aromatase activity (Richard et al., 2005;
Benachour et al., 2007; Gasnier et al., 2009; Defarge et al., 2016). Studies in other laboratories did not
report this (Kojima et al., 2004; Kojima, Takeuchi & Nagai, 2010) and particularly those that used the
EDSP battery of validated tests for the androgen receptor–mediated and steroidogenesis.
The differences observed in the in vitro studies with positive results and those with negative
results may be due to confounding by the glucocorticoid receptor interference in the cell line used in
the non-validated assays; the MDA-MB453-kb2 cell line has a high glucocorticoid-receptor content in
addition to androgen-receptor content.
Additional steroidogenic mechanisms of interest include a noted effect upon the post
transcriptional expression of the StAR in mouse testicular Leydig cells (Walsh et al., 2000) This was
also reported for P450scc, the enzyme responsible for the conversion of cholesterol to pregnenolone
and for initiating the synthesis of all steroid hormones cells (Walsh et al., 2000). However, another in
vitro Leydig cell model reported no effect of glyphosate on basal or recombinant human chorionic
gonadotrophin (rhCG) (Forgacs et al., 2012).
In conclusion, there is no convincing evidence of a potential interaction between glyphosate
and the androgen receptor pathway. Decreases in sperm count in the subchronic rat study (1678
mg/kg bw per day) and a delay in preputial separation (at 1234 mg/kg bw per day in the two-
generation reproduction study in rats) were observed at a dose that was above the limit dose (1000
mg/kg bw per day), and therefore of low physiological relevance.
However there is plausible but equivocal evidence that glyphosate and glyphosate
coformulants affect the steroidogenesis pathway, via P450scc and StAR. This requires further
investigation.
Thyroid pathway
No relevant in vitro or mammalian in vivo reports on the effect of glyphosate on the thyroid
pathway were identified in the literature, and the EDSP data had no evidence.
A handful of reports describe the effect of glyphosate on the negative metamorphosis of frog
and tadpole species, including a 2014 report that identified alterations in genes encoding thyroid
hormone receptor beta in brain, glucocorticoid receptor in tail and deiodinase enzyme in brain and tail
(Lanctot et al., 2014), suggesting that glyphosate formulations have the potential to alter mRNA
profiles during metamorphosis.
Other endocrine-related pathways

Following studies conducted in Xenopus laevis and chicken embryos, the retinoic acid–
signalling pathway has been proposed as a mechanistic pathway that is adversely affected by
glyphosate (Paganelli et al., 2010). In this study, a 1/5000 dilution of glyphosate induced reproducible
skeletal and craniofacial malformations. Developmental toxicity studies in the rabbit (Section 2.5b,
Rabbits) identified nonsignificant skeletal malformations, with the lowest NOAEL for developmental
toxicity 250 mg/kg bw per day (Bhide & Patil, 1989). The NOAEL and LOAEL for this study are
based on the available data (Bhide & Patil, 1989) as individual data were not provided. A subsequent
study NOAEL of 300 mg/kg bw per day was based on delayed ossification and an increased incidence
of fetuses with skeletal anomalies at 1000 mg/kg bw per day (Brooker et al., 1991a). However, these
effects were secondary to the observed severe maternal toxicity. Nevertheless, the retinoic-acid
pathway constitutes a data gap that requires further research.
Other receptor-mediated pathways reported in the literature, including aryl hydrocarbon
receptors and peroxisome proliferator-activated receptors, were negative.

217
Cortisol stress pathways

A study of the stress response of Rhamdia quelen fingerlings with acute exposure to a
glyphosate formulation (360 g/L) at 45, 90, 135 and 180 days did not demonstrate impairment of
cortisol release but did exert negative effects on growth and survival parameters (Koakoski et al.,
2014).

Table 47. Summary of information supporting EDSP data in relation to glyphosate and endocrine end-points
Influence
on Reference
End-point pathway Glyphosate formulation Strengths Uncertainties/considerations conclusiona conclusion Reference
Estrogen pathway
EDSP Tier 1 data Glyphosate USEPA validated assays There were no treatment-related effects on High Negative USEPA (2015)
2014/2015: Purity: 85.1–95.93% In vitro assays are well- female reproductive parameters in the existing
glyphosate Part 158 mammalian or wildlife
In vitro: Concentration range: 10−10 characterized and OECD
studies, however decreases in offspring body
ER binding; TG 455 ER to 10−3 mol/L TGs
weight were observed in one avian reproduction
STTA and Hela Assay ER STTA: uses HeLa cell study
In vivo: mammalian line which has ER α not ER
assays, i.e. uterotrophic β. ER α perturbation is more
and female pubertal strongly associated with
assays and mammalian adverse outcomes
toxicity studies
In vitro: Glyphosate Validated assay Glyphosate exerted proliferative effects only in Low Positive Thongprakaisang
ER agonism in estrogen- Purity > 98%) human hormone-dependent breast cancer, T47D et al. (2013)
dependent T47D human Accustandard cells, and not in hormone-independent breast
cancer, MDA-MB231 cells, at 10−12 to 10−6
breast cancer cells Concentration range: 10−12 mol/L in estrogen withdrawal condition, which
to 10−6 mol/L was reported to be confirmed by the inhibitory
effect of the ER antagonist ICI 182780. The
T47D cell line contains both ERα and ERβ.
While the use of ICI 182780 can exclude the
possibility of dioxin-like interference of
coformulant contaminant 1,4-dioxane with AhR
interactions affecting the ER, this study is
confounded because it was not tested with an
ERα-specific antagonist, such as
methylpiperidino pyrazole (CAS No. 289726-
02-9). This would determine the relative
activities of each ER (Evans, Gray & Wilson,
2012)
The luciferase reporter system was then also
used with combinations of genistein, an
isoflavone in soy. Phytoestrogens such as
genistein are known to overstimulate luciferase,
and also are stronger ligands for ERβ. Non-
receptor–mediated luminescence signals have

219
Influence
on Reference
been reported at phytoestrogen concentrations
higher than 1 μmol/L due to the over-activation
of the luciferase reporter gene (Kuiper et al.,
1998; Escande et al., 2006). While the dose–
response curve indicates that true activation of
the ER system occurs at lower concentrations,
luciferase expression obtained at high
concentrations of phytoestrogens or similar
compounds suspected of producing
phytoestrogen-like over-activation of the
luciferase reporter gene needs to be examined
carefully in stably transfected ERTA assay
systems. (See Annex 2 of OECD TG 455)
In vitro: Glyphosate (> 95–100%); Concentrations of pesticides that tested Med Negative Kojima et al.
hERα and hERβ whether this is a negative, which included glyphosate, are not (2004); Kojima,
(ant)agonism in reporter formulation is not reported; only the results of those that tested Takeuchi &
gene–transfected CHO specified in the paper. positive are provided. Concentration of Nagai (2010)
cells Concentrations for positively testing chemicals ranged from 10−6 to
glyphosate are not clearly 10−12 mol/L
specified, but can be Concentration of the test chemicals showing
assumed to be the same as 20% of the agonistic activity of 10–10 mol/L
those for the positive 17β-estradiol, and is given as REC 20(mol/L)
chemicals.
In vitro: Glyphosate formulations Non-validated assays, but Formulations reduced transcription of ERα and Med Parent- Gasnier et al.
hERα and hERβ transient and glyphosate parent well-recognized and reliable ERβ in HepG2 cells transiently transfected with negative (2009)
transfection into human chemical. hepatic cell line ERE, but glyphosate parent did not Formulation
hepatocarcinoma HepG2 Dilutions up to 10−7 s-positive
cells
In vivo: In this validated assay, the Med Negative USEPA (2015)
FSTRA non-treatment-responsive
decrease (only significant at
mid-treatment) in VTG was
seen in isolation in the
absence of any treatment-
related effects in the other
estrogen-related end-points
such as gonado-somatic

220
Influence
on Reference
index, gonadal staging,
fecundity and fertilization. In
addition, there were no
notable gonadal
histopathology
In vivo: Glyphosate and VTG induction in fish is a Med Negative Xie et al. (2005)
Rainbow trout VTG glyphosate plus standard measure for
assay surfactants; measured estrogenicity in
concentration of environmental regulatory
glyphosate 0.11 mg/L for toxicology that also
7 days considers the relevance to
humans (e.g. USEPA FIFRA
SAP 2009a,b, 2012).
Glyphosate did not increase
plasma VTG in levels in
juvenile rainbow trout,
glyphosates plus surfactants
were only marginally greater
than the controls, no trend,
no significance
Overall conclusion: No convincing evidence of a potential interaction with the estrogen pathway. The one in vitro study that is positive has not been reproduced by another laboratory.
Androgen pathway
EDSP Tier 1 data Glyphosate Standardized and validated Androgen-receptor binding assay is not a High Negative, USEPA (2015)
2014/2015: assays validated OECD TG but other validated but sperm
In vitro: negative; both androgen receptor assays not available in count and
for androgen-receptor 2014/2015 delay in
binding assay and the Aromatase assay: highest soluble test preputial
aromatase assay concentration of glyphosate was 10-3 mol/L separation
effects seen
In vivo mammalian The in vivo Tier 1 FSTRA and mammalian at very high
assays: Hershberger and assays (i.e. Hershberger and male pubertal doses,
male pubertal assays assays) were negative in the absence of overt > 1 000
toxicity. The only treatment-related effects mg/kg bw
observed in the Part 158 mammalian studies in per day
the absence of overt toxicity were decreases in
sperm count in the subchronic rat study (1678
mg/kg bw per day) and a delay in preputial

221
Influence
on Reference
separation at 1 234 mg/kg bw per day in the
post-1998 two-generation reproduction study in
rats (the EDSP Tier 2 study). Both effects were
observed at a dose that was above the limit dose
(1 000 mg/kg bw per day) for those studies. No
androgen-related effects were seen in the
wildlife Part 158 studies (decreases in offspring
body weight observed in one avian
reproduction study)
In vitro: Glyphosate Med Negative Kojima et al.
hAR transactivation (> 95–100%) formulation (2004); Kojima,
assay in CHO cells not specified in the paper Takeuchi &
Nagai (2010)
Concentrations given for

glyphosate are not clearly
specified, but can be
assumed to be the same as
those for the positive
chemicals.
In vitro: Glyphosate and Non-validated assays, but MDA-MB453-kb2 cell line has a high content Low Positive Gasnier et al.
hAR transient formulations well-recognized and reliable of glucocorticoid receptors in addition to (2009)
transfection into human Dilutions up to 10−7 hepatic cell line. androgen receptors
HepG2 cells, aromatase Method for aromatase The characterization of the cell line and
evaluation within the activity evaluation is also discussion of such confounding factors is not
HepG2 cells, and MDA- part of OECD TG 456 for considered in the paper. While glyphosate and
MB453-kb2 cells steroidogenesis. formulations reduced AR transcription in this
cell line, there appears to have been no control
with androgen-specific responses to exclude
glucocorticoid-specific responses
Steroidogenesis Glyphosate and Relevant cell models, but Inhibition of aromatase noted in two different Low–Med Positive Benachour et al.
In vitro: formulations limited characterization species by both parent compound and (2007)
0.01% (with 210 μmol/L provided in the paper formulations
Transformed and human
aromatase–transfected glyphosate) to 2% The aromatase assay may be subject to
cDNA in human glyphosate/glyphosate variability, e.g. due to degradation of the
embryonic kidney 293 formulation enzyme, and therefore performance criteria are
cells and placental- specified in guideline OPPTS 890.1200 to

222
Influence
on Reference
derived JEG3 cells demonstrate that the assay is functioning
correctly. This is addressed in the EDSP data,
but is not evident in the Séralini lab. papers
Ex vivo: (Benachour et al., 2007; Gasnier et al., 2009),
normal human placenta although OECD GD 150 is cited. An adequate
and equine testis response with the proficiency chemicals
econazole, fenarimol, nitrofen (inhibitors) and
atrazine (non-inhibitor) should be demonstrated
and the inhibitor 4-hydroxyandrostenedione
(formestane) used as a positive control
chemical in each experiment. While the correct
positive control was used, proficiency testing is
not reported
Compliance with the performance criteria
should be checked before evaluating results
from this assay. A positive result in GD OPPTS
890.1200 requires demonstration of inhibition
of aromatase activity that fits a 4-parameter
nonlinear regression model such that the
concentration response curve crosses 50%
inhibition. The concentration response curve
allows the determination of potency, i.e. IC50.
In some cases, variability may be due to limited
solubility of a chemical
Steroidogenesis Glyphosate and The coformulants were each tested Low–Med Positive Defarge et al.
In vitro: formulation ingredients independently and were reported to inhibit (2016)
Top dose: 100 ppm aromatase activity at concentrations 20–67%
Placenta-derived JEG3 below the no-observed-effect concentration, at
cells which levels glyphosate alone did not
significantly inhibit aromatase. (See also
comment above regarding proficiency testing of
the assay)
Steroidogenesis Glyphosate Relevant and well- No effect on basal or rhCG Med–High Negative Forgacs et al.
In vitro: 300 μmol/L characterized Leydig cell (2012)
model
BLTK1 murine Leydig
cells

223
Influence
on Reference
Steroidogenesis Glyphosate formulation Relevant and well- Statistically significant reduction (P < 0.01) of Med–High Positive Walsh et al.
In vitro: (containing 180 g/L characterized cell model (Bu)2cAMP with the glyphosate formulation (2000)
glyphosate) was observed after 2 hours of treatment.
StAR in a mouse MA-10 Statistical significance (P < 0.01) was also
Leydig tumour cell reported for P450scc, the enzyme responsible
for the conversion of cholesterol to
pregnenolone and for initiating the synthesis of
all steroid hormones
Overall conclusion: There is no convincing evidence of a potential interaction between glyphosate and the androgen-receptor pathway. Decreases in sperm count in the subchronic rat study
(1 678 mg/kg bw per day; USEPA 2015) and a delay in preputial separation at 1234 mg/kg bw per day in the two-generation reproduction study in rats (the EDSP Tier 2 study) were observed at
a dose that was above the limit dose (1000 mg/kg bw per day) and therefore of low physiological relevance. However, there is plausible evidence that glyphosate and glyphosate coformulants
affect the steroidogenesis pathway, via P450scc and StAR. Further investigation is needed.
Thyroid
EDSP Tier 1 data Glyphosate Relevant and validated test No convincing evidence of potential interaction High Negative USEPA 2015
2014/2015: methods of glyphosate
In vitro:
No assays conducted.
In vivo test battery: There
were no treatment-related
effects on T4 and TSH,
thyroid weights or
thyroid histopathology in
the male pubertal assay in
the absence of overt
toxicity. No thyroid-
related effects were
observed in the female
pubertal assay. There
were no developmental
effects or alterations in
thyroid histopathology in
the amphibian
metamorphosis assay. No
thyroid-related effects
were noted in any of the
Part 158 studies.

224
Influence
on Reference
Overall conclusion: There is no convincing evidence of a potential interaction with the thyroid pathway for glyphosate
Other endocrine mechanisms
Retinoid system 360 pg and 5 000 pg of Whole vertebrate models, Experimental design and hypothesis based on Med–High Positive: Paganelli et al.
In vivo Xenopus laevis glyphosate (Sigma) two species medical observations of craniofacial defects increase in (2010)
embryo model and with malformations observed in humans endogenous
chicken embryos residing in areas chronically exposed to retinoic-acid
glyphosate formulations. Suspected to be activity
resulting from a dysfunctional retinoic-acid or
Sonic hedgehog pathway. Further investigation
is needed
Cortisol Glyphosate formulation Stress response of Rhamdia Stress responses important but difficult variable Med Negative for Koakoski et al.
In vivo fish study 360 g/L quelen fingerlings acute to control for, as stress is induced from impaired (2014)
exposure at 45, 90, 135 and handling, etc. This study included appropriate cortisol
Rhamdia quelen 180 days controls for stress confounders release, but
fingerlings impaired
growth and
survival
Hypolipidaemia and Glyphosate formulation No increase in number or size or peroxisomes Med Negative Vainio et al.
peroxisome proliferation 300 mg/kg single daily (1983)
In vivo rat dose for 2 weeks, 5
animals/dose per group
AhR induction Glyphosate (95–100% Relevant and recognized Med Negative Takeuchi et al.
purity) assay (2008)
In vitro: Assay performed at
concentrations of ≤ 10−5
Mouse hepatoma mol/L
Hepa1c1c7 cells AhR
Luciferase reporter gene
transcriptional assay
In vitro Glyphosate Review, insufficient detail given. Concentration Low Negative Kojima et al.
mPPARα, mAhR, hPXR tested not given for negative test chemicals (2004); Kojima,
Takeuchi &
Nagai (2010)
Overall conclusion: Suggestion of adverse effect upon retinoic-acid pathways. Further investigation required.

225
AhR: aryl hydrocarbon receptor; AR: androgen receptor; CAS: Chemical Abstracts Service; CHO: Chinese hamster ovary; EDSP: Endocrine Disruptor Screening Program; ER: estrogen
receptor; ERTA: estrogen receptor transcriptional activation; FSTRA: fish short-term reproduction assay; GD: guideline; hAR: human androgen receptor; HepG2: hepatocellular carcinoma;
IC50: median inhibitory concentration; no.: number; OECD: Organisation for Economic Co-operation and Development; PPAR: peroxisome proliferator-activated receptor; PXR: pregnane X
receptor; rhCG: recombinant human chorionic gonadotrophin; StAR: steroidogenic acute regulatory protein; T4: thyroxine; TG: test guideline; TSH: thyroid-stimulating hormone; VTG:
vitellogenin
a
High: line of evidence could be sufficient on its own to be almost sure of entry (approaching 100% likelihood); Med: contributes importantly towards increasing likelihood; Low: minor
contribution towards increasing likelihood.

226
(g) Microbiological effects

Bacteria
The herbicidal action of glyphosate is generated by chelating manganese required in the
reduction of the flavin mononucleotide cofactor 5-enolpyruvylshikimate 3-phosphate synthase
(EPSPS) (Cerdeira & Duke, 2006). Since bacteria have EPSPS and produce amino acids via the
shikimate pathway, there is potential for glyphosate residues to disrupt microbes in the human
gastrointestinal tract. However, no studies have specifically addressed whether glyphosate affects the
microbiota in the human gastrointestinal tract or in mouse and rat animal models. What is known is
that selected bacterial pathogens and probiotic bacteria from dairy cows and poultry can be affected
differently by residual levels of glyphosate.
The minimum inhibitory concentration (MIC) of glyphosate on the growth and viability of
poultry microbiota and pathogens was determined in triplicate in 24-well microtitre plates. Just 100
µL of the tested bacteria (105 colony-forming units [cfu] per mL) was added to 900 µL broth media
containing different concentrations of glyphosate (0.075, 0.15, 0.30, 0.60, 1.20, 2.40 or 5.0 mg/mL).
Plates containing glyphosate and bacteria were incubated at 37 ºC. MIC values were determined by
quantitative analysis of bacteria on agar plates.
Clostridium perfringens, Salmonella gallinarum, S. typhimurium and S. enteritidis were
highly resistant to glyphosate (MIC of 5 mg/mL). Lactobacillus casei, L. buchneri, L. harbinensis,
Staphylococcus aureus, S. lentus and S. haemolyticus were moderately resistant to glyphosate (MIC
0.60–0.30 mg/mL). All other tested bacteria including Enterococcus faecalis, E. faecium, Bacillus
badius, B. cereus and Bifidobacterium adolescentis were highly sensitive to glyphosate, with MIC
values ranging from 0.15 to 0.075 mg/mL (Table 48). Pathogenic E. coli and E. coli 1917 strain
Nissle were also found to be resistant to glyphosate (MIC of 1.2 mg/mL).
In summary, most of the tested pathogenic bacteria were highly resistant to glyphosate;
however, most other tested bacteria were moderate to highly susceptible (Shehata et al., 2013b).
Table 48. Inhibitory effects of glyphosate on different bacteria

Bacterial count a
Treated with glyphosate Not treated with
Genus/species MIC (mg/mL) at MIC glyphosate
Bacillus badius 0.15 2.24 ± 0.49 8.90 ± 0.44
B. cereus 0.3 2.75 ± 0.68 8.08 ± 0.12
Bacteriodes vulgatus 0.6 3.54 ± 0.31 7.37 ± 0.10
Bifidobacterium adolescentis 0.075 3.87 ± 0.50 8.67 ± 0.48
Campylobacter coli 0.15 3.07 ± 0.50 9.00 ± 0.70
C. jejuni 0.15 3.90 ± 0.50 9.54 ± 0.97
Clostridium perfringens 5.0 3.37 ± 0.89 8.30 ± 0.28
C. botulinum type A 1.2 4.00 ± 0.50 8.16 ± 0.32
C. botulinum type B 1.2 3.56 ± 0.45 7.60 ± 057
E. coli 1.2 3.15 ± 0.24 8.00 ± 0.34
E. coli 1917 strain Nissle 1.2 2.35 ± 0.24 7.26 ± 0.21
Enterococcus faecalis 0.15 2.00 ± 0.45 8.49 ± 0.58
E. faecium 0.15 2.01± 0.34 7.06 ± 0.95
Lactobacillus buchneri 0.6 4.00 ± 0.88 8.00 ± 0.34
L. casei 0.6 4.74 ± 0.56 8.28 ± 0.35
L. harbinensis 0.6 5.30 ± 0.44 8.40 ± 0.32

227
Bacterial count a
Treated with glyphosate Not treated with
Genus/species MIC (mg/mL) at MIC glyphosate
Riemerella anatipestifer 0.15 4.00 ± 0.50 7.88 ± 0.50
Salmonella enteritidis 5.0 2.35 ± 0.26 8.28 ± 0.16
S. gallinarum 5.0 2.15 ± 0.33 8.68 ± 0.20
S. typhimurium 5.0 2.75 ± 0.68 8.03 ± 0.16
Staphylococcus aureus 0.3 5.74 ± 0.58 9.00 ± 0.10
S. haemolyticus 0.3 5.74 ± 0.32 8.08 ± 0.16
S. lentus 0.3 3.90 ± 0.44 8.08 ± 0.14
MIC: minimum inhibitory concentration; SD: standard deviation
a
Mean of n = 3 quantitative bacterial counts expressed as reciprocal log10 ± SD.
Source: Shehata et al. (2013b)
An evaluation of the effects of Roundup and its glyphosate ingredients on the growth and
viability of three food-associated microorganisms widely used as starters in traditional and industrial
dairy technologies found that glyphosate inhibited the growth of Lactobacillus delbrueckii subsp.
bulgaricus at a concentration of 1 mg/mL and Lactococcus lactis subsp. cremoris, which was more
sensitive to glyphosate, with an MIC of 0.312 mg/mL (Table 49). The fungus Geotrichum candidum
was more sensitive, with an MIC of 0.100 mg/mL (Clair et al., 2012).
Table 49. Effect of Roundup on three food-associated microorganisms

Concentration of
glyphosate in
Microorganism strain Roundup (g/L) MIC (ppm) MMC (ppm)
G. candidum ATCC 204307 400 100 1000
450 625 1000
L. lactis subsp. cremoris ATCC 19257 450 312 625
L. delbrueckii subsp. bulgaricus CFL1 450 1000 1250
MIC: minimum inhibitory concentration; MMC: minimum microbicidal concentration; ppm: parts per million
MIC and MMC measured after 24-hour incubation in growth media supplemented with Roundup or equivalent amount of
glyphosate.
Source: Clair et al. (2012)
The minimal agricultural use of the herbicide is 10 000 ppm.
In a study of the impact of glyphosate on poultry microbiota and the production of botulinum
neurotoxin during ruminal fermentation, ruminal microbiota were characterized by fluorescence in
situ hybridization technique using 16S rRNA/23S rRNA-targeted oligonucleotide probes. After
incubation with 0, 1, 10 or 100 µg/mL glyphosate in rumen fluids from donor cows, the cell counts of
Ruminococcus albus and R. flavefaciens were significantly lower in the presence of 1 µg/mL
glyphosate; Streptococcus spp. cell counts were significantly lower with 100 µg/mL glyphosate, and
cell counts of the phylum Euryarchaeota were significantly lower on exposure to 10 and 100 µg/mL.
In contrast, cell counts of Clostridium histolyticum and Lactobacilli and Enterococci were
significantly higher with 100 µg/mL glyphosate. The study authors noted that more bacterial species
were inhibited when cows were fed a crude fibre-rich diet than a lower-fibre diet, indicating a possible
inhibitory effect on the microbiota responsible for fibre degradation (Ackermann et al., 2015).

228
In a study of the toxicity of glyphosate to the most prevalent Enterococcus spp. in the
gastrointestinal tract, the lowest concentration of glyphosate and Roundup to show bactericidal or
bacteriostatic effects was determined in 96-well microtitre plates. Serial dilutions of glyphosate from
10–0.001 mg/mL were made in nutrient broth. Enterococcus isolates were added at a final
concentration of 104 cfu/mL, and the test plates with diluted glyphosate and Enterococcus incubated
overnight at 37 °C before plating aliquots on citrate azide tween carbonate agar. Bacterial growth on
each agar plate was evaluated.
Glyphosate and Roundup at 0.1–10 mg/mL inhibited the growth of E. faecalis but not of
C. botulinum or the production of botulinum neurotoxin (Table 50). The study authors proposed that
glyphosate may be a significant factor in the observed increased risk of C. botulinum infection in
cattle in Germany over the past 10 to 15 years (Krüger et al., 2013). Glyphosate toxicity to
Enterococcus spp. leads to an imbalance in the gut favouring overgrowth of Clostridium spp. because
the common, beneficial bacteria, Enterococcus spp., suppress Clostridium growth in the
gastrointestinal tract (Krüger et al., 2013; Shehata et al., 2013a,b).
Table 50. Effect of glyphosate and Roundup on the growth of C. botulinum type B and E. faecalis
Glyphosate Roundup formulation
Herbicide C. botulinum C. botulinum
concentration type B BoNT E. faecalis type B BoNT E. faecalis
(mg/mL) (cfu/mL) a (ng/mL) b (cfu/mL) c (cfu/mL) a (ng/mL) (cfu/mL)
0 6.9 ± 0.34 300 ± 47 8.2 ± 0.87 6.9 ± 0.34 270 ± 120 8.2 ± 0.87
0.1 5.3 ± 0.78 312 ± 20 0 5.1 ± 0.78 337 ± 50 0
1 5.4 ± 0.45 319 ± 60 0 3.3 ± 0.80 0 0
10 3.2 ± 0.43 0 0 3.0 ± 0.65 0 0
BoNT: botulinum neurotoxin; cfu: colony-forming unit; ELISA: enzyme-linked immunosorbent assay; SD: standard
deviation
a
C. botulinum type B (104/mL) cultured anaerobically in reinforced clostridial medium containing different concentrations
of glyphosate or herbicide formulation for 5 days. C. botulinum quantified using the most probable number estimation
method. Data express as reciprocal log10.
b
C. botulinum type B quantified by ELISA.
c
E. faecalis cultured aerobically in reinforced clostridial medium containing different concentrations of glyphosate or
herbicide formulation for 8 hours and quantified on citrate-acid-tween-carbonate agar. Data expressed as reciprocal log10 ±
SD.
Source: Krüger et al. (2013)
The neutralization ability of the antimicrobial effect of glyphosate by different humic acids
was investigated by determining the MIC of glyphosate for different bacteria in different
concentrations (0.25, 0.5 and 1.0 mg/mL) of humic acid. The MIC values of glyphosate for E.
faecalis, B. badius and B. adolescentis were 0.3, 0.3 and 0.15 mg/mL, respectively. Humic acids
neutralized the antimicrobial effect of glyphosate in different patterns. The WH67/2, WH67/4/3 and
WH67/4 humic acids at 1 mg/mL showed the highest degree of neutralization of the antimicrobial
effect of glyphosate. The MIC values of glyphosate for E. faecalis, B. badius and B. adolescentis in
the presence of 1 mg/mL WH67/2, WH67/3, and WH67/4 humic acids were more than 2.4 mg/mL,
while the MIC values in the presence of other humic acids ranged from 0.3 to 0.6 mg/mL (Shehata et
al., 2014). Sorption of the glyphosate to humic acids varied, depending upon their macromolecular
structure, but overall, these compounds neutralized the antimicrobial effect of glyphosate (Piccolo et
al., 1995, 1996).

229
Rats
Toxicokinetics of glyphosate after single 100 mg/kg intravenous and 400 mg/kg oral doses
were studied in rats. The oral bioavailability of glyphosate was 23.21% (Anadón et al., 2009). This
was lower than the oral bioavailability in studies in which [ 14C]glyphosate was administered orally at
10 mg/kg, when approximately 30–36% of the dose was absorbed (Howe, Chott & McClanahan,
1988; Ridley & Mirley, 1988; Brewster, Warren & Hopkins, 1991). A National Toxicology Program
study showed that approximately 19–23% of the 1000 mg/kg dose was absorbed, as determined from
urinary excretion data (Chan & Mahler, 1992). Conversely, when a single oral dose of glyphosate (6–
9 mg/kg) was administered to New Zealand White rabbits, 80% of the test material appeared in the
faeces (Colvin & Miller, 1973c). Glyphosate is poorly metabolized in rats, and the metabolite AMPA
represented 6.49% of the parent drug plasma concentration. A similar metabolic characterization was
indicated by Brewster et al. (1991). The production of this metabolite could have been the result of
intestinal microbial action (Rueppel et al., 1977; Mueller et al., 1981). Taken together, the fraction of
the oral dose of glyphosate bioavailable to intestinal microorganisms could range from 70–80% and
be microbiologically active. The microbiological activity of the minor metabolite AMPA has not been
determined.
Humans
A review of the published scientific literature found no specific information on whether
glyphosate bioaccumulates or affects the microbiota in the human gastrointestinal tract. There are no
data that show measurements of the amount of glyphosate residues in human gastrointestinal tract.
However, several pharmacokinetic, toxicokinetic and bioavailability studies indicate that glyphosate
is poorly absorbed after oral administration.
A review of the literature does not indicate that intestinal bacteria generally found in the
human gastrointestinal tract have been tested for the ability to degrade glyphosate. However, the
microbial capacity for glyphosate degradation has been shown in terrestrial and aquatic environments
(Balthazor & Hallas, 1986; Rueppel et al., 1977; Sprankle, Meggitt & Penner, 1975; Mueller et al.,
1981; Franz et al., 1997; Zaranyika & Nyandoro, 1993; Kryuchkova et al., 2014). Glyphosate is
metabolized by several bacteria in soil to give sarcosine, which is then converted to glycine and
ammonia by sarcosine oxidase. An alternative metabolic pathway involves the formation of AMPA
by glyphosate oxidoreductase, which is found in colon tissue in rats (Brewster et al., 1991). Therefore,
based on the enzymatic repertoire of the intestinal microbiota, there is potential for these
microorganisms to metabolize glyphosate.
There are no specific studies on the effects of glyphosate on the mammalian gut microbiota in
mouse, rat, rabbit or humans, that is, there is a lack of in vivo studies: all reports are on in vitro tests.
In addition, there are no data on the microbiological activity of the glyphosate metabolites, for
example, AMPA.
Many of the chronic and long-term in vivo studies reviewed in this monograph reported that
high doses of glyphosate have an impact upon the gastrointestinal tract. While not uncommon with
administration of high-dose chemical substances, this merits further investigation as glyphosate is
known to be poorly absorbed in mammalian models and alterations in gut microbiota profiles,
specifically reductions in the beneficial microbiota and increases in pathogenic bacteria, are known to
affect the early initiation and progression of the multistep processes in carcinogenesis (Viljoen et al.,
2015).
Evidence from livestock species indicates that pathogenic bacteria are more resistant to
glyphosate, while beneficial microbiota are more sensitive, and thus more vulnerable (Shehata et al.,
2013b). There is also evidence of intestinal metabolism of glyphosate to AMPA in the colon tissue of
rats (Brewster, Warren & Hopkins, 1991).
While plausible mechanistic links could be postulated between chromosome breakage, Bcl-2
and p53, adverse gut microbiome profiles in relation to glyphosate formulations (including the

230
solvent/contaminant 1,4-dioxane), the (nonsignificant) association seen between glyphosate exposure

and non-Hodgkin lymphoma (McDuffie et al., 2001) and mechanisms of action of several proteins
closely associated with non-Hodgkin lymphoma (NHL) pathogenesis (Song et al., 2016), there are
major knowledge gaps in addressing this question. This is because the available information does not
specifically address measurement of glyphosate residues in the (gastro)intestinal tract or whether
glyphosate adversely affects the normal functioning of the microbiota in the human gastrointestinal
tract or the gastrointestinal tract of experimental mammalian models.
2.7 Studies on metabolites: AMPA

AMPA is the only identified metabolite found in the urine and faeces of orally treated rats. It
was reviewed by the JMPR in 1997. The Meeting established an acceptable daily intake (ADI) of 0–
0.3mg/kg bw (sum of glyphosate and AMPA) based on a NOAEL of 31 mg/kg bw per day, the
highest dose tested in a 26-month study of toxicity in rats with glyphosate.
(a) Acute toxicity of AMPA

Mice
In an acute oral toxicity study, five male and five female ICR(Crj:CD-1) mice were orally
dosed with AMPA (purity 99.33%) at 5000 mg/kg bw. The test material was administered as a 25%
suspension in 1% CMC sodium solution at 20 mL/kg bw. There were no deaths and no signs of
toxicity. All mice gained weight on days 0–7; one male and two females had slight weight losses on
days 7–14. There were no observed abnormalities at necropsy.
The oral LD50 of AMPA in male and female mice was greater than 5000 mg/kg bw (Komura,
1996).
Rats
In an acute oral toxicity study, five male and five female Wistar-derived Alpk:APfSD(SPF)
albino rats were orally dosed with 5000 mg/kg bw AMPA (assumed purity 100%). The test material
was administered as a 50% suspension in 0.5% aqueous polysorbate 80 at a constant dose volume of
10 mL/kg bw.
There were no deaths. Signs of toxicity included diarrhoea, chromodacryorrhea, piloerection,
stains around the nose and ungroomed appearance, with recovery by day 5. All the rats but one male
gained weight on days 1–8; two males and three females had weight losses on days 8–15. No
abnormalities were observed at necropsy.
The oral LD50 of AMPA in male and female rats was greater than 5000 mg/kg bw (Leah,
1988).
In a study of acute oral toxicity, five male and five female Sprague Dawley rats were
administered AMPA (purity 99.2%) in 0.5% CMC as a single dose at 5000 mg/kg bw by gavage.
Clinical signs, observed 4 hours after dosing, included piloerection, diarrhoea, subdued
behaviour, hunched appearance and soiled anal and perigenital areas. All the animals had normal
body-weight gain throughout the experiment. No abnormalities were detected at necropsy after 14
days of observation.
The acute oral LD50 of AMPA in rats was greater than 5000 mg/kg bw (Cuthbert & Jackson,
1993a).

231
In a study of acute dermal toxicity, five male and five female Sprague Dawley rats were
treated with a single 2000 mg/kg bw dose of AMPA (purity 99.2%). The test material was evenly
spread on a 5 × 5 cm dressing moistened with distilled water that was then placed on the shaved back
of each rat. The patch was covered with an occlusive dressing and kept in contact with the skin for 24
hours. At the end of the exposure period the patch was removed and the exposed skin wiped with
distilled water to remove any excess test material.
There were no mortalities after a single dermal application of AMPA at 2000 mg/kg bw and
no clinical signs or abnormalities were noted at necropsy. Thus, the acute dermal LD50 of AMPA to
rats must be greater than 2000 mg/kg bw (Cuthbert & Jackson, 1993b).
In an acute dermal toxicity study, 2000 mg AMPA (purity 98.0%) suspended in 0.5% aqueous
hydroxypropylmethylcellulose gel was applied at a volume of 10 mL/kg to five male and five female
CD/Crl:CD rats as an occluded exposure for 24 hours. There were no deaths, no signs of toxicity, no
dermal irritation and no observed abnormalities at necropsy.
The dermal LD50 of AMPA was greater than 2000 mg/kg (Leuschner, 2002a).
Guinea pigs
The sensitization potential of AMPA (purity 99.2%) was investigated by means of the
Magnusson–Kligman Maximization Test in guinea pigs. A group of 20 female Dunkin Hartley guinea
pigs were intradermally injected with AMPA at 10% w/v in CMC; 6 days later, 25% w/v in 0.5%
CMC was topically applied. Challenge was at a concentration of 25% w/v in CMC.
At challenge, none of the test or control group animals showed a positive response. There was
no evidence from the test results that AMPA is a sensitizer in guinea pigs (Cuthbert & Jackson,
1993c).
In a Magnusson–Kligman (maximization test) dermal sensitization study, 10 male Dunkin

Hartley guinea pigs were injected with 5% AMPA (purity 98.0%) on day 0, had their application site
skin treated with sodium lauryl sulfate on day 6, and then were topically treated with 2 mL of a 50%
suspension of AMPA in aqua ad iniectabilia on day 7. They were challenged (along with five
negative control animals) with 2 mL of a 50% suspension of AMPA in aqua ad iniectabilia on day
21. There was no resultant skin irritation in any guinea pig.
The evidence from the test results was that AMPA was a non-sensitizer in this assay
(Leuschner, 2002b).
(b) Short-term toxicity studies of AMPA

In a short-term toxicity study, groups of five male and five female Sprague Dawley rats were
administered AMPA (purity 99.2%) in CMC at concentrations of 0, 10, 100, 350 or 1000 mg/kg bw
per day by oral gavage for 28 days.
There were no treatment-related effects on mortality, clinical signs, body weight, body-weight
gains, feed or water consumption or macroscopic findings. There were slight but statistically
significant increases in kidney weights in males at 350 and 1000 mg/kg bw per day compared with
control group (by 7% and 8%, respectively). Histological examinations revealed a very slight
reduction in serous secretion in the mandibular salivary gland of one high-dose male. Whether the
minor salivary gland findings is related to treatment is equivocal.
The NOAEL is 100 mg/kg bw per day based on an increase in kidney weights seen at 350
mg/kg bw per day and greater (Heath, Strutt & Iswariah, 1993).

232
In a 90-day toxicity study, groups of 10 male and 10 female Sprague Dawley rats were
administered AMPA (purity 99.2%; in CMC) at a concentrations of 0, 10, 100 or 1000 mg/kg bw per
day by gavage for 13 weeks. Blood samples were taken from all animals during week 13 for
investigation of haematology and clinical chemistry parameters. An ophthalmoscopic examination
was undertaken on all animals during pre-trial and on all control and high-dose animals during week
12. All surviving animals were necropsied at termination as were all pre-terminal decedents.
Histological examination was carried out on selected tissues from all control and high-dose animals
and all pre-terminal decedents and on the kidneys, liver, lungs, submaxillary salivary gland,
sublingual salivary gland and parotid salivary gland of all other animals.
There was no treatment-related effect on mortality, clinical signs, body weight, body-weight
gain, feed consumption, water consumption, haematology and clinical chemistry parameters,
ophthalmoscopic examination, organ weights, macroscopic findings and histological examination.
The NOAEL in this 90-day gavage toxicity in rats with AMPA was 1000 mg/kg bw per day (Strutt et
al., 1993).
Table 51. Summary of acute toxicity studies of AMPA

Purity LD50
Species Strain Sex Route (%) (mg/kg bw) Reference
Mouse (Crj:CD-1) M+F Oral 99.33 > 5 000 Komura (1996)
Rat Alpk:APfSD, M+F Oral 100 > 5 000 Leah (1988)
Wistar (assumed)
Rat Sprague M+F Oral 99.2 > 5 000 Cuthbert &

Dawley Jackson (1993a)
Rat Sprague M+F Dermal 99.2 > 2 000 Cuthbert &
Dawley Jackson (1993b)
Rat CD/Crl:CD M+F Dermal 98.0 > 2 000 Leuschner
(2002a)
Guinea pig Dunkin F Sensitization 99.2 Negative Cuthbert &

Hartley (Magnusson–Kligman Jackson (1993c)
Maximization Test)
Guinea pig Dunkin M Sensitization 98.0 Negative Leuschner
Hartley (Magnusson–Kligman (2002b)
Maximization Test)
LD50: median lethal dose
(c) Genotoxicity of AMPA

A much smaller number of studies have been conducted on the glyphosate metabolite,
AMPA, as well as the plant metabolites, N-acetyl-glyphosate and N-acetyl-AMPA. The results are
shown in Tables 33, 34 and 35. The in vivo studies (Jensen, 1993c; Kier & Stegeman, 1993; Manas et
al., 2009b; see Table 35) investigated the ability of AMPA to induce micronuclei in the bone marrow
erythrocytes of mice and have largely been negative although a modest positive response was reported
by Manas (2009b) when AMPA was administered by intraperitoneal injection to male mice.
Studies by other investigators using the more relevant oral route of administration did not
show an increase in micronuclei in either male or female mice.
In the in vitro studies, increases in mutation in bacteria were not seen for AMPA or the
acetylated metabolites. Both positive (Manas et al., 2009b) and negative (Jensen, 1993b,c; Roustan et
al., 2014) results were reported in studies of chromosome aberrations and DNA damage for AMPA.
AMPA was negative in two studies of unscheduled DNA synthesis in isolated rat hepatocytes (Bakke,

233
1991; Nesslany, 2002). Studies of chromosome aberrations and gene mutation in mammalian cells
using the acetylated metabolites were negative.
(d) Developmental toxicity of AMPA

In a developmental toxicity study, AMPA (purity 99.2%) suspended in CMC was
administered to 10 copulated Sprague Dawley female rats per dose by oral gavage at concentrations of
0, 100, 350 or 1000 mg/kg bw per day from days 6 through 16 of gestation. On day 20 of gestation,
the dams were terminated, pregnancy status determined and numbers of corpora lutea, implantations
and live fetuses recorded. All live fetuses were weighed, sexed and examined for external, visceral
and skeletal abnormalities.
There were no mortalities or treatment-related clinical throughout the study. Body-weight
gain and feed consumption of the test animals were similar to those of the controls. There were no
notable intergroup differences in the incidence of intrauterine deaths or in mean fetal weights.
Examination of fetuses for developmental abnormalities and variations of the viscera and skeleton
(including state of ossification) showed no intergroup differences.
The NOAEL for maternal and developmental toxicity was 1000 mg/kg bw per day, the
highest dose tested (Hazelden, 1992).
2.8 Studies on metabolites: N-acetyl-glyphosate and N-acetyl-AMPA

Metabolism studies in genetically modified soya beans and maize containing the glyphosate-
N-acetyltransferase gene demonstrated the formation of new metabolites not observed in conventional
crops. The major metabolite in the new maize and soya bean varieties was N-acetyl-glyphosate (which
may be degraded to glyphosate in the rat), whereas glyphosate, N-acetyl-AMPA and AMPA were
found in low concentrations in the edible parts of the crops. N-Acetyl glyphosate and N-acetyl-AMPA
were reviewed by the JMPR in 2011. The Meeting (2011) concluded that the group ADI of 0–1 mg/kg
bw established by the 2004 JMPR for glyphosate and AMPA may also be applied to N-acetyl-
glyphosate and N-acetyl-AMPA as the available toxicological data showed that these plant
metabolites have no greater toxicity than the parent glyphosate. The 2004 JMPR decided that an acute
reference dose (ARfD) for glyphosate was unnecessary. The 2011 JMPR confirmed that it is not
necessary to establish an ARfD for N-acetyl-glyphosate or N-acetyl-AMPA in view of their low acute
toxicity and the absence of any toxicological effects that would be likely to be elicited by a single
dose.
(a) Biotransformation of N-acetyl-glyphosate (company code IN-MCX20)

A total of 45 male Crl:CD(SD)IGS BR rats were each administered a single oral dose of free
acid at 15 mg eq/kg bw of [14C]N-acetyl glyphosate (sodium salt; purity 84.3%, radiochemical purity
99.2%) in water. Blood was collected from four animals pre dose and at 0.5, 1, 2, 4, 8, 12, 48 and 72
hours post dose. Excreta were collected from five animals at specified intervals through 168 hours
post dose. Plasma, excreta and carcasses were analysed for radioactive content. Selected samples of
plasma, urine and faeces were analysed for unchanged parent compound and metabolites.
The mean total recovery was 95.5%, with 66.1% (61.3% within 12 hours of dosage) in urine,
26.4% (25.8% within 48 hours of dosage) in faeces, 2.79% in cage wash and wipe, and 0.23% in
residual carcass. More than 90% of the total radioactivity was eliminated 48 hours post dose. Cmax in
blood and plasma were 2.93 and 5.31 µg eq/g at 1 and 2 hours post dose, respectively. Radioactivity
was eliminated from blood and plasma with half-life values of 20.1 and 15.6 hours, respectively.
Comparison of blood and plasma AUC values indicates that 14C-labelled N-acetyl-glyphosate
distributed preferentially into plasma.
Unchanged 14C-labelled N-acetyl-glyphosate recovered in urine and faeces represented over
99% of the administered radioactivity. A metabolite, glyphosate, was detected in faeces and

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represented less than 1% of the total radioactivity. Plasma radioactivity consisted entirely of
unchanged 14C-labelled N-acetyl-glyphosate (Cheng & Howard, 2004).
(b) Acute toxicity of N-acetyl-glyphosate and of N-acetyl-AMPA

N-Acetyl glyphosate (purity 84.3% sodium salt, equivalent to 67.4% free acid) was suspended
in water and administered to five male and five female Crl:CD(SD) IGS BR fasted (17–20 hours) rats
at a dose of 5000 mg/kg bw of free acid, administered as a constant dose volume of 10 mL/kg bw.
One female was found dead at 6 hours after administration, and one female and one male
were found dead the following day. Signs of toxicity (seen in all rats) included slight hypoactivity,
irregular respiration, liquid faeces, soft faeces, light-brown perineal staining, squinted eyes and brown
nasal crust. All survivors were normal 3 days after dosing. Necropsy findings of decedents included
mottled or discoloured lungs, discoloured (black) liver, soft stomach, yellow fluid or gel-like clear
liquid in stomach, fluid in abdominal cavity, fluid in duodenum, jejunum and ileum.
The LD50 of N-acetyl glyphosate in rats was greater than 5000 mg/kg bw of free acid
(Vegarra, 2004).
N-Acetyl AMPA (purity 97%) suspended in deionized water was administered by oral gavage
at a constant dose volume of 20 mL/kg bw at 5000 mg/kg to three Crl (CD)SD female rats.
There were no deaths. Signs of toxicity included diarrhoea, dark eyes, lethargy, high posture,
stained fur/skin, wet fur, ataxia and/or hyperreactivity. All the rats had fully recovered 3 days after
dosage. All the rats gained weight on days 0–7 and 7–14. There were no dose-related abnormalities at
necropsy.
The LD50 of N-acetyl-AMPA in rats was greater than 5000 mg/kg bw based on the signs of
toxicity (Carpenter, 2007).
(c) Subacute toxicity of N-acetyl-glyphosate

Five groups of young adult male and female Crl:CD(SD) rats (10/sex per group) were fed
diets containing 0, 180, 900, 4500 or 18 000 ppm N-acetyl-glyphosate sodium salt (purity 81.8%)
(equal to 0, 11.3, 55.7, 283 and 1157 mg/kg bw per day, respectively, for males and 0, 13.9, 67.8, 360
and 1461 mg/kg bw per day, respectively, for females) for 95 days (males) or 96 days (females).
No adverse effects on body weights or nutritional parameters were observed. The slight
decrease in body weight (92% of the control) in the high-dose animals was not considered adverse
since statistical significance was not achieved. Statistically significant lower overall mean body-
weight gain (86% of control) was observed in males at 18 000 ppm but it was not considered adverse
as it was not associated with a statistically significant difference in mean final body weight or in
overall mean feed consumption of feed efficiency.
There were neither any treatment-related deaths nor any clinical, ophthalmological or
neurobehavioural observations. There were no adverse effects on clinical pathology parameters, organ
weights, gross pathology or microscopic pathology in male or female rats. The NOAEL for male and
female rats was 18 000 ppm, equivalent to 1157 mg/kg bw per day in males and females, respectively
(MacKenzie, 2007).
A supplemental report to the 90-day MacKenzie (2007) study tested dietary disodium N-
acetyl-N-(phosphonomethyl)glycine (purity 63%, expressed as the weight per cent on a free acid
basis).
Pooled urine samples for male rat groups I (control), III (180 ppm), V (900 ppm), VII (4500
ppm) and IX (18 000 ppm) were collected on day 82 and for female rats groups II (control), IV (180

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ppm), VI (900 ppm), VIII (4500 ppm) and X (18 000 ppm) on day 83 for analysis of IN-MCX20 (N-
acetyl-glyphosate) and its possible metabolites, IN-B2856 (glyphosate) and IN-EY252 (N-acetyl
AMPA). On the same days, plasma samples from individual rats were collected for the same analyses.
Concentrations of IN-MCX20 (N-acetyl-glyphosate) in the urine increased with the increasing
dietary levels of N-acetyl-N-(phosphonomethyl)glycine. Concentrations of IN-B2856 and IN-EY252
were above the limit of detection at higher dietary levels (900–18 000 ppm) but at or below the limit
of detection at 180 ppm. In addition, the concentrations of these metabolites were much higher in
urine samples from male rats than from female rats at 4500 and 18 000 ppm. Neither IN-MCX20 nor
its metabolites were detected in urine of control rats.
Concentrations of IN-MCX20 (N-acetyl-glyphosate) also increased in the plasma samples
with increasing dietary levels of N-acetyl-N-(phosphonomethyl)glycine. Concentrations of IN-
MCX20 were less than 1.0 µg/mL for males and females in the 180 ppm dietary group, but increased
from a mean of about 2 µg/mL up to about 14.0 µg/mL for the other dietary groups. Little to no IN-
B2856 (glyphosate) or IN-EY252 (N-acetyl AMPA) was detected in plasma at all dietary levels.
These results confirm that IN-MCX20 (N-acetyl-glyphosate) is metabolized in rats to small
quantities of IN-B2856 (glyphosate) and IN-EY252 (N-acetyl AMPA) (Shen, 2007).
(d) Genotoxicity of N-acetyl-glyphosate and of N-acetyl-AMPA

A few studies have been conducted on the genotoxicity of the glyphosate metabolite, N-
acetyl-glyphosate. The results are shown in Tables 36, 37 and 38.
The in vivo studies shown in Table 35 (Murli, 2004; Donner, 2006; Glatt, 2006) that
investigated the ability of N-acetyl-glyphosate to induce micronuclei in the bone marrow erythrocytes
of mice and gene mutations and chromosomal aberrations in CHO cells were negative.
Increases in mutation were also not seen in the in vitro studies.
A smaller number of studies have been conducted on the plant metabolites, N-acetyl-AMPA.
The results are shown in Tables 33, 34 and 35. The in vivo study (Donner, 2007; Table 35)
investigating the ability of N-acetyl-AMPA to induce micronuclei in the bone marrow erythrocytes of
mice was negative.
In the in vitro studies, increases in mutation in bacteria were not seen; nor were gene
mutations in Chinese hamster cells (Glatt, 2007) or chromosomal aberrations in human peripheral
blood lymphocytes (Gudi & Rao, 2007).
2.9 Studies on other formulation ingredients

Several publications have reported that glyphosate formulation ingredients and possible
contaminants have a greater toxicity than the active ingredient, glyphosate.
Although it was pertinent to consider the toxicity of the known formulants, a detailed review
and exhaustive analysis could not be undertaken due to lack of time and confidentiality constraints;
producers often consider formulation ingredients proprietary and hence confidential and obtaining this
information can be problematic. Nevertheless, based on the reports listed below, it is apparent that
some of the formulants may have a greater toxicity than the active ingredient, glyphosate.
Polyethoxylated tallow amine (polyoxyethyleneamine [POEA]; MON 0818; CAS No. 61791-
26-2 (tallow); POE n =15)
In a 30-day oral toxicity study, MON 0818 (purity and lot number not reported) was
administered to groups of 10 male and 10 female Sprague Dawley rats in the diet at concentrations of

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0, 800, 2000 or 5000 ppm (equal to 0, 51.7, 122.8 and 268.7 mg/kg bw per day for males and 0, 63.2,
159.9 and 324.8 mg/kg bw per day for females).
All the treated rats survived until scheduled termination. Soft stools were observed from three
high-dose males on four occasions and from eight high-dose females on 23 occasions. Body weight,
body-weight gain and feed consumption of high-dose male and female rats were significantly reduced
during the study; this was consistent with poor diet palatability. Feed consumption of mid-dose male
rats was statistically decreased during the first week of treatment, as was total body weight at the end
of the study; however, the final body weight was decreased by only 7% relative to controls. No
treatment-related effects were found in mid-dose female rats or in low-dose male and female rats. The
absolute and relative organ weights of high-dose male and female rats were decreased consistent with
the markedly decreased body weight. Prominent or enlarged lymphoid aggregates in the colon of five
high-dose female rats were observed at necropsy.
Because a description of the test material, its lot number, its purity and its concentration,
homogeneity and stability in the diet were not provided or determined, an estimate of the dose
inducing treatment-related effects on male and female rats cannot be made. In addition, very limited
in-life observations and, with the exception of selected organ weights and gross pathology, no post-
termination studies or observations were made (Ogrowsky, 1989). As a result, this study was deemed
unacceptable and it could not be used to establish a NOAEL or LOAEL.
In a subchronic oral toxicity study of MON 0818 in Sprague Dawley rats, the test material
was administered in the diet ad libitum to three groups of 10 male and 10 female rats for 90 days.
Target test diet concentrations were 0, 500, 1500 or 4500 ppm (equal to 0, 33.0, 99.3 and 291.6 mg/kg
bw per day in males and 0, 39.9, 123.1 and 356.6 mg/kg bw per day in females). A similar, concurrent
control group of rats were fed the basal diet only.
Exposure at 1500 and 4500 ppm resulted in statistically and toxicologically significant
effects. Toxicity observed at 4500 ppm consists of clinical signs (soft stools, three incidences in two
males and 86 incidences in all females) observed from day 16 through day 92 of the study, decreased
mean body weights throughout the study (from 12–20% in males and 8–18% in females), and
decreased mean total body-weight gains in males (31%) and females (35%). Feed consumption was
also significantly reduced throughout most of the study (13 weeks for males and 10 weeks for
females), particularly during the first week of the study (32% decrease in males and 27% decrease in
females). Since a feed efficiency assessment was not conducted, it is not possible to determine if the
decreases in body weights, body-weight gains, and feed consumption were due, in part, to the
unpalatability of the diet. Statistically significant changes in haematological parameters observed in
females may be a result of the inflammation observed in the intestines. Statistically significant
changes in clinical chemistry parameters and organ weights observed in high-dose males and females
are likely a result of decreased feed consumption/nutrient absorption and body weight.
At both 1500 and 4500 ppm, microscopic examination conducted at necropsy revealed
lesions, including hypertrophy and/or vacuolation of histiocytes in the lamina propria of the ileum in
all high-dose males and females, and four mid-dose males and four mid-dose females; hypertrophy
and/or vacuolation of histiocytes in the lamina propria of the jejunum in four high-dose males, seven
high-dose females and one mid-dose female; sinus histiocytosis in nine high-dose males, six high-
dose females and two mid-dose males and females; and accumulation of macrophage aggregates in
the cortex and medullary cords of the mesenteric lymph node in eight high-dose males, seven high-
dose females and two mid-dose females. These inflammatory changes are likely the cause of the soft
stools observed during the study and are considered treatment-related.
No statistically significant treatment-related effects on body weight, body-weight gain, feed
consumption, haematological/clinical chemistry parameters and organ weights were observed at the
low-dose level of 500 ppm. In addition, no gross abnormalities or histopathological findings related to
treatment were observed at this dose level.

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Based on treatment-related inflammatory changes at 1500 ppm (equal to 99.3 mg/kg bw per
day), the NOAEL for MON 0818 was 500 ppm (equal to 33.0 mg/kg bw per day). The LOAEL was
1500 ppm (equal to 99.3 mg/kg bw per day) based on irritation in the intestines and colon
(hypertrophy and vacuolation of histiocytes in the lamina propria of the jejunum and ileum, and
histiocytosis and accumulation of macrophage aggregates in the mesenteric lymph node (Stout, 1990).
In a screening study, the potential reproductive toxicity and developmental (prenatal and
postnatal) toxicity of MON 0818 (purity 69–73%) was evaluated in CD (Sprague Dawley) rats
through two successive generations. The study was designed to evaluate the effects of MON 0818 on
male and female reproduction within the scope of a screening study. The study was extended to a two-
generation study when a decrease in live litter size was observed at the high-dose level. MON 0818
was administered orally via the diet to three groups of 20 male and 20 female CD rats. Target test diet
concentrations were 0, 100, 300 or 1000 ppm (corrected for purity to doses equal to 0, 4.4, 13.4 and
44.5 mg/kg bw per day for males and 0, 9.6, 16.1 and 54.0 mg/kg bw per day for females). A similar
concurrent control group of rats were fed the basal diet only. At approximately 10 weeks of age, the
F0 animals were dosed via diet for at least 70 days prior to mating and then to termination (males) or
lactation day 21 (females). All F0 adults were terminated following selection of the F1 generation on
postnatal day 21.
Parents for the F1 generation were selected from the weaned F1 litters. Between postnatal day
21 or 22 and 70, the weanling F1 animals (3 per sex/litter, if possible) were administered the test diet
on a mg/kg bw basis (so not to overexpose the rapidly growing F1 animals) at target concentrations of
0, 6, 18 or 61 mg/kg bw per day for the F1 males and 0, 7, 22 or 74 mg/kg bw per day for the F1
females. Beginning on postnatal day 70, the F1 animals selected for breeding from the control and
high-dose groups only (2 per sex/litter) were administered the test diet at a constant concentration (0
or 1000 ppm) for at least 80–88 days prior to mating. The selected F1 males continued to receive the
test diet throughout mating and until termination (after lactation day 4). The selected F1 females
continued to receive the test diet throughout mating, gestation and lactation, until termination (after
lactation day 4).
Mortality and clinical signs, body weights, body-weight gains, feed consumption,
reproductive function, fertility and mating performance, absolute and relative organ weights,
macroscopic abnormalities at necropsy and histopathological findings were recorded for all
parental/adult animals. In addition, blood samples for testosterone and/or thyroid hormone
concentration determinations were collected from one F1 male and one F1 female per litter at the
scheduled necropsy. Sperm evaluation (motility and morphology) was also performed on all F 1 male
animals at termination. Litter size, viability, clinical signs, body weights, body-weight gains,
developmental (sexual and physical) parameters, and macroscopic abnormalities at necropsy were
recorded for the F1 and F2 pups.
Survival and clinical conditions, mean body weights and feed consumption (pre-mating,
gestation, and lactation), reproductive performance, mean organ weights, and macroscopic and
microscopic morphology of the F0 and F1 parental generations were unaffected at all dose levels.
Treatment-related effects were also not seen in estrous cyclicity, spermatogenic end-points and
testosterone and thyroid hormone levels of the F1 generation or in the clinical signs, mean body
weights and developmental landmarks of the F1 and F2 pups, as well as the litter viability and
postnatal survival of the F2 litters.
Potential treatment-related effects were observed in litter loss, increased mean number of
unaccounted-for implantation sites and decreased mean number of pups born, live litter size and
postnatal survival from birth to lactation day 4 in the high-dose F0 females and F1 litters. These effects
were limited to a small number of litters, were not always statistically significant and were not
reproduced in the F2 litters. However, the increased (statistically significant) mean number of
unaccounted-for implantation sites exceeded the maximum mean value in the laboratory historical
control data. While not statistically significant, the corresponding reduced number of pups born and

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live litter size, as well as the reduced postnatal survival, were at or below the limits observed in the
laboratory historical control data.
The LOAEL of MON 0818 for reproductive toxicity and offspring toxicity in rats was 1000
ppm (equal to 44.5 mg/kg bw per day) based on litter loss, increase mean number of unaccounted-for
implantation sites and decreased mean number of pups born, live litter size and postnatal survival
from birth to lactation day 4. The NOAEL for reproductive and offspring toxicity was 300 ppm (equal
to 13.4 mg/kg bw per day). The NOAEL for parental systemic toxicity was 1000 ppm (equal to 44.5
mg/kg bw per day). A LOAEL for parental systemic toxicity was not determined (Knapp, 2007).
In a combined repeated-dose toxicity study with the reproduction/developmental toxicity

screening test, MON 8109 (coco amine ethoxylates, CAS No. 61791-31-9, (coco); Ave POE n = 2);
purity 100%) or MON 0818 (purity 100%) was administered to 12 Crl:CD(SD) rats/sex per dose in
the diet at dose levels of 0, 30, 100, 300 or 2000 ppm MON 8109 or 1000 ppm MON 0818. The mean
compound intake for MON 8109 was 0, 2, 8, 23 and 134 mg/kg bw per day for males and 0, 3, 9, 26
and 148 mg/kg bw per day for females. The mean compound intake for MON 8108 was 0, 2, 8, 23
and 76 mg/kg bw per day for males and 0, 3, 9, 26 and 86 mg/kg bw per day for females. Males were
fed the test or basal diets for a total of 71–72 days, and the females were fed the test or basal diets for
a total of 69–72 days. Functional observational battery and locomotor activity data were recorded for
six males per group near the end of diet administration and for six females per group on lactation day
4. Parental animals were terminated approximately 2.5 weeks after lactation day 4, and offspring were
terminated on lactation day 4.
There was no treatment-related mortality. One female in the 1000 ppm MON 0818 group was
found dead with dystocia on lactation day 1 and another was euthanized in extremis on gestation day
30 and found to have a ruptured uterus. Increased incidences of red material around the nose,
reddened nose and reddened mouth at 2000 ppm MON 8109 in males and females were treatment-
related. Mean body-weight losses were noted at 2000 ppm MON 8109 in male and females during the
first week of test diet administration. Lower mean body weight and/or body-weight gain with
corresponding reduction in feed consumption were usually observed in the animals from this group
throughout the study. Absolute and relative organ-weight values that were statistically different from
the corresponding control were not treatment related as this difference was due to the significantly
lower body weight of the 2000 ppm MON 8109–treated animals. The females from this group had a
lower number of implantation sites and lower live litter size. The offspring of these females had lower
postnatal survival on postnatal day 0, postnatal day 0–1, postnatal days 1–4 and birth to postnatal day
4 compared to the control group. No effect of treatment was observed in male and female mating and
fertility, male copulation and female conception indices, gestation length, functional observational
battery, locomotor activity, haematology or serum chemistry. No test-substance–related findings were
noted in the 30, 100 or 300 ppm MON 8109 or 1000 ppm MON 0818 group males, females or
offspring.
The parental systemic LOAEL was 2000 ppm for MON 8109 (equal to 134 mg/kg bw per
day), based on clinical findings and decreased mean body weight, body-weight gain and feed
consumption. The parental systemic NOAEL was 300 ppm for MON 8109 (equal to 23 mg/kg bw per
day).
The reproductive/developmental LOAEL was 2000 ppm MON 8109 (equal to 134 mg/kg bw
per day) based on decreased postnatal survival, lower live litter size on postnatal day 0, lower number
of pups born and lower number of implantation sites. The reproductive NOAEL is 300 ppm MON
8109 (equal to 23 mg/kg bw per day).
A parental LOAEL for MON 0818 was not demonstrated in this study. The parental NOAEL
was 1000 ppm for MON 0818 (equal to 76 mg/kg bw per day).
The reproductive/developmental LOAEL for MON 0818 was not demonstrated in this study.
The reproductive NOAEL was 1000 ppm MON 0818 (equal to 76 mg/kg bw per day) (Knapp, 2008;
Nord, 2008).

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In a developmental toxicity study, MON 0818 (purity 100%) was administered in Mazola
Corn Oil to 25 Charles River Crl:CDBr female rats per dose by gavage at dose levels of 0 (corn oil
only), 15, 100 or 300 mg/kg bw per day from gestation day 6 through 15. On gestation day 20, all
surviving females were terminated for developmental examination. The developmental parameters
noted included the number of viable fetuses, early and late resorptions, total implantations and total
corpora lutea and the sex and weight of fetuses and external, visceral and skeletal examinations of all
fetuses.
Six of the 25 high-dose females died during gestation days 6–15. Clinical signs observed in
the high-dose females included rales (12/25), laboured respiration (3/25), yellow uro- (15/25) or
anogenital (14/25) matting and mucoid faeces (22/25) compared to none for the control animals. Few
to no clinical signs were observed in the mid-dose and low-dose females. High-dose females weighed
significantly (P < 0.01) less than the controls from study day 9 until termination at study day 20.
High-dose females also gained 59% less weight compared to controls during treatment (days 6–16).
Body weight was similar to controls in the low- and mid-dose groups. Gravid uterine weight was not
affected by treatment in any of the groups. High-dose females ate statistically (P < 0.01) less feed
compared to the control rats, with the most significant decrease (55% less than controls) on days 6–9
before gradually improvement to comparability with controls by day 16. Overall, the high-dose group
ate 29% less than the controls during days 6–16. Feed consumption for the low-dose and mid-dose
females was comparable to that of controls throughout the study, except for days 6–9 when the mid-
dose group had a statistically significant (P < 0.05) decrease. No treatment-related effects were
observed on liver weight or gross pathology at necropsy in any of the treated dams.
No treatment-related differences were observed in the mean number of corpora lutea,
implantations, live fetuses or resorptions or mean fetal weight. On external examination, the mean
number of fetal malformations from the high-dose dams appeared to be high but most were observed
in a single fetus and a dose–response relationship was not observed. On visceral examination of the
fetuses from the high-dose group, one fetus was missing a urinary bladder, one had stenosis of the
right carotid artery and two had situs inversus, but these were not considered treatment related as there
was no dose–response relationship for the situs inversus and the others were within the historical
control data range. Vertebral anomalies with or without rib anomalies were observed in one fetus in
the high-dose group but this was within the range of historical control data. No malformations were
observed in the low- or mid-dose groups. Several skeletal variations in the sternebrae and ribs were
identified but they were observed in both the control and treated groups at similar incidences and are
not considered treatment related.
The maternal toxicity LOAEL for MON 0818 in rats was 300 mg/kg bw per day, based on
increased mortality, clinical signs and decreased body weight, body-weight gain and feed
consumption. The maternal NOAEL for MON 0818 was 100 mg/kg bw per day.
The developmental toxicity LOAEL for MON 0818 in rats could not be determined as no
effects were associated with treatment. The developmental toxicity NOAEL for MON 0818 is 300
mg/kg bw per day (Holson, 2006).
In independent trials of the reverse gene mutation assay in bacteria, strains TA1535, TA1537,
TA98 and TA100 of S. typhimurium were exposed to MON 0818 (purity not stated). In the first trial,
all tester strains were exposed to 0.001, 0.003, 0.01, 0.03 or 0.1 mg/plate with S9 activation and
0.0003, 0.001, 0.003, 0.01 or 0.03 mg/plate without S9 activation. (The S9-fraction was obtained from
Aroclor 1254–induced male Sprague Dawley rat liver.) A repeat assay was performed on TA1535 and
TA1537 (±S9) using the same concentrations as in in trial one. Because cytotoxicity was not observed
with all tester strains, test material concentrations were adjusted for the subsequent mutagenicity trials
(trials 3 and 4). Concentrations of MON 0818 from 0.01–1.0 mg/plate with S9 activation and 0.003–
0.3 mg/plate without S9 activation were tested in strain TA98; 0.001–0.10 mg/plate with and without

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S9 activation in TA100; 0.001–0.1 mg/plate without S9 in TA1535; 0.003–0.3 mg/plate with S9

activation and 0.001–0.1 mg/plate without S9 activation in TA1537.
No evidence of mutagenicity was observed in trial 1. A statistically significant (P < 0.01)
increase in the number of revertant colonies was observed at 0.03 mg/plate (−S9) in TA98 and 0.0003
mg/plate in TA1535 (−S9); however, the increases were less than twofold and not concentration
dependent. When the strains were retested in trials 3 and 4, cytotoxicity was seen at 0.3 mg/plate and
above with S9 activation and 0.1 mg/plate and above without S9 activation in TA98; 0.03 mg/plate
and above with and without S9 activation in TA100; at 0.1 mg/plate without S9 activation in TA1535;
and at 0.1 mg/plate and above with and without S9 activation in TA1537. Although slight increases in
the number of revertants were seen at non-cytotoxic concentrations of 0.01 and 0.1 mg/plate with S9
activation in TA98, the increases were less than twofold greater than the solvent controls and did not
satisfy the criteria for a positive response. No concentration-dependent increase in the number of
revertant colonies was observed in any of tester strains with or without S9 activation.
Overall, no evidence of mutagenicity was observed at non-cytotoxic concentrations with or
without S9 activation.
MON 0818 was tested up to cytotoxic concentrations in all strains, but failed to induce a
mutagenic response in this test system. The positive controls induced the expected mutagenic
responses in the appropriate strain (Stegeman & Li, 1990).
In a bone marrow micronucleus assay, adult male and female ICR(Crl:CD-1) mice (5/sex per
dose) were treated once via intraperitoneal injection with 0 or 100 mg/kg MON 0818, which was
estimated to be about 61% of the LD50 (batch/lot no. PIT-8907-757-I; purity 100%, prepared in corn
oil). Bone marrow cells were harvested at 24 and 48 hours following dosing and scored for
micronucleated polychromatic erythrocytes. Cyclophosphamide (60 mg/kg) served as the positive
control.
No deaths or overt signs of clinical toxicity or cytotoxicity of bone marrow were observed at
this dose. Although no toxicity was seen at 100 mg/kg, the selected level was considered acceptable in
accordance with the high dose recommended by the USEPA Gene-Tox Program (i.e. when a dose that
is not less than 50% of the LD50 is used to define the maximum tolerated dose) for the micronucleus
assay (Mavournin et al., 1990). Administration of 60 mg/kg cyclophosphamide caused a significant
(P < 0.01) induction of micronucleated polychromatic erythrocytes in both sexes. There was no
significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow
after any harvest time up to the maximum tolerated dose (Stegeman & Kier, 1998).
N,N-bis-(2-hydroxyethyl) alkylamine; synthetic ethoxylated amine, ATMER 163 (CAS No.

70955-14-5; C13-C15; ave POE n =2)
In a 90-day oral gavage toxicity study, ATMER 163 (100% a.i. assumed and batch/lot no. not
reported) was administered to 20 Sprague Dawley (Crl:CD[SD]BR) rats per sex per dose at
concentrations of 0, 15, 30 or 150 mg/kg bw per day. Deionized water was administered to controls.
Numerous clinical signs were observed in animals at 150 mg/kg bw per day. The most
notable signs were wheezing and salivation in all high-dose animals and in some at 30 mg/kg bw per
day. Other clinical signs observed in both sexes at 150 mg/kg bw per day included blood crust and/or
red discharge (nose), dyspnoea, rhinorrhea, opaque eyes, redness, hunched posture, thinness, urine
stains, rough hair, desquamation and an increased incidence of alopecia. Two males at 30 mg/kg bw
per day as well as four males and one female at 150 mg/kg bw per day died during the study.
Statistically significant body weight and body-weight gain deficits were observed in both sexes at 150
mg/kg bw per day; overall body-weight gains were 30.5% and 15.3% lower than control values in
males and females, respectively. Statistically significant decreased feed consumption was seen at 150
mg/kg bw per day in males only. An ophthalmoscopic assessment revealed posterior subcapsular
cataracts in males at 30 and 150 mg/kg bw per day and in females at 150 mg/kg bw per day while

241
complete cataracts were found only at 150 mg/kg bw per day in both sexes. Increased mean values for
platelet count, white blood cell count, segmented neutrophil count and lymphocyte count were seen at
the 150 mg/kg bw per day dose in both males and females; all of the increases were statistically
significant except the increased lymphocyte count in males. These findings are often associated with
tissue inflammation which, together with other relevant findings, was observed in the lungs and
stomach of both sexes at this dosage. The only noteworthy treatment-related gross pathology findings
were in the nonglandular stomach and eyes. The findings in the nonglandular stomach, desquamation
and alteration of mucosa, were primarily found in both sexes at 150 mg/kg bw per day, although some
alterations of mucosa were also seen in animals at 30 mg/kg bw per day. Opaque eyes, seen in both
sexes at 150 mg/kg bw per day, were consistent with the ophthalmoscopic findings of complete
cataracts. Treatment-related histopathological findings included inflammation in the lungs of both
sexes at 150 mg/kg bw per day and the nonglandular stomach of both sexes at 30 and 150 mg/kg bw
per day. The inflammation in lungs might have been due to inadvertent aspiration since previous
studies have established that ATMER 163 is a primary irritant. Dose-related incidences of acanthosis
in the nonglandular stomach were seen in males and females at 30 and 150 mg/kg bw per day. The
only noteworthy finding in the glandular stomach was suppurative inflammation in two females at
150 mg/kg bw per day. In addition, microscopic assessment showed cataracts, mostly bilateral, in the
eyes of both sexes at 150 mg/kg bw per day.
There were no toxicologically significant treatment-related effects based on assessment of
clinical chemistry and limited assessment of organ weights. Urine analysis was not conducted.
The LOAEL for ATMER 163 in Sprague Dawley rats was 30 mg/kg bw per day based on
increased mortality, salivation and posterior subcapsular cataracts in males as well as wheezing and
macro- and microscopic changes in the nonglandular stomach of both sexes. The NOAEL is 15 mg/kg
bw per day (Zoetis, 1991).
In a subchronic 90-day oral toxicity study, ATMER 163 (purity 100%) was administered via
capsule to three groups of four male and four female beagle dogs for 13 weeks at concentrations of
15, 30, or 100 mg/kg bw per day. A similar concurrent control group was given empty capsules.
There were no unscheduled deaths during the study. All the dogs survived until scheduled
termination. Exposure at 100 mg/kg bw per day resulted in statistically and toxicologically significant
effects. Clinical signs of toxicity included increased incidence of salivation, emesis and soft faeces
(noted with mucus alone or mucus and bile-like material). Salivation was observed in all males and
females beginning in week 3 of the study (six animals) and continuing over 5–11 weeks. Emesis was
also observed in all of the males and females and was first observed during the first 2 weeks of the
study in seven animals and continued over 1–11 weeks. Soft mucoid faeces were observed in three
males and all the females over 2 to 7 weeks; soft mucoid or bile-particle–containing faeces were
observed in the high-dose animals (three males and two females) over 1–3 weeks. All of these clinical
signs are considered treatment related based on the high frequency of occurrence and clear dose–
response relationship. In addition, mean alanine transaminase levels in females were significantly
increased (154%) relative to controls. Microscopic examination at necropsy revealed increased
pigment accumulation in the Kupffer cells and bile canaliculi in the livers of all high-dose females.
The increased pigment accumulation was not observed in any of the treated males or in the low- and
mid-dose females. Other microscopic findings were observed, but were not dose related or were found
also found in control animals.
The statistically significant increase (22%) in mean erythrocyte counts observed in high-dose
females was within the historical control range. The significant increases (6%) in mean calcium levels
observed in the mid- and high-dose females were small, and the observed significant decrease (23%)
in mean blood urea nitrogen levels in the mid-dose males did not follow a dose–response pattern. All
of the changes are considered incidental to treatment.

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No statistically significant effects on body weight, body-weight gain, feed consumption or

organ weights were observed at any dose level. In addition no gross abnormalities or
ophthalmological changes related to treatment were observed.
The NOAEL for ATMER 163 in rats was 30 mg/kg bw per day based on the clinical signs
seen at 100 mg/kg bw per day. The LOAEL was 100 mg/kg bw per day based on clinical signs
(increased incidence of salivation, emesis, and soft faeces (with mucus alone or mucus and bile-like
material) in males and females, increased alanine transaminase levels in females, and an increased
incidence of pigment accumulation in the Kupffer cells and bile canaliculi in the livers of females
(Osheroff, 1991).
Armoblen 557 (CAS No. 68213-26-3 (Tallow, POE n = 5/12)

In a four-week oral toxicity study, Armoblen 557 (purity unknown) was administered daily by
gavage to groups of five male and five female CD rats at concentrations of 0, 15, 75 or 200 mg/kg bw
per day.
All the rats survived until scheduled termination. Salivation in males and females at 75 and
200 mg/kg bw per day was probably due to the taste of the test material and was not considered
toxicologically significant. Rales reported in one to three high-dose females were not associated with
other effects seen at necropsy and was therefore not considered toxicologically significant. The brown
staining around the muzzle occasionally seen in females at 75 mg/kg bw per day and males and
females at 200 mg/kg bw per day was also not considered toxicologically significant. Mean body
weight was decreased in males (11–17% lower than controls) and females (4–7% lower than controls)
at 200 mg/kg bw per day. Overall body-weight gain was decreased in males at 75 mg/kg bw per day
(13% lower than controls) and in males and females at 200 mg/kg bw per day (27% and 14% lower
than controls, respectively). Overall feed consumption for high-dose females was decreased (10%
lower than controls), while it was decreased in high-dose males during week 1 only. Overall feed
conversion efficiency was decreased in males at 75 and 200 mg/kg bw per day (13 and 23% lower
than controls, respectively).
Changes in haematology and clinical chemistry parameters were either not treatment related
or not toxicologically significant. Increases in the absolute and relative adrenal weights in males and
females at 200 mg/kg bw per day were not accompanied by microscopic findings and were not
considered toxicologically significant.
Based on decreased body weight, body-weight gain and food-conversion efficiency, a
LOAEL of 200 mg/kg bw per day and a NOAEL of 75 mg/kg bw per day was established for
Armoblen 557 in male CD rats. A LOAEL for Armoblen 557 in female CD rats was not established.
The NOAEL in female CD rats was 200 mg/kg bw per day (Higgs, 1994).
MON 59112
In three independent reverse gene mutation assays, S. typhimurium strains TA1535, TA1537,
TA98 and TA100 and E. coli WP2 uvrA were exposed to MON 59112 (assumed 100% purity) in
deionized water at concentrations of 0, 1, 3.33, 10, 33.3, 100 or 333 g/plate with and without S9
activation for the S. typhimurium strains and 0, 10, 33.3, 100, 333, 1000 or 3330 μg/plate with and
without S9 activation for WP2 uvrA. The S9-fraction was derived from male Sprague Dawley rats
induced with Aroclor 1254.
MON 59112 was tested up to cytotoxic concentrations in all strains (≥ 100 μg/plate +S9 and
≥ 33.3 μg/plate −S9 for S. typhimurium TA1535, TA1537, TA100 and TA98; ≥ 3330 μg/plate +S9
and ≥1000 μg/plate –S9 for WP2 uvrA) but failed to induce a mutagenic response in this test system.
The positive controls induced the expected mutagenic responses in the appropriate strain. There was
no evidence of induced mutant colonies over background (Lawlor, 2000).

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In a bone marrow micronucleus assay, adult male and female ICR(Crl:CD-1) mice were
treated once via oral gavage with MON 59112 (lot no. GLP-9708-8157-I) emulsified in corn oil.
Doses of 0, 375, 750 or 1500 mg/kg bw were administered to groups of six male mice and doses of 0,
500, 1000 or 2000 mg/kg bw were administered to groups of six female mice. Bone marrow cells
were harvested from the first five survivors at 24 hours (all dose groups) and 48 hours (1500 or 2000
mg/kg bw) following dosing. The harvested bone marrow cells were scored for micronucleated
polychromatic erythrocytes and the ratio of polychromatic to normochromatic erythrocytes.
Cyclophosphamide (80 mg/kg bw) served as the positive control.
Based on the findings of no substantial differences in the toxicological response of the male
or female mice, only the females were administered the limit dose of 2000 mg/kg bw. Two males in
the 1500 mg/kg bw and one female in the 2000 mg/kg bw treatment groups died before the scheduled
termination. Other toxic signs included hypoactivity, hunched posture, squinted eyes, rough hair coats
and faecal stains (1500 mg/kg bw males) and hunched posture and urine stains (2000 mg/kg bw
females). There were also significant reductions in the polychromatic to normochromatic erythrocyte
ratio for the high-dose males but not the high-dose females. Administration of 80 mg/kg bw
cyclophosphamide caused a significant (P < 0.01) induction of micronucleated polychromatic
erythrocytes in both sexes. There was, however, no significant increase in the frequency of
micronucleated polychromatic erythrocytes in any treatment group at either harvest time (Myhr,
2000).
Five glyphosate coformulants were tested for activation of the steroidogenic enzyme
aromatase in an in vitro assay (Defarge et al., 2016).
The coformulants tested may have different CAS numbers as the formulations differ. Those
tested were (1) pure polyethoxylated tallow amine (POEA; POE-15, CAS No.: 61791-26-2, trade
name Emulson AG GPE 3SS) and formulated polyethoxylated tallow amine (POEA/F; CAS No.:
61791-26-2, trade name Emulson AG GPE 3/SSM) form containing 70% of POE-15; (2) alkyl
polyglucoside (APG; CAS No.: 383178-66-3/110615-47-9, trade name Plantapon LGC); (3) a mixture
of alkyl (C8–10) polyoxyethylene ether phosphates and polyoxyethylene alkyl ether phosphate (POE-
APE; CAS Nos.: 68130-47-2 and 50769-39-6, trade name Rolfen Bio); and (4) quaternary ammonium
compound (QAC, CAS No.: 66455-29-6, trade name Emulson AG CB 30; and (5) alkyl polyglycoside
(CAS No. 110615-49-9, trade name Plantapon LGC).
Aromatase activity was measured by tritiated water release in human JEG3 cells (for
discussion on this assay, see Section 2.6f, Table 50). Mitochondrial succinate dehydrogenase activity
and membrane integrity were assayed after a 24-hour exposure to assess cytotoxic effects.
The concentrations tested for succinate dehydrogenase activity were derived based on those
concentrations reported to be used in glyphosate formulations which can differ according to different
formulations: for example, POEA (9 ppm); POEA (18 ppm); APG (800 ppm); POE-APE (100 ppm);
and QAC (100 ppm). Statistically significant differences from the controls were determined by a
Kruskal–Wallis nonparametric test followed by a post hoc test using significant levels. Aromatase
assays were performed at 2.5 ppm of POEA, 120 ppm of APG. The authors report that aromatase
activity was decreased by the coformulant alone (POEA, −43%; P < 0.01) and slightly by the
formulation of the active ingredient plus the coformulant (−25%; P < 0.05). Formestane (4-
hydroxyandrost-4-ene-3,17-dione), a known aromatase inhibitor, was used as a positive control to
demonstrate the specificity of the effect.
1,4-Dioxane
1,4-Dioxane is used primarily as a solvent in the manufacture of chemicals and as a
laboratory reagent; it has been noted as being a trace contaminant of glyphosate formulations.

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1,4-Dioxane has been classified by the IARC as “possibly carcinogenic to humans (Group
2B)” (IARC, 1987) and, in the National Toxicology Program’s fourteenth edition report on
carcinogens, as “reasonably anticipated to be a human carcinogen” (NTP, 2016).
Studies in rodents show liver tumours to be consistently reported after chronic oral exposure
to 1,4-dioxane. A weight-of-evidence evaluation re-examined mouse liver slides from the 1978
National Cancer Institute bioassay of 1,4-dioxane in drinking water. This re-examination clearly
identified dose-related non-neoplastic changes in the liver; specifically, a dose-related increase in the
hypertrophic response of hepatocytes, followed by necrosis, inflammation and hyperplastic
hepatocellular foci. While 1,4-dioxane does not cause point mutations, DNA repair or initiation, it
appears to promote tumours and stimulate DNA synthesis. The weight of the evidence suggests that
1,4-dioxane causes liver tumours in rats and mice through cytotoxicity followed by regenerative
hyperplasia. A reference dose (RfD) of 0.05 mg/kg day was proposed to protect against regenerative
liver hyperplasia based on a benchmark dose approach (Dourson et al., 2014).
FD&C Blue No. 1

FD&C Blue No. 1 is a blue colourant used in glyphosate formulations. As literature on this
compound is sparse, it was run through predictive expert system software (Derek Nexus 5.0.1, Nexus
2.1.0, Lhasa Ltd., Leeds, United Kingdom) in February 2016. The parent compound indicated
plausible toxicity with respect to chromosome damage in vitro in mammals due to an alert match with
triarylmethane salt and irritation of the eye in mammals due to an alert matched with 4,4'-
methylenedianiline.
Availability of supplementary Toxcast/Tox 21 data

In addition to supplementary literature review, Toxcast and Tox21 data searches were
conducted on 29 April 2016 for glyphosate coformulants. The Tox21 toolbox
(http://ntp.niehs.nih.gov/results/tox21/tbox/index.html) was utilized to access the databases and
acquire the data. Data were obtained for two of the glyphosate coformulants: 1,4-dioxane and FD&C
Blue No 1. Other typical coformulants were not tested.
In a broad sweep of testing (including AhR, FXR, PPARs, VDR, MMP, p53, NFkB, GR), for
FD&C Blue No. 1, the AC50 (µmol/L) results were positive for estrogenic agonist (1 assay only:
1.00E-4) and antagonist activity (1 assay only: 4.79), AR antagonist activity (4.76), aromatase
inhibition (1.00E-4), TR antagonism (1.00E-4) and retinoic acid–receptor-related orphan receptor
antagonism (1.00E-4). For 1,4-dioxane, the AC50 (µmol/L) results were negative across all assays.
3.1 Occupational exposure: Biomonitoring studies
Occupational exposure to glyphosate can occur via dermal and inhalation routes. However, in
vitro and in vivo percutaneous absorption studies suggest that dermal penetration of glyphosate
formulation is very limited and that exposure through inhalation is minimal due to the low vapour
pressure of glyphosate.
Both passive dosimetry and biomonitoring have been used as techniques to assess exposure.
Biomonitoring results represent systemic (internal) exposure, whereas passive dosimetry results
quantify external deposition. There is general agreement that biological measurements obtained
through biomonitoring provide the most relevant information for safety assessments (Franklin, Muir
& Moody, 1986; Chester & Hart, 1986).
The Farm Family Exposure Study was a biomonitoring study supported by seven agricultural
companies. In this study, eligible farm families from Minnesota and South Carolina were randomly

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selected from a roster of licensed private pesticide applicators. Participant families consisted of a
farmer, their spouse and at least one child between the ages of 4 and 17 years; lived on the farm; and
planned to apply one of the target pesticides (glyphosate, chlorpyrifos, 2,4-D) to at least 10 acres (4.1
hectares) of land within 1 mile (1.6 kilometres) of their house. For each family member, geometric
means were calculated for 24-hour composite urinary samples, with a 1 ppb limit of detection, the day
before, the day of and for 3 days after the pesticide application. For the farmers, the peak geometric
mean concentrations were 3 ppb for glyphosate, 64 ppb for 2,4-D and 19 ppb for the primary
chlorpyrifos metabolite. For the spouses and children, the percentage with detectable values varied by
chemical, although the average values for each chemical did not vary during the study period. The
applicators had the highest urine pesticide concentrations, children had much lower values and
spouses had the lowest values. Exposure to family members was largely, though not exclusively,
determined by the degree of direct contact with the application process. The exposure profile varied
for the three chemicals for each family member (Mandel et al., 2005).
As part of the Farm Family Exposure Study, urinary glyphosate concentrations were
evaluated for 48 farmers, their spouses and their 79 children (4–18 years of age). The study authors
stated that they evaluated 24-hour composite urine samples for each family member the day before,
the day of and for 3 days after a glyphosate application. On the day of application, 60% of the farmers
had detectable levels of glyphosate in their urine on the day of application. The geometric mean
concentration was 3 ppb, the maximum value was 233 ppb, and the highest estimated systemic dose
was 0.004 mg/kg. Those farmers who did not use rubber gloves had higher geometric mean urinary
concentrations than the other farmers (10 ppb vs 2.0 ppb). For spouses, 4% had detectable levels in
their urine on the day of application; their maximum value was 3 ppb. For children, 12% had
detectable glyphosate in their urine on the day of application, with a maximum concentration of 29
ppb. All but one of the children with detectable concentrations had helped with the application or
were present during herbicide mixing, loading or application. None of the systemic doses estimated in
this study approached the USEPA reference dose for glyphosate of 2 mg/kg bw per day (Acquavella
et al., 2004).
Some earlier biomonitoring studies were performed on silvicultural workers who sprayed a
glyphosate formulation in a variety of forestry and tree farming activities. In one study, the United
States Department of Agriculture’s Forest Service, in collaboration with Monsanto and the University
of Arkansas, sponsored a study to investigate exposure to glyphosate of workers at two forestry
nurseries (Phipps Nursery in Oregon and Ashe Nursery in Massachusetts) where glyphosate was used
for weed control. Urine samples were collected from the weeders and scouts prior to working with
glyphosate and for an eight-month period thereafter. Continuous total urine sampling was conducted
for the first 12 consecutive weeks of the study, after which a 24-hour sample was collected each
Wednesday for the next five months.
Of the 355 daily urine samples analysed, none were found to contain quantifiable levels of
glyphosate. The limit of quantification was 10 ppb (Lavy et al., 1992).
A separate collaborative study conducted by the United States Department of Agriculture

(USDA) Forestry Service, Georgia Tech Research Institute and Monsanto examined the effects of
exposure to glyphosate on applicators using a hand-held directed spray foliar application at three sites
maintained by the USDA Forestry Service. Urinary samples were collected for 5 days after exposure.
Of the 96 urine samples analysed, five were found to contain quantifiable levels of glyphosate. The
highest glyphosate measure was 14 ppb and the highest estimated internal dose was 0.0006 mg/kg
body weight (Cowell & Steinmetz 1990).

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Two other studies have been conducted to measure exposure of forestry workers to
glyphosate during normal silvicultural applications. In the Finnish study (Jauhiainen et al., 1991),
urine samples were collected at the end of each day from workers spraying glyphosate for 5
consecutive days in August 1988. In addition, each worker had an ECG; underwent haematology,
clinical chemistry and pulmonary function tests and a general clinical examination (including blood
pressure, pulse rate and pressure craft of hands); and was interviewed for a health questionnaire on the
first day and last day. All urine samples had less than detectable concentrations of glyphosate. There
were no statistically significant differences in the findings of the medical examinations conducted
before and after exposure (Jauhiainen et al., 1991).
The Canadian study of forestry workers following normal silviculture uses of glyphosate was
conducted over two growing seasons (in 1986) and involved 45 workers conducting various
operations. Glyphosate was not detected in the majority of urine samples. For the two flagmen and the
operator, glyphosate concentrations in all urine samples were less than 0.03 ppm (the limit of
quantitation). In contrast, 14 of 33 urine samples from the mixer and two urine samples for the
foreman contained glyphosate concentrations greater than 0.03 ppm. Maximum glyphosate
concentrations in the foreman’s and mixer’s urine were 0.043 and 0.055 ppm, respectively. In the
follow-up study in 1987, glyphosate concentrations in urine of exposed workers were very low. In the
majority of samples, glyphosate was not detectable. In those samples with detectable levels of
glyphosate, concentrations were less than 0.1 ppm in all cases and typically less than 0.035 ppm
(Centre de Toxicologie du Quebec, 1988).
3.2 Occupational exposure: Epidemiological studies with specific reference to cancer outcomes
epidemiological studies on malathion (and diazinon and glyphosate) are described in “Section 2.2:
Methods for the evaluation of epidemiological evidence for risk assessment” of the Meeting report9.
Identification of compound/cancer sites and screening of papers

This assessment was limited to studies of cancer outcomes; numerous studies have assessed
risks for neurodevelopmental, neurodegenerative and reproductive outcomes, among other health
outcomes. Restricting the assessment to cancer outcomes was partly driven by reasons of feasibility: a
clinically relevant adverse effect size (or an acceptable level of risk) for a non-cancer outcome must
be defined, and the methodologies for hazard identification and characterization based on
observational epidemiological findings of non-carcinogenic adverse effects are less well-established
than those for cancer (Clewell & Crump, 2005; Nachman et al., 2011).
epidemiological studies on glyphosate (and malathion and diazinon) are described in “Section 2.2:
Methods for the evaluation of epidemiological evidence for risk assessment” of the Meeting report10.
The IARC monographs on glyphosate, malathion and diazinon refer to a total of 45
epidemiological studies. Two studies published since the IARC monographs, which evaluated at least
one of malathion, diazinon or glyphosate in relation to cancer outcomes, were also identified (Lerro et
al., 2015; Koutros et al., 2015).
9
Pesticide residues in food 2016: Special session of the joint FAO/WHO meeting on pesticide residues
May 2016: Report 2016 (http://www.who.int/foodsafety/areas_work/chemical-risks/jmpr/en/).
10
Pesticide Residues in Food 2016: Special session of the joint FAO/WHO meeting on pesticide
residues May 2016: Report 2016 (http://www.who.int/foodsafety/areas_work/chemical-risks/jmpr/en/).

