Food and Chemical Toxicology 43 (2005) 1141–1177
www.elsevier.com/locate/foodchemtox
Review
Criteria for the safety evaluation of flavoring substances
The Expert Panel of the Flavor and Extract Manufacturers
Association
Robert L. Smith a, Samuel M. Cohen b, John Doull c, Victor J. Feron d, Jay I. Goodman e,
Lawrence J. Marnett f, Ian C. Munro g, Philip S. Portoghese h,
William J. Waddell i, Bernard M. Wagner j, Timothy B. Adams k,*
a
Division of Biomedical Sciences Section of Molecular Toxicology, Imperial College School of Medicine, South Kensington, London SW7 2AZ,
United Kingdom
b
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA
c
Department of Pharmacology and Toxicology, University of Kansas Medical Center, Kansas City, KS 66103, USA
d
TNO Nutrition, Toxicology, Utrechtseweg 48, The Netherlands
e
Department of Pharmacology and Toxicology, Michigan State University, B440 Life Science Building, East Lansing, MI 48824, USA
f
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
g
CanTox Health Sciences International, Mississauga, Canada, ON L5N 2X7
h
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
i
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA
j
New York University, School of Medicine, New York, New York Bernard M. Wagner, Associates, Millburn, NJ 07078, USA
k
Scientific Secretary to the FEMA Expert Panel, Flavor and Extract Manufacturers Association, 1620 I Street,
NW Suite 925, Washington, DC 20006, USA
Received 8 September 2004; accepted 26 November 2004
Abstract
The current status of the GRAS evaluation program of flavoring substances operated by the Expert Panel of FEMA is discussed.
The Panel maintains a rigorous rotating 10-year program of continuous review of scientific data related to the safety evaluation of
flavoring substances. The Panel concluded a comprehensive review of the GRAS (GRASa) status of flavors in 1985 and began a
second comprehensive review of the same substances and any recently GRAS materials in 1994. This second re-evaluation program
Abbreviations: ABS, chromosomal aberration; ADH, alcohol dehydrogenase; ALD, aldehyde dehydrogenase; AE, anethole epoxide; CYP450,
Cytochrome P450; CHO, Chinese hamster ovary; CoA, coenzyme A; CoE, Council of Europe; CCK, cholecystokinin; DDA, detailed dietary
analysis; DNA, deoxyribonucleic acid; DSB, double strand breaks; EC, European Commission; ECETOC, European Centre for Ecotoxicology and
Toxicology of Chemicals; EFSA, European Food Safety Authority; EH, epoxide hydrolase; E. coli, Escherichia coli; EPA, Environmental Protection
Agency; F, female; FAA, Food Additives Amendment; FDA, United States Food and Drug Administration; FEMA, The Flavor and Extract
Manufacturers Association; FSM, robust stochastic model; GSH, glutathione; GST, glutathione-S-transferase; GRAS, Generally Recognized as
Safe; GRASa, GRAS affirmed; GRASr, GRAS reaffirmed; IARC, International Agency for Research on Cancer; IOFI, International Organization
of the Flavor Industry; i.p., intraperitoneal; LD50, median lethal dose; M, male; MLA, mouse lymphoma cell assay; MRCA, Market Research
Corporation of America; NAS, National Academy of Science; NCI, National Cancer Institute; NFC, natural flavor complex; NOAEL, no
observable adverse effect level; NOEL, no observed effect level; NR, not reported; NTP, National Toxicology Program; MW, molecular weight;
OECD, Organisation for Economic Cooperation and Development; PADI, possible average daily intake; PCI, per capita intake; PPARa,
peroxisome proliferator-activated receptor; PE, polychromatic erythrocytes; ppm, parts per million; PTS, Primary Toxicity Screen; S. typhimurium,
Salmonella typhimurium; SCE, sister chromatid exchanges; SCF, European Scientific Committee for Food; SLR, Scientific Literature Review;
TAMDI, theoretical added maximum daily intake.
*
Corresponding author. Tel.: +1 202 293 5800; fax: +1 202 463 8998.
E-mail address: tadams@therobertsgroup.net (T.B. Adams).
0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2004.11.012
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R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
of chemical groups of flavor ingredients, recognized as the GRAS reaffirmation (GRASr) program, is scheduled to be completed in
2005.
The evaluation criteria used by the Panel during the GRASr program reflects the significant impact of advances in biochemistry,
molecular biology and toxicology that have allowed for a more complete understanding of the molecular events associated with
toxicity. The interpretation of novel data on the relationship of dose to metabolic fate, formation of protein and DNA adducts,
enzyme induction, and the cascade of cellular events leading to toxicity provides a more comprehensive basis upon which to evaluate
the safety of the intake of flavor ingredients under conditions of intended use. The interpretation of genotoxicity data is evaluated in
the context of other data such as in vivo animal metabolism and lifetime animal feeding studies that are more closely related to
actual human experience. Data are not viewed in isolation, but comprise one component that is factored into the PanelÕs overall
safety assessment.
The convergence of different methodologies that assess intake of flavoring substances provides a greater degree of confidence in
the estimated intake of flavor ingredients. When these intakes are compared to dose levels that in some cases result in related chemical and biological effects and the subsequent toxicity, it is clear that exposure to these substances through flavor use presents no
significant human health risk.