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The 45 publications referred to in the IARC monographs and the two publications since
(Lerro et al., 2015; Koutros et al., 2015) covered a total of 48 compound/cancer site combinations.
The current evaluation focuses on the six compound/cancer site combinations for which IARC
identified positive associations from the body of epidemiological evidence, that is, those associations
noted in Section 6.1 of the monographs, and which underpin the IARC’s evaluation of “limited
evidence” in humans for the carcinogenicity of malathion, diazinon and glyphosate. The definition for
limited evidence of carcinogenicity used by the IARC is as follows: “A positive association has been
observed between exposure to the agent and cancer for which a causal interpretation is considered by
the Working Group to be credible, but chance, bias or confounding could not be ruled out with
reasonable confidence” (IARC, 2015).
The compound/cancer site combination for glyphosate was NHL. The evaluation of the
relevant publications is summarized in Table 52.
During the identification of relevant publications, stand-alone analyses for specific subtypes
of NHL (of which there are many subtypes) were noted. The risk was not evaluated separately for
subtypes of NHL as there was insufficient evidence (too few studies or small numbers of cases), or for
other haematopoietic and lymphoid tumours as the positive associations identified by the IARC were
for total NHL.
Overview of studies included in evaluation

The IARC monograph on malathion (IARC, 2015) already provides a good overview of the
epidemiological studies which have assessed pesticide exposures and cancer risk. Therefore, only a
brief summary (largely based on the IARC monograph) of the studies contributing to the current
evaluation is provided here to give context.
The Agricultural Health Study (AHS) is a prospective cohort study of pesticide applicators
(predominantly farmers; n ≈ 52 000) and their spouses (n ≈ 32 000) from Iowa and North Carolina,
United States of America, enrolled in 1993–1997. The AHS has examined a range of cancer
outcomes, and published updated analyses with longer periods of follow-up (e.g. Beane Freeman et
al., 2005; De Roos et al., 2005; Koutros et al., 2013; Alavanja et al., 2014; Jones et al., 2015; Lerro et
al., 2015). Information on participants’ use of 50 pesticides and other determinants of exposure was
collected retrospectively via baseline and two follow-up questionnaires. Cumulative lifetime exposure
estimates were calculated. Validation studies have been conducted to assess the reliability and
accuracy of exposure intensity scores (a component of the exposure assessment) (Coble et al., 2005;
Hines et al., 2008; Thomas et al., 2010). The impact of exposure misclassification in this study was to
bias risk estimates towards null (Blair et al., 2011).
The United States Midwest case–control studies are three population-based case–control
studies of cancer conducted in Nebraska (Zahm et al., 1990), Iowa and Minnesota (Brown et al., 1990;
Cantor et al., 1992), Kansas (Hoar et al., 1986), which have subsequently been pooled (748
cases/2236 controls) for analysis of NHL in white males only (Waddell et al., 2001; De Roos et al.,
2003; Lee et al., 2004). Information on participants’ occupational use of pesticides was collected
retrospectively via questionnaire. There were some differences in case ascertainment and exposure
assessment methods between the three studies. For 39% of the pooled study population, proxy
respondents were used (Waddell et al., 2001), for whom recall of specific pesticide use could be
problematic and subject to recall bias which may differ for cases and controls. De Roos et al. (2003)
(same study population as Waddell et al., 2001) performed an extensive evaluation and adjustment for
other pesticides.
The Cross-Canada Case–control Study of Pesticides and Health is a population-based case–
control study of haematopoietic cancers in men diagnosed during 1991–1994 across six Canadian
provinces (McDuffie et al., 2001). It includes 517 NHL cases and 1506 controls. A questionnaire was
administered by post, followed by a telephone interview for those who reported pesticide exposure of
10 hours/year or more, and for a 15% random sample of the remainder. The study was not restricted
to pesticide exposure experienced by a specific occupational group (McDuffie et al., 2001). Further

248
analyses stratified by asthma/allergy status – to assess possible effect modification by immune system
modulation – have been conducted (Pahwa et al., 2012). The study has a large sample size and
detailed information of pesticide exposures; however, the proportion exposed to pesticides was low.
These three sets of studies were deemed as high quality and highly informative by the IARC
Working Group (IARC, 2015).
A number of other case–control studies of pesticide exposure and cancer risk were included in
this evaluation: the Florida Pest Control Worker study (Pesatori et al., 1994); nested case–control
studies within the United Farm Workers of America cohort study (Mills, Yang & Riordan, 2005); a
population-based case–control study of prostate cancer in British Columbia, Canada (Band et al.,
2011); and case–control studies of NHL/haematopoietic cancers from Sweden (Hardell et al., 2002,
Eriksson et al., 2008), and France (Orsi et al., 2009). The IARC Working Group (IARC, 2015) noted
substantial limitations in these studies, either in relation to exposure assessment, scope for and
variation in exposure misclassification, lack of detail reported in publication which hindered
interpretation, lack of specificity due to high correlations between use of different pesticides, and
limited power.
Strengths and limitations of studies included in evaluation

The included studies predominantly examined the occupational pesticide exposures of
farmers and other pesticide applicators, with the vast majority of research being on males only. None
of the studies assessed exposure via food consumption or ambient exposure from agriculture (e.g.
spray drift). The scientific evidence available is therefore limited in its generalizability and the extent
to which it can be translated to general population exposure scenarios and levels that would be
associated with pesticide residues. Nonetheless, these observational epidemiological studies provide
insight into real-world exposure scenarios, and allow for observation of the species of interest
(humans) over long follow-up time periods relevant to cancer.
The number of high quality studies is relatively small. Typically the number of exposed cases
in studies is small, particularly when evaluating specific pesticides, which limits study power.
Relatively few studies have assessed exposure quantitatively, meaning the epidemiological
evidence available to inform/establish dose–response relationships is very limited. Exposure
misclassification is a potential issue for all studies. This is expected to be largely non-differential for
cohort studies (i.e. the AHS), resulting in attenuation of risk estimates. All except one of the studies
included are case–control studies, and these may be affected by recall bias, that is, cases and controls
recall past pesticide exposure with differing accuracy, leading to differential exposure
misclassification which can bias risk estimates either towards or away from the null. As a cohort
study, the AHS avoids recall bias.
Given that studies focused on occupational exposures among farmers/pesticide applicators, it
is unlikely that they were exposed to only one specific pesticide. As a result, confounding, possible
effect modification and additive/multiplicative effects due to co-exposures are all concerns. However,
many studies were able to adjust risk estimates for other pesticide co-exposures, which yields more
accurate risk estimates.
There are some issues in terms of comparing studies and evaluating the consistency of
evidence overall. Results of studies may appear heterogeneous, but usually there are too few studies to
really assess consistency and heterogeneity. Exposure assessment methods and referent groups vary
between studies.
Finally, changes in disease classifications (particularly NHL) or screening/diagnosis rates
(prostate cancer) over time may limit comparability between studies.

249
Publication bias
A formal analysis of publication bias was not undertaken because the number of studies (risk
estimates from non-overlapping study populations) available were few and funnel plot tests for
asymmetry should be used only where there are at least 10 studies because otherwise statistical power
is insufficient to distinguish true asymmetry from chance (Higgins & Green, 2011; Sterne et al.,
2011). Other formal objective statistical tests require an even larger number of studies, typically at
least 30, to achieve sufficient statistical power (Lau et al., 2006). As a result, publication bias cannot
be fully excluded. However, given the very considerable resources invested in these types of (large,
difficult exposure assessment) studies, it is unlikely that results would go unpublished.
Summary of evidence for an association between glyphosate and NHL

This evaluation considered several aspects of each study and of all the studies combined,
including factors which decrease the level of confidence in the body of evidence, including risk of
bias, unexplained inconsistency, and imprecision, and factors which increase the level of confidence,
including large magnitude of effect, a dose–response relationship, residual confounding and
consistency (Guyatt et al., 2008; Morgan et al., 2016).
The risk estimates findings for each study are summarized in Table 52, and findings for non-
quantitative exposure assessment (predominantly ever- vs never-use) are shown in the forest plot
below.
Table 52. Results of Tier 1 evaluation and summary of publications by glyphosate/cancer site
Study/ Location Glyphosate / NHL Reference
Meta-analysis Qualitative exposure only – ever-/never-use of glyphosate Schinasi & Leon
Meta risk ratio: 1.5 (95% CI: 1.1–2.0) (2014)
Meta-analysis includes McDuffie et al. (2001); Hardell et al. (2002); De Roos et
al. (2003, 2005a); Eriksson et al. (2008); and Orsi et al. (2009). Ns for each
meta-analysis not presented
Agricultural Quantitative exposure (cumulative exposure days; intensity-weighted De Roos et al. (2005)
Health Study cumulative exposure days [years of use  days/year  estimated intensity level]:
in tertiles)
Ever-use 1.1 (0.7–1.9)
LED 1–20.0 1.0 (ref.)
LED 21–56 0.7 (0.4–1.4)
LED 57–2678 0.9 (0.5–1.6)
P for trend 0.73
IW-LED 0.1–79.5 1.0 (ref.)
IW-LED 79.6–337.1 0.6 (0.3–1.1)
IW-LED 337.2–18241 0.8 (0.5–1.4)
P for trend = 0.99
Total N = 54 315 (49 211/36 823, depending on the analysis), with 92 incident
NHL cases (for ever-use; and 61 for analysis based on tertiles of exposure)
United States The study population overlaps with that of De Roos et al. (2003). See comment Lee et al. (2004)
Midwest case– below
control studies Qualitative – ever/never (analysis stratified by asthmatics vs non asthmatics)
Non-asthmatics: 1.4 (0.98–2.1)
Asthmatics: 1.2 (0.4–3.3)
Total N = 3208 (872 NHL cases, 2336 controls).

N = 53/91 glyphosate-exposed NHL cases/controls for non-asthmatics and 6/12
glyphosate-exposed NHL cases/controls for asthmatics

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The study population overlaps with Lee et al. (2004) and total N is smaller, but De Roos et al. (2003)
as an exception this study was not excluded in the assessment of consistency of
risk estimates as it provides overall risk estimates which are comparable with
other studies, while Lee et al. (2004) only provides risk estimates stratified by
asthma diagnosis
Risk estimates – aOR (95% CI)
From a logistic regression model: Exposed 2.1 (1.1–4.0)
From the hierarchical regression model: Exposed 1.6 (0.9–2.8)
Both adjusted for other pesticides
Total N =2 583 (650 NHL cases, 1 933 controls).

N = 36 exposed cases; N = 61 controls
Excluded – as this study is pooled in De Roos et al. (2003) and Lee et al. (2004) Cantor et al. (1992)
Qualitative exposure only – ever-/never-use of glyphosate
Risk estimates – OR (95% CI)

Ever-use = 1.1 ( 0.7–1.9)
Total N = 1867 (622 cases, 1245 controls)

Cross-Canada Quantitative exposure – days of use per year (3 categories – cutpoints are McDuffie et al. (2001)
Study of given).
Pesticides and
Health Risk estimates – OR (95% CI)
Ever-use: 1.2 (0.83–1.74)
Unexposed 1.0 (ref.)

>0–<=2 days/year 1.0 (0.63–1.57)
> 2 days/year 2.12 (1.20–3.73)
P trend = NR
Total N = 2 023
517 cases, 1 506 controls (overall)
N = 51 exposed cases, 133 exposed controls
Sweden – note Quantitative exposure – days of use per year (2 categories – cutpoints are Eriksson et al. (2008)
that there is some given).
overlap between
Eriksson et al. Risk estimates – aOR (95% CI)
(2008), Hardell et Ever-use: 2.02 (1.10–3.71)
al. (2002) and
Hardell & Risk estimates – aOR (95% CI)
Eriksson (1999) Non-farmers: 1.0 (ref.)
 10 days/year: 1.69 (0.7–4.07)
> 10 days/year: 2.36 (1.04–5.37)
P trend = NR

N = 29 exposed cases; N = 18 exposed controls
Qualitative exposure only – ever-/never-use of glyphosate. A pooled analysis of Hardell et al. (2002)
Nordström et al. (1998) (NHL subtype only, not evaluated separately here) and
Hardell & Eriksson (1999)

Ever-use: 1.85 (0.55–6.20)
Total N = 1 656 (515 cases, 1 141 controls)

N = 8 exposed cases; N = 8 exposed controls.
Exclude as this study is pooled in Hardell et al. (2002). Qualitative exposure Hardell & Eriksson
only – ever-/never-use of glyphosate (1999)

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France Qualitative – ever-/never-use of glyphosate Orsi et al. (2009)

Ever-use: 1.0 (0.5–2.2)
N = 12 exposed cases; N = 24 exposed controls
(The researchers report evaluating quantitative duration with respect to median

duration of exposure among exposed controls as never exposed; duration
< median; duration > median, but neither the median cutpoint nor ORs/test for
trend results are presented in the paper, so this study cannot contribute any
information for quantitative risk assessment.)
aOR: adjusted odds ratio; aRR: adjusted risk ratio; CI: confidence interval; IW-LED: intensity-weighted lifetime exposure
days, defined as number of years of use  number of days used per year  personal protective equipment use reduction factor
 intensity level score (a unit-less score which reflects a combination of self-reported pesticide exposure modifiers, e.g.
pesticide mixing status, application method, equipment repair activities); LED: lifetime exposure days, defined as number of
years of use  number of days used per year; NHL: non-Hodgkin lymphoma; N: sample size; NR: not reported; OR: odds
ratio; ref.: reference
The maximally adjusted risk estimates were extracted.
The Glyphosate / NHL evaluation included seven studies (McDuffie et al., 2001; Hardell et
al., 2002; De Roos et al., 2003; Lee et al., 2004; De Roos et al., 2005; Eriksson et al., 2008; Orsi et al.,
2009) and one meta-analysis (Schinasi & Leon, 2014). Three studies used quantitative exposure
metrics, although, the units differed: lifetime exposure days and intensity-weighted lifetime exposure
days (De Roos et al., 2005) and days of use per year (McDuffie et al., 2001; Eriksson et al., 2008).
The AHS found no evidence of elevated risk of NHL or exposure–response associated with
glyphosate exposure (De Roos et al., 2005). Elevated risks were reported in various case–control
studies. De Roos et al. (2003) reported significant elevated risk of NHL associated with ever- versus
never-use of glyphosate (OR: 2.1 [1.1–4.0] and a borderline nonsignificant OR (1.6 [0.9–2.8]) with an
alternative Bayesian hierarchical model) from the United States Midwest pooled case–control studies.
There was no evidence of effect modification by asthma diagnosis in the United States Midwest
pooled case–control studies (Lee et al., 2004). Ever-use of glyphosate was not associated with risk of
NHL in the Cross-Canada Case–control Study of Pesticides and Health, but using glyphosate for
longer than 2 days per year was associated with a significant elevated risk (OR: 2.12; 95% CI: 1.20–
3.73), although there was no indication of an exposure–response relationship across exposure
categories (McDuffie et al., 2001). Eriksson et al. (2008) reported significant elevated risk of NHL
associated with ever-use (OR: 2.02 [1.10–3.71]) and use of glyphosate for longer than 10 days per
year (OR: 2.36 [1.04–5.37]) and indicate an exposure–response relationship. A pooled study of two
Swedish case–control studies reported a nonsignificant elevated risk of NHL for ever-use of (OR:
1.85 [0.55–6.2]); however, with only eight exposed cases, this study had limited power to detect
associations (Hardell et al., 2002). Orsi et al. (2009) found no evidence of association. Schinasi &
Leon (2014) reported a meta risk ratio of 1.5 (95% CI: 1.1–2.0) for ever- versus never-use of
glyphosate. The meta-analysis included the AHS (De Roos et al., 2005) and five out of the six case–
control studies included in this evaluation (McDuffie et al., 2001; Hardell et al., 2002; De Roos et al.,
2003; Eriksson et al., 2008; Orsi et al., 2009).

252
Fig. 3. Forest plot for risk estimates of NHL associated with glyphosate, from studies with
qualitative exposure categories
Overall, there is some evidence of a positive association between glyphosate exposure and risk of
NHL from the case–control studies and the overall meta-analysis. However, it is notable that the
AHS, which is the only cohort study and is large and of high quality, found no evidence of association
at any exposure level.
Comments
Biochemical aspects
In studies with radiolabelled glyphosate in rats, glyphosate was rapidly absorbed from the
gastrointestinal tract following oral intake, but only to a limited extent (about 20–30%) (McEwen,
1995). Elimination was fast and virtually complete within 72–168 hours, with the majority being
excreted during the first 48 hours (McEwen, 1995). Most of the excretion occurred in faeces, largely
as unabsorbed dose, and in the urine. Biliary excretion of glyphosate was negligible. Less than 1% of
the administered dose was retained in tissues 168 hours post-administration. Highest residues were
detected in bone, followed by kidney and liver (Powles, 1992b; Ridley & Mirly, 1988). This pattern
of absorption, distribution and elimination was independent of dose, treatment regimen and sex of the
test animals. Peak plasma concentrations of radiolabel were observed at 6 and 2 hours after
administration in male and female rats, respectively (McEwen, 1995). The estimated half-life for
whole-body elimination of the radiolabel was about 5.9–8.3 hours (McEwen, 1995).
There was very little biotransformation of glyphosate; the only metabolite, AMPA, accounted
for 0.2–0.7% of the administered dose in excreta; the rest was unchanged glyphosate (Macpherson,
1996).
Toxicological data
Glyphosate has low acute oral toxicity in mice (LD50 > 2000 to > 10 000 mg/kg bw; no
lethality at 2000 mg/kg bw) (Shirasu & Takahashi, 1975)and rats (LD 50 5600 mg/kg bw) (Heenehan,
1979a), low acute dermal toxicity in rats (LD50 > 2000 mg/kg bw) (Cuthbert & Jackson, 1989b;
Komura, 1995c; Doyle, 1996b; Talvioja, 2007b; Do Amaral Guimaraes, 2008b; Haferkorn, 2009b,
2010c,d; Simon, 2009b) and rabbits (LD50 > 5000 mg/kg bw) (Heenehan, 1979b; Blaszcak, 1988b;

253
Reagan, 1988a), and low acute inhalation toxicity in rats (LC50 > 5.48 mg/L). Glyphosate was not
irritating to the skin of rabbits (Heenehan, 1979c; Reagan & Laveglia, 1988b; Hideo, 1995a; Doyle,
1996c; Arcelin, 2007c; Talvioja, 2007c; Canabrava Frossard de Faria, 2008a; You, 2009c; Leuschner,
2009a,c, 2010a). Glyphosate produced moderate to severe eye irritation in rabbits, with irreversible
corneal opacity in one study as a consequence of the low pH of the test material in solution (Arcelin,
2007d; Blaszcak, 1988d; Hideo, 1995b; Johnson, 1997; Merkel, 2005e; Talvioja, 2007d; You, 2009d).
Glyphosate was not sensitizing in guinea pigs (Doyle, 1996d; Haferkorn, 2009d, 2010f,g; Hideo,
1995c; Lima Dallago, 2008; Merkel, 2005f; Richeux, 2006; Talvioja, 2007e; Simon, 2009d; You,
2009e) or mice (Betts, 2007; Török-Bathó, 2011) as determined by the Magnusson–Kligman
maximization test, the Buehler test and the local lymph node assay.
In short-term studies of toxicity in different species, the most notable effects were clinical
signs related to gastrointestinal irritation, decreased body weight, salivary gland changes (hypertrophy
and increase in basophilia of cytoplasm of acinar cells), histological findings in the caecum and
hepatotoxicity.
In short-term studies in mice, reduced body weight was seen at a dietary concentration of
50 000 ppm (equal to 9710 mg/kg bw per day) (Tierney & Rinehart, 1979). The NOAEL for
decreased body weight was 10 000 ppm (equal to 1221 mg/kg bw per day) (Kuwahara, 1995). Effects
on the salivary glands were observed in mice in only one study out of four, at 6250 ppm (equal to
1065 mg/kg bw per day) (Chan & Mahler, 1992). The NOAEL for the salivary gland effects in mice
was 3125 ppm (equal to 507 mg/kg bw per day) (Chan & Mahler, 1992). The overall NOAEL in
short-term studies in mice was 3125 ppm (equal to 507 mg/kg bw per day), and the overall LOAEL
was 6250 ppm (equal to 1065 mg/kg bw per day).
In 90-day toxicity studies in rats, common findings included soft faeces, diarrhoea, reduced
body-weight gain and decreased food utilization at dietary concentrations of 20 000 ppm (equal to
1262.1 mg/kg bw per day) and above. The lowest NOAEL was 371.9 mg/kg bw per day. A decrease
in urine pH was frequently noted owing to the acidic nature of the compound and excretion as
glyphosate in the urine. In two 90-day dietary toxicity studies, an increase in caecum weight (at
10 000 ppm, equal to 569 mg/kg bw per day) and histological findings in the caecum (at 50 000 ppm,
equal to 3706 mg/kg bw per day) (Kinoshita, 1995; Coles et al., 1996) were observed. In rats, effects
on the salivary gland were seen in two out of seven 90-day studies starting at 12 500 ppm (equal to
811 mg/kg bw per day). The NOAELs for effects on the salivary gland were 300 and 410 mg/kg bw
per day. The overall NOAEL in short-term studies in rats was 300 mg/kg bw per day, and the overall
LOAEL was 10 000 ppm (equal to 569 mg/kg bw per day).
In four 90-day toxicity studies in dogs, the most notable effects were loose stools, decreased
body weight and reduced feed consumption (Hodge, 1996; Yoshida, 1996; Prakash, 1999; Gaou,
2007). In one study, there were no treatment-related effects at doses up to 40 000 ppm (equal to 1015
mg/kg bw per day) (Yoshida, 1996). The lowest NOAEL and LOAEL were 300 mg/kg bw per day
and 1000 mg/kg bw per day, respectively.
Seven 1-year toxicity studies in dogs are available. In one study, changes in faeces were
observed at 100 mg/kg bw per day and above. The NOAEL was 30 mg/kg bw per day (Teramoto,
1998). However, these results were not reproduced in four other studies with administration via
capsules at 300 or 500 mg/kg bw per day (Reyna & Ruecker, 1985; Goburdhun, 1991; Haag, 2008).
In the remaining six studies, the NOAELs ranged from 8000 ppm (equal to 182 mg/kg bw per day;
Nakashima, 1997) to 500 mg/kg bw per day (Reyna, 1985; Haag, 2008), and the LOAELs ranged
from 30 000 ppm (equal to 926 mg/kg bw per day; Brammer, 1996) to 1000 mg/kg bw per day
(Goburdhun, 1991).
The overall NOAEL in the 90-day and 1-year toxicity studies in dogs was 15 000 ppm (equal
to 448 mg/kg bw per day), and the overall LOAEL was 30 000 ppm (equal to 926 mg/kg bw per day).
The Meeting compiled the tumour incidence data for all relevant mouse and rat studies in
order to undertake statistical analysis and investigate any potential pattern of occurrence across
studies. In addition, incidences of tumours of lymphatic tissues were summarized, as these were

254
identified as possible targets of relevance from the review of epidemiological cancer studies.
However, the Meeting recognized that the relationship between tumours of lymphatic tissues in
rodents and humans has not been clearly established.
Nine carcinogenicity studies in mice were available. Two studies were considered to be of
insufficient quality to be included in the assessment (Bhide, 1988; Vereczkey & Csanyi, 1982, revised
1992). Effects such as loose stools, reduced body weights and decreased feed consumption were noted
in most of the studies (Pavkov & Turnier, 1987; Atkinson et al., 1993a; Sugimoto, 1997; Takahashi,
1999a). The overall NOAEL for systemic toxicity in mice was 1600 ppm (equal to 153 mg/kg bw per
day), and the overall LOAEL was 8000 ppm (equal to 787 mg/kg bw per day).
The Meeting concluded that there is equivocal evidence of induction of lymphomas in male
mice in three out of seven studies (Sugimoto, 1997; Kumar, 2001; Wood et al., 2009a) and in female
mice in one out of seven studies (Takahashi, 1999a) at high doses (5000–40 000 ppm, equal to 814–
4348 mg/kg bw per day). The Meeting also noted that in the other three studies in which even higher
doses (up to 50 000 ppm, equal to 7470 mg/kg bw per day) had been used, no effect was observed.
The Meeting concluded that there is some indication, by a trend test and not by pairwise
comparison, of induction of kidney adenomas in male mice in four out of seven studies (Knezevich &
Hogan, 1983; Sugimoto, 1997; Takahashi, 1999a; Kumar, 2001). The Meeting noted that the
increases were marginal and occurred at the highest dose only and that other studies that used
appreciably higher doses did not find any excess. However, the Meeting noted that kidney adenomas
are uncommon in male mice.
Eleven combined chronic toxicity and carcinogenicity studies in rats were available (Lankas,
1981; Pavkov & Wyand, 1987; Strout & Ruecker, 1990; Atkinson et al., 1993b; Milburn, 1996;
Suresh, 1996; Bhide, 1997; Enomoto, 1997; Takahasi, 1999a,b; Brammer, 2001; Wood et al., 2009b).
One study was considered to be inadequate for carcinogenicity assessment due to its exposure
duration (12 months). Toxicities variously reported in some of these studies included increased
incidences of clinical signs, reduced body weights, degenerative lens changes (cataracts) in males,
microscopic findings in the salivary gland, increased incidence of basophilia of parotid acinar cells,
and microscopic findings in liver, prostate and kidneys. The overall NOAEL for systemic toxicity in
rats was 100 mg/kg bw per day, and the overall LOAEL was 300 mg/kg bw per day.
The Meeting discussed the increased incidence of a variety of tumours observed in one or, in
one case, two of the 10 studies in rats. The Meeting concluded that these findings were incidental,
based on the following considerations:
 interstitial cell tumours of the testes: occurred in only one study (Lankas, 1981); and other studies
that used appreciably higher doses did not find any excess;
 pancreatic islet cell adenoma: occurred in only one study in males only (Strout & Ruecker, 1990);
other studies that used appreciably higher doses did not find any excess; there was no dose–
response relationship; and the incidence in controls was unusually low (less than the lower bound
of the historical control data); the Meeting also noted that there was a negative dose–response
relationship in females;
 thyroid C-cell tumours: occurred in only one study (Strout & Ruecker, 1990); other studies that
used appreciably higher doses did not find any excess; and these tumours are considered not to be
relevant for humans;
 skin keratoma: occurred in two studies in males only; other studies that used appreciably higher
doses did not find any excess; in one study (Strout & Ruecker, 1990), there was no dose–response
relationship; and in the other study, only the test for trend was statistically significant, not the
pairwise test at any dose (Enomoto, 1997); and
 lymphoma (in spleen and kidney): no evidence of induction in any of the studies.
The Meeting concluded that there is no reliable evidence for treatment-related tumours in rats
at doses up to 32 000 ppm (equal to 1750 mg/kg bw per day).

255
The Meeting concluded that glyphosate is not carcinogenic in rats but could not exclude the
possibility that it is carcinogenic in mice at very high doses.
Glyphosate and its formulation products have been extensively tested for genotoxic effects
using a variety of tests in a wide range of organisms. While no mutational effects have been detected
in bacterial test systems, DNA damage and chromosomal effects have commonly been seen in cell
culture models and in organisms that are phylogenetically distant from humans. However, these
effects have not been seen in vivo in orally treated mammalian models. The overall weight of
evidence indicates that administration of glyphosate and its formulation products at doses as high as
2000 mg/kg bw by the oral route, the route most relevant to human dietary exposure, was not
associated with genotoxic effects in an overwhelming majority of studies conducted in mammals, a
model considered to be appropriate for assessing genotoxic risks to humans.
The Meeting concluded that glyphosate is unlikely to be genotoxic at anticipated dietary
exposures.
Seven reproductive toxicity studies in rats were available. No evidence of reproductive
toxicity was observed at doses up to 30 000 ppm (equal to 1983 mg/kg bw per day). In one study, an
increased incidence of histopathological findings in the parotid (both sexes) and submaxillary salivary
glands in females was observed in both generations at 10 000 ppm (equal to 668 mg/kg bw per day).
The NOAEL was 3000 ppm (equal to 197 mg/kg bw per day) (Brooker et al., 1992). In a separate
study, an increased incidence of loose stools and caecum distension was observed in both generations
at 30 000 ppm (equal to 2150 mg/kg bw per day), and the NOAEL was 6000 ppm (equal to 417
mg/kg bw per day) (Takahashi, 1997). Slight reductions in pup weight or weight gain were observed
in most studies, but were confined to very high, parentally toxic dose levels (Moxon, 2000;
Takahashi, 1997). In addition, a significant delay in sexual maturation in male pups (F 1) was seen at
15 000 ppm (equal to 1063 mg/kg bw per day) (Dhinsa, 2007). The overall NOAEL for parental
toxicity was 6000 ppm (equal to 417 mg/kg bw per day), and the overall LOAEL was 10 000 ppm
(equal to 668 mg/kg bw per day). The overall NOAEL for offspring toxicity was 6000 ppm (equal to
417 mg/kg bw per day), and the overall LOAEL was 10 000 ppm (equal to 985 mg/kg bw per day).
No evidence of teratogenicity was observed in four developmental toxicity studies in rats at
doses up to 3500 mg/kg bw per day. There was some variation in the extent of toxicity observed in the
four studies. The lowest NOAEL for maternal toxicity was 300 mg/kg bw per day, based on loose
stools and reduced body weights seen at 1000 mg/kg bw per day (Hatakenaka, 1995). The lowest
NOAEL for embryo and fetal toxicity was 300 mg/kg bw per day, based on delayed ossification and
an increased incidence of fetuses with skeletal anomalies observed at 1000 mg/kg bw per day.
Seven developmental toxicity studies in the rabbit were available. Maternal toxicity was
primarily manifested as an increased incidence of soft stool and diarrhoea at doses of 175 mg/kg bw
per day and above. The overall NOAEL for maternal toxicity was 100 mg/kg bw per day. In three
studies, the occurrences of a variety of low-incidence fetal effects (e.g. cardiac malformation, absent
kidney) were slightly increased at higher dose levels (Bhide & Patil, 1989; Brooker et al., 1991b;
Suresh, 1993c). These increases are considered secondary to maternal toxicity. The overall NOAEL
for embryo and fetal toxicity was 250 mg/kg bw per day (Bhide & Patil, 1989), based on effects at
450 mg/kg bw per day. The Meeting considered that these effects were secondary to local irritation
from unabsorbed glyphosate in the colon administered by gavage dosing and concluded that they were
not relevant for establishing health-based guidance values.
The Meeting concluded that glyphosate is not teratogenic.
Glyphosate was tested in a range of validated in vivo and in vitro assays for its potential to
interact with the endocrine system. The studies that the Meeting considered adequate for the
evaluation clearly demonstrate that there is no interaction with estrogen or androgen-receptor
pathways or thyroid pathways.
There was no evidence of neurotoxicity in an acute neurotoxicity study in rats at doses up to
2000 mg/kg bw. The NOAEL for systemic toxicity was 1000 mg/kg bw, based on a single death and
general signs of toxicity at 2000 mg/kg bw (Horner, 1996a). In a 90-day neurotoxicity study in rats,

256
no evidence of neurotoxicity or systemic toxicity was seen at doses up to 20 000 ppm (equal to 1546.5
mg/kg bw per day) (Horner, 1996b).
No evidence of immunotoxicity was seen in a 28-day dietary study in female mice at doses up
to 5000 ppm (equal to 1448 mg/kg bw per day) (Haas, 2012).
Effects on the salivary glands were observed in several repeated-dose toxicity studies in rats.
The pH of glyphosate in solution is low, and it has been shown that exposure to organic acids can
cause such changes in salivary glands. Therefore, the changes are likely secondary to the effects
caused by the pH of the test compound in solution.
In many of the long-term repeated-dose studies reviewed, glyphosate was reported to have an
impact on the gastrointestinal tract at high doses. Although this is not uncommon with high-dose
chemical substance administration, this was investigated further, as glyphosate is known to be poorly
absorbed in mammalian models, and alterations in gut microbiota profiles, specifically reductions in
the beneficial microbiota and increases in pathogenic bacteria, are known to have impacts on
carcinogenesis. There is evidence from livestock species that pathogenic bacteria are more resistant to
glyphosate, whereas beneficial microbiota are more sensitive, and thus more vulnerable.
This is an emerging area of scientific investigation. The extent to which glyphosate adversely
affects the normal functioning of the microbiota in the human gastrointestinal tract or the
gastrointestinal tract of mammalian models is unclear. However, it is unlikely, given the available
information on MIC values, that this would occur from glyphosate residues in the diet.
Toxicological data on metabolites and/or degradates

AMPA is the only identified metabolite found in the urine and faeces of orally treated rats.
AMPA was of low acute oral and dermal toxicity in rats (LD50 > 5000 [Leah, 1988; Cuthbert &
Jackson, 1993a] and > 2000 mg/kg bw [Leuschner, 2002a], respectively) and was not sensitizing in
guinea pigs, as determined by the Magnusson–Kligman maximization test. In a 90-day study of
toxicity in rats, the NOAEL was 1000 mg/kg bw per day, the highest dose tested (Strutt et al., 1993).
AMPA administered orally in mammalian test systems showed no evidence of genotoxicity (Leah,
1988; Cuthbert & Jackson, 1993a; Komura, 1996). Only negative results were seen in studies in vitro
(Callander, 1988b; Jensen, 1993a; Akanuma, 1996). The Meeting concluded that AMPA is unlikely to
be genotoxic in vivo by the oral route.
In a study of developmental toxicity in rats, no evidence for embryo or fetal toxicity was
observed; the NOAEL for maternal and embryo/fetal toxicity was 1000 mg/kg bw per day, the highest
dose tested.
Following single gavage administration of radiolabelled N-acetyl-glyphosate, a plant-specific
metabolite, at 15 mg/kg bw in rats, about 66.1% of the administered dose was excreted in urine
(61.3% within 12 hours post dosing), 26.4% in faeces (25.8% within 48 hours post dosing), 2.79% in
cage wash and wipe, and 0.23% in residual carcass. Radioactivity was eliminated rapidly from blood
and plasma, with half-life values of 20.1 and 15.6 hours, respectively. Unchanged [14C]N-acetyl-
glyphosate recovered in urine and faeces represented over 99% of the administered radioactivity.
Glyphosate, a metabolite of N-acetyl-glyphosate, was detected in faeces and represented less than 1%
of the total radioactivity (Cheng & Howard, 2004).
The acute oral toxicity LD50 of N-acetyl-glyphosate in rats is greater than 5000 mg/kg bw,
expressed as the free acid (Vegarra, 2004). In a 90-day toxicity study in rats, the NOAEL was 18 000
ppm (equal to 1157 mg/kg bw per day) (MacKenzie, 2007).
N-Acetyl-glyphosate was tested for genotoxicity in vitro and in vivo in an adequate range of
assays; it was not found to be genotoxic in mammalian or microbial test systems.
The Meeting concluded that N-acetyl-glyphosate is unlikely to be genotoxic.
N-Acetyl-AMPA, another plant-specific metabolite, was of low acute oral toxicity; the LD50
was greater than 5000 mg/kg bw in rats (Carpenter, 2007).

257
N-Acetyl-AMPA was tested for genotoxicity in vitro and in vivo in an adequate range of
assays; it was not found to be genotoxic in mammalian or microbial test systems.
The Meeting concluded that N-acetyl-AMPA is unlikely to be genotoxic.
Human data
Routine medical surveillance of workers in production and formulation plants revealed no
adverse health effects attributable to glyphosate. In operators applying glyphosate products, cases of
eye, skin and/or respiratory tract irritation have been reported. Acute intoxication was reported in
humans after accidental or intentional ingestion of concentrated glyphosate formulations, resulting in
gastrointestinal, cardiovascular, pulmonary and renal effects and, occasionally, death. The acute
toxicity of glyphosate formulations was likely caused by the surfactant in these products (JMPR,
2004).
Several epidemiological studies on cancer outcomes following occupational exposure to
glyphosate were available. The evaluation of these studies focused on the occurrence of NHL, as
outlined in Section 2.2 of the Meeting report. One meta-analysis and one prospective cohort study, the
AHS, with a large sample size and detailed exposure assessment, were available. Cohort studies are
considered a powerful design, as recall bias is avoided. All other studies were case–control studies,
usually retrospective, which are more prone to recall and selection biases.
The AHS cohort study found no evidence of a positive association of NHL with glyphosate
exposure or an exposure–response relationship (De Roos et al., 2005). Elevated risks were reported in
various case–control studies. A significant elevated risk of NHL associated with ever- versus never-
use of glyphosate (OR = 2.1; 95% CI = 1.1–4.0) was reported (De Roos et al., 2003). Ever-use of
glyphosate was not associated with risk of NHL in the Cross-Canada Case–control Study of Pesticides
and Health (McDuffie et al., 2001), but when analysing days of use per year, there was a significant
elevated risk in the highest usage category (OR = 2.12; 95% CI = 1.20–3.73; for > 2 days/year
glyphosate use). There was, however, no indication of an exposure–response relationship across
exposure usage categories (McDuffie et al., 2001). In another case–control study, a significant
increased risk of NHL associated with ever-use (OR = 2.02; 95% CI = 1.10–3.71) as well as the
highest usage category (OR = 2.36; 95% CI = 1.04–5.37; for greater than 10 days/year glyphosate
use) was observed, with some suggestion of an exposure–response gradient (Eriksson et al., 2008).
Two smaller case–control studies with few exposed cases and limited statistical power reported a
nonsignificant elevated risk (Hardell et al., 2002) and no association (Orsi et al., 2009), respectively,
for risk of NHL and ever-use of glyphosate. The meta-analysis, including the AHS, found a
significant 50% excess risk ratio for ever- versus never-use of glyphosate (Schinasi & Leon, 2014).
Overall, there is some evidence of a positive association between glyphosate exposure and
risk of NHL from the case–control studies and the overall meta-analysis. However, it is notable that
the AHS (De Roos et al., 2005), which is the only cohort study and is large and of high quality, found
no evidence of association at any exposure level.
In view of the absence of carcinogenic potential in rodents at human-relevant doses and the
absence of genotoxicity by the oral route in mammals, and considering the epidemiological evidence
from occupational exposures, the Meeting concluded that glyphosate is unlikely to pose a
carcinogenic risk to humans via exposure from the diet.
The Meeting concluded that the existing database on glyphosate was adequate to characterize
The Meeting reaffirmed the group ADI for the sum of glyphosate, AMPA, N-acetyl-
glyphosate and N-acetyl-AMPA of 0–1 mg/kg bw on the basis of the NOAEL of 100 mg/kg bw per
day for effects on the salivary gland in a long-term study of toxicity and carcinogenicity in rats and

258
application of a safety factor of 100. The Meeting noted that these effects may be secondary to local
irritation due to the low pH of glyphosate in solution, but was unable to establish this unequivocally.
The Meeting concluded that it was not necessary to establish an ARfD for glyphosate,
AMPA, N-acetyl-glyphosate and N-acetyl-AMPA in view of their low acute toxicity, the absence of
relevant developmental toxicity in rats and rabbits that could have occurred as a consequence of acute
exposure, and the absence of any other toxicological effect that would be elicited by a single dose.
Levels relevant to risk assessment of glyphosate

Mouse Eighteen- to 24-month Toxicity 1 600 ppm, equal to 8 000 ppm, equal to
studies of toxicity and 153 mg/kg bw per dayc 787 mg/kg bw per day
carcinogenicitya,b
Carcinogenicity The Meeting could not exclude the possibility
that glyphosate is carcinogenic in mice at very
high doses.
Rat Acute neurotoxicity studya Neurotoxicity 2 000 mg/kg bwc –
Two-year studies of toxicity Toxicity 100 mg/kg bw per day 300 mg/kg bw per day
and carcinogenicityb
Carcinogenicity 32 000 ppm, equal to –
1 750 mg/kg bw per
dayc
Two-generation studies of Reproductive toxicity 30 000 ppm, equal to –
reproductive toxicitya,b 1 983 mg/kg bw per
dayc
Parental toxicity 6 000 ppm, equal to 10 000 ppm, equal to
417 mg/kg bw per day 668 mg/kg bw per day
Offspring toxicity 6 000 ppm, equal to 10 000 ppm, equal to
Developmental toxicity Maternal toxicity 300 mg/kg bw per day 1 000 mg/kg bw per
studiesb,d day
Embryo and fetal 300 mg/kg bw per day 1 000 mg/kg bw per
toxicity day
Rabbit Developmental toxicity Maternal toxicitye 100 mg/kg bw per day 175 mg/kg bw per day
studiesb,d
Embryo and fetal 250 mg/kg bw per day 450 mg/kg bw per day
toxicitye
Dog Thirteen-week and 1-year Toxicity 15 000 ppm, equal to 30 000 ppm, equal to
studies of toxicityb,f 448 mg/kg bw per day 926 mg/kg bw per day
AMPA
Rat Thirteen-week study of Toxicity 1 000 mg/kg bw per –
toxicityd dayc
Developmental toxicity Maternal toxicity 1 000 mg/kg bw per –
studyd dayc
Embryo and fetal 1 000 mg/kg bw per –
toxicity dayc
a
b
c
d
e
Secondary to local irritation of the colon.
f
Capsule administration.

259

0–1 mg/kg bw (for sum of glyphosate, N-acetyl-glyphosate, AMPA and N-acetyl-AMPA)

Unnecessary
human exposure
Critical end-points for setting guidance values for exposure to glyphosate

Rate and extent of oral absorption Rapidly, but only to a limited extent (about 20–30%)
Dermal absorption About 1–3%
Distribution Widely distributed (low levels occurring in all tissues)
Potential for accumulation No evidence of accumulation
Rate and extent of excretion Rapid and nearly complete in 48 h (about 20–30% in urine and
about 60–70% in faeces)
Metabolism in animals Very limited (< 0.7%), by hydrolysis leading to AMPA
Toxicologically significant compounds in animals Parent compound, AMPA, N-acetyl-glyphosate, N-acetyl-AMPA
and plants
Acute toxicity
Rat, LD50, oral 5 600 mg/kg bw
Rat, LC50, inhalation > 5.48 mg/L
Rabbit, dermal irritation Not irritating
Rabbit, ocular irritation Moderately to severely irritating
Guinea-pig, dermal sensitization Not sensitizing (Magnusson and Kligman test, Buehler test)
Mouse, dermal sensitization Not sensitizing (local lymph node assay)
Short-term studies of toxicity
Target/critical effect Clinical signs (loose stools, diarrhoea), liver, salivary glands and
reduced body weights
Lowest relevant oral NOAEL 300 mg/kg bw per day (90 days; rat)
Lowest relevant dermal NOAEL > 5 000 mg/kg bw per day (21 days; rabbit)
Lowest relevant inhalation NOAEC No data
Long-term studies of toxicity and carcinogenicity
Target/critical effect Reduced body weights, loose stools, liver (toxicity), salivary glands
(organ weight, histology), eye (cataracts, lens fibre degeneration)
Lowest relevant NOAEL 100 mg/kg bw per day (2 years; rat)
Carcinogenicity Not carcinogenic in rats; could not exclude possibility of
carcinogenicity in mice at very high dosesa
Genotoxicity

260

No genotoxic potential via oral route in mammalsa
Target/critical effect Reduced body weights and delayed development (absence of
maternal toxicity)
Lowest relevant parental NOAEL 417 mg/kg bw per day (rat)
Lowest relevant offspring NOAEL 417 mg/kg bw per day (rat)
Lowest relevant reproductive NOAEL 1 983 mg/kg bw per day (rat)
Target/critical effect Slight increase in malformations at maternally toxic doses
Lowest relevant maternal NOAEL 100 mg/kg bw per day (rabbit)b
Lowest relevant embryo/fetal NOAEL 250 mg/kg bw per day (rabbit)b
Neurotoxicity
Acute neurotoxicity NOAEL 2 000 mg/kg bw, highest dose tested
Subchronic neurotoxicity NOAEL 1 547 mg/kg bw per day, highest dose tested
Developmental neurotoxicity NOAEL No data
Other toxicological studies
Immunotoxicity No immunotoxicity; NOAEL 1 448 mg/kg bw per day, highest dose
tested (28 days; mouse)
Studies on toxicologically relevant metabolites Toxicological studies on AMPA, N-acetyl-glyphosate and N-acetyl-
AMPA reveal the metabolites to be less toxic than the parent
compound
Human data
Medical surveillance of workers in plants producing and
formulating glyphosate did not reveal any adverse health effects. In
operators applying glyphosate products, cases of eye, skin and/or
respiratory irritation have been reported. Cases of acute intoxication
have been observed after accidental or intentional ingestion of
glyphosate formulation.
a
Unlikely to pose a carcinogenic risk to humans via exposure from the diet.
b
Secondary to local irritation of the colon.
Summary
Value Study Safety factor
ADI 0–1 mg/kg bw Two-year studies of toxicity (rat) 100
ARfD Unnecessary – –
References
Ackermann W, Coenen M, Schrodl W, Shehata AA, Krüger M (2015) The influence of glyphosate on the
microbiota and production of botulinum neurotoxin during ruminal fermentation. Curr Microbiol..
70(3):374–82.
Acquavella JF, Alexander BH, Mandel JS, Gustin H, Baker B, Chapman P et al. (2004). Glyphosate
biomonitoring for farmers and their families: results from the Farm Family Exposure Study. Environ Health
Perspec.112:321–6.

261
Acquavella JF, Weber JA, Cullen MR, Cruz OA, Martens MA, Holden LR et al. (1999). Human ocular effects
from self-reported exposure to Roundup herbicides. Hum Exp Toxicol. 18: 479–86.
Akanuma M (1995a). HR-001: Reverse mutation test. The Institute of Environmental Toxicology, Tokyo,
Japan. Data owner: Arysta LifeScience. Study no.: IET 94-0142, dated 3 April 1995. Unpublished study.
Akanuma M (1995b). HR-001: DNA repair test (Rec-Assay). The Institute of Environmental Toxicology,
Tokyo, Japan. Laboratory project ID IET 94-0141, dated 14 March 1995. Sponsored by Sankyo Co. Ltd.,
Tokyo, Japan. Unpublished study.
Akanuma M (1996). AMPA reverse mutation test. Institute of Environmental Toxicology, Tokyo, Japan.
Laboratory project ID IET 96-0076. Sponsored by Sankyo Co., Ltd., Tokyo, Japan. Unpublished study.
Alavanja MC, Dosemeci M, Samanic C, Lubin J, Lynch CF, Knott C et al. (2004). Pesticides and lung cancer
risk in the agricultural health study cohort. Am J Epidemiol. 160(9):876–85.
risk and insecticide, fungicide and fumigant use in the agricultural health study. PLoS ONE. 9:e109332.
doi:10.1371/journal.pone.0109332.
Alison RH, Capen CC, Prentice DE (1994). Neoplastic lesions of questionable significance to humans. Toxicol
Pathol. 22;179–86.
Allen SL (1996). Glyphosate acid: Comparison of salivary gland effects in three strains of rat. Unpublished
report no. CTL/P/5160, study no. PR 1029, dated 18 August 1996, from Zeneca Agrochemicals, Central
Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Submitted to WHO by
Syngenta Crop Protection AG, Basel, Switzerland.
Alvarez-Moya C, Silva MR, Ramirez CV, Gallardo DG, Sanchez RL, Aguirre AC et al. (2014). Comparison of
the in vivo and in vitro genotoxicity of glyphosate isopropylamine salt in three different organisms. Genet
Mol Biol. 37(1):105–10.
Amer SM, Aly FAE, Farghaly AA, Ibrahim AAE (2006). In vitro and in vivo evaluation of the genotoxicity of
the herbicide glyphosate in mice. B Natl Res Cent (Cairo). 31:427–46.
Anadón A, Martínez-Larrañaga MR, Martinez MA, Castellano VJ, Martinez M, Martin MT et al (2009).
Toxicokinetics of glyphosate and its metabolite aminomethyl phosphonic acid in rats. Toxicol Lett. 190(1):
91–5.
Arcelin G (2007a). Glyphosate technical material acute oral toxicity in the rat (Up and Down procedure). RCC
Ltd. Toxicology, Fullinsdorf, Switzerland. Laboratory report no. B02755, Syngenta task no. T007035-05,
dated February 2007. February 2007. Unpublished report.
Arcelin G (2007b). Glyphosate technical material acute dermal toxicity study in rats. RCC Ltd. Toxicology,
Fullinsdorf, Switzerland. Laboratory report no. T007036-05 dated February 2007. Unpublished report.
Arcelin G (2007c). Glyphosate technical material: Primary skin irritation study in rabbits (4-hour semi-occlusive
application). RCC Ltd., Toxicology, Füllinsdorf, Switzerland. Data owner: Syngenta report no.:
R61837/1010, dated 8 February 2007. Unpublished study.
Arcelin G (2007d). Glyphosate technical material: Primary eye irritation study in rabbits. RCC Ltd., Toxicology,
Füllinsdorf, Switzerland. Data owner: Syngenta report no.: B02788, dated 26 March 2007. Unpublished
study.
Ashby J, Tennant RW, Zeiger E, Stasiewicz S (1989) Classification according to chemical structure,
mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity
by the U.S. National Toxicology Program. Mutat Res. 223(2):73–103.
Atkinson C, Martin T, Hudson P, Robb D (1993a). Glyphosate: 104 week dietary carcinogenicity study in mice.
Unpublished report No. 7793. IRI project No. 438618, dated 12 April 1991. Inveresk Research
International, Tranent, Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Atkinson C, Perry CJ, Hudson P, Snodgrass E (1989). Glyphosate: 4 Week dietary toxicity study in rats.
Inveresk Research International, Tranent, Scotland, UK. Report no.: 5626. Cheminova report no.: 49 GLY.
Atkinson C, Strutt AV, Henderson W, Finch J, Hudson P (1993b). Glyphosate: 104 week combined chronic
feeding/oncogenicity study in rats with 52 week interim kill (results after 104 weeks.). Unpublished report
no. 7867. IRI project no. 438623, dated 7 April 1993. From Inveresk Research International, Tranent,
Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.

262
Axelrad JC, Howard CV, McLean WG (2003). The effects of acute pesticide exposure on neuroblastoma cells
chronically exposed to diazinon. Toxicology. 185:67–78.
Bakke JP (1991). Evaluation of the potential of AMPA to induce unscheduled DNA synthesis in the in vitro
hepatocyte DNA repair assay using the male F-344 rats. Monsanto Report No. SR-91-234. Unpublished
study.
pesticides in British Columbia farmers. Prostate. 71(2):168–83. doi:10.1002/pros.21232.
Baldrick P, Reeve L (2007). Carcinogenicity evaluation: comparison of tumor data from dual control groups in
the CD-1 mouse. Toxicol Pathol. 35(4):562–9.
Balthazor TM, Hallas LE (1986). Glyphosate-degrading microorganisms from industrial activated sludge. Appl
Environ Microbiol. 51(2):432–4.
Epidemiol. 162:1070–9.
Benachour N, Sipahutar H, Moslemi S, Gasnier C, Travert C, Séralini GE (2007). Time- and dose-dependent
effects of Roundup on human embryonic and placental cells. Arch Environ Contam Toxicol. 53(1):126–33.
doi:10.1007/s00244-006-0154-8.
Betts CJ (2007). Glyphosate technical material: Skin sensitisation (local lymph node assay in the mouse).
Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Data
owner: Syngenta report no.: R61837/1004, dated 9 February 2007. Unpublished study.
Bhide MB (1988). Carcinogenicity and chronic toxicity study of glyphosate (technical) in mice. Excel Industries
Ltd., Bombay, India.
Bhide RM (1997). Combined chronic toxicity/carcinogenicity study of glyphosate technical in Sprague Dawley
Rat. Indian Institute of Toxicology, Pune, India. Study no.: 1231. Sankyo Co. Ltd., Japan. Unpublished
study.
Bhide MB, Patil UM (1989). Rabbit teratology study with glyphosate technical. Indian Institute of Toxicology,
Sanpada, New Bombay, India. Data owner: Excel. Study no.: IIT project no. 1086, November 3, 1989.
Unpublished study.
Blair A, Thomas K, Coble J, Sandler DP, Hines CJ, Lynch CF et al. (2011). Impact of pesticide exposure
misclassification on estimates of relative risks in the Agricultural Health Study. Occup Environ Med.
68(7):537–41. doi:10.1136/oem.2010.059469.
Blakley BR (1997). Effect of roundup and tordon 202C herbicides on antibody production in mice. Vet Hum
Toxicol. 39(4):204–6.
Blaszcak DL (1988a). Acute oral toxicity study in rats. Test material: glyphosate wet cake. Unpublished report,
Bio/dynamics project no. 4885-88, Monsanto reference no. BD-88-114, dated 20 September 1988. From
Bio/dynamics Inc., East Millstone, NJ, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels,
Belgium.
Blaszcak DL (1988b). Acute dermal toxicity study in rabbits. Test material: glyphosate wet cake. Unpublished
report, Bio/dynamics project no. 4886-88, Monsanto reference no. BD-88-114, dated 20 September 1988.
From Bio/dynamics Inc., East Millstone, NJ, USA. Submitted to WHO by Monsanto Int. Services SA,
Brussels, Belgium.
Blaszcak DL (1988c). Primary dermal irritation study in rabbits (4–hour exposure/semi-occlusive covering).
Test material: glyphosate wet cake. Unpublished report. Bio/dynamics project no. 4887-88. Monsanto
reference no. BD-88-114, dated 20 September 1988. From Bio/dynamics Inc., East Millstone, NJ, USA.
Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium.
Blaszcak DL (1988d). Eye irritation study in rabbits. Test material: Glyphosate wet cake. Unpublished report,
Bio/dynamics project No. 4888-88, Monsanto reference No. BD-88-114, dated 20 September 1988, from
Bio/dynamics Inc., East Millstone, NJ, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels,
Belgium.

263
Blaszcak DL (1998e). MON 77945: Primary eye irritation study in rabbits. Unpublished report, study no. 97-
1728, Monsanto reference no. HU-97-242, dated 24 March 1998, from Huntingdon Life Sciences, East
Millstone, NJ, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium.
Bolognesi C, Carrasquilla G, Volpi S, Solomon KR, Marshall ER (2009). Biomonitoring of genotoxic risk in
agricultural workers from five Colombian regions: association to occupational exposure to glyphosate. J
Toxicol Environ Health A. 72(15–16):986–97.
Bolognesi C, Bonatti S, Degan P, Gallerani E, Peluso M, Rabboni R et al (1997). Genotoxic activity of
glyphosate and its technical formulation roundup. J Agric Food Chem. 45(5):1957–62.
Bonassi S, Fenech M, Lando C, Lin YP, Ceppi M, Chang WP et al. (2001). HUman MicroNucleus project:
international database comparison for results with the cytokinesis-block micronucleus assay in human
lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of
micronuclei. Environ Mol Mutagen. 37(1):31-45.
Bonnette KL (2001). A primary eye irritation study in rabbits with MON 78623. SLI study no. 3044.813,
Monsanto study no. SB-2000-248, dated 6 August 1979, from Springborn Laboratories Inc., Spencerville,
OH, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium. Unpublished report.
Bonnette K (2004). An acute nose-only inhalation study in rats with MON 78623. Charles River Laboratories
Inc., Spencerville, OH, USA. Laboratory report no. 3044.969, Monsanto study no. SB-2003-116.
Unpublished report.
Botham PA (1996). First revision to glyphosate acid: 90 Day feeding study in rats. Unpublished report no.
CTL/P/1599, study no. PR 0663, dated 7 November 1996, from Zeneca Agrochemicals, Central Toxicology
Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Submitted to WHO by Syngenta Crop
Protection AG, Basel, Switzerland.
Bradberry SM, Proudfoot AT, Vale JA (2004). Glyphosate poisoning. Toxicol Rev. 23(3):159-67.
Brammer A (1996). Glyphosate acid: 1 Year dietary toxicity study in dogs. Unpublished report no. CTL/P/5079,
study no. PD 1006, dated 24 September 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory,
Alderley Park, Macclesfield, Cheshire, England, UK. Submitted to WHO by Syngenta Crop Protection AG,
Basel, Switzerland.
Brammer A (2001). Glyphosate acid: Two year dietary toxicity and oncogenicity study in rats. Unpublished
report no. CTL/PR1111, study no. PR1111, dated 15 March 2001, from Zeneca Agrochemicals, Central
Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Submitted to WHO by
Syngenta Crop Protection AG, Basel, Switzerland.
Branch DK (1981). Primary eye irritation of MON 0139 to rabbits. Unpublished report, study no. 800260,
DMEH project no. ML-80-261, dated 17 March 1981, from Monsanto Environmental Health Laboratory,
St. Louis, MO, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium.
Brooker AJ, John DM, Anderson A, Dawe IS (1991a). The effect of glyphosate on pregnancy of the rat
(incorporates preliminary investigation). Unpublished report No. CHV 43 & 41/90716, dated 14 October
1991, from Huntingdon Research Centre Ltd., Huntingdon, England, UK. Submitted to WHO by
Cheminova A/S, Lemvig, Denmark.
Brooker AJ, Brennan C, John DM, Anderson A, Dawe IS (1991b). The effect of glyphosate on pregnancy of the
rabbit (incorporates preliminary investigations). Unpublished report no. CHV 45 & 39 & 40/901303, dated
14 October 1991, from Huntingdon Research Centre Ltd., Huntingdon, England, UK. Sub- mitted to WHO
by Cheminova A/S, Lemvig, Denmark.
Brooker AJ, Myers DP, Parker CA, Offer JM, Singh H, Anderson A et al. (1992). The effect of dietary
administration of glyphosate on reproductive function of two generations in the rat. Unpublished report no.
CHV 47/911129, dated 14 May 1992, from Huntingdon Research Centre Ltd., Huntingdon, England, UK.
Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
other agricultural risk factors for leukemia among men in Iowa and Minnesota. Cancer Res, 50(20):6585–
91.
Brewster DW, Warren J, Hopkins WE 2nd (1991). Metabolism of glyphosate in Sprague–Dawley rats: Tissue
distribution, identification, and quantitation of glyphosate-derived materials following a single oral dose.
Fundam Appl Toxicol. 17(1):43–51.

264
Busch B (1987a). Primary eye irritation study of MON-8722 in New Zealand White rabbits. Unpublished report,
FDRL study no. 9307A, Monsanto study no. FD-86-430, dated 29 January 1987, from Food & Drug
Research Laboratories, Waverly, NY, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels,
Belgium.
Busch B (1987b). Primary eye irritation study of MON-8750 in New Zealand White rabbits. Unpublished
report, FDRL study no. 9308A, Monsanto study no. FD-86-431, dated 30 January 1987, from Food & Drug
Research Laboratories, Waverly, NY, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels,
Belgium.
Callander RD (1988a). ICIA 0224 - An evaluation of mutagenic potential using S. typhimurium and E. coli. ICI
Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Report no.
CTL/P/2203, dated 20 February 1988. Sponsored by ICI Agrochemicals. Submitted by Syngenta, Basel,
Switzerland. Unpublished study.
Callander, RD (1988b) Aminomethyl phosphonic acid: An evaluation of the mutagenic potential using S.
typhimurium and E. coli. Unpublished report no. CTL/P/2206, study No. YV2280 and YV2281, dated 21
September 1988, from ICI Agrochemicals, Central Toxicology Laboratory, Alderley Park, Macclesfield,
Cheshire, England, UK. Submitted to WHO by Syngenta Crop Protection AG, Basel, Switzerland.
Callander RD (1993). TMSC – An evaluation of mutagenic potential using S. typhimurium and E. coli. Zeneca
Agrochemicals, Central Toxicology Laboratory, Alderley Park Macclesfield, Cheshire, England, UK.
Report no. CTL/P/3992, dated 2 April 1993. Sponsored by ICI Agrochemicals, submitted by Syngenta,
Basel. Unpublished study.
Callander RD (1996). Glyphosate acid: An evaluation of mutagenic potential using S. typhimurium and E. coli.
Unpublished report no. CTL/P/4874, study no. YV3611, dated 16 February 1996, from Zeneca
Agrochemicals, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK.
Submitted to WHO by Syngenta Crop Protection AG, Basel, Switzerland.
Canabrava Frossard de Faria B (2008a). Acute dermal irritation/corrosion study in rabbits with glyphosate
technical. Bioagri Laboratorios, Sao Paulo, Brazil. Laboratory report no. RF – 3996.311.476.07, dated 23
September 2008. Unpublished report.
Canabrava Frossard de Faria BC (2008b). Acute eye irritation/corrosion study in rabbits with glyphosate
technical. Bioagri Laboratórios, Sao Paulo, Brazil. Data owner: Helm AG. Report no.: RF-3996.312.599.07,
dated 12 September 2008. Unpublished study.
52:2447–55.
Carpenter C (2007) IN-EY252: Acute oral toxicity study in rats-up-and-down procedure. Unpublished report no.
DuPont-22229. DuPont Haskell Laboratory, Newark, DE, USA. Unpublished study.
Carter L (2009). Acute inhalation toxicity study in rats. Stillmeadow, Inc., Sugar Land, TX, USA. Laboratory
report no. 12107-08. Unpublished report, dated 9 March 2009.
Carvalho Marques MF (1999). A micronucleus study in mice for glyphosate técnico. Nufarm Bioagri
Laboratorios, Sao Paulo, Brazil. Data owner: Nufarm study no.: RF-G12.79/99, dated 27 December 1999.
Unpublished study.
Centre de Toxicologie du Quebec (1988). Etude de l’exposition professionnelle des travailleurs forestiers
exposés au glyphosate [Study of occupational exposure of forestry workers exposed to glyphosate]. Québec
(QC): Centre de Toxicologie du Quebec; August 1988. (In French)
Cerdeira A, Duke S (2006). The current status and environmental impacts of glyphosate-resistant crops: a
review. J Environ Qual. 35(5):1633–58.
Chan PO, Mahler JF (1992). NTP technical report on toxicity studies of glyphosate (CAS No. 1071-83-6)
administered in dosed feed to F344/N rats and B6C3F1 mice. National Toxicology Program, Research
Triangle Park, NC, USA. NTP Toxicity Report Series No. 16, NIH Publication 92-3135, dated July 1992.
Chandra M, Frith CH (1994). Spontaneous renal lesions in CD-1 and B6C3F1 mice. Exp Toxicol Pathol.
46:189–98.

265
Chandra M, Riley MG, Johnson DE (1992). Spontaneous neoplasms in aged Sprague-Dawley rats. Arch
Toxicol. 66:496–502.
Chen L, Xie M, Bi Y, Wang G, Deng S Liu Y (2012). The combined effects of UV-B radiation and herbicides
on photosynthesis, antioxidant enzymes and DNA damage in two bloom-forming cyanobacteria. Ecotoxicol
Environ Saf. 80:224–30.
Cheng T, Howard S (2004). Mass balance, metabolism, and pharmacokinetics of [ 14C]N-acetyl-glyphosate
following administration of a single oral dose to rats. Unpublished report no. Covance 7535-100, amended
report. Covance Laboratories, Inc., Vienna, VA, USA. Submitted to WHO by EI du Pont de Nemours and
Company, Wilmington, DE, USA.
Chester G, Hart TB (1986). Biological monitoring of a herbicide applied through backpack and vehicle sprayers.
Toxicol Lett. 33:137–49.
Chruscielska K, Brzezinski J, Kita K, Kalhorn D, Kita I, Graffstein B et al. (2000a). Glyphosate - Evaluation of
chronic activity and possible far-reaching effects. Part 1. Studies on chronic toxicity. Pestycydy (Warsaw).
3–4:11–20.
Chruscielska K, Graffstein B, Szarapinska-Kwaszewska J, Brzezinski J, Kalhorn D (2000b). Glyphosate:
Evaluation of chronic activity and possible far-reaching effects. Part 2. Studies on mutagenic activity.
Pestycydy (Warsaw). 3–4: 21–5.
Clair E, Linn L, Travert C, Amiel C, Séralini GE, Panoff JM (2012). Effects of Roundup and glyphosate on
three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus
delbrueckii subsp. bulgaricus. Curr Microbiol. 64:486–91.
Clay P (1996). Glyphosate acid: L5178Y TK+/− mouse lymphoma gene mutation assay. CTL/P/4991, study no.
VV0123, dated 24 May 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley Park,
Macclesfield, Cheshire, England, UK. Submitted to WHO by Syngenta Crop Protection AG, Basel,
Switzerland. Unpublished report.
Clegg ED, Cook JC, Chapin RE, Foster PM, Daston GP (1997). Leydig cell hyperplasia and adenoma
formation: Mechanisms and relevance to humans. Reprod Toxicol. 11;107–21.
Clewell HJ, Crump KS (2005). Quantitative estimates of risk for noncancer endpoints. Risk Anal. 25:285–9.
Coble J, Arbuckle T, Lee W, Alavanja M, Dosemeci M (2005). The validation of a pesticide exposure algorithm
using biological monitoring results. J Occup Environ Hyg. 2:194–201.
Coble J, Thomas KW, Hines CJ, Hoppin JA, Dosemeci M, Curwin B et al. (2011). An updated algorithm for
estimation of pesticide exposure intensity in the Agricultural Health Study. Int J Environ Res Public Health.
8(12):4608–22.
Coles LJ, Thomas ON, Bartlett AJ, Brooks PN (1996). Technical glyphosate: Ninety day sub-chronic oral
(dietary) toxicity study in the rat. SafePharm Laboratories Limited, Shardlow, Derbyshire, England, UK.
SPL project no. 434/016. Sponsored by by Mastra Industries Sdn. Bhd., Port Klang, Malaysia and Maruzen
Kako Co., Ltd., Tokyo, Japan. Unpublished study.
Coles RJ, Doleman N (1996). Glyphosate technical: Oral gavage teratology study in the rabbit. SafePharm
Laboratories Limited, Shardlow, Derbyshire, England, UK. Data owner: Nufarm, SPL project no.: 434/020.
Unpublished study.
Colvin LB, Miller JA (1973a). CP 67573 Residue and metabolism. Part 8: the gross metabolism of N-
phosphonomethylglycine-14C (CP 67573-14C) in the laboratory rat following a single dose. Unpublished
report No. 297, study No. 9-23-760.06-7863, dated 15 June 1973, from Monsanto Commercial Products
Co., Agricultural Division, Research Department. Submitted to WHO by Monsanto Int. Services SA,
Brussels, Belgium.
Colvin LB, Miller JA (1973b). CP 67573 Residue and Metabolism. Part 13: The dynamics of accumulation and
depletion of orally ingested N-phosphonomethylglycine-14C, Monsanto report MSL-309.
Colvin LB, Miller JA (1973c). CP 67573 Residue and metabolism. Part 9: The metabolism of N-
phosphonmethylglycine-14C (CP 67573-14C) in the rabbit. Unpublished report no. 298, study no. 9-23-
760.06-7863, dated 20 June 1973, from Monsanto Commercial Products Co., Agricultural Division.

266
Cook JC, Klinefelter GR, Hardisty JF, Sharpe RM, Foster PM (1999). Rodent Leydig cell tumorigenesis: A
review of the physiology, pathology, mechanisms, and relevance to humans. Crit Rev Toxicol. 29:169–261.
Costa KC (2008). Evaluation of the mutagenic potential of glyphosate technical micronucleus assay in mice
Bioagri Laboratories, Sao Paulo, Brazil. Data owner: HAG. Report no.: RF - 3996.402.395.07, dated 29
September 2008. Unpublished study.
Cowell JE, Steinmetz JR (1990). Assessment of forestry nursery workers exposure to glyphosate during normal
operations. Monsanto report no. MSL-9655. Monsanto Co., St. Louis, MO, USA.
Cross MF (1988). ICIA 0224: Assessment of mutagenic potential using L5178Y mouse lymphoma cells. ICI
Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Report no.
CTL/P/2228, dated 2 August 1988. Sponsored by ICI Agrochemicals. Unpublished study.
Cuthbert JA, Jackson D (1989a). Glyphosate technical: Acute oral toxicity (limit) test in rats. Unpublished
report no. 5883, IRI project no. 243268, dated 22 June 1989, from Inveresk Research International,
Musselburgh, Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Cuthbert JA, Jackson D (1993a). AMPA: Acute oral toxicity (limit) test in rats. Unpublished report no. 8763,
IRI project no. 552409, dated 28 January 1993, from Inveresk Research International, Tranent, Scotland,
UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Cuthbert JA, Jackson D (1993b). AMPA: Acute dermal toxicity (limit) test in rats. Unpublished report no. 8764,
IRI project no. 552409, dated 28 January 1993, from Inveresk Research International, Tranent, Scotland,
UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Cuthbert JA, Jackson D (1993c). AMPA: Magnusson–Kligman maximisation test in guinea pigs. Unpublished
report no. 8765, IRI project no. 552409, dated 28 January 1993, from Inveresk Research International,
Tranent, Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Cuthbert JA, D Jackson (1989b). Glyphosate technical: Acute dermal toxicity (limit) test in rats. IRI Project No.
243268. Inveresk Research International, Musselburgh, Scotland, UK. Laboratory report no. 5884.
Unpublished Report.
Dallago BS (2008). Acute inhalation toxicity test of glyphosate technical in rats (Rattus norvegicus). Bioagri
Laboratories, Sao Paulo, Brazil. Laboratory report no. RF – 3996.309.377.07. Unpublished report, dated 11
September 2008.
Davies DJ (1996a). Glyphosate acid: excretion and tissue retention of a single oral dose (10 mg/kg) in the rat.
Unpublished report no. CTL/P/4940, dated 26 April 1996, from Zeneca Agrochemicals, Central Toxicology
Davies DJ (1996b). Glyphosate acid: excretion and tissue retention of a single oral dose (1000 mg/kg) in the rat.
Unpublished report no. CTL/P/4942, dated 19 June 1996, from Zeneca Agrochemicals, Central Toxicology
Davies DJ (1996c). Glyphosate acid: excretion and tissue retention of a single oral dose (10 mg/kg) in the rat
following repeat dosing. Unpublished report no. CTL/P/4944, dated 22 May 1996, from Zeneca
Agrochemicals, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK.
Submitted to WHO by Syngenta Crop Protection AG, Basel, Switzerland.
Davies DJ (1996d). Glyphosate acid: whole body autoradiography in the rat (10 mg/kg). Unpublished report No.
CTL/P/4943, dated 10 June 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley
Park, Macclesfield, Cheshire, England, UK. Submitted to WHO by Syngenta Crop Protection AG, Basel,
Switzerland.
Davies DJ (2003). Glyphosate SL (360 g/L) formulation (A12798Q). In vitro absorption through human
epidermis CTL/JV1732/Regulatory/Report. Syngenta UK Limited, Fulbourn, Cambridgeshire, England,
UK. CTL study no.: JV1732, Document no. CTL/JV1732/REG/REPT, Unpublished study.
De Roos AJ, Blair A, Rusiecki JA, Hoppin JA, Svec M, Dosemeci M et al. (2005). Cancer incidence among
glyphosate-exposed pesticide applicators in the Agricultural Health Study. Environ Health Perspect.
113:49–54.

267
Med. 60:E11. doi:10.1136/oem.60.9.e11.
Decker J (2007). Glyphosate technical (NUP) 05068): 4-Hour acute inhalation toxicity study in rats. RCC Ltd,
Toxicology, Fullinsdorf, Switzerland. Lab report no. RCC study no.: B05068). Unpublished report dated 2
April 2007.
Defarge N, Takács E, Lozano VL, Mesnage R, Spiroux de Vendômois J, Séralini GE et al. (2016). Co-
formulants in glyphosate-based herbicides disrupt aromatase activity in human cells below toxic levels. Int
J Environ Res Public Health. 13(3): 264. doi.org/10.3390/ijerph13030264.
Dhinsa NK, Watson P, Brooks PN (2007). Glyphosate technical: Dietary two generation reproduction study in
the rat. SafePharm Laboratories Limited, Shardlow, Dernyshire, England, UK. Data owner: Nufarm. SPL
project no.: 2060/0013, amended 8 April 2008 and 8 August 2008. Unpublished study.
Dideriksen LH (1991). Assessment of acute oral toxicity of “glyphosate technical” to mice. Unpublished report.
Laboratory report no. 12321, dated 7 January 1991, from Scantox A/S, Lille Skensved, Denmark.
Dimitrov BD, Gadeva PG, Benova DK, Bineva MV (2006). Comparative genotoxicity of the herbicides
Roundup, Stomp and Reglone in plant and mammalian test systems. Mutagenesis. 21(6):375–82.
Do Amaral Guimarães SD (2008a). Acute oral toxicity study in Wistar Hannover rats for glyphosate technical.
Bioagri Laboratórios, Sao Paulo, Brazil. Data owner: Helm AG report no.: RF-3996.305.475.07, dated 16
Do Amaral Guimaraes S (2008b). Acute dermal toxicity study in Wistar Hannover rats for glyphosate technical.
Bioagri Laboratórios, Sao Paulo, Brazil. Lab report no. RF-3996.310.456.07, dated 4 July 2008.
Unpublished report.
Donath C (2010). Reverse mutation assay using bacteria (Salmonella typhimurium) with Glyphosate TC. BSL
Bioservice, Planegg, Germany. BSL Bioservice study no. 104039, dated 18 October 2010. Sponsored by
Helm AG, Hamburg, Germany. Unpublished study.
Donath C (2011a). Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with
Glyphosate Technical. BSL Bioservice, Planegg, Germany. BSL Bioservice study no. 111608, dated 18
May 2011. Sponsored by Cheminova A/S, Lemvig, Denmark.
Donath C (2011b). Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with
March 2011. Sponsored by Cheminova A/S, Lemvig, Denmark.
Donath C (2011c). Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with
May 2011. Sponsored by Cheminova A/S, Lemvig, Denmark. Unpublished study.
Donner EM (2006). IN-MCX20: Mouse bone marrow micronucleus test. Unpublished report no. DuPont-20154,
DuPont Haskell Laboratory, Newark, DE, USA. Submitted to WHO by EI du Pont de Nemours and
Company, Wilmington, DE, USA.
Donner EM (2007). IN-EY252: Mouse bone marrow micronucleus test. Unpublished report no. DuPont-22226.
DuPont Haskell Laboratory, Newark, DE, USA. Submitted to WHO by EI du Pont de Nemours and Com-
pany, Wilmington, DE, USA.
Dosemeci M, Alavanja MC, Rowland AS, Mage D, Zahm SH, Rothman N et al. (2002). A quantitative
approach for estimating exposure to pesticides in the Agricultural Health Study. Ann Occup Hyg. 46:245–
60.
Dourson M, Reichard J, Nance P, Burleigh-Flayer H, Parker A, Vincent M et al. (2014). Mode of action analysis
for liver tumors from oral 1,4-dioxane exposures and evidence-based dose response assessment. Regul
Toxicol Pharmacol. 68(3):387-401. doi: 10.1016/j.yrtph.2014.01.011.
Doyle CE (1996a). Glyphosate acid: acute oral toxicity study in rats. Unpublished report no. CTL/P/4660, study
No. AR5959, dated 23 August 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley
Switzerland.

268
Doyle CE (1996b). Glyphosate acid: Acute dermal toxicity study in the rat. Central Toxicology Laboratory,
Alderley Park, Macclesfield, Cheshire, England, UK. Laboratory report no. CTL/P/4664. Unpublished
report, dated 23 August 1996.
Doyle CE (1996c). Glyphosate acid: skin irritation to the rabbit. Unpublished report no. CTL/P/4695, study no.
EB4365, dated 23 August 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley
Switzerland.
Doyle CE (1996d). Glyphosate acid: Skin sensitisation to the guinea pig. Unpublished report no. CTL/P/4699,
study No. GG6427, dated 23 August 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory,
Basel, Switzerland.
Durward R (2006). Glyphosate technical: Micronucleus test in the mouse. SafePharm Laboratories Ltd,
Shardlow, Derbyshire, England, UK. Data owner: Nufarm report no.: 2060/014, dated 8 February 2006.
Unpublished study.
Enami T, Nakamura H (1995). Acute toxicity study of MON 0139 by oral administration in mice. Bozo
Research Center Inc., Tokyo, Japan. Data owner: Monsanto, Monsanto report no.: XX-95-205 dated 5
October 1995. Unpublished study.
Enomoto K (1997). HR-001: 24-Month oral chronic toxicity and oncogenicity study in rats. Institute of
Environmental Toxicology, Tokyo, Japan. Arysta Life Sciences. Study no.:IET 94-0150. Also referred to as
Arysta Life Sciences, 1997. Unpublished study.
Erexson GL (2003). In Vivo mouse micronucleus assay with MON 78634. Covance Laboratories Inc., Vienna,
VA, USA. Monsanto study no. CV-2002-189, dated 24 April 2003. Unpublished report.
doi:10.1002/ijc.23589.
Escande A, Pillon A, Servant N, Cravedi JP, Larrea F, Muhn P et al. (2006). Evaluation of ligand selectivity
using reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem Pharmacol. 71:1459–69.
Eskenazi B, Harley K, Bradman A, Weltzien E, Jewell NP, Barr DB et al. (2004). Association of in utero
organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population.
Environ Health Perspect. 112: 1116–24.
Evans N, Gray LE, Wilson VS (2012). Validation of T47D-KBluc cell assay for detection of estrogen receptor
agonists and antagonists. Society of Toxicology (SOT) Annual Meeting, San Francisco, CA, USA. 11–15
March 2012.
Flügge C (2009a). Mutagenicity study of glyphosate TC in the Salmonella typhimurium reverse mutation assay
(in vitro). LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Hamburg, Germany. Report
no.: LPT 23916, dated 30 April 2009. Sponsored by Helm AG, Hamburg, Germany. Unpublished study.
Flügge C (2009b). Micronucleus test of Glyphosate TC in bone marrow cells of the CD rat by oral
administration. LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Hamburg, Germany.
Sponsored by Helm AG, Hamburg, Germany. Report no.: LPT 23917, dated 18 May 2009. Unpublished
study.
Flügge C (2010). Mutagenicity study of glyphosate TC in the Salmonella typhimurium reverse mutation assay
(in vitro). LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Hamburg, Germany. Report
no.: LPT 24880, dated 25 January 2010. Sponsored by Helm AG, Hamburg, Germany. Unpublished study.
Forgacs AL, Ding Q, Jaremba RG, Huhtaniemi IT, Rahman NA, Zacharewski TR (2012). BLTK1 murine
Leydig cells: a novel steroidogenic model for evaluating the effects of reproductive and developmental
toxicants. Toxicol Sci. 127(2):391–402. doi:10.1093/toxsci/kfs121.
Fox V (1998). Glyphosate acid: In vitro cytogenetic assay in human lymphocytes. Unpublished report no.
CTL/P/6050, study no. SV0777, dated 29 October 1998, from Zeneca Agrochemicals, Central Toxicology
Fox V, Mackay JM (1996). Glyphosate acid: mouse bone marrow micronucleus test. Unpublished report no.
CTL/P/4954, study no. SM0796, dated 21 March 1996, from Zeneca Agrochemicals, Central Toxicology

269
Franklin CA, Muir NI, Moody RP (1986). The use of biological monitoring in the estimation of exposure during
the application of pesticides. Toxicol Lett. 33:127–36.
Franz JI, Mao MK, Sikorski JA (1997). Glyphosate: a unique global herbicide. ACS Monograph Series no. 189.
Washington (DC): American Chemical Society. 1–678.
Gaou I (2007). 13-Week toxicity study by oral route (capsule) in beagle dogs. CIT, Evreux, France. Laboratory
study no. 29646 TTC, dated 1 June 2007. Submitted by Nufarm Asi Sdn Bhd, Selangor, Malaysia.
Unpublished study.
Gasnier C, Dumont C, Benachour N, Clair E, Chagnon MC, Séralini GE (2009). Glyphosate-based herbicides
are toxic and endocrine disruptors in human cell lines. Toxicology. 262:184–91. doi:10.1016/j.
tox.2009.06.006.
Gava MA (2000). Evaluation of the mutagenic potential of the test substance GLIFOSATO IPA TECNICO
NUFARM by micronucleus assay in mice. Unpublished report no. RF-G12.022/00, dated 13 July 2000.
Nufarm do Brasil LTDA, Curitiba, Brazil.
George J, Prasad S, Mahmood Z, Shukla Y (2010). Studies on glyphosate-induced carcinogenicity in mouse
skin: A proteomic approach. J Proteomics. 73:951–64.
Glatt CM (2006). IN-MCX20: In vitro mammalian cell gene mutation test (CHO/HGPRT). Unpublished report
no. DuPont-20155. DuPont Haskell Laboratory, Newark, DE, USA. Submitted to WHO by EI du Pont de
Nemours and Company, Wilmington, DE, USA.
Glatt CM (2007). IN-EY252: In vitro mammalian cell gene mutation test (CHO/HGPRT). Unpublished report
no. DuPont-22224. DuPont Haskell Laboratory, Newark, DE, USA. Submitted to WHO by EI du Pont de
Goburdhun R (1991). Glyphosate: 52 Week oral toxicity study in dogs. Unpublished report no. 7502, IRI project
no. 642675, dated 12 April 1991, from Inveresk Research International, Tranent, Scotland, UK. Submitted
to WHO by Cheminova A/S, Lemvig, Denmark.
Goburdhun R, Oshodi RO (1989). Glyphosate: Oral maximum tolerated dose study in dogs. Unpublished report
no. 5660, IRI project no. 640683, dated 22 June 1989, from Inveresk Research International, Musselburgh,
Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Griffiths DJ (2009). Glyphosate tech: Acute inhalation toxicity (nose only) study in the rat. Harlan Laboratories
Ltd., Shardlow, Derbyshire, England, UK. Laboratory report no. 2743/0001. Unpublished Report. 23 June
2009.
Griffiths K, Mackay J (1993). TMSC An evaluation in the in vitro cytogenetic assay in human lymphocytes.
Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England,
UK. Report no. CTL/P/4072, dated 13 August 1993. Sponsored by Zeneca Agrochemicals. Unpublished
study.
Grisolia CK (2002). A comparison between mouse and fish micronucleus test using cyclophosphamide,
mitomycin C and various pesticides. Mutat Res. 518(2):145–50.
Gudi R, Rao M (2007). IN-EY252: In vitro mammalian chromosome aberration test in human peripheral blood
lymphocytes. Unpublished report no. DuPont-22225. BioReliance, Rockville, MD, USA. Submitted to
WHO by EI du Pont de Nemours and Company, Wilmington, DE, USA.
Guilherme S, Gaivao I, Santos MA, Pacheco M (2012a). DNA damage in fish (Anguilla anguilla) exposed to a
glyphosate-based herbicide – elucidation of organ-specificity and the role of oxidative stress. Mutat Res.
743(1-2):1–9.
Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso-Coello P et al. (2008). GRADE: an emerging
consensus on rating quality of evidence and strength of recommendations. BMJ. 336:924–6.
Haag V (2008) 52-Week toxicity study by oral route (capsule) in beagle dogs. CIT, Evreux, France, Laboratory
study no. 29647 TTC. Sponsored by Nufarm Asia Sdn Bhd, Selangor, Malaysia. Unpublished study.
Haas MC (2010). An 8-week oral (diet and gavage) toxicity study of citric acid in male rats, WIL Research
Laboratory, Ashland, OH, USA. Laboratory project no. WIL-50361, Sponsor Monsanto Company on behalf
of glyphosate task force, dated 8 January 2010. Unpublished study.

270
Haas MC (2012). Glyphosate – A 28-day oral (dietary) immunotoxicity study in female B6C3F1 mice. WIL
Research Laboratories, LLC, Ashland, OH, USA, ImmunoTox, Inc., Virginia Bio Technology Research
Park, Richmond, VA, USA. Project no.: WI-10-460 (study no.: WIL-50393). Data owner: Monsanto,
Unpublished study.
Hadfield N (2012a). Glyphosate acid: In vitro absorption through abraded rabbit skin using [ 14C]glyphosate.
Dermal Technology Laboratory Ltd., Keele, Staffordshire, England, UK. Study no.: JV2182, Report no.:
JV2182-REG. Date: 2012-04-18. Unpublished study.
Hadfield N (2012b). Glyphosate: Glyphosate 360IPA salt (CA2273): In vitro penetration through human
epidermis using 14C-glyphosate. Dermal Technology Laboratory Ltd., Keele, Staffordshire, England, UK.
Study no.: JV2147, Report no.: JV2147-REG. Unpublished study.
Haferkorn J (2009a). Acute oral toxicity study of glyphosate TC in rats. LPT Laboratory of Pharmacology and
Toxicology, GmbH & Co., Hamburg, Germany. Laboratory report no. 23910, dated 16 June 2009.
Unpublished report.
Haferkorn J (2009b). Acute dermal toxicity study of glyphosate TC in CD rats. Laboratory report no 23912.
LPT Laboratory of Pharmacology and Toxicology, GmbH & Co., Hamburg, Germany. Unpublished report,
dated 16 June 2009
Haferkorn J (2009c). Acute inhalation toxicity study of glyphosate TC in rats.. LPT Laboratory of
Pharmacology and Toxicology GmbH & Co., Hamburg, Germany. Laboratory report no. 23911.
Unpublished report, dated 9 November 2009.
Haferkorn J (2009d). Examination of glyphosate TC in the skin sensitization test in guinea pigs according to
Magnusson and Kligman (Maximisation test). LPT Laboratory of Pharmacology and Toxicology GmbH &
Co., Hamburg, Germany. Laboratory report no. 23915. Unpublished report, dated 19 May 2009.
Haferkorn J (2010a). Acute oral toxicity study of glyphosate TC in rats. LPT Laboratory of Pharmacology and
Toxicology GmbH & Co., Hamburg, Germany. LPT Laboratory report no. 24602, dated 19 February 2010.
Unpublished report.
Haferkorn J (2010b). Acute oral toxicity study of glyphosate TC in rats. LPT Laboratory of Pharmacology and
Toxicology GmbH & Co., Hamburg, Germany. Laboratory report no. 24874. Unpublished report, dated 6
January 2010.
Haferkorn J (2010c). Acute dermal toxicity study of glyphosate TC in CD rats. LPT Laboratory of
Pharmacology and Toxicology GmbH & Co., Hamburg, Germany. Lab report no. 24876, dated 6 January
2010.
Haferkorn J (2010d). Acute dermal toxicity study of glyphosate TC in CD rats. LPT Laboratory of
Pharmacology and Toxicology GmbH & Co., Hamburg, Germany. Lab report No. 24604, dated 19
February 2010. Unpublished report.
Haferkorn J (2010e). Acute inhalation toxicity study of glyphosate TC in rats. LPT Laboratory of Pharmacology
and Toxicology GmbH & Co., Hamburg, Germany. Lab. report no. 24603, dated 3 June 2010. Unpublished
report.
Haferkorn J (2010f). Acute inhalation toxicity study of glyphosate TC in rats. LPT Laboratory of Pharmacology
and Toxicology GmbH & Co., Hamburg, Germany. Lab. report no. 24875, dated 3 June 2010. Unpublished
report.
Haferkorn J (2010g). Examination of glyphosate TC in the skin sensitization test in guinea pigs according to
Magnusson and Kligman (Maximisation Test). LPT Laboratory of Pharmacology and Toxicology GmbH &
Co., Hamburg, Germany. Laboratory report no. 24607, dated 19 April 2010. Unpublished report.
Haferkorn J (2010h). Examination of glyphosate TC in the skin sensitisation test in guinea pigs according to
Magnusson and Kligman (maximisation test). LPT Laboratory of Pharmacology and Toxicology GmbH &
Co., Hamburg, Germany. Data owner: Helm AG. Report no.: 24879, dated 22 January 2010. Unpublished
study.
Hardell L, Eriksson M (1999). A case-control study of non-Hodgkin lymphoma and exposure to pesticides.
Cancer. 85(6):1353–60.
Hardell L, Eriksson M, Nordstrom M (2002). Exposure to pesticides as risk factor for non-Hodgkin's lymphoma
and hairy cell leukemia: pooled analysis of two Swedish case-control studies. Leuk Lymphoma. 2002
May;43(5):1043–9.

271
Hardisty JF (2013). Pathology Working Group review of the histopathologic changes in the kidney: a combined
chronic toxicity/carcinogenicity study of AK-01 bulk substance [glyphosate] by dietary administration in
rats. Nippon Experimental Medical Research Institute Co., Ltd, Agatsuma, Gunma, Japan. Study no.: H-
95053, EPL project no.: 911-004, dated 7 October 2013. Unpublished study.
Hatakenaka N (1995). HR-001: Teratogenicity study in rats. Institute of Environmental Toxicology, Tokyo,
Japan. Data owner: Arysta LifeScience. Study no.: IET 94-0152. Unpublished study.
Hazelden KP (1992). AMPA: Teratogenicity study in rats. Unpublished report no 7891, IRI project no. 490421,
dated 29 December 1992, from Inveresk Research International, Tranent, Scotland, UK. Submitted to WHO
by Cheminova A/S, Lemvig, Denmark.
Heath J, Strutt A, Iswariah V (1993). AMPA: 4 Week dose range finding study in rats with administration by
gavage. Unpublished report no. 7803, IRI project no. 450860, dated 16 March 1993, from Inveresk
Research International, Tranent, Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Hecker M, Hollert H, Cooper R Vinggaard AM, Akahori Y, Murphy M et al. (2011). The OECD validation
program of the H295R steroidogenesis assay: phase 3. Final inter-laboratory validation study. Environ Sci
Pollut Res. 18:503–15.
Heenehan PR (1979a). Acute oral toxicity study in rats. Compound: glyphosate technical. Unpublished report
no. BDN-77-428, Bio/dynamics project no. 4880-77, dated 6 August 1979, from Bio/dynamics Inc., East
Heenehan PR (1979b). Acute dermal toxicity study in rabbits. Compound: glyphosate technical. Unpublished
report, no. BDN-77-428, Bio/dynamics project no. 4881-77, dated 6 August 1979, from Bio/dynamics Inc.,
East Millstone, NJ, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium.
Heenehan PR (1979c) Primary dermal irritation study in rabbits. Compound: glyphosate technical. Unpublished
report no. BDN-77-428, Bio/dynamics project No. 4883-77, dated 6 August 1979, from Bio/dynamics Inc.,
East Millstone, NJ, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium.
Heenehan PR (1979d). Rabbit eye irritation study. Compound: Glyphosate technical. Unpublished report no.
BDN-77-428, Bio/dynamics project no. 4882-77, dated 6 August 1979, from Bio/dynamics Inc., East
Heenehan PR, Rinehart WE, Braun WG (1979). Acute oral toxicity study in rats. Compound: glyphosate
technical. Unpublished report no. BDN-77-428, Bio/dynamics project No. 4880-77, dated 6 August 1979,
from Bio/dynamics Inc., East Millstone, NJ, USA. Submitted to WHO by Monsanto Int. Services SA,
Brussels, Belgium.
Heydens WF, Healy CE, Hotz KJ, Kier LD, Martens MA, Wilson AG et al. (2008). Genotoxic potential of
glyphosate formulations: Mode-of-action investigations. J Agric Food Chem. 56(4):1517–23.
Hideo U (1995a). HR-001: Primary dermal irritation study in rabbits. Institute of Environmental Toxicology,
Tokyo, Japan. Laboratory report no. IET 95-0035, dated 28 June 1995. Unpublished report.
Hideo U (1995b). HR-001: Primary eye irritation study in rabbits. Institute of Environmental Toxicology,
Tokyo, Japan. Report no.: IET 95-0034, Data owner: Arysta LifeScience, dated 29 June 1995. Unpublished
study.
Hideo U (1995c). HR-001: Dermal sensitisation study in guinea pigs. Institute of Environmental Toxicology,
Tokyo, Japan. Study no.: IET 95-0036, Data owner: Arysta LifeScience, dated 28 June 1995. Unpublished
study.
Higgins JP, Green S (editors). Cochrane handbook for systematic reviews of interventions version 5.1.0. The
Cochrane Collaboration, 2011. (www.cochrane-handbook.org.
http://handbook.cochrane.org/chapter_10/10_4_3_1_recommendations_on_testing_for_funnel_plot_asymm
etry.htm, accessed May 2016).
Higgs P (1994). Armoblen 557: Toxicity study by oral (gavage) administration to CD rats for four weeks:
Pharmaco LSR LTD, Suffox, UK, Final report. Project no.: AKL/247, 94/AKL247/0280, 94/0280, dated 4
July 1994. Unpublished study prepared by AKZO Chemicals BV, Amersfoort, Netherlands,
Hines CJ, Deddens JA, Jaycox LB, Andrews RN, Striley CAF, Alavanja MC (2008). Captan exposure and
evaluation of a pesticide exposure algorithm among orchard pesticide applicators in the Agricultural Health
Study. Ann Occup Hyg. 52:153–66. doi:10.1093/annhyg/men001.

272
Hoar SK, Blair A, Holmes FF, Boysen CD, Robel RJ, Hoover R et al. (1986). Agricultural herbicide use and
risk of lymphoma and soft-tissue sarcoma. JAMA. 256(9):1141–7.
doi:10.1001/jama.1986.03380090081023.
Hodge MCE (1996). First revision to glyphosate acid: 90 Day oral toxicity study in dogs. Unpublished report
no. CTL/P/1802, study no. PD 0674, dated 14 November 1996, from Zeneca Agrochemicals, Central Tox-
icology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Submitted to WHO by Syngenta
Crop Protection AG, Basel, Switzerland.
Hojo H. (1995). HR-001: A teratogenicity study in rabbits. Institute of Environmental Toxicology, Kodaira-shi,
Tokyo, Japan. Data owner: Arysta Life Sciences. Study no.: IET 94-0153. Unpublished study.
Holson J (2006). A developmental toxicity study of (inert ingredient) in rats. WIL Research Laboratories, Inc.,
Ashland, OH, Lab Project no.: WI/89/388, WIL/50097, 1663. Sponsor by Monsanto Company, dated 31
July 2006. Unpublished study.
Honarvar N (2005). Glyphosate technical – Micronucleus assay in bone marrow cells of the mouse. RCC
Cytotest Cell Research GmbH, Rossdorf, Germany. Data owner: Syngenta report no.: 1158500, dated 9
June 2008. Unpublished study.
Honarvar N (2008). Glyphosate technical – Micronucleus assay in bone marrow cells of the mouse. RCC
Cytotest Cell Research GmbH, Rossdorf, Germany. Data owner: Syngenta. Report no.: 1158500, dated 9
June 2008. Unpublished study.
Horner SA (1996a). Glyphosate acid: Acute neurotoxicity study in rats. Unpublished report no. CTL/P/4866,
study no. AR5968, dated 11 March 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory,
Basel, Switzerland.
Horner SA (1996b). Glyphosate acid: Subchronic neurotoxicity study in rats. Unpublished report no.
CTL/P/4867, study No. PR1009, dated 11 March 1996, from Zeneca Agrochemicals, Central Toxicology
Howe RK, Chott RC, McClanahan RH (1988). The metabolism of glyphosate in Sprague-Dawley rats. Part II.
Identification, characterisation and quantitation of glyphosate and its metabolites after intravenous and oral
administration. Unpublished report no. MSL-7206, study no. 206300, dated February 1988, from Monsanto
Environmental Health Laboratory, St. Louis, MO, USA and Monsanto Life Sciences Research Center,
Chesterfield, MO, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium.
Howe CM, Berrill M, Pauli BD, Helbing CC, Werry K, Veldhoen N (2004). Toxicity of glyphosate-based
pesticides to four North American frog species. Environ Toxicol Chem. 23(8):1928–38.
IARC (1987). 1,4-Dioxane. In: IARC monographs on the evaluation of carcinogenic risks to humans. Volume
71 Supplement 7. Overall evaluations of carcinogenicity: An updating of IARC monographs, Volumes 1 to
42. Lyon: International Agency for Research on Cancer. pp. 201.
(http://monographs.iarc.fr/ENG/Monographs/suppl7/Suppl7.pdf; accessed 8 November 2016)
Inoue T (2004). Micronucleus study in mice with AK-01 Technical. Kobuchisawa Laboratories, Fuji Biomedix
Co. Ltd, Japan. Study no. FBM 03-8152 (2), dated 11 December 2004. Submitted by TAC Group, Japan.
Unpublished study.
International Agency for Research on Cancer (2015). Volume 112: Some organophosphate insecticides and
herbicides: tetrachlorvinphos, parathion, malathion, diazinon and glyphosate. IARC Working Group. Lyon:
IARC Monogr Eval Carcinog Risk Chem Hum; 3–10 March 2015.
International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for
Human Use (2011). ICH Harmonised Tripartite Guideline: Guidance on genotoxicity testing and data
interpretation for pharmaceuticals intended for human use. S2(R1). Current Step 4 version dated 9
November 2011. Geneva: ICH.
Jauhiainen A, Räsänen K, Sarantila R, Nuutinen J, Kangas J (1991). Occupational exposure of forest workers to
glyphosate during brush saw spraying work. Am Ind Hyg Assoc J. 52:61–4.
Jensen JC (1991a). Mutagenicity test: Ames Salmonella assay with glyphosate, batch 206-JaK-25-1. Laboratory
report no. 12323, dated 10 September 1991, from Scantox A/S, Lille Skensved, Denmark. Submitted to
WHO by Cheminova A/S, Lemvig, Denmark. Unpublished report.

273
Jensen JC (1991b). Mutagenicity test: in vitro mammalian cell gene mutation test with glyphosate, batch 206-
JaK-25-1. Unpublished report. Laboratory report no. 12325, dated 10 September 1991, from Scantox A/S,
Lille Skensved, Denmark. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Jensen JC (1991c). Mutagenicity test: micronucleus test with glyphosate, batch 206-JaK-25-1. Unpublished
report. Laboratory report no. 12324, dated 12 September 1991, from Scantox A/S, Lille Skensved,
Denmark. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Jensen JC (1993a). Mutagenicity test: Ames Salmonella test with AMPA, batch 286-JRJ-73-4. Unpublished
report. Laboratory report no. 13269, dated 18 February 1993, from Scantox A/S, Lille Skensved, Denmark.
Jensen JC (1993b). Mutagenicity test: In vitro mammalian cell gene mutation test performed with mouse
lymphoma cells (L5178Y). Test compound: AMPA, batch 286-JRJ-73-4. Unpublished report. Laboratory
report no. 13270, dated 18 February 1993, from Scantox A/S, Lille Skensved, Denmark. Submitted to
WHO by Cheminova A/S, Lemvig, Denmark.
Jensen JC (1993c). Mutagenicity test: Micronucleus test with AMPA, batch 286-JRJ-73-4. Unpublished report
Laboratory report no. 13268, dated 18 February 1993, from Scantox A/S, Lille Skensved, Denmark.
Johnson AJ (1996). Glyphosate acid: Acute delayed neurotoxicity study in the domestic hen. Unpublished report
No. CTL/C/3122, project no. ISN 361/960244, dated 23 August 1996, from Huntingdon Life Sciences Ltd.,
Huntingdon, England, UK. Submitted to WHO by Syngenta Crop Protection AG, Basel, Switzerland.
Johnson AJ (1982). Glyphosate: 21-Day dermal toxicity study with rabbits. International Research and
Development Corporation. Sponsored by Monsanto, St. Louis, MO, USA. Monsanto no.: IR-81-195.
Unpublished study.
Johnson IR (1997). Glyphosate acid: Eye irritation to the rabbit. Unpublished report no. CTL/P/5138, study no.
FB5378, dated 18 March 1997, from Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley
Switzerland.
Jones E (1999). Potassium salt of glyphosate: Mouse bone marrow micronucleus test. Zeneca Agrochemicals,
Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, United Kngdom. Report
no. CTL/P/6244, Sponsored by Zeneca Agrochemicals, Surrey, England, UK. Unpublished study.
among pesticide applicators exposed to the organophosphate insecticide diazinon in the Agricultural Health
Study: an updated analysis. Occup Environ Med. 72:496-503. doi: 10.1136/oemed-2014-102728.
Kamijo Y, Takai M, Sakamoto T (2016). A multicenter retrospective survey of poisoning after ingestion of
herbicides containing glyphosate potassium salt or other glyphosate salts in Japan. Clin Toxicol (Phila).
54:147–51. http://dx.doi.org/10.3109/15563650.2015.1121271.
Kennelly JC (1990). ICIA 0224 :Assessment for the induction of unscheduled DNA synthesis in rat hepatocytes
in vivo. ICI Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, UK. Report
no. CTL/P/2789, dated 7 February 1990. Sponsored by ICI Agrochemicals. Submitted by Syngenta, Basel,
Kier LD (1978). Final report on Salmonella mutagenicity assay of glyphosate. Unpublished report no. LF-78-
161, dated 23 October 1978. From Monsanto Environmental Health Laboratory, St. Louis, MO, USA.
Kier LD, Kirkland DJ (2013). Review of genotoxicity studies of glyphosate and glyphosate-based formulations.
Crit Rev Toxicol. 43(4):283–315.
Kier LD, Stegeman SD (1993). Mouse micronucleus study of AMPA. Report no. MSL-13243, dated 8
December 1993. Monsanto Environmental Health Laboratory, St. Louis, MO, USA. Unpublished report.
Kier LD, Flowers LJ, Huffman MB (1992). Mouse micronucleus study of RODEO herbicide formulation. EHL
study no. 91201/91205, dated 25 February 1992. Monsanto Environmental Health Laboratory, St. Louis,
MO, USA. Unpublished report.
Kier LD, Stegeman SD, Costello JG, Schermes S (1992). Ames/Salmonella mutagenicity assay of RODEO.
EHL study no. 91184, dated 7 February 1992. Monsanto Environmental Health Laboratory, St. Louis, MO,
USA. Unpublished report.

274
Kinoshita M (1995). HR-001: 13-Week subchronic oral toxicity study in rats. The Institute of Environmental
Toxicology, Tokyo, Japan. Laboratory project ID IET-94-0138, dated 20 July 1995. Sponsored by Sankyo
Co., Ltd., Tokyo, Japan. Unpublished study.
Knapp J (2007). Reproduction/developmental toxicity screening study of MON 0818 in Rats. WIL Research
Laboratories, LLC., Ashland, OH, USA. Study no. WIL-50282, dated 4 January 2007. Unpublished study.
Knapp JF (2008). A combined 28-day repeated dose oral (dietary) toxicity study with the
reproduction/developmental toxicity screening test of MON 8109 and MON 0818 in rats. WIL Research
Laboratories, LLC., Ashland, OH, USA. Study no. WIL-50337, dated 3 April 2008. Unpublished study.
Knezevich A, Hogan G (1983). A chronic feeding study of glyphosate (Roundup Technical) in mice: Project no.
77-2061: Monsanto report BDN-77- 420, dated 21 July 1983. Unpublished study.
Knowles SL, Mookherjee CR (1996). [14C]Glyphosate: Absorption, distribution, metabolism and excretion
following oral administration to the rat. Corning Hazleton Europe, Harrogate, North Yorkshire, England,
UK. Data owner: Nufarm. Unpublished report no.: 1413/2-1011, dated 23 October 1996.
Koakoski G, Quevedo RM, Ferreira D, Oliveira TA, da Rosa JG, de Abreu MS et al. (2014). Agrichemicals
chronically inhibit the cortisol response to stress in fish. Chemosphere. 112:85–91.
doi.org/http://dx.doi.org/10.1016/j.chemosphere.2014.02.083.
Koichi E (1995). HR-001: Acute inhalation toxicity study in rats. Institute of Environmental Toxicology, Tokyo,
Japan. Laboratory project ID IET 94-0155. Unpublished report.
Kojima H, Takeuchi S, Nagai T (2010). Endocrine disrupting potential of pesticides via nuclear receptors and
aryl hydrocarbon receptor. J Health Sci. 56(4):374–86. doi:10.1248/jhs.56.374.
Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K (2004). Screening for estrogen and androgen
receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells.
Environ Health Perspect. 112(5):524–31. doi:10.1289/ehp.6649.
Koller VJ, Furhacker M, Nersesyan A, Misik M, Eisenbauer M, Knasmueller S (2012). Cytotoxic and DNA-
damaging properties of glyphosate and Roundup in human-derived buccal epithelial cells. Arch Toxicol.
86:805-13.
Komura H (1995a). HR-001: Acute oral toxicity study in mice. USEPA FIFRA Guideline Subdivision F, 81-1.
Institute of Environmental Toxicology, Tokyo, Japan. Laboratory project ID no. IET 94-0133, dated 20
February 1995. Unpublished report.
Komura H (1995b). HR-001: Acute oral toxicity study in rats. Institute of Environmental Toxicology, Tokyo,
Japan. Laboratory Project no. IET 94-0134, dated 20 February 1995. Unpublished report..
Komura H (1995c). HR-001: Acute dermal toxicity study in rats. Institute of Environmental Toxicology, Tokyo,
Japan. Laboratory project ID IET 94-0154, dated 14 March 1995. Unpublished report.
Komura H (1996). AMPA: Acute oral toxicity study in mice. Institute of Environmental Toxicology, Tokyo,
Japan. Laboratory project ID no. IET 96-0075, dated 11 November 1996. Sponsor: Sankyo Co., Ltd, Tokyo,
Japan. Unpublished study.
Koureas M, Tsezou A, Tsakalof A, Orfanidou T, Hadjichristodoulou C (2014). Increased levels of oxidative
DNA damage in pesticide sprayers in Thessaly Region (Greece). Implications of pesticide exposure. Sci
Total Environ. 496:358–64.
doi:10.1093/aje/kws225.
Koutros S, Silverman DT, Alavanja MC, Andreotti G, Lerro CC, Heltshe S et al. (2015). Occupational exposure
to pesticides and bladder cancer risk. Int J Epidemiol. 45(3):792–805. doi: 10.1093/ije/dyv195.
Krüger M, Shehata AA, Schrödl W, Rodloff (2013). Glyphosate suppresses the antagonistic effect of
Enterococcus spp. on Clostridium botulinum. Anaerobe. 20:74–8.
Kryuchkova YV, Burygin GL, Gogoleva NE, Gogolev YV, Chernyshova MP, Makarov OE et al. (2014).
Isolation and characterization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7.
Microbiol Res. 169(1):99–105. doi: 10.1016/j.micres.2013.03.002.

275
Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT et al. (1998). Interaction of
estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology. 139:4252–63.
Kumar DP (2001). Carcinogenicity study with glyphosate technical in swiss albino mice. Toxicology
Department, Rallis Research Centre, Rallis India Limited, Bangalore, India. Data owner: Feinchemie
Schwebda GmbH. Study no.: Toxi:1559.CARCI-M. Unpublished study.
Kumar S, Khodoun M, Kettleson EM, McKnight C, Reponen T, Grinshpun SA et al. (2014). Glyphosate-rich air
samples induce IL-33, TSLP and generate IL-13 dependent airway inflammation. Toxicology. 325:42–51.
Kuwahara M (1995). HR-001: 13-Week subchronic oral toxicity study in mice. Institute of Environmental
Toxicology, Tokyo, Japan. Laboratory report no.: IET 94-0136, dated 24 April 1995. Data owner: Arysta
LifeScience. Unpublished study.
Lanctot C, Navarro-Martin L, Robertson C, Park B, Jackman P, Pauli BD et al. (2014). Effects of glyphosate-
based herbicides on survival, development, growth and sex ratios of wood frog (Lithobates sylvaticus)
tadpoles. II: agriculturally relevant exposures to Roundup WeatherMax® and Vision® under laboratory
conditions. Aquat Toxicol. 154:291–303. http://dx.doi.org/10.1016/j.aquatox.2014.05.025.
Lankas GR (1981). A lifetime feeding study of glyphosate (ROUNDUP Technical) in rats. Bio/dynamics Inc.,
East Millstone, NJ, USA. Data owner: Monsanto. Study/project no.: 77-2062 (BDN 77-416). Unpublished
study.
Lau J, Ioannidis JP, Terrin N, Schmid CH, Olkin I (2006). The case of the misleading funnel plot. BMJ.
333(7568):597–600.
Lavy TL, Cowell JE, Steinmetz JR, Massey JH (1992). Conifer seedling nursery worker exposure to glyphosate.
Arch Environ Contam Toxicol. 22:6–13.
Lawlor TE (2000). Mutagenicity test with MON 59112 in the Salmonella-Escherichia coli/mammalian-
microsome reverse mutation assay with a confirmatory assay. Covance Laboratories Inc., Vienna, VA,
USA. Covance study no. 19022-0-409OECD; Monsanto study no. HL-97-235, dated 22 May 2000.
Unpublished study.
Leah AM (1988). Aminomethyl phosphonic acid: acute oral toxicity to the rat. Unpublished report no.
CTL/P/2266, study no. AR4690, dated 26 August 1988, from ICI Agrochemicals, Central Toxicology
Lee WJ, Cantor KP, Berzofsky JA, Zahm SH (2004). Non hodgkin’s lymphoma among asthmatics exposed to
pesticides. Int J Cancer. 111:298–302.
Lee HI, Chen KW, Chi CH, Huang JJ, Tsai LM (2000). Clinical presentations and prognostic factors of a
glyphosate-surfactant herbicide intoxication: a review of 131 cases. Acad Emerg Med. 7:906–10.
Lerro CC, Koutros S, Andreotti G, Friesen MC, Alavanja MC, Blair A et al. (2015). Organophosphate
Leuschner PJ (2002a). Acute toxicity of AMPA (aminomethyl phosphonic acid) in CD rats by dermal
administration-Limit test. LPT Laboratory of Pharmacology, Hamburg, Germany. LPT report no. 16168/02.
Sponsored by Agan Chemical Manufacturers Ltd., Ashdod, Israel.
Leuschner PJ (2002b). Examination of AMPA (aminomethyl phosphonic acid) in the skin sensitization test in
guinea pigs according to Magnusson and Kligman (Maximisation test). LPT Laboratory of Pharmacology,
Hamburg, Germany. LPT report no. 16169/02. Sponsored by Agan Chemical Manufacturers Ltd., Ashdod,
Israel.
Leuschner PJ (2009a). Acute dermal irritation/corrosion test (Patch Test) of glyphosate TC in rabbits. LPT
Laboratory of Pharmacology and Toxicology GmbH & Co., Hamburg, Germany. Laboratory report no.
LPT report no. 24877, dated 27 November 2009. Unpublished report.
Leuschner PJ (2009b). Acute eye irritation/corrosion test of glyphosate TC in rabbits. LPT Laboratory of
Pharmacology and Toxicology GmbH & Co., Hamburg, Germany. LPT report no. 24878, dated 27
November 2009. Unpublished report.

276
Leuschner PJ (2009c). Acute dermal irritation/corrosion test (patch test) of glyphosate TC in rabbits. LPT
Laboratory of Pharmacology and Toxicology GmbH & Co., Hamburg, Germany. LPT report no. 23913,
dated 30 April 2009. Unpublished report.
Leuschner J. (2009d). Acute eye irritation/corrosion test of glyphosate TC in rabbits. LPT Laboratory of
Pharmacology and Toxicology, GmbH & Co. KG, Hamburg, Germany. Data owner: Helm AG. Report no.:
LPT 23914, dated 30 April 2009. Unpublished study.
Leuschner PJ (2010a). Acute dermal irritation/corrosion test (Patch Test) of glyphosate TC in rabbits. LPT
Laboratory of Pharmacology and Toxicology GmbH & Co., Hamburg, Germany. LPT report no. 24605,
dated 6 January 2010. Unpublished report.
Leuschner J (2010b). Acute eye irritation/corrosion test of glyphosate TC in rabbits. LPT Laboratory of
Pharmacology and Toxicology GmbH & Co. Hamburg, Germany. Data owner: Helm AG. LPT report no.
24606 - 185, dated 6 January 2010. Unpublished study.
Li P, Long TJ (1988). An evaluation of the genotoxic potential of glyphosate. Fundam Appl Toxicol. 10:537–
46.
Lima Dallago BS (2008). Skin sensitisation test for glyphosate technical in guinea pigs. Buehler Test. Bioagri
Laboratórios, Sao Paulo, Brazil. Data owner: Helm AG report no.: RF-3996.318.431.07, dated 30
Lioi MB, Scarfi MR, Santoro A, Barbieri R, Zeni O, Di Berardino D et al. (1998a). Genotoxicity and oxidative
stress induced by pesticide exposure in bovine lymphocyte cultures in vitro. Mutat Res. 403(1-2):13–20.
Lioi MB, Scarfi MR, Santoro A, Barbieri R, Zeni O, Salvemini F et al. (1998b). Cytogenetic damage and
induction of pro-oxidant state in human lymphocytes exposed in vitro to gliphosate, vinclozolin, atrazine,
and DPX-EP636. Environ Mol Mutagen. 32:39–46.
Lopez MS, Monroy CM, Sicard DM, Groot H (2005). [Cytotoxicity and genotoxicity of human cells exposed in
vitro to glyphosate]. Biomédica: revista del Instituto Nacional de Salud. 25:335-45 (in Spanish).
Lueken A, Juhl-Strauss U, Krieger G, Witte I (2004). Synergistic DNA damage by oxidative stress (induced by
H2O2) and nongenotoxic environmental chemicals in human fibroblasts. Toxicol Lett. 147:35–43.
MacKenzie SA (2007) IN-MCX20: Subchronic toxicity 90-day feeding study in rats. Unpublished report no.
DuPont-19008. DuPont Haskell Laboratory, Newark, DE, USA. Submitted to WHO by EI du Pont de
Nemours and Company, Wilmington, DE, USA, dated 22 February 2007. Unpublished study.
Macpherson D (1996). Glyphosate acid: biotransformation in the rat. Unpublished report no. CTL/P/5058, dated
28 June 1996, from Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley Park, Macclesfield,
Cheshire, England, UK. Submitted to WHO by Syngenta Crop Protection AG, Basel, Switzerland.
Maibach HI (1983). (a) Elimination of 14C-glyphosate in Rhesus monkeys following a single parenteral dose. (b)
Percutaneous absorption of 14C-glyphosate in Roundup formulation in Rhesus monkeys following a single
topical dose. Unpublished report No. MA-81-349, dated 1 April 1983, from University of California,
School of Medicine; San Francisco, CA, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels,
Belgium.
Majeska JB (1982). SC-0224 Mutagenicity evaluation in bone marrow cytogenetic analysis in rats. Report No.
T-10884, dated 9 September 1982. The In Vitro Toxicology Section, Environmental Health Center, Stauffer
Chemical Company, Farmington, CT, USA. Submitted by Syngenta, Basel, Switzerland. Unpublished
study.
Majeska JB (1985). Mutagenicity evaluation in Chinese Hamster Ovary Cytogenic assay. The In Vitro
Toxicology Section, Environmental Health Center, Stauffer Chemical Company, Farmington, CT, USA.
Report no. T-12663 SC-0224, dated 18 December 1985. Submitted by Syngenta, Basel, Switzerland.
Unpublished study.
Majeska JB (1986). SC-0224 (Lot no. JHC 8865-20-1) mutagenicity evaluation in bone marrow micronucleus.
The In Vitro Toxicology Section, Stauffer Chemical Company, Farmington, CT, USA. Report no. T-12589,
dated 8 April 1986. Submitted by Syngenta. Unpublished study.
Manas FL, Peralta L, Raviolo J, Ovando HG, Weyers A, Ugnia L et al. (2009a). Genotoxicity of glyphosate
assessed by the comet assay and cytogenetic tests. Environ Toxicol Pharmacol. 28(1):37–41.

277
Manas F, Peralta L, Raviolo J, Ovando HG, Weyers A, Ugnia L et al. (2009b). Genotoxicity of AMPA, the
environmental metabolite of glyphosate, assessed by the Comet assay and cytogenetic tests. Ecotoxicol
Environ Saf. 72(3):834–7.
Mandel JS, Alexander BH, Baker BA, Acquavella JF, Chapman P, Honeycutt R (2005). Biomonitoring for farm
families in the Farm Family Exposure Study. Scan J Work Environ Health. 31(Suppl 1): 98–104.
Matheson DW (1982). SC-0224 (Lot no. 6841-48-3) Mutagenicity evaluation in bone marrow
micronucleuscytogenetic analysis in rats. The In Vitro Toxicology Section, Stauffer Chemical Company,
Farmington, CT, USA. Report no. T-10884. Submitted by Syngenta. Unpublished study.
Matsumoto K (1995) HR-001: In Vitro Cytogenetics Test. The Institute of Environmental Toxicology, Tokyo,
Japan. Report no. IET 94-0143. Sponsored by Sankyo Co., Tokyo, Japan.
Mavournin KH, Blakey DH, Cimino MC, Salamone MF, Heddle JA (1990). The in vivo micronucleus assay in
mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency
Gene-Tox Program. Mutation Res. 239:29–80.
McEwen AB (1995). HR-001: Metabolism in the rat. Huntingdon Research Centre Ltd., Huntingdon,
Cambridgeshire, England, UK. Data owner: Arysta Lifescience SAS, Report No.: SNY 332/951256, dated
16 August 1995. Unpublished study no. ASB2012-11379.
Mecchi MS (2004). Salmonella–Escherichia coli/mammalian-microsome reverse mutation assay with a
confirmatory assay with N-acetyl-glyphosate. Unpublished report no. Covance 7535-101. Covance
Laboratories, Inc., Vienna, VA, USA. Submitted to WHO by EI du Pont de Nemours and Company,
Wilmington, DE, USA.
Merkel DJ (2005a). Acute oral toxicity up and down procedure in rats. Product Safety Laboratories, Dayton, NJ,
USA. Laboratory study no. 15274, dated 4 April 2005. Unpublished report.
Merkel DJ (2005b). Acute dermal toxicity study in rats – Limit Test. Product Safety Laboratories, Dayton, NJ,
USA. Laboratory report no. 15275, dated 4 April 2005. Unpublished report.
Merkel DJ (2005c). Acute inhalation toxicity study in rats – Limit Test. Product Safety Laboratories, Dayton,
NJ, USA. Laboratory report no. 15276, dated 4 April 2005. Unpublished report
Merkel DJ (2005d). Primary skin irritation study in rabbits. Product Safety Laboratories, Dayton, NJ, USA.
Laboratory report no. 15278, dated 14 April 2005. Unpublished report.
Merkel D (2005e) Eye irritation/corrosion effects in rabbits (Oryctolagus cuniculus) of glyphosate 95 TC
Product Safety Laboratories, Dayton, NJ, USA. Data owner: Helm AG, report no.: PSL 15277 dated 4 April
2005. Unpublished study.
Merkel D (2005f). Glyphosate acid technical – Dermal sensitization in guinea pigs (Buehler Method). Product
Safety Laboratories, Dayton, NJ, USA. Data owner: Helm AG report no.: PSL 15279, dated 4 April 2005.
Unpublished study.
Milburn GM (1996). Glyphosate acid: One year dietary toxicity study in rats. Unpublished report no.
CTL/P/5143. Study no. PR 1012, dated 2 October 1996. From Zeneca Agrochemicals, Central Toxicology
(UFW), 1988–2001. Cancer Causes Control. 16(7):823–30. doi:10.1007/s10552-005-2703-2.
Ming Z, Ting H, Yiping Y, Caigao Z, Lan G, Wang A et al. (2014). Cytotoxicity of glyphosate to GC-1 mice
spermatogonium and antagonistic effects of N-acetylcysteine. Asian J Ecotoxicol. 9(1):159–66.
doi:10.7524/AJE.1673-5897. 20130906001.
Miyaji CK (2008): Evaluation of the mutagenic potential of the test substance Glyphosate Technical by reverse
mutation assay in Salmonella typhimurium (Ames Test). Bioagri Laboratorios, Sao Paulo, Brazil. Study no.
3996.401.392.07, RF-3996.401.392.07, dated 15 September 2008. Sponsored by Jingma Chemicals Co.,
Ltd, Zhejiang, China. Unpublished study.

278
Mizuyama K (1987). Irritating effect of glyphosate, surfactant and roundup on stomach and small intestine in
dogs. University of Tsukuba, Clinic medicine, Tsukuba, Japan. Unpublished study.
Mladinic M, Berend S, Vrdoljak AL, Kopjar N, Radic B, Zeljezic D (2009). Evaluation of genome damage and
its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro. Environ Mol Mutagen.
50(9):800–7.
Moore GE (1999). NUP5a99 62% glyphosate MUP: Acute oral toxicity study in rats – Limit test Product Safety
Labs, NJ, USA study no.: 7907, dated 16 September 1999, Unpublished study.
Morgan RL, Thayer KA, Bero L, Bruce N, Falck-Ytter Y, Ghersi D et al. (2016). GRADE: Assessing the
quality of evidence in environmental and occupational health. Environ Int. 92–3:611–6. doi:
10.1016/j.envint.2016.01.004.
Moxon ME (1996a). Glyphosate acid: Developmental toxicity study in the rat. Unpublished report
No.CTL/P/4819, study No. RR0690, dated 27 March 1996, CTL/P/4819/amendment-001, dated 20
November 2002, from Zeneca Agrochemicals, Central Toxicology Laboratory, Alderley Park,
Macclesfield, Cheshire, England, UK. Submitted to WHO by Syngenta Crop Protection AG, Basel,
Moxon ME (1996b). Glyphosate acid: Developmental toxicity study in the rabbit. Unpublished report no.
CTL/P/5009, study no. RB0709, dated 2 July 1996, from Zeneca Agrochemicals, Central Toxicology
Moxon ME (2000). Glyphosate acid: Multigeneration reproduction toxicity study in rats. Unpublished report no.
CTL/P/6332, study no. RR0784, dated 16 June 2000, from Zeneca Agrochemicals, Central Toxicology
Mueller MM, Rosenberg C, Siltanen H, Wartiovaara T (1981). Fate of glyphosate and its influence on nitrogen-
cycling in two Finnish agriculture soils. Bull Environ Contamin Toxicol. 27:724–30.
Murli H (2004). Chromosomal aberrations in Chinese hamster ovary (CHO) cells. Unpublished report no.
Covance 7535-102. Covance Laboratories, Inc., Vienna, VA, USA. Submitted to WHO by EI du Pont de
Myhr BC (2000). Mutagenicity test on MON 59112 in the in vivo mouse micronucleus assay. Covance
Laboratories, Inc., Covance, Vienna, VA, USA. Project no.: Covance 19022-0-455OECD, dated 31 May
2000. MRID 46930503. Unpublished study.
Nachman KE, Fox MA, Sheehan MC, Burke TA, Rodricks JV, Woodruff TJ (2011). Leveraging epidemiology
to improve risk assessment. Open Epidemiol J. 4:3–29.
Nagy K (2011). Glyphosate technical – Acute inhalation toxicity study (nose-only) in the rat. LAB Research
Ltd., Szabadsagpuszta, Hungary. Lab report no. 11/054-004P, dated 6 June 2011. Unpublished report.
Nakashima N (1997). HR-001: 12-month oral chronic toxicity study in dogs. Institute of Environmental
Toxicology, Tokyo, Japan. Study no.: IET 94-0157. Data owner: Arysta LifeScience, dated 20 May 1997.
Unpublished study.
Nascimento A, Grisolia CK (2000). Analise Comparativa Entre os Testes de Micronucleos em Camundongos E
em Eritrocitos Perifericos de Oreochromis niloticus na Avaliacao do Potencial Mutagenico dos Agrotoxicos
Deltametrian, Dicofol, Glifosato E Imazapyr. Pesticidas R. Ecotoxicol. E Meio Ambiente, Curitiba, v. 10, p.
41-48 jan/dez 2000.
Navarro CD, Martinez CB (2014). Effects of the surfactant polyoxyethylene amine (POEA) on genotoxic,
biochemical and physiological parameters of the freshwater teleost Prochilodus lineatus. Comp Biochem
Physiol C Toxicol Pharmacol. 165:83–90.
Naydenova E, Troev K, Topashka-Ancheva M, Hagele G, Ivanov I, Kril A (2007). Synthesis, cytotoxicity and
clastogenicity of novel alpha-aminophosphonic acids. Amino Acids. 33(4):695–702.
Negro Silva LF (2009). Glyphosate A17035A-mammalian erythrocyte micronucleus test. TECAM Technologia
Ambiental Sao Roques Ltd. Sao Roque, SP, Brazil. Report no. RL 7459/2008-14.0MN-B, Study no.
7459/2008-14.0MN, dated January 2009. Sponsored by Syngenta Protecao de Cultivos, Ltda., Sao Paulo,
Brazil. Unpublished study.

279
Negro Silva LF (2011). Glyphosate: Glyphosate SL (A13013Z)-mammalian erythrocyte micronucleus test.

TECAM Technologia Ambiental Sao Roque Ltda, Sao Roque, SP, Brasil. Report no. RL69575MN-B,
February 18, 2011. Sponsored by Syngenta Protecao De Cultivos LTDa, Sao Paulo-SP, Brazil, Submitted
by Syngenta, Basel, Switzerland. Unpublished study.
Nesslany F (2002). Measurement of unscheduled DNA synthesis (UDS) in rat hepatocyte using an in vitro
procedure with AMPA (aminomethylphosphonic acid). Institut Pasteur DElille, Genetic Toxicology
Laboratory, Lille Cedex. Report no. IPL-R 020625/AMPA (aminomethylphosphonic acid)/Calliope SAS.
Sponsored by Mrs. Florence Leconte. Unpublished study.
Nippon Experimental Medical Research Institute (2012). Responses to Inquiries from Pesticide Expert Panel,
Food Safety Commission of Japan (held on 20 December 2012).
Nord PJ (2008). A combined 28-day repeated dose oral (dietary) toxicity study with the
reproduction/developmental toxicity screening test of MON 8109 and MON 0818 in rats- Sub-report on
analysis of dietary formulations. Monsanto, St. Louis, MO, USA. Study no.: WI-2007-013, dated 17
January 2008. Unpublished study.
Nordström M, Hardell L, Magnuson A, Hagberg H, Rask-Andersen A. Occupational exposures, animal
exposure and smoking as risk factors for hairy cell leukaemia evaluated in a case-control study. Br J
Cancer. 1998 Jun;77(11):2048–52.
NTP (2016). Report on Carcinogens, Fourteenth Edition. 1,4-Dioxane CAS No. 123-91-1. Research Triangle
Park, NC: National Toxicology Program. (http://ntp.niehs.nih.gov/ntp/roc/content/profiles/dioxane.pdf;
accessed 3 December 2016).
OECD (2014). OECD guidelines for testing of chemicals Section 4: Mammalian erythrocyte micronucleus test.
TG 474. Paris: Organisation for Economic Cooperation and Development; pp. 21. doi:
http://dx.doi.org/10.1787/9789264224292-en.
Ogrowsky D (1989). Four-week feeding study of (inert ingredient) in Sprague-Dawley rats. Monsanto
Environmental Health Laboratory, St. Louis, MO, USA. Project no.: ML/88/273, MSL/9238, 1663, dated
31 July 2006. Unpublished study.
Omoike OE, Lewis RC, Meeker JD (2015). Association between urinary biomarkers of exposure to
organophosphate insecticides and serum reproductive hormones in men from NHANES 1999–2002. Reprod
Toxicol. 53:99–104.
Orsi L, Delabre L, Monnereau A, Delval P, Berthou C, Fenaux P et al. (2009). Occupational exposure to
pesticides and lymphoid neoplasms among men: results of a French case-control study. Occup Environ
Med. 66(5):291–8. doi: 10.1136/oem.2008.040972.
Osheroff M. (1991). 13-Week subchronic toxicity study in dogs with ATMER 163. Hazelton Washington, Inc.,
Vienna, VA, USA. HWA study no. 564-164, dated 18 April 1991 (study completion date). Unpublished
study.
Paganelli A, Gnazzo V, Acosta H, López SL, Carrasco AE (2010). Glyphosate-based herbicides produce
teratogenic effects on vertebrates by impairing retinoic acid signalling. Chem Res Toxicol. 23(10):1586–95.
doi:10.1021/tx1001749.
Cancer. 131(11):2650–9. doi:10.1002/ijc.27522.
Parker RM (1993). 90 Day range finding study of glyphosate in rats. TSI Redfield Laboratories, Redfield, AR,
USA. Laboratory project no. 011-0001. Submitted/sponsored by by Alkaloida Co. Ltd., Hungary.
Unpublished study.
Patel NN (2012). Micronucleus test of glyphosate TGAI in mice. Jai Research Foundation, Toxicology and
Environmental Research Consulting (TERC), Valsad, Gujarat India. Study no. 120709, 485-1-06-4696,
dated 13 September 2012. Sponsored by Dow AgroScience LLC, Indianapolis, IN, USA. Unpublished
study.
Pavkov KL, Turnier JC (1987). Two-year chronic toxicity and oncogenicity dietary study with SC-0224 in mice.
Stauffer Laboratory Farmington, CT, USA. Study no. T-11813. Unpublished study. Submitted by Syngenta,
Basel, Switzerland.

280
Pavkov KL, Wyand S (1987). Two-year chronic toxicity and oncogenicity dietary study with SC-0224 in rats.
Stauffer Laboratory, Farmington, CT, USA. Study no. T-11082. Unpublished study. Submitted by
Syngenta, Basel, Switzerland.
Paz-y-Mino C, Sanchez ME, Arevalo M, Munoz MJ, Witte T, De-la-Carrera GO, Leone PE (2007). Evaluation
of DNA damage in an Ecuadorian population exposed to glyphosate. Genet Mol Biol. 30(2):456–60.
Paz-y-Mino C, Munoz MJ, Maldonado A, Valladares C, Cumbal N, Herrera C et al. (2011). Baseline
determination in social, health, and genetic areas in communities affected by glyphosate aerial spraying on
the northeastern Ecuadorian border. Rev Environ Health. 26(1):45–51.
Perry CJ, Atkinson C, Strutt A, Hudson P, Jones M (1991a). Glyphosate: 13 Week dietary toxicity study in
mice. Unpublished report no 7024, IRI project no. 437918, dated 7 March 1991, from Inveresk Research
International, Tranent, Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Perry CJ, Atkinson C, Strutt A, Henderson W, Hudson P (1991b). Glyphosate: 13 Week dietary toxicity study in
rats. Unpublished report no. 7136, IRI project no. 437876, dated 7 March 1991, from Inveresk Research
International, Musselburgh, Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig, Denmark.
Pesatori AC, Sontag JM, Lubin JH, Consonni D, Blair A (1994). Cohort mortality and nested case-control study
5(4):310–8. doi:10.1007/BF01804981.
Piccolo A, Celano G, Conte P (1996). Adsorption of glyphosate by humic substances. J Agric Food Chem.
44:2442–6.
Piccolo A, Gatta L, Campanella L (1995). Interactions of glyphosate herbicide with a humic acid and its iron
complex. Ann Chim. 85:31–40.
Piesova E (2004) The influence of different treatment length on the induction of micronuclei in bovine
lymphocytes after exposure to glyphosate. Folia Veterinaria. 48(3):130–4.
Piesova E (2005). The effect of glyphosate on the frequency of micronuclei in bovine lymphocytes in vitro. Acta
veterinaria (Beograd). 55(2):101–9. doi: 10.2298/AVB0503101P.
Pinto PJ (1996). Glyphosate acid: 21 Day dermal toxicity study in rats. Central Toxicology Laboratory, Alderley
Park, Macclesfield, Cheshire, England, UK. Report no.: CTL/P/4985, dated 24 June 1996. Data owner:
Syngenta. Unpublished study.
Powles P (1992a). 14C-Glyphosate: Absorption and distribution in the rat – preliminary study. Unpublished
report no. 6365-676/1, dated 10 June 1992, from Hazleton UK, Harrogate, England, UK. Submitted to
Powles P (1992b). 14C-Glyphosate: absorption, distribution, metabolism, and excretion in the rat. Unpublished
report no. 7006-676/2, dated 30 June 1992, from Hazleton UK, Harrogate, England, UK. Submitted to
Prakash PJ (1999). Subchronic (90 day) oral toxicity study with glyphosate technical in beagle dogs. Test
compound: Glyphosate technical. Rallis Research Center, Rallis India Limited, Banglore, India. Study no.
1816. Sponsored by M/s Feinchemie Schwebda GmbH, Koln, Germany. Unpublished study.
Prasad S, Srivastava S, Singh M, Shukla Y (2009). Clastogenic effects of glyphosate in bone marrow cells of
swiss albino mice. J Toxicol. 2009:308985. doi: 10.1155/2009/308985.
Raipulis J, Toma MM, Balode M (2009). Toxicity and genotoxicity testing of Roundup. Proceedings of the
Latvian Academy of Sciences, Section B. Nat Exact Appl Sci. 63(1/2):29–32.
Rank J, Jensen AG, Skov B, Pedersen LH, Jensen K. (1993). Genotoxicity testing of the herbicide Roundup and
its active ingredient glyphosate isopropylamine using the mouse bone marrow micronucleus test,
Salmonella mutagenicity test, and Allium anaphase-telophase test. Mutat Res. 300(1):29–36.
Rattray NJ (1996). Glyphosate Acid: 4-Hour Acute Inhalation Toxicity Study in Rats. Central Toxicology
Laboratory, Alderley Park Macclesfield, Cheshire, England, UK. Laboratory report no. CTL/P/4882.
Unpublished report, dated 29 April 1996.
Reagan EL (1988a). Acute dermal toxicity study of glyphosate batch/lot/nbr. No. XLI-55 in New Zealand White
rabbits. Unpublished report, FDRL study No. 88.2053.008, Monsanto study No. FD-88-29, dated 8 June
1988, from Feed & Drug Research Laboratories, Waverly, NY, USA. Submitted to WHO by Monsanto Int.
Services SA, Brussels, Belgium.

281
Reagan EL (1988b). Primary eye irritation study of glyphosate batch/lot/nbr No. XLI-55 in New Zealand White
rabbits. Unpublished report, FDRL study No. 88.2053.009, Monsanto study No. FD-88-29, dated 8
June1988, from Food & Drug Research Laboratories, Waverly, NY, USA. Submitted to WHO by Monsanto
Int. Services SA, Brussels, Belgium.
Reagan EL, Laveglia J (1988a). Acute oral toxicity study of glyphosate batch/lot/NBR No. XLI-55 in Sprague-
Dawley rats. Feed and Drug Research Laboratory, Waverly, NY, USA. FDRL study no. 88.2053.007,
Monsanto study no. FD-88-29. Submitted by Monsanto Company, St. Louis, MO, USA, 8 June 1988.
Unpublished study.
Reagan EL, Laveglia J (1988b). Primary dermal irritation study of glyphosate batch/lot/NBR No. XLI-55 in
New Zealand White Rabbits. Feed and Drug Research Laboratories, Waverly, NY, USA. Data owner:
Monsanto, Monsanto Report No.: FD-88-29, dated 8 June 1988. Unpublished study.
Reagan EL, Laveglia J (1988c). Primary irritation study of glyphosate, Guideline 81-4: Primary eye irritation.
Rabbit Feed and Drug Research Laboratories, Waverly, NY, USA. Data owner: Monsanto, Monsanto
Report No.: FD-88-2053.009 (FD-88-29), dated 28 September 1988. Unpublished study.
Reyna MS (1990). Glyphosate: Two generation reproduction feeding study with glyphosate in Sprague-Dawley
Rats. Monsanto Company Environmental Health Laboratory, MO, USA. Monsanto unpublished report no.
ML-88106, Unpublished Study.
Reyna MS, Ruecker FA (1985). Twelve-month study of glyphosate administered by gelatin capsule to beagle
dogs, Monsanto report ML-83-137.
Reyna MS, Thake DC (1989). Range finding study of glyphosate administered in feed to Sprague-Dawley rats.
Unpublished report No. ML-88-272, study No. 88181, dated April 1989, from Monsanto Environmental
Health Laboratory, St. Louis, MO, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels,
Belgium.
Riberri do Val R (2007): Bacterial reverse mutation test (Ames Test) for Glifosato Téchnico Helm TECAM
Technologia Ambiental Ltda., Brazil. Report no.: 3393/2007-2.0AM-B, dated 13 December 2007.
Sponsored by Helm do Brasil Mercantil LTDA, Sao Paulo, Brazil. Unpublished study.
Richard S, Moslemi S, Sipahutar H, Benachour N, Seralini GE (2005). Differential effects of glyphosate and
roundup on human placental cells and aromatase. Environ Health Perspect. 113(6): 716–20.
doi: 10.1289/ehp.7728.
Richeux F (2006). Glyphosate technical: Skin sensitisation in the guinea pig – Magnusson and Kligman
maximisation method. Data owner: Nufarm. Study no.: SMK-PH-05/2018, Report no.: 2060/009, dated 13
January 2006. GLP: Unpublished study.
Ridley WP, Mirly K (1988). The metabolism of glyphosate in Sprague-Dawley rats. Part I. Excretion and tissue
distribution of glyphosate and its metabolites following intravenous and oral administration. Unpublished
report, study no. 86139, project no. ML-86-438, dated March 1988, from Monsanto Environmental Health
Laboratory, St. Louis, MO, USA. Submitted to WHO by Monsanto Int. Services SA, Brussels, Belgium.
Ridley WP (1983). A study of the plasma and bone marrow levels of glyphosate following intraperitoneal
administration in the rat. Unpublished report, study No. 830109, project no. ML-83-218, dated 24 October
1988, from Monsanto Environmental Health Laboratory, St. Louis, MO, USA. Submitted to WHO by
Monsanto Int. Services SA, Brussels, Belgium.
Rodrigues HG, Penha-Silva N, deAraujo M, Nishijo H, Aversi-Ferreira TA (2011). Effects of Roundup
pesticide on the stability of human erythrocyte membranes and micronuclei frequency in bone marrow cells
of Swiss mice. Open Biol J. 4:54–9.
Rodwell DE (1980). Dominant lethal study in mice. Unpublished report, study no. 401-064, IR-79-014, dated 16
April 1980, from International Research and Development Corporation, Mattawan, MI, USA. Submitted to
WHO by Monsanto Int. Services SA, Brussels, Belgium.
Rossberger S (1994). DNA repair test with primary rat hepatocytes. ANAWA Munchen AG, Planegg, Germany.
Report no. 931564, Study completion 18 March 1994. Sponsored by Feinchemie Schwebda GmbH.
Unpublished study.
Roth M (2012). Glyphosate technical - Micronucleus assay in bone marrow cells of the mouse. Harlan Cytotest
Cell Research GmbH, Rossdorf, Germany. Report no. 1479200, dated 28 September 2012. Sponsored by
Syngenta Ltd., Jealott’s Hill International Research Centre, Berkshire, England, UK. Unpublished study.

282
Roustan A, Aye M, De Meo M, Di Giorgio C (2014). Genotoxicity of mixtures of glyphosate and atrazine and
their environmental transformation products before and after photoactivation. Chemosphere. 108:93–100.
Rueppel M, Brightwell BB, Schaefer J, Marvel JT (1977). Metabolism and degradation of glyphosate in soil and
water. J Agric Food Chem. 25:517–28.
Sawada Y, Nagai Y, Ueyama M, Yamamoto I (1988). Probable toxicity of surface-active agent in commercial
herbicide containing glyphosate. Lancet. 1(8580):299.
Health. 11(4):4449–527. doi:10.3390/ijerph110404449.
Schreib G (2010). Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with
glyphosate technical. BSL Bioservice Scientific Laboratories GmbH, Planegg, Germany. Report no.:
102025, dated 18 June 2010. Unpublished study.
Schreib G (2012). Reverse mutation assay using bacteria (Salmonella typhimurium) with Glyphosate Tech. BSL
Bioservice study no., 126159, dated 17 December 2012. Sponsor: Industrias Afrsa, S. A., Paterna, Spain.
Schreib G (2015). Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with
Glyphosate technical. BSL Bioservice study no. 150099, dated 27 March 2015. Sponsor: Cheminova A/S,
Levmvig, Denmark. Unpublished study.
Schroeder RE, Hogan GK (1981). Glyphosate: A three generation reproduction study in rats with glyphosate.
Bio/Dynamics, Inc., East Millstone, NJ, USA. Bio/Dynamics Unpublished report no. BDN-77-417, dated
31 March 1981. Unpublished study.
Séralini GE, Clair E, Mesnage R, Gress S, Defarge N, Manuela Malatesta M et al. (2014). Republished study:
Long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Environ Sci
Europe, 26(1):1–14.
Shehata AA, Kühnert M, Haufe S, Krüger M (2014). Neutralization of the antimicrobial effect of glyphosate by
humic acid in vitro. Chemosphere. 104:258–61.
Shehata A, Schrödl W, Neuhaus J, Krüger M (2013b). Antagonistic effect of different bacteria on Clostridium
botulinum types A, B, C, D and E in vitro. Vet Rec. 172(2):47.
Shehata AA, Schrödl W, Aldin AA, Hafez HM, Krüger M (2013a). The effect of glyphosate on potential
pathogens and beneficial members of poultry microbiota in vitro. Curr Microbiol. 66(4):350–8.
Shen ZA (2007). IN-MCX20: Subchronic toxicity 90-day feeding study in rats. Unpublished report no. DuPont-
19008, Supplement No. 1. DuPont Haskell Laboratory, Newark, DE, USA. Submitted to WHO by EI du
Pont de Nemours and Company, Wilmington, DE, USA.
Shirasu Y, Takahashi K (1975). Acute toxicity of Roundup (correction: CP67573) in mice. Unpublished report
no. ET-19-105, dated 5 March 1975. Institute of Environmental Toxicology, Tokyo, Japan. Submitted to
WHO by Monsanto Int. Services SA, Brussels, Belgium.
Simon C (2009a). Acute oral toxicity study in rats. Harlan Laboratories Ltd., Fullinsdorf, Switzerland.
Laboratory report no. Harlan Laboratories Study C22864, dated 2 April 2009. Unpublished Report.
Simon C (2009b). Acute dermal toxicity study in rats. Harlan Laboratories Ltd., Fullinsdorf, Switzerland.
Laboratory report no. Harlan Laboratories Study C22875. Unpublished report. 02 April 2009.
Simon C (2009c). Expert statement glyphosate technical: Primary eye irritation study in rat Harlan Laboratories
Ltd., Füllinsdorf, Switzerland. Data owner: Excel report no.: C22897, dated 23 January 2009. Unpublished
study.
Simon C (2009d). Glyphosate technical: Contact hypersensitivity in albino guinea pigs – maximization test.
Harlan Laboratories Ltd. Füllinsdorf, Switzerland. Data owner: Excel report no.: C22908, dated 15 May
Sivikova K, Dianovsky J (2006). Cytogenetic effect of technical glyphosate on cultivated bovine peripheral
lymphocytes. Int J Hyg Environ Health. 209:15–20.
Sokolowski A (2007a). Salmonella typhimurium and Escherichia coli reverse mutation assay with glyphosate
technical (NUP-05068). RCC Ltd., Itingen, Switzerland. Data owner: Nufarm. RCC study no.: 1061401,
dated 16 March 2007. Unpublished study.

283
Sokolowski A (2007b). Salmonella typhimurium and Escherichia coli reverse mutation assay with glyphosate
Sokolowski A (2007c) Salmonella typhimurium and Escherichia coli reverse mutation assay with glyphosate
Sokolowski A (2009). Salmonella typhimurium and Escherichia coli reverse mutation assay. Harlan Cytotest
Cell Research GmbH, Rossdorf, Germany. Data owner: Syngenta report no.: 1264500, dated 18 December
Sokolowski A (2010). Salmonella typhimurium and Escherichia coli reverse mutation assay with solution of
glyphosate TC spiked with glyphosine. Harlan Cytotest Cell Research GmbH (Harlan CCR), Rossdorf,
Germany. Data owner: HAG, report no.: 1332300, dated 7 April 2010. Unpublished study.
Song W, Liu MG, Zhang JB, Zhang JJ, Sun MM, Yu QK (2016). Mechanism of action of EBV, Bcl-2, p53, c-
Myc and Rb in non-Hodgkin's lymphoma. Eur Rev Med Pharmacol Sci. 20(6):1093–7.
Sprankle P, Meggitt WF, Penner D (1975). Adsorption, mobility, and microbial degradation of glyphosate in
soil. Weed Sci. 23(3):229–34.
Stegeman SD, Kier LD (1998). Mouse micronucleus screening assay of MON 0818. Monsanto Co.
Environmental Health Laboratory (EHL), St.Louis, MO, USA. Project no.: ML-89-463, EHL-89182, R.D.
no. 1663, dated 26 March 1998. Unpublished study.
Stegeman SD, Li AP (1990). Ames/Salmonella mutagenicity assay of MON 0818. Monsanto Co. Environmental
Health Laboratory, St. Louis, MO, USA. Project no. ML-89-461; Study no. 89178, dated 12 November
1990; Registrant submission dated 31 July 2006. Unpublished study.
Sterne JA, Sutton AJ, Ioannidis JP, Terrin N, Jones DR, Lau J et al. (2011). Recommendations for examining
and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. BMJ.
22;343:d4002. doi: http://dx.doi.org/10.1136/bmj.d4002.
Stout L. (1990) Ninety-day study of (inert ingredient) administered in feed to albino rats. Project no:
ML/89/359, MSL/10468, 1663, dated 14 September 1990. Unpublished study prepared by Monsanto
Company.
Stout LD, Johnson CW (1987). 90-Day study of glyphosate administered in feed to Sprague/Dawley rats.
Unpublished report no. ML-86-351, study no. EHL 86128, dated 30 November 1987, from Monsanto
Environmental Health Laboratory, St. Louis, MO, USA. Submitted to WHO by Monsanto Int. Services SA,
Brussels, Belgium.
Stout LD, Ruecker FA (1990). Chronic study of glyphosate administered in feed to albino rats. Unpublished
report no. MSL-10495. Job/project no. ML-87-148/EHL 87122, dated 22 October 1990, from Monsanto
Environmental Health Laboratory, St. Louis, MO, USA. Submitted to WHO by Monsanto Int. Services SA,
Brussels, Belgium.
Strutt AV, Atkinson C, Hudson P, Snodgrass E (1993). AMPA: 13 Week toxicity study in rats with
administration by gavage. Unpublished report no. 7866, IRI project no. 450876, dated 16 April 1993, from
Inveresk Research International, Tranent, Scotland, UK. Submitted to WHO by Cheminova A/S, Lemvig,
Denmark.
Stump DG (2012a). A Hershberger assay of glyphosate administered orally in peripubertal
orchidoepididymectomized rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory report no.:
WIL-843003, dated 6 January 2012. Unpublished study.
Stump DG (2012b). A uterotrophic assay of glyphosate administered orally in ovariectomized rats. WIL
Research Laboratories, LLC, Ashland, OH, USA. Laboratory report no.: WIL-843002, dated 6 January
Stump DG (2012c) A pubertal development and thyroid function assay of glyphosate administered orally in
intact juvenile/peripubertal male rats. WIL Research Laboratories, LLC, Ashland, OH, USA. Laboratory
project ID: WIL-843005, April 10, 2012. Unpublished study.
Stump DG (2012d). A pubertal development and thyroid function assay of glyphosate administered orally in
intact juvenile/peripubertal female rats. WIL Research Laboratories, LLC, Ashland, Ohio, USA. Laboratory
project ID: WIL-843007, dated 10 April 2012. Unpublished study.

284
Sugimoto K (1997). HR-001: 18-month oral oncogenicity study in mice. The Institute of Environmental
Toxicology, Tokyo, Japan. Laboratory project ID IET 94-0151, Sankyo Co., Ltd., Tokyo,
Japan.Unpublished study. Also referred to as Arysta Life Sciences, 1997.
Suresh TP (1993a). Mutagenicity – Micronucleus test in Swiss Albino mice. Rallis India Limited, Banglore,
India. Study no. TOXI:889-MUT-CH.MN, dated 6 May 1993. Sponsor: M/s Feinchemie Schwebda GmbH,
Schwebda, Germany. Unpublished study.
Suresh TP (1993b). Two generation reproduction study in Wistar Rats. Toxicology Department, Rallis India
Ltd., Rallis Agrochemical Research Station, Bangalore, India. Data owner: Feinchemie Schwebda GmbH,
Study no.: TOXI 885-RP-G2, dated 27 August 1993. Unpublished study.
Suresh TP (1993c). Teratogenicity study in rabbits – Test compound: Glyphosate technical (FSG 03090 H/05
March 1990). Rallis India Limited, Rallis Agrochemical Research Station, Bangalore, India. Data owner:
Feinchemie Schwebda GmbH. Study no.: TOXI: 884-TER-RB, amended 18 June 1994. Unpublished study.
Suresh TP (1994). Genetic toxicology – In vivo mammalian bone marrow cytogenetic test - Chromosomal
analysis. Rallis India Limited, Banglore, India. Study no. TOXI:890-MUT-CH.AB, dated 22 January 1994.
Sponsor: M/s Feinchemie Schwebda GmbH, Schwebda, Germany. Unpublished study.
Suresh TP (1996). Combined chronic toxicity and carcinogenicity study with glyphosate technical in Wistar
Rats. Rallis Research Centre, Rallis India Ltd., Bangalore, India. Data owner: Feinchemie Schwebda
GmbH, Study no.: 886.C.C-R., dated 20 July 1996. Unpublished study.
Takahashi M (1997) HR-001: A two-generation reproduction study in rats. Institute of Environmental
Toxicology, Tokyo, Japan. Data owner: Arysta Life Sciences, Study no.: IET 96-0031, dated 19 June 1997.
Unpublished study.
Takahashi M (1999a). Oral feeding carcinogenicity study in mice with AK-01, Nippon Experimental Medical
Research Institute Co. Ltd., Agatsuma, Gunma, Japan. Technical project no. H-95056, 3303-58
Takahashi M (1999b). A combined chronic toxicity/carcinogenicity study of AK-01 bulk substance by dietary
administration in rats. Nippon Experimental Medical Research Institute Co. Ltd, Agatsuma, Gunma, Japan.
Project no. H-95053. Unpublished study.
Takeuchi S, Iida M, Yabushita H, Matsuda T, Kojima H (2008). In vitro screening for aryl hydrocarbon receptor
agonistic activity in 200 pesticides using a highly sensitive reporter cell line, DR-EcoScreen cells, and in
vivo mouse liver cytochrome P450-1A induction by propanil, diuron and linuron. Chemosphere. 74(1):155–
65. doi: 10.1016/j.chemosphere.2008.08.015.
Talbot AR, Shiaw MH, Huang JS, Yang SF, Goo TS, Wang SH et al (1991). Acute poisoning with a
glyphosate-surfactant herbicide (‘Round-up’): a review of 93 cases. Hum Exp Toxicol. 10:1–8.
Talvioja K (2007a). Glyphosate technical (NUP05068): Acute oral toxicity in rats. RCC Ltd. Toxicology,
Füllinsdorf, Switzerland. Data owner: Nufarm, Report no.: BO2272, dated 1 March 2007. Unpublished
report.
Talvioja K (2007b). Glyphosate technical (NUP 05068): Acute dermal toxicity study in rats. RCC Ltd.
Toxicology, Fullinsdorf, Switzerland. RCC study no.: B02283 Glyphosate Technical (NUP 05068), dated
January 2007. Unpublished report.
Talvioja K (2007c). Glyphosate technical (NUP 05069): Primary skin irritation study in rabbits (4-hour semi-
occlusive application). RCC Ltd. Toxicology, Fullinsdorf, Switzerland. RCC study no. B02294, dated 15
January 2007. Unpublished report.
Talvioja K (2007d). Glyphosate technical (NUP 05068): Primary eye irritation study in rabbits (4-hour semi-
occlusive application). RCC Ltd. Toxicology, Fullinsdorf, Switzerland. RCC study no. B02305. Data
owner: Nufarm Asia Sdn Bhd, Selangor, Malaysia, dated 5 March 2007. Unpublished report.
Talvioja K (2007e). Glyphosate technical (NUP 05068): Contact hypersensitivity in albino guinea pigs,
Maximization test. RCC Ltd. Toxicology, Fullinsdorf, Switzerland. RCC study no. B02316. Data owner:
Nufarm Asia Sdn Bhd, Selangor, Malaysia, dated 8 March 2007. Unpublished report.
Tasker EJ, Rodwell DE, Jessup DC (1980a). Glyphosate: Teratology study in rats with technical glyphosate.
International Research and Development Corp., MI, USA. Monsanto unpublished report no. IR-79-016,
March 21, 1980. Unpublished study.

285
Tasker EJ, Rodwell DE, Jessup, DC (1980b). Technical glyphosate: Teratology study in rabbits. International
Research and Development Corporation, MI, USA. Monsanto report no.: IR-79-016, dated 29 February
Tavaszi J (2011a). Glyphosate technical: Acute oral toxicity study in the rat (Up and Down procedure) LAB
Research Ltd., Szabadságpuszta, Hungary. Data owner: Syngenta report no.: 10/218-001P, dated 15 April
Tavaszi J (2011b). Glyphosate technical: Acute eye irritation study in rabbits. LAB Research Ltd.,
Szabadságpuszta, Hungary. Data owner: Syngenta report no.: 10/218-005N, dated 13 May 2011.
Unpublished study.
Teramoto I (1998). A 12-month chronic toxicity study of AK-01 bulk substance by oral gavage administration
in dogs. Nippon Experimental Medical Research Institute Co., Ltd., Agatsuma, Gunma, Japan Project no.
H-95059.
Thomas KW, Dosemeci M, Coble JB, Hoppin JA, Sheldon LS, Chapa G et al. (2010). Assessment of a pesticide
exposure intensity algorithm in the Agricultural Health Study. J Expo Sci Environ Epidemiol. 20:559–69.
Thompson P (2014). Glyphosate: Reverse mutation assay ‘Ames Test’ using Salmonella typhimurium and
Escherichia coli. Harlan Laboratories Ltd., Derbyshire, England, UK. Harlan study no.: 41401854, dated 18
July 2014. Sponsor: Albaugh Europe Sarl, Lausanne, Switzerland. Unpublished study.
Thompson PW (1996) Technical glyphosate: Reverse mutation assay “Ames test” using Salmonella
typhimurium and Escherichia coli. SafePharm Laboratories, Shardlow, Derbyshire, England, UK. Data
owner: Nufarm. SPL project no.: 434/014, dated 20 February 1996. Unpublished study.
Thongprakaisang S, Thiantanawat A, Rangkadilok N, Suriyo T, Satayavivad J (2013). Glyphosate induces
human breast cancer cells growth via estrogen receptors. Food Chem Toxicol. 59:129–36. doi:10.1016/j.
fct.2013.05.057.
Tierney WJ, Rinehart WE (1979). A three month feeding study of glyphosate (Roundup Technical) in mice.
Monsanto report BDN-77-419, dated 31 December 1979. Unpublished study.
Tominack RL, Conner P, Yamashita M (1989). Clinical management of Roundup herbicide exposure. Jpn J
Toxicol. 33:553.
Tominack RL, Yang GY, Tsai WJ, Chung HM,Deng, JF (1991). Taiwan National Poison Center survey of
glyphosate-surfactant herbicide ingestions. J Toxicol Clin Toxicol. 29:91–109.
Tornai A (1994). Glyphosate technical - Repeated dose twenty-eight day toxicity study in rabbits, Institute of
Toxicology, Keszthely, Hungary. Testing facility code GLY-94-410/N, Sponsored by Alkaloida Co. Ltd.,
Tiszavasvari, Hungary. Report no. MUF 214/94. Unpublished study.
Török-Bathó M (2011). Glyphosate technical: Local lymph node assay in the mouse. LAB Research Ltd.,
Szabadságpuszta, Hungary. Data owner: Syngenta. Report no.: 10/218-037E, dated 21 April 2011.
Unpublished study.
USEPA-SAP (US Environmental Protection Agency-FIFRA Scientific Advisory Panel) (2009a). An effects-
based expert system to predict estrogen receptor binding affinity for food use inert ingredients and
antimicrobial pesticides: Application in a prioritization scheme for endocrine disruptor screening, meeting
materials (https://www.regulations.gov/#!docketDetail;D=EPA-HQ-OPP-2009-0322, accessed 8 November
2016).
USEPA-SAP (US Environmental Protection Agency-FIFRA Scientific Advisory Panel) (2009b). An effects-
based expert system to predict estrogen receptor binding affinity for food use inert ingredients and
antimicrobial pesticides: application in a prioritization scheme for endocrine disruptor screening, Meeting
minutes. EPA-HQ-OPP-2009-0322, 70 pp.
USEPA-SAP (US Environmental Protection Agency-FIFRA Scientific Advisory Panel) (2013). Scientific issues
associated with prioritizing the universe of endocrine disruptor screening program (EDSP) chemicals using
computational toxicology tools EPA-HQ-OPP-2012-0818. May 2013.
Vainio H, Linnainmaa K, Kähönen M, Nickels J, Hietanen E, Marniemi J et al. (1983). Hypolipidemia and
peroxisome proliferation induced by phenoxyacetic acid herbicides in rats. Biochem Pharmacol.
32(18):2775–9. doi:10.1016/0006-2952(83)90091-6.

286
Vargas AAT (1996). The Salmonella typhimurium reverse mutation by GLIFOS. BioAgri (Biotecnologia
Agricola Ltda.), Sao Paulo, Brazil. On behalf of Cheminova; BioAgri Report G.1.1 - 050/96. Dated 12
October 1996–23 December 1996. Unpublished study.
Van de Waart EJ (1995). Evaluation of the ability of glyfosaat to induce chromosome aberrations in cultured
peripheral human lymphocytes (with independent repeat). NOTOX B.V’s-Hertogenbosch, Netherlands.
Notox project no. 141918, dated 30 June 1995. Unpublished report.
Vegarra MM (2004). Acute oral toxicity study in rats with N-acetyl-glyphosate, sodium salt (acute toxic class
method). Unpublished report no. Covance 7535-103. Covance Laboratories, Inc., Vienna, VA, USA.
Submitted to WHO by EI du Pont de Nemours and Company, Wilmington, DE, USA.
Vereczkey L, Csanyi E (1992). 18-month carcinogenicity study of glyphosate in mice (revised version).
Vigfusson NV, Vyse ER (1980). The effect of the pesticides, Dexon, Captan and Roundup, on sister-chromatid
exchanges in human lymphocytes in vitro. Mutat Res. 79(1):53–7.
Viljoen KS, Dakshinamurthy A, Goldberg P, Blackburn JM (2015). Quantitative profiling of colorectal cancer-
associated bacteria reveals associations between fusobacterium spp., enterotoxigenic Bacteroides fragilis
(ETBF) and clinicopathological features of colorectal cancer. PLoS One. 10:e0119462.
http://dx.doi.org/10.1371/journal.pone.0119462.
Cancer Causes Control. 12:509–17. doi:10.1023/A:1011293208949.
Wagner VO, Klug ML (2007). IN-EY252: bacterial reverse mutation assay. Unpublished report no. DuPont-
22227. BioReliance, Rockville, MD, USA. Dated 23 July 2007. Submitted to WHO by EI du Pont de
Wallner B (2010). Reverse mutation assay using bacteria (Salmonella typhimurium) with Glyphosate TC. BSL
Bioservice Scientific Laboratories GmbH, Planegg, Germany. Data owner: Helm AG, Report no.: BSL
101268, dated 8 April 2010. Unpublished study.
Walsh LP, McCormick C, Martin C, Stocco DM (2000). Roundup inhibits steroidogenesis by disrupting
steroidogenic acute regulatory (StAR) protein expression. Environ Health Perspect. 108(8):769–76.
doi:10.1289/ ehp.00108769.
Wang G, Deng S, Li C, Liu Y, Chen L, Hu C (2012). Damage to DNA caused by UV-B radiation in the desert
cyanobacterium Scytonema javanicum and the effects of exogenous chemicals on the process.
Chemosphere. 88(4):413–7.
Ward RJ (2010a). 450 g/L Glyphosate SL formulation (MON 79545). In vitro absorption of glyphosate through
human epidermis. Dermal Technology Laboratory Ltd., Keele, Staffordshire, England, UK. Study no.:
JV2083, Report no.: JV2083-REG, dated 19 February 2010. Unpublished study.
Ward RJ (2010b). 480 g/L Glyphosate SL formulation (MON 79351). In vitro absorption of glyphosate through
human epidermis. Dermal Technology Laboratory Ltd., Keele, Staffordshire, England, UK. Study no.:
JV2085, Report no.: JV2085-REG, dated 19 February 2010. Unpublished report.
Wilga PC (2012). Glyphosate: Human recombinant aromatase assay. CeeTox, Inc., Kalamazoo, MI, USA.
Laboratory study no.: 6500V-100334AROM, dated 9 March 2012. Unpublished study.
Williams GM, Kroes R, Munro IC (2000). Safety evaluation and risk assessment of the herbicide Roundup and
its active ingredient, glyphosate, for humans. Regul Toxicol Pharmacol. 31:117–65.
Willoughby JA (2012a). Glyphosate: Androgen receptor binding (rat prostate cytosol) screening assay. CeeTox,
Inc., Kalamazoo, MI, USA. Laboratory study no.: 6500V-100334ARB, dated 8 March 2012. Unpublished
study.
Willoughby JA (2012b). Glyphosate: Estrogen receptor binding (rat uterine cytosol). CeeTox, Inc., Kalamazoo,
MI, USA. Laboratory study no.: 6500V-100364ERB, dated 8 March 2012. Unpublished study.
Willoughby JA (2012c). Estrogen receptor transcriptional activation (human cell line (HeLa-9903)) screening
assay with glyphosate. CeeTox, Inc., Kalamazoo, MI, USA. Laboratory report no.: 6500V-100334ERTA,
dated 8 March 2012. Unpublished report.
Wnorowski, G. 1999. Acute inhalation toxicity study in rats – Limit Test. Product Safety Labs, East Brunswick,
NJ, USA. Laboratory report no. 7909, dated 18 October 1999. Unpublished report.

287
Wood E. (1996). Glyphosate technical: Pharmacology screening study in the rat. SafePharm Laboratories Ltd.,
Shardlow, Derbyshire, England, UK. Data owner: Nufarm, Study no.: 434/021, dated 28 June 1996.
Unpublished study.
Wood E, Dunster J, Watson P, Brooks P (2009a). Glyphosate technical: Dietary carcinogenicity study in the
mouse. Harlan Laboratories Limited, Shardlow, Derbyshire, England, UK. SPL project no.: 2060-0011,
dated 22 April 2009. Unpublished study.
Wood E, Dunster J, Watson P, Brooks P (2009b). Glyphosate technical: Dietary combined chronic
toxicity/carcinogenicity in the rat. Harlan Laboratories Ltd., Shardlow, Derbyshire, England, UK. Study
no.: 2060-0012, dated 8 May 2009. Unpublished study.
Wright NP (1996). Technical glyphosate: Chromosome aberration test in CHL cells in vitro. SafePharm
Laboratories, Shardlow, Derbyshire, England, UK. Data owner: Nufarm. SPL project no.: 434/015, dated
13 March 1996. Unpublished study.
Xie L, Thrippleton K, Irwin MA, Siemering GS, Mekebri A, Crane D et al. (2005). Evaluation of estrogenic
activities of aquatic herbicides and surfactants using an rainbow trout vitellogenin assay. Toxicol Sci.
87(2):391–8. doi:10.1093/toxsci/kfi249.
Xu Y (2008a). In vivo mouse bone marrow micronucleus assay. Covance Laboratories Inc., Vienna, VA, USA.
Monsanto study no. CV-08-031, dated 07 October 2008. Unpublished report.
Xu Y (2008b). In vivo mouse bone marrow micronucleus assay. Covance Laboratories Inc., Vienna, VA, USA.
Monsanto study no. CV-08-031, dated 03 December 2008. Unpublished report.
Xu Y (2008c). In vivo bone marrow micronucleus assay with MON 76171. Covance Laboratories Inc., Vienna,
VA, USA. Monsanto study no. CV-2007-103, dated 30 December 2008. Unpublished report.
Xu Y (2009a). In vivo mouse bone marrow micronucleus assay with MON 79991. Covance Laboratories Inc.,
Vienna, VA, USA. Monsanto study no. CV-2007-083, dated 23 January 2009. Unpublished report.
Xu Y (2009b). In vivo mouse bone marrow micronucleus assay with MON 76138. Covance Laboratories Inc.,
Vienna, VA, USA. Monsanto study no. CV-2007-095, dated 9 February 2010. Unpublished report.
Xu Y (2010). Amendment 1 to final report: In vivo mouse bone marrow micronucleus assay. Covance
Laboratories Inc., Vienna, VA, USA. Monsanto study no. CV2005-120, dated 1 December 2010. Amended
report issued Dec. 1, 2010. Unpublished report.
Xu Y (2011). Amended final report: In vivo mouse micronucleus assay with MON 78239. Covance Laboratories
Inc., Vienna, VA, USA. Monsanto study no. CV-2002-187, dated February 2011. Unpublished report.
Yanagimoto Y (1991). Bacterial reverse mutation assay with AK-01 technical. Life Sciences Laboratory, Osaka,
Japan. Study code: 91-VII-0101, dated 28 September 1991. Submitted by TAC Group, Japan. Unpublished
study.
Yanagimoto Y (1992a). In vitro mammalian cytogenetic assay with AK-01 Technical. Life Sciences Laboratory,
Osaka, Japan. Study code: 91-VII-0903, dated 13 January 1992. Submitted by TAC Group, Japan.
Unpublished study.
Yanagimoto Y (1992b). Bacterial DNA repair assay with AK-01 technical. Life Sciences Laboratory, Osaka,
Japan. Study code: 91-VII-0902, dated 10 January 1992. Submitted by TAC Group, Japan. Unpublished
study.
Yoshida A (1996). HR-001: 13 Week oral subchronic toxicity study in dogs. Institute of Environmental
Toxicology, Tokyo, Japan. Laboratory project ID IET 94-0158, dated 9 July 1996. Sponsored by Sankyo
Co. Ltd., Tokyo, Japan. Unpublished study.
You J (2009a). Acute oral toxicity study (UDP) in rats. OPPTS No 870.110. Stillmeadow Inc., Sugar Land, TX,
USA. Laboratory study no. 12170.08, dated 11 March 2009. Unpublished study.
You J (2009b). Acute dermal toxicity study in rats. Stillmeadow Inc., Sugar Land, TX, USA. Laboratory report
no. 12171-08, dated March 2009. Unpublished study.
You J (2009c). Acute dermal irritation study in rabbits. Stillmeadow, Inc., Sugar Land, TX, USA. Laboratory
report no. 12173-08, dated 11 March 2009. Unpublished study.
You J (2009d). Glyphosate - Acute eye irritation study in rabbits. Stillmeadow Inc., Sugar Land, TX, USA.
Laboratory report no. 12172-08, dated 11 March 2009. Data owner: Helm AG. Unpublished study.

288
You J (2009e). Glyphosate – Skin sensitization study in guinea pigs. Buehler test. Stillmeadow Inc., Sugar
Land, TX, USA. Data owner: Helm AG report no. 12174-08, dated 11 March 2009. Unpublished study.
Zaccaria CB (1996). A micronucleus study in mice for the product GLIFOS. BioAgri (Biotecnologia Agricola
Ltda.), Piracicaba, Sao Paulo, Brazil, on behalf of Cheminova; BioAgri Report G.1.2 - 060/96. Dates of
experimental work: 08 October 1996–19 November 1996. Unpublished study.
Zahm SH, Weisenburger DD, Babbit PA, Saal RC, Vaught JB, Cantor KP et al. (1990). A case control study of
non-Hodgkin’s lymphoma and agricultural factors in Eastern Nebraska. Epidemiology. 1:349–56.
doi:10.1097/00001648-199009000-00004.
Zaranyika MF, Nyandoro MG (1993). Degradation of glyphosate in the aquatic environment - an enzymatic
kinetic-model that takes into account microbial-degradation of both free and colloidal (or sediment) particle
adsorbed glyphosate. J Agric Food Chem. 41(5): 838–42.
Zelenak V (2011a). Glyphosate technical: Acute dermal toxicity study in rats; Final report amendment 1. LAB
Research Ltd., Szabadságpuszta, Hungary. Data owner: Syngenta. Report no.: 10/218-002P, dated 13 April
Zelenak V (2011b). Glyphosate: Glyphosate technical-Primary skin irritation study in rabbits. Final Report
Amendment I. LAB Research Ltd., Szabadságpuszta, Hungary. Data owner: Syngenta. Report no.: 10/218-
006N, dated 13 April 2011. Unpublished study.
Zoetis T (1991). Subchronic toxicity study in rats with AMTER 163. Hazelton Washington, Inc., Vienna, VA,
USA. HWA study no. 564-162, dated 6 May 1991 (study completion date). Unpublished study.
Zoriki Hosomi R (2007). Mammalian erythrocyte micronucleus test for Glifosato Téchnico Helm TECAM
Tecnologia Ambiental Ltda., São Paulo, Brazil. Data owner: HAG report no.: 3393/2007-3.0MN-B, dated
13 December 2007. Unpublished study.

289
Appendix 1(a). Results of in vitro genotoxicity with glyphosate in nonmammalian species

GLP
End-point Test object Concentration Purity % (Yes/No) Results Reference
Chromosome Allium root cells w/o S9; 720– Glyphosate No Negative Rank et al.
damage 2880 µg/L isopropylamine (1993)
(96%)
Chromosome Allium root cells w/o S9; 720– Roundup No Positive Rank et al.
alterations 2 880 µg/L (1993)
calculated as
glyphosate
isopropylamine
Chromosome Allium cepa 0.036– 0.146% Springbok, No Positive Asita &
alterations onion root tips glyphosate Makhalemele
isopropylamine (2008)
formulation (48%)
Chromosome Trigonella 0.1–0.5% Glyphosate No Positive Siddiqui et al.
alterations foenum- (2012)
graecumn root
tips
Chromosome Allium cepa 3% Glyphosate No Positive Frescura et al.
alterations onion root tips (2013)
Micronucleus Allium cepa 35. 70, 105, 140, Glyphosate No Equivocal De Marco et al.
onion root tips 350, 700. 1050 formulation (21%) (1992)
and 1400 µg/g
DNA strand Spiderwort plant w/o S9; Glyphosate No Positive Alvarez-Moya
breaks (Comet Tradescantia 0.000 7–0.7 isopropylamine et al. (2011)
assay) stamen hair mmol/L (96%)
nuclei
DNA strand Oyster 0.5; 1.0; 1.5; Glyphosate No Negative Akcha, Spagnol
breaks (Comet spermatozoa 2.5; 5.0 µg/L & Rouxel
assay) (2012)
DNA strand Oyster 0.5; 1.0; 1.5; Roundup No Negative Akcha, Spagnol
assay) active ingredient (2012)
DNA strand Frog 4.6–37 mg Roundup SL– No Positive Meza-Joya,
breaks (Comet (Eleutherodactyl a.e./cm2 Cosmoflux 411F Ramirez-Pinilla
assay) us johnstonei) (360 g/L & Fuentes-
blood cells glyphosate) Lorenzo (2013)
DNA strand Tilapia w/o S9; Glyphosate No Positive Alvarez-Moya
breaks (Comet (Oreochromis 0.000 7– 0.7 isopropylamine et al. (2014)
assay) niloticus) mmol/L (96%)
erythrocytes
DNA strand Spiderwort plant w/o S9; Glyphosate No Inconclusi Alvarez-Moya
breaks (Comet Tradescantia 0.000 7–0.7 isopropylamine ve et al. (2014)
assay) stamen hair mmol/L (96%)
nuclei
S9: 9000 × g supernatant fraction

290
Appendix 1(b). Results of in vivo genotoxicity with glyphosate, Roundup and other formulations in
nonmammalian species
GLP
End-point Test object Concentration Purity (%) (Yes/No) Results Reference
Glyphosate
Mutation Drosophila larvae 0.1 ppm Pondmaster N/S Positive Kale et al.
(1995)
Mutation Drosophila larvae 1 ppm Roundup N/S Positive Kale et al.
(1995)
Mutation Drosophila 0.1–10 mmol/L Glyphosate No Weak Kaya et al.
standard cross (96%) positive (2000)
Mutation Drosophila high 0.1–10 mmol/L Glyphosate No Negative Kaya et al.
bioactivation cross (96%) (2000)
DNA strand Spiderwort plant w/o S9; 0.000 7– Glyphosate No Positive Alvarez-Moya
breaks (Comet Tradescantia 0.7 mmol/L isopropylamine et al. (2011)
assay) stamen hair nuclei (96%)
DNA strand Oyster 0.5; 1.0; 1.5; Glyphosate No Negative Akcha, Spagnol
assay) (2012)
DNA strand European eel 17.9 35.7 µg/L Glyphosate No Positive Guilherme et al.
breaks (Comet (Anguilla anguilla) (2012a)
assay) blood cells
DNA strand Nile tilapia w/o S9; 0.000 7– Glyphosate No Positive Alvarez-Moya
breaks (Comet Oreochromis 0.7 mmol/L isopropylamine et al. (2014)
assay) niloticus (96%)
erythrocytes
DNA strand Spiderwort plant w/o S9; 0.0007– Glyphosate No Weak Alvarez-Moya
breaks (Comet Tradescantia 0.7 mmol/L isopropylamine positive et al. (2014)
assay) stamen hair nuclei (96%) /inclusive
DNA damage Zebrafish (Danio 5 & 10 mg/L Glyphosate No Positive Lopes et al.
rerio) sperm (2014)
DNA strand Sabalo fish 0.48 & 2.4 mg/L Glyphosate No Positive Moreno, Sofia
breaks (Comet (Prochilodus & Martinez
assay) lineatus) (2014)
erythrocytes and
gill cells
Mutation (sex- Drosophila 1 ppm Roundup No Positive Kale et al.
linked standard cross (1995)
recessive
lethal)
Mutation (sex- Drosophila 0.1 ppm Pondmaster No Positive Kale et al.
linked standard cross (1995)
recessive
lethal)
Chromosomal Plant meristems of 0.05–1% Roundup No Negative Dimitrov et al.
aberrations Crepis capillaris (> 90% purity) (2006)
Chromosome Mitotic plant 0.1–2% Roundup No Positive Truta et al.
abnormalities meristems of (2011)
Hordeum vulgare
Micronucleus Nile tilapia fish 42–170 mg/kg Glyphosate N/S Negative Nascimento &
Oreochromis bw (Roundup 69) Grisolia (2000)
niloticus
erythrocytes

291
GLP
Micronucleus Tilapia rendalli 42–170 mg/kg Roundup (480 No Positive Grisolia (2002)
peripheral bw g/L)
erythrocytes
Micronucleus Plant meristems of 0.05–1% Roundup No Negative Dimitrov et al.
Crepis capillaris (> 90% purity) (2006)
Micronucleus The freshwater 5, 10 and 15 Roundup (480 No Positive Cavas & Konen
goldfish ppm g/L) (2007)
(Carassius
auratus)
erythrocytes
Micronucleus Neotropical fish 10 mg/L Roundup No Negative Cavalcante,
(Prochilodus (41%) Martinez &
lineatus) Sofia (2008)
erythrocytes and
gill cells
Micronucleus Caiman latirostris 50–1 750 µg/egg Roundup No Positive Poletta et al.
erythrocytes (66.2%) (2009)
Micronucleus European eel 58 & 116 µg/L Roundup No Negative Guilherme et al.
(Anguilla anguilla) (30.8%) (2010)
blood cells
Micronucleus Caiman latirostris 3% Roundup No Positive Poletta et al.
erythrocytes (66.2%) (2011)
Micronucleus Brazilian 0.006 mL/L Roundup No Positive Rossi et al.
and Nuclear freshwater fish (2011)
abnormalities Astyanax sp.
Micronucleus The fish 6.67 μg/L Roundup No Negative De Castilhos,
Corydoras (48%) Ghisi & Cestari
paleatus (2013)
erythrocytes
Micronucleus Guppy (Poecilia 0, 1.41, 2.83, Roundup No Positive De Souza Filho
reticulata) gill 4.24 and 5.65 Transorb et al. (2013)
erythrocytes µL/L (64.8%)
Micronucleus Caiman latirostris 2.5–21 mg/L Roundup No Positive López Gonzáles
erythrocytes et al. (2013)
Micronucleus Ten spotted live- 22.9–68.8 mg/L Glyphosate No Positive Vera-Candioti,
bearer fish formulation Soloneski &
Cnesterodon Credit (48%) Larramendy
decemmaculatus (2013)
erythrocytes
Micronucleus Ten spotted fish 3.9–11.8 mg/L Glyphosate No Weak Vera-Candioti,
Cnesterodon formulation positive Soloneski &
decemmaculatus Panzer (48%) Larramendy
erythrocytes (2013)
Micronucleus Indian skittering 1–8 mg a.e./L Roundup No Positive Yadav et al.
frog (Euflictis (41%) (2013)
cyanophlyctis)
tadpole
erythrocytes
Micronucleus Earthworm 47–432 µg cm–2 Glyphosate No Positive Muangphra,
(Pheretima formulation Kwankua &
peguana) (36%) Gooneratne
coelomocytes (2012)
Micronucleus Channa punctatus 8.1–24.4 mg/L Roundup No Positive Nwani et al.
blood cells (41%) (2014)

292
GLP
Micronuclei Black lentil beans Not specified Glyphosate No Positive Singh &
and meiotic Vigna mungo Srivastava
anomalies (2014)
DNA strand Bullfrog (Rana 1.69–27 mg/L Roundup (356 No Positive Clements,
breaks (Comet catesbeiana) g/L) Ralph & Petras
assay) tadpoles (1997)
DNA strand Freshwater 2.5 and 5 mg/L Roundup No Negative Conners &
breaks (Comet mussels (18%) Black (2004)
assay) (Utterbackia
imbecillis)
DNA strand Freshwater 5, 10 and 15 Roundup (480 No Positive Cavas & Konen
breaks (Comet goldfish ppm g/L) (2007)
assay) (Carassius
auratus)
erythrocytes
DNA strand Neotropical fish 10 mg/L Roundup No Weak Cavalcante,
breaks (Comet (Prochilodus (41%) positive Martinez &
assay) lineatus) Sofia (2008)
erythrocytes and
gill cells
DNA strand Caiman latirostris 50–1 750 µg/egg Roundup No Positive Poletta et al.
breaks (Comet erythrocytes (66.2%) (2009)
assay)
DNA strand European eel 58 and 116 µg/L Roundup No Positive Guilherme et al.
breaks (Comet (Anguilla anguilla) (30.8%) (2010)
assay) blood cells
DNA strand Caiman latirostris 3% Roundup No Positive Poletta et al.
breaks (Comet erythrocytes (66.2%) (2011)
assay)
DNA strand Snail 10 mg/L Roundup No Positive Mohamed
breaks (Comet (Biomphalaria (48%) (2011)
assay) alexandrina)
haemocytes
DNA strand Oyster 0.5; 1.0; 1.5; Roundup No Negative Akcha, Spagnol
assay) active ingredient (2012)
breaks (Comet (Anguilla anguilla) (30.8%) (2012b)
assay) gill and liver cells
breaks (Comet (Anguilla anguilla) (30.8%) (2012a)
assay) blood cells
DNA strand Guppy (Poecilia 0, 1.41, 2.83, Roundup No Positive De Souza Filho
breaks (Comet reticulata) gill 4.24 and 5.65 Transorb et al. (2013)
assay) erythrocytes µL/L (64.8%)
DNA strand Frog 0.5–1.7 mg Roundup SL– No Positive Meza-Joya,
breaks (Comet (Eleutherodactylus a.e./cm2 Cosmoflux Ramirez-Pinilla
assay) johnstonei) blood 411F (360 g/L & Fuentes-
cells glyphosate) Lorenzo (2013)
DNA strand Fish Corydoras 6.67 μg/L Roundup No Positive De Castilhos,
breaks (Comet paleatus (48%) Ghisi & Cestari
assay) erythrocytes (2013)

293
GLP
DNA strand Freshwater clam 2 and 10 ppm Roundup No Negative Dos Santos &
breaks (Comet (Corbicula Martinez (2014)
assay) fluminea)
haemocytes
DNA strand Common carp 2 mg/L Roundup (480 No Positive Gholami-
breaks (Comet (Cyprinus carpio) g/L) Seyedkolaei et
assay) erythrocytes al. (2013)
DNA strand Channa punctatus 3.25–6.51 mg/L Roundup No Positive Nwani et al.
breaks (Comet blood and gill cells (41%) 2013
assay)
DNA strand Earthworm 15 and 30 Roundup FG No Positive Piola et al.
breaks (Comet (Eisenia andrei) µg/cm-1 (71%) (2013)
assay) coelomocytes
DNA strand Earthworm 15–240 µg/cm-1 Glyphosate No Negative Piola et al.
breaks (Comet (Eisenia andrei) formulation (2013)
assay) coelomocytes (85.4%)
DNA strand Ten spotted live- 3.9 mg/L Glyphosate No Positive Vera-Candioti
breaks (Comet bearer fish formulation Soloneski &
assay) Cnesterodon Panzer (48%) Larramendy
decemmaculatus (2013b)
erythrocytes
DNA strand Ten spotted live- 22.9 mg/L Glyphosate No Positive Vera-Candioti,
breaks (Comet bearer fish formulation Soloneski &
assay) Cnesterodon Credit (48%) Larramendy
decemmaculatus (2013b)
erythrocytes
DNA strand European eel 116 µg/L Roundup No Positive Guilherme et al.
breaks (Comet (Anguilla anguilla) (30.8%) (2014a)
assay) blood cells
DNA strand European eel 58 and 116 µg/L Roundup No Positive Marques et al.
breaks (Comet (Anguilla anguilla) (30.8%) (2014)
assay) liver cells
DNA strand Sabalo fish 1 and 5 mg/L Roundup No Positive Moreno, Sofia
breaks (Comet (Prochilodus Transorb (480 & Martinez
assay) lineatus) g/L) (2014)
erythrocytes and
gill cells
DNA strand Earthworm 47–432 µg cm–2 Glyphosate No Negative Muangphra
breaks (Comet (Pheretima formulation Kwankua &
assay) peguana) (36%) Gooneratne
coelomocytes (2012)
DNA strand Tambaqui 10–15 mg/L Roundup (360 No Positive Braz-Mota et al.
breaks (Comet (Colossoma g/L) (2015)
assay) macropomum) fish
DNA breakage Neotropical fish 0.15–1.5 mg/L Polyoxyethylen N/S Positive Navarro &
(Comet assay) Prochilodus e amine Martinez (2014)
lineatus blood cells
AMPA
Micronucleus European eel 11.8, 23.6 µg/L N/A No Negative Guilherme et al.
(Anguilla anguilla) (2014b)
blood cells
DNA strand European eel 11.8, 23.6 µg/L N/A No Positive Guilherme et al.
breaks (Comet (Anguilla anguilla) (2014b)
assay) blood cells

294
GLP
Nuclear European eel 11.8, 23.6 µg/L N/A N/S Positive Guilherme et al.
abnormalities (Anguilla anguilla) (2014b)
blood cells
AMPA: aminomethylphosphonic acid; bw: body weight; GLP: good laboratory practice; N/A: not applicable; N/S: not
stated; ppm: parts per million; S9: 9000 × g supernatant fraction
References to Appendix 1
Akcha F, Spagnol C, Rouxel J (2012). Genotoxicity of diuron and glyphosate in oyster spermatozoa and
embryos. Aquat Toxicol. 104–13.
Alvarez-Moya C, Silva MR, Arambula AR, Sandoval AI, Vasquez HC, Gonzalez Montes RM (2011).
Evaluation of genetic damage induced by glyphosate isopropylamine salt using Tradescantia bioassays.
Genet Mol Biol. 34(1):127–30.
Asita AO, Makhalemele R (2008). Genotoxicity of chlorpyrifos, alpha-thrin, efekto virikop and Springbok to
onion root tip cells. J Biotechnol. 7(23):4244–50.
Braz-Mota S, Sadauskas-Henrique H, Duarte FM, Val AL, Almeida-Val VM (2015). Roundup exposure
promotes gills and liver impairments, DNA damage and inhibition of brain cholinergic activity in the
Amazon teleost fish Colossoma macropomum. Chemosphere. 135:53–60.
Cavalcante DG, Martinez CB, Sofia SH (2008). Genotoxic effects of Roundup on the fish Prochilodus lineatus.
Mutat Res. 655(1-2):41–6.
Cavas T, Konen S (2007). Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish
(Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay.
Mutagenesis. 22(4):263–8.
Clements C, Ralph S, Petras M (1997). Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the
alkaline single-cell gel DNA electrophoresis (comet) assay. Environ Mol Mutagen. 29(3):277–88.
Conners DE, Black MC (2004). Evaluation of lethality and genotoxicity in the freshwater mussel Utterbackia
imbecillis (Bivalvia: Unionidae) exposed singly and in combination to chemicals used in lawn care. Arch
Environ Contam Toxicol. 46(3):362–71.
De Castilhos Ghisi N, Cestari MM (2013). Genotoxic effects of the herbicide Roundup® in the fish Corydoras
paleatus (Jenyns 1842) after short-term, environmentally low concentration exposure. Environ Monit
Assess. 185(4):3201–7.
De Marco A, De Simone C, Raglione M, Testa A, Trinca S (1992). Importance of the type of soil for the
induction of micronuclei and the growth of primary roots of Vicia faba treated with the herbicides atrazine,
glyphosate and maleic hydrazide. Mutat Res. 279:9–13.
De Souza Filho J, Sousa CC, Da Silva CC, De Saboia-Morais SM, Grisolia CK (2013). Mutagenicity and
genotoxicity in gill erythrocyte cells of Poecilia reticulata exposed to a glyphosate formulation. Bull
Environ Contam Toxicol. 91(5):583–7.
Dos Santos KC, Martinez CB (2014). Genotoxic and biochemical effects of atrazine and Roundup®, alone and
in combination, on the Asian clam Corbicula fluminea. Ecotoxicol Environ Saf. 100:7–14.
Frescura VD, Kuhn AW, Laughinghouse HD, Paranhos JT, Tedesco SB (2013). Post-treatment with plant
extracts used in Brazilian folk medicine caused a partial reversal of the antiproliferative effect of glyphosate
in the Allium cepa test. Biocell. 37:23–8.
Gholami-Seyedkolaei SJ, Mirvaghefi A, Farahmand A, Kosari AA, Gholami-Seyedkolaei SJ, Gholami-
Seyedkolaei SJ (2013). Optimization of recovery patterns in common carp exposed to roundup using
response surface methodology: evaluation of neurotoxicity and genotoxicity effects and biochemical
parameters. Ecotoxicol Environ Saf. 98:152–61.

295
Guilherme S, Gaivao I, Santos MA, M Pacheco M (2010). European eel (Anguilla anguilla) genotoxic and pro-
oxidant responses following short-term exposure to Roundup–a glyphosate-based herbicide. Mutagenesis.
25(5):523–30.
Guilherme S, Santos MA, Barroso C, Gaivao I, Pacheco M (2012b). Differential genotoxicity of roundup
formulation and it constituents in blood cells of fish (Anguilla anguilla): Considerations on chemical
interactions and DNA damaging mechanisms. Ecotoxicol. 21:1381–90.
Guilherme S, Santos MA, Gaivao I, Pacheco M (2014a). Are DNA-damaging effects induced by herbicide
formulations (Roundup® and Garlon®) in fish transient and reversible upon cessation of exposure? Aquat
Toxicol. 155:213–21.
Guilherme S, Santos MA, Gaivao I, M. Pacheco M (2014b). DNA and chromosomal damage induced in fish
(Anguilla anguilla L.) by aminomethylphosphonic acid (AMPA)–the major environmental breakdown
product of glyphosate. Environ Sci Pollut Res Int. 21(14):8730–9.
Kale PG, Petty BT, Walker S, Ford JB, Dehkordi N, Tarasia S et al. (1995). Mutagenicity testing of 9 herbicides
and pesticides currently used in agriculture. Environ Mol Mutagen. 25(2):148–53.
Kaya B, Creus A, Yanikoglu A, Cabre O, Marcos R (2000). Use of the Drosophila wing spot test in the
genotoxicity testing of different herbicides. Environ Mol Mutagen. 36(1):40–6.
Lopes FM, Varela Junior AS, Corcini CD, da Silva AC, Guazzelli VG, Tavares G et al (2014). Effect of
glyphosate on the sperm quality of zebrafish Danio rerio. Aquat Toxicol. 155:322–6.
López González EC, Latorre MA, Larriera A, Siroski PA, Poletta GL (2013). Induction of micronuclei in broad
snouted caiman (Caiman latirostris) hatchlings exposed in vivo to Roundup (glyphosate) concentrations
used in agriculture. Pesticide Biochem Physiol. 105:131–4.
Marques A, Guilherme S, Gaivao I, Santos MS, Pacheco M (2014). Progression of DNA damage induced by a
glyphosate-based herbicide in fish (Anguilla anguilla) upon exposure and post-exposure periods–insights
into the mechanisms of genotoxicity and DNA repair. Comp Biochem Physiol C Toxicol Pharmacol.
166:126–33.
Meza-Joya FL, Ramirez-Pinilla MP, Fuentes-Lorenzo JL (2013). Toxic, cytotoxic, and genotoxic effects of a
glyphosate formulation (Roundup®SL-Cosmoflux®411F) in the direct-developing frog Eleutherodactylus
johnstonei. Environ Mol Mutagen. 54(5): 362–73.
Mohamed AH (2011). Sublethal toxicity of Roundup to immunological and molecular aspects of Biomphalaria
alexandrina to Schistosoma mansoni infection. Ecotoxicol Environ Saf. 74(4): 754–60.
Moreno NC, Sofia SH, Martinez CB (2014). Genotoxic effects of the herbicide Roundup Transorb and its active
ingredient glyphosate on the fish Prochilodus lineatus. Environ Toxicol Pharmacol. 37(1):448–54.
Muangphra P, Kwankua W, Gooneratne R (2012). Genotoxic effects of glyphosate or paraquat on earthworm
coelomocytes. Environmental Toxicol. 29:612–20. doi: 10.1002/tox.21787.
Nwani CD, Nagpure NS, Kumar R, Kushwaha B, Lakra WS (2013). DNA damage and oxidative stress
modulatory effects of glyphosate-based herbicide in freshwater fish, Channa punctatus. Environ Toxicol
Pharmacol. 36(2):539–47.
Nwani CD, Nagpure NS, Kumar R, Kushwaha B, Kumar P, Lakra WS (2014). Induction of micronuclei and
nuclear lesions in Channa punctatus following exposure to carbosulfan, glyphosate and atrazine. Drug
Chem Toxicol. 37(4):370–7.
Piola L, Fuchs J, Oneto ML, Basack S, Kesten E, Casabé N (2013) Comparative toxicity of two glyphosate-
based formulations to Eisenia andrei under laboratory conditions. Chemosphere. 91(4):545–51.
Poletta GL, Larriera A, Kleinsorge E, Mudry MD (2009). Genotoxicity of the herbicide formulation Roundup
(glyphosate) in broad-snouted caiman (Caiman latirostris) evidenced by the Comet assay and the
Micronucleus test. Mutat Res. 672(2):95–102.
Poletta GL, Kleinsorge E, Paonessa A, Mudry MD, Larriera A, Siroski PA (2011). Genetic, enzymatic and
developmental alterations observed in Caiman latirostris exposed in ovo to pesticide formulations and
mixtures in an experiment simulating environmental exposure. Ecotoxicol Environ Saf. 74(4):852–9.
Rossi SC, Dreyer da Silva M, Piancini LD, Oliveira Ribeiro CA, Cestari MM, Silva de Assis HC (2011).
Sublethal effects of waterborne herbicides in tropical freshwater fish. Bull Environ Contam Toxicol.
87:603–7.

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Siddiqui S, Meghvansi MK, Khan SS (2012). Glyphosate, alachor and maleic hydrazide have genotoxic effect
on Trigonella foenum-graecum L. Bull Environ Contam Toxicol. 88(5):659–65.
Singh N, Srivastava A (2014). Biomonitoring of genotoxic effect of glyphosate and pendimethalin in Vigna
mungo populations. Cytologia 79:173–80.
Truta E, Vochita G, Rosu CM, Zamfirache MM, Olteanu Z (2011). Evaluation of Roundup-induced toxicity on
genetic material and on length growth of barley seedlings. Acta Biol Hung. 62(3):290–301.
Vera-Candioti J, Soloneski S, Larramendy ML (2013). Single-cell gel electrophoresis assay in the ten spotted
live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842), as bioassay for agrochemical-induced
genotoxicity. Ecotoxicol Environment Saf. 98:368–73.
Yadav SS, Giri S, Singha U, Boro F, Giri A (2013). Toxic and genotoxic effects of Roundup on tadpoles of the
Indian skittering frog (Euflictis cyanophlyctis) in the presence and absence of predator stress. Aquat
Toxicol. 132-133:1–8.

297
MALATHION
Matthew O’Mullane1, Carl Cerniglia2, Virissa Lenters3, Rachel B. Smith4, Raymond Tice5,
Mireille B. Toledano4 and Alan Boobis6
1
Australian Pesticides and Veterinary Medicines Authority, Kingston, Australia
2
Division of Microbiology, United States Food and Drug Administration, Jefferson, United States of
America (USA)
3
Norwegian Institute of Public Health, Oslo, Norway11
4
Department of Epidemiology and Biostatistics, School of Public Health, Faculty of Medicine,
Imperial College London, London, United Kingdom
5
National Institute of Environmental Health Science, National Institutes of Health, USA (retired)
6
Centre for Pharmacology and Therapeutics, Faculty of Medicine, Imperial College London, London,
England, United Kingdom
Explanation ...........................................................................................................................................298
Evaluation for acceptable intake ...........................................................................................................298
1. Biochemical aspects ..........................................................................................................................3
1.1 Absorption, distribution and excretion ........................................................................................3
1.2 Biotransformation ........................................................................................................................5
2. Toxicological studies .........................................................................................................................8
2.1 Acute toxicity .............................................................................................................................8
2.2 Short-term studies of toxicity .....................................................................................................9
2.3 Long-term studies of toxicity and carcinogenicity ...................................................................24
2.4 Genotoxicity .............................................................................................................................32
(a) In vitro studies ....................................................................................................................45
(b) In vivo studies.....................................................................................................................49
(c) Observations in humans ......................................................................................................50
2.5 Reproductive and developmental toxicity ................................................................................41
(a) Single-generation and multigeneration studies ...................................................................41
(b) Developmental toxicity.......................................................................................................42
2.6 Special studies. .........................................................................................................................45
(a) Neurotoxicity ......................................................................................................................45
(b) Cholinesterase inhibition ....................................................................................................49
(c) Immunotoxicity...................................................................................................................50
(d) In silico toxicity predictions ...............................................................................................51
(e) Intestinal microbiota effects ................................................................................................52
(f) Studies on metabolites or impurities ...................................................................................56
(g) Studies on cholinesterase inhibition ...................................................................................57
3. Observations in humans ..................................................................................................................57
3.1 Dosing studies in volunteers ......................................................................................................57
3.2 Occupational exposure studies ..................................................................................................60
3.3 Poisoning case reports ...............................................................................................................60
3.3 Epidemiological studies ............................................................................................................61
Comments……………………………………………………………………………………..………. .68
Toxicological evaluation ........................................................................................................................74
References ..............................................................................................................................................77
Appendix 1. Mode of action analysis – rodent liver tumours .................................................................74
Appendix 2. Mode of action analysis – rodent nasal tumours ................................................................74
11
Formerly at the Division of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht
University, Utrecht, the Netherlands

298
Explanation
Malathion is the International Organization for Standardization–approved common name for
S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethyl phosphorothioate (International Union of Pure and
Applied Chemistry), with the Chemical Abstracts Service (CAS) number 121-75-5. The chemical
structure of malathion is shown in Fig. 1.
Malathion is a non-systemic organophosphorus insecticide whose mode of pesticidal action is
the inhibition of cholinesterase activity. It is used to control insects on agricultural crops and stored
commodities and for vector control.
The toxicity of malathion was evaluated by the Joint FAO/WHO Meeting on Pesticide
Residues (JMPR) in 1963, 1965, 1966, 1997 and 2003. Malathion was listed in the periodic review
programme of the Codex Committee on Pesticide Residues (CCPR) but was not yet scheduled for
review. The compound was reviewed by the present Meeting following the recommendation of an
electronic task force of the World Health Organization Core Assessment Group on Pesticide Residues
that it be re-evaluated due to public health concerns identified by the International Agency for
Research on Cancer (IARC) and the availability of a significant number of new studies.
The current Meeting evaluated all previously submitted toxicological data in addition to new
published and unpublished toxicological studies and published epidemiological studies on cancer
outcomes. All critical unpublished studies contained certificates of compliance with good laboratory
practice (GLP), unless otherwise specified. Human volunteer studies were conducted according to the
Declaration of Helsinki or equivalent ethical standards.
Fig. 1. Chemical structure of malathion
O
O
O S
P O
S O

In an absorption, distribution, metabolism and excretion study, Reddy, Freeman & Cannon
(1989) administered [2,3-14C]malathion (> 98% radiochemical purity) in corn oil as a single gavage
dose of 40 or 800 mg/kg body weight (bw), or 15 daily gavage doses of unlabelled malathion (purity
94.6%) at 40 mg/kg bw followed by a single gavage dose of 40 mg/kg bw radiolabelled malathion, to
groups of five Sprague Dawley (Crl:CD [BR]) rats per sex. Urine and faeces were collected at 4, 8,
12, 24, 48 and 72 hours after dosing. The rats were terminated after 72 hours, and blood and tissues
collected. Expired air was not collected during the definitive phase of the study because excretion of
radioactivity via exhaled carbon dioxide was less than 1% of the administered radioactive dose in a
preliminary study. Radioactivity was quantified in excreta, blood and tissues by liquid scintillation
counting. Metabolites were analysed in excreta by high-performance liquid chromatography (HPLC)
and gas chromatography/mass spectrometry.
The cumulative mass balance of radioactivity is summarized in Table 1. Recovery of
radioactivity was greater than 90%, with the majority (76–88%) detected in urine and relatively low

299
levels detected in faeces (6–14%). Based on the concentration of radioactivity in urine and remaining
in the carcass, gastrointestinal absorption was estimated to be at least 77% in males and 86% in
females. The majority of radioactivity was excreted in urine within 24 hours of dosing. Less than 1%
of radioactivity was detected in tissues, with the highest proportions detected in the liver, skin, fat and
gastrointestinal tract (GIT) (Table 2).
Table 1. Cumulative mass balance of radioactivity in rats following oral dosing with [14C]malathion
Mean % of administered radioactivity
40 mg/kg bw (single dose) 800 mg/kg bw (single dose) 40 mg/kg bw (repeated dose)
Sample Males Females Males Females Males Females
Urine (including cage wash)
0–4 h 12.4 44.9 14.7 25.3 33.8 47.7
0–8 h 68.0 69.2 33.0 49.4 66.8 69.4
0–12 h 77.4 81.0 48.3 67.1 75.8 79.3
0–24 h 81.4 85.4 62.5 81.2 82.3 86.6
0–48 h 83.2 87.5 73.6 84.1 83.9 87.9
0–72 h 83.8 88.0 76.2 85.2 84.6 88.4
Faeces
0–4 h – – 0.005a – – –
a
0–8 h – – 0.005 – – –
0–12 h 2.8 1.6 2.2 1.9 0.3 0.0
0–24 h 9.4 4.8 10.5 5.3 5.5 4.2
0–48 h 10.7 5.8 12.9 6.4 6.5 5.7
0–72 h 11.0 5.9 13.7 6.6 6.8 5.8
Total
0–4 h 12.4 44.9 14.7 25.3 33.8 47.7
0–8 h 68.0 69.3 32.7 49.4 66.8 69.3
0–12 h 80.2 82.7 50.5 69.1 75.9 79.3
0–24 h 90.8 90.3 73.0 86.5 87.8 90.9
0–48 h 93.9 93.3 86.5 90.5 90.5 93.5
0–72 h 94.8 94.0 89.9 91.9 91.4 94.2
Carcass 0.36 0.46 0.67 0.50 0.46 0.24
bw: body weight; (–) indicates no faeces produced
a
Mean of two rats.
Source: Reddy, Freeman & Cannon (1989)

300
Table 2. Levels of radioactivity in tissues 72 hours after oral dosing with [14C]malathion
Mean % of administered radioactivity
40 mg/kg bw (single dose) 800 mg/kg bw (single dose) 40 mg/kg bw (repeated dose)
Tissue Males Females Males Females Males Females
Blood 0.042 0.032 0.033 0.020 0.046 0.034
Plasma 0.025 0.015 0.019 0.011 0.026 0.200
Erythrocytes 0.011 0.012 0.011 0.008 0.011 0.010
Liver 0.204 0.127 0.193 0.106 0.252 0.110
Kidneys 0.016 0.012 0.015 0.009 0.020 0.015
Lungs 0.002 0.002 0.002 0.002 0.002 0.002
Brain 0.007 0.005 0.003 0.002 0.006 0.005
Heart 0.001 0.001 0.001 0.000 0.001 0.001
Spleen 0.002 0.001 0.002 0.001 0.002 0.002
Testes 0.008 – 0.004 0.000 – –
Ovaries – 0.001 – 0.000 0.006 0.000
Uterus – 0.001 – 0.000 – 0.001
Adrenals 0.000 0.000 0.000 0.000 0.000 0.000
Fat 0.110 0.080 0.084 0.039 0.074 0.044
Skin 0.102 0.091 0.134 0.062 0.123 0.065
Muscle 0.039 0.031 0.037 0.020 0.048 0.023
Bone 0.055 0.038 0.046 0.028 0.059 0.036
GIT 0.053 0.062 0.158 0.058 0.044 0.021
bw: body weight; GIT: gastrointestinal tract
Results expressed as means.
Source: Reddy, Freeman & Cannon (1989)
In a human volunteer study conducted by Wester at al. (1983), 20 µL undiluted

[14C]malathion (purity not specified) was applied to the ventral forearm of five males (4.6 cm2 total
area; 5 mg/cm2) followed by repeated administration of unlabelled malathion. This procedure was
repeated eight days later when radioactivity was first detected in urine. The absorption of radioactivity
was 4.48% of the applied dose after the first application and 3.53% after the second application; there
was no statistically significant difference between these two estimates of dermal absorption.
An in vitro study undertaken by de Ligt (2004) to examine the absorption of [ 14C]malathion

by human and rat skin used flow-through diffusion cells. The test material comprised an undiluted
formulation containing 440 g/L malathion or an aqueous field-strength dilution of this formulation
containing 1.5 g/L malathion. The exposure time was 8 hours, reflecting a typical day’s work, and the
post-exposure period was 16 hours. The mean flux constant through human skin was 0.281 µg/cm per
hour for the undiluted formulation and 0.081 µg/cm per hour for the diluted formulation. The flux
constant through rat skin was 3.372 and 0.765 µg/cm per hour, respectively. Estimated dermal
absorption through human skin was 1.44% and 8.74% for the undiluted and diluted formulation,
respectively. Estimated dermal absorption through rat skin was 3.05% and 56.89% for the undiluted
and diluted formulation, respectively.
In an in vitro dermal absorption study by Moody et al. (2007), samples of human breast or leg
skin (n = 5) were exposed to [14C]malathion (purity > 95%) at concentrations of 2 mmol/L,

301
20 mmol/L or 200 mmol/L for 30 minutes. This was followed by a 6.5-hour collection period. The
mass balance of radioactivity is shown in Table 3. Estimates of dermal absorption ranged from 8–
20.7%.
Table 3. Mass balance of radioactivity following dermal exposure to [14C]malathion

% of administered radioactivity
Parameter 2 mmol/L 20 mmol/L 200 mmol/L
Total absorption into receiver (%) 11.6 3.0 0.7
Total absorption into skin depot (%) 9.1 5.0 9.6
Total absorption (%) 20.7 8.0 10.3
Skin wash (%) 60.2 85.8 67.4
Charcoal trap (%) 0.8 0.2 0.2
Donor chamber wash (%) 1.8 2.8 1.8
Total mass balance 83.5 96.9 79.7
Results expressed as means.
Source: Moody et al. (2007)
In the study by Reddy, Freeman & Cannon (1989) described in section 1.1 of this monograph,
the major metabolites detected in urine (> 80% of urinary radioactivity) were malathion α-
monocarboxylic acid, and malathion β-monocarboxylic acids (MMCA) and malathion dicarboxylic
acid (MDCA). The remaining urinary radioactivity comprised desmethyl malathion, O,O-dimethyl
phosphorothioic acid, fumaric acid, 2-mercaptosuccinic acid, O,O-dimethyl phosphorodiothioic acid,
monoethyl fumarate and malaoxon. Malaoxon was found only in urine samples and accounted for less
than 2% of total urinary radioactivity. The metabolite profile in faeces was qualitatively comparable
to urine.
In a published study, Buratti et al. (2005) characterized the metabolism of malathion (10–
500 µmol/L) by human liver microsomes and the role of cytochrome P450 (CYP) enzymes in
generating malaoxon. At low malathion concentrations ( 50 µmol/L), CYP1A2 catalysed malaoxon
formation, whereas CYP2B6 and 3A4 played a more significant role at higher concentrations of
malathion ( 400 µmol/L).
In a published study, Buratti & Testai (2005) determined that human microsomes efficiently
metabolized malathion to malathion monocarboxylic acid (MMCA) via hepatic carboxylesterase
activity and that isomalathion was a potent non-competitive inhibitor of this process (inhibitory
constant [Ki] = 0.6 µmol/L).
Takeuchi et al. (2006) screened 200 pesticides for in vitro peroxisome proliferator–activated
receptor (PPAR)α and PPARγ activity using an in vitro reporter gene assays in CV-1 monkey kidney
cells transiently transfected with the expression plasmid for mouse PPARα or PPARγ. Takeuchi et al.
(2008) also screened 200 pesticides for in vitro aryl hydrocarbon receptor activity using an in vitro
luciferase reporter gene assay in mouse hepatoma Hepa1c1c7 cells stably transfected with a dioxin-
responsive element (DR-EcoScreen cells). Malathion (purity >95%) showed no activity in either
assay.

302
Groups of three female Holtzmann rats were administered a single gavage dose of
73 µmol/kg bw malathion (unspecified purity), MMCA (purity 89.4%), MDCA (purity 99.6%),
dimethyl dithiophosphate (purity 97.9%), dimethyl thiophosphate (purity 98.7%) or dimethyl
phosphate (purity 98%) in corn oil. Urine and faeces were collected every 24 hours for eight days.
The results are summarized in Table 4. The authors concluded that low-level human dietary and non-
occupational urine biomonitoring studies will be confounded by preformed pesticide biomarkers used
to infer potential human pesticide exposure. This has profound implications for epidemiology studies
where biomarker excretion is used as a surrogate for organophosphate exposures that cannot be
related to a particular insecticide residue (Chen et al. 2013).
Table 4. Proportion of metabolites in urine following administration of a single dose of malathion

or malathion metabolites to rats
% of urinary metabolite
Treatment MMCA MMDA DMP DMTP DMDTP Total
Malathion 11.0 45.5 0.78 6.0 6.8 70.2
MMCA 8.7 15.1 0.53 2.9 4.8 32.0
MDCA 0.0 36.3 0.05 5.5 3.8 45.6
DMP 0 0 84.5 0 0 84.5
DMTP 0 0 8.3 46.7 0 55.0
DMDTP 0 0 1.1 3.9 40.8 45.9
DMDTP: dimethyl dithiophosphate; DMP: dimethyl phosphate; DMTP: dimethyl thiophosphate; MDCA: malathion
dicarboxylic acid; MMCA: malathion monocarboxylic acid
Results expressed as mean % of administered dose; n = 3.
Source: Chen et al. (2013).
Human volunteer studies described in section 3.1 of this monograph have analysed
metabolites in urine and blood. In the study by Gillies & Dickson (2000), where male and female
volunteers received a single oral dose of 0.5–15 mg/kg bw, blood samples were collected from high-
dose and control participants prior to dosing and then at various times up to 72 hours after dosing.
Similarly, urine was collected from all participants to analyse malathion metabolites. No malathion or
malaoxon were detectable in plasma (limit of detection = 102 and 99.8 ng/mL, respectively).
Supplementary analysis of urinary metabolites by Aston (2000) indicated that the majority of
metabolites were excreted within 12 hours of dosing, with total clearance by 24 hours. MMCA was
the main urinary metabolite followed by dimethyl thiophosphate, MDCA, dimethyl phosphate and
dimethyl dithiophosphate; the total concentration of metabolites in urine was proportional to the dose.
In a second human study (Jellinek, Schwartz & Connolly Inc., 2000), where volunteers also
received a single oral dose of malathion up to 15 mg/kg bw, plasma was analysed for malaoxon and
other major metabolites. Urine was collected prior to dosing and at 0–12, 12–24 and 24–48 hours. No
malathion or malaoxon was detected in plasma (limit of detection = 100 ng/mL). Approximately 90%
of the dose was excreted in urine within 12 hours, with excretion completed by 24–48 hours. The
major urinary metabolites were MMCA and MDCA (30% and 12% of the administered dose,
respectively). Other urine metabolites accounting for approximately 30% of the administered dose,
included dimethyl thiophosphate, dimethyl phosphate and dimethyl dithiophosphate (Aston, 2000).
The disposition kinetics of malathion and its metabolites in humans (MMCA, MDCA,
dimethyl dithiophosphate, dimethyl thiophosphate and dimethyl phosphate) following oral, dermal or
intravenous exposure was estimated using a heuristic toxicokinetic model, based on data from

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Feldmann & Maibach (1974) and Jellinek, Schwartz & Connolly Inc. (2000). The time taken to
recover 50% of the absorbed dose of malathion as urinary metabolites was about 4 hours following
oral administration, 11.8 hours following dermal administration and 3.2 hours following intravenous
administration. When simulating a single oral exposure, approximately 80–89% of the absorbed dose
was eliminated from the body within 12 hours; after a single dermal application, 29–53% of the
absorbed dose was estimated to be excreted during the same period. Assuming a continuous 8-hour
dermal exposure scenario, the model estimated that 52–80% of the absorbed dose would be eliminated
from the body within 24 hours and 84–98% within 48 hours (Bouchard et al., 2003).
The proposed metabolic pathway of malathion in rats is shown in Fig. 2:
 oxidative desulfuration of malathion in the liver to generate malaoxon, which may be excreted
in urine or further metabolized by phosphatases;
 hydrolysis of malaoxon by phosphatases to yield O,O-dimethyl phosphorothioic acid or
hydrolysis of malathion to yield O,O-dimethyl phosphorodithioate;
 hydrolysis of the carboxyester by tissue or plasma carboxylesterases, resulting in - and -
monocarboxylic acid or dicarboxylic acid (major pathway);
 dealkylation, probably by glutathione-S-transferases;
 glutathione-dependent demethylation to yield S-methyl-glutathione and the corresponding
desmethyl phosphate compound.
Fig. 2. Proposed metabolic pathway of malathion in rats

O
O O
O O R= H, CH 2CH3
C OCH2CH3
C OH C OR
CH3O P SH CH3O P S CH
HS CH CH
OCH3 OCH3 CH2
O, O-Dimethyl- CH2 CH
phosphorothiolate Malaoxon
C OCH2CH3
C OH C OH
O
O O
O 2-Mercapto- fumaric acid
S S succinic acid
C OCH2CH3
CH3O P SH O O
CH3O P S CH
O
OCH3 HO C C OH
OCH3 CH2 S
O, O-Dimethyl- C OH
Malathion H C S C H
phosporodithioate
C OCH2CH3 CH3O P S CH
H2 C C H2
O OCH3 CH2
O HO C C OH
MCA-beta
S C OCH2CH3 C OCH2CH3
O O
O 2-mercaptosuccinic
CH3O P S CH acid disulphide
OH CH2
Desmethyl O O
Malathion C OCH2CH3 S C OH S C OH
O CH3O P S CH CH3O P S CH
OCH3 CH2 OCH3 CH2
MCA-alpha DCA
C OCH2CH3 C OH
O O

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2.1 Acute toxicity
The results of acute toxicity tests on malathion, including skin and eye irritation and skin
sensitization studies, are summarized in Table 5. In acute oral dosing studies in rats, a range of
clinical signs were observed consistently at doses above 1260 mg/kg bw: piloerection, decreased
locomotor activity, ataxia, comatose condition, ptosis, respiratory depression, sedation, tremors,
clonic convulsions, salivation, lacrimation and red staining around the eyes and mouth (Terrell, 1978;
Terrell,1979a,b; Kynoch, 1986a; Fischer, 1991a,b; Kuhn, 1996; Moore, 2002, 2003). These clinical
signs started after 1 to 24 hours, with survivors recovering 1–5 days after dosing (Fischer, 1991a,b;
Moore, 2003). In acute dermal toxicity studies in rats, no clinical signs or dermal irritation occurred at
2000 mg/kg bw (Kynoch, 1986b; Bollen, 2003a). In an acute dermal study in rabbits, decreased
locomotor activity and salivation, and irritation at the application site occurred at and above 5000
mg/kg bw (Parke, 1978). In a whole-body inhalation study in rats, partial closing of the eyes,
excessive salivation, abnormal respiration and body posture occurred at 5.2 mg/L (Jackson, 1986).
Nose-only exposure to malathion aerosols resulted in ruffled fur, hunched posture, salivation and a
red secretion from the nose at 5.2 mg/L; rats recovered by one hour after exposure (Decker, Knuppe
& Ullrich, 2003).
Table 5. Results of studies of the acute toxicity of malathion

LD50
Purity (mg/kg bw) or
Species Strain Sex Route (%) Vehicle LC50 (mg/L) Reference
Rat SD M+F Oral NS Undiluted 5 000 Terrell
(1978)a
Rat SD M+F Oral NS Undiluted 3 800 (males) Terrell
4 400 (females) (1979a)a
Rat SD M+F Oral NS Undiluted 3 200 (males) Terrell

3 700 (females) (1979b)a
Rat SD (CD) M+F Oral 96–98 Undiluted 5 400 (males) Kynoch

5 700 (females) (1986a)
5 500
(combined)
Rat Crl:CD[SD] BR M+F Oral 94.6 Undiluted 1 768 (males) Fischer
1 539 (females) (1991a)
1 649
(combined)
Rat Crl:CD[SD] BR M+F Oral 96.8 Undiluted 6 156 (males) Fischer
4 061 (females) (1991b)
5 000
(combined)
Rat HSD:SD M+F Oral 99.1 Undiluted 8 227 Kuhn
(combined) (1996)
Rat SD M+F Oral 92.2 (0.44 Undiluted 1 857 Moore
IM) (2002)
Rat SD M+F Oral 96.0 Undiluted 2 382 Moore
(2003)
Rat SD (CD) M+F Dermal 96–98 Undiluted > 2 000 Kynoch
(1986b)
Rat BrlHan:WIST@Mol M+F Dermal 92.2 (0.44 Sesame oil > 2 000 Bollen
IM) (2003a)

305
LD50
Rabbit New Zealand White M+F Dermal NS Undiluted 8 790 Parke
(1978)a
Rat COBS Wistar M+F Inhalation 96–98 NS > 5.2 Jackson
(whole body) (1986)
Rat HanBrl:WIST(SPF) M+F Inhalation 96.3 (0.43 Undiluted > 5.20 Decker,
(nose only) IM) Knuppe &
Ullrich,
(2003)
Rabbit New Zealand White NS Dermal 96–98 Undiluted Slightly Liggett &
irritation irritating Parcell
(1985a)
Rabbit New Zealand White F Dermal 92.2 (0.43 Undiluted Not irritating Bollen
irritation IM) (2003b)
Rabbit New Zealand White NS Eye irritation 96–98 Undiluted Slightly Liggett &
irritating Parcell
(1985b)
Rabbit New Zealand White F Eye irritation 92.2 (0.43 Undiluted Slightly Bollen
IM) irritating (2003c)
Guinea- Hartley/Dunkin F Skin 96–98 75% w/v Not sensitizing Kynoch &
pig sensitization in acetone Smith
(Buehler) (1986)
Guinea- CBA/Jlbm F Skin 92.2 (0.43 50% w/v Sensitizing Bollen
pig sensitization IM) in sesame (2003d)
(Maximization) oil or
undiluted
Mice CBA/Jlbm F Skin 96.3 (0.44 25, 50 and Not sensitizing Wang-Fan
sensitization IM) 100% in (2003)
(LLNA) acetone
olive oil
(4:1 v/v)
Mice CBA/J F Skin 96.0 10–100% Not sensitizing Lowe
sensitization in acetone (2011a)
(LLNA) olive oil
(4:1 v/v)
bw: body weight; GLP: good laboratory practice; F: female; IM: isomalathion; LC50: median lethal concentration; LD50:
median lethal dose; LLNA: local lymph node assay; M: male; v/v: volume per volume; NS: not specified; w/v: weight per
volume
a
Study predates GLP and modern test guidelines.

Rats
In a 14-day range-finding study (Barnett Jr, 2011a), malathion (purity 96%) in corn oil was
administered by gavage to groups of eight Crl:CD[SD] adult rats per sex at 0, 800 or 1000 mg/kg bw
per day for up to 10 days. Observations for death and clinical signs were made throughout the study,
with body weight and feed consumption recorded daily. Female rats were terminated on day 4 and
male rats on day 11. The rats were necropsied and their kidney and liver weights recorded. There were
no deaths or treatment-related clinical signs. Body-weight gain was reduced at 800 and 1000 mg/kg
bw per day in males (−9.8% and −19.8%, respectively) and at 1000 mg/kg bw per day in females
(−44.4%). A slight reduction in feed consumptions was noted at these same doses (−4.7% and −7.1%,
respectively, in males; −5.9% and −8.5%, respectively, in females). Terminal body weights were
comparable across all groups. Liver weight was increased at 800 and 1000 mg/kg bw per day in males

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(+24.4% and +25.0%, respectively) and females (+8.3% and +11%, respectively). Some variations in
kidney weights were noted but these were not clearly treatment related.
Barnett Jr (2012c) undertook a 14-day range-finding study in juvenile rats to determine

suitable doses for a subsequent pubertal endocrine disruptor screening assay (Barnett, 2012d). Groups
of eight Crl:CD[SD] rats per sex were administered malathion (purity 96.0%) in corn oil at 0, 250,
450 or 600 mg/kg bw per day from postnatal days 23–36 in males and 22–35 in females. There were
no deaths. Salivation (graded as slight to extreme) was observed in at least one male at every dose and
in all females at 450 and 600 mg/kg bw per day. There were no effects on body weight, feed
consumption, the occurrence of macroscopic abnormalities, brain or liver weights. In males,
erythocyte and brain acetylcholinesterase activity was significantly lower (P < 0.01 or 0.05) than the
control at every dose (−67.4% to −79.6% and −9.3% to −16.0%, respectively), while in females
erythrocyte and brain acetylcholinesterase activity was significantly lower than the control at 450 and
600 mg/kg bw per day (−56.5% to −77.7% and −6.7% to −14.6%, respectively).
In a 28-day repeat-dose toxicity study by Barnett Jr (2012a), malathion (purity 95.8%) was
admixed in the diet and fed ad libitum to groups of Crl:CD[SD] rats (15/sex) at concentrations of 0,
100, 500, 5000 or 10 000 parts per million (ppm) (equal to 0, 9.2, 46.1, 457.5 and 947.8 mg/kg bw per
day in males and 0, 9.4, 47.4, 461.3 and 910.1 mg/kg bw per day in females). Rats were observed
daily for deaths and clinical signs. Body weight and feed consumption were recorded daily. Following
termination on day 29, the rats were necropsied and blood and brain tissue collected to analyse
acetylcholinesterase activity. In addition, the nasal passages, the nasal cavity and the neck and
associated organs and tissues were examined. The liver and kidneys were weighed and, along with the
nasal cavity, processed and retained for possible histopathological examination. There were no deaths
or treatment-related clinical signs. Absolute body weight was 9% to 11% lower than the control
(P < 0.01) at 10 000 ppm in males throughout the exposure period, while overall body-weight gain
was 20% lower (P < 0.01) than the control. In high-dose males, feed consumption was significantly
lower than the control during the first week of exposure (−11%; P < 0.01). Overall feed conversion
efficiency was 14% lower than the control (P < 0.1) in high-dose males. Body weight was unaltered in
females, while feed consumption was significantly lower than the control during the last week of
exposure in high-dose females (−11%; P < 0.05).
Erythrocyte acetylcholinesterase activity was significantly lower (P < 0.01) than the control at
500, 5000 and 10 000 ppm in males (−22.3%, −82.6% and −88.6%, respectively) and at every dose in
females (−13.8%, −29.0%, −82.9% and −88.6% at 100, 500, 5000 and 10 000 ppm, respectively).
Brain acetylcholinesterase activity was significantly lower (P < 0.01 or 0.05) than the control at 500,
5000 and 10 000 ppm in males (−7.2%, −21.5% and −21.2%, respectively) and at 5000 and 10 000
ppm in females (−25.0 and −47.8%, respectively; P < 0.01); the significant reduction in brain
acetylcholinesterase in males at 500 ppm was not considered toxicologically significant as it was less
than 10%. Benchmark dose (BMD) modelling was applied to the erythrocyte acetylcholinesterase data
using an exponential model. The estimated dose for a 20% inhibition (BMD20) was 45.6 mg/kg bw per
day in males and 42.9 mg/kg bw per day in females. The BMD10 for brain acetylcholinesterase
inhibition was 215.8 mg/kg bw per day in males and 159.2 mg/kg bw per day in females.
There were no treatment-related macroscopic abnormalities. Absolute and relative liver
weights were significantly increased (P < 0.01 or 0.05) at 5000 (males: 23 and 30%, respectively;
females 12% and 10%, respectively) and 10 000 ppm (males: 31% and 48%, respectively; females:
21% and 27%, respectively). Histopathological examination revealed minimal and mild hepatocellular
degeneration, a combination of cellular hypertrophy and clumping of basophilic material in the
cytoplasm in the livers of two males at 10 000 ppm. The study authors proposed that this finding may
have been related to the increases in organ weight, but due to the small number of male rats affected,
this change was considered equivocal. At 5000 and 10 000 ppm, relative paired kidney weights were
significantly increased (P < 0.01; +15 to +20%) but were not accompanied by any microscopic
changes. Histopathological examination of nasal tissue revealed goblet cell depletion on the nasal

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septum (graded as minimal to marked) and minimal to moderate hyperplasia of the olfactory
epithelium (consisting of increased numbers of nuclei) at 10 000 ppm (Table 6). The authors proposed
that these findings were the result of continued nasal exposure to malathion in the diet.
Table 6. Histopathological findings in nasal tissue in rats exposed to malathion in the diet for 28
days
No. of findings per dietary concentration
Males Females
Finding 0 ppm 10 000 ppm 0 ppm 10 000 ppm
No. of animals 15 15 15 15
Nose, Level 2 – Goblet cell depletion
Minimal 0 1 0 4
Mild 0 3 0 6
Moderate 0 9 0 4
Marked 0 2 0 0
Total 0 15 0 14
Nose, Level 3 – Hyperplasia of the olfactory epithelium
Minimal 0 3 0 6
Mild 0 12 0 9
Total 0 15 0 15
Minimal 0 0 0 2
Mild 0 3 0 9
Moderate 0 12 0 4
Total 0 15 0 15
Minimal 0 0 0 1
Mild 0 2 0 5
Moderate 0 12 0 8
Total 0 14 0 14
Results expressed as the absolute number of rats with the finding.
Source: Barnett Jr (2012a)
The no-observed-adverse-effect level (NOAEL) was 500 ppm (equal to 46.1 mg/kg bw per
day in males and 47.4 mg/kg bw per day in females) based on the inhibition of erythrocyte and brain
acetylcholinesterase activity at 5000 ppm (equal to 457.5 mg/kg bw per day in males and 461.3 mg/kg
bw per day females).
Malathion (purity 96.4%) was admixed in the diet at concentrations of 0, 50, 100, 500, 10 000
or 20 000 ppm and fed ad libitum to CDF(F-344)/CrlBr rats (5/sex per dose) for 29 or 30 days (equal
to 0, 5.1, 10.4, 51.9, 1036 and 2008 mg/kg bw per day in males and 0, 5.7, 11.6, 57.6, 1134 and 2193
mg/kg bw per day in females, respectively). Rats were observed daily for mortality and clinical signs.
Body weight and feed consumption were recorded weekly. Blood was sampled pretreatment and at

308
termination to analyse haematology and clinical chemistry parameters, including analysis of plasma
cholinesterase (ChE) and erythrocyte acetylcholinesterase activities. Ophthalmoscopy was performed
pretreatment and prior to termination. Following termination, the rats were necropsied and selected
organs weighed (adrenals, brain, kidneys and liver) and histopathology performed on tissues from
control and high-dose rats. In the remaining groups, histopathology was performed on the kidneys,
liver and lungs. Brain acetylcholinesterase activity was analysed in all the rats.
There were no deaths and no treatment-related clinical signs. In high-dose males, absolute
body weight was 12% lower (P < 0.05) than the control during the first week of exposure, while in
females it was 10% and 8% lower (P < 0.05) than the control during the first and second weeks of
exposure. In males at the highest dose, body-weight gain was 29–64% lower than the control
(P < 0.01 or 0.05) during the first three weeks of exposure, while in females, body-weight gain was
27–72% lower than the control (P < 0.01 or 0.05) over the entire exposure period.
There were no treatment-related ophthalmological findings. At 10 000 and 20 000 ppm, mean
corpuscular volume (males only), mean corpuscular haemoglobin (both sexes) and mean corpuscular
haemoglobin concentration (females only) were significantly lower (P < 0.01 or 0.05) than the
control. However, as the magnitude of these reductions was small (2–5%) and within the normal
range, they are not considered treatment related. Alkaline phosphatase (ALP) was significantly lower
than the control at 10 000 and 20 000 ppm (−24% and −27%, respectively, in males; −31% and −43%,
respectively, in females); however, reduced ALP alone is not toxicologically relevant. At these same
doses, total protein was 13–15% (P < 0.01) higher than the control and total albumin was 14–19%
(P < 0.05) higher than the control. However, these increases are unlikely to be toxicologically
relevant. In males, plasma cholinesterase activity was 28% and 59% lower (P < 0.01) than the control
at 10 000 and 20 000 ppm, respectively, while in females it was 45% and 78% lower (P < 0.05).
Although erythrocyte acetylcholinesterase activity was significantly lower (P < 0.01 or 0.05) than the
control at these same doses, the magnitude (< 20%) would not normally be considered toxicologically
significant. In males, brain acetylcholinesterase activity was 11% and 26% lower (P < 0.05 or 0.01)
than the control at 10 000 and 20 000 ppm, while in females it was 17% and 28% lower (P < 0.01),
respectively.
Relative kidney weight was significantly higher (P < 0.01) than the control at 10 000 ppm
(males only; +18%) and 20 000 ppm (both sexes; +32% in males and +27% in females); there was no
difference in absolute kidney weight. In males, absolute and relative liver weight was significantly
higher (P < 0.01) than the control at 20 000 ppm (+28% and +26%, respectively). In females, absolute
and relative liver weight was significantly higher (P < 0.01) than the control at 10 000 (+30% and
+29%, respectively) and 20 000 ppm (+38% and +52%, respectively). There were no treatment-
related macroscopic findings. Histopathology revealed centrilobular hypertrophy of hepatocytes at
10 000 (four rats/sex per group) and 20 000 ppm (all rats).
The NOAEL was 500 ppm (equal to 51.9 mg/kg bw per day in males and 57.6 mg/kg bw per
day in females) for the inhibition of brain acetylcholinesterase activity at 10 000 ppm (equal to 1036
mg/kg bw per day in males and 1134 mg/kg bw per day in females) (Daly, 1993a).
In a 90-day toxicity study by Daly (1993b), malathion (purity 96.4%) was admixed in the diet
at concentrations of 0, 100, 500, 5000, 10 000 or 20 000 ppm and fed ad libitum to CDF(F-344)/CrlBr
rats (10/sex per dose) (equal to 0, 7, 34, 340, 680 and 1390 mg/kg bw per day, respectively, in males
and 0, 8, 39, 384, 784 and 1597 mg/kg bw per day in females, respectively). The rats were observed
daily for mortality and clinical signs. Body weight and feed consumption were recorded weekly.
Blood was sampled at termination and analysed for haematology and clinical chemistry parameters,
including plasma and erythrocyte cholinesterase activities. Following termination, the rats were
necropsied, organs weighed and tissues histopathologically examined. The brains were also sampled
to analyse acetylcholinesterase activity.
One high-dose male was found dead on day 20, following emaciation, laboured breathing,
anogenital staining, decreased feed consumption and tremors; autopsy was unremarkable. There were

309
no other deaths. Anogenital staining was also observed in four males and six females at the highest
dose. Adverse effects on body weight and feed consumption were confined to the highest dose.
Absolute body weight was 10–12% lower than the control throughout most of the study, reaching
statistical significance (P < 0.01 or 0.05) during week 1 (both sexes), weeks 8–13 (males) and 6–13
(females). With the exception of reduced feed consumption in high-dose females during week 1
(−17%; P < 0.01), feed consumption was significantly elevated (P < 0.01 or 0.05) throughout most of
the exposure period.
Key haematological, clinical chemistry, organ weight and histopathological findings are
presented in Table 7. There was a significant (P < 0.01) dose-related reduction in mean corpuscular
volume and mean corpuscular haemoglobin at and above 5000 ppm (4–10%) of the control. Plasma
cholinesterase and erythrocyte and brain acetylcholinesterase activities were significantly lower
(P < 0.01 or 0.05) than the control at and above 5000 ppm (up to 17–91%, 58–72% and 9–44% of the
control, respectively), with the reduction in erythrocyte acetylcholinesterase in 500 ppm males less
than 20% and therefore not considered toxicologically significant. Gamma-glutamyltransferase
(GGT) was significantly increased (P < 0.01) in males at 20 000 ppm and in females at 10 000 and 20
000 ppm.
At the highest dose, terminal body weight was approximately 10% lower than the control
(P < 0.01 or 0.05) and thus relative brain (both sexes) and testes (males) weights were increased.
Absolute and relative kidney weights were significantly increased (P < 0.01 or 0.05) at and above
10 000 and 5000 ppm, respectively. Microscopic examination of the kidneys revealed an increase in
the severity but not the incidence of chronic nephropathy in males at and above 5000 ppm. In males,
absolute and relative liver weights were significantly higher (P < 0.01 or 0.05) than the control at and
above 5000 ppm (28–37% and 26–44%, respectively), while in females increases occurred at 10 000
and 20 000 ppm (26% and 11–28%, respectively). Periportal hypertrophy of hepatocytes was
observed microscopically at 10 000 and 20 000 ppm in males and at and above 5000 ppm in females.
Hypocellularity of the femur bone marrow was observed microscopically in three high-dose females
and in the bone marrow of the sternum of four high-dose females.
The NOAEL was 500 ppm (equal to 34 mg/kg bw per day in males and 39 mg/kg bw per day
in females) for the inhibition of erythrocyte and brain acetylcholinesterase activity at 5000 ppm (equal
to 340 mg/kg bw per day in males and 384 mg/kg bw per day in females).
Table 7. Effect of 90 days of dietary exposure to malathion in rats

Measure per dietary concentration
Parameter 0 ppm 100 ppm 500 ppm 5 000 ppm 10 000 ppm 20 000 ppm
MCV (pg)
Males 50.0 49.5 48.9 47.4** (−5%) 46.6** (−7%) 44.8** (−10%)
Females 54.5 53.1 53.1 53.3 52.1** (−4%) 50.1** (−8%)
MCH (g/dL)
Males 17.3 17.1 17.0 16.6** (−4%) 16.2** (−6%) 15.8** (−9%)
Females 19.2 18.8 18.8 18.5** (−4%) 18.3** (−5%) 17.6** (−8%)
Plasma ChE (IU/L)
Males 0.575 0.571 0.562 0.478* (−17%) 0.387** (−33%) 0.201**(−65%)
Females 0.339 0.337 0.328 0.190* (−44%) 0.0971** (−71%) 0.0311**(−91%)
Erythrocyte AChE (IU/mL)
Males 1.1 1.0 0.9** (−18%) 0.4** (−64%) 0.4** (−64%) 0.3** (−72%)
Females 1.2 0.9 0.9 0.5** (−58%) 0.4** (−67%) 0.4** (−67%)
Brain AChE (IU/g)

310

Parameter 0 ppm 100 ppm 500 ppm 5 000 ppm 10 000 ppm 20 000 ppm
Males 11.4 10.8 11.2 10.4* (−9%) 9.9** (−13%) 9.1** (−20%)
Females 11.5 11.7 11.2 10.3* (−10%) 9.5** (−17%) 6.4** (−44%)
GGT (IU/L)
Males 0 0 0 0 1 5*
Females 0 0 0 1 3** 10**
Terminal bw (g)
Males 264.9 262.2 264.3 268.8 253.2 234.0**(−12%)
Females 159.9 155.1 154.2 151.5 153.2 144.4* (−9%)
Brain weight – relative (%)
Males 6.79 6.85 6.76 6.69 7.06 7.50** (+10%)
Females 10.51 10.63 10.88 10.98 10.67 11.33** (+8%)
Kidney weight – absolute (g)
Males 2.05 2.05 2.10 2.22 2.33* (+4%) 2.55** (+24%)
Females 1.32 1.29 1.30 1.37 1.40 1.50** (+13%)
Kidney weight – relative (%)
Males 7.74 7.84 7.93 8.26** (+7%) 9.22** (+19%) 10.88** (+29%)
Females 8.27 8.32 8.46 9.02** (+9%) 9.13** (+10%) 10.35** (+26%)
Liver weight – absolute (g)
Males 7.43 7.39 7.83 9.49** (+28%) 9.93** (+34%) 11.71** (+37%)
Females 4.81 4.48 4.53 4.77 5.10 6.08** (+26%)
Liver weight – relative (g)
Males 2.80 2.82 2.96 3.53** (+26%) 3.92** (+29%) 5.00** (+44%)
Females 3.01 2.89 2.94 3.14 3.33** (+11%) 4.20** (+28%)
Testes weight – relative (%)
Males 1.47 1.48 1.45 1.46 1.52 1.63* (+11%)
Histopathology – periportal hypertrophy of hepatocytes (n = 10)
Males 0 0 0 0 1 9
Females 0 0 0 1 4 10
Histopathology – hypocellularity of bone marrow (n = 10)
Males 0 0 0 0 0 0
Females 0 0 0 0 0 4
AChE: acetylcholinesterase; bw: body weight; ChE: cholinesterase; GGT: gamma-glutamyltransferase; IU: International
Unit; MCH: mean corpuscular haemoglobin; MCV: mean corpuscular volume; ppm: parts per million; *: P < 0.05; **:
P < 0.01
Results expressed as the mean, with the % increase (+) or decrease (−) relative to the control in parentheses.
Source: Daly (1993b)
In a 90-day repeat-dose toxicity study by Barnett Jr (2012b), malathion (purity 95.8%) was
admixed in the diet and fed ad libitum to groups of Crl:CD[SD] rats at concentrations of 0, 100, 500,
5000 or 10 000 ppm. The study was divided into two parts: (1) groups of 10 rats per sex allocated to
the subchronic phase of the study; and (2) groups of 15 rats per sex allocated to the analysis of
acetylcholinesterase activity. The doses achieved in the subchronic phase of the study were 0, 7.2,

311
35.0, 353.6 and 733.8 mg/kg bw per day in males and 0, 7.5, 35.9, 363.1 and 719.0 mg/kg bw per day
in females, while the doses achieved in the acetylcholinesterase phase were 0, 6.2, 31.4, 311.8 and
635.3 mg/kg bw per day in males and 0, 6.6, 33.8, 335.5 and 680.3 mg/kg bw per day, respectively.
Rats were observed regularly for deaths and clinical signs. Body weight and feed consumption were
recorded daily during the first week of exposure and weekly thereafter. Ophthalmological
examinations were performed prior to exposure and in the week prior to termination. On day 91, the
10 rats per sex per group in the subchronic phase of the study were terminated and their blood
haematology and clinical chemistry parameters analysed. Rats were necropsied and selected organs
weighed. Tissues, including bone marrow and tissue from the nasal cavity and turbinates, were
histopathologically examined. The remaining 15 rats per sex per group were processed to analyse
erythrocyte and brain acetylcholinesterase activity.
Subchronic phase: There were no deaths or treatment-related clinical signs. In males, overall
(day 1–85) body-weight gains were 17% and 15% lower than the control (P < 0.05) at 5000 and
10 000 ppm, respectively, while in females overall body-weight gain was significantly lower
(P < 0.05) than the control at 10 000 ppm (−25%). At 10 000 ppm, feed consumption was
significantly lower than the control (P < 0.01) in both sexes during the first week of dosing (−20% in
males, −13% in females). Overall feed conversion efficiency was significantly lower (P < 0.01 or
0.05) than the control at 5000 and 10 000 ppm in males (−8% at both doses) and 10 000 ppm in
females (−15%). There were no treatment-related ophthalmological findings. In males, there were no
significant intergroup differences in haematological parameters, while in females, significant changes
in platelet volume (+12%, P < 0.05), MCV (−4%, P < 0.05) and MCH (−6%, P < 0.05) at the highest
dose were within the normal range for age- and sex-matched rats and therefore not considered
adverse. At 10 000 ppm, GGT was significantly higher than the control (P < 0.05) in both sexes,
while cholesterol was increased at 5000 and 10 000 ppm in males (+50% and +84%, respectively;
P < 0.05) and 10 000 ppm in females (+68%; P < 0.05). Slight though significant (P < 0.05) increases
in total protein (+9%), albumin (+7%) and globulin (+12%) occurred at 5000 and 10 000 ppm without
a change in the albumin to globulin ratio in males.
There were no treatment-related macroscopic abnormalities. In males, absolute liver weight
was 28% higher than the control at 10 000 ppm (P < 0.01), while relative liver weight was increased
at 5000 (+20%, P < 0.01) and 10 000 ppm (+44%, P < 0.01). At 10 000 ppm, absolute and relative
paired kidney weights were significantly increased by 23% and 38%, respectively (P < 0.01).
Absolute paired epididymides weights were significantly reduced at 5000 (−12%, P < 0.01) and 10
000 ppm (−10%, P < 0.05). Absolute prostate weights were decreased at 5000 (−20%, P < 0.05) and
10 000 ppm (−22%, P < 0.01). Relative paired testes weight was 19% higher than the control
(P < 0.01) at 10 000 ppm. None of these changes in organ weights were accompanied with any
corroborating microscopic changes. In females, organ weight changes were confined to the highest
dose and included increased relative liver weight (+29%, P < 0.01) and relative paired kidney weight
(+29%, P < 0.01). At and above 500 ppm, minimal to mild depletion of goblet cells in the nasal cavity
occurred (Table 8). Small numbers of cells with abundant non-staining cytoplasm were interspersed
where goblet cells were depleted. Minimal to moderate hyperplasia of olfactory epithelium was also
noted at these same doses consisting of increased numbers of nuclei (Table 8). There were no
treatment-related effects in the bone marrow.

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Table 8. Histopathological findings in the nasal tissue of rats exposed to malathion in the diet for
90 days
No. of findings per dietary concentration
Males Females
100 500 5 000 10 000 100 500 5 000 10 000
Finding 0 ppm ppm ppm ppm ppm 0 ppm ppm ppm ppm ppm
No. of animals 10 10 10 10 10 10 10 10 10 10
examined
Goblet cell depletion – nose Level 2
Minimal 0 0 5 1 3 0 0 3 4 1
Mild 0 0 0 7 2 0 0 2 3 2
Moderate 0 0 0 2 4 0 0 0 1 6
Marked 0 0 0 0 0 0 0 0 0 1
Total 0 0 5 10 9 0 0 5 8 10
Hyperplasia of olfactory epithelium – nose Level 3
Minimal 0 0 0 6 7 0 0 0 5 3
Mild 0 0 0 3 3 0 0 0 4 6
Moderate 0 0 0 0 0 0 0 0 0 1
Total 0 0 0 9 10 0 0 0 9 10
Minimal 0 0 0 0 0 0 0 0 1 0
Mild 0 0 0 7 7 0 0 0 4 5
Moderate 0 0 0 3 3 0 0 0 5 5
Total 0 0 0 10 10 0 0 0 10 10
Results expressed as the number of rats with the finding.
Source: Barnett Jr (2012b)
Cholinesterase phase: There were no treatment-related deaths. Treatment-related clinical

signs were confined to high-dose females, where a significant increase (P < 0.01) in urine-stained
abdominal fur occurred. Absolute body weight was 9–12% lower than the control (P < 0.01)
throughout the study at 10 000 ppm in males, with overall (day 1–91) body-weight gain 16%
(P < 0.01) lower than the control. Absolute body weights were unremarkable in females, while body-
weight gain was significantly lower than the control during the first week of exposure (−32%,
P < 0.01). Feed consumption was significantly lower (P < 0.01 or 0.05) than the control at various
times (−10%) in males at 10 000 ppm, with overall feed consumption significantly lower than the
control (−8%, P < 0.05). Overall feed conversion efficiency was also significantly lower than the
control in high-dose males (−9%, P < 0.01). In females at 10 000 ppm, feed consumption was
reduced only during the first week of exposure (−13%, P < 0.01), while feed conversion efficiency
was also reduced during the first week (−24%, P < 0.01). There were no treatment-related
In males, erythrocyte acetylcholinesterase activity was significantly lower than the control at
and above 500 ppm (P < 0.01), while toxicologically significant inhibition occurred at 5000 and 10
000 ppm (−16.3%, −72.9% and −85.5% at 500, 5000 and 10 000 ppm, respectively). In females,
erythrocyte acetylcholinesterase activity was significantly lower (P < 0.01 or 0.05) than the control at
every dose but was toxicologically significant at and above 500 ppm (−10.2%, −19.6%, −79.2% and
−85.6% at 100, 500, 5000 and 10 000 ppm, respectively). Brain acetylcholinesterase activity was

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significantly lower (P < 0.01) than the control at 5000 and 10 000 ppm in both sexes (−17.3% and
−18.1% in males and −21.7% and −49.5% in females, respectively). BMD modelling was applied to
these data using an exponential model recommended by the United States Environmental Protection
Agency (USEPA). The estimated BMD20 of erythrocyte acetylcholinesterase was 48.7 mg/kg bw per
day for males rats and 42.0 mg/kg bw per day for females. The estimated BMD10 for brain
acetylcholinesterase activity was 174.6 mg/kg bw per day for male rats and 118.4 mg/kg bw per day
for female rats.
The NOAEL was 100 ppm (equal to 7.2 mg/kg bw per day in males and 7.5 mg/kg bw per
day in females) for goblet cell depletion in the nasal cavity and the inhibition of erythrocyte
acetylcholinesterase activity at 500 ppm (equal to 35.0 mg/kg be per day in males and 35.9 mg/kg bw
per day in females). The lower NOAEL in this study compared to other 90-day studies of toxicity is
considered an outlier due to the likelihood that it resulted from inhalational exposure to the test
compound in feed.
In a 13-week inhalational toxicity study by Beattie (1994), groups of 15 Crl:CD[SD]BR rats

per sex were exposed (whole-body) to aerosols of malathion (purity 94%) for six hours per day, five
days per week for 13 weeks at nominal concentrations of 0, 0.24, 1.10 or 4.94 mg/L (analytical
concentrations of 0, 0.1, 0.45 and 2.0 mg/L, respectively). These concentrations were based on the
results of a 2-week range-finding study (Beattie 1993) that tested nominal concentrations of 0, 1.56,
6.29 or 13.81 mg/L (analytical concentrations of 0.56, 1.58 and 4.23 mg/L, respectively). The rats
were observed daily for mortality and clinical signs. Body weight and feed consumption were
recorded weekly. Ophthalmology was performed pretreatment and during week 13. Blood and urine
were sampled during week 13 to analyse haematology, clinical chemistry and urine analysis
parameters, including plasma and erythrocyte cholinesterase activities. Following termination, the rats
were necropsied, organs weighed and tissues histopathologically examined, with attention paid to
effects on the nasal cavities, bronchi, lungs, skin and eyes. Brains were also sampled to analyse
acetylcholinesterase activity.
There were no deaths. Treatment-related clinical signs included excessive salivation and oily
fur, which occurred at all doses and affected up to 2, 5 and 15 rats, at 0.1, 0.45 and 2.0 mg/L,
respectively, in both sexes. Red staining of the muzzle, lower jaw, periorbital region, cranial region
and ventral cervical region were observed at every concentration, but there is uncertainty about
whether these finding were due to malathion exposure or to high concentrations of aerosols. There
was no treatment-related effect on body weight or feed consumption. Ophthalmology and
haematology were unremarkable. Toxicologically significant inhibition of erythrocyte
acetylcholinesterase occurred at 0.45 (−22% in males, P > 0.05; −27% in females, P < 0.05) and
2.0 mg/L (−43% in males, P > 0.05; −44% in females, P < 0.05). Brain acetylcholinesterase activity
was significantly lower than the control only in high-dose females (−41%, P < 0.01). In high-dose
males and females, serum cholesterol was 33% and 31% higher than the control (P < 0.01),
respectively. There were some changes in urine analysis parameters at the highest dose, including
increased white blood cells and epithelial cells in males and lower urine pH (pH 6 or 6.5 versus pH 6–
7 in the control) and increased uric acid crystals in females.
There were no treatment-related macroscopic findings. Absolute liver weight was
significantly higher (P < 0.05) than the control at the highest dose (+14% in males and +15% in
females), with a corresponding increase (P < 0.05) in relative liver weight (+20% in males and +14%
in females). Absolute kidney weight was 9% higher than the control (P < 0.05) in high-dose females.
As the magnitude of these differences was small and, in the absence of any treatment-related
microscopic changes in these organs, these differences in liver and kidneys weights were not
considered adverse. Histopathology revealed a high incidence of laryngeal hyperplasia and
degeneration and/or hyperplasia of the olfactory epithelium in the nasal cavity, at all doses, with a
dose-related increase in severity (Table 9). A NOAEL was not determined because treatment-related
changes in the respiratory tract occurred at all doses.

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Table 9. Histopathological findings in rats exposed to aerosols of malathion for 13 weeks

No. of findings per aerosol concentration
Parameter 0 mg/L 0.1 mg/L 0.45 mg/L 2.0 mg/L
Laryngeal hyperplasia
Males
Incidence 0/15 13/15 15/15 15/15
a
Severity – 1.1 2.4 2.9
Females
Incidence 0/15 15/15 15/15 15/15
a
Severity – 1.4 2.7 2.5
Degeneration and/or hyperplasia of the olfactory epithelium
Males
Incidence 1/15 15/15 15/15 14/15
a
Severity 0.1 1.6 1.7 2.6
Females
Incidence 1/15 10/15 15/15 14/15
a
Severity 0.1 0.7 1.6 2.6
No.: number
Results expressed as number of animals with the finding / number of animals examined.
Severity expressed as the mean histopathological grading: 1 – slight, 2 – mild, 3 – moderate, 4 – severe.
Source: Beattie (1994)
Rabbits
In a dermal toxicity study by Moreno (1989), groups of six New Zealand White rabbits per
sex were exposed to malathion (purity 94%) for six hours per day, five days per week for three weeks
at doses of 0, 50, 300 or 1000 mg/kg bw per day. Other than irritation at the application site, the only
treatment-related effect was reduced cholinesterase activity. At 300 mg/kg bw per day, erythrocyte
acetylcholinesterase activity was 26% lower than the control (P < 0.01) in females, while at the
highest dose, plasma, erythrocyte and brain cholinesterase activities were significantly lower than the
control in both sexes (plasma: −57% in males, −48% in females; erythrocytes: −74% in males, −66%
in females; cerebrum: −66% in males, −53% in females; cerebellum: −41% in males, −49% in
females. The NOAEL was 300 mg/kg bw per day for the inhibition of erythrocyte
acetylcholinesterase activity and 1000 mg/kg bw per day for the inhibition of brain
A 21-day dermal toxicity study was conducted in Hra:NZW(SPF) rabbits by Barnett (2006d)
to generate cholinesterase data suitable for BMD modelling. Groups of 10 rabbits per sex were
exposed to malathion (purity 96%) for 6 hours per day via clipped skin under an occlusive dressing at
doses of 0, 75, 100, 150 or 500 mg/kg bw per day. Following the exposure period, the application site
was washed with water. The rabbits were observed daily for mortality and clinical signs. The
application site was examined for signs of irritation immediately after washing. Ophthalmological
examinations were performed prior to commencing the study and at termination in rabbits from the
control and high-dose groups. The rabbits were terminated on day 22, necropsied and blood and brain
acetylcholinesterase activity analysed. Blood haematology and clinical chemistry parameters was also
analysed. Organs were weighed, and tissues from the control and high-dose groups examined
histopathologically.
There were no treatment-related deaths or clinical signs. Irritation was observed at the
application site: erythema (every dose), flaking (every dose), oedema (grade 1) and scabs (500 mg/kg

315
bw per day). There was no treatment-related effect on body weight, feed consumption, haematology
or clinical chemistry parameters, or on the occurrence of macroscopic or microscopic findings.
Absolute and relative spleen weights were significantly higher than the control at 500 mg/kg bw per
day but were unaccompanied by any other treatment-related effects. Plasma cholinesterase activity
was significantly lower (P < 0.01) than the control at 500 mg/kg bw per day (−32% in males and
−37.1% in females). In males, erythrocyte acetylcholinesterase was significantly lower (P < 0.01)
than the control at every dose but only the reduction at the highest dose was considered
toxicologically significant (−17.5%, −18.4%, −19.4% and −60.9% at 75, 100, 150 or 500 mg/kg bw
per day, respectively). In females, statistically and toxicologically significant inhibition of erythrocyte
acetylcholinesterase occurred at 150 and 500 mg/kg bw per day (−24.2% and −71.2%, respectively;
P < 0.01). Brain acetylcholinesterase activity was inhibited only at 500 mg/kg bw per day (−21.9% in
males and −23.0% in females; P < 0.01). The NOAEL was 150 mg/kg bw per based on the inhibition
of brain acetylcholinesterase activity at 500 mg/kg bw per day.
Dogs
In a 28-day range-finding study by Fischer et al. (1988), malathion (purity 92.4%) in gelatine
capsules was administered orally to groups of three beagle dogs of each sex at doses of 0, 125, 250 or
500 mg/kg bw per day. Dogs were observed daily for mortality and clinical signs. Body weight was
recorded weekly and feed consumption daily. Blood was sampled pretreatment and on days 15 and 19
(termination) to analyse haematology, clinical chemistry parameters and plasma and erythrocyte
cholinesterase activities. Ophthalmoscopy was not performed. Following termination, dogs were
necropsied, organs weighed and histopathology performed. Brain acetylcholinesterase was not
analysed.
One high-dose male died on day 24, following anorexia and listlessness. Clinical signs
occurred in 1 to 4 dogs at every dose and included increased diarrhoea, loose stools and mucoid
faeces. In addition, at the highest dose emesis, anorexia and depression occurred sporadically. These
clinical signs began from day 1 and generally persisted throughout the dosing period. At 500 mg/kg
bw per day, body weight was 18% (P < 0.05) lower than the control during week 3, with body-weight
gain lower during weeks 1–2 and 2–3 (−4 g versus 0 and +2 g, respectively, in the control). Also at
500 mg/kg bw per day, feed consumption was significantly lower (P < 0.05) than the control during
week 1 (−26%), 2 (−42%) and 3 (−26%). There were no treatment-related haematology findings.
Plasma and erythrocyte cholinesterase activities were significantly lower (P < 0.05) than the
control at 250 and 500 mg/kg bw per day (plasma: −17% at termination at 250 mg/kg bw per day and
−20% on day 15 at 500 mg/kg bw per day; erythrocytes: −32% on day 15 at both 250 and 500 mg/kg
bw per day). At termination, serum albumin was significantly lower than the control (P < 0.05) at 250
and 500 mg/kg bw per day (−11% and −32%, respectively). At 500 mg/kg bw per day, absolute uterus
and ovary weight was 58% lower (P < 0.05) than the control. There were no treatment-related
macroscopic or histopathological findings.
In a one-year toxicity study by Shellenberger & Billups (1987), malathion (purity 95%) in
gelatine capsules was administered orally to groups of six beagle dogs per sex at doses of 0, 62.5, 125
or 250 mg/kg bw per day. Dogs were observed daily for mortality and clinical signs. Body weight was
recorded weekly and feed consumption daily. Blood and urine were sampled pretreatment and after 6
weeks, 3 months and 6 months to analyse haematology, clinical chemistry or urine analysis
parameters, including plasma and erythrocyte cholinesterase activities. Ophthalmoscopy was
performed pretreatment and prior to termination. Following termination, dogs were necropsied,
organs weighed and histopathology performed and brain acetylcholinesterase analysed.
There were no treatment-related deaths and clinical signs. There were no significant
intergroup differences in body weight although high-dose dogs had body weights approximately 0.5–1
kg lower in the second half of the exposure period. In high-dose males, feed consumption was

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significantly lower (P < 0.05, 10–23%) than the control at various times starting at week 11. There
were no treatment-related ophthalmological effects.
Significant reductions (P < 0.05) in erythrocyte counts (−9% to −18% in males and −14% to
−17% in females), haemoglobin (−10% to −16% in both sexes) and haematocrit (−12% to −15% in
both sexes) occurred consistently at the highest dose, with erythrocytes also significantly lower
(P < 0.05), than the control at 125 mg/kg bw per day in females at 3 and 6 months (−11% or −10%,
respectively). Platelet counts were 21–44% higher than the control (P < 0.05) throughout the study
and at every dose in males at 6 weeks and in females at termination, but still within the normal range
for beagle dogs (200–500  109/L). Albumin was 10–12% lower than the control (P < 0.05) at the
highest dose in males and 13–19% lower in females at every sampling point. In females, albumin was
8% or 9% lower than the control (P < 0.05) at 62.5 and 125 mg/kg bw per day during week 6 only. In
high-dose females, the albumin to globulin ratio was 21% and 15% lower than the control (P < 0.05)
at 6 weeks and 6 months, respectively. Lactate dehydrogenase was approximately threefold higher
than the control (P < 0.05) in high-dose males at 3 months and in high-dose females at termination.
Serum calcium was 7–10% lower than the control (P < 0.05) in high-dose males at 6 months and
high-dose females at every sampling point. There was no treatment-related effect on urine analysis
parameters.
The results of cholinesterase analysis are summarized in Table 10. Plasma and erythrocyte
cholinesterase activities were significantly lower (P < 0.05) than the control at every dose. While the
inhibition of erythrocyte acetylcholinesterase activity was greater than 20%, there was a flat dose-
related increase in the level of inhibition and no significant reduction in brain acetylcholinesterase
activity; on this basis the inhibition of erythrocyte acetylcholinesterase activity is considered to be of
marginal toxicological significance.
Table 10. Effects of one-year of capsular exposure of dogs to malathion on cholinesterase activity
Measure of cholinesterase activity per dietary concentration
0 62.5 125 250
Parameter mg/kg bw per day mg/kg bw per day mg/kg bw per day mg/kg bw per day
Plasma ChE (mU/mL)
Males
6 weeks 1 918 1 401* (−27%) 1 480* (−23%) 1 348* (−30%)
3 months 1 850 1 348* (−27%) 1 384* (−25%) 1 321* (−29%)
6 months 1 907 1 421* (26%) 1 442* (−24%) 1 421* (_26%)
12 months 1 837 1 361 (−26%) 1 333 (−27%) 1 474 (−20%)
Females
6 weeks 1 995 1 551* (−22%) 1 489* (−25%) 1 254* (−37%)
3 months 1 912 1 468* (−23%) 1 326* (−31%) 1 244* (−35%)
6 months 2 098 1 629* (−22%) 1 545* (−26%) 1 304* (−38%)
12 months
Erythrocyte AChE (mU/mL)
Males
6 weeks 4 090 3 273* (−18%) 3 257* (−20%) 3 163* (−23%)
3 months 4 053 3 181* (−22%) 2 987* (−26%) 2 978* (−27%
6 months 3 727 2 950* (−21%) 2 900* (−22%) 2 753* (−26%)
12 months 3 790 2 793* (−26%) 2 860* (−25%) 2 810* (−26%)
Females

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0 62.5 125 250
Parameter mg/kg bw per day mg/kg bw per day mg/kg bw per day mg/kg bw per day
6 weeks 3 960 3 113* (−21%) 3 077* (−22%) 3 023* (−24%)
3 months 3 915 3 047* (−22%) 2 987* (−24%) 2 913* (−26%)
6 months 3 717 2 913* (−22%) 2 760* (−26%) 2 550* (−31%)
12 months 3880 2 820* (−27%) 2 813* (−28%) 2 800* (−28%)
Brain AChE – cerebrum (mU/mL)
Males 1 050 997 (−5%) 983 (−6%) 1 004 (−4%)
Females 888 885 823 (−7%) 885
Brain AChE – cerebellum (mU/mL)
Males 2 602 2 831 2 677 2 190 (−16%)
Females 2 390 2 704 2 383 2 123 (−11%)
AChE: acetylcholinesterase; bw: body weight; ChE: cholinesterase; *: P < 0.05
Source: Shellenberger & Billups (1987)
There were no treatment-related macroscopic findings. In males, absolute kidney weight was
25% (P < 0.05) and 47% (P < 0.05) higher than the control at 125 and 250 mg/kg bw per day,
respectively, with a corresponding increase in relative kidney weight (23% and 58%, respectively).
Relative liver weight increased by 33% (P < 0.05) in high-dose males. In females, relative kidney
weight increased (P < 0.05) by 17% and 70% at 125 and 250 mg/kg bw per day, respectively.
Relative liver weight increased (P < 0.05) at every dose (34%, 31% and 51% at 62.5, 125 and 250
mg/kg bw per day, respectively). These increases in organ weights were not accompanied by any
histopathological findings.
The NOAEL was 125 mg/kg bw per day for reduced body weight and haematological
changes at 250 mg/kg bw per day.

Mice
In a pre-GLP study conducted by the National Cancer Institute (NCI, 1978), malathion (purity
> 95%) was admixed in the diet at concentrations of 8000 or 16 000 ppm and fed ad libitum to groups
of B6C3F1 mice (50/sex per group) for 80 weeks (equivalent to doses of 1200 and 2400 mg/kg bw
per day, respectively). Concurrent control groups comprised 10 untreated mice per sex, while matched
controls from similar bioassays comprised 40 mice per sex for statistical comparisons. Following the
exposure period, the mice were observed for a further 14 or 15 weeks. There was no treatment-related
effect on survival. There were no treatment-related clinical signs observed during the first year of
exposure, while alopecia, rough and discoloured fur, reduced feed consumption, hyperexcitability and
abdominal distension occurred with increasing frequency in treated mice. Five high-dose females
displayed generalized body tremors from weeks 71–79. Graphically presented data indicated that
mean body weight was lower than the control at both doses throughout the exposure period. In males,
the incidence of hepatocellular carcinoma was consistent across all groups (2/10 [20%], 7/48 [15%]
and 11/49 [22%] at 0, 8000 and 16 000 ppm, respectively) while there was a slight increase in
neoplastic nodules in the liver of high-dose males (6/49 versus 3/49 in the pooled control). While
combining the incidence of hepatocellular carcinoma and nodules resulted in a significant linear trend
when either the matched control (P = 0.019) or pooled control (P = 0.019) was used, pairwise
comparisons of either neoplasm were not statistically significant. In addition, the incidence of these
findings was consistent with historical control data from the same laboratory where the incidence of

318
spontaneous liver tumours in males was 19%. On the basis of these findings, the authors concluded
that malathion was not carcinogenic in mice. Rueber (1985) re-examined the slides from this study
and concluded that malathion caused an increase in neoplasms in the liver of male mice. However,
this conclusion is not considered reliable because of the absence of methodological detail on how the
slides were re-examined. The United States National Toxicology Program (NTP) (Huff et al., 1985)
also re-evaluated the same slides and confirmed the conclusion of the original study authors. A
NOAEL for chronic toxicity was not determined because clinical signs during the second year of
exposure and reduced body weight occurred at both doses. Overall this study was considered not
acceptable for the evaluation of carcinogenicity because of the small number of concurrent control
animals.
Malathion (purity 96.4%) was admixed in the diet at concentrations of 0, 100, 800, 8000 or
16 000 ppm and fed ad libitum to groups of 65 B6C3F1 (BR) mice per sex. Ten mice per sex per
group were terminated after 12 months, and all the survivors after 18 months. The doses achieved
over 18 months were, respectively, 0, 17, 143, 1476 and 2978 mg/kg bw per day in males and 0, 21,
167, 1707 and 3448 mg/kg bw per day in females. Mice were observed daily for mortality and clinical
signs. Body weight and feed consumption were recorded weekly for 14 weeks, twice weekly to week
26 and monthly thereafter. Blood was sampled at 12 and 18 months to analyse haematology and
clinical chemistry parameters, including the analysis of plasma and erythrocyte cholinesterase
activities. In mice assigned for termination after 12 months, blood was also collected at 9 months to
analyse erythrocyte acetylcholinesterase activity. Following scheduled termination at 12 and 18
months, the mice were necropsied, organs weighed and tissues histopathologically examined. Brain
acetylcholinesterase was analysed in all mice.
Survival was comparable across all groups and there were no adverse, treatment-related
clinical signs. At 8000 and 16 000 ppm, absolute body weight was significantly lower than the control
(P < 0.01) over the entire period of exposure; at the end of the study, absolute body weight was 3%,
3%, 14% and 20% lower than the control in males and 0%, 0%, 10% and 16% lower than the control
in females at 100, 800, 8000 and 16 000 ppm, respectively. At these same doses, feed consumption
was also significantly lower than the control (P < 0.01 or 0.05), most consistently from week 30
(mean feed consumption over the exposure period was 2% and 6% lower than the control in males
and 5.4% and 12.5% lower than the control in females at 8000 and 16 000 ppm, respectively).
There was no treatment-related effect on haematological parameters. The results of
cholinesterase analyses are presented in Table 11. Plasma cholinesterase was significantly lower
(P < 0.01 or 0.05) than the control at 8000 and 16 000 ppm in males, and at and above 800 ppm in
females. Statistically and toxicologically significant inhibition of erythrocyte acetylcholinesterase
activity occurred in both sexes at and above 800 ppm. Brain acetylcholinesterase activity was
inhibited by more than 20% at 8000 and 16 000 ppm; however, only inhibition at the highest dose at
termination was statistically significant.
Table 11. Effects of 18 months of dietary exposure to malathion on cholinesterase activity in mice
Parameter 0 ppm 100 ppm 800 ppm 8 000 ppm 16 000 ppm
Plasma ChE (µmol/mL per min)
Males
12 months 8.8 8.3 6.8 (−23%) 1.1* (−88%) 0.6* (−93%)
18 months 2.1 2.3 1.6 (−24%) 0.2** (−91%) 0.1** (−95%)
Females
12 months 10.8 10.8 8.9* (−18%) 1.2** (−89%) 0.6** (−94%)
18 months 2.5 2.2 (−12%) 1.6* (−36%) 0.2** (−92%) 0.1** (−96%)
Erythrocyte AChE (µmol/mL per min)

319

Males
9 months 2.4 2.6 1.5 (−28%) 0.7* (−71%) 0.7* (−71%)
12 months 4.9 4.5 (−8%) 3.1* (−37%) 1.7** (−65%) 1.5** (−69%)
18 months 3.9 3.3 (−15%) 2.2 (−44%) 0.4** (−90%) 0.3** (−92%)
Females
9 months 1.8 1.8 1.1** (−39%) 0.7* (−61%) 0.6** (−67%)
12 months 4.8 4.9 3.1 (−35%) 1.7** (−65%) 1.5** (−69%)
18 months 3.6 2.5 (−31%) 1.5* (−58%) 0.3** (−92%) 0.3** (−92%)
Brain AChE (µmol/L per g per min)
Males
12 months 15.6 17.2 17.4 14.9 (−4%) 11.9 (−24%)
18 months 16.2 16.3 15.1 (−7%) 12.4 (−23%) 10.2** (−37%)
Females
12 months 16.2 16.8 15.8 17.7 13.0 (−20%)
18 months 15.2 13.7 (−10%) 14.8 (−3%) 12.2 (−20%) 8.7** (−43%)
AChE: acetylcholinesterase; ChE: cholinesterase; min: munite; ppm: parts per million; *: P < 0.05; **: P < 0.01
Results expressed as the mean, with the % decrease (−) relative to the control in parentheses.
Source: Slauter (1994)
Selected macroscopic and microscopic findings are summarized in Table 12. No treatment-
related macroscopic abnormalities were observed in mice terminated after 12 months. Necropsy
revealed an increased incidence of liver masses, liver nodules and tan or yellow foci (mainly graded
as slight) at the highest dose. In mice terminated after 12 and 18 months, absolute and relative liver
weights were increased in males at 8000 and 16 000 ppm, and in females at 16 000 ppm. In high-dose
mice terminated after 18 months, absolute heart weight was significantly lower than the control in
both sexes (−21% in males and −19% in females). Hypertrophy of hepatocytes occurred
microscopically at 8000 and 16 000 ppm in mice terminated after 12 and 18 months. In mice
terminated after 12 months, the hypertrophy was graded as trace to moderate, while in mice
terminated after 18 months, it was graded as mild to trace at 8000 ppm and moderate to severe at
16 000 ppm. The incidence of hypertrophy was outside of the performing laboratory’s historical
control range (0–4.76% over 18 months in both sexes).
The incidence of hepatocellular adenomas was increased at 8000 and 16 000 ppm in both
sexes, which was outside the performing laboratory’s historical control range (14.29–21.74% in males
and 0–10.24% in females over 18 months). In males, the occurrence of liver carcinomas was
significantly higher in the test mice than in the controls (P < 0.05) at the lowest dose and second-
highest dose; however, these increases were not considered treatment related for the following
reasons:
 There was no dose–response relationship.
 The non-dose-related increase in carcinomas was not corroborated in females where
equivalent increases in liver hypertrophy and liver adenomas occurred as in males.
 Liver adenomas and carcinomas are common age-related tumours observed in dietary studies
in untreated control B6C3F1 male mice. Published data (Haseman, Hailey & Morris, 1998)
indicate that the combined incidence of these tumours is 10–68%, with the range for
adenomas 4–60% and for carcinomas 6–29%.

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In females terminated after 12 months, early disappearance of the x-zone of the inner cortex
of the adrenals occurred in all mice at 8000 and 16 000 ppm. In females terminated after 18 months,
multifocal mineralization in the cortex of the kidney occurred at 8000 and 16 000 ppm (1/55, 6/52,
8/52, 32/53 and 36/51 at 0, 100, 800, 8000 and 16 000 ppm, respectively). Due to severe nasal toxicity
in a 24-month rat study (Daly, 1996a), the USEPA requested that the nasal turbinate tissue in the
current study be histopathologically examined. This examination by Swenberg (1999c) detected no
neoplastic lesions. Non-neoplastic lesions identified at 8000 and 16 000 ppm consisted of
degeneration and loss of cellularity of the olfactory epithelium, loss of olfactory nerves in the
submucosa, increased glandular secretion due to the retention of mucus and atrophy of the olfactory
epithelium adjacent to the retained mucus. This nasal toxicity occurred at both 12 and 18 months.
The NOAEL was 800 ppm (equal to 143 mg/kg bw per day in males and 167 mg/kg bw per
day in females) for the inhibition of brain acetylcholinesterase activity at 8000 ppm (equal to 1476
mg/kg bw per day in males and 1707 mg/kg bw per day in females). The NOAEL for carcinogenicity
was 800 ppm (equal to 143 mg/kg bw per day in males and 167 mg/kg bw per day in females) for the
occurrence of liver adenomas at 8000 ppm (equal to 1476 mg/kg bw per day in males and 1707 mg/kg
bw per day in females) (Slauter, 1994).
Table 12. Macroscopic and microscopic findings in mice after dietary exposure to malathion
a
Liver masses – 18 month necropsy
Males 0/50 8/51 (16%) 4/48 (8%) 5/54 (9%) 18/50 (36%)
Females 1/55 (2%) 0/52 3/52 (6%) 2/53 (4%) 10/51 (20%)
a
Liver nodules - 18 month necropsy
Males 5/50 (10%) 2/51 (4%) 3/48 (6%) 10/54 (19%) 19/50 (38%)
Females 1/50 (2%) 2/51 (4%) 0/48 9/54 (17%) 29/50 (58%)
a
Tan or yellow liver foci - 18 month necropsy
Males 0/50 0/51 1/48 (2%) 2/54 (4%) 18/50 (36%)
Females 0 0 0 2/54 (4%) 9/50 (18%)
b
Absolute liver weight (g) – 12 months
Males 1.62 1.71 1.78 1.98** (+22%) 2.38** (+47%)
Females 1.55 1.68 1.56 1.66 1.92** (+24%)
b
Relative liver weight (%) – 12 months
Males 5.15 5.19 5.42 6.95** (+35%) 8.30** (+61%)
Females 5.22 5.39 5.22 6.21** (+19%) 7.56** (+45%)
b
Males 1.90 2.90 1.96 2.26** (+19%) 2.66** (+40%)
Females 1.93 1.77 1.96 1.92 2.18
b
Males 5.59 6.15 5.82 7.51** (+34%) 9.38** (+68%)
Females 6.19 5.76 6.26 6.90 8.51** (+37%)
a
Hypertrophy of hepatocytes – 12 months
Males 0/10 0/10 0/10 7/10 10/10
Females 0/10 0/10 0/10 5/10 10/10
a

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Males 0/50 1/51 0/48 1/54 3/50
Females 0/55 0/52 0/52 53/53 51/51
a
Hepatocellular adenoma – 18 months
Males 1/50 6/51 2/48 13/54* 49/50**
Females 0/55 1/52 0/52 9/53* 42/51**
a
Hepatocellular carcinoma – 18 months
Males 0/50 6/51* 2/48 6/54* 1/50
Females 1/55 0/52 2/52 1/53 2/51
a
Results expressed as the number of animals with the finding / number of animals examined per group, with the % increase
(+) or decrease (−) relative to the control in parentheses, or as the mean, with the % increase (+) or decrease (−) relative to
the control in parentheses.
b
Results expressed as absolute weight (g) or relative weight (%) with the % increase (+) relative to the control in
parentheses.
Rats
In a pre-GLP study conducted by the National Cancer Institute (NCI, 1978), malathion (purity
> 95%) was admixed in the diet at concentrations of 4700 or 8150 ppm and fed ad libitum to groups
of Osborne–Mendel rats (50/sex per group) for 80 weeks (equivalent to doses of 1200 and 2400
mg/kg bw per day, respectively). Concurrent control groups comprised 15 untreated rats per sex,
while matched controls from similar bioassays comprised 40 rats per sex. Following the exposure
period, the rats were observed for a further 30 weeks and then terminated after 109 weeks. There was
no treatment-related effect on survival. During the second year of treatment, an increase in clinical
signs, including rough hair coats, pale mucous membranes, dermatitis, ataxia, alopecia and
haematuria, was reported. Graphically presented data showed that body weights of treated females
were lower than the control. The treated rats had an increase in proliferative lesions of the thyroid
(Table 13). The Cochran–Armitage test or pairwise comparisons using Fisher’s exact test found no
significant increase in tumours in males. In females, the Cochran–Armitage test found a significant
dose-related increase in thyroid follicular cell adenomas and carcinomas (P = 0.026), but pairwise
comparisons with Fisher’s exact test found no significant differences. On the basis of these findings,
the authors concluded that malathion was not carcinogenic in rats. Rueber (1985) re-examined the
slides from this study and concluded that the incidence of neoplasms across all tissues was increased
in the treated rats. However, this conclusion is not considered reliable because of the absence of
methodological detail on how the slides were re-examined. The NTP (Huff et al., 1985) also re-
evaluated the same slides and confirmed the original interpretation that malathion is not carcinogenic
in rats. Overall this study was considered not acceptable for the evaluation of carcinogenicity because
of the small number of concurrent control animals.

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Table 13. Thyroid lesions in rats after dietary exposure to malathion

No. and incidence per dose
Males Females
Lesion Control Low dose High dose Control Low dose High dose
C-cell 5/48
0/14 1/41 (2.4%) 3/47 (6.4%) 0/15 3/49 (6.1%)
hyperplasia (10.4%)
C-cell
0/14 1/41 (2.4%) 3/47 (6.4%) 0/15 1/48 (2.1%) 2/49 (4.1%)
adenoma
Follicular cell
1/14 (7.1%) 7/41 (17%) 8/47 (17%) 0/15 3/48 (6.3%) 0/49
hyperplasia
Follicular cell
1/14 (7.1%) 1/41 (2.4%) 1/47 (2.1%) 0/15 0/48 1/49 (2%)
adenoma
Follicular cell
0/14 2/41 (4.9%) 6/47 (12.8%) 0/15 0/48 3/49 (6.1%)
carcinoma
No.: number
Results expressed as the number of rats with the lesion / number of rats examined, with the incidence (%) in parentheses.
Source: NCI (1978)
In a subsequent pre-GLP study by the National Cancer Institute (NCI, 1979a), malathion
(purity > 95%) was admixed in the diet at concentrations of 2000 or 4000 ppm and fed ad libitum to
groups of F344 rats (49 or 50/sex per group) for 103 weeks (equivalent to doses of 100 and 200 mg/kg
bw per day, respectively). The rats were then observed for a further 2 or 3 weeks. Concurrent control
groups comprised 50 untreated rats per sex. No signs of toxicity were observed in females. There was
a slight reduction in survival in males at the highest dose (88%, 86% and 80% survival at 0, 2000 and
4000 ppm, respectively), with a significant (P = 0.001) dose-related trend. There were no treatment-
related neoplastic lesions. Treatment-related non-neoplastic lesions included chronic inflammation of
the stomach in both sexes (4.1%, 13% and 23% in males and 0%, 4.5% and 8.5% in females at 0,
2000 and 4000 ppm, respectively), gastric ulcers in males (2%, 20% and 32% at 0, 2000 and
4000 ppm, respectively) and fatty metamorphosis of the liver in females (0, 12 and 19% at 0,
2000 and 4000 ppm, respectively). The authors concluded that malathion was not carcinogenic.
Rueber (1985) re-examined the slides and concluded that the total incidence of benign and malignant
neoplasms across all tissues increased in treated males. However, this conclusion is not considered
reliable because of the absence of methodological detail on how the slides were re-examined. The
NTP (Huff et al., 1985) also re-evaluated the same slides and confirmed the original interpretation that
malathion is not carcinogenic in rats.
In a pre-GLP study, malathion (purity 92.1%) was admixed in the diet at concentrations of 0,
100, 1000 or 5000 ppm and fed ad libitum to groups of 50 Sprague Dawley rats per sex for 24 months
(doses estimated to be equal to 0, 5, 50 and 250 mg/kg bw per day, respectively). The rats were
observed daily for mortality and clinical signs. Body weight and feed consumption were recorded
weekly to week 13 and then at 24, 53, 79 and 103 weeks. Blood and urine were sampled at 3, 6, 12
and 24 months to analyse haematology and clinical chemistry or urine analysis parameters, including
plasma and erythrocyte cholinesterase activities. Brain acetylcholinesterase was not analysed.
Following scheduled termination, the mice were necropsied, organs weighed and tissues
histopathologically examined.
There were no treatment-related deaths or clinical signs. At 1000 and 5000 ppm, body weight
was significantly lower (P < 0.05) than the control throughout the study (up to 10% lower than the
control in males and 5% in females at both doses). There was no treatment-related effect on feed
consumption or haematology and urine analysis parameters. Erythrocyte acetylcholinesterase activity
was significantly lower (P < 0.05) than the control at 1000 and 5000 ppm (−6% to −18%, −25% to

323
−42 and −45% to −82% in males and −7% to −13, −20% to −45 and −37% to −71% in females at 100,
1000 and 5000 ppm, respectively). There were no significant intergroup differences in plasma
cholinesterase activity. There were no treatment-related macroscopic findings including any increase
in palpable masses in treated groups; the incidence of palpable masses was 12%, 6%, 14% and 12% in
males and 30%, 48%, 38% and 20% in females at 0, 5, 50 and 250 mg/kg bw per day, respectively.
Absolute and relative liver weights were 14% and 26% higher, respectively, than the control
(P < 0.05) in high-dose males; this was accompanied by periportal hepatocellular hypertrophy and
cystic hepatocellular degeneration observed microscopically. There was no evidence of a treatment-
related increase in neoplastic or non-neoplastic lesions although there was a slight increase in the
occurrence of benign fibroadenomas of the mammary gland in low- and mid-dose females (Table 14).
However, there was no increase at the highest dose (where the incidence was reduced) and neither the
study authors nor Seely (1991) determined the findings to be statistically significant. Further,
mammary gland fibroadenomas are a common age-related tumour in female Sprague Dawley rats and
can occur in approximately 50% of control rats (Dinse et al., 2010).
Table 14. Incidence of mammary neoplasms in female Sprague Dawley rats after dietary exposure
to malathion
No. and incidence per dietary concentration
Neoplasm 0 ppm 100 ppm 1 000 ppm 5 000 ppm
Adenoma 2/50 (4%) 4/50 (8%) 1/50 (2%) 22/50 (4%)
Adenocarcinoma 2/50 (4%) 0/50 (0%) 2/50 (4%) 0/50 (0%)
Fibroadenoma 8/50 (16%) 9/50 (18%) 15/50 (30%) 5/50 (10%)
Mixed tumour 0/50 (0%) 1/50 (2%) 0/50 (0%) 0/50 (0%)
Results expressed as the number of rats with the finding / number of female rats examined, with the incidence (%) in
parentheses.
Source: Seely (1991).
The NOAEL was 100 ppm (estimated to be equal to 5 mg/kg bw per day) for the inhibition of
erythrocyte acetylcholinesterase activity at 1000 ppm (estimated to be equal to 50 mg/kg bw per day)
(Rucci, Becci & Parent, 1980; Seely, 1991). The NOAEL for carcinogenicity was 5000 ppm
(estimated to be equal to 250 mg/kg bw per day), the highest tested dose.
Malathion (purity 96.4%) was admixed in the diet at concentrations of 0, 100, 500, 6000 or
12 000 ppm and fed ad libitum to groups of CDF(F-344)/CrlBr rats (90/sex per dose) for 24 months.
The low-dose group was reduced to 50 ppm from day 113 because of the inhibition of erythrocyte
acetylcholinesterase activity. Thirty-five rats per sex per group were assigned to the chronic phase of
the study and terminated after 12 months, while 44 rats per sex per group were assigned to the
oncogenicity phase of the study and terminated after 24 months. The rats were observed daily for
mortality and clinical signs. Body weight and feed consumption were recorded at regular intervals.
Ophthalmology was performed pretreatment and at 3, 6 and 12 months. Electroretinograms and fundic
photographs were recorded at 3, 6 and 12 months (chronic phase) and at 24 months (oncogenicity
phase). Blood was sampled at 6 and 12 months (chronic phase – 10 rats/sex per group) and 18 and 24
months (chronic phase – all rats) to analyse haematology and clinical chemistry parameters, including
cholinesterase activity. Urine was collected at 6, 12, 18 and 24 months for urine analysis. Following
death or scheduled termination, the rats were necropsied and their organs weighed, tissues
histopathologically examined and brain acetylcholinesterase activity analysed.
The doses achieved over 24 months were 0, 7, 29, 359 and 729 mg/kg bw per day in males
and 0, 8, 35, 415 and 868 mg/kg bw per day in females at 0, 100, 500, 6000 or 12 000 ppm,

324
respectively. From day 113, the achieved low dose was 2 mg/kg bw per day in males and 3 mg/kg bw
per in females.
Survival was adversely affected by treatment at 6000 and 12 000 ppm from 14 months in
males and towards the end of the study in females; after 24 months, survival was 67%, 75%, 53%,
26% and 0% in males and 69%, 74%, 75%, 62% and 36% in females at 0, 100, 500, 6000 and 12 000
ppm, respectively. The most common apparent cause of death was chronic nephropathy or
mononuclear cell leukaemia. The only treatment-related clinical sign was yellow anogenital staining
in high-dose females. Body weight was significantly lower (P < 0.05) than the control at 6000 and
12 000 ppm (up to 11.1% and 16.8 % lower in males and 5.1% and 16.8% lower in females,
respectively), while feed consumption increased (up to 9.7% and 19.6% in males and 8.8% and 25.6%
in females, respectively). There were no treatment-related ophthalmological or urine analysis
findings.
At 6000 and 12 000 ppm, significantly reduced haemoglobin, haematocrit, mean corpuscular
volume and mean corpuscular haemoglobin and increased platelet counts, cholesterol and GGT
occurred over the majority of the 24-month exposure period (Table 15). At 6000 and 12 000 ppm,
significant reductions (P < 0.01 or 0.05) in aspartate aminotransferase (males only; −50% to −58% at
12 and 18 months), alanine transaminase (females only; −26% at 12 months) and alkaline phosphatase
(both sexes at 6, 12 and 18 months; −24% to −36% and −25% to −45% at 6000 and 12 000 ppm,
respectively) were of uncertain toxicological significance. Plasma cholinesterase was significantly
lower than the control (P < 0.01 or 0.05) over 24 months at 6000 and 12 000 ppm (males: −17% to
−64% and −43% to −53%, respectively; females: −38% to −61% and −70% to −89%, respectively).
Acetylcholinesterase activity was significantly lower than the control (P < 0.01 or 0.05) over 24
months at 6000 and 12 000 ppm (males: −43% to −48% and −48% to −58%, respectively; females:
−44% to −58% and −51% to −66%, respectively). Toxicologically significant inhibition of
erythrocyte acetylcholinesterase activity occurred in females at 3 months (−30%, P < 0.01). Brain
acetylcholinesterase activity was significantly lower than the control (P < 0.01 or 0.05) at 6000 and
12 000 ppm (males: −12% to −31% and −16% to −19%, respectively; females: −12% to −18% and
−28% to −67%, respectively).
Table 15. Effects of 24 months of dietary exposure to malathion on haematology and clinical
chemistry parameters in rats
Parameter 0 ppm 100 or 50 ppm 500 ppm 6 000 ppm 12 000 ppm
Hb (g/dL)
Males
6 months 15.5 15.5 15.5 14.7** (−5%) 14.6* (−6%)
12 months 15.6 15.6 15.8 14.6** (−6%) 13.8** (−12%)
18 months 15.1 15.2 15.4 14.2 12.7** (−16%)
24 months 14.5 14.5 14.0 12.3* (−15%) No survivors
Females
6 months 14.9 14.7 14.9 14.3* (−4%) 14.3** (−4%)
12 months 15.6 15.4 15.6 15.0** (−4%) 15.2
18 months 15.2 14.8 15.6 14.9 15.2
24 months 14.0 13.5 13.6 12.1 13.4
Hct (%)
Males
6 months 43.4 43.5 43.4 41.7* (−4%) 41.4* (−5%)

325

12 months 44.2 44.6 54.2 42.3* (−4%) 40.2** (−9%)
18 months 44.4 44.3 45.1 42.0 37.8**
24 months 40.2 40.2 38.9 34.5 No survivors
Females
6 months 40.9 40.5 40.9 39.4* (−4%) 39.5* (−3%)
12 months 44.3 43.7 44.1 42.7 43.3
18 months 43.6 42.7 44.7 42.6 43.3
24 months 39.6 38.3 37.8 34.5 38.6
6
Erythrocytes (10 /µL)
Males
12 months 9.15 9.30 9.28 8.92 8.52** (−7%)
Females
12 months 8.24 8.09 8.43 8.21 8.43
3
Platelets (10 /µL)
Males
6 months 590 612 595 679 721** (+22%)
12 months 584 578 538 637 694** (+19%)
18 months 553 579 610 688* (+24%) 830** (+50%)
24 months 621 526 556 764 No survivors
Females
6 months 627 635 624 630 640
12 months 560 529 569 555 615** (+10%)
18 months 478 533 489 573** (+20%) 596** (+25%)
24 months 509 460 455 512 631* (+24%)
MCV (fL)
Males
6 months 47.8 47.2 47.7 46.0** (−4%) 45.7** (−4%)
12 months 48.2 48.0 48.8 47.4 47.1* (+2%)
18 months 53.0 51.4 51.9 50.3 50.8
Females
6 months 51.1 51.3 51.1 50.3** (−2%) 49.5** (−3%)
12 months 52.2 52.1 52.0 51.0** (−2%) 50.2** (−4%)
18 months 53.0 52.7 53.1 51.9** (−2%) 51.4** (−3%)
MCH (pg)
Males
6 months 17.1 16.8* 17.0 16.3** (−5%) 16.1** (−6%)
12 months 17.0 16.7 17.1 16.4** (−4%) 16.2** (−5%)
18 months 18.1 17.6 17.7 17.1** (−6%) 17.0** (−6%)
Females

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6 months 18.6 18.6 18.6 18.3** (−2%) 17.9** (−4%)
12 months 18.4 18.3 18.4 18.0** (−2%) 17.7** (−4%)
18 months 18.4 18.1 18.6 18.1 18.0
24 months 19.3 19.8 20.3 19.1 17.9** (−7%)
Cholesterol (mg/dL)
Males
6 months 78 80 81 129** (+65%) 163** (+109%)
12 months 94 90 93 134* (+44%) 211** (+124%)
18 months 153 144 137 224 500** +227%)
24 months 218 222 263 522** (+139%) No survivors
Females
6 months 99 99 102 121** (+22%) 147** (+48%)
12 months 130 123 127 151** (+16%) 172** (+32%)
18 months 126 142 157 231** (+83%) 286** (+127%)
24 months 263 162 284 341 430** (+63%)
GGT (IU/L)
Males
6 months 0 0 0 2 7
12 months 0 0 0 1* 4**
18 months 1 1 1 5** 13**
24 months 3 2 6 15** No survivors
Females
6 months 1 0 1 2** 6**
12 months 1 1 1 2 4**
18 months 1 1 1 3* 1
24 months 0 1 0 8** 3
Hb: haemoglobin; Hct: haematocrit; GGT: gamma-glutamyltransferase; IU: International Unit; MCH: mean corpuscular
haemoglobin; MCV: mean corpuscular volume; ppm: parts per million; *: P < 0.05; **: P < 0.01
Source: Daly (1996a)
Necropsy revealed emaciation and irregular kidney surface in high-dose decedents. Absolute
and relative liver and kidney weights in rats terminated after 12 and 24 months were significantly
increased (P < 0.01) in both sexes at 6000 and 12 000 ppm (Table 16). Absolute and relative spleen
weights were significantly increased in high-dose males terminated after 12 months; in the absence of
similar changes at other times or in females, or any microscopic changes, these increases was not
considered treatment related. Changes in thyroid/parathyroid weight occurred inconsistently over time
and between sexes and is therefore unlikely to be treatment related (Table 16).

327
Table 16. Effects of 24 months of dietary exposure to malathion on organ weights in rats
Kidney weight (g)
Males
12 months 2.539 2.587 2.565 2.884** (+14%) 3.282** (+29%)
24 months 3.767 3.245 3.570 4.193** (+11%) No survivors
Females
12 months 1.653 1.561 1.675 1.795** (+9%) 1.864** (+13%)
24 months 2.262 2.286 2.409 2.760** (+22%) 3.090** (+37%)
Relative kidney weight (%)
Males
12 months 6.99 7.19 7.17 8.35** (+10%) 10.14** (+45%)
Females
12 months 7.85 7.77 8.15 8.79** (+12%) 9.78** (+25%)
24 months 0.94 0.91 0.98 1.20** (+32% 1.62** (+72%)
Liver weight (g)
Males
12 months 11.798 11.422 11.613 14.440** (+22%) 16.056** (+36%)
Females
12 months 7.096 7.096 6.810 7.644 8.255** (+16%)
24 months 10.168 10.295 10.921 13.187** (+30%) 13.315** (+31%)
Relative liver weight (%)
Males
12 months 3.25 3.17 3.23 4.18** (+29%) 4.96** (+53%)
Females
12 months 3.37 3.29 3.30 3.74** (+11%) 4.32** (+28%)
24 months 4.23 4.08 4.42 5.59** (+32%) 6.82** (+61%)
Spleen weight (g)
Males
12 months 0.711 0.709 0.711 0.773 0.877* (+23%)
Females
12 months 0.568 0.507 0.536 0.538 0.549
24 months 0.974 1.001 1.413 8.890 0.903
Relative spleen weight (%)
Males
12 months 1.96 1.97 1.99 2.24* 2.70** (+38%)

328

Females
12 months 2.71 2.37 2.61 2.64 2.89
24 months 4.07 4.12 5.95 3.83 4.73
Thyroid/parathyroid weight (g)
Males
12 months 0.0211 0.0227 0.0226 0.0242* (+15%) 0.0264** (+25%)
Females
12 months 0.0178 0.0174 0.0179 0.0198 0.0181
24 months 0.0381 0.0276 0.0302 0.0349** (−8%) 0.0281
Relative thyroid/parathyroid weight (%)
Males
12 months 5.82 6.31 6.34 7.00** (+20%) 8.15** (+40%)
Females
12 months 8.49 8.16 8.74 9.68* (+14%) 9.51
24 months 1.61 1.11 1.21 1.51** (−6%) 1.48** (−8%)
Non-neoplastic findings: Table 17 summarizes the non-neoplastic histopathological findings in

nasoturbinal and nasopharyngeal tissues. A range of treatment-related changes occurred at 6000 and
12 000 ppm in both sexes, including dilated mucosal glands (the majority graded as slight), subacute
or chronic inflammation of the nasal mucosa (the majority graded as slight to moderate), degeneration
of the epithelium (the majority graded as moderate to moderately severe), epithelium cysts in the
nasal mucosa (mainly graded as minimal to slight) and glandular and epithelium hyperplasia (mainly
graded as slight). Chronic nephropathy occurred with similar frequency across all dose groups,
including the control, with a slight increase in the severity of nephropathy at the highest dose
Table 17. Histopathological findings in the nasal tissue of rats exposed to malathion in the diet for
24 months
No. per dietary concentration
No. of animals 90 90 90 90 90
Nasal mucosa (olfactory) – glands dilated
Males 2 1 0 31 27
Females 2 1 0 38 33
Nasal mucosa (olfactory) – subacute/chronic inflammation
Males 6 1 7 52 35
Females 0 3 2 42 20
Nasal mucosa (olfactory) – epithelium degeneration

329

Males 4 2 5 66 69
Females 2 2 1 69 66
Nasal mucosa (olfactory) – epithelium cysts
Males 0 0 0 43 55
Females 0 0 0 58 62
Nasal mucosa (olfactory) – glandular hyperplasia
Males 0 0 0 17 18
Females 0 0 0 24 14
Nasal mucosa (olfactory) – epithelium hyperplasia
Males 0 0 0 42 51
Females 0 0 0 57 54
Nasal mucosa (olfactory) – olfactory epithelium replaced by ciliated and non-ciliated columnar epithelial cells
Males 6 1 7 43 43
Females 2 2 1 50 25
Nasal mucosa (olfactory) – hyperplasia of ciliated and non-ciliated columnar epithelial cells
Males 3 1 4 18 22
Females 2 1 0 33 21
Nasal mucosa (respiratory) – subacute/chronic inflammation
Males 10 2 12 41 21
Females 7 4 5 34 10
Nasal mucosa (respiratory) – glands dilated
Males 18 0 13 28 24
Females 8 4 6 14 20
Nasal mucosa (respiratory) – hyperplasia
Males 13 2 12 44 41
Females 7 3 7 44 33
Nasal lumen – cell/cell debris/metachromatic basophilic amorphous material
Males 15 5 22 69 63
Females 10 7 9 64 58
Nasopharynx – epithelial hyperplasia
Males 10 0 15 22 14
Females 4 1 14 26 21
Source: Daly (1996a).
Neoplastic findings: Neoplasms observed microscopically in nasoturbinal tissue included an adenoma

in one male at 6000 ppm and a carcinoma in one male at 12 000 ppm. Spontaneous neoplasms are
uncommon in nasoturbinal tissue of F344 rats; this occurrence has not been observed by the
performing laboratory in six previous studies (0/238 males and 0/241 females). Published historical
control data (Haseman, Arnold & Eustis, 1990) indicated that the incidence of nasal adenomas is 0–
2% in control males (in gavage studies only) and of nasal carcinomas is also 0–2% (dietary studies).

330
In males, liver adenomas and carcinomas occurred with similar frequency across all groups
(adenomas: 2.9%, 3.6%, 5.5%, 3.6% and 1.4% at 0, 100/50, 500, 6000 and 12 000 ppm, respectively;
carcinoma: 1.4%, 3.6%, 1.8%, 1.8% and 0% at 0, 100/50, 500, 6000 and 12 000 ppm, respectively). In
females, the incidence of liver adenomas was significantly increased (P < 0.01 or 0.05) at 6000 and
12 000 ppm (0%, 1.8%, 1.8%, 5.5% and 4.3% at 0, 100/50, 500, 6000 and 12 000 ppm, respectively),
while the incidence of liver carcinomas was significantly increased (P < 0.05) at the highest dose (0%,
1.8%, 1.8%, 0% and 4.3% at 0, 100/50, 500, 6000 and 12 000 ppm, respectively). The occurrence of
liver adenomas in females was within the performing laboratory’s historical control range (0–5.4%),
while the occurrence of carcinomas was outside its historical control range (0–2.4%).
A number of subsequent independent histopathological re-evaluations closely examined the
microscopic findings in the liver (females), pituitary and uterus (females), and nasal tissue (both
sexes).
 Hardisty (2000) confirmed the treatment-related increase in hepatocellular adenomas at 6000
and 12 000 ppm but determined that no hepatocellular carcinomas were present at any dose in
females.
 Swenberg (1999a) confirmed that there were no treatment-related neoplasms in the uterus or
pituitary glands.
 Swenberg (1999b) confirmed that dietary exposure to malathion at 6000 or 12000 ppm caused
significant nasal toxicity characterized by olfactory epithelial degeneration, hyperplasia and
cyst formation, goblet cell hyperplasia, congestion, oedema and inflammation. The pattern of
distribution of these changes was noted to be unusual for a dietary study and was more
consistent with inhalational exposure. No treatment-related increases in neoplasms were
apparent in the nasoturbinal and nasopharyngeal tissues. A total of four nasal epithelial cell
tumours were observed, one in each of the two highest doses of each sex; all were well-
differentiated adenomas and each occurred at a different site.
Swenberg (1999b) noted a large squamous cell carcinoma arising from the alveolus of the
tooth in one low-dose female; the absence of a similar neoplasm at substantially higher doses in
females (up to two orders of magnitude higher) or at any dose in males indicates that this finding was
not treatment related. Swenberg (1999b) stated that there was no relationship between the tumour of
the tooth or any other neoplasm, including a squamous cell carcinoma in the palate of one high-dose
female and a small squamous cell papilloma of the palate, because these neoplasms arise from
different tissues and cannot be combined. The published historical range for squamous cell carcinoma
or papilloma of the palate in control female F344 rats in dietary studies is 0–4% (Haseman, Hailey &
Morris, 1998).
Bolte (1999a,b) examined additionally prepared slides and concluded that the carcinoma
originally observed in the respiratory epithelium of one high-dose male was in fact an adenoma of the
respiratory epithelium.
The NOAEL was 500 ppm (equal to 29 mg/kg bw per day in males and 35 mg/kg bw per day
in females) for reduced erythrocyte parameters, inhibition of brain acetylcholinesterase activity and
the occurrence of nasal toxicity at 6000 ppm (equal to 359 mg/kg bw per day in males and 415 mg/kg
bw per day in females) (Daly, 1996a). The NOAEL for carcinogenicity was also 500 ppm based on
the increase in nasal adenomas at 6000 ppm (Daly, 1996a, 1999).
In a published study, Cabello et al. (2001) examined the potential of malathion to cause
mammary tumours in an experimental rat tumour model. In the first experiment, groups of five female
Sprague Dawley rats (16 days old) were administered eserine (0.33 mg/kg bw twice per day),
parathion (2.25 mg/kg bw twice per day), malathion (170 mg/kg bw twice per day), atropine (2.25
mg/kg bw twice per day), serine plus atropine or malathion (twice per day at the doses already
described) plus atropine (twice per day at the doses already described) either subcutaneously or
intraperitoneally for five days. The rats were terminated 16 hours after the final injection. Mammary

331
tissue was histopathologically examined. There were no treatment-related effects on the density of
terminal end buds or alveolar buds per mm2.
In a second experiment, the protocol was repeated using slightly older female rats (39 days
old). Acetylcholinesterase was analysed in blood sampled from each rat although no results were
presented. Rats were terminated 16 hours after the final injection. Mammary tissue was
histopathologically examined. In malathion-treated rats there was a significant increase (P < 0.05) in
the density of terminal end buds (12.04 versus 3.30/mm2 in the control), while the density of alveolar
buds decreased from 20.80 to 2.50/mm2.
In a third experiment, 70 rats per group (39 days old) received subcutaneous injections twice
daily for five days of the treatments at the doses described. Rats were palpated weekly for tumours
and terminated after 28 months. Mammary tissue was histopathologically examined. There were no
deaths over the 28 months of the study. The incidence of mammary tumours increased in malathion-
treated rats (17/70 versus 0/70 in the control). The tumours were described as adenocarcinomas and
“grossly nodular and encapsulated, with areas of cribiform or papillary patterns”. As mammary
tumours are a common age-related neoplasm in this particular rat strain, the absence of any tumours in
controls, the lack of any effect on survival and the absence of further details of the classification of the
tumours makes it difficult to interpret these findings. Overall this study is not considered relevant to
the risk assessment of malathion because it used dose routes not relevant to possible human
exposures.
2.4 Genotoxicity
An extensive range of in vitro and in vivo genotoxicity tests have been conducted on
malathion and its metabolites or impurities. General details of the mythology used to evaluate these
studies (along with those on diazinon and glyphosate) are described in “Section 2: General
Considerations” of the JMPR meeting report12.
Genotoxicity tests in prokaryotes and lower eukaryotes (Table 18)
In a cell-free assay using DNA isolated from Escherichia coli K-12, malathion at 0.1 mg/mL
induced DNA breakage (Griffin & Hill, 1978). Testing was in the absence of metabolic activation
only.
In multiple studies, malathion and malathion formulations were reported negative for
mutagenicity, with or without metabolic activation, in multiple strains of Salmonella typhimurium
(generally some combination of TA97a, TA98, TA100, TA102, TA1535, TA1536, TA1537, TA1538)
and/or various strains of E. coli (K-12, WP2 uvr) (Dean, 1972; Mohn, 1973; Hyun & Lee, 1976;
USEPA, 1977, 1990; Haworth et al., 1983; Pednekar, Gandhi & Netrawali, 1987; Traul, 1987; Wong
et al., 1989; USEPA, 1990; Bowles, 2005; Taylor, 2008a,b,c; Beevers 2009; Thompson, 2013;
Schreib, 2015b). In contrast, Shiau et al. (1980) reported positive mutagenic activity at 300 μg/plate in
S. typhimurium strain 1535 and in Bacillus subtilis strain TKJ6321 but not TKJ5211 in the absence of
metabolic activation only. Shiau et al. (1980) also reported positive results for a DNA damage assay
with and without metabolic activation in various strains of B. subtilis, while Venkat et al. (1995)
reported positive results for the SOS test (a DNA damage response test) in E. coli; the test was
conducted without metabolic activation only. In contrast, USEPA (1977) reported malathion as
negative in the B. subtilis rec assay and for differential survival in DNA repair–deficient E. coli, both
of which were tested without metabolic activation only.
12
Pesticide residues in food 2016: Special session of the joint FAO/WHO meeting on pesticide
residues. May 2016: Report 2016 (http://www.who.int/foodsafety/areas_work/chemical-risks/jmpr/en/).

332
In lower eukaryotes, malathion was negative, with and without metabolic activation, in a
mutational assay in Schizosaccharomyces pombe (Gilot-Delhalle et al., 1983) and for mitotic
recombination in Saccharomyces cerevisiae (USEPA, 1977).
Malaoxon was negative for mutagenicity in S. typhimurium strains TA97, TA98, TA100 and
TA1535 (Zeiger et al., 1988) as well as TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA
(Schreib, 2015a); both studies were conducted with and without metabolic activation. Isomalathion,
O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate and O,S,S-trimethyl
phosphorodithioate were negative for mutagenicity in S. typhimurium strains TA97, TA98 and
TA100, with and without metabolic activation (Imamura & Talcott, 1985).

333
Table 18. In vitro genotoxicity tests in prokaryotes and lower eukaryotes

Results
End-point Test object Concentration Purity −S9 +S9 Comments Reference
Malathion
DNA breakage DNA isolated from 0.1 mg/mL in hexane NS Positive Not tested Inadequate due to a lack of Griffin & Hill
(Acellular) E. coli K-12 reporting detail (e.g. (1978)
exposure duration)
Reverse mutation S. typhimurium 1.6, 8, 40, 200, 1 000 & Batch/lot no. 1050- Negative Negative GLP & TG compliant Beevers (2009)
(Prokaryote) TA98, TA100, 5 000 μg/plate in OSJ-15A, 99.1%
TA102, TA1535, DMSO w/w malathion,
TA1537 0.25% w/w
MeOOSPS-
triester, 0.3% w/w
isomalathion,
0.08% w/w
malaoxon
Reverse mutation S. typhimurium 50, 150, 500, 1 500 & Batch/lot no. Negative Negative GLP & TG compliant Bowles (2005)
(Prokaryote) TA98, TA100, 5 000 μg/plate in 9010501, 96.0%
TA102, TA1535, DMSO w/w malathion,
TA1537 0.25%
isomalathion
Reverse mutation S. typhimurium 10 μL/plate in DMSO NS Negative Negative Inadequate Hyun & Lee.
(Prokaryote) TA98, TA100, Only maximum (1976)
TA1535, TA1538 concentration tested reported
and quantitative results not
provided
Reverse mutation E. coli WP2 uvr NS 97.4% Negative Not tested Inadequate Dean (1972)
(Prokaryote) Semiquantitative paper disk
method; details not provided
Reverse mutation S. typhimurium 1, 5, 10, 50, 100 & 500, Technical grade Negative Not tested Solvent not specified but USEPA (1977)
(Prokaryote) TA100, TA1535, 1 000 μg/plate from America likely DMSO
TA1537, TA1538 E. Cyanamid; lot
coli WP2 40216006.300

334
Results
+
Rec assay B. subtilis H17 Rec , 1 mg/disk Technical grade Negative Not tested Solvent not specified but USEPA (1977)
(Prokaryote) M45 Rec- from America likely DMSO
Cyanamid; lot
40216006.300
Differential survival E. coli W3110, 1 mg/disk Technical grade Negative Not tested Solvent not specified but USEPA (1977)
due to DNA damage P3478 from America likely DMSO
(Prokaryote) Cyanamid; lot
40216006.300
Reverse mutation S. typhimurium 1, 3.3, 10, 33, 100, 333, NS Negative Negative Solvent not specified but Haworth et al.
(Prokaryote) TA98, TA100, 1 000, 3 333 & 10 000 likely DMSO (1983)
TA1535, TA1537 μg/plate S9 from liver of induced
male rats plus Syrian
hamster
Reverse mutation S. typhimurium 10, 100 & 1 000 NS Negative Negative – Hyun & Lee
(Prokaryote) TA98, TA100, μg/plate (1976)
TA1535, TA1537
Reverse mutation S. typhimurium 10, 100, 1 000 & 5 000 Technical grade Negative Negative – Machado (1996)
(Prokaryote) TA97a, TA98, nL/plate in DMSO 1 103 g/L
TA100, TA1535
Forward mutation E. coli K-12/galRS18 200 mmol/L NS Negative Not tested Inadequate Mohn (1973)
(Prokaryote) Resistance to 5-methyl-DL-
tryptphan, solvent not
provided, data not provided
Reverse mutation S. typhimurium 33 & 1 650 mg/L in NS Negative Negative Metabolic activation by S9 Pednekar, Gandhi
(Prokaryote) TA97a, TA98, DMSO or caecal microbial extract & Netrawali
TA100 (0.1 mL per plate), (1987)
compound added in 0.2 mL

335
Results
Reverse mutation S. typhimurium Experiment I except Batch/lot no. Negative Negative GLP & TG compliant Schreib (2015b)
(Prokaryote) TA98, TA100, TA102 – 0.033, 0.10, 9010501/ME+H2,
TA102, TA1535, 0.33, 1.04, 2.6 & 5.2 95.7% w/w
TA1537 μL/plate malathion, 0.19%
Experiment I TA 102 w/w MeOOOPS-
and Experiment II - triester, 0.83%
0.031 6, 0.100, 0.316, w/w MeOOSPS-
1.0, 2.5 & 5.0 μL/plate triester, >0.34%
in DMSO w/w isomalathion,
<0.02% malaoxon
Forward mutation B. subtilis TKJ5211, 5–300 μg/plate in NS. Sample Positive: (300 Negative Inadequate Shiau et al. (1980)
(Prokaryote) TKJ6321 DMSO obtained from μg/plate), Semiquantitative disk assay
American TKJ6321 measuring His+, Met+
Cyanamid Negative (300 mutations
μg/plate),
TKJ4211
Reverse mutation S. typhimurium 5–300 μg/plate in NS Positive (300 Negative Inadequate Shiau et al. (1980)
(Prokaryote) TA98, TA100, DMSO Sample obtained µg/plate) in Semiquantitative disk assay
TA1535, TA1536, from American TA1535 only
TA1537, TA1538 Quantitative data not
Cyanamid presented
DNA damage B. subtilis (15 strains) 5–300 μg/plate in NS Negative Not tested Inadequate Shiau et al. (1980)
(Prokaryote) DMSO Sample obtained Semiquantitative disk assay
from American Quantitative data not
Cyanamid presented
Rec assay B. subtilis H17 Rec+, NS NS. Sample Negative Not tested Inadequate Shirasu et al.
(Prokaryote) M45 Rec- provided by Semiquantitative disk assay (1976)
Japanese
government Quantitative data not
presented

336
Results
Reverse mutation S. typhimurium 1.5, 5, 15, 150, 500, Batch/lot no. Negative Negative GLP & TG compliant Thompson (2013)
(Prokaryote) TA98, TA100, 1 500, 5 000 μg/plate in D2014-OSJ-MLT-
TA1535, TA1537 DMSO 01-S, 95.8% w/w
E. coli WP2 uvrA malathion, 0.50%
w/w MeOOOPS-
triester, 1.72%
w/w MeOOSPS-
triester, 0.39%
isomalathion,
0.073% w/w
malaoxon
Reverse mutation S. typhimurium 100, 500, 1 000, 2 500 Batch/lot no. AC Negative Negative GLP & TG compliant Traul (1987)
(Prokaryote) TA98, TA100, and 5 000 μg/plate in 4870-54B, 95.2 %
TA1535, TA1537 DMSO w/w malathion
and E. coli WP2
uvrA
DNA damage - SOS E. coli PQ37 NS. DMSO or sodium Reference grade Positive Not tested Inadequate due to lack of Venkat et al.
test (Prokaryote) taurocholate micells as provided by details (1995)
vehicles USEPA Started with stock solution at
1:100; activity was 2708
units/umol in DMSO, 3765
units/umol in taurocholate,
5th most active of 47
pesticides
Reverse mutation S. typhimurium 80–400 ppm, vehicle Technical grade Negative Negative Inadequate due to lack of Wong et al. (1989)
(Prokaryote) TA98, TA102, not specified from American details
TA1535, TA1537 Cynamid No quantitative data; vehicle
not specified
Mitotic S. cerevisiae D3 5% w/v or v/v Technical grade Negative Negative Solvent not specified but USEPA (1977)
recombination from America likely DMSO
(Yeast) Cyanamid; lot
40216006.300
Forward mutation S. pombe (ade6) 30, 91 and 182 mmol/L 99% Negative Negative S9 from induced male mice Gilot-Delhalle et
(Yeast) in 5% ethanol al. (1983)

337
Results
Malaoxon
Reverse mutation S. typhimurium 100, 333, 1 000, 3 333 94.4% Negative Negative Summary paper. Activation Zeiger et al. (1988)
(Prokaryote) TA97, TA98, TA100, and 10 000 μg/plate in using S9 from rat and Syrian
TA1535 DMSO hamster
Isomalathion
Reverse mutation S. typhimurium 10, 100 and 1 000 > 99% Negative Negative However, alkylates Imamura & Talcott
(Prokaryote) TA97, TA98, TA100 μg/plate in DMSO nitrobenzylpyridine, which is (1985)
a measure of compound
reactivity
MDCA
Reverse mutation S. typhimurium 1.6, 8, 40, 200, 1 000 Batch No. 621- Negative Negative GLP & TG compliant Taylor, 2008b
(Prokaryote) TA98, TA100, and 5 000 μg/plate in Bse-81A, 98.8%
TA102, TA1535, DMSO purity
TA1537
Desmethyl malathion
Reverse mutation S. typhimurium Exp 1: 1.6, 8, 40, 200, Batch 972-OSJ- Negative Negative GLP & TG compliant Taylor, 2008c
(Prokaryote) TA98, TA100, 1 000, 5 000 μg/plate; 41C, 45.9%
TA102, TA1535, Exp 2: 31.25, 62.5,125, malathion as free
TA1537 250, 500, 1 000, 2 000, acid
5 000 μg/plate in water
Desmethyl-malathion monocarboxylic acid, potassium salt
Reverse mutation S. typhimurium 31.6, 100, 316, 1 000, 77.6% w/w, Batch Negative Negative GLP & TG compliant Donath (2012)
(Prokaryote) TA98, TA100, 2 500 and 5 000 676-Bse-16A
TA1535, TA1537 μg/plate in DMSO
and E. coli
WP2 uvrA
Desmethyl-malaoxon dicarboxylic acid, trisodium salt
Reverse mutation S. typhimurium 0.031 6, 0.100, 0.316, 23.9% w/w, Batch Negative Negative GLP & TG compliant Schreib (2015a)
(Prokaryote) TA98, TA100, 1.0, 2.5 and 5.0 No. P1334-CSO-
TA1535, TA1537 μL/plate in distilled 15-filtered
and E. coli water
WP2 uvrA

338
Results
MMCA
Reverse mutation S. typhimurium 1.6, 8, 40, 200, 1 000 92.2%, Batch No. Negative Negative GLP & TG compliant Taylor (2008a)
(Prokaryote) TA98, TA100, and 5 000 μg/plate in 676-BSe-8A,
TA102, TA1535, DMSO mixture of alpha
TA1537 and beta isomers
O,O,O-trimethyl phosphorothioate
Reverse mutation S. typhimurium 10, 100 & 1 000 > 99% Negative Negative However, alkylates Imamura & Talcott
reactivity
O,O,S-trimethyl phosphorothioate
Reverse mutation S. typhimurium 10, 100 & 1 000 > 99% Negative Negative However, alkylates Imamura & Talcott
reactivity
O,S,S-trimethyl phosphorodithioate
Reverse mutation S. typhimurium 10, 100 & 1 000 > 99% Negative Negative However, alkylates Imamura &Talcott
reactivity
DMSO: dimethylsulfoxide; GLP: good laboratory practice; MDCA: malathion dicarboxylic acid; MMCA: malathion monocarboxylic acid; NS: not specified; ppm: parts per million; S9: 9000 ×
g supernatant from induced male rat; −S9: without metabolic activation; +S9: with metabolic activation; TG: test guideline; USEPA, United States Environmental Protection Agency; v/v:
volume per volume; w/w: weight per weight

339
Genotoxicity tests in non-human mammalian cells (Table 19)

The results of in vitro genotoxicity studies conducted on malathion, metabolites and
impurities in non-human mammalian cells are summarized in Table 19.
Malathion was reported to induce DNA–protein crosslinks in isolated rat lymphocytes when
tested in the absence of metabolic activation only (Ojha & Gupta, 2014).
Malathion was reported as positive for the induction of DNA damage, as measured by the
alkaline and neutral versions of the comet assay, in isolated rat lymphocytes in the absence of
metabolic activation only (Ojha & Gupta, 2014; Ojha & Srivastava 2014) and as measured by the
alkaline comet assay in metabolically competent rat hepatoma cells (Bianchi et al., 2015) or in rat
adrenal gland PC12 cells tested in the absence of metabolic activation only (Lu et al., 2012). Two of
these positive studies concluded that reactive oxygen species were involved in the induction of
damage (Lu et al., 2012; Ojha & Srivastava, 2014). Supportive of this hypothesis, malathion appears
to selectively induce markers of oxidative stress in Tox21/ToxCast high-throughput screening assays
(USEPA interactive Chemical Safety for Sustainability Dashboard, 2016).
Malathion was reported to induce DNA damage as measured by the induction of sister
chromatid exchanges in Chinese hamster ovary (CHO) and V79 cells tested in the absence of
metabolic activation only (Chen et al., 1981; Nishio & Uyeki, 1981), and positive for inducing sister
chromatid exchanges in CHO cells in the absence and presence of metabolic activation (Galloway et
al. 1987). Szekely, Goodwin & Delaney (1992) reported negative sister chromatid exchange results
for malathion tested in V79 cells but did report a concentration-related increase in polyploidy. A
malathion formulation was negative for the induction of unscheduled DNA synthesis in rat primary
hepatocytes (Pant, 1989).
Malathion was reported to induce chromosomal aberrations in CHO cells in the presence but
not the absence of metabolic activation (Galloway et al., 1987). A malathion formulation was positive
for the induction of micronuclei in Chinese hamster lung cells, tested in the absence of metabolic
activation only (Ni et al., 1983) but negative for micronuclei induction in metabolically competent rat
hepatoma cells (Bianchi, Mantovani & Marin-Morales, 2015).
Malathion and malathion formulations were positive for the induction of small (clastogenic
response) and large (mutagenic response) colonies in the mouse lymphoma (L5178Y) assay, with and
without metabolic activation (Edwards, 2001b).
In studies with malaoxon, Ivett et al. (1989) reported a significant increase in sister chromatid
exchanges but not chromosomal aberrations in CHO cells, with or without metabolic activation (the
sister chromatid exchange response was much weaker with metabolic activation). Nishio & Uyeki
(1981) also reported an increase in sister chromatid exchanges when testing in the absence of
metabolic activation only; these investigators also reported that malaoxon was more potent than
malathion in this assay.
Malaoxon was also reported to induce DNA damage as measured by the alkaline comet assay
in rat adrenal gland PC12 cells when tested in the absence of metabolic activation only (Lu et al.,
2012). It was also mutagenic in mouse lymphoma (L5178Y) cells in the absence but not the presence
of metabolic activation (Myhr & Caspary, 1991). In this study, there seemed to be a preference for the
induction of small colonies, generally considered to be indicative of chromosome damage rather than
gene mutations.

340
Table 19. In vitro genotoxicity tests in non-human mammalian cells

Results
Malathion
DNA damage Hepatoma tissue 0.000 9, 0.009 &, 0.09 50% (500 CE from Not Positive Decreasing damage with increasing Bianchi,
(alkaline comet culture (rat HTC mg per 5 mL in Dipil Chemical applicable (0.009 mg/5 concentration Mantovani &
assay) cells) distilled water Industry Ltd - mL) Benzo(a)pyrene used as positive Marin-Morales
Massaranduba, SC, control to show that cells were (2015)
Brazil) metabolically competent
Chromosome Hepatoma tissue 0.000 9, 0.009 & 0.09 50% (500 CE from Not Negative Criteria for concentration selection Bianchi,
damage culture (rat HTC mg per 5 mL in Dipil Chemical applicable not specified Mantovani &
(micronuclei) cells) distilled water Industry Ltd - Cytokinesis block assay Marin-Morales.
Massaranduba, SC, (2015)
Brazil) Benzo(a)pyrene used as positive
control to show that cells were
metabolically competent
DNA damage (SCE) Chinese hamster 10, 20, 40 & 80 μg/mL 94% Positive (40 Not tested 80 μg/mL cytotoxic Chen et al.
V79 cells in DMSO μg/mL) (1981)
Mutation (colony Mouse lymphoma −S9: 125, 250, 500, 96.0% w/w, Positive Positive GLP and TG compliant Edwards (2001b)
formation) (L5178Y) cells 1 000, 1 500, 1 800, Batch/lot no.: (2 000 (2 000 Small but significant increases in
2 000 μg/mL 9010501, 0.19% µg/mL) µg/mL) small, large and total colonies
+S9 250, 500, 1 000, w/w MeOOOPS-
triester, 0.83% Positive increases associated with
1 800, 2 000, 2 200 decreases of 89% (−S9) and 64%
μg/mL in DMSO w/w MeOOSPS-
triester, 0.15% and 78% (+S9) in relative total
w/w isomalathion, growth
<0.02% malaoxon
Chromosome CHO cells −S9 25, 50, 76 & 101 NS Negative Positive −S9 = 101 μg/mL cytotoxic, positive Galloway et al.
damage μg/mL (303 μg/mL) concentration-related response (1987)
(chromosomal +S9 303, 352 & 402
aberrations) μg/mL in DMSO
DNA damage (SCE) CHO cells −S9 4.03, 12.1 & 40.3 NS Positive Positive (298 Response generally concentration- Galloway et al.
μg/mL (40.3 μg/mL) related (1987)
+S9 121, 298, 351 & μg/mL)
403 μg/mL in DMSO

341
Results
DNA damage Rat adrenal gland 20 & 40 mg/L in 95% Positive (40 Not tested Increased intracellular ROS levels Lu et al. (2012)
(alkaline comet PC12 cells DMSO mg/mL) and reduced catalase, superoxide
assay) dismutase
DNA damage (SCE) CHO cells 0.03, 0.1, 0.3 & 1.0 99% Positive (0.3 Not tested Concentration–response relationship Nishio & Uyeki
mmol/L in DMSO mmol/L) (1981)
Mutation (colony Mouse lymphoma S9 = 6 trials with NS Equivocal Negative −S9: 6 trials resulted in 1 NTP study
formation) (L5178Y) cells concentrations 1.25– inconclusive, 1 positive, 2 equivocal reported in
60 μg/mL and 2 negative trials CEBS with
+S9 = 2 trials of 5–60 Tested to excessive cytotoxic accession
μg/mL; in ethanol concentrations number 002-
02380-0020-
0000-7 (2017)
DNA damage Lymphocytes 1/10 (0.52), 1/4 (1.3) NS Positive Not tested Duration of exposure, 2, 4, 8, or 12 Ojha & Gupta
(alkaline & neutral (isolated from male LC50 (mg/L) in DMSO (0.52 hours; 4-hour LC50 > 5.2 mg/L, (2014)
comet assay) Wistar rats) mg/mL) for alkaline measures DNA SSB &
both SSB alkali-labile sites, neutral measures
and DSB DSB
DNA damage Lymphocytes 1/10 (0.52), 1/4 (1.3) NS Positive Not tested Duration of exposure, 2, 4, 8, or 12 Ojha & Gupta
(DNA–protein (isolated from male LC50 (mg/L) in DMSO (0.52 hours; 4-hour LC50 > 5.2 mg/L, (2014)
crosslink) Wistar rats) mg/mL) assay based on binding of SDS to
proteins, and lack of binding to
DNA
DNA damage Lymphocytes Alkaline 1/20 (0.26), NS Positive Not tested Duration of exposure, 2, 4 hours; 4- Ojha &
(alkaline & neutral (isolated from male 1/10 (0.52), 1/8 (0.65), alkaline 1/20 hour LC50 > 5.2 mg/L, alkaline Srivastava,
comet assay) Wistar rats) 1/4 (1.3) LC50 (mg/L) LC50 (0.26 measures DNA SSB, neutral (2014)
Neutral 1/4 LC50 (1.3 mg/L) @ 2 measures DSB, Fpg–Endo enzyme
mg/L) in DMSO hours; treatment indicates presence of
neutral 1/4 ROS-generated damage, using
LC50 (1.3 comet assay
mg/L)

342
Results
DNA damage Primary hepatocytes 0.02,0.04, 0.08 & 0.12 94% w/w, Not Negative GLP study Pant (1989)
(UDS) from male SD rats μL/mL in DMSO Batch/lot no.: AC applicable
18-hour exposure 6015-136, 0.1 %
w/w MeOOOPS-
triester, 0.4 % w/w
MeOOSPS-
triester, 0.2 % w/w
isomalathion, 0.1%
w/w malaoxon
DNA damage (SCE) Chinese hamster 10, 20 & 30 μg/mL in > 99% Negative Not tested Selection of concentrations based on Szekely,
V79 cells ethanol effects on cell growth. Strong Goodwin &
increase in polyploidy at 20–40 Delaney (1992)
mg/L
Chromosome CHO cells −S9 187, 249, 377 94.4% Negative Negative Most trials harvested at 10–12 hours Ivett et al. (1989)
damage +S9 = trial 1: 502, (not current protocol), one of two
(chromosomal 1 256, 2 512 μg/mL in +S9 trials with delayed harvest
aberrations) DMSO; trial 2: 1 998, weekly positive; overall call
2 498, 3 010 μg/mL in negative. Concentrations tested
DMSO; trial 3: 3 000, based on range-finding studies
3 250, 3 500 μg/mL in
DMSO
Malaoxon
DNA damage (SCE) CHO cells S9 Trial 1 = 16.7, 50, 94.4% Positive (50 Positive NTP study, no second generation Ivett et al. (1989)
167, 500; trial 2 = 50, µg/L) (1670 µg/L) metaphase cells to score at
124, 168, 251 concentrations of 251 μg/mL and
+S9 = 167, 500, 1670, higher (−S9) and 5 000 μg/mL (+S9)
5 000 μg/mL in –
DMSO
DNA damage Rat PC12 adrenal 20 & 40 mg/L in 95% Positive (20 Not tested Increased intracellular ROS levels Lu et al. (2012)
(alkaline comet gland cells DMSO mg/L) and reduced catalase, superoxide
assay) dismutase

343
Results
Mutation (colony Mouse lymphoma −S9: 2 trials with 94.4% Positive Negative Relative total growth decreased by Myhr &
formation) (L5178Y) cells concentrations of 25– (100 µg/mL) ~60% at LEC without S9; no growth Caspary, (1991)
400 μg/mL at highest concentrations in each
+S9: 3 trials with trial; increase in both large and
concentrations of 50– small colonies, with some
300 μg/mL in ethanol preference for small colonies
DNA damage (SCE) CHO 0.03, 0.1, 0.3 & 1.0 96% Positive (0.1 Not tested Concentration–response Nishio & Uyeki
cells mmol/L in DMSO mmol/L) relationship; malaoxon induced (1981)
higher level of SCEs than malathion
CHO: Chinese hamster ovary; CEBS: Chemical Effects in Biological Systems; DMSO: dimethylsulfoxide; DSB: double strand break; GLP: good laboratory practice; HTC: hepatoma cell; LC 50:
median lethal concentration; NS: not specified; ROS: reactive oxygen species; SCE: sister chromatid exchange; SDS: sodium dodecyl sulfate; SSB: single strand breaks; S9: 9000 × g
supernatant; −S9: without metabolic activation; +S9: with metabolic activation; TG: test guideline; UDS: unscheduled DNA synthesis; NTP: National Toxicology Program (USA)

344
Genotoxicity tests in human cells (Table 20)

In assays that used human cells to assess DNA damage, malathion induced 8-hydroxy-2'-
deoxyguanosine (8-Oxo-dG) adducts in isolated peripheral blood mononuclear cells, damage that was
associated with oxidative stress (Ahmed et al., 2011). Supportive of this hypothesis, malathion
appears to selectively induce markers of oxidative stress in Tox21/ToxCast high-throughput screening
assays (USEPA interactive Chemical Safety for Sustainability Dashboard, 2016). Malathion was
positive in the alkaline comet assay using the hepatocellular carcinoma cell line HepG2, albeit at a
very high concentration that was associated with lipid peroxidation (Moore, Yedjou & Tchounwou,
2010), but negative using the same assay platform in isolated lymphocytes (Blasiak et al., 1999).
These studies were conducted in the absence of metabolic activation. In primary mucosal epithelial
cells from human tonsil tissue, malathion induced DNA damage as measured by the alkaline comet
assay (Tisch, Faulde & Maier, 2007); studies were conducted in the absence of metabolic activation
only. In the absence of metabolic activation, malathion consistently induced sister chromatid
exchanges in phytohaemagglutinin (PHA)-stimulated lymphocytes in whole blood cultures (Herath et
al., 1989; Garry et al., 1990; Balaji & Sasikala, 1993), in a lymphoid cell line (LAZ-007) (Sobti,
Krishan & Pfaffenberger, 1982), and in fetal lung fibroblasts (Nicholas, Vienne & van den Berghe,
1979). Malathion was also active in inducing sister chromatid exchanges in the two studies that
evaluated the response in the presence of metabolic activation (Sobti, Krishan & Pfaffenberger, 1982;
Garry et al., 1990). Malathion was negative for inducing unscheduled DNA synthesis in WI-38 cells
(USEPA, 1977).
In the absence of metabolic activation, malathion consistently induced chromosomal
aberrations in PHA-stimulated human lymphocytes cultured as whole blood or after isolation (Walter,
Czajkowska & Lipecka, 1980; Herath et al., 1989; Garry et al., 1990; Balaji & Sasikala, 1993;
Edwards, 2001a; Lloyd, 2009) and in LAZ-007 cells, a lymphoid cell line (Sobti, Krishan &
Pfaffenberger. 1982). In the presence of metabolic activation, some studies reported a positive
clastogenic effect (Garry et al. 1990; Lloyd, 2009), while others were negative (Sobti, Krishan &
Pfaffenberger. 1982; Edwards 2001a). Malathion was reported to be negative for clastogenicity in the
haematopoietic cell line B411-4 (tested in the absence of metabolic activation only) (Huang, 1973).
Malathion induced micronuclei in PHA-stimulated lymphocytes cultured both as whole blood
(positive by slope analysis) and after isolation (testing was in the absence of metabolic activation
only); the compound was more potent in whole blood cultures (Titenko-Holland et al., 1997).
Malathion also induced micronuclei in the hepatoma cell line HepaRG, which express liver-like
metabolism (Josse et al., 2014), but not in Molt-4 T-lymphocytes in the absence of metabolic
activation (Szekely, Goodwin & Delaney, 1992).
Malathion was reported to induce mutations at the hypoxanthine-guanine
phosphoribosyltransferase (HPRT) locus in isolated T-cells (Pluth et al., 1996); a subsequent
expanded evaluation concluded that mutations arose preferentially at G:C basepairs (Pluth et al.,
1998).
Malaoxon and isomalathion induced DNA damage in isolated lymphocytes in the absence of
metabolic activation only, as measured by the alkaline comet assay (Blasiak et al., 1999). Further, a
follow-up study concluded that the malaoxon-mediated damage was likely induced by reactive
oxygen species (ROS) (Blasiak & Stankowska, 2001). Isomalathion was reported also to induce
micronuclei in the hepatoma cell line HepaRG (Josse et al., 2014).

345
Table 20. In vitro genotoxicity tests in human cells

Results
Malathion
DNA damage Peripheral blood 5, 10, 20, 50 & NS (technical Positive (20 Not tested Treatment for 6, 12, 24 hours. Adducts measured Ahmed et al.
(8-Oxo-dG mononuclear 100 μmol/L grade) µmol/L) by ELISA, duration- and concentration-dependent (2011)
adducts) cells (isolated) Vehicle response. Malondialdehyde concentrations were
unspecified also increased. Effect attenuated by N-
acetylcysteine & curcumin
Chromosome Lymphocytes 0.2, 0.2, 2 & NS Positive Not tested Treatment started 0, 24, 48 hours after mitogen Balaji &
damage (male – whole 20 μg/mL in 1% (2 µg/mL) stimulation, termination at 72 hours, most active Sasikala (1993)
(chromosomal blood, PHA acetone when administered at 0 time, concentration–
aberrations) stimulated) response relationship, gaps excluded from
analysis. Mitotic index reduced by 71% at 2
μg/mL
DNA damage Lymphocytes 0.2, 0.2, 2 & NS Positive Not tested Treatment at 0, 24, 48 hours, termination at 72 Balaji &
(SCE) (male – whole 20 μg/mL in (20 µg/mL) hours, concentration-dependent response obtained Sasikala (1993)
blood, PHA acetone using all 3 protocols but most active when
stimulated) administered at 0 time
DNA damage Lymphocytes 25, 75 & 200 ≥ 99.8% Negative Not tested Inadequate publication. One hour incubation, Blasiak et al.
(alkaline comet (isolated) μmol/L in ethanol rationale for concentration selection not provided (1999)
assay)
Chromosome Lymphocytes −S9 150, 300 & Batch/lot no.: Positive (450 Negative GLP- and TG-compliant study Edwards
damage (male – whole 450 μg/mL 9010501, 96.0% μg/mL) 2 donors, whole blood, positive in one donor at (2001a)
(chromosomal blood, PHA +S9 300, 600 & w/w malathion, maximum concentration, associated with 50%
aberrations) stimulated) 900 μg/mL 0.19% w/w depression in mitotic index
MeOOOPS-
DMSO vehicle triester, 0.83%
w/w MeOOSPS-
triester, 0.15%
isomalathion,
< 0.02%
malaoxon

346
Results
−3 −4
DNA damage Human fetal lung −S9: 10 , 10 , Technical grade Negative Negative Solvent not specified USEPA (1977)
(UDS) fibroblasts (WI- 10−5, 10−6, 10−7 (America summarized in
38) mol/L; Cyanamid; lot Waters et al.
+S9: 10−3, 10−4, 40216006.300) (1980)
10−5 mol/L
Chromosome Lymphocytes (M 66, 83, 132 & 660 NS Positive Positive Exposed cells in G0, concentration-dependent Garry et al.
damage – whole blood, μg/mL in DMSO increase (1990)
(chromosomal PHA stimulated)
aberrations)
DNA damage Lymphocytes (M 83, 132 or NS Positive by Positive by Exposed cells in G0, positive is based on Garry et al.
(SCE) – whole blood, 660 μg/mL in trend test trend test concentration-dependent increase but does not (1990)
PHA stimulated) DMSO indicate if any concentration statistically increased
over control
Chromosome Lymphocytes 5, 20, 40 & > 98% Positive Not tested Treatment for 4 and 24 hours, no positive control, Herath et al.
damage (whole blood, 50 μg/mL in (20 µg/mL) when gaps excluded, active at 20 μg/mL at 24 (1989)
(chromosomal PHA stimulated) DMSO hour only but not at higher concentrations. Mitotic
aberrations) index at 24 hour not reduced significantly
DNA damage Lymphocytes (M 5, 20 & 50 μg/mL > 98% Positive Not tested Treatment for 24 hours, concentration-related Herath et al.
(SCE) – whole blood), in DMSO (20 µg/mL) response (1989)
PHA stimulated
Chromosome Human 50 & 100 μg/mL 95% Negative Not tested Inadequate publication, lacking critical details. Huang (1973)
damage haematopoietic in DMSO Multiple sample times: 6, 12, 24, 50 hours
(chromosomal cell line B411-4
aberrations)
Chromosome HepaRG 10, 25 or 50 NS Not Positive Absence of clear dose–response over Josse et al.
damage hepatoma cell μmol/L in DMSO applicable (10 µmol/L) concentration range tested but all 3 concentrations (2014)
(micronucleus line significant
formation)

347
Results
Chromosome Lymphocytes (M −S9: 125, 500 & Batch/lot no.: Positive Positive GLP- and TG-compliant study Lloyd (2009)
damage – whole blood, 600 μg/mL 1050-OSJ-15A, (500 μg/mL) (900 μg/mL) Malathion spiked to the specification limit with
(chromosomal PHA stimulated) +S9: 600, 900 & 99.1% w/w relevant impurities; used pooled blood of 3 male
aberrations) 1 000 μg/mL malathion, 0.25% donors
DMSO vehicle w/w MeOOSPS-
triester, 0.3% Mitotic index reduced by 32% (−S9) and 14%
w/w (+S9) at LEC
isomalathion,
0.08% w/w
malaoxon
DNA damage HepG2 6, 12, 18 & 98.2% Positive Not tested Treatment for 48 hours, cell viability decreased Moore, Yedjou
(alkaline comet hepatocellular 24 mmol/L in (24 mmol/L) by > 70%, induced malondialdehyde at 6 mmol/L, & Tchounwou
assay) carcinoma cell DMSO a measure of lipid peroxidation (2010)
line
DNA damage Fetal lung 2.5, 5, 10, 20 & 99% Positive Not tested Treatment either 1 at 4 hours or 2 at 4 & 24 Nicholas,
(SCE) fibroblasts 40 μg/mL in (20 μg/mL) hours, termination at 72 hours. Induced Vienne & van
ethanol concentration-related response den Berghe
(1979)
Mutation T-lymphocytes 30, 50, 80, 150, 97–99% Positive (50, Not tested Multiple mutants from different cultures Pluth et al.
(HPRT mutation) (isolated) 300, 450 & 450 & aggregated. Specific deletion more common than (1996)
600 μg/mL in 600 μg/mL in control mutants. 6 of 84 (7.1%)
DMSO but not 80, HPRT mutants arising in in vitro malathion-
150 or treated human T-lymphocytes were characterized
300 μg/mL) by specific genomic deletions in a 125-bp region
of exon 3
Mutation T-lymphocytes 10–650 μg/mL in 97–99% Positive Not tested Extended evaluation of HPRT mutations 24 from Pluth et al.
(HPRT mutation) (isolated) DMSO control, 77 from 6 in vitro treatments with cells (1998)
from 4 male donors, mutations induced
preferentially at G:C basepairs
DNA damage Lymphoid cells −S9: 0.02, 0.2, 2 NS Positive Positive Concentration-induced response Sobti, Krishan
(SCE) (LAZ-007) & 20 μg/mL (0.2 μg/mL) (20 μg/mL) & Pfaffenberger
+S9: 20 μg/mL in (1982)
ethanol

348
Results
Chromosome T-lymphocytes 7.5, 15, 30, 60, 80 > 99% Negative Not tested Higher concentrations cytotoxic Szekely,
damage (Molt-4) & 120 μg/mL in Goodwin &
(micronucleus ethanol Delaney (1992)
formation)
DNA damage Tonsil specimens 0.05, 0.1, 0.5, 99.2% Positive Not tested Inadequate publication lacking critical Tisch, Faulde &
(alkaline comet 0.75 & 1.0 information on cell samples used as tonsil Maier (2007)
assay) mmol/L in specimens taken from 85 patients; publication in
DMSO German
Chromosome Lymphocytes 5, 20, 50 & 95% Positive (by Not tested Impurities include malaoxon, Kinetochore- Titenko-Holland
damage (whole blood and 100 μg/mL in slope analysis negative micronuclei (malathion mostly induced et al. (1997)
(micronucleus isolated), PHA- DMSO whole blood, chromosome breakage). No concentration–
formation) stimulated 75 μg/mL response relationship in micronucleus formation
isolated cells) in whole blood culture
Chromosome Lymphocytes 10, 30, 50 & 99% Positive Not tested Treatment 24 hours before PHA – 96-hour Walter,
damage (isolated, PHA 70 μg/mL in (10 µg/mL) cultures; 4, 18 and 48 hours after PHA, 72 hours Czajkowska &
(chromosomal stimulated) ethanol cultures. Analysis includes gaps. Active with Lipecka (1980)
aberrations) maximum exposure duration & maximum
response at lowest concentration
Malaoxon
DNA damage Lymphocytes 25, 75 & 200 > 98% Positive Not tested Concentration-dependent response, extent of Blasiak &
(alkaline comet (isolated) μmol/L in ethanol (25 μmol/L) damage reduced by pretreatment with α- Stankowska
assay) 1 hour exposure tocopherol and treatment with catalase; detected (2001)
presence of Fpg sites, indicative of ROS
DNA damage Lymphocytes 25, 75 & 200 ≥ 99.8% Positive Not tested Concentration-dependent response, more potent Blasiak et al.
(alkaline comet (isolated) μmol/L in ethanol (75 μmol/L) than malathion or isomalathion. Damage repaired (1999)
assay) 1 hour incubation within 1 hour
Isomalathion
DNA damage Lymphocytes 25, 75 & 200 ≥ 99.8% Positive Not tested Concentration-dependent response, more potent Blasiak et al.
(alkaline comet (isolated) μmol/L in ethanol (25 μmol/L) than malathion but less than malaoxon. Damage (1999)
assay) 1 hour incubation repaired within 1 hour

349
Results
Chromosome HepaRG 0.05, 5, 10, 25 & NS Not Positive Concentration-dependent response. Also induced Josse et al.
damage hepatoma cell 50 μmol/L in applicable (25 μmol/L) oxidative stress as assessed by ROS formation (2014)
(micronucleus line DMSO
formation)
DMSO: dimethylsulfoxide; ELISA: enzyme-linked immunosorbent assay; Fpg: formamidopyrimidine-DNA-glycosylase; HepG2:hepatocellular carcinoma; HPRT: hypoxanthine-guanine
phosphoribosyltransferase locus; LEC: lowest effective concentration; 8-Oxo-dG: 8-hydroxy-2'-deoxyguanosine; NS: not specified; PHA: phytohaemagglutinin; ROS: reactive oxygen species;
S9: 9000 × g supernatant; −S9: without metabolic activation; +S9: with metabolic activation; SCE: sister chromatid exchange; TG: test guideline; w/w: weight per weight; UDS: unscheduled
DNA synthesis

350
(b) In vivo studies

Genotoxicity tests in nonmammalian systems (Table 21)
In laboratory studies, malathion or products containing malathion tested positive for the
induction of chromosomal aberrations or micronuclei in a number of different species, including
plants (Allium root tips) (Hoda & Sinha, 1991; Kumar, Khan & Sinha, 1995); Tradescantia early
meiotic pollen mother cells (but only under certain conditions; Ma et al., 1983); the erythrocytes of
tadpoles of Indian skittering frogs (Euflictis cyanophlyctis) (Giri et al., 2012); erythrocytes of several
species of fish (Channa punctatus (Bloch)) (Kumar et al., 2010); Oreochromis niloticus (also head
kidney cells, Kandiel et al., 2014); and in the erythrocytes of Japanese quail (Coturnix japonica)
(Hussain et al., 2015).
In Drosophila melanogaster, malathion by feeding and/or injection was reported negative for
the induction of chromosome damage and sex-linked recessive lethals in germ cells by Valencia
(1981) and Velázquez et al. (1987) or wing-spot mutations by Osaba et al. (1999), but reported
positive by feeding for sex-linked recessive lethals and dominant lethals by Hoda & Sinha (1991) and
Kumar, Khan & Sinha (1995).
Malathion also induced DNA damage in lymphocytes and cells of the gill and kidney sampled
from Channa punctatus (Bloch), as measured by the alkaline comet assay (Kumar et al., 2010) and in
liver cells of O. niloticus, as measured by DNA fragmentation assay (Kandiel et al. 2014). Malathion
was reported to not induce DNA damage in Litopenaeus stylirostris (shrimp), measured using the
alkaline unwinding assay (Galindo Reyes et al., 2002) or 8-Oxo-dG adducts in the DNA of liver cells
of seabream (Sparus aurata) (Rodríguez-Ariza et al., 1999).
Malaoxon in food induced an increase in reciprocal translocations and sex-linked recessive
lethals in D. melanogaster but not for sex-linked recessive lethals when administered by injection
(Foureman et al., 1994).

351
Table 21. In vivo genotoxicity tests in nonmammalian systems

End-point Test object Concentration or dose Purity Results Comments Reference
Malathion
Chromosome damage E. cyanophlyctis (Indian 0.5, 1.0, 2.0 mg/L 50% EC Positive The 96-hour LC50 for malathion Giri et al. (2012)
(micronuclei) skittering frog) Treatments at 24, 48, 72 commercial (0.05 mg/L) was 3.588 mg/L. Dose-dependent
(erythrocytes) and 96 hours formulation increase
Mutation (sex-linked D. melanogaster (Muller-5) 0.006% in feed Positive Inadequate publication due to lack Hoda & Sinha
recessive lethals) of protocol details (1991)
Mutation (dominant D. melanogaster (Oregon 0.006% in feed Positive Inadequate publication due to lack Hoda & Sinha
lethals) R) (germ cells) of protocol details (1991)
Chromosome damage Allium cepa (onion) 50, 100, 200, 400 & Not specified Positive Mostly clastogenic but some Hoda & Sinha
(Chromosomal (root tips) 800 ppm effects on malsegregation (1991)
aberrations) 24 hour treatment, water
suspension
Chromosome damage C. japonica (Japanese 20–120 mg/kg bw per day 95% Positive (60 Increased at all sample times; also Hussain et al.
(micronuclei in quail, male) in corn oil mg/kg bw per frequency of binucleate (2015)
erythrocytes) (erythrocytes) Administered via crop day) erythrocytes increased
tubing daily for 17, 34 and
51 days
Chromosome damage O. niloticus (fish - Nile Acute: 5 ppm 57% Positive Study of limited value since 96- Kandiel et al.
(micronuclei in tilapia) Chronic: 1 ppm hour LC50 = 5 ppm (2014)
erythrocytes)
Fish maintained in water
Chromosome damage containing malathion for
(Chromosomal aberrations 96 hours (acute) or 10
in head kidney cells) days (chronic)
Chromosome damage
(DNA fragmentation in
liver cells)
Mutation (dominant D. melanogaster (Oregon 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 50% Positive (2.5 Dose–response relationship Kumar, Khan &
lethal) R) (germ cells) 3.5, 4.0, 4.5 & 5.0 μg/L commercial μg/L) Sinha (1995)
In feeding solution grade

352

Mutation (sex-linked D. melanogaster (Oregon 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 50% Positive (4.0 Dose–response relationship Kumar, Khan &
recessive lethals) R) 5,5, 6.0, 6.5, 7.0 & 7.5 commercial μg/L) Sinha (1995)
(Germ cells) μg/L grade
In feeding solution
Chromosomal damage A. cepa (onion) 40, 42.5, 45, 47.5, 50, 50% Positive (50 Dose–response relationship Kumar, Khan &
(chromosomal (root tips) 52.5, 55, 57.5. 60 & 62.5, commercial μg/L) Sinha (1995)
aberrations) 65 mg/L grade
In feeding solution
Chromosome damage Channa punctatus (Bloch), 0.59, 0.74, 1.48 ppm 50% Positive (0.59 LC50 5.93 ppm, dose–response Kumar et al.
(micronuclei in fish Fish maintained in water commercial ppm 1/10th observed (2010)
erythrocytes) containing malathion with grade LC50)
DNA damage (alkaline sampling on days 0, 1, 3,
comet assay – gill & 7, 15, 22 and 29
kidney cells, lymphocytes Semi-static system, water
(isolated)) changed every second day
Chromosome damage Tradescantia clones 03 & a) 5.5–4125 ppm; Positive Positive results variable and Ma et al. (1983)
(micronucleus formation) 4430 absorption of malathion (~0.255%) associated with high levels of
(early meiotic pollen /water mixture (± DMSO toxicity
mother cells) and/or S9) through the
stem
b) 0.110–0.435%;
spraying a malathion
/water mixture onto the
plant cuttings in enclosed
chambers
c) 0.616%; spraying a
malathion/water mixture
on an open population of
plants in the greenhouse
d) 0.026–0.561%;
absorption of malathion
fumes through the leaves
and buds
Most treatments were
administered for 6 hours,
followed by a 24-hour
recovery time

353

Wing-spot test D. melanogaster (standard 0.000 1%, 0.000 25%,  95% Negative Active only at one intermediate Osaba et al. (1999)
and NORR strains) Somatic 0.000 5%, 0.007 5% dose so response likely not relevant
cells standard cross; 0.007 5%,
0.001% NORR cross in
3% Tween 80 plus 3%
ethanol
In feeding solution
DNA damage (alkali Litopenaeus stylirostris Unspecified Unspecified Negative Inadequate publication given Galindo Reyes et
sensitive adducts or strand (shrimp) larvae Larvae maintained in absence of critical details; LC50 = al. (2002)
breaks) water changed daily for 4 34.2 mg/L
days, damage detected by
alkaline unwinding assay
DNA damage (adduct) (8- Sparus aurata (seabream) 6.38 mg/kg bw, IP × 1 NS Negative Publication lacks specific details Rodríguez-Ariza et
Oxo-dG) (liver) sampled 7 days later on dose selection and single dose al. (1999)
only tested. 8-OH-dG measured by
HPLC-EC
Mutation (sex-linked D. melanogaster (Canton- 0.25 & 0.5 ppm Technical grade Negative Solvent not specified Valencia (1981)
recessive lethals) S) In feeding solution from America
(Germ cells) Cyanamid
Chromosome damage (sex D. melanogaster (males 0.5 ppm Technical grade Negative Solvent not specified Valencia (1981)
chromosome loss and non- with rearranged Y In feeding solution from America
disjunction) chromosome) (germ cells) Cyanamid
Mutation (sex-linked D. melanogaster (MRA) 50 ppm adult feeding 50% dissolved Negative Velázquez et al.
recessive lethals) (Germ cells) 10, 25 ppm adult injection in DMSO then (1987)
diluted in 5%
100 ppm larval feeding sucrose to give a
final DMSO
concentration of
0.1%
Chromosome damage (sex D. melanogaster (Ring-X) 5 & 10 ppm adult feeding 50% dissolved Negative Velázquez et al.
chromosome loss and non- (germ cells) 5 ppm adult injection in DMSO then (1987)
disjunction) diluted in 5%
7, 10 & 20 ppm larval sucrose to give a
feeding final DMSO
concentration of
0.1%

354

Malaoxon
Chromosome damage D. melanogaster (Canton- 5 ppm in water 94.4% Positive Foureman et al.
(reciprocal translocation) S) (germ cells) (1994)
Mutation (sex-linked D. melanogaster (Canton- 5 ppm feeding 94.4% Positive (feed) Foureman et al.
recessive lethals) S) (germ cells) 2 ppm injection, in water Negative (1994)
(injection)
bw: body weight; 8-Oxo-dG: 8-hydroxy-2ʹ-deoxyguanosine; DMSO: dimethyl sulfoxide; HPLC-EC: high pressure liquid chromatography-electrochemical-electrochemical detection; IP:
intraperitoneal; LC50: mean lethal concentration; LD50: median lethal dose; LED: lowest effective dose; NS: not specified; ppm: parts per milion

355
Genotoxicity tests in mammalian systems (Table 22)

Malathion was positive for DNA damage measured using the alkaline comet assay in blood
leukocytes of rats treated by intraperitoneal injection for 5 days (Moore, Patlolla & Tchounwou,
2011); in cells of the liver, brain, kidney and spleen of rats treated orally once or once a day for 60
days (Ojha et al., 2013); and in blood leukocytes and brain cells of rats treated once only or once a
day for 28 days via intraperitoneal injection (Réus et al. 2008). Malathion was negative for DNA
damage in a rat hepatocyte unscheduled DNA synthesis assay when administered by gavage (Meerts,
2003). Malathion was reported to induce sister chromatid exchanges in bone marrow cells of mice
treated acutely by intraperitoneal injection (Giri et al., 2002) or for 24 weeks when fed a diet of
malathion formulation–treated feed (Amer et al., 2002). Giri et al. (2002) also reported an increase in
sperm head shape abnormalities in mice treated via an intraperitoneal injection over 5 days and
sampled at 35 days.
A number of in vivo rodent studies report malathion and malathion formulations as
clastogenic. Increased chromosome damage has been reported in bone marrow cells of various strains
of gavage-administered mice (Giri et al., 2002) or mice chronically administered for 7 days (Kumar,
Khan & Sinha, 1995), 10 days (Hoda & Sinha, 1991), by intraperitoneal injection (Dulout, Pastori &
Olivero, 1983; Giri et al., 2002) or by skin painting (Salvadori et al., 1988). Positive clastogenic
results are reported also for primary spermatocytes of mice treated dermally (Salvadori et al. 1988), in
bone marrow cells of rats treated acutely via intraperitoneal injection (Moore, Patlolla & Tchounwou.,
2011), in spleen cells of mice treated once via an intraperitoneal injection (Amer et al., 1996) as well
as in Syrian hamsters treated with a formulation via intraperitoneal injection (Dzwonkowska &
Hübner, 1986). Malathion has also been reported positive for clastogenicity in bone marrow cells of
mice treated via intraperitoneal injection for 35 days (Abraham et al., 1997), in primary spermatocytes
of mice maintained on treated water for 50 and 100 days (Bulsiewicz et al., 1976) and in bone marrow
cells, spleen cells and spermatocytes of mice fed for 6 and 12 weeks with a malathion formulation–
treated grain that had been stored for 24 weeks (Amer et al. 2002). Related to these positive effects
with chromosomal aberrations, Giri, Giri & Sharma (2011) reported an increase in the frequency of
mouse bone marrow micronucleated polychromatic erythrocytes (MN-PCE) when malathion was
administered orally or by intraperitoneal injection acutely, as did Dulout et al. (1982) for mice treated
acutely via intraperitoneal injection or by skin painting. Abraham et al. (1997) reported positive
findings for the induction of bone marrow MN-PCE in mice treated via intraperitoneal injection for 35
days and sampled weekly. Rats treated once a day for 28 days by intraperitoneal injection exhibited an
increased frequency of micronucleated erythrocytes but not in MN-PCE following a single injection
(Réus et al., 2008). Because the spleen of rats (as opposed to that of mice) efficiently removes
micronuclei from erythrocytes, these findings are suspect.
Other investigators reported negative findings for the induction of chromosomal aberrations
by malathion or malathion-containing products in bone marrow cells and spermatogonia of mice
treated via intraperitoneal injection (Degraeve & Moutschen, 1984); in bone marrow cells,
spermatogonia and primary spermatocytes of mice maintained on treated drinking water for 5 days
per week for 7 weeks (Degraeve, Chollet & Moutschen, 1984a), in primary spermatocytes of mice
sampled 10–15 days after a single intraperitoneal injection (Degraeve, Chollet & Moutschen, 1984b),
in bone marrow cells of mice dosed by intraperitoneal injection or gavage (Kurinniy 1975; NTP,
2016) and in bone marrow cells of rats treated by gavage (Gudi, 1990). Malathion was also reported
negative in studies that evaluated the induction of micronuclei in bone marrow erythrocytes of mice
treated acutely by intraperitoneal injection with malathion (Navarro, 1995).
Malathion was negative in the mouse dominant lethal test when administered once orally by
gavage (USEPA 1977), by a single intraperitoneal injection (Degraeve & Moutschen, 1984), or in
drinking water for seven weeks (Degraeve, Chollet & Moutschen, 1984a).
Malaoxon was reported as negative for the induction of chromosomal aberrations and sister
chromatid exchanges in the bone marrow cells of male mice following a single intraperitoneal
injection (NTP, 2016).

356
Table 22. In vivo genotoxicity tests in mammalian systems

End-point Test object Dose Purity Result Comments Reference
Malathion
Chromosomal damage Male Swiss albino mice 1/15th LD50, IP daily for 35 NS Positive Inadequate publication. Significant Abraham et al.
(chromosomal (bone marrow) days increase in frequency of aberrant (1997)
aberrations) Vehicle not specified cells (no P value calculated);
increase directly proportional to
Terminated at weekly intervals treatment duration; level returned to
during treatment and during a control value within one week after
35 day recovery period end of treatment
Chromosome damage Female Syrian hamster 240, 480, 1200 & 2400 mg/kg Sadofos 30 Positive at lowest Limited study since positive at Dzwonkowska
(chromosomal (bone marrow) bw, IP  1 (30%) dose only lowest dose tested only, gaps & Hübner (1986)
aberrations) Vehicle not specified excluded. LD50 = 2400 mg/kg bw
Sampled at 24 hours
Chromosome damage Mice, unspecified strain 30 mg/kg bw in DMSO, IP  1 100% Positive Inadequate publication due to lack of Amer et al.
(chromosomal (spleen cells) Sampled at 6, 24, 48 hours (synthesized) (excluding gaps) detail on sample processing and (1996)
aberrations) at all sampling scoring criteria. Single dose
times represented 1/8-1/10 LD50
Chromosome damage BALB/c mouse (bone 115, 230 & 460 mg/kg bw in 95.5% Positive Data analysis limited due to lack of Dulout, Pastori
(chromosomal marrow) corn oil, IP  1 (excluding gaps) pairwise comparisons & Olivero
aberrations) Terminated at 6, 12 & 24 hours based on dose– (1983)
response
relationship
Chromosome damage Male CFW mice 0.3% solution of Sadofos-30 NS Positive Inadequate publication. Gaps Bulsiewicz et al.
(chromosomal (primary containing ~30% malathion included in the analysis and the (1976)
aberrations) spermatocytes) administered in water increase was in events related to
Gavage & oral in water for 50 chromosome number (polyploidy,
or 100 days univalents)
Mutation (dominant Male ICR/SIM mice 1 250, 2 500 & 5 000 mg/kg Technical grade Negative Limited study. Mice maintained on USEPA (1977)
lethal) (testis) bw in corn oil, feeding from America diet for 7 weeks, with the amount summarized in
Cyanamid; lot ingested not specified Waters et al.
40216006.300 (1980)
Chromosome damage Male Q strain mice 300 mg/kg bw, IP  1 > 99% Negative Inadequate publication with missing Degraeve &
(chromosomal (bone marrow, Solvent not specified critical protocol and data analysis Moutschen
aberrations) spermatogonia) information (1984)
Sampled 12, 24, 36 hours

357

Mutation (dominant Male Q strain mice 300 mg/kg bw, IP  1 > 99% Negative Inadequate study given testing of Degraeve &
lethal) (testis) Solvent not specified single dose although stated to be a Moutschen
maximum dose, no information (1984)
provided. No increase in pre- or
postimplantation fetal lethality
Chromosome damage Male Q strain mice 8 ppm in drinking water 99% Negative Inadequate study. No positive Degraeve,
(chromosomal (bone marrow, 5 days per week for 7 weeks control Chollet &
aberrations) spermatogonia, primary Moutschen
spermatocytes) (1984a)
Mutation (dominant Male Q strain mice 8 ppm in drinking water 99% Negative Inadequate study. No positive Degraeve,
lethal) (testis) 5 days per week for 7 weeks control Chollet &
Moutschen
(1984a)
Chromosome damage Male Q strain mice 300 mg/kg bw, IP  1 99% Negative Limited study given use of single Degraeve,
(chromosomal (primary Solvent unspecified dose although stated to be the Chollet &
aberrations) spermatocytes) maximum dose possible and the lack Moutschen
Sampled at 10–11, 12–13, 14– of detail (1984b)
15 days
Chromosome damage Strain 615 mice (sex 0.8, 0.4, 0.2, 0.1 × LD50, IP 99% Negative In Chinese, English abstract Ni et al. (1993)
(chromosomal unspecified) once per day for 4 days
aberrations) (bone marrow)
Chromosome damage Male Swiss (Rockland) 120, 240 & 480 mg/kg bw in 99.5% Positive (120 Questionable study given the results. Dulout et al.
(chromosomal mice corn oil, IP  1, dermal  1 mg/kg bw) For dermal, applied to abdomen (1982)
aberrations) (bone marrow) Sampled at 48 hours (single housed). More active by
dermal than IP. No dose–response
Chromosome damage Male & female Swiss 234, 468 & 701 μL/kg bw in Technical Negative Inadequate study since positive Navarro (1995)
(chromosomal albino mice corn oil, IP  2 grade, purity control induced a significant
aberrations) (bone marrow) Terminated 24 hours after 2nd 1 103 g/L increase in MN-NCE at 48 hours;
treatment magnitude only slightly lower than
that induced in PCE. Doses were 25,
50, 75% of the LD50
Chromosome damage Male & female Swiss 0.2 μg/kg bw per day for 10 NS Positive Inadequate publication given lack of Hoda & Sinha
(chromosomal albino mice (bone days, gavage information on scoring criteria. (1991)
aberrations) marrow) Solvent not specified. Sexes mixed, effect reduced by
concurrent treatment with vitamin C
Terminated on day 11

358

Chromosome damage Male & female Swiss 2.5, 5 & 10 mg/kg bw  1, 2.0 NS Positive (2.5 Inadequate publication, sexes mixed Giri et al. (2002)
(chromosomal albino mice (bone mg/kg bw  5, IP mg/kg bw single without providing details. 10 mg/kg
aberrations) marrow) Distilled water vehicle treatment, 2.0 bw maximum sublethal dose.
mg/kg bw per day Appropriately, gaps excluded from
Sampled at 24- and 48-hour repeated analysis
intervals treatment)
Chromosome damage Male & female Swiss 5 mg/kg bw  1, 2.0 mg/kg bw NS Positive Inadequate publication, sexes mixed. Giri et al. (2002)
(chromosomal albino mice (bone  5, gavage Appropriately, gaps excluded from
aberrations) marrow) Distilled water vehicle analysis
Sampled at 24- and 48-hour
intervals
Chromosome damage Male Swiss albino mice 1/15th LD50, IP daily for 35 NS Positive Inadequate publication. Statistically Abraham et al.
(chromosomal (bone marrow) days significant increase in frequency of (1997)
aberrations) Solvent not specified MN formation (no P value
calculated); increase directly
Terminated at weekly intervals proportional to treatment duration;
during treatment and during a level returned to control value within
35-day recovery period 1 week of treatment. Does not
indicate cell type scored but cites
paper that scored PCE
Mutation (sperm head Male Swiss albino mice 2.5, 5 & 10 mg/kg bw, IP  5 at NS Positive (2.5 Giri et al. (2002)
abnormalities) (sperm) 24-hours intervals mg/kg bw)
Distilled water vehicle
Sampled at 35 days
DNA damage (SCE) Male & female Swiss 2.5, 5 & 10 mg/kg bw, IP  1 NS Positive (2.5 Inadequate publication. Sexes mixed Giri et al. (2002)
albino mice (bone Distilled water vehicle mg/kg bw) with no details
marrow)
Sampled at 24 hours
Chromosome damage Male & female Swiss 2.5, 5, 10 mg/kg bw  1, 5.0 95% Positive Inadequate publication. Sexes mixed Giri, Giri &
(micronuclei) albino mice (bone mg/kg bw  2, IP with no details. Dose–response Sharma (2011)
marrow) Distilled water vehicle evident
Sampled after 24 or 48 hours

359

Chromosome damage Male & female Swiss 5 mg/kg bw in distilled water, 95% Positive Inadequate study: used IP solvent Giri, Giri &
(micronuclei) albino mice (bone gavage  1 or  2 control and sexes mixed with no Sharma (2011)
marrow) Sampled at 24 hours details
Chromosome damage Swiss albino mice, sex 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, Technical grade Positive (1.5 Inadequate publication given lack of Kumar, Khan &
(chromosomal unspecified 4.0, 4.5, 5.0, 5.5 & 6.0 mg/kg formulation of mg/kg bw) information on scoring criteria and Sinha (1995)
aberrations) (bone marrow) bw per day for 7 days, gavage 50% gaps included in the analysis. Dose-
Terminated at 24 hours dependent response
Chromosome damage Male Swiss Webster Single treatment: 500, 1 000 & Commercial Positive (multiple Gaps excluded from analysis, Salvadori et al.
(chromosomal mice 1 500 mg/kg bw, dermal malathion treatments = bone highest response at lowest dose. (1988)
aberrations) (bone marrow, Multiple treatments: 25, 500 & (Malatol 100 marrow, 250 Single-dose administration gave
spermatocytes) 1000 mg/kg bw per day for 10 CE, lot no. mg/kg bw; negative results. Also induced
days, dermal 4263-01; primary increase in univalents in primary
Cyanamid spermatocytes, spermatocytes
Corn oil vehicle Quimica do 500 mg/kg bw)
Terminated 24 hours after last Brasil Ltda.)
treatment
Chromosome damage Male white Swiss mice 8.36, 25.08 & 41.80 mg/kg in 57% (Keminof, Positive Inadequate study. Gaps excluded in Amer et al.
(chromosomal (bone marrow, wheat grain for 6 or 12 weeks, Denmark) bone marrow analysis; unclear how (2002)
aberrations) spermatocytes, spleen with treated wheat grain stored aberrations in spermatocytes were
cells) for 4, 12 or 24 weeks scored, lack of information on spleen
cell cultures. Positive results also
obtained with treated grain stored for
24 weeks; negative results with mice
fed with grain stored for 4 weeks, no
dose–response
DNA damage (SCE) Male white Swiss mice 8.36, 25.08 & 41.80 mg/kg in 57% (Keminof, Positive Negative results with mice fed with Amer et al.
(spleen cells) wheat grain for 6 or 12 weeks Denmark) grain stored for 4 weeks, no dose– (2002)
with treated grain stored for 4, response
12 or 24 weeks

360

Chromosome damage Male & female SD rats 0.4 (500), 0.8 (1 000), 1.6 Batch/lot no.: Negative GLP- and TG-compliant study. Gudi (1990)
(chromosomal (bone marrow) (2 000) mL(mg)/kg bw in corn AC 6015-136, Frequency of chromosomal
aberrations) oil, gavage 94.0 % w/w aberrations in control and treated
Terminated at 12, 24 & 48 malathion, 0.1 animals unreasonable low: 5
hours % w/w chromatid-type aberrations in 5 950
MeOOOPS- cells of control and treated animals
triester, 0.4 % suggesting scoring criteria issues
w/w and/or number of cells scored
MeOOSPS- underpowered
triester, 0.2 %
w/w
isomalathion.
0.1% w/w
malaoxon
Chromosome damage Male SD rats 2.5, 5, 10 & 20 mg/kg bw in 98.2% Positive (5 mg/kg Inadequate publication, no Moore, Patlolla
(chromosomal (bone marrow) DMSO, IP  5 at 24-hour bw per day) information on types of & Tchounwou
aberrations) intervals chromosomal aberrations detected or (2011)
if gaps included or excluded from
analysis
DNA damage (alkaline Male SD rats 2.5, 5, 10 & 20 mg/kg bw in 98.2% Positive (2.5 Not clear what cell type scored since Moore, Patlolla
comet assay) (leukocytes) DMSO IP  5 at 24-hour mg/kg bw per text says leukocytes and & Tchounwou
intervals day) lymphocytes in different sections (2011)
DNA damage (alkaline Male Wistar rats 687.5 mg/kg bw, gavage NS Positive Dose = 1/2 reported LD50 of 1375 Ojha et al.
comet assay) (liver, brain, kidney, Terminated at 24, 48, 72 hours mg/kg bw, all tissues affected, for (2013)
spleen cells) (acute) acute, greatest increase in damage at
24 hours, more damage in chronic
23 mg/kg bw per day for 60
days, gavage
Terminated at 24 hours
DNA damage (alkaline Male Wistar rats 25, 50, 100, 150 mg/kg bw in NS Positive (at and 150 mg/kg bw = 1/9th the LD50 Réus et al.
comet assay) (hippocampus cells & 0.9% NaCl above 50 mg/kg (2008)
leukocytes) IP injection  1 day (acute) or  bw per day for
28 days (chronic) 28 days)

361

Chromosome damage Male Wistar rats 25, 50, 100, 150 mg/kg bw in NS Positive (150 Questionable chronic study given Réus et al.
(micronuclei) (erythrocytes) 0.9% NaCl mg/kg bw per day screening of MN from blood by rat (2008)
IP injection  1 day (acute) or  for 28 days) spleen. 150 mg/kg bw = 1/9 the
28 days (chronic) LD50; acute - scored MN in PCE;
chronic, scored MN in total
erythrocytes
DNA damage (UDS) Male Wistar rats 500, 1 000 & 1 500 mg/kg bw 96% Negative GLP- and TG-compliant study Meerts (2003)
(hepatocytes) in corn oil
Malaoxon
Chromosome damage Male B6C3F1 mice 7.5, 15, 30 mg/kg bw, IP × 1 > 95% Negative Potential solubility issues given low NTP (2016)
(chromosomal Terminated at 17 hours solubility of compound in water
aberrations)
5, 10 & 20 mg/kg bw, IP  1
Terminated at 24 hours
Phosphate-buffered saline
vehicle
DNA damage (SCE) Male B6C3F1 mice 7.5, 15 & 30 mg/kg bw in > 95% Negative Potential solubility issues given low NTP (2016)
phosphate-buffered saline solubility of compound in water
bw: body weight; CEBS: Chemical Effects in Biological Systems; DMSO: dimethyl sulfoxide; GLP: good laboratory practice; IP: intraperitoneal; LD50: median lethal dose; MN: micronuclei;
MN-NCE: micronucleated normochromatic erythrocyte; NS: not specified; PCE: polychromatic erythrocyte; NTP: United States National Toxicology Program; SCE: sister chromatid exchange;
TG: test guideline; UDS: unscheduled DNA synthesis

362
(c) Observations in humans

Compared to a control population, 14 patients treated for acute intoxication with a malathion-
based product exhibited increased levels of chromosome damage in mitogen-stimulated lymphocytes
cultured from their blood shortly after admittance to the hospital (van Bao et al., 1974).
In workers exposed selectively to malathion in the California Mediterranean Fruit Fly
Eradication Program, the frequency of erythrocyte glycophorin A mutations was not increased
significantly (the number of participants evaluated was very low) and neither was there a significantly
increased frequency of micronuclei in lymphocytes isolated from blood and mitogen-stimulated to
proliferate in vitro (Titenko-Holland et al., 1997; Windham et al., 1998). Pluth et al. (1996) reported a
spectrum of HPRT mutations on T-lymphocytes from a single worker exposed to malathion and other
pesticides similar to that obtained when T-lymphocytes were exposed to malathion in vitro.
Workers exposed to a combination of malathion and chlorpyrifos exhibited an increased
frequency of micronuclei in isolated lymphocytes (Omari, 2011).
Workers exposed to multiple pesticides, including malathion, have been reported as having
increased levels of DNA damage in unstimulated leukocytes or isolated lymphocytes compared to
controls, as measured by the alkaline comet assay (Garaj-Vrhovac & Zeljezic, 2000, 2001; Singh et
al., 2011; Benedetti et al., 2013; Varona-Uribe et al., 2016) and by the increased frequencies of sister
chromatid exchanges in mitogen-stimulated lymphocytes (Rupa et al., 1988, 1991; De Ferrari et al.,
1991; Garaj-Vrhovac & Zeljezic, 2001, 2002).
Exposed workers were also reported as exhibiting increased frequencies of chromosomal
aberrations (Yoder, Watson & Benson, 1973; Páldy et al., 1987; Rupa et al., 1988, 1989; De Ferrari et
al., 1991; Garaj-Vrhovac & Zeljezic, 2001, 2002) and micronuclei (Garaj-Vrhovac & Zeljezic 2001,
2002; Benedetti et al., 2013) in mitogen-stimulated blood lymphocytes. In contrast, Lucero et al.
(2000) reported a lack of significant increase in the frequency of micronuclei in buccal epithelial cells
and mitogen-stimulated lymphocytes sampled from exposed workers, while Davies et al. (1998)
reported the same negative finding for the frequency of micronuclei in mitogen-stimulated blood
lymphocytes in a population of British Columbia female seasonal farmworkers.
Pluth et al. (1996) reported a spectrum of HPRT mutations in T-lymphocytes from a single
worker exposed to malathion and other pesticides similar to that obtained when T-lymphocytes from
unexposed individuals were exposed to malathion in vitro.
The results of observations in humans are summarized in Table 23.

363
Table 23. Genotoxicity observations in humans exposed to malathion

End-point Tissue or cell type Exposure conditions Response/significance Comments Reference
Chromosome Lymphocytes (whole 60 workers actively engaged in the Negative 20 on-site control, 4/group. Singaravelu,
damage blood, PHA stimulated) processing unit and in direct contact with Inadequate publication lacking critical Mahalingam &
(chromosomal malathion. Participants were classified details. Negative if gaps excluded. Muthu (1998)
aberrations) depending on the period of exposure (0–5, Exposure not likely to have been
60–10, 11–15, 15–20, > 20 years; number of limited to malathion
participants = 7, 8, 25, 12 and 8,
respectively)
Chromosome Lymphocytes (isolated, 38 (29 male, 9 female) malathion-exposed Negative P values after 6 months of exposure vs Titenko-Holland et
damage PHA stimulated, workers involved in the Mediterranean Fruit control group (n = 16, 9 male, 7 al. (1997)
(micronuclei) cytochalasin B) Fly Eradication Program, California female), malathion diacid levels in
urine ranged from not detected (< 5
ppb) to 2 200 ppb, kinetochore status
did not differ. Exposure to multiple
agents
Chromosome Lymphocytes (whole 14 patients with acute intoxication with a Positive (P < 0.001) P values for intoxicated group vs van Bao et al.
damage blood, PHA stimulated) malathion-based formulation: blood analyses control group (n = 15). Exposure to (1974)
(chromosomal immediately (3–6 days), and 1 and 6 months multiple agents
aberrations) after intoxication
Mutation Erythrocytes 1992: 9 male and female workers; 1993: 10 Negative Very limited number/group: 1 in Windham et al.
(glycophorin A male and female workers in the California 1992, 4 in 1993; some workers also (1998)
assay) Mediterranean Fruit Fly Eradication Program use diazinon
Chromosome Lymphocytes (isolated, Sept 1993: 24 male and female workers; Dec Negative Sept 1993: 10 male and female Windham et al.
damage PHA stimulated, 1993: 14 male and female workers in the controls; Dec 1993: 6 male and female (1998)
(micronuclei) cytochalasin B) California Mediterranean Fruit Fly controls; malathion diacid levels in
Eradication Program; some workers also urine ranged from not detected (< 5
used diazinon ppb) to 2 200 ppb, expanded analysis
from Titenko-Holland et al. (1997)
Chromosome Lymphocytes (isolated, 23 healthy Jordanian non-smoking workers Positive (P < 0.01 at 8 P values for exposed group after 8 Omari (2011)
damage PHA stimulated, with varied durations of exposure (3–30 months of exposure; months of exposure vs control group
(micronuclei) cytochalasin B) years). Malathion was used together with P < 0.05 after 8 months of (n = 22), also sampled 8 months after
chlorpyrifos no exposure) no exposure; lower levels of MN but
still elevated. Exposure to multiple
agents

364

DNA damage Leukocytes (whole 81 soybean workers (65 male, 16 female) Positive (P < 0.001 for Compared to 46 controls (19 male, 27 Benedetti et al.
(alkaline comet blood) exposed to 6 herbicides, 14 insecticides males, P < 0.05 for female), no correlation for age and (2013)
assay) (including malathion) and 5 fungicides females) exposure time. Exposure to multiple
throughout the growing season agents
Chromosome Epithelial cells (buccal) 81 soybean workers (65 male, 16 female) Positive (P < 0.001 for Compared to 46 controls (19 male, 27 Benedetti et al.
damage exposed to 6 herbicides, 14 insecticides males, P < 0.05 for female), no correlation for age and (2013)
(micronuclei) (including malathion) and 5 fungicides females) exposure time. Exposure to multiple
throughout the growing season agents
Chromosome Lymphocytes (isolated, 18 British Columbia seasonal farmworkers Negative Compared to 21 age-matched female Davies et al. (1998)
damage PHA stimulated, (female) exposed to the herbicides simazine, controls; trend for increased response
(micronuclei) cytochalasin B) paraquat, napropamide and glyphosate, the with increasing work duration.
fungicides captan and triforine and the Exposure to multiple agents
insecticides diazinon, malathion, carbofuran
and endosulfan
Chromosome Lymphocytes (isolated, (A) 32 healthy individuals exposed to Positive (P < 0.01 for A vs Response not confounded by age or De Ferrari et al.
damage PHA stimulated) pesticides while working in the flower C, P < 0.05 for B vs C) smoking habit. Observed also (1991)
(chromosomal industry; (B) 32 individuals exposed to increased frequencies of aneuploid
aberrations) pesticides while working in the flower and polyploid cells. Exposure to
industry and hospitalized for bladder cancer multiple agents
but not yet treated; (C) 31 matched controls.
Exposure included 18 nitro-organic
herbicides and fungicides, 9 nitro-organic
fungicides, 12 organophosphate and
organothiophosphate insecticides, 4
hydrocarbon-derivative herbicides and 5
inorganic fungicides and insecticides
DNA damage Lymphocytes (isolated, (A) 28 healthy individuals exposed to Positive (P < 0.01 for A vs Response not confounded by age or De Ferrari et al.
(SCE) PHA stimulated) pesticides while working in the flower C, P < 0.001 for B vs C) smoking habit. Exposure to multiple (1991)
industry; (B) 14 individuals exposed to agents
pesticides while working in the flower
industry and hospitalized for bladder cancer
but not yet treated; and (C) 15 matched
controls. Exposure included 18 nitro-organic
herbicides and fungicides, 9 nitro-organic
fungicides, 12 organophosphate and
organothiophosphate insecticides, 4
hydrocarbon-derivative herbicides and 5
inorganic fungicides and insecticides

365

DNA damage Leukocytes (whole 10 workers (7 male, 3 female) in pesticide Positive (P < 0.001) P values for exposed group after 8 Garaj-Vrhovac &
(alkaline comet blood) production simultaneously exposed to a months of exposure vs control group Zeljezic (2000)
assay) complex mixture of pesticides (atrazine, (n = 10, 7 male, 3 female); values
alachlor, cyanazine, 2,4- reduced after 8 months of non-
dichlorophenoxyacetic acid and malathion) exposure. Exposure to multiple agents
Chromosome Lymphocytes (whole 20 workers (17 male, 3 female) in pesticide Positive (P < 0.001) P values for exposed group after 8 Garaj-Vrhovac &
damage blood, PHA stimulated) production simultaneously exposed to a months of exposure vs 20 controls (12 Zeljezic (2001)
(chromosomal complex mixture of pesticides (atrazine, male, 8 female); gaps excluded, values
aberrations) alachlor, cyanazine, 2,4- reduced but still elevated vs controls
dichlorophenoxyacetic acid, malathion) after 8 months on non-exposure.
Exposure to multiple agents
DNA damage Leukocytes (whole 20 workers (17 male, 3 female) in pesticide Positive (P < 0.001) P values for exposed group after 8 Garaj-Vrhovac &
(alkaline comet blood) production simultaneously exposed to a months of exposure vs 20 controls (12 Zeljezic (2001)
assay) complex mixture of pesticides (atrazine, male, 8 female); values reduced but
alachlor, cyanazine, 2,4- still elevated vs controls after 8
dichlorophenoxyacetic acid, malathion) months on non-exposure. Expanded
from the Garaj-Vrhovac & Zeljezic,
2000 study. Exposure to multiple
agents
Chromosome Lymphocytes (whole 20 workers (17 male, 3 female) in pesticide Positive (P < 0.05) P values for exposed group after 8 Garaj-Vrhovac &
damage blood, PHA stimulated, production simultaneously exposed to a months of exposure vs 20 controls (12 Zeljezic (2001)
(micronuclei) cytochalasin B) complex mixture of pesticides (atrazine, male, 8 female), values reduced but
dichlorophenoxyacetic acid, malathion) months on non-exposure. Exposure to
multiple agents
DNA damage Lymphocytes (whole 20 workers (17 male, 3 female) in pesticide Positive (P < 0.001) P values for exposed group after 8 Garaj-Vrhovac &
(SCE) blood, PHA stimulated) production simultaneously exposed to a months of exposure vs 20 controls (12 Zeljezic (2001)
complex mixture of pesticides (atrazine, male, 8 female); values reduced but
dichlorophenoxyacetic acid, malathion) months on non-exposure. Exposure to
multiple agents
Chromosomal Lymphocytes (isolated, 10 workers (7 male, 3 female) in pesticide Positive (P < 0.05) P value for exposed workers vs 20 Garaj-Vrhovac &
aberrations PHA stimulated) production simultaneously exposed to a controls (12 male, 8 female). Exposed Zeljezic (2002)
complex mixture of pesticides (atrazine, population from Garaj-Vrhovac &
alachlor, cyanazine, 2,4- Zeljezic (2000) study, control from
dichlorophenoxyacetic acid, malathion) 2001 study. Exposure to multiple
agents

366

DNA damage Leukocytes 10 workers (7 male, 3 female) in pesticide Positive (P < 0.01) P value for exposed workers vs 20 Garaj-Vrhovac &
(alkaline comet production simultaneously exposed to a controls (12 male, 8 female). Exposed Zeljezic (2002)
assay) complex mixture of pesticides (atrazine, population from Garaj-Vrhovac &
agents
Chromosome Lymphocytes (isolated, 10 workers (7 male, 3 female) in pesticide Positive (P < 0.05) P value for exposed workers vs 20 Garaj-Vrhovac &
damage PHA stimulated, production simultaneously exposed to a controls (12 male, 8 female). Exposed Zeljezic (2002)
(micronuclei) cytochalasin B) complex mixture of pesticides (atrazine, population from Garaj-Vrhovac &
agents
Chromosome Lymphocytes (isolated, 64 greenhouse workers (male) exposed to 16 Negative P value for exposed workers vs 50 Lucero et al. (2000)
damage PHA stimulated, pesticides including malathion (usage controls (male) matched for smoking,
(micronuclei) cytochalasin B) 12.5%), 1 bactericide and 9 fungicides mean age of control 6 years greater.
Chromosome Epithelial cells (buccal) 59 greenhouse workers (male) exposed to 16 Negative P value for exposed workers vs 49 Lucero et al. (2000)
damage pesticides including malathion (usage controls (male) matched for smoking,
(micronuclei) 12.5%),1 bactericide and 9 fungicides mean age of control 6 years greater.
Chromosome Lymphocytes (isolated, 80 workers (male) exposed to ~80 different Positive (P < 0.001) Compared to 24 controls (male); Páldy et al. (1987)
damage PHA stimulated) kinds of formulations from groups of organic increasing damage generally with
(chromosomal phosphates, dithiocarbamates, nitro increasing duration of exposure; blind
aberrations) compounds, triazines, urea compounds, scoring. Exposure to multiple agents
phthalimides, organochlorines,
phenoxyacetic acids, pyrethroids,
carbamates, heterocyclic compounds and
sulfur- and copper-containing chemicals
Chromosome Lymphocytes (isolated, 25 vegetable-garden workers (male), Positive (P < 0.05) P value for exposed workers, Rupa et al. (1988)
damage PHA stimulated) smokers and alcohol consumers, exposed to independent of years worked, vs
(chromosomal seven pesticides including malathion control I (20 healthy male non-
aberrations smokers and non-alcohol consumers)
or control II (10 healthy male smokers
and alcohol consumers); analysis
based on all aberrations, including
gaps, response independent of years
worked. Exposure to multiple agents

367

DNA damage Lymphocytes (whole 25 vegetable-garden workers (male), Positive (P < 0.05) P value for exposed workers, Rupa et al. (1988)
(SCE) blood, PHA stimulated) smokers and alcohol consumers, exposed to independent of years worked, vs
seven pesticides including malathion control I (20 healthy male non-
smokers and non-alcohol consumers)
or control II (10 healthy male smokers
and alcohol consumers); response
independent of years worked.
Chromosome Lymphocytes (whole 50 smoking cotton-field workers (male; Positive (P < 0.05) Frequency independent of years Rupa, Reddy &
damage blood, PHA stimulated) based on Rupa et al., 1991) exposed to 11 worked, total number of aberrations Reddi (1989)
(chromosomal pesticides including malathion (50% purity) compared to 20 non-smoking male
aberrations) controls and 27 smoking male
controls. Analysis excluded gaps.
DNA damage Lymphocytes (whole 61 non-smoking, cotton-field workers (male) Positive (P < 0.05) P value for pesticide applicators vs 45 Rupa et al. (1991)
(SCE) blood, PHA stimulated) regularly exposed to 11 pesticides including controls (male), increasing frequency
malathion with exposure duration. Exposure to
multiple agents
DNA damage Lymphocytes (isolated) 70 male and female workers spraying Positive (P < 0.001) P value vs 70 matched controls; Singh et al. (2011)
(alkaline comet pesticides for community health programmes exposure to multiple agents
assay) in Delhi, India, exposed to pirimiphos-
methyl, chlorpyrifos, temephos and
malathion
DNA damage Leukocytes 223 rice field workers (98% male) in Positive (95% CI: 2.34– Cross-sectional study, 31 pesticides Varona-Uribe et al.
(alkaline comet Colombia 21.60) were quantified in blood, serum and (2016)
assay) urine. Maximum-likelihood factor
analysis identified 8 different
mixtures. Robust regressions were
used to explore associations between
the factors identified and the comet
assay. The mixture of pirimiphos-
methyl, malathion, bromophosmethyl
and bromophosethyl (but not
malathion alone) was associated with
increased tail length

368

Chromosome Lymphocytes (whole 16 workers (male) exposed to 17 pesticides Positive (no P value) No statistics provided vs 16 male Yoder, Watson &
damage blood, PHA stimulated) including malathion, sampled at midwinter controls but a 5-fold increase in Benson (1973)
(chromosomal ebb in spraying operations and again during damage between sampling times and
aberrations) the peak spraying 3.5-fold greater than concurrent
control samples; slides scored blind.
DNA damage Lymphocytes (whole 20 workers (17 male, 3 female) working in Positive (P < 0.001) P value by Mann–Whitney test for Zeljezic & Garaj-
(SCE) blood, PHA stimulated) pesticide production simultaneously exposed exposed group vs control group (n = Vrhovac (2002)
to 5 pesticides (atrazine, alachlor, cyanazine, 20, 12 male, 8 female), increase was
2,4-dichlorophenoxyacetic acid, malathion) present at the beginning of 8-month
exposure, at the end of 8-month
exposure and 8 months later. Also,
significant increase in high frequency
cells (> 95 percentile of pooled
distribution). Exposure to multiple
agents
MN: micronuclei; PHA: phytohaemagglutinin; ppb: parts per billion; SCE: sister chromatid exchange.

369

(a) Single-generation and multigeneration studies
In a two-generation reproductive toxicity study by Schroeder (1990), malathion (purity 94%)
was admixed in the diet at 0, 550, 1700 or 1800 ppm and fed ad libitum to two parental generations of
Sprague Dawley–derived COBSCD rats (25/sex per dose) and their offspring through premating,
mating, gestation and lactation. Observations for mortalities and clinical signs were made daily, with
more detailed clinical examinations performed on each rat weekly. Body weight and feed
consumption were recorded weekly. Pups were examined, weighed, counted and sexed on postnatal
days 0, 4, 7, 14 and 21. Standard reproduction, offspring and litter parameters were recorded or
calculated. At scheduled termination, the rats were necropsied and their organs weighed and tissues
The doses achieved during premating, gestation and lactation are summarized in Table 24.
There were no treatment-related mortalities, clinical signs, effects on body weight or feed
consumption, macroscopic or microscopic findings in parental rats. There were no effects on
reproduction parameters or reproductive tissues. In F1A litters, pup weights were 14% lower
(P < 0.05) than the control on day 21 of lactation at 5000 and 7500 ppm. In F1B litters, pup weights
were 10.7% lower than the control on day 21 of lactation. In F2A litters, pup weights were 10% lower
than the control (P < 0.05) at 7500 ppm. In F2B litters, pup weights were significantly lower
(P < 0.05) than the control on day 7, 14 and 21 of lactation (−15.2%, −17.0% and −19.8%,
respectively) at 5000 ppm and on day 21 of lactation (−13.7%; P < 0.05) at 7500 ppm.
The NOAEL for both reproductive toxicity and parental toxicity was 7500 ppm (equal to
595 mg/kg bw per day in males and 655 mg/kg bw per day in females), the highest tested dose. The
NOAEL for offspring toxicity was 1700 ppm (equal to 130 mg/kg bw per day in males and 152 mg/kg
bw per day in females) for reduced pup weights at 5000 ppm (equal to 393 mg/kg bw per day in males
and 438 mg/kg bw per day in females).
Table 24. Achieved doses of malathion in rats

Achieved dose in mg/kg bw per day
Males Females
1 700 5 000 7 500 1 700 5 000 7 500
Phase 550 ppm ppm ppm ppm 550 ppm ppm ppm ppm
Premating
F0 42.71 131.79 393.54 594.98 49.91 151.89 438.09 655.05
F1 42.61 129.97 394.41 628.04 51.23 154.25 464.71 751.84
Gestation
F0 – F1A – – – – 47.35 143.44 420.16 629.91
F0 – F1B – – – – 44.08 135.70 393.67 599.78
F1 – F1A – – – – 45.68 139.00 418.57 685.40
F1 – F1B – – – – 39.58 124.12 383.84 582.20
ppm: parts per million; F0: parental generation; F1A: first generation –first litter; F1B: first generation –second litter
Results expressed as the mean achieved dose in mg/kg bw per day
Source: Schroeder (1990)
Uzun et al. (2009) administered malathion (purity not specified) in corn oil by gavage to
groups of six sexually mature male Wistar rats for 4 weeks at 0 or 27 mg/kg bw per day in the

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presence or absence of 200 mg/kg bw per day vitamin C and vitamin E. The rats were terminated after
4 weeks and their reproductive tissues dissected to analyse testicular sperm counts, epididymal sperm
motility and epididymal sperm morphology. The reproductive organs were not weighed or
histopathologically examined. Blood was also sampled at termination to analyse luteinizing hormone
(LH), follicle-stimulating hormone (FSH) and testosterone. Sperm counts and sperm motility were
significantly lower than the control (P < 0.05) in malathion-treated rats (−22% and −36%
respectively), with vitamin co-treatment having a marginal effect on these parameters (−13% and
−26% lower than the control, respectively). Abnormal sperm morphology was significantly increased
(P < 0.05) in malathion-treated rats (2.72% versus 1.71% in the control), with vitamin co-treatment
also having a marginal effect on these parameters (1.97% versus 1.71% in the control). Significant
reductions (P < 0.05) in FSH (−17%), LH (−32%) and testosterone (−27%) occurred in malathion-
treated rats, while vitamin co-treatment had no effect. The authors observed “fewer spermatogenic
cells in some of the seminiferous tubules [in malathion-treated rats], and necrosis in some
seminiferous tubules and oedema in interstitial tissue”. Pictures of representative histopathological
changes were shown, but there was no quantitative evaluation of these microscopic changes. This
study is considered to have limited value for risk assessment purposes because of the small group size,
the use of only one dose of malathion, the absence of organ weight data and the absence of
histopathological examination of reproductive tissues.
Geng et al. (2015) administered malathion (purity 95%) in corn oil by gavage at 0, 33.75, 54
or 108 mg/kg bw per day for 60 days to groups of 10 male Wistar rats (age unspecified; 80–100 g
bw). Body weights were recorded weekly while feed consumption was not recorded. Following
termination, the testes were weighed. Sperm counts, motility and morphology were recorded. Blood
samples were collected at termination to analyse serum LH, FSH and testosterone concentrations.
Acetylcholinesterase activity was not analysed. The following testicular enzymes were analysed in
homogenized left testis samples: acid phosphatase (ACP), lactate dehydrogenase, succinate
dehydrogenase (SDH) and GGT. Histopathology was performed on the right testis. The seminal
vesicle, epididymis or prostate were not microscopically examined. Apoptosis was analysed in testes
by terminal deoxynucleotidyl transferase nick end labelling (TUNEL) assay. Bax and Bcl-2 protein
expression was analysed in testes using immunohistochemistry.
There were no deaths and no clinical signs. Terminal body weight and overall body-weight
gain was significantly lower (P < 0.05) than the controls at the highest dose (−17% and −24%,
respectively). Effects on the testes, sperm characteristics, hormone levels and testicular enzymes are
summarized in Table 25. The authors stated that the testes appeared small and mauve in colour at 54
and 108 mg/kg bw per day. While absolute testis weight was approximately 20% lower than the
control (P < 0.01) at 54 and 108 mg/kg bw per day, relative testis weight was consistent across all
groups. Sperm counts were significantly reduced (P < 0.01) at the highest dose while sperm motility
was decreased and dismorphology rates were increased at 54 and 108 mg/kg bw per day. Serum LH
was reduced at 54 and 108 mg/kg bw per day, while serum FSH and testosterone were reduced at 108
mg/kg bw per day. Changes in testicular enzymes include reduced acid phosphatase (108 mg/kg bw
per day) and GGT (54 and 108 mg/kg bw per day) and increased lactate dehydrogenase (108 mg/kg
bw per day). There was no treatment-related change in succinate dehydrogenase. The results of the
histopathological examination of the testes were not quantified; the authors stated that at 54 and 108
mg/kg bw per day “severe alterations in the seminiferous tubules including the loss, derangement and
sloughing of the spermatogenic cells, vacuolization in Sertoli cell cytoplasm and destruction of Sertoli
cell cytoskeleton” occurred. Graphically presented data showed a significant dose-related increase
(P < 0.01) in apoptosis in spermatogenic cells at every dose of malathion. Bax protein levels were
significantly lower than the control (P < 0.05) at 54 and 108 mg/kg bw per day, while at these same
doses, Bcl-2 proteins levels were significantly increased (P < 0.01).
A number of factors confound the interpretation of these observations, including uncertainty
of the age of the rats and the possibility that the effects were secondary to the reduction in body-
weight gain. In addition, there were limited methodological details surrounding the histopathological
examination of the testes including how slides were evaluated and graded.
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Table 25. Effect of malathion on testicular parameters in rats

Measure per dose
0 mg/kg bw per 33.75 mg/kg bw per 54 mg/kg bw per 108 mg/kg bw per
Parameter day day day day
Terminal body weight (g) 402.10 380.40 345.5 334.9* (−17%)
Testes weight (g) 4.26 3.80 3.43** (+19%) 3.36** (+21%)
Relative testes weight (%) 0.011 0.010 0.010 0.010
9
Sperm counts (10 ) 9.26 9.19 7.36 5.54** (−40%)
Sperm motility (%) 78.50 73.70 41.60** (−47%) 36.10** (−54%)
Dismorphology rates (%) 0.24 0.37 0.57** (+140%) 0.63** (+160%)
LH (mIU/mL) 1.08 0.73 0.52** (−52%) 0.49** (−55%)
FSH (mIU/mL) 2.05 1.79 1.53 1.25** (−39%)
Testosterone (nmol/L) 3.35 2.94 2.67 1.91* (−43%)
ACP (U/g protein) 1.46 1.31 1.20 1.13** (−23%)
LDH (U/g protein) 0.19 0.24 0.25 0.31** (+63%)
SDH (U/mg protein) 96.81 100.21 84.42 87.36
GGT (U/g protein) 1.22 1.19 0.95** (−22%) 0.82** (−33%)
ACP: acid phosphatase; FSH: follicle-stimulating hormone; GGT: gamma-glutamyltransferase; IU: International Unit; LDH:
lactate dehydrogenase; LH: luteinizing hormone; SDH: succinate dehydrogenase; U: enzyme unit; *: P < 0.05; **: P < 0.01
Results expressed as the mean, with the % increase (+) or decrease (−) relative to the concurrent control in parentheses.
Source: Schroeder (1990)
In a uterotrophic assay by Barnett Jr (2011b), groups of eight ovariectomized female

Crl:CD[SD] rats were administered by gavage malathion (purity 96.0%) in corn oil at 0, 100, 300 or
1000 mg/kg bw per day for three days. A positive control group of eight rats was administered
5 µg/kg bw per day 17α-ethynyl estradiol (purity 99%) as two subcutaneous injections of 2.5 µg/kg
bw for three days. Estrous cycling was examined for five days prior to commencing dosing. The rats
were observed twice daily for mortality and clinical signs. Body weight and feed consumption were
recorded daily. Vaginal patency was recorded twice on the day of termination. The rats were
terminated 24 hours after the final dose, and blood and brain acetylcholinesterase activity analysed.
Each rat was necropsied, and the uterus, liver and brain weights recorded. There were no deaths or
treatment-related clinical signs. At 1000 mg/kg bw per day, there was a significant loss of body
weight on day 2 –3 (−0.8 g; P < 0.05). The positive control group also had a significant loss of body
weight on day 2–3 (−2.7 g; P < 0.01). Overall body-weight gain from day 1–4 was −14%, −3.2% and
−35.5% lower than the control at 100, 300 and 1000 mg/kg bw per day, respectively. At 1000 mg/kg
bw per day, daily feed consumption was significantly lower than the control on days 2, 3 and 4
(−10%, −8% and −6%, respectively; P < 0.01 or 0.05). Vaginal patency was confirmed at
termination. There were no treatment-related macroscopic findings. Mean absolute liver weight was
increased at 1000 mg/kg bw per day (+24%; P < 0.01), while relative liver weight was increased at
300 (+10%, P < 0.05) and 1000 mg/kg bw per day (+25%, P < 0.01). The absolute and relative
weights of the wet and blotted uteri in malathion-dosed rats were comparable to the vehicle control
group values. Toxicologically and statistically significant inhibition of erythrocyte
acetylcholinesterase occurred at every dose of malathion (−20.2%, −38.3% and −68.7% at 100, 300
and 1000 mg/kg bw per day, respectively; P < 0.01), while brain acetylcholinesterase activity was
significantly lower (P < 0.01) than the control at 1000 mg/kg bw per day (−15.7%). On the basis of
these findings, malathion was not positive for estrogenic activity in the uterotrophic bioassay at doses
up to the limit dose of 1000 mg/kg bw per day.
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A Hershberger assay was conducted by Barnett Jr (2011c) to determine the effect of

malathion on the weights of the following androgen-dependent tissues in castrate-peripubertal male
rats: ventral prostate, seminal vesicle plus fluids and coagulating glands, levator ani-bulbocavernosus
muscle, paired Cowper’s glands and the glans penis. In Phase 1 of the study, to evaluate the potential
androgenic activity of malathion, malathion (purity 96.0%) in corn oil was administered by gavage at
0, 100, 300 or 1000 mg/kg bw per day for 10 days to groups of eight male Crl:CD[SD] rats. A
positive control group of eight rats was administered 0.4 mg/kg bw per day testosterone propionate
subcutaneously for 10 days.
In Phase 2 of the study, which evaluated the potential antiandrogenic activity of malathion,
groups of eight male rats were administered by gavage with malathion in corn oil at 0, 100, 300 or
1000 mg/kg bw per day for 10 days. A positive control group of eight rats was administered by
gavage 3 mg/kg bw per day flutamide in corn oil for 10 days. All groups received a subcutaneous
dose of 0.4 mg/kg bw per day of testosterone propionate for 10 days to enable the detection of
potential antiandrogenic activity. Observations for mortality and clinical signs were made daily. Body
weight and feed consumption were recorded daily. Preputial separation was evaluated prior to
commencing dosing. Following termination, the rats were necropsied, and blood and brain
acetylcholinesterase activity analysed (Phase 1 only) and selected organs weighed.
Phase 1: There were no treatment-related deaths or clinical signs. There were no significant
intergroup differences in absolute body weight, while body-weight gain was significantly lower than
the control at 1000 mg/kg bw per day from days 3–4 (−89%, P < 0.01) and 4–5 (−59%, P < 0.05);
overall body-weight gain (days 1–11) was 15% lower than the control but this difference was not
statistically significant. Feed consumption was significantly lower than the control at 1000 mg/kg bw
per day at days 3–4 (−9%, P < 0.05), 4–5 (−10%, P < 0.05) and 7–8 (−19%, P < 0.01); overall feed
consumption was 9.4% lower (P < 0.05) than the control. There were no treatment-related
macroscopic findings. In the positive control, there were significant increases (P < 0.01) in the weight
of the levator ani-bulbocavernosus muscle, seminal vesicles with coagulating glands and fluid,
Cowper’s glands, ventral prostate and glans penis. In malathion-dosed rats, no change in androgen-
dependent organ weights occurred. Absolute liver weight increased by 29% (P < 0.01) at 1000 mg/kg
bw per day, while relative liver weight increased by 9% (P < 0.01) and 32% (P < 0.01) at 300 and
1000 mg/kg bw per day, respectively. Similarly, absolute paired kidney weight increased by 18%
(P < 0.05) at 1000 mg/kg bw per day, while relative kidney was increased by 7% (P < 0.05) and 21%
(P < 0.01) at 300 and 1000 mg/kg bw per day, respectively. Erythrocyte acetylcholinesterase activity
was significantly lower (P < 0.01) than the control at every dose of malathion (−21.6%, −61.2% and
−88.1% at 100, 300 and 1000 mg/kg bw per day, respectively). Brain acetylcholinesterase activity
was significantly lower than the control at 1000 mg/kg bw per day (−39.1%, P < 0.01).
Phase 2: There were no treatment-related deaths or clinical signs. There were no significant
intergroup differences in absolute body weight, while overall body-weight gain was 14% lower than
the control (P < 0.01), at 1000 mg/kg bw per day. Feed consumption was comparable across all
groups. There were no treatment-related macroscopic findings. In malathion-dosed rats, no change in
androgen-dependent organ weights occurred. At 1000 mg/kg bw per day, absolute and relative liver
weights increased (+21% and +27%, respectively; P < 0.05), while relative paired kidney weight also
increased (+13%, P < 0.01).
Based on these findings, malathion did not show an androgenic or antiandrogenic response in
male rats.
As part of a screen for endocrine disruption potential by the USEPA, Palmer (2011a)
examined the effect of malathion on the reproduction of the fathead minnow (Pimephales promelas).
Groups of 24 fish were exposed to malathion (purity 96.0%) for 21 days at concentrations of 0, 0.10.
0.32 or 1.00 mg/L (analytical concentrations of 0, 0.08, 0.25 and 0.82 mg/L, respectively). The fish
were observed daily for survival, adverse signs, fecundity (egg production) and fertility
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(determination of fertile versus non-fertile eggs). At termination, the fish were weighed and their
length recorded. Secondary sex characteristics were recorded (pigmentation patterns, tubercles,
fatpads and ovipositors). Blood was collected to analyse serum vitellogenin. Each fish was
macroscopically examined and gonadal sex determined. Their gonads were weighed and
There was no effect on survival. At the highest concentration, eight fish showed various signs
of toxicity including bruising on the body, loss of colour or no colour in the tail, erratic swimming
behaviour and/or lethargy. There was no treatment-related effect on fish growth, fecundity or fertility.
At the highest dose in males, mean fatpad and tubercle score were significantly lower (P < 0.05) than
the control (2.3 versus 4.0, and 11.4 versus 23.8, respectively). There was no effect on the
gonadosomatic index in males, while a slight increase occurred in females (1.4 versus 11.9 in the
control). There were no treatment-related effects on vitellogenin. Microscopic examination detected
an increased presence of diploid spermatogonia with a decreased presence of spermatocysts
containing spermatocytes (primary and secondary) and spermatids in the testis germinal epithelium in
males at the highest concentration. In females at this same concentration, there was a slight though
significant increase (P < 0.05) in atresia in the ovaries.
In an amphibian metamorphosis assay, African Clawed Frog tadpoles (Xenopus laevis) were
exposed to malathion (purity 96.0%) for 21 days at concentrations of 0, 0.04, 0.13 and 0.40 mg/L
(analytical concentrations of 0, 0.03, 0.11 and 0.32 mg/L. There were no treatment-related effects on
tadpole growth or development (Palmer 2011b, c).
Wagner (2011) examined the potential of malathion (purity 96%) to affect the steroidogenic
pathway in the H295R human adrenocarcinoma cell line. Malathion in dimethyl sulfoxide (DMSO)
was tested at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μmol/L (3 replicates per plate)
in three independent assays. Testosterone and estradiol were analysed by HPLC/MS-MS. Following
the 48-hour incubation, precipitation was noted at 100 μmol/L in two assays, while cytotoxicity did
not exceed 20% at any concentration. A statistically significant reduction (P < 0.05; −19%) in
testosterone concentration occurred at a malathion concentration of 100 µmol/L) in one assay but at
no other concentrations or in the other independent assays; precipitation was observed at 100 µmol/L
malathion in those other two assays. Similarly, estradiol was significantly increased (P < 0.05; +12%)
at 10 µmol/L in one assay but at no other concentrations or in the other independent assays. In this
same assay, precipitation occurred at the highest dose of 100 µmol/L. In the only assay where
precipitation did not occur, estradiol did not significantly increase at 10 or 100 µmol/L (+14 and
+13%, respectively; P > 0.05). Based on these results the authors concluded that malathion caused an
equivocal response in the steroidogenesis assay. However, as these changes were both small and
inconsistent across the three assays, they are unlikely to represent a treatment-related effect.
Wilga (2011) evaluated the potential of malathion (purity 96%) in DMSO to inhibit aromatase
activity using human CYP19 and P450 reductase recombinant microsomes at concentrations from
10−10 to 10−3 mol/L. Three independent assays were performed, with each concentration tested in
triplicate. The positive control substance, 4-hydroxyandrostenedione (purity 99.6%) was included in
each assay at concentrations from 10−10 to 10−5 mol/L. Over the three assays, malathion decreased
aromatase activity at 10-4 to 10−3 mol/L. As slight precipitation occurred at 10−3 mol/L in the first
assay, the highest concentration in subsequent assays was reduced to 10−3.5 mol/L. At this
concentration, malathion inhibited aromatase activity by 64.6% of control activity. Using the
USEPA’s interpretation procedure for aromatase inhibition, malathion was classified as equivocal;
however, the authors concluded that malathion was not an inhibitor of aromatase activity.
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Willoughby (2011a) evaluated the potential of malathion (purity 96%) in DMSO to act as an
agonist of human estrogen receptor alpha (hERα) in a transcriptional activation assay using the hERα-
HeLa-9903 cell line at concentrations of 10−11, 10−10, 10−9, 10−8, 10−7, 10−6, 10−5 and 10−4 mol/L. Each
concentration was tested in replicates of six wells per plate at 24-hour-long exposures. In addition,
two replicates per plate tested the positive control hERα antagonist, ICI 182,780. In a preliminary
assessment of cytotoxicity and precipitation, cytotoxicity occurred at 10 -4 mol/L. In two valid
independent assays, malathion did not result in an increase in luciferase activity at any of the viable
concentrations tested. On this basis the author concluded that malathion is not an agonist of hERα.
Willoughby (2011b) evaluated the potential of malathion (purity 96%) to act as an agonist of
human androgen receptors in rat ventral prostate tissue homogenate at concentrations of 10 −10, 10−9,
10−8, 10−7, 10−6, 10−5, 10−4 and 10−3 mol/L. Two positive controls were included in each of the three
binding assays in replicates of 3: a positive control, R1881 (purity 98%) (10−11 to 10−6 mol/L), and a
weak positive control, dexamethasone (purity 99%) (10−10 to 10−3 mol/L). A solvent (DMSO) control
(6 replicates) was also included in each assay. In the first assay, the mean specific binding of
1 nmol/L [3H]-R1881 was 51.6% at 10−3 mol/L malathion; dexamethasone had a logIC50 of
−4.3 mol/L while the logIC50 of R1881 was −9.9 mol/L. In the second assay, the mean specific
binding of 1 nmol/L [3H]-R1881 was 50.4% at 10−3 mol/L malathion; dexamethasone had a logIC50 of
−4.4 mol/L, while the logIC50 of R1881 was −9.0 mol/L. In the third assay, the mean specific binding
of 1 nmol/L [3H]-R1881 was greater than 75% at every soluble concentration of malathion;
dexamethasone had a LogIC50 of −4.6 mol/L while the logIC50 of R1881 was −9.0 mol/L. The authors
classified malathion as equivocal in the first two assays and a non-binder in the third. On this basis
malathion was classified as equivocal for binding to the androgen receptor.
Barnett Jr (2012d) undertook a pubertal development and thyroid function study to determine
the potential of malathion to interact with the endocrine system. In the main study, groups of 20
peripubertal Crl:CD[SD] rats per sex were administered malathion (purity 95.8%) in corn oil by
gavage at 0, 250, 500 or 1000 mg/kg bw per day from postnatal days 23–54 (males) and 22–43
(females). The highest dose was reduced to 750 mg/kg bw per day due to overt toxicity in females.
Additional groups of eight rats per sex were dosed similarly to analyse acetylcholinesterase activity.
The rats were observed daily for mortality and clinical signs. Body weight and feed consumption were
recorded daily. Once vaginal patency was observed, daily vaginal smears were taken to determine the
stage of the estrous cycle. Thyroid hormones (thyroxine, thyroid stimulating hormone and
testosterone) and clinical chemistry parameters were analysed in blood samples collected at
termination. Erythrocyte and brain acetylcholinesterase activities were analysed in the additional
groups of eight rats following termination. All rats were necropsied, selected organs weighed and
tissues histopathologically examined.
Treatment-related deaths occurred at 500 and 750 mg/kg bw per day in the males in the main
study (2 and 14 rats, respectively), with an additional high-dose male terminated in extremis. At 500
mg/kg bw per day, the deaths occurred on postnatal days 45 and 50, while at 750 mg/kg bw per day,
the deaths occurred between postnatal days 25 and 53. Clinical signs observed prior to death included
urine-stained abdominal fur, slight-to-severe excess salivation, prostrate behaviour, tremors,
lacrimation, decreased motor activity, ataxia, loss of righting reflex, chromodacryorrhea, slight
dehydration, ungroomed coat, dyspnoea, coldness to touch and hunched posture. Similar clinical signs
were observed in survivors at these same doses. Among the females, treatment-related deaths
occurred at the highest dose (two rats, which died on postnatal days 25 and 41), with clinical signs
consistent with those observed in males. In both sexes, there was no treatment-related effect on body
weight or feed consumption, sexual maturation (preputial separation or vaginal patency) or estrous
cycling.
The results of the analysis of clinical chemistry parameters and hormone levels are
summarized in Table 26. In the male rats, testosterone was decreased at every dose; in the absence of
other antiandrogenic findings, the toxicological relevance of this decrease is unclear. In addition,
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thyroxine was reduced at every dose but did not follow a dose–response relationship. There was no
treatment-related effect on TSH levels. A number of significant changes in clinical chemistry
parameters occurred, although some of these did not follow a dose–response relationship or were
inconsistent in males and females; the most consistent findings were increased cholesterol and
decreased alkaline phosphatase, triglycerides, albumin, globulin, albumin to globulin ratio, calcium
and potassium.
Table 26. Effect of malathion on clinical chemistry parameters, hormone levels and organ weights
in prepubertal rats
Measure and per cent change per dose of malathion
Males Females
0 250 500 750/1000 250 500 750/1000
mg/kg mg/kg mg/kg mg/kg 0 mg/kg mg/kg mg/kg mg/kg
Parameter day day day day day day day day
Testosterone 2.15 0.86** 0.48** 0.21** – – – –
(ng/mL) (−60%) (−78%) (−90%)
5.12 7.24** 6.92** 6.76** 4.11 4.89* 4.75 5.12**
T4 (µg/dL)
(+41%) (+35%) (+32%)
TSH (ng/mL) 2.78 4.01 3.41 3.26 1.89 2.26 2.49 2.14
AST (IU/L) 212.1 195.4 154.0** 143.1** 216.0 234.1 213.4 210.4
ALP (IU/L) 514.1 524.6 415.2* 330.0** 402.5 397.6 388.8 297.5**
Total bilirubin 0.159 0.133** 0.111** 0.101** 0.154 0.128** 0.107** 0.113
(mg/dL)
Cholesterol (mg/dL) 90.6 95.7 101.1* 108.3** 92.4 103.2* 104.4* 109.0**
Triglycerides 465.0 312.1** 164.8** 150.0** 157.3 141.6 93.8** 74.5**
(mg/dL)
Albumin (g/dL) 3.39 3.20** 3.18** 3.21* 3.38 3.26* 3.12** 3.10**
Globulin (g/dL) 2.28 2.47** 2.67** 2.66** 2.16 2.41 2.27* 2.40**
Albumin : globulin 1.49 1.30** 1.20** 1.21** 1.57 1.45** 1.38** 1.29**
ratio
Glucose (mg/dL) 124.6 123.1 146.7** 174.0** 145.1 132.6** 135.8* 142.6
BUN (mg/dL) 15.1 14.3 13.4 13.3 17.5 15.5** 14.5** 13.4**
Creatinine (mg/dL) 0.26 0.26 0.27 0.25 0.25 0.23 0.21** 0.20**
Calcium (mg/dL) 10.50 10.18** 10.15** 10.18** 10.82 10.75 10.53* 10.41**
Potassium (mmol/L) 6.65 6.33 6.16** 6.05** 7.27 7.10 6.53** 6.16**
Chloride (mmol/L) 93.8 94.8 95.6* 97.0* 97.7 96.5 96.6 95.6
Absolute liver 15.44 17.00 18.72** 18.64* 8.82 9.31 9.37 10.24**
weight (g) (+10%) (+21%) (+21%) (+6%) (+6%) (+16%)
Relative liver 4.70 5.18** 5.75** 5.91** 5.04 5.22 5.34** 5.76**
weight (%) (+10%) (+22%) (+26%) (+4%) (+6%) (+14%)
Absolute kidney 2.49 2.63 2.79** 2.74 1.64 1.70 1.76* 1.84**
weight (g) (+12%) (+7%) (+12%)
Relative kidney 0.76 0.81** 0.86** 0.88** 0.94 0.95 1.01** 1.04**
weight (%) (+7%) (+13%) (+16%) (+7%) (+11%)
ALP: alkaline phosphatase; AST: aspartate aminotransferase; BUN: blood urea nitrogen; IU: International Unit; T4:
thyroxine; TSH: thyroid stimulating hormone; *: P < 0.01; **: P < 0.001
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Source: Barnett Jr (2012d)
Significant increases (P < 0.01 or 0.05) in absolute and/or relative liver and kidney weights
occurred at and above 250 mg/kg bw per day in males and 500 mg/kg bw per day in females (Table
26). None of these increases were accompanied by any microscopic changes, and the majority were of
a magnitude that would not be considered toxicologically significant. In males, there was significant
decrease (P < 0.01) in the absolute weight of the levator ani-bulbocavernosus complex at 500 and
750/1000 mg/kg bw per day (−14% and −17%, respectively). These decreases were not considered
treatment related as there was no dose–response relationship, there were no corroborating effects on
other androgen-responsive organs, and the mean values were within the historical control range. There
were no treatment-related histopathological findings in males. In females, there was a significant
(P < 0.05) increase in the number of primary follicles in both left and right ovaries combined at
750/1000 mg/kg bw per day (28 primary follicles versus 20 in the control); however, this difference
was not considered treatment-related as no increase occurred in the left and right ovary separately.
Cholinesterase study: Deaths and clinical signs occurred at the highest dose (six males and
four females were found dead on postnatal days 24–49 and 23–25, respectively). The clinical signs
were consistent with those that occurred during the main study. There were no effects on body weight,
feed consumption or the occurrence of macroscopic abnormalities. Toxicologically and statistically
significant (P < 0.01) inhibition of erythrocyte acetylcholinesterase occurred at 250 and 500 mg/kg
bw per day in males (−79.2% and −87.7%, respectively; apart from the dead rats, no data were
available at the highest dose) and at every dose in females (−74.1%, −89.1% and −97.3% at 250, 500
and 750/1000 mg/kg bw per day, respectively). Brain acetylcholinesterase activity was significant
lower (P < 0.01) than the control in males at 250 and 500 (−17.4% and −34.6%, respectively; apart
from the dead rats, no data were available at the highest dose) and in females at every dose (−8.9%,
−23.6% and −42.1% at 250, 500 and 750/1000 mg/kg bw per day, respectively).
Based on these findings, malathion did not cause any antiandrogenic, estrogenic or
antiestrogenic effects in rats or any effect on pubertal development or thyroid function up to 750
mg/kg bw per day.
In a published in vitro study by Kjeldsen, Ghisari & Bonefeld-Jørgensen (2013), malathion
(purity > 93%) and a number of other pesticides were screened for their potential to interact with the
human estrogen or androgen receptors or to interfere with aromatase activity. The estrogen receptor
transactivation assay was conducted in human breast carcinoma MVLN cells, the androgen receptor
transactivation assay was conducted in hamster ovary CHO-K1 cells and the aromatase activity assay
was conducted in human choriocarcinoma JEG-3 cells. Malathion weakly induced estrogen receptor
transactivation at a concentration of 1  10−5 mol/L (+113% relative to the solvent control). In the
presence of 25 pmol/L estradiol, the same concentration of malathion had no effect on estrogen
receptor transactivation. In contrast, the positive control (estradiol) induced estrogen receptor
transactivation at a concentration of 6.3  10−12 mol/L (+248% relative to the solvent control). The
conclusion reached was that malathion had no effect on androgen receptor transactivation or on
aromatase activity.

Rats
In a pilot study by Lochry (1988), groups of eight pregnant Crl:CD[SD]BR rats were
administered malathion (unspecified purity) in corn oil by gavage at doses of 0, 300, 600, 800 or 1000
mg/kg bw per day from gestation days 6–15. The dams were observed daily for clinical signs, with
body weight recorded throughout this period; feed consumption was not reported, and there was no
statistical analysis of body weight data. On gestation day 20, the surviving dams were terminated and
necropsied; the following parameters were recorded: corpora lutea counts, total resorptions, number
of implantations, live fetuses, dead fetuses and pup sex ratio. Fetuses were examined for external,
visceral and skeletal abnormalities. At the highest dose, three dams died on gestation days 11 or 12,
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with clinical signs (salivation, urine-stained abdomen and chromorrhinorrhea) and body-weight loss
(6–26 g in two dams) prior to death. In survivors, clinical signs were observed at and above
600 mg/kg bw per day: salivation (one, four and eight dams at 600, 800 and 1000 mg/kg bw per day,
respectively) and urine-stained abdominal fur (four dams at 1000 mg/kg bw per day). Mean body
weight was generally lower than the control at and above 600 mg/kg bw per day (up to 5% lower than
the control at the highest dose), with body-weight gain reduced over the first few days of dosing at
800 and 1000 mg/kg bw per day (body-weight gain was −6 g at the highest dose versus +9.1 g in the
control) from days 6–9. There were no treatment-related macroscopic findings or effects on litter
parameters or incidence of external, visceral or skeletal abnormalities in fetuses.
In the main developmental toxicity study by Lochry (1989), groups of 25 pregnant
Crl:CD[SD]BR rats were administered malathion (purity 94%) in corn oil by gavage at doses of 0,
200, 400 or 800 mg/kg bw per day from gestation days 6–15. Dams were observed daily for clinical
signs, with body weight and feed consumption recorded throughout this period and from gestation
days 16–20. On gestation day 20, the surviving dams were terminated and necropsied, and the
following parameters recorded: gravid uterine weight, corpora lutea counts, total resorptions, number
of implantations, live fetuses, dead fetuses and pup sex ratio. Fetuses were examined for external,
visceral and skeletal abnormalities. There were no treatment-related deaths. Treatment-related clinical
signs were confined to the highest dose and included urine staining of the abdomen in five dams and
chromodacryorrhea and chromorrhinorrhea in one dam. Body-weight gain was significantly lower
(P < 0.01 or 0.05) than the control at the highest dose over days 6–9 (+11 versus +14.8 g) and 6–12
(+27.3 versus +33.8 g), with a compensatory increase observed from days 16–20 (+73.4 versus
+62.0 g). Feed consumption during the dosing period was also significantly lower (P < 0.01) than the
control (66.8 versus 70.7 g). There were no treatment-related macroscopic findings or effects on litter
parameters or on the incidence of external, visceral or skeletal abnormalities in fetuses. The NOAEL
for maternal toxicity was 400 mg/kg bw per day for clinical signs and reduced body-weight gain and
feed consumption at 800 mg/kg bw per day. The NOAEL for embryo and fetal toxicity was 800
mg/kg bw per day, the highest tested dose.
Rabbits
In a range-finding study, Siglin, Voss & Becci (1985) administered malathion (purity 92.4%)
in corn oil by gavage to groups of five pregnant New Zealand White rabbits at doses of 0, 25, 50, 100,
200 or 400 mg/kg bw per day from gestation days 6–18. The dams were observed daily for clinical
signs, and body weight was recorded on gestation days 0, 6, 12, 15, 18, 24 and 29. Feed consumption
was not recorded. On gestation day 29, the surviving dams were terminated and necropsied, and the
following parameters recorded: gravid uterine weight; ovary weight; corpora lutea counts; number of
implantations, resorptions, live fetuses and dead fetuses; and pup sex ratio. The fetuses were
examined for external, visceral and skeletal abnormalities. Maternal deaths occurred at 200 and 400
mg/kg bw per day (four and two dams, respectively) between gestation days 7 and 17, with clinical
signs consisting of decreased activity, tremors and salivation also at these doses in the majority of the
rabbits. There was no effect on body weight or body-weight gain, on litter parameters or on the
incidence of external, visceral or skeletal abnormalities in fetuses and there were no treatment-related
In the main study by the same authors (Siglin, Voss & Becci, 1985), groups of 20 pregnant
New Zealand White rabbits were administered malathion (purity 92.4%) in corn oil by gavage at
doses of 0, 25, 50 or 100 mg/kg bw per day from gestation days 6–18. The dams were observed daily
for clinical signs. Body weight was recorded on gestation days 0, 6, 12, 15, 18, 24 and 29. Feed
consumption was not recorded. On gestation day 29, the surviving dams were terminated and
necropsied, and the following parameters recorded: gravid uterine weight; ovary weight; corpora lutea
counts; number of implantations, resorptions, live fetuses and dead fetuses; and pup sex ratio. The
fetuses were examined for external, visceral and skeletal abnormalities.
There were no treatment-related deaths or clinical signs. There was no significant difference
in mean body weight between treated and control dams. At 50 and 100 mg/kg bw per day, mean
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body-weight gain was significantly lower (P < 0.05) than the control over gestation days 6–18 (−0.03
g versus 0.19 g in the control). One control dam, one mid-dose dam and two high-dose dams aborted
their litters on gestation days 21–26. At 50 and 100 mg/kg bw per day, the number of resorption sites
and the percentage of resorptions was increased but not significantly or dose-relatedly (Table 27).
Further, an expert review of the resorption data (Robinson, 2002) concluded that these slightly higher
resorption values were within the historical control range (0–43%) and the apparent increase was not
corroborated by the live litter size or the number of late resorptions, which were unaffected by
treatment. This would not be expected if the compound induced an increased resorption rate. Based on
these considerations, the expert review concluded that there was no evidence of a toxicologically
significant embryo-lethal effect (Robinson, 2002). There were no treatment-related macroscopic
findings in the dams or external, visceral or skeletal abnormalities in fetuses. The NOAEL for
maternal toxicity was 25 mg/kg bw per day for reduced body-weight gain at 50 mg/kg bw per day.
The NOAEL for embryo and fetal toxicity was 100 mg/kg bw per day, the highest tested dose. These
findings indicate that malathion is not teratogenic.
Table 27. Effect of malathion on litter resorptions in rabbits

No. or % measure per dose
100 mg/kg bw per
Parameter 0 mg/kg bw per day 25 mg/kg bw per day 50 mg/kg bw per day day
Resorption sites 0.9  1.2 0.7  1.1 2.3  2.8 2.0  2.7
Per cent resorptions 15.6  26.9 12.3  20.7 29.2  34.2 28.4  34.9
(%)
Postimplantation
loss (%) 24.7 + 31.2 14.9 + 21.4 29.2 + 34.2 30.0 + 34.4
Total live fetuses 5.6  3.0 7.8  3.2 5.8  3.6 6.0  3.3
(no.)
Results expressed as the mean ± 1 standard deviation.
Source: Siglin, Voss & Becci (1985)
2.6 Special studies

(a) Neurotoxicity
Fletcher (1989) examined the potential of malathion to cause delayed peripheral neuropathy
in White Leghorn hens. In the acute toxicity phase of the study, the LD50 was estimated to be
775 mg/kg bw (range: 610–984 mg/kg bw). In the atropine sulfate sufficiency phase of the study,
efficacious doses of atropine were determined to be 1.3 times the LD50 on day 1 and 1.1 times the
LD50 on day 21. In the main phase of the study, a group of 60 hens received a single intramuscular
injection of atropine sulfate (10 mg/kg bw), followed one hour later by a single gavage dose of
malathion (purity 93.6%) in water at 1.3 times the LD50. Atropine (30 mg/kg bw) was administered at
0.5, 1, 3 and 50 hours after dosing. Twenty-one days after dosing, a second dose of malathion was
given (1.1 times the LD50) following this same procedure. Vehicle (water) and positive control
(500 mg/kg bw tri-o-tolyl phosphate) groups comprised 15 hens.
Thirty-nine hens died within 15 days of the first dose of malathion, with seven of the
remaining hens dying within 7 days of the second dose. One control hen died on day 33 and all
positive control hens were terminated on day 16 in a moribund condition. Clinical signs were
observed in malathion-treated hens within approximately one hour of dosing and included general
weakness, ataxia, inability to stand, sitting on haunches, diarrhoea, paralysis of legs and wings, and
pale comb. Similar signs occurred after the second dose of malathion and resolved 5 or 6 days after
dosing. No clinical signs were observed in negative controls. The positive controls showed signs of
moderate to severe ataxia, inability to stand, paralysis of legs and wings, general weaknesses and
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sitting on haunches by day 10. Body-weight gain over days 1–21 was significantly lower (P < 0.05)
than the control in malathion-treated hens (−234 versus −72 g, respectively). Reduced feed
consumption occurred for approximately week after the first dose of malathion (5–65 g/day versus
73–112 g/day in the vehicle control). Feed consumption was also lower in the positive control group
(41–86 g/day). Necropsy and histopathology revealed no treatment-related effects on nervous tissue.
In the positive control group, neural lesions were observed histopathologically (axonal degeneration
and demyelination, hyperplasia of Schwann cells, increased glial cell proliferation). There was no
evidence that malathion caused delayed peripheral neuropathy.
In an acute neurotoxicity study by Lamb (1994a), groups of 27 Crl:CD®BR rats of each sex
received a single gavage dose of malathion (purity 96.2%) in corn oil at 0, 500, 1000 or 2000 mg/kg
bw. These doses were based on the range-finding study by Nemec (2000), where the time to peak
effect was estimated to be 15 minutes. In each group, seven rats per sex were allocated to the
neuropathology evaluation and 20 to the analysis of cholinesterase. Observations were made daily for
deaths and clinical signs. Body weights were recorded on day 1 and weekly thereafter. Feed
consumption was not recorded. A functional observational battery and motor activity assessment were
performed at 15 minutes and 7 and 14 days after dosing in 12 rats per sex per group. Plasma,
erythrocyte and brain cholinesterase activities were analysed in samples collected from five rats per
sex per dose pretreatment and at 15 minutes, 7 days and 15 days (termination) after dosing. The
remaining rats were terminated on day 15, necropsied and the nervous system examined
One high-dose male was terminated in a moribund condition seven days after dosing.
Salivation occurred within 15 minutes of dosing in every treated group (two males in each group; one,
one and four females at 500, 1000 and 2000 mg/kg bw, respectively). Additional clinical signs at the
highest dose included yellow material around the uro/anogenital region (six males and nine females),
red material around the nose and mouth (two males and seven females) and decreased defecation
(three males) or small faeces (13 females). There was no treatment-related effect on body weight.
Salivation was observed at 15 minutes after dosing (males: 0, 0, 1 and 4 of 12 rats at 0, 500, 1000 and
2000 mg/kg bw per day, respectively; females: 0, 0, 1 and 2 of 12 rats at 0, 500, 1000 and 2000 mg/kg
bw per day). No other treatment-related observations were made during the functional observational
battery. In high-dose males, a significantly lower ambulatory activity (P < 0.05) was observed 15
minutes after dosing (321 versus 422 counts in the control during subsession 1; total counts were 653
versus 940, respectively). There was no treatment-related effect on plasma cholinesterase or brain
acetylcholinesterase activity in either sex. At the highest dose, erythrocyte acetylcholinesterase
activity was significantly lower than the control only in females 7 days after dosing (−39%, P < 0.05);
erythrocyte acetylcholinesterase was 40% lower than the control in males but the difference was not
statistically significant. There was no treatment-related effect on brain weights or macroscopic or
microscopic findings in nervous tissue.
The NOAEL was 1000 mg/kg bw for reduced erythrocyte acetylcholinesterase activity in
females and reduced ambulatory activity in males at 2000 mg/kg bw.
In a 13-week neurotoxicity study by Lamb (1994b), groups of 25 Crl:CD BR rats of each sex
were fed diets containing 0, 50, 5000 or 20 000 ppm malathion (purity 96.4%) ad libitum. The
achieved doses were 0, 4, 352 and 1486 mg/kg bw per day in males and 0, 4, 395 and 1575 mg/kg bw
per day in females, respectively. Observations were made daily for deaths and clinical signs, with
body weight and feed consumption recorded weekly. A functional observational battery and motor
activity assessment (10 rats/sex per dose) were performed pretreatment and during weeks 4, 8 and 13.
Plasma, erythrocyte and brain cholinesterase activities were analysed in samples collected from five
rats per sex per dose and during weeks 3, 7 and 13. Following scheduled termination on day 91, all
the rats were necropsied and brain weights recorded. Nervous tissue was examined
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There were no deaths. The only treatment-related clinical sign was the presence of yellow or
orange material around the ano/uro-genital region and on the tail of high-dose rats (nine males and
eight females). At 20 000 ppm, mean body weight was 9–20% (P < 0.01 or 0.05) and 9–13%
(P < 0.01) lower than the control in males and females, respectively, throughout the exposure period.
Also at 20 000 ppm, body-weight gain was significantly lower (P < 0.01) than the control in males
during the first week of exposure (−79%) and in females during weeks 0–1 (−57%), 4–5 (−41%), 10–
11 (−71%). At the highest dose, cumulative body-weight gain to week 12 was significantly lower
(P < 0.05) than the control in males (−15%), while cumulative body-weight gain to week 13 was
significantly lower (P < 0.01) than the control in females (−24%). Mean feed consumption was also
reduced at the highest dose in males during weeks 0–1 (−19%, P < 0.01), 6–7 (−10%, P < 0.05) and
in females throughout most of the exposure period (−9 to −20%, P < 0.01 or 0.05). There were no
treatment-related functional observational battery findings or effects on locomotor activity.
The effect of malathion on cholinesterase activity is summarized in Table 28. Significant
inhibition (P < 0.01 or 0.05) of plasma cholinesterase activity occurred at 5000 and 20 000 ppm in
males during week 3 but only at 20 000 ppm in females or at other sampling points. Toxicologically
and statistically significant inhibition of erythrocyte cholinesterase activity occurred at 5000 and 20
000 ppm in both sexes, and at every sampling point. Acetylcholinesterase activity in different brain
regions was significantly lower than in the control at 20 000 ppm more consistently in females than
males. There was no treatment-related effect on brain weights or macroscopic or microscopic findings
in nervous tissue.
The NOAEL was 5000 ppm (equal to 352 mg/kg bw per day in males and 395 in females)
based on the inhibition of brain acetylcholinesterase activity, clinical signs and reduced body weight
at 20 000 ppm (equal to 1486 mg/kg bw per day in males and 1575 mg/kg bw per day in females).
Table 28. Effect of malathion on cholinesterase in rats over 13 weeks of dietary exposure
Cholinesterase activity per dietary concentration
Parameter 0 ppm 50 ppm 5 000 ppm 20 000 ppm
Plasma ChE (IU/mL)
Males
Week 3 0.36 0.320 0.292* (−18%) 0.170* (−53%)
Week 7 0.309 0.309 0.248 0.142** (−54%)
Week 13 0.303 0.277 0.266 0.150** (−51%)
Females
Week 3 0.933 1.176 0.92 0.42** (−55%)
Week 7 1.777 1.325 1.250 (−30%) 0.430** (−76%)
Week 13 1.517 2.257 1.291 0.453* (−70%)
Erythrocyte AChE (IU/mL)
Males
Week 3 0.88 0.90 0.43** (−51%) 0.41** (−53%)
Week 7 0.80 0.79 0.31** (−61%) 0.26** (−68%)
Week 13 1.06 1.04 0.54** (−49%) 0.39** (−63%)
Females
Week 3 0.99 1.04 0.49** (−51%) 0.36** (−64%)
Week 7 0.90 0.87 0.42** (−53%) 0.39** (−57%)
Week 13 1.22 1.23 0.62** (−49%) 0.39** (−68%)
Brain hippocampus (IU/g)
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Males
Week 3 3.33 3.19 2.96 2.70
Week 7 5.88 5.71 5.99 4.8
Week 13 5.61 4.99 5.50 6.15
Females
Week 3 3.26 3.16 3.33 2.36** (−44%)
Week 7 6.34 6.32 5.54 3.94** (−38%)
Week 13 5.02 5.70 4.56 2.68** (−47%)
Brain olfactory (IU/g)
Males
Week 3 5.46 6.67 6.39 4.77
Week 7 12.98 13.18 12.32 11.51
Week 13 12.67 13.62 13.26 8.30* (−34%)
Females
Week 3 7.23 6.56 6.62 5.00* (−31%)
Week 7 12.45 15.50 12.60 9.09* (−27%)
Week 13 16.70 12.46 11.78 8.27** (−50%)
Mid brain (IU/g)
Males
Week 3 5.24 5.19 5.07 4.34
Week 7 7.27 7.27 6.37 6.08
Week 13 9.69 9.51 9.02 7.4** (−24%)
Females
Week 3 4.68 5.20 4.76 3.84
Week 7 7.67 6.03 6.66 4.99** (−35%)
Week 13 8.84 9.86 8.86 5.30** (−40%)
Brainstem (IU/g)
Males
Week 3 4.40 4.40 4.89 3.67
Week 7 7.28 7.79 6.72 6.47
Week 13 8.56 8.09 8.03 7.06** (−18%)
Females
Week 3 4.35 4.78 4.15 3.38
Week 7 7.13 8.01 6.92 5.95
Week 13 8.71 9.10 7.34 5.61** (−36%)
Brain cerebellum (IU/g)
Males
Week 3 2.62 2.33 2.28 2.50
Week 7 3.04 3.16 2.91 2.96
Week 13 3.80 3.48 3.39 3.32
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Females
Week 3 2.50 2.65 2.49 2.0* (−20%)
Week 7 3.09 2.85 2.99 2.60
Week 13 3.67 3.86 3.68 2.50** (−32%)
Brain cortex (IU/g)
Males
Week 3 5.15 5.18 5.21 4.57
Week 7 8.32 8.78 7.65 6.12** (−26%)
Week 13 10.02 10.08 10.74 7.72
Females
Week 3 5.85 5.47 4.81** (−18%) 4.00** (−32%)
Week 7 9.13 7.75 7.28 5.46**
Week 13 10.47 10.29 9.20 4.92** (−53%)
AChE: acetylcholinesterase; ChE: cholinesterase; IU: International Unit; *: P < 0.05; **: P < 0.01
Source: Lamb (1994b)
In a range-finding study by Fulcher (2002a) designed to establish appropriate doses for both a
developmental neurotoxicity study and a cholinesterase study, groups of 15 pregnant Crl:CD BR rats
were administered malathion (purity 96%) in corn oil by gavage from gestation day 6–20 or day 10 of
lactation. Pups (3/sex per group from 10 litters) were dosed from postnatal days 11–21. In the initial
phase of the study, doses of 0, 7.5, 750 or 1000/1250 mg/kg bw per day were administered to the
dams and doses of 0, 7.5, 200 or 450 mg/kg bw per day were administered to the pups. Due to overt
toxicity, these doses were reduced to 0, 7.5, 35, 75 and 150 mg/kg bw per day in Phase 2 of the study.
Observations for mortality and clinical signs, body weight and feed consumption were recorded
regularly throughout the study. Standard litter and offspring parameters were recorded. Plasma,
erythrocyte and brain cholinesterase activities were analysed in samples collected on gestation day 20
in dams and postnatal day 21 in pups. A limited range of clinical chemistry parameters were analysed
in both dams and pups (sodium, potassium, bicarbonate, chloride and calcium). Following scheduled
termination (gestation day 20 for dams and postnatal day 21 for pups), the rats were necropsied and
any macroscopic abnormalities processed for histopathological examination.
In Phase 2 of the study, there were no deaths. The dams salivated at and above 35 mg/kg bw
per day, while no clinical signs occurred in pups. There was no treatment-related effect on body
weight or feed consumption. Litter, offspring and clinical chemistry parameters were unremarkable.
There were no treatment-related macroscopic findings. Erythrocyte acetylcholinesterase activity was
significantly lower than the control in the dams at 75 (−33%, P < 0.05) and 150 mg/kg bw per day
(−59%, P < 0.01), while plasma cholinesterase and brain acetylcholinesterase were unaffected by
treatment. In dams terminated on gestation day 20, plasma cholinesterase was significantly lower than
the control (P < 0.01 or 0.05) at and above 35 mg/kg bw per day (−9% to −11%), whereas there was
no effect on erythrocyte or brain acetylcholinesterase activities. In pups terminated on postnatal day
21, erythrocyte acetylcholinesterase activity was significantly lower than the control at every dose
(−19%, −30%, −42% and −58% in males and −12%, −25%, −41% and −65% in females at 7.5, 35, 75
and 150 mg/kg bw per day, respectively), whereas brain acetylcholinesterase activity was
significantly lower than the control at 75 (−9% in males and −7% in females, P < 0.01) and 150
mg/kg bw per day (−18% in males and −19% in females, P < 0.01). There were no significant
intergroup differences in plasma cholinesterase activity in male pups while in the female pups plasma
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cholinesterase was 16 (P < 0.01) and 26% (P < 0.01) lower than the control at 75 and 150 mg/kg bw
per day, respectively.
In a developmental neurotoxicity study that was not guideline compliant, groups of at least 21
pregnant Crl:CD BR rats were administered malathion (purity 96%) in corn oil by gavage at doses of
0, 5, 50 or 150 mg/kg bw per day from gestation day 6 to day 10 of lactation. Offspring (seven per
litter) were also given comparable doses of malathion by gavage from postnatal day 11–21. The doses
were based on the results of the range-finding study by Fulcher (2002a) and the cholinesterase study
by Fulcher (2001). Observations for mortalities and clinical signs were made daily, with body weight
and feed consumption recorded regularly. The behaviour of 10 dams per group was assessed on
gestation days 12 and 18 and postnatal days 4 and 10. The following reproduction parameters were
determined: gestation index; gestation length; postimplantation and survival index; live birth index;
viability index; and lactation index. The following offspring parameters were determined: number of
live and dead offspring; individual body weights; sex; clinical signs and the time of vaginal opening
and balanopreputial separation. On postnatal day 4, the litters were culled to eight pups and each litter
assigned to an assessment of behaviour (postnatal days 4, 11, 21, 35, 45 and 60 in 10 pups/sex per
group), motor activity (postnatal days 13, 17 and 22 in one pup/sex per litter), auditory startle
response habituation and auditory startle pre-pulse inhibition (postnatal days 23 and 60 in one pup/sex
per litter), and learning and memory (postnatal days 23 or 24, and 61 or 62 in one pup/sex per litter).
The dams were terminated after weaning on postnatal day 20 or 21 and necropsied; the weights of the
brain, pituitary and sex organs were recorded. The offspring were terminated on postnatal day 11 (10
offspring/sex per group), 21 (10 offspring/sex per group) or 65 (all remaining offspring), necropsied,
brain weight and brain morphometry recorded and nervous tissue histopathologically examined.
There were no treatment-related deaths. In the dams, there was an increase in post-dosing
salivation at the highest dose (5, 4, 3 and 20 dams at 0, 5, 50 and 150 mg/kg bw per day) but there was
no effect on body weight, feed consumption or reproduction parameters or on treatment-related
behavioural abnormalities, macroscopic findings or effect on brain weight. There was no treatment-
related effect on litter parameters and no effect on survival or body weight in offspring. At 150 mg/kg
bw per day, four offspring from the same litter exhibited clinical signs after 7–9 days of exposure to
malathion (postnatal day 17–19), including whole-body tremors, underactivity, prostrate posture,
partially closed eyelids and abnormal gait. Abnormalities observed in offspring at 150 mg/kg bw per
day during the functional observational battery included failure to show an immediate surface righting
reflex (5 females versus 1 female in the control on postnatal day 11). In offspring there was no effect
on brain weight or the incidence of neuropathological findings.
The NOAEL for both maternal toxicity and offspring toxicity was 50 mg/kg bw per day for
clinical signs at 150 mg/kg bw per day (Fulcher, 2002b; Reiss, 2004).
Myers (2003, 2004) examined the potential of malathion to affect the thickness of the corpus
callosum in rat pups. Dams were administered malathion (unspecific purity and vehicle) by gavage
from gestation days 6–10 followed by direct dosing of offspring (20/sex per dose) from postnatal days
11–21 at 0, 5, 50 or 150 mg/kg bw per day. Ten offspring per sex per dose were terminated on
postnatal day 21 while the remainder were observed undosed until postnatal day 65. Histopathologic
examination of standard sections of brain tissue showed no treatment-related effect on the thickness of
the corpus callosum.
(b) Cholinesterase inhibition

Rodriguez et al. (1997) compared the potential of enantiomers of malaoxon to inhibit
different cholinesterases, including erythrocyte and human plasma cholinesterase. Rat and electric eel
erythrocyte acetylcholinesterase were the most sensitive to malaoxon, with bovine and human
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erythrocyte acetylcholinesterase less sensitive; inhibitory potency varied approximately 100-fold

(Table 29). In all experiments, R-malaoxon was more potent than S-malaoxon.
Table 29. Interspecies comparison of cholinesterase inhibition by enantiomers of malaoxon

Species/cholinesterase R-malaoxon S-malaoxon R : S ratio Racemic malaoxon
Rat erythrocyte AChE 1.52  10 5
3.00  10 4
5 1.01  105
Sheep red blood cell AChE 1.96  105 8.70  103 22.5 1.54  105
Electric eel erythrocyte AChE 9.39  105 5.79  104 16.2 4.74  105
Human plasma ChE 1.46  104 4.24  103 3.4 7.78  103
Human erythrocyte AChE 1.27  105 1.09  104 11.6 6.5  104
AChE: acetylcholinesterase; ChE: cholinesterase
Results are expressed as the mean bimolecular reaction constants (ki; mol/L per minute).
Source: Rodriguez et al. (1997)
In non-guideline studies (Fulcher, 2001, 2003) designed to examine the effect of malathion on
cholinesterase activity, groups of nine pregnant Crl:CD BR rats were administered malathion (purity
96%) in corn oil by gavage at 0, 5, 50 or 150 mg/kg bw per day from gestation days 6–20. Three
hours after dosing on gestation day 20, eight dams were terminated and plasma, erythrocyte and brain
cholinesterase activities of the dams and fetuses analysed. Additional groups of 10 pregnant rats were
administered malathion by gavage at the same doses from gestation day 6 to postnatal day 10.
Offspring in eight litters per group were then administered comparable doses of malathion from
postnatal days 11– 21. Selected offspring were terminated on postnatal days 4, 21 and 60, and plasma,
erythrocyte and brain cholinesterase activities analysed. In an additional part of the study, pups from
eight control dams received a single gavage dose of malathion on postnatal day 11 at 0, 5, 50, 150 or
450 mg/kg bw and were terminated 2 hours later to analyse plasma, erythrocyte and brain
cholinesterase activity. Additional groups of eight male and eight female young adult rats were
administered a single gavage dose of malathion at 0, 5, 50, 150 or 450 mg/kg bw and terminated 2
hours later to analyse plasma, erythrocyte and brain cholinesterase activities. Alternatively, groups of
eight young adult rats received 11 consecutive daily doses of malathion at 0, 5, 50 or 150 mg/kg bw
per day and were terminated 2 hours after the final dose to analyse plasma, erythrocyte and brain
cholinesterase activities. Throughout all phases of the study, mortality, clinical signs, body weight,
litter and offspring parameters were recorded. Following termination, the rats were necropsied and
brain weights recorded.
There were no treatment-related deaths or clinical signs in the dams. Tremors were observed
in the offspring (5 of 16) that received a single dose of 450 mg/kg bw per day, with one rat terminated
in a moribund condition 1 hour after dosing. Body weight, litter and fetal parameters were unaffected
by treatment. There were no treatment-related macroscopic findings or effect on brain weight.
In the dams exposed to malathion from gestation days 6–20 and terminated on gestation day
20, erythrocyte acetylcholinesterase activity was significantly lower (P < 0.01) than the control at 50
and 150 mg/kg bw per day (−19% and −51%, respectively); there was no effect on plasma or brain
cholinesterase activities. In fetuses from these same dams, plasma and erythrocyte cholinesterase
activities were significantly lower (P < 0.01 or 0.05) than the control (plasma: −14% and −15%,
respectively; erythrocytes: −11% and −19%, respectively), while there was no change in brain
acetylcholinesterase activity. In pups from these same-treated dams terminated on postnatal day 4,
plasma, erythrocyte and brain cholinesterase activities were unaffected by treatment.
In offspring administered a single dose of malathion on postnatal day 11, plasma
cholinesterase activity was significantly lower (P < 0.01 or 0.05) than the control in both sexes at and
above 50 mg/kg bw per day (−19%, −36% and −54% in males and −16%, −35% and −52% in females
at 50, 150 and 450 mg/kg bw, respectively). Erythrocyte acetylcholinesterase was significantly lower
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than the control (P < 0.01 or 0.05) at every dose in males and at and above 50 mg/kg bw in females,
(−16%, −25%, −55% and −72% in males and −7%, −23%, −48% and −61% in females at 5, 50, 150
and 450 mg/kg bw, respectively). Brain acetylcholinesterase was significantly lower (P < 0.01) than
the control at 150 and 450 mg/kg bw in both sexes (−44% and −84% in males and −48% and −81% in
females, respectively).
In young adult rats administered a single dose of malathion, plasma and erythrocyte
cholinesterase activities were significantly lower (P < 0.001 or 0.01) than the control in males (−24%
and −25%, respectively), but there was no effect on brain acetylcholinesterase activity. In high-dose
females, statistically but not toxicologically significant inhibition of erythrocyte acetylcholinesterase
occurred (−17%, P < 0.001), but there was no effect on plasma or brain cholinesterase activities.
In offspring terminated on postnatal day 21 after 11 consecutive daily doses, plasma
cholinesterase activity was significantly lower (P < 0.01 or 0.05) than the control in both sexes at 50
and 150 mg/kg bw per day (−19% and −24% in males and −19% and −32% in females, respectively).
Erythrocyte acetylcholinesterase activity was significantly lower than the control in males at every
dose (−17%, −39% and −67% at 5, 50 and 150 mg/kg bw per day, respectively), while in females
significant inhibition occurred at 50 and 150 mg/kg bw per day (−34% and −68%, respectively). Brain
acetylcholinesterase activity was significantly lower than (P < 0.01) the control only at the highest
dose (−16% in both sexes).
In young adults administered 11 consecutive doses of malathion, plasma cholinesterase was
significantly lower (P < 0.05) than the control only in males at 50 and 150 mg/kg bw per day (−11%
and −13% respectively. Erythrocyte acetylcholinesterase was significantly lower (P < 0.01) than the
control at 50 and 150 mg/kg bw per day in both sexes (−20% and −43% in males, and −20% and
−48% in females, respectively). There was no effect on brain acetylcholinesterase in either sex.
In offspring terminated on postnatal day 60, there was no treatment-related effect on plasma,
erythrocyte or brain cholinesterase activities.
Barnett Jr (2006a) undertook a range-finding study in Crl:CD[SD] juvenile rats to determine

the effect of malathion or malaoxon on acetylcholinesterase activity. Groups of five rat pups per sex
were administered malathion (purity 96%) in corn oil by gavage from postnatal days 11–21 at doses
of 0, 5, 15 or 50 mg/kg bw per day or malaoxon (purity 97.7%) at doses of 0, 0.05, 0.1 or 1 mg/kg bw
per day. The rats were observed daily for mortality and clinical signs. Body weights were recorded
daily during the dosing period. The rats were terminated on postnatal day 21 and blood and brain
acetylcholinesterase activity analysed. There were no treatment-related deaths, clinical signs, effects
on body weight or macroscopic abnormalities. Brain acetylcholinesterase activity was not affected by
treatment. Toxicologically significant inhibition of erythrocyte acetylcholinesterase activity occurred
in females at 50 mg/kg bw per day malathion (−28.2%) and 1 mg/kg bw per day malaoxon (−30.4%
in males and −29.4% in females).
Barnett Jr (2006b) determined the time to peak cholinesterase inhibition in young

preweanling Crl:CD[SD] rats (20/sex per group) following repeated gavage dosing with 0 or 150
mg/kg bw per day malathion (purity 96%) or 4 mg/kg bw per day malaoxon (purity not specified) in
corn oil from postnatal days 11–21. Five malathion-treated rats per sex were terminated at 1, 2, 3 and
4 hours after dosing while five malaoxon-treated rats per sex, were terminated at 30, 60, 90 and 120
minutes after dosing to analyse erythrocyte and brain acetylcholinesterase activities. There were no
deaths. Clinical signs including whole-body tremors, reduced motor activity, prostrate positioning,
soft or liquid faeces, impaired righting reflex and dehydration, were observed only at 150 mg/kg bw
per day malathion in both sexes from postnatal days 13–18. In malathion-treated rats, toxicologically
significant inhibition of erythrocyte acetylcholinesterase activity occurred at all time points in both
sexes (−20.7% to −50.7% in males and −38.6% to −62.2% in females), with the peak effect 2 hours
after dosing. Brain acetylcholinesterase activity was inhibited at every time point (−11.2% to −14.0%
in males and −9.5% to −21.4% in females), with the peak effect 2 hours after dosing. In malaoxon-
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treated rats, inhibition of erythrocyte activity occurred at most time points (−41.0% to −62.2% in
males and −50.3% to −59.4% in females), with the peak effect 90 minutes after dosing in males and 2
hours after dosing in females. Inhibition of brain acetylcholinesterase activity occurred at most time
points in males (−4% to −15.7%) with the time of peak effect 30 minutes after dosing. In females,
brain acetylcholinesterase activity was inhibited at 30 and 60 minutes after dosing (−8.3% and −2.2%,
respectively).
In the second part of this study, the inhibition of cholinesterase activity was determined
following a single gavage dose of 0, 50, 150 or 450 mg/kg bw per day malathion on postnatal day 21
(6–10 rats per sex per group). Rats were terminated 2 hours after dosing to analyse erythrocyte and
brain acetylcholinesterase activities. There were no treatment-related deaths. Slight whole-body
tremors, miosis and urine-stained abdominal fur were observed in one high-dose female. Erythrocyte
acetylcholinesterase activity was inhibited in a dose-related manner in males (−19.6%, −27.1% and
−32.5%, respectively), while in females the level of inhibition at 150 mg/kg bw was higher than that
at 450 mg/kg bw (−13.4%, −35.5% and −22.8%, respectively). Only slight inhibition of brain
acetylcholinesterase occurred in high-dose males (−14.9%) and females (−5.7%).
Barnett Jr (2006c) undertook a further study to determine the effect of malathion or malaoxon
on acetylcholinesterase activity in Crl:CD[SD] juvenile rats. Groups of five pups per sex were
administered malathion (purity 96%) in corn oil by gavage from postnatal days 11–21 at doses of 0, 5,
25, 50 or 150 mg/kg bw per day or malaoxon (purity 97.7%) at doses of 0, 0.1, 1, 2.5 or 4 mg/kg bw
per day. The pups were observed daily for mortality and clinical signs. Body weights were recorded
daily during the dosing period. The rats were terminated on postnatal day 21, and blood and brain
acetylcholinesterase activity analysed. There were no treatment-related deaths. At 150 mg/kg bw per
day malathion, clinical signs were observed in both sexes: these included tremors, decreased motor
activity, impaired righting reflex, splayed forelimbs and pale extremities. No clinical signs occurred in
pups exposed to malaoxon. There was no effect on body weight. Erythrocyte acetylcholinesterase
activity was significantly lower (P < 0.01 or 0.05) than the control at and above 25 mg/kg bw per day
malathion in both sexes; however, toxicologically significant inhibition occurred only at 50 and 150
mg/kg bw per day (−15.1%, −34.1% and −54.1 % in males and −17.6%, −30.1% and −51.7% in
females at 25, 50 and 150 mg/kg bw per day, respectively). Erythrocyte acetylcholinesterase activity
was significantly lower (P < 0.01 or 0.05) than the control at and above 1 mg/kg bw per day
malaoxon in both sexes; however, toxicologically significant inhibition occurred only at 2.5 and 4
mg/kg bw per day (−14.1%, −45.8% and −51.1 % in males and −13.5%, −34.7% and −45.3% in
females at 1, 2.5 and 4 mg/kg bw per day, respectively). Brain acetylcholinesterase activity was
significantly lower than the control only at 150 mg/kg bw per day malathion (−14.5% and −16.8% in
males and females, respectively) and was unaffected by malaoxon. The NOAEL for erythrocyte
cholinesterase inhibition was 25 mg/kg bw per day for malathion and 1 mg/kg bw per day for
malaoxon.
Pratt (2006) compared the acute oral toxicity and inhibition of acetylcholinesterase activity of
malathion and desmethyl malathion in female CD rats. In a pilot experiment, groups of five female
rats were administered a single gavage dose of malathion (purity 96.8%) in DMSO at doses of 1500
or 2000 mg/kg bw. Blood was sampled prior to dosing and at 2 and 24 hours after dosing to analyse
erythrocyte acetylcholinesterase activity. At 2000 mg/kg bw malathion, all the rats exhibited
salivation immediately after dosing and piloerection approximately 4 hours after dosing; two rats
died. Acetylycholinesterase activity was inhibited by 10–71%. At 1500 mg/kg bw malathion, there
were no deaths. Salivation was observed immediately after dosing in all rats, with piloerection from
30 minutes after dosing. Acetylcholinesterase activity was inhibited by 42–59% at 2 hours after
dosing and approximately 50% at 24 hours after dosing.
In the main study, groups of 10 females rats were administered a single dose of malathion
(purity 96.8%) or desmethyl malathion (purity 90–92.7%) by gavage at 1500 mg/kg bw in DMSO. A
control group of 10 rats were dosed with the vehicle alone. Deaths and clinical signs were recorded
387
daily until termination on day 14. Body weight was recorded prior to dosing and at termination. Blood
was sampled prior to dosing and at 2 and 24 hours after dosing to analyse erythrocyte
acetylcholinesterase activity. There were no treatment-related deaths. The rats dosed with malathion
displayed salivation and piloerection from approximately 30 minutes after dosing. Salivation ceased
by one hour after dosing while piloerection ceased by day 2. No clinical signs were observed in rats
dosed with desmethyl malathion. The rats were terminated on day 14 and necropsied. There was no
effect on body weight and no treatment-related macroscopic findings. Both malathion and desmethyl
malathion caused a signification reduction in acetylcholinesterase activity, with the magnitude and
duration of the reduction higher with malathion (Table 30).
Table 30. Effect of malathion or desmethyl malathion on erythrocyte acetylcholinesterase activity

in female rats
Erythrocyte AChE activity
1 500 mg/kg bw desmethyl
Time Control 1 500 mg/kg bw malathion malathion
Pre-dose 2 330  197 2 285  192 2 320  203
2 hours 2 365  228 1 205  80 (−49%)** 1 585  78 (−33%)*
24 hours 2 248  198 1 770  99 (−21%)** 2 023  116 (−10%)*
AChE: acetylcholinesterase; bw: body weight; U: enzyme unit; SD: standard deviation; *: P < 0.01 compared with the
control; **: P < 0.001 compared with desmethyl malathion
Results expressed as the mean activity (U/L) ± 1 SD, with the % decrease (−) relative to the control in parentheses.
Source: Pratt (2006)
Using data from 2-year rat chronic toxicity and carcinogenicity studies on malathion (Daly,
1996a) and malaoxon (Daly, 1996b), Reiss (2006a) undertook BMD modelling on erythrocyte and
brain acetylcholinesterase inhibition. Data from 90-day, 180-day, 1-year and 2-year sampling times
were analysed while feed consumption data were reanalysed to obtain more accurate dose estimates.
Two BMD models were used based on USEPA methodology: a basic model consisting of an
exponential declining curve and an expanded model incorporating saturable metabolism. The data
from the four sampling points were meta-analysed jointly. The results are summarized in Table 31.
The authors concluded that the use of a BMD20 for erythrocyte acetylcholinesterase activity would be
protective for the inhibition of brain acetylcholinesterase activity at the 10% level by at least a factor
of 2 for malathion and 20 for malaoxon. The authors also proposed toxicity adjustment factors (TAFs)
for malaoxon from 37–38 in males and 65–69 in females for the inhibition of erythrocyte
Table 31. BMDs for erythrocyte and brain acetylcholinesterase activities from 2-year rat chronic
toxicity and carcinogenicity studies on malathion and malaoxon
Erythrocyte AChE
Compound Sex Brain AChE BMD10 Inhibition level (%) BMD
Malathion Male 231.1 10 42.8
15 67.5
20 95.2
Female 337.7 10 46.2
15 73.3
20 104.0
Male 52.0 10 1.1
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Erythrocyte AChE
Malaoxon
15 1.8
20 2.6
Female 74.7 10 0.71
15 1.1
20 1.5
AChE: acetylcholinesterase; BMD: benchmark dose; BMD10: estimated benchmark dose for a 10% inhibition; F: female; M:
male
Source: Reiss (2006a)
Reiss (2006b) undertook a similar analysis of erythrocyte and brain acetylcholinesterase

activity after repeated oral dosing of rat pups with malathion or malaoxon from postnatal days 11–21.
The results are summarized in Table 32. The authors concluded that the use of a BMD20 for
erythrocyte acetylcholinesterase activity would be protective for the inhibition of brain
acetylcholinesterase activity at the 10% level by at least a factor of 2.8 for malathion and 3.6 for
malaoxon. The authors also proposed TAFs of 30 in males and 26–28 in females for the inhibition of
erythrocyte acetylcholinesterase activity by malaoxon.
Table 32. BMDs for erythrocyte and brain AChE activities in rat pups
Erythrocyte AChE
a
Malathion Male 91.2 10 12.7
15 20.1
20 28.1
a
Female 85.7 10 13.6
15 21.5
20 30.3
b
Malaoxon Male >4 10 0.43
15 0.67
20 0.93
b
Female >4 10 0.53
15 0.82
20 1.1
AChE: acetylcholinesterase; BMD: benchmark dose; BMD10: estimated benchmark dose for a 10% inhibition; bw: body
weight
a
Brain AChE data were insufficient to estimate a BMD: no inhibition occurred at 50 mg/kg bw per day while inhibition
greater than 10% occurred at the next highest dose of 150 mg/kg bw per day.
b
Brain AChE data were insufficient to estimate a BMD because inhibition was less than 10% at the highest dose of 4 mg/kg
bw per day.
Source: Reiss (2006b)
Stannard (2006a) examined the time to peak effect on erythrocyte and brain
acetylcholinesterase activities of a single gavage dose of malathion in young preweanling
Crl:CD®(SD) IGS BR rats. In the first experiment, four groups of rats (6/sex) were administered a
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single gavage dose of 150 mg/kg bw malathion (purity 96%) in corn oil on postnatal day 11 and
terminated at 10, 20, 30 or 40 minutes after dosing. In a second experiment, which incorporated a
control group of five rats per sex, four groups of rats (6/sex) were administered a single gavage dose
of 150 mg/kg bw malathion on postnatal day 11 and terminated at 30, 50, 60 or 80 minutes after
dosing. In a third experiment, three groups of rats (5/sex) were administered a single gavage dose of
150 mg/kg bw malathion and terminated at 60, 90 or 120 minutes after dosing. Assessment of mean
erythrocyte and brain acetylcholinesterase activity over the three experiments indicated that the time
to peak effect was approximately 50–60 minutes after dosing. However, due to a number of
limitations the authors concluded that the study objective had not been met. In a repeat study
(Stannard, 2006b), six groups of preweanling Crl:CD®(SD) IGS BR rats (8/sex) were administered a
single gavage dose of 150 mg/kg bw malathion (purity 96%) in corn oil on postnatal day 11 and one
group terminated at 30, 60, 90, 120, 240 or 360 minutes after dosing. A concurrent control group
comprised 16 rats per sex. Blood and brain acetylcholinesterase activity were analysed. The level of
inhibition of erythrocyte acetylcholinesterase activity was 72–57% after 30–60 minutes and 36–37%
for brain acetylcholinesterase at 60 minutes. The time to peak effect on erythrocyte and brain
acetylcholinesterase activities was 60 minutes.
A range-finding study by Stannard (2006c) assessed the effects of a single dose of malaoxon
(purity 97.7%) in corn oil on acetylcholinesterase activity administered on postnatal day 11 to juvenile
Crl:CD®(SD) IGS BR rats (up to 6/sex per dose). Three experiments collectively tested doses ranging
from 0.1 to 30 mg/kg bw. Successive reductions in the dose were made across the three experiments
as overt signs of toxicity (deaths and clinical signs within 30 minutes) and large reductions in
erythrocyte and brain acetylcholinesterase occurred at 20 and 30 mg/kg bw. Toxicologically
significant inhibition of erythrocyte acetylcholinesterase activity (i.e. > 20% inhibition) occurred at
and above 0.5 mg/kg bw, while brain acetylcholinesterase activity inhibition occurred at and above 5
mg/kg bw. In the main study by Stannard (2006d), the time to peak effect on erythrocyte and brain
acetylcholinesterase activities was examined following a single gavage dose of 7 mg/kg bw malaoxon
(purity 97.7%) in corn oil to juvenile Crl:CD®(SD) IGS BR rats on postnatal day 11 (six groups of
eight rats/sex). One group of rats per sex was terminated at 20, 40, 60, 90, 120 and 240 minutes after
dosing, and erythrocyte and brain acetylcholinesterase activities analysed in all the rats, including a
control group of 16 rats per sex. There were no mortalities or treatment-related clinical signs. The
time to peak effect was determined to be 20 minutes, with the maximum level of inhibition of
erythrocyte acetylcholinesterase activity at 48% in males and 49% in females, while brain
acetylcholinesterase activity was inhibited by 46% in both sexes.
The effect of a single dose of malathion or malaoxon on acetylcholinesterase activity in
juvenile Crl:CD®(SD) IGS BR rats was examined by Stannard (2006e). Both compounds were
administered in corn oil by gavage on postnatal day 11 under the following conditions: groups of 12
rats per sex received malathion (purity 96%) at 0, 5, 15, 40 or 60 mg/kg bw; groups of 12 males
received malaoxon (purity 97.7%) at 0, 0.5, 1.0, 2.0 or 3.5 mg/kg bw; groups of 12 females received
malaoxon (purity 97.7%) at 0, 0.1, 0.25, 0.75 or 2.0 mg/kg bw. The rats that received malaoxon were
terminated after 20 minutes and those that received malathion were terminated after 60 minutes.
Blood and brain acetylcholinesterase activity was analysed. In malathion-dosed rats, there were no
deaths or clinical signs. Toxicologically significant inhibition of erythrocyte and brain
acetylcholinesterase activity occurred in both sexes at 40 and 60 mg/kg bw (Table 33). In malaoxon-
dosed rats, there were no deaths or clinical signs. Toxicologically significant inhibition of erythrocyte
and brain acetylcholinesterase activity occurred in males at 2.0 and 3.5 mg/kg bw, and in females at
0.75 and 2.0 mg/kg bw (Table 33).
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Table 33. Inhibition of acetylcholinesterase in juvenile rats following an acute oral dose of
malathion or malaoxon
AChE activity per treatment per dose
Males Females
Erythrocyte AChE Erythrocyte AChE
Treatment (U/L) Brain AChE (U/L) (U/L) Brain AChE (U/L)
Malathion
0 mg/kg bw 2 250 4 529 2 252 4 500
5 mg/kg bw 2 102 (6.6%) 4 050 (−10.6%) 2 084 (−7.5%) 3 917 (−13.0%)
15 mg/kg bw 1 979 (−12.0%) 3 854 (−14.9%) 1 994 (−11.5%) 3 779 (−16.0%)
40 mg/kg bw 1 306 (−41.9%) 3 629 (−19.9%) 1 429 (−36.5%) 3 300 (−26.7%)
60 mg/kg bw 973 (−56.8%) 3 075 (−32.1%) 783 (−65.2%) 2 725 (−39.4%)
Malaoxon
0 mg/kg bw 2 225 4 313 2 205 4 338
0.1 mg/kg bw – – 2 115 (−4.1%) 4 242 (−3.3%)
0.24 mg/kg bw – – 1 914 (−13.2%) 4 029 (−8.2%)
0.5 mg/kg bw 2 148 (−3.5%) 4 242 (−1.6%) – –
0.75 mg/kg bw – – 1 394 (−36.8%) 3 638 (−17.1%)
1.0 mg/kg bw 2 007 (−9.8%) 4 133 (−4.2%) – –
2.0 mg/kg bw 1 429 (−35.8%) 3 850 (−10.7%) 865 (−60.8%) 3 054 (−30.4%)
3.5 mg/kg bw 1 046 (−53.0%) 3 408 (−21.0%) – –
AChE: acetylcholinesterase; bw: body weight; U: enzyme unit
Source: Stannard (2006e)
Barnett Jr (2007) performed an acute range-finding study in juvenile Crl:CD[SD] rats to

determine the effect of malaoxon on acetylcholinesterase activity. Groups of five rat pups per sex
were administered malaoxon (purity 97.7%) in corn oil by gavage on postnatal day 11 at doses of 0,
3.5, 7 or 10 mg/kg bw. Blood and brain samples were collected approximately 20 minutes after dosing
to analyse acetylcholinesterase activity. No deaths or clinical signs were observed. There was a dose-
related reduction in erythrocyte acetylcholinesterase activity, which was toxicologically significant at
7 and 10 mg/kg bw (−18.1%, −33.0% and −42.8% in males and −19.5%, −35.7% and −51.3% in
females at 3.5, 7 and 10 mg/kg bw). In both sexes, toxicologically significant inhibition of brain
acetylcholinesterase occurred only at 10 mg/kg bw (−27%).
Barnett Jr (2008a) examined the time to peak effect on cholinesterase activity of an acute oral
dose of malathion or malaoxon in juvenile Crl:CD[SD] rats. Pups were administered malathion
(purity 96.0%) in corn oil by gavage on postnatal day 11 at a dose of 0 (32/sex) or 150 mg/kg bw
(56/sex) or to malaoxon (purity 97.7%) at 10 mg/kg bw (56/sex). Eight pups per sex were terminated
at 20, 40, 60, 80, 100, 120 and 150 minutes to analyse erythrocyte and brain acetylcholinesterase
activities. Five male pups and nine female pups administered malathion displayed whole-body
tremors at 18–62 minutes after dosing. One female pup dosed with malaoxon displayed whole-body
tremors 36 minutes after dosing. In malathion-treated rats, the inhibition of erythrocyte
acetylcholinesterase activity ranged from −24.2% to −44.3% in males and −30.0% to −53.6% in
females, with the time to peak effect of 40–60 minutes. The inhibition of brain acetylcholinesterase
activity ranged from −20.0% to −36.4% in males and −25.2% to −55.5% in females, with the time to
peak effect of 60–80 minutes. In malaoxon-treated rats, the inhibition of erythrocyte
391
acetylcholinesterase activity ranged from −24.2% to −44.3% in males and −30.0% to −53.6% in
females, with the time to peak effect of 40–60 minutes. The inhibition of brain acetylcholinesterase
activity ranged from −18.6% to −30.9% in males and −5.9% to −32.7% in females, with the time to
peak effect of 60–80 minutes.
Barnett Jr (2008b) examined the time to peak effect on cholinesterase activity in juvenile
Crl:CD[SD] rats following an acute oral dose of malaoxon. In a range-finding experiment, rat pups
received a single gavage dose of 12.5 (five/sex) or 15 mg/kg bw (15/sex) malaoxon (purity 96.0%) in
corn oil on postnatal day 11 and were observed for up to 4 hours after dosing. In the main experiment,
groups of 40 pups per sex were dosed with 12.5 mg/kg bw malaoxon on postnatal day 11. A vehicle
control group comprised eight pups per sex. At 10, 30, 60, 90 and 240 minutes after dosing, four pups
per sex were terminated and their blood and brain acetylcholinesterase activity analysed. In the range-
finding experiment, deaths occurred in males (one and five pups at 12.5 and 15 mg/kg bw,
respectively) and females (three pups at 15 mg/kg bw). Decreased motor activity was observed in one
male at 12.5 mg/kg bw, while decreased motor activity, salivation and loss of righting reflex were
observed in two males and three females at 15 mg/kg bw. In the main experiment, there were no
deaths, while five males and seven females had slight or moderate tremors 23–87 minutes after
dosing. One other male had intermittent whole-body twitches at 30 minutes after dosing. Erythrocyte
acetylcholinesterase was inhibited at every time point, with maximum inhibition 60 minutes after
dosing in males (−78.4% compared to the control) and 30–90 minutes in females (−75.8% to −79.1%
compared to the control). Brain acetylcholinesterase activity was inhibited in both sexes, reaching
maximum inhibition at 60 minutes in males (−54.6% compared to the control) and 90 minutes in
females (−49.8% compared to the control).
Barnett Jr (2008c) examined the time to peak effect of malathion on cholinesterase activity in
juvenile Crl:CD[SD] rats. Rat pups were administered a single gavage dose of malathion (purity
96.0%) in corn oil at 0 (8 pups/sex) or 150 mg/kg bw (40 pups/sex) on postnatal day 11. At 30, 60, 80,
100 and 150 minutes after dosing, eight pups per sex were terminated and blood and brain
acetylcholinesterase activity analysed. All pups survived to scheduled termination. Clinical signs were
observed 16–143 minutes after dosing and included whole-body tremors (10 males, 6 females),
whole-body and head tremors (five males, five females), whole-body tremors and body jerks, (1
male), body jerks (2 females), head tremors (1 male, 1 female) and whole-body and head tremors, and
body jerks (1 female). Erythrocyte acetylcholinesterase was inhibited in both sexes at every time point
(−47.2% to −75.8% in males and −55.3% to −78.0% in females) reaching a maximum at 60 minutes
after dosing. Brain acetylcholinesterase activity was inhibited in both sexes at every time point
(−29.3% to −69.6% in males and −36.4% to −68.7% in females), reaching a maximum at 60 minutes
after dosing.
Barnett Jr (2008d) compared the effect of malathion and malaoxon on erythrocyte and brain
acetylcholinesterase activities at the time of peak effect in juvenile Crl:CD[SD] rats. On postnatal day
11, groups of 12 pups per sex were administered a single gavage dose of malathion (purity 96.0%) in
corn oil at 0, 10, 25, 50, 100 or 150 mg/kg bw. Separate groups of 12 pups per sex received malaoxon
(purity 97.7%) at 0, 1.0, 3.5, 7.0, 10.0 or 12.5 mg/kg bw. At 60 minutes after dosing, the pups were
terminated and blood and brain acetylcholinesterase activity analysed. In malathion-dosed rats, there
were no treatment-related deaths. Slight or moderate whole-body tremors were observed at 100 (five
males, one female) and 150 mg/kg bw (five males, seven females), while body jerks were observed at
150 mg/kg bw (3 females) at 41–57 minutes after dosing. Toxicologically relevant and statistically
significant inhibition of erythrocyte acetylcholinesterase occurred at and above 50 mg/kg bw in both
sexes, while brain acetylcholinesterase activity was significantly lower than the control at 100 and
150 mg/kg bw (Table 34). In malaoxon-dosed pups, slight whole-body tremors were observed
between 33 and 57 minutes after dosing (one male and three females at 10 mg/kg bw; two males at
12.5 mg/kg bw). Toxicologically and statistically significant inhibition of erythrocyte
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acetylcholinesterase activity occurred at and above 3.5 mg/kg bw, while brain acetylcholinesterase
was significantly lower than the control at and above 7.0 mg/kg bw in males and at 10.0 and 12.5
mg/kg bw in females (Table 25).
Table 34. Inhibition of erythrocyte and brain acetylcholinesterase in rat pups 60 minutes after a
single dose of malathion or malaoxon
Males Females
Treatment Erythrocyte (U/mL) Brain (U/g) Erythrocyte (U/mL) Brain (U/g)
Malathion
0 mg/kg bw 2.04 5.11 2.173 4.98
10 mg/kg bw 1.95 (−4.7%) 5.20 1.944 (−10.5%) 5.11
25 mg/kg bw 1.75 (−14.5%) 5.11 (−0.1%) 1.780* (−18.1%) 4.86 (−2.4%)
50 mg/kg bw 1.25** (−39.0%) 4.38 (−14.3%) 1.550** (−28.7%) 4.66 (−6.6%)
100 mg/kg bw 1.04** (−49.0%) 3.33** (−34.9%) 0.801** (−63.1%) 2.89* (−42.0%)
150 mg/kg bw 0.67** (−67.3%) 2.82** (−44.8%) 0.483** (−77.8%) 1.76** (−64.7%)
Malaoxon
0 mg/kg bw 2.32 5.14 2.127 5.14
1 mg/kg bw 1.89 (−18.4%) 5.27 1.77 (−16.8%) 5.07 (−1.5%)
3.5 mg/kg bw 1.13** (−51.3%) 4.65 (−9.6%) 1.20** (−43.8%) 4.65 (−9.5%)
7 mg/kg bw 0.63** (−72.7%) 3.82** (−25.8%) 0.73** (−65.5%) 4.31 (−16.2%)
10 mg/kg bw 0.55** (−76.3%) 2.84** (−44.8%) 0.55** (−74.0%) 2.66** (−48.3%
12.5 mg/kg bw 1.05** (−54.7%) 3.95 (−23.2%) 0.49** (−76.9%) 2.50** (−51.3%)
AChE: acetylcholinesterase; bw: body weight; U: enzyme unit; *: P < 0.01; **: P < 0.001
Source: Barnett Jr (2008d)
An acute range-finding study was conducted by Barnett Jr (2008e) in adult Crl:CD[SD] rats
to determine the time to peak effect of erythrocyte acetylcholinesterase activity inhibition by
malathion (purity 96%), desmethyl-malathion sodium (45.9% purity as the free acid), MMCA (purity
92.2%) or MDCA (purity 98.8%). The vehicle was DMSO. Rats were administered a single gavage
dose of malathion at 0, 1000, 1500 or 2000 mg/kg bw (10/sex per dose) or a single gavage dose of
MMCA (10/sex per dose), MDCA (10/sex per dose) or desmethyl-malathion sodium (5/sex per dose)
at 1500 mg/kg bw. The rats were observed for mortality and clinical signs following dosing. Blood
was sampled (five rats/sex per dose) prior to dosing and at 2 and 8 hours after dosing to analyse
erythrocyte acetylcholinesterase activity. The rats (five rats/sex per dose) were terminated after 2 or 8
hours and their brains analysed for acetylcholinesterase activity.
There were no deaths. Clinical signs were confined to malathion-treated rats (1500 and
2000 mg/kg bw) and included red perioral substance; urine-stained abdominal fur; excess salivation;
prostrate posture; decreased motor activity, fasciculations on the head, whole body and/or both
hindpaws, no pupillary response; hunched posture; gasping; rales; ataxia; lacrimation; miosis; whole-
body twitches, slight whole-body tremors; and bradypnea. The results of erythrocyte and brain
acetylcholinesterase activity are summarized in Table 35. At every dose of malathion, erythrocyte
acetylcholinesterase activity was significantly lower (P < 0.01 or 0.05; 23–69%) than the control in
both sexes. At 1500 and 2000 mg/kg bw, brain acetylcholinesterase activity was significantly lower
(P < 0.01) than the control in females 8 hours after dosing. Toxicologically significant (i.e. > 20%)
inhibition of brain acetylcholinesterase activity occurred at 2000 mg/kg bw in both sexes 2 hours after
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dosing and in males at 8 hours after dosing. Of the metabolites, only MMCA caused a significant
reduction in erythrocyte acetylcholinesterase activity 8 hours after a dose of 1500 mg/kg bw. All
remaining cholinesterase levels in the three metabolites were comparable with the controls.
Table 35. Effect of a single dose of malathion or malathion metabolites on acetylcholinesterase

activity
Erythrocyte AChE (U/mL) Brain AChE (U/g)
Treatment Males Females Males Females
Malathion
Control – 2 hours 1.03 1.24 14.68 15.05
1 000 mg/kg bw – 2 hours 0.74* (−28%) 0.96* (−23%) 15.30 (+4%) 14.45 (−4%)
1 500 mg/kg bw – 2 hours 0.59** (−43%) 0.70** (−44%) 13.98 (−5%) 11.14 (−26%)
2 000 mg/kg bw – 2 hours 0.53** (−49%) 0.38** (−69%) 9.98 (−32%) 5.29 (−65%)
Control – 8 hours 1.32 1.27 15.77 15.10
1 000 mg/kg bw – 8 hours 0.85** (−36%) 0.64** (−50%) 14.43 (−8.5%) 12.05 (−20%)
1 500 mg/kg bw – 8 hours 0.60** (−55%) 0.49** (−61%) 12.17 (−23%) 8.25** (−45%)
2 000 mg/kg bw – 8 hours 0.45** (−66%) 0.56** (−56%) 8.71 (−45%) 8.03** (−47%)
Desmethyl-malathion sodium
1 500 mg/kg bw – 2 hours Not analysed Not analysed Not analysed Not analysed
1 500 mg/kg bw – 8 hours 1.24 (−20%) 1.30 (+5%) 15.50 (−2%) 15.12 (0%)
MMCA
1 500 mg/kg bw – 2 hours 0.99 (−4%) 1.11 (−10%) 14.75 (0%) 15.20 (+1%)
1 500 mg/kg bw – 8 hours 0.94** (−29%) 1.15 (−9%) 14.23 (−10%) 14.98 (−1%)
MDCA
1 500 mg/kg bw – 2 hours 0.99 (−4%) 1.10 (−11%) 14.19 (−3%) 14.37 (−5%)
1 500 mg/kg bw – 8 hours 1.17 (−12%) 1.23 (−3%) 14.67 (−7%) 14.74 (−2%)
AChE: acetylcholinesterase; bw: body weight; MDCA: malathion dicarboxylkic acid; MMCA: malathion monocarboxylic
acid; U; enzyme unit; *: P < 0.01; **: P < 0.001
Source: Barnett (2008)
Reiss & Edwards (2008) conducted BMD modelling of brain and erythrocyte
acetylcholinesterase inhibition based on the studies of Fulcher (2001) and Barnett Jr. (2008). BMDs
for malathion were estimated for 10% and 20% response levels for brain acetylcholinesterase
cholinesterase inhibition and at 10%, 20% and 30% response levels for erythrocyte
acetylcholinesterase inhibition. Adequate dose–response data are not available for desmethyl-
malathion sodium, MMCA or MDCA. For brain acetylcholinesterase activity, the BMD10 of
malathion was estimated to be 1078 mg/kg bw in males and 739 mg/kg bw in females, while the
BMD20 was 1433 mg/kg bw and 1024 mg/kg bw, respectively. For the inhibition of erythrocyte
acetylcholinesterase activity, the BMD10 was estimated to be 181 mg/kg bw in males and 183 mg/kg
bw in females; the BMD20 was 390 mg/kg bw and 388 mg/kg bw, respectively; the BMD30 was
638 mg/kg bw and 620 mg/kg bw, respectively.
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Reiss (2008) undertook BMD modelling of erythrocyte and brain acetylcholinesterase data
from an acute oral cholinesterase study with malathion and malaoxon in juvenile rats (Barnett Jr,
2008d) to estimate an acute TAF for malaoxon. BMDs were estimated using USEPA methodologies
developed for the Organophosphate Cumulative Risk Assessment, including a simple model that
represents an exponential decline in cholinesterase levels with dose and an expanded model that
includes saturable metabolism and allows for a minimal response at low doses before beginning an
exponential decline. For erythrocyte acetylcholinesterase inhibition, the simple model provided the
best fits to the dose–response data. For brain acetylcholinesterase, the expanded model provided the
best fit for the malathion data and the simple model provided the best fit for the malaoxon data. The
BMDs and TAFs estimated for acetylcholinesterase inhibition are summarized in Table 36.
Table 36. BMDs and TAFs for AChE inhibition in juvenile rats
Compound Sex BMD10 (mg/kg bw) BMD15 (mg/kg bw) BMD20 (mg/kg bw)
Erythrocyte AChE
Malathion Male 10.8 16.0 23.6
Female 12.3 19.0 26.1
Malaoxon Male 0.50 0.77 1.1
Female 0.69 1.1 1.5
TAF Male 21.6 21.9 21.5
Female 17.8 17.3 17.4
Brain AChE
Malathion Male 41.6 – –
Female 39.6 – –
Malaoxon Male 2.8 – –
Female 3.6 – –
TAF Male 14.8 – –
Female 11.0 – –
AChE: acetylcholinesterase; BMD: benchmark dose; BMD10: estimated benchmark dose for 10% inhibition, BMD20:
estimated benchmark dose for 20% inhibition; BMD30: estimated benchmark dose for a 30% inhibition; bw: body weight;
TAF: toxicity adjustment factor
Source: Reiss (2008)
Barnett Jr (2014) examined the time to peak effect on cholinesterase activity of an acute
gavage dose of 200 mg/kg bw MDCA (purity 98.8%) in groups of 32 adult Crl:CD[SD] rats per sex.
Control groups of 16 rats per sex were dosed with the vehicle (DMSO). The rats were observed for
one hour after dosing and prior to scheduled termination (eight rats per sex per group at 2, 4, 8 and 12
hours after dosing). Blood and brain samples were analysed for acetylcholinesterase activity after
termination. There were no deaths or treatment-related clinical signs. There was no treatment-related
effect on either erythrocyte or brain acetylcholinesterase activity.
(c) Immunotoxicity
Groups of 15 female Crl:CD-1[ICR] mice were exposed to malathion (purity 96.0%) at
dietary concentrations of 0, 50, 100, 700 or 7000 ppm ad libitum for 6 weeks (equivalent to 0, 8.9,
17.6, 126.8 and 1215.8 mg/kg bw per day, respectively). An additional group of 15 mice served as the
positive control. Four days prior to termination, all the mice were administered an intravenous dose of
sheep red blood cells. Positive controls also received 50 mg/kg per day cyclophosphamide for four
days prior to sacrifice (10 mg/mL). Mortality and clinical signs were observed throughout the
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exposure period, and body weight and feed consumption recorded. acetylcholinesterase activity was
analysed in blood and brain samples collected at termination. All the mice were necropsied and the
spleen and thymus weighed. The spleen was retained for immunological evaluation. There no deaths,
treatment-related clinical signs or effects on body weight or feed consumption. There was no
treatment-related macroscopic findings or effects on spleen or thymus weights. In contrast, absolute
and relative thymus and spleen weights were significantly reduced (P < 0.01) in the positive control.
Statistically significant and toxicologically relevant inhibition of erythrocyte acetylcholinesterase
occurred at 700 and 7000 ppm (−51.2% and −87.1%, respectively; P < 0.01). There was no effect on
brain acetylcholinesterase activity. Immunological evaluation revealed no change in spleen cell
numbers or spleen immunoglobulin-M response to sheep red blood cells.
The NOAEL for immunotoxicity was 7000 ppm (equal to 1215.8 mg/kg bw per day), the
highest tested dose (Barnett Jr, 2011d).
(d) In silico toxicity predictions

The theoretical toxicity of malathion and a storage impurity, 2-mercaptosuccinic acid diethyl
ester, were estimated by Clerkin (2015) using quantitative structure–activity relationships (QSAR)
contained within the Organisation for Economic Co-operation and Development (OECD)–developed
QSAR Application Toolbox (Version 3.2; 2013). Alerts for the storage impurity were generally
comparable with malathion (Table 37). Malathion is a Cramer Class III compound while the storage
impurity is in Cramer Class I. There were no structural alerts for DNA binding using the OECD
QSAR Application Toolbox.
The storage impurity had a low-level alert for ‘keratinocyte gene expression’ that was not
present for malathion. However, as a guinea-pig maximization test had previously demonstrated
positive results with malathion, this alert for the storage impurity is of limited toxicological concern.
The estimated oral and dermal LD50 values for malathion using read-across were 4140 and
2260 mg/kg bw, respectively, while those for the storage impurity were 3870 and 3040 mg/kg bw,
respectively. The general pattern of results suggests that the impurity is of no greater toxicity than
malathion.
Table 37. In silico toxicity predictions for malathion and a storage impurity
End-point Malathion 2-Mercaptosuccinic acid diethyl ester
DNA binding by OASIS v 1.2 SN2 Radical
SN2 >> DNA alkylation Radical >> Generation of
SN2 >> DNA alkylation >> reactive oxygen species
Alkylphosphates, Radical >> Generation of
Alkylthiophosphates and reactive oxygen species >>
Alkylphosphonates Thiols
DNA binding by OECD No alert found No alert found
DPRA cysteine peptide depletion Not possible to classify according to Not possible to classify according to
these rules these rules
DPRA lysine peptide depletion Not possible to classify according to Not possible to classify according to
these rules these rules
Estrogen receptor binding Non-binder, non-cyclic structure Non-binder, non-cyclic structure
Protein binding potency Not possible to classify according to Not possible to classify according to
these rules (GSH) these rules (GSH)
Toxic hazard classification by Cramer High (Class III) Low (Class I)
(original)
Bioaccumulation – metabolism half- Very fast Very fast
lives
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End-point Malathion 2-Mercaptosuccinic acid diethyl ester

Carcinogenicity (genotox and non- No alert found No alert found
genotox) alerts by ISS
DNA alerts for Ames, MN and CA by SN2 No alert found
OASIS v1.2 SN2 >> DNA alkylation
SN2 >> DNA alkylation >>
Alkylphosphates,
Alkylthiophosphates and
Alkylphosphonates
Eye irritation/corrosion No alert found No alert found
Exclusions rules by BfR Solubility < 0.01 g/kg Solubility < 0.01 g/kg
Eye irritation/corrosion inclusion Inclusion rules not met Inclusion rules not met
rules by BfR
In vitro mutagenicity (Ames test) No alert found No alert found
alerts by ISS
In vivo mutagenicity (micronucleus) H-acceptor-path3-H-acceptor H-acceptor-path3-H-acceptor
alerts by ISS
Keratinocyte gene expression Not possible to classify according to Low gene expression
these rules Low gene expression >>
Thiols
Protein binding alerts for skin SN2 SN2
sensitization by OASIS v1.2 SN2 >> Nucleophilic SN2 >> Interchange reaction
substitution at sp3 carbon atom with sulfur-containing compounds
SN2 >> Nucleophilic SN2 >> Interchange reaction
substitution at sp3 carbon with sulfur-containing
atom >> (Thio)Phosphates compounds >> Thiols and
disulfide compounds
rtER Expert System ver.1 – USEPA No alert found No alert found
Skin irritation/corrosion exclusion No alert found No alert found
rules by BfR Solubility < 0.01 g/kg Solubility < 0.01 g/kg
Skin irritation/corrosion inclusion Inclusion rules not met Inclusion rules not met
rules by BfR
Repeated dose (HESS) Not categorized Not categorized
BfR: German Bundesinstitut für Risikobewertung; CA: chromosomal aberrations: DPRA: direct peptide reactivity assay;
GSH: glutathione; HESS: Hazard Evaluation Support System; ISS: Istituto Superiore di Sanità; OASIS: Organization for the
Advancement of Structured Information Standards; OECD: Organisation for Economic Co-operation and Development;
MN, micronuclei; ROS; reactive oxygen species; rtER: rainbow trout estrogen receptor; SN2, bimolecular nucleophilic
substitution; USEPA: United States Environmental Protection Agency
Source: Clerkin (2015)
(e) Intestinal microbiota effects

An extensive literature search indicated that malathion had no potential adverse effects on the
intestinal microbiota. Based on the mode of action, malathion would most likely not affect the
intestinal microbiota. In addition, no studies were found on the ability of intestinal microbiota to
metabolize malathion.
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(f) Studies on metabolites and impurities

Toxicity tests were conducted on malathion metabolites, the details of which are in Table 38.
Table 38. Malathion metabolites or impurities

Common name Chemical name (CAS) Structure Description
O
Malaoxon Butanedioic acid, 2- O
Rat metabolite
[(dimethoxyphosphinyl)thio]-1,4- O S
Crop metabolite (apple,
P O
diethyl ester cotton, wheat, alfalfa,
O O
lettuce)
O
O
Isomalathion Butanedioic acid, 2- O
Crop metabolite (alfalfa)
[[methoxy(methylthio)phosphinyl]thio]- S S
P O
1,4-diethyl ester
O O
O
Desmethyl Butanedioic acid, 2- OH
Rat metabolite
malathion [(mercaptomethoxyphosphinyl)thio]- O S
P O
1,4-diethyl ester cotton, wheat, alfalfa,
S O
lettuce)
O
Simulated processing
metabolite
O
Malathion Butanedioic acid, 2- O
Rat metabolite
monocarboxylic [(dimethoxyphosphinothioyl)thio]- O S
P OH
acid (MMCA) monoethyl ester cotton, wheat, alfalfa,
S O
lettuce)
Livestock metabolite
O
O
(lactating goat, laying hen)
O
O S
P O
S OH
O
Malathion Butanedioic acid, 2- O
Rat metabolite
dicarboxylic acid [(dimethoxyphosphinothioyl)thio]- O S Crop metabolite (apple,
(MDCA) monoethyl ester P OH
cotton, wheat, alfalfa,
S OH lettuce)
Livestock metabolite
O (lactating goat, laying hen)
Desmethyl- Butanedioic acid, 2- O
O
Processing metabolite –
malaoxon [(methoxyphosphinyl)thio] HO S generated by the high-
dicarboxylic acid P OH temperature hydrolysis of
O OH
MDCA
CAS: Chemical Abstracts Service
Acute toxicity
The results of acute oral toxicity tests on malathion metabolites in rats are summarized in
Table 39.
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Table 39. Results of studies of the acute toxicity of malathion metabolites and impurities
LD50
Desmethyl malathion, sodium salt
Rat CD F Oral 92.7 DMSO >2 000a Pratt
(2005)
Desmethyl-malathion monocarboxylic acid, potassium salt
Rat Crl:WI(Han) F Oral 77.6 Water >2 000 Leoni
(2012)
MMCA
Rat CD F Oral 92.2 DMSO >2 000b Sanders
(2008a)
MDCA
Rat CD F Oral 98.8 DMSO >2 000 Sanders
(2008b)
Malaoxon
Rat SD derived, F Oral 97.7 Distilled 50 Lowe
albino water (2011b)
Desmethyl-malaoxon dicarboxylic acid, trisodium salt
Rat WISTAR rats F Oral 23.9 Nil >2 000 Allingham
Crl:WI(Han) (2015)
bw: body weight; F: female; LC50: median lethal concentration; LD50: median lethal dose; DMSO: dimethyl sulfoxide;
MDCA: malathion dicarboxylic acid; MMCA: malathion monocarboxylic acid

In a 14-day range-finding study, Daly (1995) exposed Fischer 344 (CDF (F-344)/Crl BR) rats
(5/sex per group) to malaoxon (purity 96%) at dietary concentrations of 0, 10, 25, 100, 2500 or
3500 ppm. The doses achieved were 0, 1.1, 3, 12.1, 293 and 387 mg/kg bw per day in males and 0,
1.1, 3.1, 12.5, 281.6 and 294.7 mg/kg bw per day in females, respectively. Mortalities, clinical signs,
body weight and feed consumption were recorded. Haematology and clinical chemistry parameters,
including plasma and erythrocyte cholinesterase activities, were analysed in blood collected during
week 1 and/or at termination. Following termination, the rats were necropsied and their brain
acetylcholinesterase activity analysed. No histopathology was performed. At 3500 ppm, one female
was found dead on day 10 and another on day 12, with the remaining three rats terminated in a
moribund condition on day 12. Clinical signs consisting of decreased faecal volume, hunched posture,
tremors, pale appearance, anogenital staining, lethargy and emaciation were observed at and above
100 ppm in females and 2500 ppm in males. Body-weight gain was 30% lower than the control in
high-dose males over the 2 weeks and 16.5% and 49% lower than the control in females at 2500 and
3500 ppm, respectively. Feed consumption was significantly lower (P < 0.01) than the control during
the first week of exposure at 2500 and 3500 ppm (−12% and −21%, respectively, in males; −18% and
−39%, respectively, in females). There was no treatment-related effect on haematological parameters
and the majority of clinical chemistry parameters. During week 1, plasma cholinesterase activity was
significantly lower (P < 0.01 or 0.05) than the control at 100, 2500 and 3500 ppm (males: −10%,
−85% and −82%, respectively; females: −26%, −89% and −92%, respectively). At termination,
plasma cholinesterase activity was significantly lower (P < 0.01) than the control at 2500 and
3500 ppm in males (males: −88% and −91%, respectively) and at 3500 ppm in females (−93%).
During week 1, erythrocyte acetylcholinesterase activity was significantly lower (P < 0.01 or 0.05)
than the control in males at 100, 2500 and 3500 ppm (−11%, −18% and −22%, respectively) and in
females at the highest dose (−16%). At termination, erythrocyte acetylcholinesterase activity was
significantly lower (P < 0.01) than the control only in males at 2500 and 3500 ppm (−24% and −17%,
399
respectively). At termination, brain acetylcholinesterase activity was significantly lower than the
control (P < 0.01) in males at 2500 and 3500 ppm (−37% and −75%, respectively) and in females
only at 3500 ppm (−67%). There were no treatment-related macroscopic findings.
Long-term studies of toxicity

In a pre-GLP study (NCI, 1979b), groups of 50 B6C3F1 mice and 50 F344 rats per sex were
fed diets containing malaoxon (purity > 95%) at concentrations of 0, 500 or 1000 ppm for 103 weeks
(estimated to be equal to 75 and 150 mg/kg bw per day, respectively, in mice and 25 and 50 mg/kg bw
per day, respectively, in rats). Following an additional 2-week observation period, the rats were
terminated at 103–105 weeks.
In male mice, there was a significant (P = 0.028) dose-related reduction in survival (90%,
84% and 74% at 0, 500 and 1000 ppm, respectively). In the second year of the study, a treatment-
related increase in clinical signs consisted of alopecia, pale mucous membranes, abdominal distension
and hunched posture. Graphically presented data showed high-dose females as having lower body
weights than the control. There were no treatment-related neoplastic or non-neoplastic lesions.
In the rats, there was a small reduction in survival in high-dose males but this was not
statistically significant (80%, 82% and 64% at 0, 500 and 1000 ppm, respectively). There were no
treatment-related clinical signs or effects on body weight. In females, the combined incidence of
C-cell adenomas and carcinomas of the thyroid was increased (0%, 2% and 11% at 0, 500 and
1000 ppm, respectively), although this was comparable to the historical control mean of 7%.
Statistical analysis using the Cochran–Armitage test indicated that this increase was significant (P =
0.009), with pairwise comparisons using Fisher’s exact test indicating a significant increase at the
highest dose (P = 0.024). The incidence of gastric ulcers, commonly observed in the forestomach, was
higher in treated rats (4%, 12% and 15% in males and 0%, 2% and 6% in females at 0, 500 or 1000
ppm, respectively).
The authors concluded that malaoxon was not carcinogenic in rats or mice. The slides from
this study were re-examined by Rueber (1985) who concluded that there was an increase in neoplasms
at all sites and on this basis malathion was carcinogenic. However, a subsequent re-evaluation of this
study by Huff et al. (1985) confirmed the original conclusions of the study author and also concluded
that there was equivocal evidence that malaoxon had increased the incidence of C-cell neoplasms of
the thyroid.
Groups of 85 Fischer 344 (CDF (F-344)/CrlBR) rats per sex were exposed to malaoxon
(purity 96.4%) admixed in the diet at concentrations of 0, 20, 1000 or 2000 ppm (equal to 0, 1, 57 and
110 mg/kg bw per day in males and 0, 1, 68 and 140 mg/kg bw per day in females). Groups of 55 rats
per sex were retained for 24 months, while 10 rats per sex per group were terminated at 3, 6 and 12
months. Mortalities and clinical signs were recorded daily and body weight and feed consumption
throughout the study. Ophthalmology was performed pretreatment and at 12 months and termination.
Plasma, erythrocyte and brain cholinesterase activities were analysed in all rats. Clinical chemistry
and haematology parameters were analysed in the blood from all the rats terminated at 6 and 12
months and in the 10 rats per sex per group that were retained at 18 months and termination. Urine
was collected at 6, 12 and 18 months and at termination to analyse urinary parameters. All surviving
rats were terminated at 24 months. All the rats were necropsied and selected organs weighed.
Histopathological examinations were performed on controls and high-dose groups at 12 and 24
months and on rats that died or were terminated during the study. Selected tissues from animals at the
intermediate and low doses were also examined.
There was a dose-related increase in mortality that was statistically significant (P < 0.05) in
females at 1000 and 2000 ppm and in males at 2000 ppm at 24 months (Table 33). The incidence of
early deaths was also significantly increased (P < 0.01 or 0.05) at the highest dose. Additional
statistical analysis was undertaken by Nicolich (1998a) using the Thomas, Breslow and Garth
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Analyses, which confirmed the effect on survivorship. The only treatment-related clinical sign was
yellow anogenital staining in high-dose females throughout the study and in high-dose males from
week 81. At the highest dose, absolute body weight was significantly lower (P < 0.01 or 0.05) than
the control (−1.4% to −7.1% in males and −4.0% to −8.8% in females). Cumulative body-weight gain
(from week 0) was significantly lower than the control (P < 0.01 or 0.05) in high-dose males during
the first year of dosing (−31% to week 1 and then approximately −5% for the remainder of the first
year) and in high-dose females throughout the study (from −49% to week one and approximately 10%
thereafter). Cumulative body weight was also significantly lower (P < 0.01 and 0.05) than the control
in females at 1000 ppm during the early part of the study (up to 13% lower than the control). At the
highest dose, feed consumption was generally higher than the control throughout most of the study.
There were no treatment-related ophthalmological findings or effects on haematology, urine
analysis parameters and the majority of clinical chemistry parameters. Results of the analysis of
cholinesterase activity are summarized in Table 40. Plasma cholinesterase activity and erythrocyte
acetylcholinesterase activity were significantly lower (P < 0.01 or 0.05) than the control at 1000 and
2000 ppm at every sampling point. At 6 months only, erythrocyte acetylcholinesterase activity was
significantly lower than (P < 0.01) the control at 20 ppm in both sexes, but as the level of inhibition
was at 20%, this finding was not considered toxicologically significant. Brain acetylcholinesterase
activity was significantly lower than the control (P < 0.01 or 0.01) at 1000 and 2000 ppm. There were
no other effects on clinical chemistry parameters.
Table 40. Summary of findings in male and female rats exposed to malaoxon for up to 2 years
No. and per cent change compared to control per dietary concentration
Survival (%)
Males
12 months 100 98 98 100
18 months 100 91 96 91
24 months 71 65 58 47*
Females
12 months 100 98 97 97
18 months 100 93 87 78
24 months 87 76 56* 51*
Early deaths (absolute number)
Males 16 19 23 29*
Females 7 13 24** 27**
Plasma ChE activity (IU/mL)
Males
3 months 0.53 0.53 0.13** (−75%) 0.09** (−83%)
6 months 0.62 0.59 0.12** (−81%) 0.07** (−89%)
12 months 0.74 0.79 0.19* (−74%) 0.09** (−88%)
24 months 1.60 1.62 0.36** (−78%) 0.15** (−91%)
Females
3 months 2.56 2.58 0.36* (−86%) 0.14** (−95%)
6 months 3.20 3.00 0.42** (−87%) 0.14** (−96%)
12 months 3.42 3.36 0.61* (−82%) 0.20** (−94%)
401
No. and per cent change compared to control per dietary concentration
24 months 3.12 3.65 0.53* (−83%) 0.32 (−90%)
Erythrocyte AChE activity (IU/mL)
Males
3 months 1.06 0.93 0.40** (−62%) 0.45** (−58%)
6 months 1.15 0.91** (−21%) 0.39** (−66%) 0.43% (−63%)
12 months 1.25 1.08 0.49** (−61%) 0.44** (−65%)
24 months 1.25 1.12 0.68** (−46%) 0.70** (−44%)
Females
3 months 1.25 1.00 0.47** (−62%) 0.53** (−58%)
6 months 1.29 1.04** (−19%) 0.56** (−57%) 0.52** (−60%)
12 months 1.43 1.18 0.58** (−59%) 0.49** (−66%)
24 months 1.32 1.10 0.72** (−45%) 0.71** (−46%)
Brain AChE activity (IU/g)
Males
3 months 10.45 10.25 9.51 8.53** (−18%)
6 months 10.22 10.49 10.03 9.05** (−11%)
12 months 11.45 11.24 10.58 9.46** (−17%)
24 months 10.73 10.61 7.52** (−30%) 2.82** (−74%)
Females
3 months 10.57 10.38 9.34** (−12%) 2.30** (−78%)
6 months 10.29 10.43 9.64** (−6%) 3.97** (−61%)
12 months 11.27 11.27 10.67* (−5%) 4.26** (−62%)
24 months 10.77 10.63 9.26 4.40** (−62%)
AChE: acetylcholinesterase; BMD: benchmark dose; ChE: cholinesterase; ppm: parts per million; IU: International Unit; *:
P < 0.05; **: P < 0.01
The only treatment-related macroscopic abnormality was emaciation, which occurred at the
highest dose in males (9.4% versus 3.5% in the control) and at 1000 and 2000 ppm in females (14%
and 15%, respectively, versus 0% in the control). In rats terminated after 12 months, increased
absolute and relative liver weights (+22.4% and +14.8%, respectively; P < 0.01 or 0.05) and absolute
kidney weights (+10.2%, P < 0.05) occurred in high-dose males. In rats terminated after 24 months,
absolute and relative adrenals weights were increased in high-dose males (+12.6% and +33.3%,
respectively; P < 0.05) and absolute and relative spleen weights were decreased in high-dose females
(−50.5% and −45.6%, respectively; P < 0.01). None of these variations in organ weights were
accompanied with any microscopic abnormalities or evidence of organ dysfunction.
Histopathological examination revealed a number of non-neoplastic findings at 1000 and
2000 ppm (Table 41). In the stomach, mineral deposits (minimal to moderate) were detected in the
muscularis at the highest dose. In the nasal lumen, the presence of foreign material (minimal to
severe) and inflammatory cell debris was increased (minimal to moderately severe) at 1000 and
2000 ppm. In the respiratory nasal mucosa, subacute or chronic inflammation (slight to moderately
severe) and hyperplasia of goblet cells (slight to moderately severe) and hyperplasia of the respiratory
epithelium (slight to moderately severe) was increased in females at 1000 and 2000 ppm and in males
402
at 2000 ppm. In the olfactory nasal mucosa, increased degeneration of the epithelium (slight to
moderate) occurred in males at 2000 ppm and in females at 1000 and 2000 ppm. In females, there was
an increase in the replacement of the epithelium with ciliated and non-ciliated columnar epithelial
(slight to moderate severe), and hyperplasia of ciliated and non-ciliated columnar epithelial cells
(slight to moderate severe) at 1000 and 2000 ppm. In the lung, oedema (minimal to moderate),
subacute-chronic interstitial and purulent-chronic purulent inflammation (minimal to moderate) and
foreign body granulomas (minimal to moderate) occurred at 2000 ppm in males and at 1000 and 2000
ppm in females. In the middle ear, subacute (chronic active)/chronic inflammation was accompanied
by the accumulation of inflammatory cells or cells debris within the tympanic spaces at 1000 ppm in
females and at 2000 ppm in both sexes. Collectively these effects were attributable to inhaled food
particles resulting in tissue injury and inflammation to the nasal cavity, with secondary effects in the
lungs and middle ear.
Table 41. Non-neoplastic findings in male and female rats exposed to malaoxon for 2 years
Stomach – mineral deposits in the muscularis
Males 2/65 1/55 13/55 25/64
Females 0/64 0/55 5/53 23/65
Nasal lumen – presence of foreign material
Males 6/65 10/65 9/65 28/64
Females 1/65 6/63 17/64 27/65
Nasal lumen – inflammatory cell debris
Males 13/65 21/65 15/65 31/64
Females 6/65 6/63 20/64 27/65
Nasal mucosa (respiratory) – subacute (chronic active) or chronic inflammation
Males 11/65 11/65 10/65 21/64
Females 6/65 6/63 20/64 27/65
Nasal mucosa (respiratory) – epithelial hyperplasia
Males 11/65 18/65 13/65 20/64
Females 3/65 5/63 27/64 20/65
Nasal mucosa (respiratory) – epithelium squamous or squamoid metaplasia
Males 3/65 4/65 8/65 6/64
Females 0/65 1/63 6/64 5/65
Males 4/65 6/65 5/65 12/64
Females 2/65 0/63 17/64 10/65
Males 5/65 6/65 7/65 7/64
Females 2/65 2/63 11/64 10/65
Males 5/65 2/65 4/65 7/64
Females 1/65 1/63 11/64 7/65
Lung – oedema
403

Males 5/65 5/55 9/55 16/65
Females 1/64 3/55 22/55 17/65
Lung – inflammation of the interstitium
Males 12/65 9/55 12/55 23/65
Females 14/64 15/55 29/55 34/65
Lung – purulent/chronic purulent inflammation or abscess(es)/chronic abscess(es)
Males 4/65 2/55 7/55 17/65
Females 2/64 2/55 22/55 19/65
Lung – granulomatous inflammation/granulomas
Males 8/65 3/55 11/55 12/65
Females 2/64 6/55 29/55 29/65
Middle ear (tympanic cavity/epithelial lining) – subacute (chronic active)/chronic inflammation/inflammatory cells/cell
debris
Males 8/54 5/16 7/22 15/58
Females 2/54 3/8 17/20 19/50
No.: number; ppm: parts per million; *: P < 0.05; **: P < 0.01
Results expressed as the absolute number of rats / number of rats examined.
Source: Daly (1996b).
There was a significant dose-related trend in mononuclear cell leukaemia in males (P = 0.03)
based on either the Cox’s test and Gehan-Breslow test, but pairwise comparisons did not reveal any
significant differences (20%, 22%, 35% and 25% at 0, 20, 1000 and 2000 ppm, respectively).
Subsequently, Nicolich (1998b) undertook additional statistical analysis using the method Peto et al.
(1980) described; this indicated that mononuclear cell leukaemia was increased in high-dose males.
However, as the incidences were within the historical control range of both the NTP (10–72%) and
the performing laboratory (15–36%), and as there was no dose–response relationship, this significant
increase is not considered treatment related.
The NOAEL for chronic toxicity was 20 ppm (equal to 1 mg/kg bw per day in both sexes)
based on mortality and the inhibition of brain acetylcholinesterase activity at 1000 ppm (equal to
57 mg/kg bw per day in males and 68 mg/kg bw per day in females). The NOAEL for carcinogenicity
was 2000 ppm (equal to 110 mg/kg bw per day in males and 140 mg/kg bw per day in females), the
highest test dose (Daly, 1996b).
Genotoxicity
The results of genotoxicity studies on malathion metabolites are described in section 2.4.
(g) Studies on cholinesterase inhibition

Comparative studies on the inhibition of acetylcholinesterase activity in juvenile and adult
rats by malathion metabolites are described in section 2.6(b).
404
3.1 Dosing studies in volunteers
A randomized double-blind placebo-controlled ascending single-dose study was undertaken
in human volunteers to determine the NOAEL for the inhibition of plasma and erythrocyte
cholinesterase activities. The participants (38 men and 10 women, aged 18–50 years) were given a
gelatine capsule containing malathion (purity 95.8%) over seven sessions at 0, 0.5, 1.5, 5, 10 or
15 mg/kg bw. In the first session, three men were given malathion at a dose of 0.5 mg/kg bw. In the
second session, another three men were given malathion at a dose of 1.5 mg/kg bw. Subsequently,
seven men were given malathion at a dose of 5.0 mg/kg bw. Three and four men received malathion
at a dose of 10 mg/kg bw in two separate sessions. Over three separate sessions, three male, four male
and seven female participants all received doses of 15 mg/kg bw. In each session, one or more
participants received a placebo, which contained lactose.
The participants were kept under close observation from before dosing until 72 hours after
dosing. Any symptoms or clinical signs were recorded and blood pressure, pulse rate, respiratory rate
and body temperature monitored from the day before dosing, immediately before dosing and 2, 4, 8
and 24 hours after dosing. Twelve-lead electrocardiograms were performed 30 minutes before dosing
and 2, 4, 8 and 24 hours after dosing, and single channel continuous electrocardiograms were
performed from 30 minutes before dosing until 4 hours after dosing. Blood collected at screening,
prior to dosing and 24 hours after dosing was analysed for haematological and clinical chemistry
parameters. Urine was collected at screening and 24 hours after dosing for urine analysis. Plasma
cholinesterase activity and erythrocyte acetylcholinesterase activity were analysed in blood samples
collected on days 9, 7, 5, 2 and 1 and 30 minutes prior to dosing, and then at 1, 2, 4, 8, 12, 24 and 48
hours and days 4, 7 and 14 after dosing.
There were no treatment-related clinical effects, changes in haematological or clinical
chemistry parameters or inhibition of plasma or erythrocyte cholinesterase activities. The NOAEL
was 15 mg/kg bw, the highest tested dose (Gillies & Dickson, 2000).
Healthy male and female volunteers ingested a single gelatine capsule containing malathion
(purity 95.8%) at a dose of 0, 0.5, 1.5, 5.0, 10.0 or 15.0 mg/kg bw after a light meal. For each of the
two lowest dose groups, three volunteers received malathion and one received the placebo. All
remaining groups consisted of seven treated and three control volunteers. Volunteers were observed
closely for 48 hours in a clinic and were then followed-up for two weeks. Blood was sampled six
times prior to dosing and at 1, 2, 4, 8, 12, 24 and 48 hours and 4, 7 and 14 days after dosing to analyse
plasma and erythrocyte cholinesterase activities. There were no treatment-related adverse events or
effects on plasma or erythrocyte cholinesterase activity (Jellinek, Schwartz & Connolly Inc., 2000).
The potential of malathion to effect cutaneous vasculature was examined by applying 0.5 mL
of a 10 mg/mL malathion solution to the forearms of human volunteers for 5 hours under an occlusive
dressing. Malathion increased blood flow within the region of application, and pretreatment with
10 mmol/L atropine attenuated this effect. Iontophoretic delivery of acetylcholinesterase within 1 hour
of the removal of malathion or its vehicle caused a significant increase in blood flux at both the
malathion and control-treated sites. Malathion had no effect on the concentration-dependent blood
flux response to sodium nitroprusside. There was no treatment-related effect on nitric oxide or
histamine levels (Boutsiouki & Clough, 2004).
The efficacy and clinical effects of malathion in the treatment of head lice was examined in
school-age children (7–14 years old) and their families. Of a cohort of 629 students, 48 were found to
have live head lice. Students who had lice and/or eggs were treated with 1% malathion shampoo by
applying it to the dry scalp and hair for 10 minutes before washing it off. No estimates of the systemic
doses were calculated. The shampoo was also given to family members and students without lice on
405
request. The participants were treated twice with the shampoo. Blood was collected from
32 volunteers before treatment and after the second treatment to analyse erythrocyte
acetylcholinesterase activity. Reported side-effects among the 43 treated students included nausea
(4%), a burning sensation (7%) and irritation (2%). Pairwise comparisons indicated that mean
erythrocyte acetylcholinesterase activity was significantly decreased (P = 0.03; −7.5%) after two
applications; 26 of the 32 participants showing a decrease in erythrocyte acetylcholinesterase ranging
from −5 to −26% of pretreatment activity, with the remaining 6 students showing an increase of
between +3 and +29% (Wananukul et al., 2011).
3.2 Occupational exposure studies

In a summary report by Nielsen (1994), no poisoning incidents and no inhibition of plasma
cholinesterase was observed in workers involved in the manufacture of malathion over a 20-year
period. In a subsequent summary report by Ravn Nielsen (1999), biological monitoring of workers
employed at plants manufacturing dimethoate and malathion from 1994 to 1999 detected no work-
related reduction in plasma cholinesterase activity.
3.3 Poisoning case reports

A workman was hit on his clothes by a mixture of MP-1 (O,O-dimethyldithiophosphate) and
malathion during operations at a malathion plant in November 1998. There was no direct contact with
his skin as his clothes absorbed the chemical, and he immediately took a shower. No clinical
symptoms were noted. Subsequent assessment of cholinesterase showed no reduction in blood
cholinesterase levels and the specific activity (activity/concentration) was normal (Ravn Nielsen,
1999).
3.4 Epidemiological studies

epidemiological studies on malathion (and diazinon and glyphosate) are described in “Section 2.2:
Methods for the evaluation of epidemiological evidence for risk assessment” of the meeting report13.
This evaluation considered several aspects of each study and of all studies combined in this
evaluation, including factors that decrease the level of confidence in the body of evidence, including
risk of bias, unexplained inconsistency, and imprecision, and factors that increase the level of
confidence, including large magnitude of effect, dose–response and consistency (Guyatt et al., 2008;
Morgan et al., 2016).
The findings for each study are summarized in Table 42, with findings for non-quantitative
exposure assessment (predominantly ever-use vs never-use) shown in forest plots below.
13
residues May 2016: Report 2016 (http://www.who.int/foodsafety/areas_work/chemical-risks/jmpr/en/).
406
Table 42. Results of Tier 1 evaluation and summary of cancer epidemiological studies on
malathion
Study / Location Details Reference
Malathion/NHL
Meta-analysis Qualitative exposure only – ever-use/never-use of malathion. Schinasi & Leon
Meta risk ratio: 1.8 (95% CI: 1.4–2.2) (2014)
Meta-analysis includes Waddell et al., 2001; Mills, Yang & Riordan,

2005; Pahwa et al., 2012. Does not include AHS (i.e. Bonner et al.,
2007) or Eriksson et al., 2008. Ns for each meta-analysis not
presented
AHS Qualitative (ever/never) Lerro et al. (2015)
Ever-use: 0.64 (0.41–0.99)
AHS Quantitative exposure – LEDs and IW-LEDs given Alavanja et al.
Risk estimates – aRR (95% CI) (2014)
Ever- vs never-use 0.9 (0.8–1.1)
No exposure 1.0 (ref)
LED  8.75 0.97 (0.7–1.3)
LED >8.75–38.75 0.7 (0.5–1.1)
LED > 38.75–737.5 0.9 (0.6–1.3)
P for trend 0.63
No exposure 1.0 (ref)

IW-LED – Low 1.0 (0.7–1.4)
IW-LED – Med 0.8 (0.6–1.1)
IW-LED – High 0.9 (0.6–1.2)
P for trend 0.46
Total N = 26 737 with 269 incident NHL cases
N = 179 exposed cases (LED analysis), 178 exposed cases (IW-LED
analysis)
AHS Exclude - Alavanja et al. (2014) has longest follow-up Bonner et al. (2007)
United States Midwest The study population overlaps with Waddell et al. (2001) and total N De Roos et al.
case–control studies is smaller, but this study is not excluded as it provides more fully (2003)
adjusted risk estimates for ever-use vs never-use analyses.
From a logistic regression model: Exposed 1.1 (0.6–1.8)
From a hierarchical regression model: Exposed 1.1 (0.7–1.7)
Both adjusted for other pesticides
Total N = 2 583 (650 NHL cases, 1 933 controls)

N = 53 exposed cases and 100 exposed controls
407

United States Midwest The study population overlaps with De Roos et al. (2003) above. Waddell et al.
case–control studies Quantitative exposure – days of use per year – for Nebraska only (2001)
Exposed 1.6 (1.2–2.2)
N = 91 exposed cases and 147 exposed controls.
Restricted to direct respondent farmers:

Exposed 1.2 (0.9–1.8)
Adjusted for diazinon 1.1 (0.7–1.8)
Adjusted for fonofos 1.1 (0.7–1.6)

Non-farmers: 1.0 (ref.)
< 5 days/year: 2.1 (0.7–6.1)
5+ days/year: 1.5 (0.5–5.2)
N = 12 exposed cases and 15 exposed controls (7/8 for < 5 days and
5/7 for 5+ days for exposed cases/controls, respectively)
Cross-Canada Study of NB. Study population is almost the same as for McDuffie et al. Pahwa et al. (2012)
Pesticides and Health (2001), minus N = 4 cases excluded due to pathology review. This
study is not excluded as outcome classification is expected to be more
accurate, and thus it provides the most robust risk estimate for ever-
use vs never-use analysis.
Ever-use 1.96 (1.42–2.70)
NB. Estimates also reported stratified by no/asthma; no/allergies;
no/asthma or allergies or hay fever
Cross-Canada Study of Exclude – Analysis focuses on malathion use in combination with Hohenadel et al.
Pesticides and Health other pesticides. Multiple ‘malathion only’ ORs reported but they (2011)
vary in sample size by each ‘combination’ analysis. The largest
‘malathion only’ analysis gives the following result:
aOR for ever-use: 1.75 (1.22–2.52)
However, these analyses were conducted in a very small selected

subset of the Cross-Canada Study of Pesticides and Health with
exposure to both malathion and another pesticide
Because McDuffie et al. (2001) reports on the largest sample size,
Hohenadel et al. (2011) is excluded
Cross-Canada Study of The study population overlaps with Pahwa et al. (2012) (see above). McDuffie et al.
Pesticides and Health Quantitative exposure – days of use per year (3 categories – cutpoints (2001)
are given).

Ever-use 1.83 (1.31–2.55)
Unexposed 1.00 (ref)
>0– <=2 days/year 1.82 (1.25–2.68)
> 2 days/year 1.75 (1.02–3.03)
Total N = 2 023
517 cases, 1 506 controls (overall); 179 cases, 456 controls (with
telephone interview data, i.e. detailed pesticide information)
408

United Farm Workers Qualitative (high vs low – no cutpoints; the researchers used work Mills, Yang &
of America history and crop-exposure-matrix coupled with pounds applied) Riordan (2005)
High vs low 1.77 (0.99–3.17)
Total 131 cases, 651 controls. N exposed cases/controls not reported
Sweden Qualitative Eriksson et al.
Risk estimates – aOR (95% CI) (2008)
Ever-use 2.81 (0.54–14.7)
N = 5 and 2 exposed cases and controls, respectively (few cases; not
reported in main results but only in discussion)
Malathion/Prostate cancer
AHS Quantitative exposure – quartiles – median values for quartiles given Koutros et al.
(results for IW-LEDs shown, results for LEDs not presented) (2013)
Total prostate cancer
Non-exposed 1.0 (ref.)
Q1 IW-LED 1.03 (0.84–1.26)
Q2 IW-LED 1.13 (0.94–1.36)
Q3 IW-LED 1.11 (0.93–1.34)
Q4 IW-LED 1.08 (0.90–1.29)
P for trend 0.62
Aggressive prostate cancer

Non-exposed 1.0 (ref.)
Q1 IW-LED 1.19 (0.89–1.59)
Q2 IW-LED 1.27 (0.97–1.67)
Q3 IW-LED 1.28 (0.98–1.68)
Q4 IW-LED 1.43 (1.08–1.88)
P for trend 0.04
Total N = 54 412 (746 exposed and 328 non-exposed prostate cases;
374 and 140 exposed and non-exposed aggressive prostate cancers)
United Farm Workers Quantitative – Quartiles of exposure, based on ecological exposure Mills & Yang
of America assessment (total pesticide use in the county in which the (2003)
cases/controls were employed on a farm).
High vs low: 0.96 (0.66–1.40)
Quartile 1 (low): 1.00 (ref.)

Quartile 2: 0.93 (0.62–1.39)
Quartile 3: 1.01 (0.61–1.67)
Quartile 4 (high): 1.04 (0.59–1.85)
P for trend = 0.89

N = 129 exposed cases (i.e. High or Quartiles 2–4)
Case–control study of Quantitative – Ever vs low vs high exposure – based on median of Band et al. (2011)
prostate cancer in lifetime cumulative exposure level for controls as a cutpoint (i.e.
British Columbia < 7.67, and > 7.67). However, no units are given for this exposure
metric.
Ever exposure: 1.34 (1.01–1.78)
Unexposed: 1.0 (ref.)
Low (< 7.67): 1.18 (0.78–1.78)
High (> 7.67): 1.49 (1.02–2.18)
P for trend = 0.03
409
AHS: Agricultural Health Study; aOR: adjusted odds ratio; aRR: adjusted risk ratio; CI: confidence interval; IW-LED:
intensity-weighted lifetime-exposure days, defined as number of years of use  number of days used per year  personal
protective equipment use reduction factor  intensity level score (a unit-less score which reflects a combination of self-
reported pesticide exposure modifiers, e.g. pesticide mixing status, application method, equipment repair activities); LED:
lifetime-exposure days (defined as number of years of use  number of days used per year); N: size of sample; NHL: non-
Hodgkin lymphoma; NB.: nota bene; OR: odds ratio; Q: quartile
A. Malathion / NHL
The evaluation included seven studies (Lerro et al., 2015; Alavanja et al., 2014; Waddell et
al., 2001; Pahwa et al., 2012; McDuffie et al., 2001; Mills, Yang & Riordan, 2005; Eriksson et al.,
2008) and one meta-analysis (Schinasi & Leon, 2014). The Agricultural Health Study (AHS)14 found
no evidence of elevated risk of NHL associated with malathion exposure in either men (Alavanja et
al., 2014) or women (Lerro et al., 2015). In contrast, various case–control studies reported elevated
risks of NHL associated with use of malathion: Waddell et al. (2001) report significant elevated risk
of NHL associated with ever-use versus never-use of malathion (OR: 1.6; 95% CI: 1.2–2.2) from the
United States Midwest pooled case–control studies; however, risk estimates were attenuated and no
longer significant when a) proxy respondents were excluded, and b) analyses were mutually adjusted
for other pesticides (diazinon, fonofos). Similarly, in a further analysis of the United States Midwest
pooled case–control studies limited to just direct respondents, De Roos et al. (2003) found no
association in risk estimates adjusted for all other pesticides. Significant elevated risks of NHL were
reported from the Cross-Canada Study of Pesticides and Health for ever-use versus never-use of
malathion (OR: 1.96; 95% CI: 1.42–2.70 by Pahwa et al., 2012; McDuffie et al., 2001), and when
examining annual days of exposure. However, there was no clear exposure–response relationship
across quantitative exposure categories (McDuffie et al., 2001). [NB. McDuffie et al. (2001) and
Pahwa et al. (2012) report on the same study population, but are both included in this evaluation
because Pahwa et al. (2012) provides the most robust risk estimate for ‘ever-use’ for this population
whereas McDuffie et al. (2001) provide risk estimates for quantitative exposure categories]. Non-
significant elevated risks of NHL were reported by two other case–control studies (Mills, Yang &
Riordan, 2005; Eriksson et al., 2008). The report by Eriksson et al. (2008) is limited by the extremely
small number of exposed cases and controls (5 and 2 respectively); the resulting uncertainty around
the risk estimate reported means it adds little to the evidence base. Schinasi & Leon (2014) report a
meta risk ratio of 1.8 (95% CI: 1.4–2.2) for ever-use versus never-use of malathion, but this was based
only on Waddell et al. (2001), Mills, Yang & Riordan (2005), and Pahwa et al. (2012) and excluded
the large AHS cohort (the relevant publication at the time would have been Bonner et al. (2007) and
Eriksson et al. (2008)).
14
We thank the Agricultural Health Study, and Dr Laura Beane Freeman in particular, for providing
the additional information we requested relating to this publication.
410
Fig. 3. Forest plot for risk estimates of studies with qualitative exposure categories
B. Malathion / prostate cancer

The evaluation included three studies (Koutros et al., 2013; Band et al., 2011; Mills & Yang,
2003). There was no evidence of an association with all prostate cancer and malathion exposure in the
AHS (Koutros et al., 2013). However, Koutros et al. (2013) observed a significant excess risk of
aggressive prostate cancer (RR: 1.43; 95% CI: 1.08–1.88) in the highest exposure category (highest
quintile of intensity-weighted lifetime days of malathion exposure) along with a significant exposure–
response relationship (P for trend = 0.04). Band et al. (2011) observed a significant elevated risk of all
prostate cancer in a case–control study for ever-use (OR: 1.34; 95% CI: 1.01–1.78) and for high
exposure versus no exposure (OR: 1.49; 95% CI: 1.02–2.18) with a significant linear trend across
exposure categories (P = 0.03). Both studies were large. However, the interpretation of results by
Band et al. (2011) is limited by a potential for exposure misclassification in the assessment based job–
exposure matrix as well as the potential for confounding due to other pesticides as there was no
adjustment for their use. There was no evidence of an association between prostate cancer and
malathion exposure in the United Farm Workers of America study (Mills & Yang, 2003); however,
this study is limited by the use of ecological exposure assessment, with potential for considerable
exposure misclassification.
411
Fig. 4. Forest plot for risk estimates of studies with qualitative exposure categories
Comments
Biochemical aspects
In a study conducted in rats using [14C]malathion (Reddy, Freeman & Cannon, 1989),
gastrointestinal absorption was at least 77% in males and 86% in females. The majority (up to 90%)
of radioactivity was excreted in urine within 24 hours. Less than 1% of radioactivity was detected in
tissues, with the highest proportions in the liver, skin, fat and gastrointestinal tract. There was no
evidence that malathion or its metabolites accumulated in any tissue.
Malathion is extensively metabolized via desulfuration, oxidation, hydrolysis, dealkylation
and demethylation reactions. In particular, the oxidative desulfuration of malathion in the liver
generates malaoxon, which is a more potent inhibitor of acetylcholinesterase compared with
malathion. The major metabolites detected in rat urine (> 80% of urinary radioactivity) were α- and β-
monocarboxylic acids (MMCA) and the dicarboxylic acid (MDCA) of malathion. Other urinary
metabolites include desmethyl malathion, O,O-dimethyl phosphorothioic acid, fumaric acid, 2-
mercaptosuccinic acid, O,O-dimethyl phosphorodithioic acid, monoethyl fumarate and malaoxon.
Malaoxon was observed only in urine samples and accounted for less than 2% of total urinary
radioactivity. Similar metabolites were detected in human studies (Aston, 2000; Jellinek, Schwartz &
Connolly Inc., 2000).
Published in vitro studies have further investigated the metabolism of malathion. In human
liver microsomes, the metabolism of malathion to malaoxon was catalysed by CYP1A2, CYP2B6 or
CYP3A4, their respective contributions depending on the concentration of malathion (Buratti et al.,
2005). Isomalathion, a storage impurity, was a potent non-competitive inhibitor of hepatic
carboxylesterase activity, important for the formation of MMCA by human liver microsomes (Buratti
& Testai, 2005).
Estimates of in vitro dermal absorption through human skin ranged from 1.44% to 8.74% (de
Ligt, 2004) and from 8% to 20.7% (Moody et al., 2007). In a volunteer study (Wester et al., 1983),
dermal absorption was 4.48% following a single application and 3.53% following a second
application.
Toxicological data
Consistent with other organophosphorus insecticides, the most sensitive toxicological effect
following acute and repeated exposures to malathion is the inhibition of acetylcholinesterase activity
in erythrocytes and brain. At higher doses, cholinergic signs become evident.
412
In rats, the oral LD50 ranged from 1539 to 8227 mg/kg bw (Terrell, 1979a,b; Kynoch, 1986a;
Fischer, 1991a,b; Kuhn, 1996; Moore, 2002, 2003), the dermal LD50 was greater than 2000 mg/kg bw
(Kynoch, 1986b; Bollen, 2003a) and the inhalation LC50 was greater than 5.2 mg/L (Jackson, 1986;
Decker, Knuppe & Ullrich, 2003). The dermal LD50 in rabbits was 8790 mg/kg bw (Parke, 1978).
Malathion was slightly irritating to rabbit skin (Liggett & Parcell, 1985a) and eyes (Liggett & Parcell,
1985b; Bollen, 2003c). In a Buehler test conducted in guinea-pigs, malathion did not cause skin
sensitization (Kynoch & Smith, 1986), whereas malathion caused skin sensitization in the guinea-pig
maximization test (Bollen, 2003d). Malathion was not sensitizing in the mouse local lymph node
assay (Wang-Fan, 2003; Lowe, 2011a).
In a 14-day range-finding study conducted in juvenile rats, which tested gavage malathion
doses of 0, 250, 450 and 600 mg/kg bw per day, salivation occurred at 450 and 600 mg/kg bw per
day. In males, erythrocyte and brain acetylcholinesterase activities were reduced at every dose,
whereas in females, erythrocyte and brain acetylcholinesterase activities were reduced at 450 and 600
mg/kg bw per day.
In a 28-day repeated-dose toxicity study in rats, which tested dietary malathion concentrations
of 0, 100, 500, 5000 and 10 000 ppm (equal to 0, 9.2, 46.1, 457.5 and 947.8 mg/kg bw per day for
males and 0, 9.4, 47.4, 461.3 and 910.1 mg/kg bw per day for females, respectively), the NOAEL was
500 ppm (equal to 46.1 mg/kg bw per day) for the inhibition of erythrocyte and brain
acetylcholinesterase activities at 5000 ppm (equal to 457.5 mg/kg bw per day). Nasal toxicity,
consisting of goblet cell depletion and hyperplasia of the olfactory epithelium, was noted at the
highest dose (Barnett, 2012a).
of 0, 50, 100, 500, 10 000 and 20 000 ppm (equal to 0, 5.1, 10.4, 51.9, 1036 and 2008 mg/kg bw per
day for males and 0, 5.7, 11.6, 57.6, 1134 and 2193 mg/kg bw per day for females, respectively), the
NOAEL was 500 ppm (equal to 51.9 mg/kg bw per day) for the inhibition of brain
acetylcholinesterase activity at 10 000 ppm (equal to 1036 mg/kg bw per day) (Daly, 1993a).
The overall NOAEL from these two 1-month repeated-dose toxicity studies in rats was 500
ppm (equal to 51.9 mg/kg bw per day), with an overall lowest-observed-adverse-effect level
(LOAEL) of 5000 ppm (equal to 457.5 mg/kg bw per day).
of 0, 100, 500, 5000, 10 000 and 20 000 ppm (equal to 0, 7, 34, 340, 680 and 1390 mg/kg bw per day
for males and 0, 8, 39, 384, 784 and 1597 mg/kg bw per day for females, respectively), the NOAEL
was 500 ppm (equal to 34 mg/kg bw per day) for the inhibition of brain acetylcholinesterase activity
at 5000 ppm (equal to 340 mg/kg bw per day) (Daly, 1993b).
In a second 90-day repeated-dose toxicity study in rats, which tested dietary malathion
concentrations of 0, 100, 500, 5000 and 10 000 ppm (equal to 0, 7.2, 35.0, 353.6 and 733.8 mg/kg bw
per day for males and 0, 7.5, 35.9, 363.1 and 719.0 mg/kg bw per day for females, respectively), the
NOAEL was 100 ppm (equal to 7.2 mg/kg bw per day) for goblet cell depletion at 500 ppm (equal to
35.0 mg/kg bw per day) (Barnett, 2012b). This is considered to be an atypical result, as the effect is
likely to have arisen through non-dietary exposure.
In a 13-week neurotoxicity study in rats, which tested dietary malathion concentrations of 0,
50, 5000 and 20 000 ppm (equal to 0, 4, 352 and 1486 mg/kg bw per day for males and 0, 4, 395 and
1575 mg/kg bw per day for females, respectively), the NOAEL was 50 ppm (equal to 4 mg/kg bw per
day), based on the inhibition of erythrocyte acetylcholinesterase activity at 5000 ppm (equal to 352
mg/kg bw per day) (Lamb, 1994b).
The overall NOAEL for the 90-day (neuro)toxicity studies in rats was 500 ppm (equal to 34
mg/kg bw per day) for effects at 5000 ppm (equal to 340 mg/kg bw per day).
In a 28-day range-finding study in dogs in which malathion was administered orally in
capsules at doses of 0, 125, 250 and 500 mg/kg bw per day, inhibition of erythrocyte
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acetylcholinesterase occurred at 250 and 500 mg/kg bw per day, with deaths, cholinergic signs and
reduced body weight and feed consumption occurring at the highest dose (Fischer et al., 1988).
In a 12-month repeated-dose toxicity study in dogs in which malathion was administered
orally in capsules at doses of 0, 62.5, 125 and 250 mg/kg bw per day, the NOAEL was 125 mg/kg bw
per day for reduced body weight and haematological changes at 250 mg/kg bw per day. Inhibition of
erythrocyte acetylcholinesterase activity occurred at every dose but was of marginal toxicological
significance in the absence of brain acetylcholinesterase inhibition (Shellenberger & Billups, 1987).
In a 3-week repeated-dose dermal toxicity study in rabbits, which tested malathion doses of 0,
50, 300 and 1000 mg/kg bw per day, the NOAEL was 300 mg/kg bw per day for the inhibition of
brain acetylcholinesterase activity at 1000 mg/kg bw per day (Moreno, 1989).
In a 21-day repeated-dose dermal toxicity study in rabbits, which tested malathion doses of 0,
75, 100, 150 and 500 mg/kg bw per day, the NOAEL was 150 mg/kg bw per day for the inhibition of
brain acetylcholinesterase activity at 500 mg/kg bw per day (Barnett, 2006d).
In a 13-week repeated-dose inhalational toxicity study in which rats were exposed whole
body to an aerosol malathion concentration of 0, 0.1, 0.45 or 2.0 mg/L, a no-observed-adverse-effect
concentration (NOAEC) was not determined, as laryngeal hyperplasia and degeneration and/or
hyperplasia of the olfactory epithelium occurred at every concentration (Beattie, 1994).
In an 18-month pre-GLP study conducted in mice, which tested dietary malathion
concentrations of 0, 8000 and 16 000 ppm (equivalent to 0, 1200 and 2400 mg/kg bw per day,
respectively), a NOAEL for chronic toxicity was not identified, because clinical signs during the
second year of exposure and reduced body weight occurred at both doses. Although no treatment-
related tumours were observed, this study was considered unreliable for assessing carcinogenicity
because of the small number of concurrent control mice (n = 10) compared with the treated groups (n
= 50) (NCI, 1978).
In a second 18-month study conducted in mice, which tested dietary malathion concentrations
of 0, 100, 800, 8000 and 16 000 ppm (equal to 0, 17, 143, 1476 and 2978 mg/kg bw per day for males
and 0, 21, 167, 1707 and 3448 mg/kg bw per day for females, respectively), the NOAEL for chronic
toxicity was 800 ppm (equal to 143 mg/kg bw per day) for the inhibition of brain acetylcholinesterase
activity at 8000 ppm (equal to 1476 mg/kg bw per day). Increases in liver carcinomas in males at the
low dose and second-highest dose were not considered treatment related because of the lack of a
dose–response relationship, the lack of corroboration in females and the fact that liver carcinomas are
a common age-related tumour in this strain of mouse (B6C3F1). The NOAEL for carcinogenicity was
800 ppm (equal to 143 mg/kg bw per day) for an increased incidence of liver adenomas at 8000 ppm
(equal to 1476 mg/kg bw per day) (Slauter, 1994).
In an 80-week pre-GLP study conducted in rats, which tested dietary malathion
concentrations of 0, 4700 and 8150 ppm (equivalent to 0, 1200 and 2400 mg/kg bw per day,
respectively), it was not possible to identify a NOAEL for chronic toxicity because of the lack of
reporting detail (NCI, 1978). While there was an increase in proliferative lesions of the thyroid in both
sexes at both doses, these increases were not statistically significant in males and were significant in
females only in a trend test and not by pairwise comparison when compared with groups of pooled
controls. Overall, this study is not considered acceptable for the assessment of carcinogenicity
because of the small number of rats in the concurrent control group (15 versus 50 in the treated
groups) and the short duration of exposure.
In a subsequent 24-month pre-GLP study conducted in rats, which tested dietary malathion
concentrations of 0, 100, 1000 and 5000 ppm (equivalent to 0, 5, 50 and 250 mg/kg bw per day,
respectively, as calculated by a previous Meeting), the NOAEL was 100 ppm (equivalent to 5 mg/kg
bw per day) for the inhibition of erythrocyte acetylcholinesterase activity at 1000 ppm (equivalent to
50 mg/kg bw per day) (Rucci, Becci & Parent, 1980; Seely, 1991). The NOAEL for carcinogenicity
was 5000 ppm (equivalent to 250 mg/kg bw per day), the highest dose tested.
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In a 24-month chronic toxicity and carcinogenicity study in rats, which tested dietary
malathion concentrations of 0, 100, 500, 6000 and 12 000 ppm (equal to 0, 7, 29, 359 and 729 mg/kg
bw per day for males and 0, 8, 35, 415 and 868 mg/kg bw per day for females, respectively), the
NOAEL for chronic toxicity was 500 ppm (equal to 29 mg/kg bw per day) for reduced red cell
parameters, inhibition of brain acetylcholinesterase activity and the occurrence of nasal toxicity at
6000 ppm (equal to 359 mg/kg bw per day) (Daly, 1996a). The nasal toxicity was characterized by
olfactory epithelial degeneration, hyperplasia and cyst formation, goblet cell hyperplasia, congestion,
oedema and inflammation. Four nasal adenomas were observed, one in each sex at the two highest
doses. In females, but not males, the incidence of liver adenomas was increased slightly at 6000 and
12 000 ppm, but the incidences were within the performing laboratory’s historical control range. A
NOAEL of 500 ppm (equal to 29 mg/kg bw per day) was identified for carcinogenicity, based on the
increase in nasal adenomas at 6000 ppm (equal to 359 mg/kg bw per day) (Daly, 1996a, 1999).
The Meeting concluded that there is some evidence that malathion is carcinogenic in rats and
mice.
The Meeting noted that the mouse liver adenomas observed in the second 18-month study
(Slauter, 1994) occurred at doses exceeding the maximum tolerated dose and were not replicated in
other mouse studies. The increases in liver adenomas in rats observed in the 24-month chronic
toxicity and carcinogenicity study (Daly, 1996a) occurred only in females and were within the
performing laboratory’s historical control range. Whereas the rodent liver adenomas were coincident
with liver hypertrophy, there were no findings in these or other studies to suggest a possible mode of
action, such as liver enzyme induction or cytotoxicity. Malathion showed no peroxisome proliferator–
activated receptor alpha or gamma (Takeuchi et al., 2006) activity and also showed no aryl
hydrocarbon receptor activity (Takeuchi et al., 2008). Overall, the Meeting considered that there was
equivocal evidence to suggest a tumorigenic response in the liver, but this had a clear threshold and
was likely to be secondary to the effects on the liver of prolonged exposure to very high dietary
concentrations of malathion.
Based on consistent observations of nasal toxicity in dietary studies of various durations

ranging from 28 days to 2 years and in a short-term inhalational toxicity study, the Meeting concluded
that the formation of nasal adenomas in rats was due to a local mechanism of irritancy and
cytotoxicity caused by prolonged exposure of the nasal epithelium to high concentrations of malathion
absorbed via inhaled food particles or as a vapour arising from food. This produces a state of reactive
hyperplasia, a causative factor in tumour formation. Scenarios of prolonged, direct and excessive
exposure of human nasal tissue to malathion or malathion metabolites following ingestion of residues
is unlikely, and therefore these tumours would not occur in humans following exposure to malathion
in the diet.
Malathion has been extensively tested for genotoxicity using a broad range of in vitro and in
vivo assays. In 1997, the Meeting evaluated the available unpublished and published genotoxicity
studies and noted that the majority of studies indicated that malathion is not genotoxic, although a
small number of studies indicated that it can induce chromosomal aberrations and sister chromatid
exchanges in vitro. However, there was no evidence that malathion induced chromosomal aberrations
in vivo. Therefore, the 1997 Meeting concluded that malathion does not induce genotoxic damage in
vivo. The 2003 Meeting evaluated supplementary genotoxicity studies and found that malathion
caused chromosomal aberrations in cultured human lymphocytes and gene mutations in the mouse
lymphoma assay at cytotoxic concentrations, but did not cause unscheduled DNA synthesis in vivo in
male rats. The 2003 Meeting reaffirmed its previous conclusion that although the results of some in
vitro tests were positive, malathion was not considered to induce genotoxic damage in vivo.
In addition to the studies considered at previous meetings, the current Meeting considered a
number of new published and unpublished genotoxicity studies, including studies that involved the
assessment of genotoxic damage in exposed workers. Many of the published studies do not provide
415
adequate experimental detail, do not specify the purity of the malathion tested or were conducted on
commercial formulations, or used in vivo test systems or exposure routes less relevant to the risk
assessment of dietary residues of pesticides. The following discussion is limited to studies that
evaluated technical malathion or malathion at purities above 90% and provided adequate experimental
and data analysis details to allow interpretation of the findings.
Using standard genotoxicity test systems, malathion was not mutagenic in assays using
prokaryotes or lower eukaryotes when tested with or without metabolic activation (USEPA, 1977;
Haworth et al., 1983; Traul, 1987; Machado, 1996; Bowles, 2005; Taylor, 2008c; Beevers, 2009;
Thompson, 2013; Schreib, 2015b). In contrast, in in vitro assays using either human or non-human
cells, malathion was generally positive for the induction of (1) chromosome damage, as measured by
increased frequencies of chromosomal aberrations (Galloway et al., 1987; Herath et al., 1989; Garry
et al., 1990; Edwards, 2001a; Lloyd, 2009) or micronuclei (Titenko-Holland et al., 1997; Josse et al.,
2014); (2) mutations (Edwards, 2001b; Pluth et al., 1996, 1998); and (3) DNA damage, as measured
by increases in DNA migration in the alkaline comet assay (Lu et al., 2012) and increased frequencies
of sister chromatid exchanges (Nicholas, Vienne & van den Berghe, 1979; Chen et al., 1981; Nishio
& Uyeki, 1981; Herath et al., 1989). Negative findings were reported for the induction of micronuclei
in Molt-4 T-lymphocytes (Szekely, Goodwin & Delaney, 1992), unscheduled DNA synthesis in WI-
38 cells (USEPA, 1977) and primary rat liver hepatocytes (Pant, 1989), and mutations in a mouse
lymphoma assay (reported to be equivocal without metabolic activation and negative with metabolic
activation, in Chemical Effects in Biological Systems [CEBS]; NTP 2016).
Using in vivo nonmammalian systems, malathion was active for micronucleus induction in a
bird model (Hussain et al., 2015) and for induction of reciprocal translocations and sex-linked
recessive lethals in one Drosophila melanogaster study (Foureman et al., 1994), but not for sex-linked
recessive lethals, sex chromosome loss or wing-spot mutations in another study (Valencia, 1981).
Based on the criteria mentioned in section 2.1 of the main JMPR meeting report15, very few of
the 34 in vivo mammalian study/end-point combinations were considered adequate for this review. In
reports submitted by the sponsor, malathion was negative in a rat liver unscheduled DNA synthesis
study when administered by gavage (Meerts, 2003), in a rat bone marrow chromosomal aberration
study when administered by gavage (Gudi, 1990) and in a mouse bone marrow erythrocyte
micronucleus assay when administered intraperitoneally (Navarro, 1995). However, the unscheduled
DNA synthesis assay is insensitive for detecting genotoxic compounds; the micronucleus assay, as
conducted, suffers from concerns about scoring criteria; and the chromosomal aberration test appears
to be significantly underpowered, based on the frequency of chromosomal aberrations detected among
control and treated animals. A negative mouse dominant lethal test was also reported when malathion
was administered in feed for 7 weeks (USEPA, 1977), and a negative mouse bone marrow
chromosomal aberration study was reported in intraperitoneally treated mice (Degraeve, Chollet &
Moutschen, 1984b). In contrast, malathion-induced micronuclei and chromosomal aberrations were
reported in bone marrow immature erythrocytes and proliferating cells, respectively (Dulout et al.,
1982, 1983). A positive alkaline comet assay using blood leukocytes sampled from rats treated
intraperitoneally once a day for 5 days was reported (Moore, Patlolla & Tchounwou, 2011).
The Meeting evaluated a number of human studies that examined genotoxicity end-points.
Patients treated for acute intoxication with a malathion-based product exhibited increased levels of
chromosomal damage in lymphocytes (van Bao et al., 1974). The frequency of micronuclei and
glycophorin A mutations in erythrocytes or micronuclei in lymphocytes was not increased in workers
exposed selectively to malathion (Titenko-Holland et al., 1997; Windham et al., 1998). However,
DNA damage and chromosomal aberrations have been reported in workers exposed to a mixture of
pesticides, including malathion (Yoder, Watson & Benson, 1973; Páldy et al., 1987; Rupa et al., 1988,
1991; De Ferrari et al., 1991; Pluth et al., 1996; Garaj-Vrhovac & Zeljezic, 2000, 2001; Omari, 2011;
15
residues. May 2016: Report 2016 (http://www.who.int/foodsafety/areas_work/chemical-risks/jmpr/en/).
416
Singh et al., 2011; Benedetti et al., 2013; Varona-Uribe et al., 2016). These studies are of limited
value for examining the specific effect of malathion on genotoxicity end-points in humans.
The Meeting noted that malathion has been reported to have genotoxic activity in multiple
assay systems at multiple genetic end-points. In several studies where evaluated, reactive oxygen
species appear to have been responsible for the increased damage, as demonstrated by the detection of
malathion-induced 8-hydroxy-2′-deoxyguanosine and increased malondialdehyde concentrations in
isolated human peripheral blood mononuclear cells treated in vitro, an effect attenuated by co-
treatment with N-acetylcysteine or curcumin (Ahmed et al., 2011); by increased intracellular levels of
reactive oxygen species and reduced levels of catalase, superoxide dismutase and glutathione in rat
PC12 cells treated in vitro (Lu et al., 2012); and by the detection of oxidative damage using the comet
assay in isolated rat lymphocytes treated in vitro with malathion (Ojha & Srivastava, 2014).
Supportive of this hypothesis, malathion appears to selectively induce markers of oxidative stress in
Tox21/ToxCast high-throughput screening assays (USEPA interactive Chemical Safety for
Sustainability Dashboard, 2016). The Meeting concluded that the observed genotoxic effects occur
secondary to the formation of reactive oxygen species, which will exhibit a threshold.
The Meeting concluded that malathion is unlikely to be genotoxic at anticipated dietary
exposures.
In the multigeneration and developmental toxicity studies, cholinesterase activity was not
measured.
In a two-generation reproductive toxicity study conducted in rats, which tested dietary
malathion concentrations of 0, 550, 1700, 5000 and 7500 ppm (equal to 0, 43, 130, 393 and
595 mg/kg bw per day for males and 0, 50, 152, 438 and 655 mg/kg bw per day for females,
respectively), the NOAEL for both reproductive toxicity and parental toxicity was 7500 ppm (equal to
595 mg/kg bw per day), the highest dose tested. The NOAEL for offspring toxicity was 1700 ppm
(equal to 130 mg/kg bw per day) for reduced pup weights at 5000 ppm (equal to 393 mg/kg bw per
day) (Schroeder, 1990).
Two published studies reported potential testicular toxicity in rats exposed to malathion orally
(Uzun et al., 2009; Geng et al., 2015), but these studies had a number of methodological limitations
that reduced their utility. Further, the reported observations are not corroborated by the preceding
GLP-compliant multigenerational rat study in which no effects on the testes were observed
(Schroeder, 1990).
A variety of in vivo and in vitro assays in mammalian and nonmammalian models indicated
that malathion is unlikely to affect the endocrine system (Barnett, 2011b,c,d; Palmer, 2011a,b,c;
Wagner, 2011; Wilga, 2011; Willoughby, 2011b; Kjeldsen, Ghisari & Bonefeld-Jørgensen, 2013).
In a pilot developmental toxicity study in rats, which tested gavage malathion doses of 0, 300,
600, 800 and 1000 mg/kg bw per day from days 6 to 15 of gestation, no embryo or fetal toxicity
occurred, whereas maternal toxicity occurred at and above 600 mg/kg bw per day (Lochry, 1988). In
the main developmental toxicity study in rats, which tested gavage doses of 0, 200, 400 and 800
mg/kg bw per day from days 6 to 15 of gestation, the NOAEL for maternal toxicity was 400 mg/kg
bw per day for clinical signs and reduced body weight gain and feed consumption at 800 mg/kg bw
per day. The NOAEL for embryo and fetal toxicity was 800 mg/kg bw per day, the highest dose tested
(Lochry, 1989).
In a range-finding developmental toxicity study in rabbits, which tested gavage malathion
doses of 0, 25, 50, 100, 200 and 400 mg/kg bw per day from days 6 to 18 of gestation, no embryo or
fetal toxicity occurred, whereas maternal toxicity occurred at 200 and 400 mg/kg bw per day (Siglin,
Voss & Becci, 1985). In the main study, which tested malathion doses of 0, 25, 50 and 100 mg/kg bw
per day from days 6 to 18 of gestation, the NOAEL for maternal toxicity was 25 mg/kg bw per day for
a marginal effect on body-weight gain at 50 mg/kg bw per day. The NOAEL for embryo and fetal
toxicity was 100 mg/kg bw per day, the highest dose tested.
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The Meeting concluded that malathion is not teratogenic.
In a study conducted in hens, there was no evidence that malathion caused delayed peripheral
neuropathy (Fletcher, 1989).
In an acute neurotoxicity study in rats, which tested gavage malathion doses of 0, 500, 1000
and 2000 mg/kg bw, the NOAEL was 1000 mg/kg bw for reduced erythrocyte acetylcholinesterase
activity in females and reduced ambulatory activity in males at 2000 mg/kg bw (Lamb, 1994a).
A 13-week neurotoxicity study in rats (Lamb, 1994b) is described above together with the
other 13-week toxicity studies in rats, and an overall NOAEL is identified for these studies.
In a developmental neurotoxicity study in rats, which tested gavage malathion doses of 0, 5,
50 and 150 mg/kg bw per day from day 6 of gestation to day 10 of lactation, the NOAEL for both
maternal toxicity and offspring toxicity was 50 mg/kg bw per day for clinical signs at 150 mg/kg bw
per day (Fulcher, 2002b; Reiss, 2004).
Administration of malathion from day 6 of gestation to day 21 of lactation had no effect on
the thickness of the corpus callosum in rat pups at doses up to 150 mg/kg bw per day (Myers, 2003,
2004).
The Meeting concluded that malathion is neurotoxic.
Studies in rats have examined the time to peak effect and compared the effects of malathion
and malaoxon on the inhibition of acetylcholinesterase activity. The time to peak effect in juvenile
rats following dosing with malathion ranged from 30 to 90 minutes for the inhibition of erythrocyte
acetylcholinesterase activity and from 60 to 90 minutes for the inhibition of brain acetylcholinesterase
activity (Stannard, 2006a,b,c; Barnett, 2008a,b,c). Malaoxon was a more potent inhibitor of
acetylcholinesterase activity compared with malathion (Barnett, 2006c, 2008d). Comparison of BMDs
following acute oral dosing indicated that the TAF for malaoxon was 21.6 in males and 17.8 in
females for the inhibition of erythrocyte acetylcholinesterase activity and 14.8 in males and 11.0 in
females for the inhibition of brain acetylcholinesterase activity (Reiss, 2008). Comparison of BMDs
for the inhibition of erythrocyte acetylcholinesterase activity from chronic toxicity studies indicated
that TAFs for malaoxon ranged from 37 to 38 in males and from 65 to 69 in females (Reiss, 2006a).
In a 6-week immunotoxicity study in female rats, which tested dietary malathion
concentrations of 0, 50, 100, 700 and 7000 ppm (equal to 0, 8.9, 17.6, 126.8 and 1215.8 mg/kg bw per
day, respectively), the NOAEL for immunotoxicity was 7000 ppm (equal to 1215.8 mg/kg bw per
day), the highest dose tested (Barnett, 2011d).
The Meeting concluded that malathion is not immunotoxic.
An extensive literature search did not identify any potential adverse effects on intestinal
microbiota or any evidence that intestinal microbiota can metabolize malathion.
Toxicological data on metabolites, degradates and/or impurities

Current FAO specifications for malathion prescribe maximum limits for isomalathion (CAS
No. 3344-12-5), malaoxon (CAS No. 1634-78-2), O,O,S-trimethyl phosphorothioate (CAS No.
2953-29-9) and O,S,S-trimethyl phosphorodithioate (CAS No. 152-18-1).
Toxicity tests were conducted on malaoxon, isomalathion, desmethyl malathion, desmethyl-
malathion monocarboxylic acid, MMCA, MDCA and desmethyl-malaoxon dicarboxylic acid.
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Malaoxon
The oral LD50 in rats for malaoxon was 50 mg/kg bw (Lowe, 2011b).
In a 14-day range-finding study in rats, which tested malaoxon at dietary concentrations of 0,
10, 25, 100, 2500 and 3500 ppm (equal to 0, 1.1, 3.0, 12.1, 293 and 387 mg/kg bw per day for males
and 0, 1.1, 3.1, 12.5, 281.6 and 294.7 mg/kg bw per day for females, respectively), inhibition of
erythrocyte acetylcholinesterase activity occurred at and above 100 ppm (equal to 12.1 mg/kg bw per
day). At the two highest doses, inhibition of brain acetylcholinesterase activity and reduced body-
weight gain and feed consumption occurred (Daly, 1995).
In a 103-week carcinogenicity study conducted in mice, which tested dietary malaoxon
concentrations of 0, 500 and 1000 ppm (estimated by a previous Meeting to be equal to 0, 75 and 150
mg/kg bw per day, respectively), survival and body weight were reduced at the highest dose. There
were no treatment-related neoplastic or non-neoplastic lesions (NCI, 1979b). In a parallel study
conducted in rats, which tested the same dietary concentrations of malathion (equal to 0, 25 and 50
mg/kg bw per day, respectively), the combined incidence of C-cell adenomas and carcinomas of the
thyroid in females was increased, although this was comparable to historical control values. The
incidence of gastric ulcers, commonly observed in the forestomach, was increased in treated rats.
In a 24-month toxicity study in rats, which tested malaoxon at dietary concentrations of 0, 20,
1000 and 2000 ppm (equal to 0, 1, 57 and 110 mg/kg bw per day for males and 0, 1, 68 and
140 mg/kg bw per day for females, respectively), the NOAEL for chronic toxicity was 20 ppm (equal
to 1 mg/kg bw per day), based on mortality and the inhibition of brain acetylcholinesterase activity at
1000 ppm (equal to 57 mg/kg bw per day). The NOAEL for carcinogenicity was 2000 ppm (equal to
110 mg/kg bw per day), the highest dose tested. Similar to studies conducted on malathion,
inflammatory changes in the nasal mucosa occurred at 1000 and 2000 ppm; these changes were likely
attributable to inhaled food particles containing malaoxon, resulting in tissue injury and inflammation
of the nasal cavity, with secondary effects on the lungs and middle ear (Daly, 1996b).
The Meeting concluded that malaoxon is not carcinogenic in mice or rats.
Malaoxon was negative for mutagenicity in bacterial assays (Zeiger et al., 1988; Schreib,
2015a) and in lower eukaryotes, both with and without metabolic activation (USEPA, 1977; Gilot-
Delhalle et al., 1983). Malaoxon was reported to be active for induction of sister chromatid exchanges
but not chromosomal aberrations in CHO cells, with or without metabolic activation (Ivett et al.,
1989). An increase in sister chromatid exchanges when tested in the absence of metabolic activation
only was also reported (Nishio & Uyeki, 1981); it was also reported that malaoxon was more potent
than malathion in this assay. Malaoxon was also reported to induce DNA damage as measured by the
comet assay in rat adrenal gland PC12 cells when tested in the absence of metabolic activation only
(Lu et al., 2012) and was mutagenic in mouse lymphoma (L5178Y) cells in the absence but not the
presence of metabolic activation (Myhr & Caspary, 1991). In this study, there seemed to be a
preference for the induction of small colonies, generally considered to be indicative of chromosomal
damage rather than gene mutations.
Malaoxon induced DNA damage in isolated lymphocytes in the absence of metabolic
activation, as measured by the alkaline comet assay; studies with metabolic activation were not
conducted (Blasiak et al., 1999). Further, a follow-up study concluded that the malaoxon-mediated
damage was likely induced by reactive oxygen species (Blasiak & Stankowska, 2001). Also,
malaoxon is more potent than malathion in inducing intracellular levels of reactive oxygen species
and reducing levels of catalase, superoxide dismutase and glutathione in rat PC12 cells treated in vitro
(Lu et al., 2012). When provided in food, malaoxon induced an increase in reciprocal translocations
and sex-linked recessive lethals in D. melanogaster, but not for sex-linked recessive lethals when
administered by injection (Foureman et al., 1994). Malaoxon was reported negative for the induction
of chromosomal aberrations and sister chromatid exchanges in the bone marrow cells of male mice
following a single intraperitoneal injection (NTP, 2016).
419
The Meeting concluded that the observed genotoxic effects occur secondary to the formation
of reactive oxygen species, which will exhibit a threshold.
The Meeting concluded that malaoxon is unlikely to be genotoxic at anticipated dietary
exposures.
Other metabolites
The oral LD50 in rats was greater than 2000 mg/kg bw for desmethyl malathion, desmethyl-
malathion monocarboxylic acid, MMCA, MDCA and desmethyl-malaoxon dicarboxylic acid (Pratt,
2005; Sanders, 2008a,b; Leoni, 2012). The oral LD50 in rats for desmethyl-malaoxon dicarboxylic
acid, trisodium salt, was greater than 2000 mg/kg bw (Allingham, 2015).
There are a limited number of genotoxicity studies on other metabolites of malathion. MDCA
(Taylor, 2008b), MMCA (Taylor, 2008a), desmethyl-malathion monocarboxylic acid, potassium salt
(Donath, 2012), and desmethyl-malaoxon dicarboxylic acid, trisodium salt (Schreib, 2015a), as well
as isomalathion, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate and O,S,S-
trimethyl phosphorodithioate (Imamura & Talcott, 1985), were reported negative for bacterial
mutagenicity, with and without metabolic activation. Isomalathion induced DNA damage in isolated
lymphocytes in the absence of metabolic activation, as measured by the alkaline comet assay; studies
with metabolic activation were not conducted (Blasiak et al., 1999). Isomalathion was also reported to
induce micronuclei in the human liver–derived HepaRG cell line (Josse et al., 2014).
Using QSAR, the storage impurity, 2-mercaptosuccinic acid diethyl ester, was determined to
have no greater toxicity than malathion (Clerkin, 2015).
The potential of malathion metabolites to inhibit acetylcholinesterase activity has been
studied in rats. Comparisons of erythrocyte acetylcholinesterase activities indicated that desmethyl
malathion, MMCA and MDCA are at least 2.75-, 1.9- and 4.6-fold less potent than malathion.
Based on a comparison of the inhibitions of acetylcholinesterase activities over acute and
chronic exposure durations and a comparison of BMDs (see above), the Meeting concluded that
malaoxon is approximately 30-fold more potent than malathion.
Human data
As in laboratory animals, the inhibition of acetylcholinesterase activity is the most sensitive
adverse effect in humans exposed to malathion, mediated through the metabolite malaoxon, which is a
more potent inhibitor of acetylcholinesterase activity compared with malathion. A comparative in
vitro study (Rodriguez et al., 1997) indicated that malaoxon was a slightly less potent inhibitor (less
thatn threefold) of human compared with rat acetylcholinesterase activity.
In a study conducted in male and female volunteers, which tested single oral doses of
malathion at 0, 0.5, 1.5, 5, 10 and 15 mg/kg bw, the NOAEL was 15 mg/kg bw, the highest dose
tested, based on the absence of any adverse effects, including the inhibition of erythrocyte
acetylcholinesterase activity (Gillies & Dickson, 2000). In a subsequent study conducted in male and
female volunteers, which tested single oral doses of malathion of 0, 0.5, 1.5, 5.0, 10.0 and 15.0 mg/kg
bw, There were no treatment-related adverse events or effects on erythrocyte acetylcholinesterase
activity (Jellinek, Schwartz & Connolly Inc., 2000).
In a published study, application of malathion to the forearm of human volunteers increased
blood flow, mediated via the inhibition of acetylcholinesterase activity (Boutsiouki & Clough, 2004).
In a published non-blinded study (Wananukul et al., 2011), slight inhibition of erythrocyte
acetylcholinesterase activity occurred in most of the children following two applications of a 1%
malathion shampoo used to treat head lice.
In a 1994 summary report (Nielsen, 1994), there were no poisoning incidents and no
inhibition of plasma cholinesterase activity in workers involved in the manufacture of malathion over
420
a 20-year period. In a subsequent summary report (Ravn Nielsen, 1999), biological monitoring of
workers employed at dimethoate and malathion manufacturing plants from 1994 to 1999 detected no
reduction in plasma cholinesterase activity.
Several epidemiological studies on cancer outcomes in relation to occupational exposure to
malathion were available. The evaluation of these studies focused on the occurrence of NHL and
prostate cancer, as outlined in section 2.2 of the meeting report. One meta-analysis was available, as
well as one prospective cohort study, the AHS, with a large sample size and detailed exposure
assessment. Cohort studies are considered a powerful design, as recall bias is avoided. All other
studies were case–control studies, usually retrospective, which are more prone to recall and selection
biases.
The AHS found no evidence of a positive association of NHL with malathion exposure or of
an exposure–response relationship (Alavanja et al., 2014; Lerro et al., 2015). In contrast, various
case–control studies reported excess risks of NHL associated with use of malathion. In a large pooled
case–control study, the unadjusted estimates showed a significant increased risk of NHL (RR: 1.6;
95% CI: 1.2–2.2) associated with ever-use versus never-use of malathion (Waddell et al., 2001).
However, these were attenuated and/or no longer significant when proxy respondents were excluded
and analyses were mutually adjusted for other pesticides (Waddell et al., 2001; De Roos et al., 2003).
Significant elevated risks of NHL were reported from the Cross-Canada Study of Pesticides and
Health for ever-use versus never-use of malathion (OR: 1.96; 95% CI: 1.42–2.70) (McDuffie et al.,
2001; Pahwa et al., 2012) and when examining annual days of use, although there was no clear
exposure–response relationship across exposure categories (McDuffie et al., 2001). Non-significant
increased risks of NHL were reported by two other case–control studies (Mills, Yang & Riordan,
2005; Eriksson et al., 2008), one of which had limited statistical power based on only five exposed
cases (Eriksson et al., 2008). The meta-analysis, which did not include the AHS, found a significant
80% excess risk ratio for ever-use versus never-use of malathion (Schinasi & Leon, 2014).
Overall, there is some very weak evidence of a positive association between malathion
exposure and NHL from the case–control studies and the overall meta-analysis. However, it is notable
that the AHS (Alavanja et al., 2014), which is the only cohort study and is large and of high quality,
found no evidence of an association at any exposure level.
There was no evidence of an association with all prostate cancers and malathion exposure in
the AHS (Koutros et al., 2013). However, a significant excess risk of aggressive prostate cancer (RR:
1.43; 95% CI: 1.08–1.88) in the highest exposure category (highest quintile of intensity-weighted
lifetime days of malathion exposure), along with a significant exposure–response relationship (P for
trend = 0.04), was observed (Koutros et al., 2013). A significant elevated risk of all prostate cancer
was observed in a case–control study (Band et al., 2011) for ever-use (OR: 1.34; 95% CI: 1.01–1.78)
and for highest lifetime cumulative exposure versus those unexposed (OR: 1.49; 95% CI: 1.02–2.18).
A significant trend across exposure categories (P = 0.03) was also reported. However, interpretation
of results from this study is limited by potential for exposure misclassification in the job–exposure
matrix used for exposure assessment and by the potential for residual confounding from lack of
adjustment for other pesticide exposures (Band et al., 2011). There was no evidence of an association
between prostate cancer and malathion exposure in the United Farm Workers of America study (Mills
& Yang, 2003), which was limited by the use of ecological rather than individual-level exposure
assessment.
Overall, the evidence is suggestive of a positive association between malathion exposure and
risk of aggressive prostate cancer; however, the evidence base is limited to the one large AHS cohort
study.
Based on a consideration of the results of animal bioassays, genotoxicity assays and
epidemiological data from occupational exposures, the Meeting concluded that malathion and its
metabolites are unlikely to pose a carcinogenic risk to humans from exposure via the diet.
The Meeting concluded that the existing database on malathion was adequate to characterize
421
The current Meeting reaffirmed the acceptable daily intake (ADI) of 0–0.3 mg/kg bw per day,
based on the NOAEL of 500 ppm (equal to 29 mg/kg bw per day) in the 2-year study of toxicity and
carcinogenicity in rats for the inhibition of brain acetylcholinesterase and using a 100-fold safety
factor, established by the 1997 Meeting. The margins of exposure between this ADI and the doses
causing liver adenomas in mice and nasal adenomas in rats are 5000-fold and 1200-fold, respectively.
The current Meeting reaffirmed the acute reference dose (ARfD) of 2 mg/kg bw, based on the
NOAEL of 15 mg/kg bw for the inhibition of erythrocyte acetylcholinesterase activity in a study
conducted in male and female volunteers with the application of a 10-fold safety factor, established by
the 2003 Meeting. This ARfD is supported by the NOAEL of 15 mg/kg bw in a second study
conducted in male and female volunteers. The ARfD is considered to be a conservative value, because
human acetylcholinesterase is slightly less sensitive (< 3-fold) than rat acetylcholinesterase to
malaoxon.
The Meeting concluded that the metabolite malaoxon is approximately 30-fold more toxic
than malathion. On this basis, a 30-fold potency factor should be applied to the residue levels for use
in both the acute and chronic dietary exposure estimates for malaoxon, and these should be added to
the dietary exposures for malathion and compared with the ARfD and ADI for malathion,
respectively.
Both the ADI and ARfD are established for the sum of malathion and malaoxon (corrected for
its potency), expressed as parent malathion. The other metabolites of malathion considered by the
present Meeting are less potent than the parent compound and therefore would be covered by the ADI
and ARfD for malathion. The impurity isomalathion may need to be taken into consideration in the
risk assessment depending on its concentration in food commodities.
Levels relevant to risk assessment of malathion

Mouse Two-year study of toxicity Toxicity 800 ppm, equal to 143 8 000 ppm, equal to
and carcinogenicitya mg/kg bw per day 1 476 mg/kg bw per
day
Carcinogenicity 800 ppm, equal to 143 8 000 ppm, equal to
mg/kg bw per day 1 476 mg/kg bw per
day
Rat Acute neurotoxicity studyb Toxicity 1 000 mg/kg bw per 2 000 mg/kg bw per
day day
One-month studies of Toxicity 500 ppm, equal to 51.9 5 000 ppm, equal to
toxicitya,c mg/kg bw per day 457.5 mg/kg bw per
day
Thirteen-week studies of Toxicity 500 ppm, equal to 5 000 ppm, equal to

toxicity and neurotoxicitya,c 34 mg/kg bw per day 340 mg/kg bw per day
Two-year study of toxicity Toxicity 500 ppm, equal to 29 6 000 ppm, equal to
and carcinogenicitya mg/kg bw per day 359 mg/kg bw per day
Carcinogenicity 500 ppm, equal to 29 6 000 ppm, equal to
mg/kg bw per day 359 mg/kg bw per day
Two-generation study of Reproductive 7 500 ppm, equal to –
reproductive toxicitya,e toxicity 595 mg/kg bw per dayd
Parental toxicity 7 500 ppm, equal to –
595 mg/kg bw per dayd
422

Offspring toxicity 1 700 ppm, equal to 5 000 ppm, equal to
Developmental toxicity Maternal toxicity 400 mg/kg bw per day 800 mg/kg bw per day
studyb,e
Embryo and fetal 800 mg/kg bw per dayd –
toxicity
Developmental neurotoxicity Maternal toxicity 50 mg/kg bw per day 150 mg/kg bw per day
studyb,e
Offspring toxicity 50 mg/kg bw per day 150 mg/kg bw per day
Rabbit Developmental toxicity Maternal toxicity 25 mg/kg bw per day 50 mg/kg bw per day
studyb,e d
Embryo and fetal 100 mg/kg bw per day –
toxicity
Dog One-year study of toxicityf Toxicity 125 mg/kg bw per day 250 mg/kg bw per day
c,f d
Human Acute volunteer studies Cholinesterase 15 mg/kg bw –
inhibition
a
b
c
d
e
f
Capsule administration.

0–0.3 mg/kg bw (for sum of malathion and malaoxon, adjusted for its potency and expressed
as malathion)

2 mg/kg bw (for sum of malathion and malaoxon, adjusted for its potency and expressed as
malathion)
human exposure
Results from in vivo genotoxicity studies investigating oral dosing, because malathion
genotoxicity data are highly variable and inconsistent and there is a lack of robust in vivo rodent
studies using the oral route of exposure
Critical end-points for setting guidance values for exposure to malathion

Rate and extent of oral absorption Rapid; > 77%
Dermal absorption Estimates vary (1.44–20.7% in human skin)
Distribution Rapid tissue distribution
Potential for accumulation No potential for accumulation
Rate and extent of excretion Rapid and complete
423
Metabolism in animals Extensive; oxidation, hydrolysis, dealkylation and demethylation

reactions
Toxicologically significant compounds in animals Malathion, malaoxon, desmethyl malathion, desmethyl malaoxon,
and plants MMCA, MDCA, isomalathion
Acute toxicity
Rat, LD50, oral > 1 539 to < 8 227 mg/kg bw
Rat, LC50, inhalation > 5.2 mg/L
Rabbit, dermal irritation Slightly irritating
Rabbit, ocular irritation Slightly irritating
Guinea-pig, dermal sensitization Not sensitizing (Buehler assay)
Sensitizing (maximization assay)
Mouse, dermal sensitization Not sensitizing (local lymph node assay)
Lowest relevant oral NOAEL 51.9 mg/kg bw per day (28 days; rat)
Lowest relevant dermal NOAEL 150 mg/kg bw per day (21 days; rabbit)
Lowest relevant inhalation NOAEC < 0.1 mg/L (13 weeks; rat)
Long-term studies of toxicity and carcinogenicity
Lowest relevant NOAEL 29 mg/kg bw per day (rat)
Carcinogenicity Some evidence of carcinogenicity in mice and ratsa
Genotoxicity
Genotoxic, possibly due to the generation of reactive oxygen
speciesa
Reproduction target/critical effect No effect on reproduction
Lowest relevant parental NOAEL 595 mg/kg bw per day (rat; highest dose tested)b
Lowest relevant offspring NOAEL 130 mg/kg bw per day (rat)b
Lowest relevant reproduction NOAEL 595 mg/kg bw per day (rat; highest dose tested)b
Developmental target/critical effect Marginally reduced maternal body-weight gain
Lowest maternal NOAEL 25 mg/kg bw per day (rabbit)b
Lowest embryo/fetal NOAEL 100 mg/kg bw per day (rabbit; highest dose tested)b
Neurotoxicity
Acute neurotoxicity NOAEL 1 000 mg/kg bw
Subchronic neurotoxicity NOAEL 4 mg/kg bw per dayc
Developmental neurotoxicity NOAEL 50 mg/kg bw per dayb
Delayed neurotoxicity No evidence
Other toxicological studies
Immunotoxicity NOAEL 1 216 mg/kg bw per day (rat; highest dose tested)
Not immunotoxic
Toxicological studies on malaoxon
424
Rat, LD50, oral 50 mg/kg bw

Lowest relevant long-term NOAEL 1 mg/kg bw per day (rat)
Carcinogenicity No evidence of carcinogenicity (mouse, rat)
Genotoxicity Some evidence of genotoxicity, secondary to the formation of
reactive oxygen species
Toxicological studies on desmethyl-malathion,
sodium salt
Rat, LD50, oral > 2 000 mg/kg bw
Genotoxicity Not mutagenic in prokaryotic assays
Toxicological studies on desmethyl-malathion
monocarboxylic acid, potassium salt
Toxicological studies on MMCA
Toxicological studies on MDCA
Toxicological studies on desmethyl-malaoxon
dicarboxylic acid
Human data Acetylcholinesterase inhibition:
Acute NOAEL: 15 mg/kg bw, highest dose tested
No adverse effects in manufacturing personnel
a
Unlikely to pose a carcinogenic risk to humans from the diet.
b
c
Ninety-day neurotoxicity study in rats is covered by the overall oral NOAEL for repeated-dose studies of toxicity.
Summary
Value Studies Safety factor
ADI 0–0.3 mg/kg bw Two-year chronic toxicity and carcinogenicity study (rat) 100
ARfD 2 mg/kg bw Single-dose studies (humans) 10
ADI: acceptable daily intake; AFfD: acute reference dose
425
References
Abraham SS, Manohar BM, Sundararaj A, Thiagarajan V (1997). Genotoxicity of malathion - a sub-chronic
study in mice. Indian Vet J. 74:565–7.
Ahmed T, Pathak R, Mustafa MD, Kar R, Tripathi AK, Ahmed RS et al. (2011). Ameliorating effect of N-
acetylcysteine and curcumin on pesticide-induced oxidative DNA damage in human peripheral blood
mononuclear cells. Environ Monit Assess. 179:293–9.
risk and insecticide, fungicide and fumigant use in the Agricultural Health Study. PLoS One.
9(10):e109332. doi:10.1371/journal.pone.0109332.
Allingham P (2015). Acute oral toxicity (up and down procedure) in the rat with desmethyl malaoxon
dicarboxylic acid, trisodium salt. Study No. 153095 by Eurofins Munich. Unpublished report, Cheminova
A/S, CHA Doc. No. 1619 FYF. Submitted to WHO by FMC/Cheminova.
Amer SM, Fahmy MA, Donya SM (1996). Cytogenetic effect of some insecticides in mouse spleen. J Appl
Toxicol. 16(1):1–3.
Amer SM, Fahmy MA, Aly FA, Farghaly AA (2002). Cytogenetic studies on the effect of feeding mice with
stored wheat grains treated with malathion. Mutat Res. 513:1–10.
Aston LS (2000). Determination of residues of malathion dicarboxylic acid (DCA), malathion monocarboxylic
acid (MCA), dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), and dimethyl dithiophosphate
(DMDTP) in human urine. Study no. PTL119801 by Pacific Toxicology Laboratories. Unpublished report,
Cheminova A/S, CHA Doc. No.: 299 FYF suppl-5 (former 299 FYF Amdt-5). Submitted to WHO by
FMC/Cheminova.
Balaji M, Sasikala K (1993). Cytogenetic effect of malathion in in vitro culture of human peripheral blood.
Mutat Res. 301:13–7.
pesticides in British Columbia farmers. Prostate. 71(2):168–83.
Barnett Jr JF (2006a). Final pilot report: Oral (gavage) dose range-finding study including preliminary
cholinesterase sensitivity assessment of malathion and malaoxon in juvenile rats. Study no. TQC00011 by
Charles River Laboratories, Preclinical Services. Unpublished report, Cheminova A/S, CHA Doc. No.:
1154 FYF. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2006b). Oral (gavage) repeat dose time of peak cholinesterase depression study of malathion and
malaoxon in juvenile rats. Study no. TQC00012 by Charles River Laboratories, Preclinical Services.
Unpublished report, Cheminova A/S, CHA Doc. No.: 1155 FYF. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2006c). Oral (gavage) repeat dose comparative cholinesterase study of malathion and malaoxon in
juvenile rats. Study no. TQC00013 by Charles River Laboratories, Preclinical Services. Unpublished report,
Cheminova A/S, CHA Doc. No.: 535 FYF. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2006d). Amendment 1 for MRID 46790501: Percutaneous repeated dose 21-day toxicity study of
malathion in rabbits. Study no. TQC00016 by Charles River Laboratories Preclinical Services. Unpublished
report, Cheminova A/S, CHA Doc. No.: 541 FYF amdt-1. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2007). Oral (gavage) acute dose range-finding cholinesterase depression study of malaoxon in
juvenile rats. Study no. TQC00022; Report no. by Charles River Laboratories, Preclinical Services.
Barnett Jr JF (2008a). Amendment 1 for oral (gavage) acute dose time of peak cholinesterase depression study
of malathion and malaoxon in juvenile rats. Study no. TQC00021 Amendment 1 by Charles River
Laboratories, Preclinical Services. Unpublished report, Cheminova A/S, CHA Doc. No.: 1564 FYF amdt-1.
Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2008b). Oral (gavage) acute dose time of peak cholinesterase depression study of malaoxon in
juvenile rats. Study no. TQC00031; Report no. by Charles River Laboratories, Preclinical Services.
Barnett Jr JF (2008c). Oral (gavage) acute dose time of peak cholinesterase depression study of malathion in
426
Barnett Jr JF (2008d). Oral (gavage) acute dose comparative cholinesterase study of malathion and malaoxon in
Barnett Jr JF (2008e). Oral (gavage) acute dose range-finding study of malathion; desmethyl malathion, Na-salt;
malathion monocarboxylic acid; or malathion dicarboxylic acid in adult rats. Study no. TQC00052 by
Charles River Laboratories, Preclinical Services. Unpublished report, Cheminova A/S, CHA Doc. No.: 617
FYF. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2011a). Oral (gavage) dosage range-finding toxicity study of malathion in rats: final amended
report. Study no. 20014600 by Charles River Laboratories Preclinical Services. Unpublished report,
Cheminova A/S, CHA Doc. No.: 1057 FYF amdt-1. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2011b). Oral (gavage) uterotrophic bioassay of malathion in female rats. Study no. 20012263 by
Charles River Laboratories Preclinical Services. Unpublished report, Cheminova A/S, CHA Doc. No.: 1055
Barnett Jr JF (2011c). Oral (gavage) Hershberger bioassay of malathion in male rats. Study no. 20012264 by
Charles River Laboratories Preclinical Services. Unpublished report, Cheminova A/S, CHA Doc. No.: 1056
Barnett Jr JF (2011d). Oral (diet) repeated dose six-week immunotoxicity study of malathion in mice. Study no.
TQC00068 by Charles River Laboratories Preclinical Services. Unpublished report, Cheminova A/S, CHA
Doc. No.: 1153 FYF. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2012a). Oral (diet) repeated dose 28-day toxicity study of malathion technical in rats. Study no.
Barnett Jr JF (2012b). Oral (diet) repeated dose 90-day toxicity study of malathion technical in rats. Study no.
Barnett Jr JF (2012c). oral (gavage) repeated dose 14-day dose range-finding study of malathion in rats. Study
no. 20014321 by Charles River Laboratories Preclinical Services. Unpublished report, Cheminova A/S,
CHA Doc. No.: 1186 FYF. Submitted to WHO by FMC/Cheminova.
Barnett Jr JF (2012d). A pubertal development and thyroid function study of malathion by oral gavage in
peripubertal male and female rats. Study no. 20012266 by Charles River Laboratories Preclinical Services.
Barnett Jr JF (2014) Oral (gavage) acute time of peak effect cholinesterase study of malathion dicarboxylic acid
in adult rats. Study no. 20039196 by Charles River Laboratories, Preclinical Services. Unpublished report,
Beattie G (1993). A 2-week toxicity study of aerosolized malathion administered by whole-body inhalation
exposure to the albino rat. Study no. 90557 by Bio-Research Laboratories Ltd. Unpublished report,
Beattie G (1994). A 13-week toxicity study of aerosolized malathion administered by whole body inhalation
exposure to the albino rat. Study no. 90729 by Bio-Research Laboratories Ltd. Unpublished report,
Beevers C (2009). Reverse mutation in five histidine-requiring strains of Salmonella typhimurium. Malathion
product no. 340 spiked to the specification limit. Study no. 676/139 by Covance Laboratories Ltd.
Benedetti D, Nunes E, Sarmento M, Porto C, Dos Santos CE, Dias JF et al. (2013). Genetic damage in soybean
workers exposed to pesticides: Evaluation with the comet and buccal micronucleus cytome assays. Mutat
Res. 752:28–33.
Bianchi J, Mantovani MS, Marin-Morales MA (2015). Analysis of the genotoxic potential of low concentrations
of malathion on the Allium cepa cells and rat hepatoma tissue culture. J Enviro Sci (China). 36:102–111.
Błasiak J, Jałoszynski P, Trzeciak A, Szyfter K (1999). In vitro studies on the genotoxicity of the
organophosphorus insecticide malathion and its two analogues. Mutat Res. 445:275–83.
427
Blasiak J, Stańkowska D (2001). Genotoxicity of malaoxon: induction of oxidized and methylated bases and
protective effect of α-tocopherol. Pestic Biochem Physiol. 71:88–96.
Bollen LS (2003a). Malathion technical (fortified): Acute dermal toxicity study in the rat. Study no. 49934 by
Scantox. Unpublished report, Cheminova A/S, CHA Doc. No.: 435 FYF. Submitted to WHO by
FMC/Cheminova.
Bollen LS (2003b). Malathion technical (fortified): primary skin irritation study in the rabbit. Study no. 49935
by Scantox. Unpublished report, Cheminova A/S, CHA Doc. No.: 442 FYF. Submitted to WHO by
FMC/Cheminova.
Bollen LS (2003c). Malathion technical (fortified): Acute eye irritation/corrosion study in the rabbit. Study no.
49936 by Scantox. Unpublished report, Cheminova A/S, CHA Doc. No.: 444 FYF. Submitted to WHO by
FMC/Cheminova.
Bollen LS (2003d). Malathion technical (fortified): Test for delayed contact hypersensitivity using the guinea
pig maximisation test. Study no. 49842 by Scantox. Unpublished report, Cheminova A/S, CHA Doc. No.:
Bolte H (1999a). A 24-month oral toxicity/oncogenicity study of malathion in the rat via dietary administration:
Nasal tissue evaluation and peer review: Supplemental information for MRID 44782301. Unpublished
report, Cheminova A/S, CHA Doc. No.: 164 FYF suppl-2.1. Submitted to WHO by FMC/Cheminova.
Bolte HF (1999b). Extent and nature of pathology assessment of the oral cavity of rats from the malathion
chronic toxicity/oncogenicity study: Supplement to MRID No. 43942901. Huntingdon Life Sciences.
Unpublished report, Cheminova A/S, CHA Doc. No.: 164 FYF suppl-3. Submitted to WHO by
FMC/Cheminova.
Bonner MR, Coble J, Blair A, Beane Freeman LE, Hoppin JA, Sandler DP et al. (2007). Malathion exposure
and the incidence of cancer in the Agricultural Health Study. Am J Epidemiol. 166(9):1023–34.
Bouchard M, Gosselin NH, Brunet RC, Samuel O, Dumoulin MJ, Carrier G (2003). A toxicokinetic model of
malathion and its metabolites as a tool to assess human exposure and risk through measurements of urinary
biomarkers. Toxicol Sci. 73:182–94.
Bowles AJ (2005). Malathion technical: reverse mutation assay “Ames Test” using Salmonella typhimurium.
Study no. 0545/0354 by SafePharm Laboratories Limited. Unpublished report, Cheminova A/S, CHA Doc.
No.: 513 FYF. Submitted to WHO by FMC/Cheminova.
Bulsiewicz H, Rózewicka I, Januszewska H, Bajko J (1976). Aberrations of meiotic chromosomes induced in
mice with insecticides. Folia Morphol. 3:361–8.
Buratti FM, D’Aniello A, Volpe M, Meneguz A, Testai E (2005). Malathion bioactivation in the human liver:
The contribution of different cytochrome P450 isoforms. Drug Metab Dispos. 33(3):295–302.
Buratti FM, Testai E (2005). Malathion detoxification by human hepatic carboxylesterases and its inhibition by
isomalathion and other pesticides. J Biochem Mol Toxic. 19(6):406–14.
Boutsiouki P, Clough GF (2004). Modulation of microvascular function following low-dose exposure to the
organophosphorous compound malathion in human skin in vivo. J Appl Physiol. 97:1091-7.
Cabello G, Valenzuela M, Vilaxa A, Durán V, Rudolph I, Hrepic N et al. (2001). A rat mammary tumor model
induced by the organophosphorous pesticides parathion and malathion, possibly through
acetylcholinesterase. Environ Health Perspect. 109:471–9.
Chen L, Zhao T, Pan C, Ross J, Ginevan M, Vega H et al. (2013). Absorption and excretion of
organophosphorous insecticide biomarkers of malathion in the rat: Implications for overestimation bias and
exposure misclassification from environmental biomonitoring. Regul Toxicol Pharmacol. 65:287–93.
Chen HH, Hsueh JL, Sirianni SR, Huang CC (1981). Induction of sister-chromatid exchanges and cell cycle
delay in cultured mammalian cells treated with eight organophosphorus pesticides. Mutat Res. 88:307–16.
Clerkin D (2015). In-silico toxicology predictions for malathion and one impurity. Study no. 100563 by
Cheminova A/S. Unpublished report, Cheminova A/S, CHA Doc. No.: 1501 FYF. Submitted to WHO by
FMC/Cheminova.
Daly IW (1993a). A 28-day study of malathion in the rat via dietary administration. Study no. 92-3806 by
Bio/Dynamics Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 68 FYF. Submitted to WHO by
FMC/Cheminova.
428
Daly IW (1993b). A subchronic (3-month) oral toxicity study of malathion in the rat via dietary administration.
Study no. 92-3843 by Bio/dynamics, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 70 FYF.
Daly IW (1995). A 14-day range-finding study of malaoxon in the rat via dietary administration. Final report.
Study no. 92-3816 by Pharmaco LSR. Unpublished report, Cheminova A/S, CHA Doc. No.: 166 FYF.
Daly IW (1996a). A 24-month oral toxicity/oncogenicity study of malathion in the rat via dietary administration:
Final report. Study no. 90-3641 by Huntingdon Life Sciences. Unpublished report, Cheminova A/S, CHA
Daly IW (1996b). A 24-month oral toxicity/oncogenicity study of malaoxon in the rat via dietary administration:
Final report. Study no. 93-2234 by Huntingdon Life Sciences. Unpublished report, Cheminova A/S, CHA
Daly IW (1999). Amended report for MRID No. 43942901: a 24-month oral toxicity/oncogenicity study of
malathion in the rat via dietary administration. Study no. 90-3641 [Amendment] by Huntingdon Life
Sciences. Unpublished report, Cheminova A/S, CHA Doc. No.: 164 FYF amdt-1. Submitted to WHO by
FMC/Cheminova.
Davies HW, Kennedy SM, Teschke K, Jenny P, Quintana E (1998). Cytogenetic analysis of South Asian berry
pickers in British Columbia using the micronucleus assay in peripheral lymphocytes. Mutat Res. 416:101–
13.
de Ligt RAF (2004). In vitro percutaneous absorption of [ 14C] malathion through human and rat skin membranes
using flow-through diffusion cells. Study no. V 5299 by TNO Chemistry, Zeist. Unpublished report,
Med. 60:E11. doi:10.1136/oem.60.9.e11.
Dean BJ (1972). The mutagenic effects of organophosphorus pesticides on micro-organisms. Arch Toxicol.
30:67–74.
Decker U, Knuppe C, Ullrich A (2003). Malathion technical (fortified): 4-hour acute inhalation toxicity study in
rats. Study no. 846761 by RCC Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 443 FYF.
De Ferrari M, Artuso M, Bonassi S, Bonatti S, Cavalieri Z, Pescatore D et al. (1991). Cytogenetic biomonitoring
of an Italian population exposed to pesticides: chromosome aberration and sister-chromatid exchange
analysis in peripheral blood lymphocytes. Mutat Res. 260:105–13.
Degraeve N, Chollet MC, Moutschen J (1984a). Cytogenetic and genetic effects of subehronic treatments with
organophosphorus insecticides. Arch Toxicol. 56:66–7.
Degraeve N, Chollet MC, Moutschen J (1984b). Cytogenetic effects induced by organophosphorus pesticides in
mouse spermatocytes. Toxicol Lett. 21: 315–9.
Degraeve N, Moutschen J (1984). Genetic and cytogenetic effects induced in the mouse by an
organophosphorus insecticide: malathion. Environ Res. 34:170–4.
Dinse GE, Peddada SD, Harris SF, Elmore SA (2010). Comparison of NTP historical control tumor incidence
rates in female Harlan Sprague Dawley and Fischer 344/N rats. Toxicol Pathology. 38(5):765-75.
Donath C (2012). Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with
desmethyl malathion monocarboxylic acid, potassium salt. Study no. 120795 by BSL Bioservice Scientific
Laboratories GmbH, DEU. Unpublished report, Cheminova A/S, CHA Doc. No.: 1158 FYF. Submitted to
WHO by FMC/Cheminova.
Dulout FN, Olivero OA, von Guradze H, Pastori MC (1982). Cytogenetic effect of malathion assessed by the
micronucleus test. Mutat Res. 105:413–6.
Dulout FN, Pastori MC, Olivero OA (1983). Malathion-induced chromosomal aberrations in bone-marrow cells
of mice: dose-response relationships. Mutat Res. 122:163–7.
Dzwonkowska A, Hübner H (1986). Induction of chromosomal aberrations in the Syrian hamster by insecticides
tested in vivo. Arch Toxicol. 58:152–6.
429
Edwards CN (2001a). Malathion technical: in vitro mammalian cell gene mutation test performed with mouse
lymphoma cells (L5178Y). Study no. 40413 by Scantox. Unpublished report, Cheminova A/S, CHA Doc.
Edwards CN (2001b). Malathion technical: in vitro mammalian chromosome aberration test performed with
human lymphocytes. Study no. 40412 by Scantox. Unpublished report, Cheminova A/S, CHA Doc. No.:
324 FYF. Submitted to WHO by FMC/Cheminova. Submitted to WHO by FMC/Cheminova.
Feldmann RJ, Maibach HI (1974). Percutaneous penetration of some pesticides and herbicides in man. Toxicol
Appl Pharmacol. 28:126–32.
Fischer JE, Lowe CA, Lubas RL, Colavita JH (1988). AC 6,601: a 28-day oral toxicity study in beagle dogs.
Study no. 0852 by American Cyanamid Company. Unpublished report, Cheminova A/S, CHA Doc. No.: 40
Fischer JE (1991a). Oral LD50 study in albino rats with AC 6,601 malathion technical (Warners Production
Batch). Study no. A91-204 by American Cyanamid Company. Unpublished report, Cheminova A/S, CHA
Fischer JE (1991b). Oral LD 50 study in albino rats with AC 6,601 malathion technical (Cheminova Production
Batch). Study no. A91-205 by American Cyanamid Company. Unpublished report, Cheminova A/S, CHA
Fletcher DW (1989). 42-Day neurotoxicity study with AC 6,601 technical in mature White Leghorn hens. Study
no. BLAL 87-DN-109 by Bio-Life Associates Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 16
Foureman P, Mason JM, Valencia R, Zimmering S (1994). Chemical mutagenesis testing in Drosophila. X.
Results of 70 coded chemicals tested for the National Toxicology Program. Environ Mol Mutagen. 23:208–
27.
Fulcher SM (2001). Malathion effects on cholinesterase in the CD rat (adult and juvenile) by oral gavage
administration. Study no. CHV/067/12452 by Huntingdon Life Sciences Ltd. Unpublished report,
Fulcher SM (2002a). Malathion dose finding study in CD rats by oral gavage administration preliminary to
developmental neurotoxicity study. Study no. CHV062/010003 by Huntingdon Life Sciences Ltd.
Fulcher SM (2002b). Malathion developmental neurotoxicity study in the CD rat by oral gavage administration.
Study no. CHV/066/013331 by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova A/S, CHA
Fulcher SM (2003). Amendment 1 to the final report: malathion effects on cholinesterase in the CD rat (adult
and juvenile) by oral gavage administration. Study no. CHV/067/012452 by Huntingdon Life Sciences Ltd.
Unpublished report, Cheminova A/S, CHA Doc. No.: 330 FYF Amdt-1. Submitted to WHO by
FMC/Cheminova.
Galindo Reyes JG, Leyva NR, Millan OA, Lazcano GA (2002). Effects of pesticides on DNA and protein of
shrimp larvae Litopenaeus stylirostris of the California Gulf. Ecotoxicol Environ Saf. 53(2):191–5.
Galloway SM, Armstrong MJ, Reuben C, Colman S, Brown B, Cannon C et al. (1987). Chromosome aberration
and sister chromatid exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ Mol
Mutagen. 10 (Suppl 10):1–175.
Garaj-Vrhovac V, Zeljezic D (2000). Evaluation of DNA damage in workers occupationally exposed to
pesticides using single-cell gel electrophoresis (SCGE) assay. Pesticide genotoxicity revealed by comet
assay. Mutat Res. 469:279–85.
Garaj-Vrhovac V, Zeljezic D (2001). Cytogenetic monitoring of Croatian population occupationally exposed to
a complex mixture of pesticides. Toxicology. 165:153–62.
Garaj-Vrhovac V, Zeljezic D (2002). Assessment of genome damage in a population of Croatian workers
employed in pesticide production by chromosomal aberration analysis, micronucleus assay and Comet
assay. J Appl Toxicol. 22(4):249–55.
430
Garry VF, Nelson RL, Griffith J, Harkins M (1990). Preparation for human study of pesticide applicators: sister
chromatid exchanges and chromosome aberrations in cultured human lymphocytes exposed to selected
fumigants. Teratog Carcinog Mutagen. 10:21–9.
Geng X, Shao H, Zhang Z, Ng JC, Peng C (2015). Malathion-induced testicular toxicity is associated with
spermatogenic apoptosis and alterations in testicular enzymes and hormone levels in male Wistar rats.
Environ Toxicol Pharmacol. 39:659-67.
Gillies D, Dickson J (2000). A randomised double blind ascending single oral dose study with malathion to
determine the no effect level on plasma and erythrocyte cholinesterase activity. Study no. ICR 013177 by
Inveresk Research. Unpublished report, Cheminova A/S, CHA Doc. No.: 299 FYF. Submitted to WHO by
FMC/Cheminova.
Gilot-Delhalle J, Colizzi A, Moutschen J, Moutschen-Dahmen M (1983). Mutagenicity of some
organophosphorus compounds at the ade6 locus of Schizosaccharomyces pombe. Mutat Res. 117:139–48.
Giri A, Giri S, Sharma GD (2011). Malathion and fenvalerate induce micronuclei in mouse bone marrow cells.
Environ Mol Mutagen. 52:607–13.
Giri S, Prasad SB, Giri A, Sharma GD (2002). Genotoxic effects of malathion: an organophosphorus insecticide,
using three mammalian bioassays in vivo. Mutat Res. 514:223–31.
Giri A, Yadav SS, Giri S, Sharma GD (2012). Effect of predator stress and malathion on tadpoles of Indian
skittering frog. Aquat Toxicol. 106–107:157–63.
Griffin DE 3rd, Hill WE (1978). In vitro breakage of plasmid DNA by mutagens and pesticides. Mutat Res.
52:161–9.
Gudi R (1990). Acute test for chemical induction of chromosome aberration in rat bone marrow cells in vivo
with AC 6,601. Study no. 0125-1531 by SITEK Research Laboratories. Unpublished report, Cheminova
A/S, CHA Doc. No.: 33 FYF. Submitted to WHO by FMC/Cheminova.
Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso-Coello P et al. (2008). GRADE: an emerging
consensus on rating quality of evidence and strength of recommendations. BMJ. 336:924–6.
Hardisty JF (1998). Pathology Working Group (PWG) peer review of proliferative lesions of the liver in male
B6C3F1 mice in an 18-month oral (dietary) oncogenicity study in mice of malathion: PWG report. Study
no. 297-003 by Experimental Pathology Laboratories, Inc. Unpublished report, Cheminova A/S, CHA Doc.
No.: 94 FYF suppl-1. Submitted to WHO by FMC/Cheminova [cited in Slauter, 1994].
Hardisty JF (2000). Pathology Working Group (PWG) peer review of proliferative lesions of the liver in female
rats in a 24-month oral toxicity/oncogenicity study of malathion (MRID 43942901). Study no. 297-006 by
Experimental Pathology Laboratories, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 164 FYF
suppl-5. Submitted to WHO by FMC/Cheminova.
Haseman JK, Arnold J, Eustis SL (1990). Tumour incidences in Fischer 344 rats: NTP historical control data In:
Boorman GA, Eustis SL, Montgomery C, editors. Pathology of the Fischer rat. San Diego (CA): Academic
Press; 555–64.
Haseman JK, Hailey JR, Morris RW (1998). Spontaneous neoplasm incidence in Fischer 344 rats and B6C3F1
mice in two-year carcinogenicity studies: a National Toxicology Program update. Toxicol Pathol.
26(3):428-41.
Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983). Salmonella mutagenicity test results for 250
chemicals. Environmental Mutagenesis. 5(Suppl 1):1–142.
Herath JF, Jalal SM, Ebertz MJ, Martsolf JT (1989). Genotoxicity of the organophosphorus insecticide
malathion based on human lymphocytes in culture. Cytologia. 54:191–5.
Hoda Q, Sinha SP (1991). Minimization of cytogenetic toxicity of malathion by vitamin C. J Nutr Sci Vitaminol
(Tokyo). 37:329–39.
Hohenadel K, Harris SA, McLaughlin JR, Spinelli JJ, Pahwa P, Dosman JA et al. (2011). Exposure to multiple
pesticides and risk of non-Hodgkin lymphoma in men from six Canadian provinces. Int J Environ Res
Public Health. 8:2320–30.
Huang CC (1973). Effect on growth but not on chromosomes of the mammalian cells after treatment with three
organophosphorus insecticides. Proc Soc Exp Biol Med. 142:36–40.
431
Huff JE, Bates R, Eustis SL, Haseman JK, McConnell EE (1985). Malathion and malaoxon: Histopathology re-
examination of the National Cancer Institute’s carcinogenesis studies. Environ Res. 37:54–173.
Hussain R, Mahmood F, Khan A, Ali F (2015). Genotoxic and pathological effects of malathion in male
Japanese quail (Coturnix japonica). Pakistan J Agric Sci. 52:1149–56.
Hyun UH, Lee SY (1976). Salmonella/microsomal enzyme activation system. Korean J Microbiol. 14:128–34.
Imamura T, Talcott RE (1985). Mutagenic and alkylating activities of organophosphate impurities of
commercial malathion. Mutat Res. 155:1–6.
Ivett JL, Brown BM, Rodgers C, Anderson BE, Resnick MA, Zeiger E (1989). Chromosomal aberrations and
sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals.
Environ Mol Mutagen. 14:165–87.
Jackson GC (1986). Fyfanon (malathion) 96/98% Technical. acute inhalation toxicity study in rats. 4-hour
exposure. Study no. CHV28/8640 by Huntingdon Research Center. Unpublished report, Cheminova A/S,
Jellinek, Schwartz & Connolly Inc (2000). The effects and pharmacological disposition of a single oral dose of
malathion administered to human volunteers. Unpublished report, Cheminova A/S, CHA Doc. No.: 299
FYF suppl-4. Submitted to WHO by FMC/Cheminova.
Josse R, Sharanek A, Savary CC, Guillouzo A (2014). Impact of isomalathion on malathion cytotoxicity and
genotoxicity in human HepaRG cells. Chem Biol Interact. 209:68–76.
Kandiel MM, El-Asely AM, Radwan HA, Abbass AA (2014). Modulation of genotoxicity and endocrine
disruptive effects of malathion by dietary honeybee pollen and propolis in Nile tilapia (Oreochromis
niloticus). J Adv Res. 5(6):671–84.
Kjeldsen LS, Ghisari M, Bonefeld-Jørgensen EC (2013). Currently used pesticides and their mixtures affect the
function of sex hormone receptors and aromatase enzyme activity. Toxicol Appl Pharmacol. 272:453-64.
doi: 10.1016/j.taap.2013.06.028.
doi:10.1093/aje/kws225
Kuhn JO (1996). Fyfanon purified. Final report. Acute oral toxicity study in rats. Study no. 2586-95 by
Stillmeadow, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 159 FYF. Submitted to WHO by
FMC/Cheminova.
Kumar D, Khan PK, Sinha SP (1995). Cytogenetic toxicity and no-effect limit dose of pesticides. Food Chem
Toxicol. 33:309–14.
Kumar R, Nagpure NS, Kushwaha B, Srivastava SK, Lakra WS (2010). Investigation of the genotoxicity of
malathion to freshwater teleost fish Channa punctatus (Bloch) using the micronucleus test and comet assay.
Arch Environ Contam Toxicol. 58:123–30.
Kurinniy AI (1975). Comparative investigation of the cytogenetic effect of certain organophosphorus
insecticides. Genetika. 11:64–9.
Kynoch SR (1986a). Acute oral toxicity to rats of malathion (Fyfanon). Technical study no.
851341D/CHV33/AC by Huntingdon Research Center. Unpublished report, Cheminova A/S, CHA Doc.
Kynoch SR (1986b). Acute dermal toxicity to rats of malathion (Fyfanon). Technical study no.
851330D/CHV/34/AC by Huntingdon Research Center. Unpublished report, Cheminova A/S, CHA Doc.
Kynoch SR, Smith PA (1986). Delayed contact hypersensitivity in the guinea pig with malathion (Fyfanon).
Technical. study no. 8666D/CHV37/SS by Huntingdon Research Center. Unpublished report, Cheminova
Lamb IC (1994a). An acute neurotoxicity study of malathion in rats. Study no. WIL-206005 by Wil Research
Laboratories, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 78 FYF. Submitted to WHO by
FMC/Cheminova.
432
Lamb IC (1994b). A subchronic (13-week) neurotoxicity study of malathion in rats. Study no. WIL-206006 by
Wil Research Laboratories, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 82 FYF. Submitted
to WHO by FMC/Cheminova.
Lee WJ, Cantor KP, Berzofsky JA, Zahm SH (2004). Non Hodgkin's Lymphoma among asthmatics exposed to
pesticides. Int J Cancer. 111:298–302.
Leoni A-L (2012). Acute oral toxicity (up and down procedure) with desmethyl malathion monocarboxylic acid,
potassium salt. Study no. 120794 by BSL Bioservice Scientific Laboratories GmbH, DEU. Unpublished
report, Cheminova A/S, CHA Doc. No.: 1159 FYF. Submitted to WHO by FMC/Cheminova.
Lerro CC, Koutros S, Andreotti G, Friesen MC, Alavanja MC, Blair A et al. (2015) Organophosphate
Liggett MP, Parcell BI (1985a). Irritant effects on rabbit skin of malathion (fyfanon) technical. Study no.
851221D/CHV35/SE by Huntingdon Research Center. Unpublished report, Cheminova A/S, CHA Doc.
Liggett MP, Parcell BI (1985b). Irritant effects on the rabbit eye of malathion (fyfanon) technical. Study no.
851214D/CHV36/SE by Huntingdon Research Center. Unpublished report, Cheminova A/S, CHA Doc.
Lloyd M (2009). Induction of chromosome aberrations in cultured human peripheral blood lymphocytes.
Malathion Product no. 340 spiked to the specification limit. Study no. 676/140 by Covance Laboratories
Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 641 FYF. Submitted to WHO by
FMC/Cheminova.
Lochry EA (1988). A developmental toxicity (embryo-fetal toxicity/teratogenicity) pilot study with AC 6,601 in
rats. Study no. 101-005P; Report no. 101-005P by Argus Research Laboratories Inc. Unpublished report,
Lochry EA (1989). A developmental toxicity study with AC 6,601 in rats. Study no. 101-005; Report no. 101-
005 by Argus Research Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 10 FYF.
Lowe C (2011a). Amended final report: malathion technical - local lymph node assay (LLNA) in mice. Study
no. 32158 by Eurofins / Product Safety Laboratories. Unpublished report, Cheminova A/S, CHA Doc. No.:
Lowe C (2011b). Malaoxon: acute oral toxicity up and down procedure in rats. Study no. 32159 by Eurofins
Product Safety Laboratories. Unpublished report, Cheminova A/S, CHA Doc. No.: 948 FYF. Submitted to
Lucero L, Pastor S, Suárez S, Durbán R, Gómez C, Parrón T et al. (2000). Cytogenetic biomonitoring of
Spanish greenhouse workers exposed to pesticides: micronuclei analysis in peripheral blood lymphocytes
and buccal epithelial cells. Mutat Res. 464:255–62.
Lu XT, Ma Y, Wang C, Zhang XF, Jin Q, Huang CJ (2012). Cytotoxicity and DNA damage of five
organophosphorus pesticides mediated by oxidative stress in PC12 cells and protection by vitamin E. J
Environ Sci Health B. 47:445–54.
Ma TH, Anderson VA, Harris MM, Bare JL (1983). Tradescantia-micronucleus (Trad-MCN) test on the
genotoxicity of malathion. Environ Mutagen. 5(2):127–37.
Machado GP (1996). The Salmonella typhimurium reverse mutation by malathion técnico. Report no. G.1.1 -
276/96 by BIOPLAN - Biotecnologia Planalto S/C Ltda. Unpublished report, Cheminova A/S, CHA Doc.
Meerts IA (2003). Revised report: evaluation of the DNA repair inducing ability of fyfanon technical in male rat
hepatocytes (in vivo rat hepatocyte DNA-repair assay). Study no. 342776 by Notox B.V. Unpublished
433
(UFW), 1988–2001. Cancer Causes Control. 16(7):823–30.
Mohn G (1973). 5-Methyltryptophan resistance mutations in Escherichia coli K-12. Mutagenic activity of
monofunctional alkylating agents including organophosphorus insecticides. Mutat Res. 20:7–15.
Moody RP, Akram M, Dickson E, Chu I (2007). In vitro dermal absorption of methyl salicylate, ethyl parathion,
and malathion: First responder safety. J Toxicol Environ Health A. 70(12):985-99.
Moore GE (2002). Malathion technical (fortified): acute oral toxicity study in rats. Study no. 12352 by Product
Safety Laboratories. Unpublished report, Cheminova A/S, CHA Doc. No.: 403 FYF. Submitted to WHO by
FMC/Cheminova.
Moore GE (2003). Malathion technical: acute oral toxicity study in rats. Study no. 12893 by Product Safety
Laboratories. Unpublished report, Cheminova A/S, CHA Doc. No.: 429 FYF. Submitted to WHO by
FMC/Cheminova.
Moore PD, Yedjou CG, Tchounwou PB (2010). Malathion-induced oxidative stress, cytotoxicity, and
genotoxicity in human liver carcinoma (HepG2) cells. Environ Toxicol. 25:221–6.
Moore PD, Patlolla AK, Tchounwou PB (2011). Cytogenetic evaluation of malathion-induced toxicity in
Sprague-Dawley rats. Mutat Res. 725:78–82.
Moreno OM (1988). 21-day dermal toxicity study with AC 6,601 in rabbits. Study no. MB 88-9191 by MB
Research Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 17 FYF. Submitted to
Morgan RL, Thayer KA, Bero L, Bruce N, Falck-Ytter Y, Ghersi D et al. (2016). GRADE: Assessing the
quality of evidence in environmental and occupational health. Environ Int. 92–3:611–6. doi:
10.1016/j.envint.2016.01.004.
Myers DP (2003). Assessment of the effect of malathion on the thickness of the corpus callosum in the CD rat.
Study no. CHV/088/032005 by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova A/S, CHA
Doc. No.: 346 FYF suppl-2. Submitted to WHO by FMC/Cheminova.
Myers DP (2004). Amendment 1 to the final report: assessment of the effect of malathion on the thickness of the
corpus callosum in the CD rat. Study no. CHV/088/032005 Report Amendment No. 1 by Huntingdon Life
Sciences Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 346 FYF suppl-2, amdt-1. Submitted to
Myhr BC, Caspary WJ (1991). Chemical mutagenesis at the thymidine kinase locus in L5178Y mouse
lymphoma cells: Results for 31 coded compounds in the National Toxicology Program. Environ Mol
Mutagen. 18:51–83.
National Cancer Institute (1978). Bioassay of malathion for possible carcinogenicity. Report No. NCI-CG-TR-
24. United States Department of Health, Education and Welfare. National Institutes of Health. DHEW
Publication No. (NIH) 78-824.
National Cancer Institute (1979a). Bioassay of malathion for possible carcinogenicity. Report No. NCI-CG-TR-
National Cancer Institute (1979b). Bioassay of malaoxon for possible carcinogenicity. Report No. NCI-CG-TR-
Navarro MC (1995). A micronucleus study in mice for the product malathion technico. Study no. -; Report no.
G.1.2 - 62/95 by BIOPLAN - Biotecnologia Planalto S/C Ltda. Unpublished report, Cheminova A/S, CHA
Nemec MD (2000). A range-finding acute study of malathion in rats. The effect of malathion on cholinesterase
following acute oral exposure in the rat. Study no. WIL-206004, Wil-206007; Report no. 93-2232 by Wil
Research Laboratories, Inc., Pharmaco LSR. Unpublished report, Cheminova A/S, CHA Doc. No.: 303
Ni Z, Li S, Liu Y, Tang Y, Pang D (1993) Induction of micronucleus by organophosphorus pesticides both in
vivo and in vitro. Hua Xi Yi Ke Da Xue Xue Bao. 24:82–6.
434
Nicholas AH, Vienne M, van den Berghe H (1979). Induction of sister-chromatid exchanges in cultured human
cells by an organophosphorous insecticide: malathion. Mutat Res. 67:167–72.
Nicolich MJ (1998a). Supplemental information for MRID 43975201: statistical analysis of survivorship and
tumor incidence data for rats from a 24-month oral toxicity and oncogenicity study with malaoxon
(Huntingdon Life Sciences Study 93-2234) by Huntingdon Life Sciences. Unpublished report, Cheminova
A/S, CHA Doc. No.: 165 FYF Suppl-1. Submitted to WHO by FMC/Cheminova.
Nicolich MJ (1998b). Supplemental information No. 2 for MRID 43975201: additional statistical analysis of
testicular cell tumors and mononuclear cell leukemia in rats from a 24-month oral toxicity and oncogenicity
study with malaoxon (Huntingdon Life Sciences Study 93-2234. Study no. 93-2234 by Huntingdon Life
Sciences. Unpublished report, Cheminova A/S, CHA Doc. No.: 165 FYF Suppl-2. Submitted to WHO by
FMC/Cheminova.
Nielsen AM (1994). Medical examination of employees at Cheminova to establish the influence of malathion
production and filling on their health. Cheminova A/S, Occupational Health Service. Unpublished report,
Nishio A, Uyeki EM (1981). Induction of sister chromatid exchanges in Chinese hamster ovary cells by
organophosphate insecticides and their oxygen analogs. J Toxicol Environ Health. 8:939–46.
NTP (2016). Chemical Effects in Biological Systems (CEBS). Research Triangle Park, NC (USA): National
Toxicology Program (NTP). http://tools.niehs.nih.gov/cebs3/ui/.
Ojha A, Gupta Y (2014). Evaluation of genotoxic potential of commonly used organophosphate pesticides in
peripheral blood lymphocytes of rats. Hum Exp Toxicol. 34:390–400.
Ojha A, Srivastava N (2014). In vitro studies on organophosphate pesticides induced oxidative DNA damage in
rat lymphocytes. Mutat Res Genet Toxicol Environ Mutagen. 761:10–7.
Ojha A, Yaduvanshi SK, Pant SC, Lomash V, Srivastava N (2013). Evaluation of DNA damage and
cytotoxicity induced by three commonly used organophosphate pesticides individually and in mixture, in rat
tissues. Environ Toxicol. 28:543–52.
Omari YL (2011). Micronucleus analysis and mitotic index in a Jordanian population exposed to pesticides of
organophosphate: malathion and chlorpyrifos. Caryologia. 64:173–8.
Osaba L, Aguirre A, Alonso A, Graf U (1999). Genotoxicity testing of six insecticides in two crosses of the
Drosophila wing spot test. Mutat Res. 439:49–61.
Cancer. 131(11):2650–9.
Páldy A, Puskás N, Vincze K, Hadházi M (1987). Cytogenetic studies on rural populations exposed to
pesticides. Mutat Res. 187:127-32.
Palmer SJ, Schneider SZ, Kendall TZ, Krueger HO (2011a). Malathion: fish short term reproduction assay with
the fathead minnow (Pimephales promelas). Study no. 232A-121 by Wildlife International, Ltd.
Palmer SJ, Schneider SZ, Kendall TZ, Krueger HO (2011b). Malathion: amphibian metamorphosis range
finding assay for the detection of thyroid active substances. Study no. 232A-122 by Wildlife International,
Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 1059 FYF. Submitted to WHO by
FMC/Cheminova. Submitted to WHO by FMC/Cheminova.
Palmer SJ, Schneider SZ, Kendall TZ, Krueger HO (2011c). Malathion: amphibian metamorphosis assay. Study
no. 232A-124 by Wildlife International, Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 1060
Pant KJ (1989). Test for chemical induction of unscheduled DNA synthesis in rat primary hepatocyte cultures
by autoradiography with AC 6,601. Study no. 0125-5100 by SITEK Research Laboratories. Unpublished
Parke GS (1978). Report the acute dermal LD50 of fyfanon on New Zealand albino rabbits. Study no. 7E-7851
by Cannon Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 139 FYF. Submitted to
435
Pednekar MD, Gandhi SR, Netrawali MS (1987). Evaluation of mutagenic activities of endosulfan, phosalone,
malathion, and permethrin, before and after metabolic activation, in the Ames Salmonella test. Bull Environ
Contam Toxicol. 38:925–33.
Peto R, Pike MC, Day NE, Gray RG, Lee PN, Parish S, et al. (1980). Guidelines for simple, sensitive
significance tests for carcinogenic effects in long-term animal experiments. In: IARC Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 2, Long-term and short-term
screening assays for carcinogens: A critical appraisal. Lyon, IARC. pp. 311–426.
Pluth JM, Nicklas JA, O’Neill JP, Albertini RJ (1996). Increased frequency of specific genomic deletions
resulting from in vitro malathion exposure. Cancer Res. 56:2393–9.
Pluth JM, O’Neill JP, Nicklas JA, Albertini RJ (1998). Molecular bases of HPRT mutations in malathion-treated
human T-lymphocytes. Mutat Res. 397:137–148.
Pratt M (2005). Desmethyl-malathion: acute oral toxicity to the rat (up-and-down procedure). Study no. CHV
115/053220/AC; Report no. by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova A/S, CHA
Pratt MR (2006). Desmethyl-malathion and malathion: Comparison of toxicity and cholinesterase inhibition
potential following a single oral gavage administration to female CD rats. Study no. CHV 116/053048;
Report no. by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 524
Ravn Nielsen AM (1999). Results of the biological monitoring of employees working at the dimethoate and
malathion plants in the period 1994 to 1999, and the two occurences of spraying with mixtures of these
compounds in the same period. Report no. 475-005 by Cheminova A/S, Occupational Health Service.
Reddy V, Freeman T, Cannon M (1989). Disposition and metabolism of 14C-labeled malathion in rats
(preliminary and definitive study). Study no. 9354-B by Midwest Research Institute. Unpublished report,
Reiss R (2004). Reanalysis of auditory startle response and startle response pre-pulse inhibition data for
offspring from the malathion developmental neurotoxicity study. Sciences International, Inc. Unpublished
Reiss R (2006a). Estimation of benchmark doses for cholinesterase inhibition after chronic oral exposure to
malathion and malaoxon. Sciences International, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.:
Reiss R (2006b). Amended report for MRID 46821703: Estimation of benchmark doses for malathion and
malaoxon after repeated oral gavage esposures to rat pups. Sciences International, Inc. Unpublished report,
Reiss R (2008). Estimation of an acute toxicity adjustment factor for malaoxon: amended report for MRID
47373705. Exponent, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 1568 FYF amdt-1.
Reiss R, Edwards M (2008). Benchmark dose modeling of cholinesterase data from adult rats after acute oral
(gavage) exposure of malathion. Exponent, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 618
Réus GZ, Valvassori SS, Nuernberg H, Comim CM, Stringari RB, Padilha PT et al. (2008). DNA damage after
acute and chronic treatment with malathion in rats. J Agric Food Chem. 56:7560–5.
Robinson K (2002). Evaluation of the embryo-lethal potential of malathion in the rabbit. Clintrials Bioresearch
Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 8 FYF Amdt-1. Submitted to WHO by
FMC/Cheminova.
Rodriguez OP, Muth GW, Berkman CE, Kim K, Thompson CM (1997). Inhibition of various cholinesterases
with the enantiomers of malaoxon. Bull Environ Contam Toxicol. 58:171–6.
Rodríguez-Ariza A, Alhama J, Díaz-Méndez FM, López-Barea J (1999). Content of 8-oxodG in chromosomal
DNA of Sparus aurata fish as biomarker of oxidative stress and environmental pollution. Mutat Res.
438:97–107.
436
Rucci G, Becci PJ, Parent RA (1980). The evaluation of the chronic toxicity effects of cythion administered in
the diet to Sprague-Dawley rats for 24 consecutive months. Study no. 5436 by Food and Drug Research
Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 57 FYF. Submitted to WHO by
FMC/Cheminova.
Rueber MD (1985). Carcinogenicity and toxicity of malathion and malaoxon. Environ Res. 37:119–53.
Rupa DS, Reddy PP, Reddi OS (1989). Frequencies of chromosomal aberrations in smokers exposed to
pesticides in cotton fields. Mutat Res. 222:37–41.
Rupa DS, Reddy PP, Sreemannarayana K, Reddi OS (1991). Frequency of sister chromatid exchange in
peripheral lymphocytes of male pesticide applicators. Environ Mol Mutagen. 18:136–8.
Rupa DS, Rita P, Reddy PP, Reddi OS (1988). Screening of chromosomal aberrations and sister chromatid
exchanges in peripheral lymphocytes of vegetable garden workers. Hum Toxicol. 7:333–6.
Salvadori DM, Ribeiro LR, Pereira CA, Beçak W (1988). Cytogenetic effects of malathion insecticide on
somatic and germ cells of mice. Mutat Res. 204:283–7.
Sanders A (2008a). Malathion monocarboxylic acid: acute oral toxicity in the rat - Fixed Dose Method. Study
no. 0545/0639 by SafePharm Laboratories Unpublished report, Cheminova A/S, CHA Doc. No.: 610 FYF.
Sanders A (2008b). Malathion dicarboxylic acid: acute oral toxicity in the rat - Fixed Dose Method. Study no.
0545/0640 by SafePharm Laboratories Ltd. Unpublished report, Cheminova A/S, CHA Doc. No.: 611 FYF.
Health. 11(4): 4449–527. doi:10.3390/ijerph110404449.
Schreib G (2015a). Reverse mutation assay using bacteria (Salmonella typhimurium with Malathion technical.
Study no. 153078 by Eurofins BioPharma Product Testing Munich, DEU. Unpublished report, Cheminova
A/S, CHA Doc. No.: 1618 FYF. Submitted to WHO by FMC/Cheminova. Submitted to WHO by
FMC/Cheminova.
Schreib G (2015b). Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with
desmethyl malaoxon dicarboxylic acid, trisodium salt. Study no. 153096 by Eurofins BioPharma Product
Testing Munich, DEU. Unpublished report, Cheminova A/S, CHA Doc. No.: 1616 FYF. Submitted to
Schroeder RE (1990). A two-generation (two-litters) reproduction study with AC 6,601 to rats. Study no. 87-
3243; Report no. 87-3243 by Bio/Dynamics Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 41
FYF. Submitted to WHO by FMC/Cheminova. Submitted to WHO by FMC/Cheminova.
Seely JC (1991). Histopathologic evaluation - The evaluation of the chronic effects of AC 6,601 administered in
the diet to Sprague-Dawley rats for 24 consecutive months. Study no. 90-78 by Patco Inc. Unpublished
report, Cheminova A/S, CHA Doc. No.: 57 FYF Amdt-2. Submitted to WHO by FMC/Cheminova.
Shellenberger TE, Billups LH (1987). One-year oral toxicity study in purebred beagle dogs with AC 6,601.
Study no. 85010 by Tegeris Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 9 FYF.
Shiau SY, Huff RA, Wells BC, Felkner IC (1980). Mutagenicity and DNA-damaging activity for several
pesticides tested with Bacillus subtilis mutants. Mutat Res. 71:169–79.
Shirasu Y, Moriya M, Kato K, Furuhashi A, Kada T (1976). Mutagenicity screening of pesticides in the
microbial system. Mutat Res. 40:19–30.
Siglin JC, Voss KA, Becci PJ (1985). A range finding teratology study with AC 6,601 in rabbits (Proj.
No.:8170) + A teratology study with AC 6,601 in rabbits (Proj. No.: 8171). Study nos. 8170 + 8171 by
Food and Drug Research Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 8 FYF.
Singaravelu G, Mahalingam S, Muthu PA (1998). Effects of malathion on hemoglobin content and its
genotoxicity in occupationally exposed field workers of Vellore. J Environ Biol. 19:187–92.
437
Singh S, Kumar V, Thakur S, Banerjee BD, Chandna S, Rautela RS et al. (2011). DNA damage and
cholinesterase activity in occupational workers exposed to pesticides. Environ Toxicol Pharmacol. 31:278–
85.
Slauter RW (1994). 18-month oral (dietary) oncogenicity study in mice, test substance malathion. Study no.
668-001 by International Research and Development Corp. Unpublished report, Cheminova A/S, CHA
Sobti RC, Krishan A, Pfaffenberger CD (1982). Cytokinetic and cytogenetic effects of some agricultural
chemicals on human lymphoid cells in vitro: organophosphates. Mutat Res. 102(1):89–102.
Stannard DR (2006a). Malathion: single dose time to peak effect cholinesterase study in juvenile rats. Study no.
CHV 111/050068; Report no. by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova A/S, CHA
Stannard DR (2006b) Malathion: single dose time to peak effect cholinesterase study in juvenile rats. Study no.
CHV 0123/050182; Report no. by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova A/S,
Stannard DR (2006c). Malaoxon: single dose range finder to assess effects cholinesterase levels in juvenile rats.
Study no. CHV 110/050069; Report no. by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova
Stannard DR (2006d). Malaoxon: single dose time to peak effect cholinesterase study in juvenile rats. Study no.
CHV 112/050147; Report no. by Huntingdon Life Sciences Ltd. Unpublished report, Cheminova A/S, CHA
Stannard DR (2006e). Malaoxon and malathion: single dose comparative cholinesterase study in juvenile rats.
Study no. CHV 0121/053810; Report no. by Huntingdon Life Sciences Ltd. Unpublished report,
Swenberg JA (1999a). A 24-month oral toxicity/oncogenicity study of malathion in the rat via dietary
administration: Pituitary and uterus tissue evaluation and peer review. Unpublished report, Cheminova A/S,
CHA Doc. No.: 164 FYF suppl-1. Submitted to WHO by FMC/Cheminova.
Swenberg JA (1999b). A 24-month oral toxicity/oncogenicity study of malathion in the rat via dietary
administration: Nasal tissue evaluation and peer review. Unpublished report, Cheminova A/S, CHA Doc.
No.: 164 FYF suppl-2. Submitted to WHO by FMC/Cheminova.
Swenberg JA (1999c). An 18-month oral (dietary) oncogenicity study of malathion in mice: nasal tissue
evaluation and peer review. Unpublished report, Cheminova A/S, CHA Doc. No.: 94 FYF suppl-3.
Szekely JG, Goodwin M, Delaney S (1992). The effect of gamma-irradiation on the toxicity of malathion in
V79 Chinese hamster cells and Molt-4 human lymphocytes. Mutat Res. 280:187–93.
Takeuchi S, Matsuda T, Kobayashi S, Takahashi T, Kojima H (2006). In vitro screening of 200 pesticides for
agonistic activity via mouse peroxisome proliferator-activated receptor (PPAR)α and PPARγ and
quantitative analysis of in vivo induction pathway. Toxicol Appl Pharmacol. 217:235-44.
Takeuchi S, Iida M, Yabushita H, Matsuda T, Kojima H (2008). In vitro screening for aryl hydrocarbon receptor
agonistic activity in 200 pesticides using a highly sensitive reporter cell line, DR-EcoScreen cells, and in
vivo mouse liver cytochrome P450-1A induction by propanil, diuron and linuron. Chemosphere. 74:155-65.
Taylor H (2008a). Reverse mutation in five histidine-requiring strains of Salmonella typhimurium. Malathion
monocarboxylic acid. Study no. 676/66 by Covance Laboratories Ltd. Unpublished report, Cheminova A/S,
Taylor H (2008b). Reverse mutation in five histidine-requiring strains of Salmonella typhimurium. Malathion
dicarboxylic acid. Study no. 676/67 by Covance Laboratories Ltd. Unpublished report, Cheminova A/S,
Taylor H (2008c). Reverse mutation in five histidine-requiring strains of Salmonella typhimurium. Desmethyl
malathion, Na-salt. Study no. 676/68 by Covance Laboratories Ltd. Unpublished report, Cheminova A/S,
438
Terrell Y (1978). Acute oral LD50 in rats. Malathion technical (fyfanon). (Revised report). Study no. 7E-7850 by
Cannon Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 140 FYF. Submitted to
Terrell Y (1979a). Acute oral LD50 of fyfanon 771006 K.L. (Malathion) in Sprague-Dawley rats. Study no. 9E-
4269 by Cannon Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 141 FYF.
Terrell Y (1979b). Acute oral LD50 of fyfanon 771006 ST.L. 1 Aar (Malathion) in Sprague-Dawley rats. Study
no. 9E-4268 by Cannon Laboratories Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 142 FYF.
Thompson PW (2013). Malathion: Reverse mutation assay Ames test using Salmonella typhimurium and
Eschericia coli. Study no. 41302181 by Harlan Laboratories Ltd. Unpublished report, Cheminova A/S,
Tisch M, Faulde M, Maier H (2007). Genotoxic effects of insecticides in current use on mucosal epithelial cells
from human tonsil tissue. HNO. 55 Suppl 1:E15–22.
Titenko-Holland N, Windham G, Kolachana P, Reinisch F, Parvatham S, Osorio AM et al. (1997). Genotoxicity
of malathion in human lymphocytes assessed using the micronucleus assay in vitro and in vivo: a study of
malathion-exposed workers. Mutat Res. 388:85–95.
Traul KA (1987). Evaluation of CL 6601 in the bacterial/microsome mutagenicity test. Study no. 114 by
American Cyanamid Company. Unpublished report, Cheminova A/S, CHA Doc. No.: 15 FYF. Submitted
USEPA (1977). Evaluation of selected pesticides as chemical mutagens – in vitro and in vivo studies. EPA ID
No. 600 (1-77-028). Research Triangle Park, NC: Health Effects Research Laboratory, Office of Research
and Development, United States Environmental Protection Agency.
USEPA (1990). Memorandum. Malathion - mutagenicity data submitted under MRID No. 409393-02. EPA ID
No. 114 (057701-5). Washington (DC): Office of Pesticides and Toxic Substances, United States
Environmental Protection Agency.
Uzun FG, Kalender S, Durak D, Demir F, Kalender Y (2009). Malathion-induced testicular toxicity in male rats
and the protective effects of vitamins C and E. Food Chem Toxicol. 47:1903–8.
Valencia R (1981). Mutagenesis screening of pesticides using Drosophila. U.S. EPA Report 600/1-81-017,
Washington (DC): United States Environmental Protection Agency; 83 pp.
van Bao T, Szabó I, Ruzicska P, Czeizel A (1974). Chromosome aberrations in patients suffering acute organic
phosphate insecticide intoxication. Humangenetik. 24:33–57.
Varona-Uribe ME, Torres-Rey CH, Díaz-Criollo S, Palma-Parra RM, Narváez DM, Carmona SP et al. (2016).
Exposure to pesticide mixtures and DNA damage among rice field workers. Arch Environ Occup Health.
71:3–9.
Velázquez A, Creus A, Xamena N, Marcos R (1987). Lack of mutagenicity of the organophosphorus insecticide
malathion in Drosophila melanogaster. Environ Mutagen. 9:343–8.
Venkat JA, Shami S, Davis K, Nayak M, Plimmer JR, Pfeil R et al. (1995). Relative genotoxic activities of
pesticides evaluated by a modified SOS microplate assay. Environ Mol Mutagen. 25:67–76.
Cancer Causes Control. 12(6):509–17.
Wagner H (2011). Malathion: H295R Steroidogenesis Assay. Study no. 9148V-100372STER by CeeTox, Inc.
Walter Z, Czajkowska A, Lipecka K (1980). Effect of malathion on the genetic material of human lymphocytes
stimulated by phytohemagglutinin (PHA). Hum Genet. 53:375–81.
Wananukul S, Chatproedprai S, Tempark T, Wananukul W (2011). Clinical response and safety of malathion
shampoo for treatment of head lice in a primary school. J Med Assoc Thai. 94(4):465–9.
439
Wang-Fan W (2003). Malathion technical (fortified): Local lymph node assay (LLNA) in mice (identification of
contact allergens). Study no. 848272 by RCC Ltd, Toxicology. Unpublished report, Cheminova A/S, CHA
Waters MD, Simmon VF, Mitchell AD, Jorgenson TA, Valencia R (1980). An overview of short-term tests for
the mutagenic and carcinogenic potential of pesticides. J Environ Sci Health B. 15:867–906.
Wester RC, Maibach HI, Bucks DA, Guy RH (1983). Malathion percutaneous absorption after repeated
administration to man. Toxicol Appl Pharmacol. 68:116–9.
Wilga PC (2011). Malathion: human recombinant aromatase assay. Study no. 9148V-100372AROM by
CeeTox, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 1063 FYF. Submitted to WHO by
FMC/Cheminova.
Willoughby JA (2011a). Malathion: estrogen receptor transcriptional activation (Human Cell Line (HeLa-
9903)). Study no. 9148V-100372ERTA by CeeTox, Inc. Unpublished report, Cheminova A/S, CHA Doc.
Willoughby JA (2011b). Malathion: androgen receptor binding (rat prostate cytosol). Study no. 9148V-
100372ARB by CeeTox, Inc. Unpublished report, Cheminova A/S, CHA Doc. No.: 1062 FYF. Submitted
Windham GC, Titenko-Holland N, Osorio AM, Gettner S, Reinisch F, Haas R et al. (1998). Genetic monitoring
of malathion-exposed agricultural workers. Am J Ind Med. 33:164–74.
Wong PK, Wai CC, Liong E (1989). Comparative study on mutagenicities of organophosphorus insecticides in
Salmonella. Chemosphere. 18:2413–22.
Yoder J, Watson M, Benson WW (1973). Lymphocyte chromosome analysis of agricultural workers during
extensive occupational exposure to pesticides. Mutat Res. 21:335–40.
Zeiger E, Anderson B, Haworth S, Lawlor T, Mortelmans K (1988). Salmonella mutagenicity tests. IV. Results
from the testing of 300 chemicals. Environ Mol Mutagen. 11(Suppl 12):1–158.
Zeljezic D, Garaj-Vrhovac V (2002). Sister chromatid exchange and proliferative rate index in the longitudinal
risk assessment of occupational exposure to pesticides. Chemosphere. 46:295–303.
Appendix 1. Mode of action analysis – rodent liver tumours

INTRODUCTION
Malathion is an organophosphorus insecticide, and like all members of this chemical class, its
mechanism of toxic action is the inhibition of acetylcholinesterase (AChE) activity mediated via the
metabolite, malaoxon.
CARCINOGENICITY OF MALATHION IN ANIMALS
Nine rodent carcinogenicity studies have been conducted on either malathion or malaoxon.
Two of these studies reported an increase in liver adenomas in male and female B6C3F1 mice
(Slauter, 1994) and female CDF(F-344)/CrlBr rats (Daly, 1996a) at high dietary concentrations of
malathion. Two other studies conducted in B6C3F1 mice did not detect liver adenomas at similar
doses (NCI, 1978, 1979) while studies conducted in Osborne–Mendel rats (NCI, 1978), F344 rats
(NCI, 1979a,b), Sprague-Dawley rats (Rucci, Becci & Parent, 1980) and Fischer 344 (CD(F-
344)/CrlBR) rats (Daly 1996a) also found no liver tumours.
Only the study by Slauter (1994), conducted in B6C3F1 mice, demonstrated a clear,
treatment-related increase in adenomas at very high dietary concentrations of malathion exceeding the
maximum tolerated dose (8000 and 16 000 ppm – equal to 1476 and 2978 mg/kg bw per day in males
and 1707 and 3448 mg/kg bw per day in females). The occurrence of these adenomas was coincident
with liver masses, nodules and tan or yellow foci observed macroscopically, increased liver weight
and hypertrophy of hepatocytes – no increase in carcinomas was observed (Table A2.1). At 8000 and
16 000 ppm, absolute body weight was significantly lower than the control over the entire period of
exposure. Statistically and toxicologically significant inhibition of erythrocyte acetylcholinesterase
activity occurred in both sexes at and above 800 ppm. Brain acetylcholinesterase activity was
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inhibited by greater than 20% at 8000 and 16 000 ppm, however, only inhibition at the highest dose at
termination was statistically significant.
Table A2.1. Liver findings in B6C3F1 mice exposed to malathion in the diet for up to 18 months
No. and incidence per dietary concentration (ppm)
Liver masses – 18 month necropsy
Males 0/50 8/51 (16%) 4/48 (8%) 5/54 (9%) 18/50 (36%)
Females 1/55 (2%) 0/52 3/52 (6%) 2/53 (4%) 10/51 (20%)
Liver nodules - 18 month necropsy
Males 5/50 (10%) 2/51 (4%) 3/48 (6%) 10/54 (19%) 19/50 (38%)
Females 1/50 (2%) 2/51 (4%) 0/48 9/54 (17%) 29/50 (58%)
Tan or yellow liver foci - 18 month necropsy
Males 0/50 0/51 1/48 (2%) 2/54 (4%) 18/50 (36%)
Females 0 0 0 2/54 (4%) 9/50 (18%)
Males 1.62 1.71 1.78 1.98** (+22%) 2.38** (+47%)
Females 1.55 1.68 1.56 1.66 1.92** (+24%)
Males 5.15 5.19 5.42 6.95** (+35%) 8.30** (+61%)
Females 5.22 5.39 5.22 6.21** (+19%) 7.56** (+45%)
Males 1.90 2.90 1.96 2.26** (+19%) 2.66** (+40%)
Females 1.93 1.77 1.96 1.92 2.18
Males 5.59 6.15 5.82 7.51** (+34%) 9.38** (+68%)
Females 6.19 5.76 6.26 6.90 8.51** (+37%)
Males 0/10 0/10 0/10 7/10 10/10
Females 0/10 0/10 0/10 5/10 10/10
Males 0/50 1/51 0/48 1/54 3/50
Females 0/55 0/52 0/52 53/53 51/51
Hepatocellular adenoma – 18 months
Males 1/50 6/51 2/48 13/54* 49/50**
Females 0/55 1/52 0/52 9/53* 42/51**
Hepatocellular carcinoma – 18 months
Males 0/50 6/51* 2/48 6/54* 1/50
Females 1/55 0/52 2/52 1/53 2/51
no.: number; ppm: parts per million; *P < 0.05; **P < 0.01
Results expressed as the mean, with the % increase (+) or decrease (-) relative to the control in parentheses.
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Two other carcinogenicity studies have been conducted in B6C3F1 mice – both are pre-GLP
studies conducted by the National Cancer Institute; one on malathion (NCI, 1978) and the other on
malaoxon (NCI, 1979b). Neither study found an increase in either adenomas or carcinomas. In the
study conducted on malathion (NCI, 1978), there was a slight increase in liver nodules observed
macroscopically at the highest dietary concentration in males (16 000 ppm – equal to 2400 mg/kg bw
per day; 6/49 versus 3/49 in the control), but this increase was not statistically significant. When the
incidence of hepatocellular carcinoma and nodules was combined, a significant linear trend was
determined when either the matched control (P = 0.019) or pooled control (P = 0.019) was used;
pairwise comparisons of either neoplasm were not statistically significant. In addition, the incidence
of these findings was consistent with historical control data from the same laboratory where the
incidence of spontaneous liver tumours in males was 19%. Rueber (1985) re-examined the slides from
this study and concluded that malathion caused an increase in neoplasms in the liver of male mice.
However, this re-evaluation is considered unreliable because no methodological details were provided
in the paper. The USA National Toxicology Program (Huff et al., 1985) also re-evaluated the same
slides and confirmed the conclusion of the original study authors that there was no evidence of
carcinogenicity. Overall this study is not considered acceptable because of the small number of mice
in the concurrent control group.
Only one of the six studies conducted in rats reported an increase in liver tumours in female
CDF(F-344)/CrlBr rats (Daly 1996a). In male rats, liver adenomas and carcinomas occurred with
similar frequency across all groups. In females, the incidence of liver adenomas was significantly
increased at 6 000 and 12 000 ppm (0, 1.8, 1.8, 5.5 and 4.3% at 0, 100, 500, 6,000 and 12,000 ppm,
respectively), while the incidence of liver carcinomas was significantly increased at 12,000 ppm (0,
1.8, 1.8, 0 and 4.3%, respectively). The occurrence of liver adenomas in females was within the
performing laboratory’s historical control range (0–5.4%), while the occurrence of carcinomas was
outside the historical control range (0–2.4%). There were a number of independent re-evaluations of
the slides from this study, with Hardisty (2000) confirming the increase in hepatocellular adenomas at
6 000 and 12 000 ppm in females but determining that no hepatocellular carcinomas were present at
any dose in females. Given that the incidence of liver adenomas in females was within the performing
laboratory’s historical control range and as there was a poor dose–response relationship, the increase
in liver adenomas in females is unlikely to be treatment-related.
IS THE WEIGHT OF EVIDENCE SUFFICIENT TO ESTABLISH AN MOA IN ANIMALS?

a. Postulated MOA
The proposed mode of action (MOA) for the occurrence of liver adenomas in B6C3F1 mice is
prolonged exposure to excessive dietary concentrations of malathion leading to sustained metabolic
activity of the liver resulting in hypertrophy and the formation of benign liver adenomas.
b. Key events
 Metabolism of malathion in the liver. Malathion is metabolized to malaoxon in the liver by
the mitochondrial cyctochrome P450-monooxygenase system, microsomal carboxylesterases
and the cytosolic glutathione-S-transferases (Ketterman, 1987). The metabolite profile is
qualitatively similar in rats (Reddy, Freeman & Cannon, 1989) and humans (Jellinek,
Schwartz & Connolly Inc., 2000).
 Liver enzyme induction. In SD rats, continuous exposure to 200 mg/kg bw per day malathion
administered intraperitoneally was required to induce epoxide hydrolase and glutathione-S-
transferase activities – no induction of cytochrome P450 monooxygenase activity occurred
(Reidy et al., 1987). There is in vitro evidence of CYP1A2, 2B6 and 3A4 involvement in the
metabolism of malathion by human microsomes, with 2B6 and 3A4 playing more of a role at
high malathion concentrations (Buratti et al. 2005). Malathion showed no peroxisome
proliferator-activated receptor (PPAR)α or PPARγ (Takeuchi et al. 2006) activity and also
442
showed no aryl hydrocarbon receptor activity (Takeuchi et al. 2008). There is no evidence
available to indicate the activation of the constitutive androstane receptor or pregnane X
receptor.
 Proliferative changes in the liver. There are no short-term studies in mice to indicate the
occurrence of liver hypertrophy. In the pivotal rat study (Daly, 1996a), increased liver weight
and hepatocellular hypertrophy occurred after 12 months of exposure, with no additional
histopathological changes indicative of liver toxicity. Studies in rats consistently show
increases in liver weight and changes in liver function (increased cholesterol, total protein,
albumin and GGT). There was no evidence of proliferative changes or cytotoxicity in the
liver, including hyperplasia or neoplasia in mice or rats.
 Development of liver adenomas. Benign liver adenomas occurred in mice after two years of
dietary exposure to very high doses of malathion (i.e. greater than the maximum tolerated
dose).
c. Dose–response relationship
In the pivotal mouse study (Slauter, 1994), there was a clear dose-related increase in liver
hypertrophy, nodules or discolouration observed macroscopically and adenomas in both sexes at 8000
and 16 000 ppm (Table A2.1).
d. Temporal relationship
There is a paucity of data on the early to middle events in the postulated MOA for the
formation of liver adenomas in mice – specifically around the induction of liver enzymes in B6C3F1
mice, adverse effects on liver function and the occurrence of pre-neoplastic changes. In the pivotal rat
study (Daly, 1996a), increased liver weight and microscopic evidence of liver hypertrophy was
observed after 12 months of dietary exposure to malathion suggestive of an adaptive response to high
doses of malathion. After 2 years of dietary exposure, these same changes remained evident at the
same doses but were coincident with macroscopic changes (nodules and tan or yellow foci) in
addition to adenomas. It is noted that the liver hypertrophy was graded as more severe after 12 than 18
months. An analysis of this study by Hardisty (2000) confirmed the increase in hepatocellular
adenomas at 6000 and 12 000 ppm in females but determined that no hepatocellular carcinomas were
present at any dose in females.
e. Strength consistency and specificity of association of the tumour response with key events
Studies conducted in the same mouse strain over comparable doses and timescales did not
reproduce the treatment-related increase in liver adenomas following dosing with either malathion
(NCI, 1978) or malaoxon (NCI, 1979b). There are no data on the potentially reversibility of liver
weight increases or hypertrophy in either mice or rats.
f. Biological plausibility and coherence
Notwithstanding the absence of data to support some of the key events, the proposed MOA is
considered biologically plausible based on the liver being a target organ.
g. Other MOAs
There is no evidence to suggest other possible modes of action such as cytotoxicity, hormonal
perturbation, immunosuppression or porphyria.
h. Uncertainties, inconsistencies and data gaps
The main areas of uncertainty and/or data gaps are studies conducted in B6C3F1 mice
demonstrating increases in liver metabolism, such as the induction of CYP enzymes or proliferative
processes, including the time frame and doses over which these events might happen. Similar tumours
were not observed in mouse and rat studies conducted on malaoxon, which is more toxic than parent
malathion. There are no studies that malathion is cytotoxic to hepatocytes.
i. Assessment of the postulated MOA
443
The level of confidence in the proposed MOA is considered low based on the absence of
experimental data indicating proliferative processes in the liver of rodents, observations of precursor
lesions or corroborative evidence across similarly conducted studies on malathion or malaoxon in
mice and rats.
CAN HUMAN RELEVANCE OF THE MOA BE REASONABLY EXCLUDED ON THE BASIS OF

FUNDAMENTAL, QUALITATIVE DIFFERENCES IN KEY EVENTS BETWEEN EXPERIMENTAL ANIMALS
AND HUMANS?
Given the similar metabolic profile between humans and experimental animals, and evidence of
the involvement of CYP enzymes in metabolism by human liver microscomes, the key early
metabolic events in the proposed MOA cannot be excluded as relevant to humans. However,
scenarios of prolonged human exposure to very high doses of malathion, essential to the proposed
MOA, are considered highly unlikely because:
 the use pattern of malathion limits such prolonged, excessive exposure;
 overt inhibition of acetylcholinesterase activity occurs at lower doses and is rate-limiting for
the proposed MOA to occur in humans.

QUANTITATIVE DIFFERENCES IN EITHER KINETICS OF DYNAMIC FACTORS BETWEEN
EXPERIMENTAL ANIMALS AND HUMANS?
Hepatocellular adenomas are the most common age-related neoplasm that occurs in B6C3F1
mice and on this basis one might reasonably exclude this mouse strain as a suitable model for humans.
Published data (Haseman, Hailey & Morris, 1998) indicates that the combined incidence of these
tumours is 10–68%, with the range for adenomas and carcinomas individually being 4–60 and 6-29%,
respectively. In addition, the occurrence of liver adenomas in mice occurred only following prolonged
exposure to excessive doses of malathion and such a scenario is unlikely to occur in humans.
Furthermore, adenomas in mice occurred at doses at least an order of magnitude higher than the
lowest doses causing toxicologically significant inhibition of acetylcholinesterase activity.
CONCLUSION: STATEMENT OF CONFIDENCE, ANALYSIS AND IMPLICATIONS

The MOA for malathion-induced liver adenoma formation in B6C3F1 mice cannot be
determined with confidence and hence cannot be completely dismissed as relevant to humans.
However, the occurrence of these adenomas is considered a likely secondary effect of prolonged
exposure to excessive dietary concentrations of malathion and to have a clear threshold; on this basis
the risk of carcinogenicity in humans is negligible.
REFERENCES (ADDITIONAL TO THOSE CITED IN THE MONOGRAPH)

Ketterman AJ, Pond SM, Becker CE (1987). The effects of differential induction of cytochrome P-450
carboxylesterase and glutathione S-transferase activities on malathion toxicity in mice. Toxicol Appl
Pharmacol. 87(3):389–92.
Reidy GF, Rose HA, Stacey NH (1987). Effects of length of exposure to malathion on xenobiotic
biotransformation in male rat liver. Toxicol Lett. 38(1-2):193–9.
444
Appendix 2. Mode of action analysis – rodent nasal tumours

INTRODUCTION
Malathion is an organophosphorus insecticide, and like all members of this chemical class, its
mechanism of toxic action is the inhibition of acetylcholinesterase (AChE) activity. Malathion is
metabolized to malaoxon in the liver, which is a more potent inhibitor of AChE activity.
CARCINOGENICITY OF MALATHION IN ANIMALS

A total of nine rodent carcinogenicity studies have been conducted on either malathion or
malaoxon. In only one of these studies (Daly, 1996a), one nasal adenoma and one nasal carcinoma
were observed microscopically in male rats at 6000 ppm and 12 000 ppm, respectively (equal to 359
and 729 mg/kg bw per day, respectively). There were a number of independent re-evaluations of this
study to more closely examine the microscopic findings in nasal tissue. Swenberg (1999b) confirmed
that dietary exposure to malathion at 6000 or 12 000 ppm caused significant nasal toxicity
characterized by olfactory epithelial degeneration, hyperplasia and cyst formation, goblet cell
hyperplasia, congestion, oedema, and inflammation. No treatment-related increases in neoplasms
were apparent in nasoturbinal or nasopharyngeal tissues. A total of four nasal epithelial cell tumours
were observed, one in each of the two highest doses of each sex; all were adenomas. Bolte (1999a)
examined additionally prepared slides and concluded that the carcinoma originally observed in the
respiratory epithelium of one high-dose male was more appropriately diagnosed as an adenoma of the
respiratory epithelium. No nasal neoplasms have been observed in any other studies including the
counterpart study on malaoxon (Daly, 1996b).
Malathion-induced nasal toxicity has been observed in mice or rats consistently following
oral or inhalational exposure of various durations. These observations are summarized in Table A3.1,
with a more detailed description of each study following. While specific examination of the nasal
cavity and respiratory tract has occurred in all studies of toxicity and carcinogenicity, routine
histopathological examination of these sites has not occurred in studies conducted prior to 1996 or
conducted over less than lifetime durations.
A.3.1. Summary of nasal toxicity findings in mice and rats

Study Observations Reference
28-day dietary Goblet cell depletion on the nasal septum and hyperplasia of the Barnett Jr (2012a)
Crl:CD[SD] rats olfactory epithelium at 10 000 ppm in both sexes (equal to 947.8 and
910.1 mg/kg bw per day, respectively).
90-day dietary At and above 500 ppm (35 mg/kg bw per day in males and 35.9 mg/kg Barnett Jr (2012b)
Crl:CD[SD] rats bw per day in females), depletion of goblet cells in the nasal cavity
occurred. Small numbers of cells with abundant non-staining
cytoplasm were interspersed where there was depletion of goblet cells.
Hyperplasia of the olfactory epithelium was also noted at the same
doses consisting of increased numbers of nuclei.
13-week inhalation At every tested concentration (at and above 0.1 mg/L), laryngeal Beattie (1994)
Crl:CD[SD]BR rats hyperplasia, and degeneration and/or hyperplasia of the olfactory
epithelium occurred in the nasal cavity.
18-month dietary At 8 000 and 16 000 ppm (equal to 1 476 and 2 978 mg/kg bw per day Slauter (1994)
B6C3F1 BR mice in males and 1 707 and 3 448 mg/kg bw per day in females),
degeneration and loss of cellularity of the olfactory epithelium, loss of
olfactory nerves in the submucosa, increased glandular secretion in the
lumen due to the retention of mucus and atrophy of the olfactory
epithelium adjacent to the retained mucus were observed at both 12 and
18 months.
445
Study Observations Reference

24-month dietary At 6 000 and 12 000 ppm (equal to 359 and 729 mg/kg bw per day in Daly (1996a)
CDF[F-344]/CrlBr rats males and 415 and 868 mg/kg bw per day in females) dilated mucosal
glands, subacute or chronic inflammation of the nasal mucosa,
degeneration of the epithelium, epithelium cysts in the nasal mucosa
and glandular and epithelium hyperplasia occurred. Nasal adenomas
occurred in one male and one female each at 6 000 and 12 000 ppm.
24-month dietary The presence of foreign material and inflammatory cell debris was Daly (1996b)
Fischer 344 (CDF[F- increased at 1 000 and 2 000 ppm. In the respiratory nasal mucosa,
344]/CrlBR) rats subacute or chronic inflammation and hyperplasia of goblet cells and
hyperplasia of the respiratory epithlelium was increased in females at
1 000 and 2 000 ppm and in males at 2 000 ppm. In the olfactory nasal
mucosa, increased degeneration of the epithelium occurred in males at
2 000 ppm and in females at 1 000 and 2 000 ppm. In females, there
was an increase in the replacement of the epithelium with by ciliated
and non-ciliated columnar epithelial, and hyperplasia of ciliated and
non-ciliated columnar epithelial cells at 1 000 and 2 000 ppm. In the
lung, oedema, subacute-chronic interstitial and purulent-chronic
purulent inflammation and foreign body granulomas occurred at 2 000
ppm in males and at 1 000 and 2 000 ppm in females. In the middle ear,
subacute inflammation was accompanied by the accumulation of
inflammatory cells or cells debris within the tympanic spaces at 1 000
ppm in females and at 2 000 ppm in both sexes.
bw: body weight; ppm: parts per million
In a 28-day repeat-dose toxicity study in Crl:CD[SD] rats (Barnett Jr, 2012a), which tested
dietary concentrations of malathion at 0, 100, 500, 5000 or 10 000 ppm (equal to 0, 9.2, 46.1, 457.5
and 947.8 mg/kg bw per day in males and 0, 9.4, 47.4, 461.3 and 910.1 mg/kg bw per day), minimal
to marked goblet cell depletion on the nasal septum and minimal to moderate hyperplasia of the
olfactory epithelium (consisting of increased numbers of nuclei) occurred at 10 000 ppm (Table
A3.2). The authors proposed that these findings were the result of continued nasal exposure to
malathion in the diet.
Table A3.2. Nasal finding in rats exposed to malathion for 28 days in the diet
Males Females
N 15 15 15 15
Nose, Level 2 – Goblet cell depletion
Minimal 0 1 0 4
Mild 0 3 0 6
Moderate 0 9 0 4
Marked 0 2 0 0
Total 0 15 0 14
Minimal 0 3 0 6
Mild 0 12 0 9
Minimal 0 0 0 2
Mild 0 3 0 9
Moderate 0 12 0 4
446
Males Females
Total 0 15 0 15
Minimal 0 0 0 1
Mild 0 2 0 5
Moderate 0 12 0 8
Total 0 14 0 14
Results expressed as the absolute number of rats with the finding.
Source: Barnett Jr (2012a)
In a 90-day repeat-dose toxicity study in Crl:CD[SD] rats (Barnett Jr, 2012b), which tested
dietary concentrations of malathion of 0, 100, 500, 5000 or 10 000 ppm (equal to 0, 7.2, 35.0, 353.6
and 733.8 mg/kg bw per day in males and 0, 7.5, 35.9, 363.1 and 719.0 mg/kg bw per day in females),
minimal to mild depletion of goblet cells in the nasal cavity was observed at and above 500 ppm
(Table A3.3). Small numbers of cells with abundant non-staining cytoplasm were interspersed where
there was depletion of goblet cells. Minimal to moderate hyperplasia of olfactory epithelium was also
noted at these same doses consisting of increased numbers of nuclei.
Table A3.3. Microscopic nasal findings in rats

Males Females
Finding 0 100 500 5 000 10 000 0 100 500 5 000 10 000
N 10 10 10 10 10 10 10 10 10 10
Goblet cell depletion – nose Level 2
Minimal 0 0 5 1 3 0 0 3 4 1
Mild 0 0 0 7 2 0 0 2 3 2
Moderate 0 0 0 2 4 0 0 0 1 6
Marked 0 0 0 0 0 0 0 0 0 1
Total 0 0 5 10 9 0 0 5 8 10
Minimal 0 0 0 6 7 0 0 0 5 3
Mild 0 0 0 3 3 0 0 0 4 6
Moderate 0 0 0 0 0 0 0 0 0 1
Total 0 0 0 9 10 0 0 0 9 10
Minimal 0 0 0 0 0 0 0 0 1 0
Mild 0 0 0 7 7 0 0 0 4 5
Moderate 0 0 0 3 3 0 0 0 5 5
Total 0 0 0 10 10 0 0 0 10 10
Source: Barnett Jr (2012b)
447
In a 13-week inhalational toxicity study in Crl:CD[SD]BR rats (Beattie, 1994), which tested
nominal concentrations of 0, 0.24, 1.10 or 4.94 mg/L (analytical concentrations of 0, 0.1, 0.45 and 2.0
mg/L, respectively), laryngeal hyperplasia and degeneration and/or hyperplasia of the olfactory
epithelium in the nasal cavity occurred at all doses, with a dose-related increase in severity (Table
A3.4).
Table A3.4. Microscopic findings in rats exposed to aerosols of malathion for 13 weeks
Aerosol concentration (mg/L)
Parameter 0 0.1 0.45 2.0
Laryngeal hyperplasia
Males (n = 15)
Incidence 0 13 15 15
Severity – 1.1 2.4 2.9
Females (n = 15)
Severity – 1.4 2.7 2.5
Degeneration and/or hyperplasia of the olfactory epithelium
Males (n = 15)
Severity 0.1 1.6 1.7 2.6
Females (n = 15)
Severity 0.1 0.7 1.6 2.6
Source: Beattie (1994)
In an 18-month study of toxicity and carcinogenicity conducted in B6C3F1 BR mice (Slauter,

1994; re-examination by Swenberg, 1999c) which tested dietary concentrations of 0, 100, 800, 8000
and 16 000 ppm (equal to 0, 17, 143, 1476 and 2978 mg/kg bw per day in males and 0, 21, 167, 1707
and 3448 mg/kg bw per day in females), degeneration and loss of cellularity of the olfactory
epithelium, loss of olfactory nerves in the submucosa, increased glandular secretion in the lumen due
to the retention of mucus and atrophy of the olfactory epithelium adjacent to the retained mucus
occurred at 8000 and 16 000 ppm. This nasal toxicity was observed at both 12 and 18 months.
In a 24-month study of toxicity and carcinogenicity conducted in CDF[F-344]/CrlBr rats

(Daly, 1996a), which tested dietary concentrations of malathion at 0, 100, 500, 6000 or 12 000 ppm
(equal to 0, 7, 29, 359 and 729 mg/kg bw per day in males and 0, 8, 35, 415 and 868 mg/kg bw per
day in females at 0, 100, 500, 6000 or 12 000 ppm, respectively), dilated mucosal glands (the majority
graded as slight), subacute or chronic inflammation of the nasal mucosa (the majority graded as slight
to moderate), degeneration of the epithelium (the majority graded as moderate to moderately severe),
epithelium cysts in the nasal mucosa (mainly graded as minimal to slight), and glandular and
epithelium hyperplasia (mainly graded as slight) occurred at 6000 and 12 000 ppm (Table A3.5).
448
Table A3.5. Histopathological findings in nasal tissue

Dietary concentration (ppm)
Parameter 0 100 or 50 500 6 000 12 000
N 90 90 90 90 90
Nasal mucosa (olfactory) – glands dilated
Males 2 1 0 31 27
Females 2 1 0 38 33
Nasal mucosa (olfactory) – subacute/chronic inflammation
Males 6 1 7 52 35
Females 0 3 2 42 20
Males 4 2 5 66 69
Females 2 2 1 69 66
Nasal mucosa (olfactory) – epithelium cysts
Males 0 0 0 43 55
Females 0 0 0 58 62
Nasal mucosa (olfactory) – glandular hyperplasia
Males 0 0 0 17 18
Females 0 0 0 24 14
Nasal mucosa (olfactory) – epithelium hyperplasia
Males 0 0 0 42 51
Females 0 0 0 57 54
Males 6 1 7 43 43
Females 2 2 1 50 25
Males 3 1 4 18 22
Females 2 1 0 33 21
Nasal mucosa (respiratory) – subacute/chronic inflammation
Males 10 2 12 41 21
Females 7 4 5 34 10
Nasal mucosa (respiratory) – glands dilated
Males 18 0 13 28 24
Females 8 4 6 14 20
Nasal mucosa (respiratory) – hyperplasia
Males 13 2 12 44 41
Females 7 3 7 44 33
Nasal lumen – cell/cell debris/metachromatic basophilic amorphous material
Males 15 5 22 69 63
Females 10 7 9 64 58
Nasopharynx – epithelial hyperplasia
Males 10 0 15 22 14
449

Parameter 0 100 or 50 500 6 000 12 000
Females 4 1 14 26 21
Neoplasms observed microscopically in nasoturbinal tissue included an adenoma in one male

at 6000 ppm and a carcinoma in one male at 12 000 ppm. The occurrence of spontaneous neoplasms
of nasoturbinal tissue is a rare finding in F344 rats and one not observed by the performing laboratory
in six previous studies (0/238 males and 0/241 females). In addition, in eight National Toxicology
Program studies only six neoplasms were detected in approximately 4000 control males. There were a
number of independent pathological re-evaluations conducted after this study was completed to more
closely examine the microscopic findings in nasal tissue. Swenberg (1999b) confirmed that dietary
exposure to malathion at 6000 or 12 000 ppm caused significant nasal toxicity characterized by
olfactory epithelial degeneration, hyperplasia and cyst formation, goblet cell hyperplasia, congestion,
oedema, and inflammation. No treatment-related increases in neoplasms were apparent in the
nasoturbinal and nasopharyngeal tissues. A total of four nasal epithelial cell tumours were observed,
one in each of the two highest doses of each sex; all were adenomas. Bolte (1999a) examined
additionally prepared slides and concluded that the carcinoma originally observed in the respiratory
epithelium of one high-dose male was more appropriately diagnosed as an adenoma of the respiratory
epithelium.
A 24-month study of toxicity and carcinogenicity was conducted in Fischer 344 (CDF (F-
344)/CrlBR) rats, which tested dietary concentrations of malaoxon of 0, 20, 1000 or 2000 ppm (equal
to 0, 1, 57 and 110 mg/kg bw per day in males and 0, 1, 68 and 140 mg/kg bw per day in females).
Nasal findings are summarized in Table A3.6. In the nasal lumen, the presence of foreign material
(minimal to severe) and inflammatory cell debris was increased (minimal to moderately severe) at
1000 and 2000 ppm. In the respiratory nasal mucosa, subacute or chronic inflammation (slight to
moderately severe) and hyperplasia of goblet cells (slight to moderately severe) and hyperplasia of the
respiratory epithlelium (slight to moderately severe) was increased in females at 1000 and 2000 ppm
and in males at 2000 ppm. In the olfactory nasal mucosa, increased degeneration of the epithelium
(slight to moderate) occurred in males at 2000 ppm and in females at 1000 and 2000 ppm. In females,
there was an increase in the replacement of the epithelium with ciliated and non-ciliated columnar
epithelial (slight to moderate severe), and hyperplasia of ciliated and non-ciliated columnar epithelial
cells (slight to moderate severe) at 1000 and 2000 ppm. In the lung, oedema (minimal to moderate),
subacute-chronic interstitial and purulent-chronic purulent inflammation (minimal to moderate) and
foreign body granulomas (minimal to moderate) occurred at 2000 ppm in males and at 1000 and 2000
ppm in females. In the middle ear, subacute (chronic active)/chronic inflammation was accompanied
by the accumulation of inflammatory cells or cells debris within the tympanic spaces at 1000 ppm in
females and at 2000 ppm in both sexes. Collectively these effects were attributable to inhaled food
particles resulting in tissue injury and inflammation to the nasal cavity, with secondary effects in the
lungs and middle ear.
450
Table A3.6. Non-neoplastic findings in rats exposed to malaoxon for 2 years

Parameter 0 20 1 000 2 000
Nasal lumen – presence of foreign material
Males 6/65 10/65 9/65 28/64
Females 1/65 6/63 17/64 27/65
Nasal lumen – inflammatory cell debris
Males 13/65 21/65 15/65 31/64
Females 6/65 6/63 20/64 27/65
Nasal mucosa (respiratory) – subacute (chronic active) or chronic inflammation
Males 11/65 11/65 10/65 21/64
Females 6/65 6/63 20/64 27/65
Nasal mucosa (respiratory) – epithelial hyperplasia
Males 11/65 18/65 13/65 20/64
Females 3/65 5/63 27/64 20/65
Nasal mucosa (respiratory) – epithelium squamous or squamoid metaplasia
Males 3/65 4/65 8/65 6/64
Females 0/65 1/63 6/64 5/65
Males 4/65 6/65 5/65 12/64
Females 2/65 0/63 17/64 10/65
Males 5/65 6/65 7/65 7/64
Females 2/65 2/63 11/64 10/65
Males 5/65 2/65 4/65 7/64
Females 1/65 1/63 11/64 7/65
Lung – oedema
Males 5/65 5/55 9/55 16/65
Females 1/64 3/55 22/55 17/65
Lung – inflammation of the interstitium
Males 12/65 9/55 12/55 23/65
Females 14/64 15/55 29/55 34/65
Lung – purulent/chronic purulent inflammation or abscess(es)/chronic abscess(es)
Males 4/65 2/55 7/55 17/65
Females 2/64 2/55 22/55 19/65
Lung – granulomatous inflammation/granulomas
Males 8/65 3/55 11/55 12/65
Females 2/64 6/55 29/55 29/65
Middle ear (tympanic cavity/epithelial lining) – subacute (chronic active)/chronic inflammation/inflammatory cells/cell
debris
Males 8/54 5/16 7/22 15/58
451

Parameter 0 20 1 000 2 000
Females 2/54 3/8 17/20 19/50
*P < 0.05; **P < 0.01
Results expressed as the absolute number of rats / number of rats examined.
IS THE WEIGHT OF EVIDENCE SUFFICIENT TO ESTABLISH A MOA IN ANIMALS?

a. Postulated MOA
The proposed MOA for the occurrence of nasal adenomas in rats is direct exposure to
malathion vapours when present in feed or to inhalation of food particles containing malathion. Direct
and repeated contact of malathion or its metabolites with nasal tissue results in irritation, which over
prolonged periods causes inflammation, pre-neoplastic changes and tumour formation.
b. Key events
 Distribution of malathion or malathion metabolites to nasal tissue. It is considered unlikely
that malathion or its metabolites could accumulate directly in nasal tissue following systemic
exposure via the diet. The Reddy, Freeman & Cannon (1989) study showed that malathion
undergoes extensive metabolism, is rapidly excreted and does not accumulate in any tissue.
However, no studies have specifically examined the distribution of malathion or its
metabolites in nasal tissue. The vapour pressure of malathion is relatively low and therefore it
is unlikely that rats would inhale malathion vapours from feed. The most likely exposure
pathway is by inhaling malathion-containing food particles. Evidence to support this exposure
pathway comes from 2-year rat studies on malathion (Daly, 1996a) and malaoxon (Daly,
1996a), where the occurrence of inflammatory and hyperplastic changes was coincident with
the presence of inhaled food particles or debris in the nasal passage. Similar tissue changes
observed in rats following direct inhalational exposure to malathion aerosols confirm that
direct exposure of nasal tissue to malathion causes hyperplastic changes (Beattie 1994).
 Irritation of nasal tissue by repeated exposure to malathion. Studies conducted in rats
indicated that malathion was only slightly irritating to rabbit skin and eyes. The respiratory
and olfactory epithelium of rats contains high concentration of carboxylesterases that could
metabolize malathion to MMCA and MDCA. Prolonged irritation by either of these two
metabolites could induce a reactive hyperplasia. The absence of nasal tumours in rats exposed
continuously to malaoxon is consistent with this hypothesis because the metabolism of
malaoxon does not involve the formation of these acids.
 Development of inflammatory changes in nasal tissue. Goblet cell depletion of the nasal
septum and hyperplasia of the olfactory epithelium was observed following 28 days of dietary
exposure (Barnett Jr, 2012a). Similar changes were also observed in subchronic studies
(Barnett Jr, 2012b; Beattie, 1994). Longer-term exposure to malathion or its metabolites
results in more severe changes including dilated mucosal glands, chronic inflammation,
epithelial degeneration, epithelium cysts and squamous metaplasia (Daly, 1996a,b).
 Development of benign nasal adenomas. Continuous exposure of nasal tissue to malathion or
its metabolites results in the formation of benign adenomas at high doses in rats as a
secondary effect of continuous inflammation and regeneration of nasal tissue.
c. Dose–response relationship
In the pivotal rat study, single adenomas occurred in both sexes at the two highest doses. Nasal
tumours are an apparently rare finding in rats and therefore the modest dose–response relationship is
considered unremarkable. In relation to nasal toxicity preceding the possible development of
452
adenomas, there is a clear and consistent, dose-related increase in the numbers of animals affected in
addition to the severity of nasal toxicity.
d. Temporal relationship
Over time there was an increase in the severity of nasal toxicity (first evident after 28 days of
dietary exposure) as the duration of dietary exposure increased to the point where the development of
adenomas occurred only after two years of continuous exposure to malathion.
e. Strength consistency and specificity of association of the tumour response with key events
In the pivotal rat study (Daly, 1996a), the occurrence of nasal adenomas was coincident with
nasal toxicity. Where histopathological examination of nasal tissue was incorporated into the study
protocol, malathion-induced nasal toxicity was observed consistently across multiple studies where
rats or mice were exposed to malathion in the diet or via inhalation from 28 days to 24 months.
f. Biological plausibility and coherence
Notwithstanding the absence of data demonstrating that direct exposure of nasal tissue is
necessary to induce nasal toxicity (rather than systemic exposure), the proposed MOA is considered
biologically plausible based on the consistency of observations and the increase in severity of toxicity
over prolonged periods of exposure.
g. Other MOAs
No other modes of action are proposed
h. Uncertainties, inconsistencies and data gaps
The main data gaps relate to tissue distribution and metabolism studies indicating that
malathion or its metabolites do not preferentially distribute to nasal tissue systemically. Such data
would support the hypothesis that direct and prolonged exposure of the upper respiratory tract to
inhaled food particles containing malathion is necessary for tumour development.
i. Assessment of the postulated MOA
The level of confidence in the proposed MOA is considered moderate to high based on the
consistency of nasal toxicity observed across studies of various durations. The MOA is qualitatively
possible in humans, though quantitatively unlikely due to functional and anatomical differences in the
respective respiratory systems (Frederick et al., 2002). There is no risk from human dietary exposure
as there is negligible potential for prolonged and direct contact with nasal tissue.

FUNDAMENTAL, QUALITATIVE DIFFERENCES IN KEY EVENTS BETWEEN EXPERIMENTAL ANIMALS
AND HUMANS?
No. It is plausible that direct contact of human nasal tissue with malathion over lifetime
exposures could induce nasal toxicity.

QUANTITATIVE DIFFERENCES IN EITHER KINETICS OF DYNAMIC FACTORS BETWEEN
EXPERIMENTAL ANIMALS AND HUMANS?
The human olfactory epithelium is better protected from vapours of organic esters than is rat
olfactory epithelium due to differences in nasal anatomy, nasal and systemic metabolism, systemic
physiology and airflow (Frederick et al., 2002).
453
CONCLUSION: STATEMENT OF CONFIDENCE, ANALYSIS AND IMPLICATIONS

The MOA for malathion-induced nasal adenomas in rats was considered quantitatively
implausible for humans on the basis that nasal tissue would not be exposed directly to the prolonged
and excessive doses of malathion necessary to induce tumours in rats.
REFERENCES (ADDITIONAL TO THOSE CITED IN THE MONOGRAPH)

Frederick CB, Lomax LG, Black KA, Finch L, Scribner HE, Kimbell JS et al. (2002). Use of a hybrid
computational fluid dynamics and physiologically based inhalation model for interspecies dosimetry
comparisons of ester vapours. Toxicol Appl Pharmacol. 183:23-40.
454
ANNEX 1
Reports and other documents resulting from previous Joint Meetings of the
FAO Panel of Experts on Pesticide Residues in Food and the
Environment and WHO Core Assessment Group on Pesticide Residues
1. Principles governing consumer safety in relation to pesticide residues. Report of a meeting of a WHO
Expert Committee on Pesticide Residues held jointly with the FAO Panel of Experts on the Use of
Pesticides in Agriculture. FAO Plant Production and Protection Division Report, No. PL/1961/11; WHO
Technical Report Series, No. 240, 1962.
2. Evaluation of the toxicity of pesticide residues in food. Report of a Joint Meeting of the FAO Committee
on Pesticides in Agriculture and the WHO Expert Committee on Pesticide Residues. FAO Meeting
Report, No. PL/1963/13; WHO/Food Add./23, 1964.
3. Evaluation of the toxicity of pesticide residues in food. Report of the Second Joint Meeting of the FAO
Committee on Pesticides in Agriculture and the WHO Expert Committee on Pesticide Residues. FAO
Meeting Report, No. PL/1965/10; WHO/Food Add./26.65, 1965.
4. Evaluation of the toxicity of pesticide residues in food. FAO Meeting Report, No. PL/1965/10/1;
WHO/Food Add./27.65, 1965.
5. Evaluation of the hazards to consumers resulting from the use of fumigants in the protection of food.
FAO Meeting Report, No. PL/1965/10/2; WHO/Food Add./28.65, 1965.
6. Pesticide residues in food. Joint report of the FAO Working Party on Pesticide Residues and the WHO
Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 73; WHO Technical Report
Series, No. 370, 1967.
7. Evaluation of some pesticide residues in food. FAO/PL:CP/15; WHO/Food Add./67.32, 1967.
8. Pesticide residues. Report of the 1967 Joint Meeting of the FAO Working Party and the WHO Expert
Committee. FAO Meeting Report, No. PL:1967/M/11; WHO Technical Report Series, No. 391, 1968.
9. 1967 Evaluations of some pesticide residues in food. FAO/PL:1967/M/11/1; WHO/Food Add./68.30,
1968.
10. Pesticide residues in food. Report of the 1968 Joint Meeting of the FAO Working Party of Experts on
Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies,
No. 78; WHO Technical Report Series, No. 417, 1968.
1969.
Pesticide Residues and the WHO Expert Group on Pesticide Residues. FAO Agricultural Studies, No.
84; WHO Technical Report Series, No. 458, 1970.
1970.
15. 1970 Evaluations of some pesticide residues in food. AGP:1970/M/12/1; WHO/Food Add./71.42, 1971.
455
17. 1971 Evaluations of some pesticide residues in food. AGP:1971/M/9/1; WHO Pesticide Residue Series,
No. 1, 1972.
19. 1972 Evaluations of some pesticide residues in food. AGP:1972/M/9/1; WHO Pesticide Residue Series,
No. 2, 1973.
21. 1973 Evaluations of some pesticide residues in food. FAO/AGP/1973/M/9/1; WHO Pesticide Residue
Series, No. 3, 1974.
23. 1974 Evaluations of some pesticide residues in food. FAO/AGP/1974/M/11; WHO Pesticide Residue
Series, No. 4, 1975.
Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Plant Production and
Protection Series, No. 1; WHO Technical Report Series, No. 592, 1976.
25. 1975 Evaluations of some pesticide residues in food. AGP:1975/M/13; WHO Pesticide Residue Series,
No. 5, 1976.
26. Pesticide residues in food. Report of the 1976 Joint Meeting of the FAO Panel of Experts on Pesticide
Residues and the Environment and the WHO Expert Group on Pesticide Residues. FAO Food and
Nutrition Series, No. 9; FAO Plant Production and Protection Series, No. 8; WHO Technical Report
Series, No. 612, 1977.
27. 1976 Evaluations of some pesticide residues in food. AGP:1976/M/14, 1977.
28. Pesticide residues in food – 1977. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide
Residues and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production
and Protection Paper 10 Rev, 1978.
29. Pesticide residues in food: 1977 evaluations. FAO Plant Production and Protection Paper 10 Suppl.,
1978.
Residues and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production
and Protection Paper 15, 1979.
1979.
Residues in Food and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant
Production and Protection Paper 20, 1980.
1980.
1981.
456
37. Pesticide residues in food: 1981 evaluations. FAO Plant Production and Protection Paper 42, 1982.
43. Pesticide residues in food – 1984 evaluations. FAO Plant Production and Protection Paper 67, 1985.
Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant
45. Pesticide residues in food – 1985 evaluations. Part I. Residues. FAO Plant Production and Protection
Paper 72/1, 1986.
46. Pesticide residues in food – 1985 evaluations. Part II. Toxicology. FAO Plant Production and Protection
Paper 72/2, 1986.
Paper 78, 1986.
Paper 78/2, 1987.
Paper 86/1, 1988.
Paper 86/2, 1988.
Paper 93/1, 1988.
Paper 93/2, 1989.
Paper 100, 1990.
Paper 100/2, 1990.
457
Production and Protection Paper 102, Rome, 1990.
Paper 103/1, Rome, 1990.
61. Pesticide residues in food – 1990 evaluations. Part II. Toxicology. World Health Organization,
WHO/PCS/91.47, Geneva, 1991.
Paper 113/1, Rome, 1991.
Paper 118, Rome, 1993.
Paper 124, Rome, 1994.
Paper 131/1 and 131/2 (2 volumes), Rome, 1995.
Residues in Food and the Environment and the WHO Core Assessment Group. FAO Plant Production
and Protection Paper 133, Rome, 1996.
Paper 137, 1996.
76. Pesticide residues in food – 1995 evaluations. Part II. Toxicological and environmental. World Health
Organization, WHO/PCS/96.48, Geneva, 1996.
and Protection Paper, 140, 1997.
Paper, 142, 1997.
79. Pesticide residues in food – 1996 evaluations. Part II. Toxicological. World Health Organization,
458
Paper, 146, 1998.
Paper, 152/1 and 152/2 (two volumes).
Paper, 157, 2000.
Paper, 165, 2001.
WHO/PCS/01.3, 2001.
Paper, 171, 2002.
WHO/PCS/02.1, 2002.
Paper, 175/1 and 175/2 (two volumes).
WHO/PCS, 2003.
Paper, 177, 2004.
WHO/PCS, 2004.
459
Paper, 182, 2005.
WHO/PCS, 2005.
Paper, 184, 2006.
WHO/PCS/07.1, 2006.
Paper, 189/1 and 189/2 (two volumes), 2007.
109. Pesticide residues in food – 2006 evaluations. Part II. Toxicological. World Health Organization, 2008.
Paper, 192, 2008.
Paper, 194, 2009.
Paper, 198, 2010.
Paper, 206, 2011.
Residues in Food and the Environment and the WHO Core Assessment Group on Pesticide Residues.
FAO Plant Production and Protection Paper, 211, 2012.
Paper, 206, 2012.
460
Paper, 216, 2013.
Paper, 220, 2014.
Paper, 222, 2015.
135. Pesticide residues in food – 2016. Report of the Special Session of the Joint Meeting of the
FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core
Assessment Group on Pesticide Residues. FAO Plant Production and Protection Paper, 227, 2016.
136. Pesticide residues in food – 2016. Report of the Joint Meeting of the FAO Panel of Experts on
Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on
Pesticide Residues. FAO Plant Production And Protection Paper, 229, 2016.
This volume contains toxicological monographs that were prepared by the 2016 Joint
FAO/WHO Meeting on Pesticide Residues (JMPR), which met in Geneva on 9–13 May
2016.
The monographs in this volume summarize the safety data on three pesticides that could
leave residues in food commodities. These pesticides are diazinon, glyphosate and
malathion. The data summarized in the toxicological monographs served as the basis for
the acceptable daily intakes and acute reference doses that were established by the
Meeting.
This volume and previous volumes of JMPR toxicological evaluations, many of which were
published in the FAO Plant Production and Protection Paper series, contain information
that is useful to companies that produce pesticides, government regulatory officers,
industrial testing laboratories, toxicological laboratories and universities.
ISBN 978 92 4 165532 3

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Pesticide residues in

Sponsored jointly by FAO and WHO

Special Session of the Joint Meeting of the

Geneva, Switzerland, 9–13 May 2016

Cataloguing-in-Publication data are available at http://apps.who.int/iris.

© WHO and FAO, 2017

List of participants .......................................................................................................................... iv

Toxicological monographs and monograph addenda ...................................................................... 1

Geneva, 8–13 May 2016

Number of Hits after screening for

(diazinon OR glyphosate OR PubMed (limited to humans; 11

Methodology of epidemiological studies

(a) Identification of compound/cancer sites and screening of papers

(b) Overview of studies included in evaluation

(c) Strengths and limitations of studies included in evaluation

(d) Publication bias

DIAZINON 2–88 JMPR 2016

3. Observations in humans ..................................................................................................................59

DIAZINON 2–88 JMPR 2016

Fig. 1. Structure of diazinon

Evaluation for acceptable intake

(a) Oral route

DIAZINON 2–88 JMPR 2016

DIAZINON 2–88 JMPR 2016

(b) Dermal route

(a) In vitro studies

DIAZINON 2–88 JMPR 2016

(b) In vivo studies

A similar metabolic pattern was found when

Cows, sheep and other mammalian and nonmammalian species

DIAZINON 2–88 JMPR 2016

Fig. 2. Proposed metabolic pathway for diazinon in the rat

DIAZINON 2–88 JMPR 2016

Table 1. Summary of acute toxicity studies of diazinon

(a) Dermal irritation

(b) Ocular irritation

DIAZINON 2–88 JMPR 2016

(c) Dermal sensitization

2.2 Short-term studies of toxicity

(a) Oral administration

DIAZINON 2–88 JMPR 2016

DIAZINON 2–88 JMPR 2016

Table 3. Maximum inhibition of cholinesterase activity in rats given diets containing

DIAZINON 2–88 JMPR 2016

In a 3-month toxicity study, groups of 15 male and 15 female Sprague Dawley

DIAZINON 2–88 JMPR 2016

Macroscopic inspection of organs at necropsy showed no gross abnormalities, although the

DIAZINON 2–88 JMPR 2016

DIAZINON 2–88 JMPR 2016

Mean per cent change in cholinesterase activity (%)

DIAZINON 2–88 JMPR 2016

DIAZINON 2–88 JMPR 2016

DIAZINON 2–88 JMPR 2016

Erythrocyte and brain acetylcholinesterase activities are summarized in Table 8. Erythrocyte

Table 8. Summary of acetylcholinesterase activities in a 90-day oral toxicity study of diazinon in

DIAZINON 2–88 JMPR 2016

DIAZINON 2–88 JMPR 2016

Table 9. Summary of a 90-day oral toxicity study of diazinon in dogs

DIAZINON 2–88 JMPR 2016

Thoracic spinal 3 +10 −27 −65 −77 +8 −39** −33* −49**