2005 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Historical background to the safety evaluation of flavor materials and the formation of the FEMA expert panel
1.2. Scope of flavoring substances reviewed by the expert panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GRAS, GRASa, and GRASr evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. GRAS process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. GRASa and GRASr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. GRASr group assessments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Formation, function, and composition of the FEMA expert panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Criteria for the GRAS evaluation of flavoring substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Estimate of intake of flavor ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. The relationship of intake to threshold of toxicologic concern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Natural occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Chemical structure and interaction with biologically important macromolecules . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Metabolism and disposition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6. Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.1. Carcinogenicity studies on flavoring ingredients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.2. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
For more than four decades, the Expert Panel of
FEMA [Flavor and Extract Manufacturers Association
(of the United States)] has maintained a program to
evaluate the safety evaluation of flavor ingredients for
their intended use in food. During this period, the Panel
has evaluated and regularly re-evaluated the ‘‘safety-inuse’’ of more than 1700 chemically identified substances
used as flavor ingredients in foods. The PanelÕs determination of whether a flavor ingredient is ‘‘generally recognized as safe’’ (GRAS), is always made with the distinct
limitation that the substance is concluded to be safe
‘‘as a flavoring substance under conditions of intended
use in food’’.
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Flavoring substances are defined by their action on
the human senses. The flavoring effect of a substance
is determined by its impact on membrane receptors of
the human gustatory and olfactory systems. The partial
or complete saturation of these receptors provides the
physiologic response to a flavoring agent. Optimum response frequently occurs at extremely low levels of exposure. Concentrations of individual flavoring ingredients
added to food are, almost without exception, <0.1%
with the majority at <0.001%. These levels simulate
concentrations of flavoring substances as they occur
naturally in traditional foods. Therefore, the intentional
addition of flavoring substances to food is, in almost all
cases, associated with the property of self-limitation.
Within the context of low exposure, the Panel performs
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
a rigorous evaluation of the available scientific data and
evaluates the safe use of substances as flavor ingredients.
The principal objective of the safety evaluation of flavoring substances (i.e., GRAS program) by the Panel
is ‘‘to protect the consumerÕs health in the context of flavor use and to provide the scientific basis for helping the
flavor industry maintain a pattern of self-regulation’’
(FEMA Expert Panel, 19861). To realize this objective
the multi-disciplinary Panel relies on expert judgment
achieved through decades of experience evaluating
flavoring substances. All decisions of the Panel concerning the safety of flavoring substances must be
unanimous.
1.1. Historical background to the safety evaluation of
flavor materials and the formation of the FEMA expert
panel
Historically, the safety evaluation of flavor substances has not been seen as a high priority issue. The
reasons for this are fairly obvious. Firstly, the safety
assessment of other groups of socially used chemicals
such as drugs, pesticides, major food additives and environmental substances were seen as a more pressing priority and would inevitably consume restricted resources.
Secondly, there were widespread perceptions that food
flavors posed no significant safety issues as exposure levels were very low, bordering in many cases on the trivial,
and quite frequently there was a long history of human
exposure to these materials as naturally occurring components of traditional foods.
The first move to regulate the safety of food flavors
appears to be that of the US Food and Drug Administration in 1958. This Governmental Agency issued the
first so called GRAS List (‘‘Generally Recognized as
Safe’’) of flavor substances in 1958. This list accommodated some 188 traditional herbs and spices and was
compiled in response to the then recently passed Food
Additives Amendment (FAA) of the Federal Food and
Drug and Cosmetic Act.
A second GRAS list was published by the FDA in
1960 and this comprised some 27 single chemical entities, mainly synthetic in nature. There was little scientific
information concerning the safety of these materials.
The statutory basis for those flavors in use before 1958
is that they had a long history of ‘‘common use in food’’
as defined in the FAA.
The passage of the 1958 Amendment and the publication of the two FDA GRAS Lists of flavor materials
elicited within the US Flavor Industry a perception that
future developments would inevitably require the gathering and systematic evaluation of information relating
1
FEMA Expert Panel (1986) Private communication.
1143
to safety aspects such as chemical identity and specifications, conditions of use and scientific data on metabolism and toxicology.
Through the combined initiatives of Dr. Bernard L.
Oser and Dr. Richard Hall it was proposed that a scientifically independent group of Experts (The Expert Panel) should be formed and they should be given the
remit of: (a) attempting to extract what judgments they
could from the data available for flavor materials; (b)
specify what additional studies would be needed; and
(c) make GRAS decisions.
The legal basis for the existence and operation of the
FEMA Expert Panel is embedded in the phraseology of
the Food Additives Amendment of 1958 which defines a
‘‘food additive’’ as ‘‘any substance the intended use of
which results or may reasonably be expected to result
in its becoming a component of food if such substance
is not generally recognized, among experts qualified by
scientific training and experience to evaluate its safety,
as having been shown to be safe under the conditions
of its intended use’’. The three conditions specified in
the Amendment which underpin and define the operation of the Expert Panel are: ‘‘general recognition’’,
‘‘qualified experts’’ and ‘‘conditions of use’’. When this
program began, it was without precedent. The flavor
industry, a small segment of the entire food industry,
not only originated this program, but has continuously
and strongly supported its existence and activities (Hallagan and Hall, 1995).
The FEMA Expert Panel first met in 1960 and since
then has regularly met 3–4 times a year for the purpose
of assessing the safety of flavor materials. The GRAS
determinations made by the Panel are published in Food
Technology and to date, there are 21 GRAS publications covering over 2000 substances. Membership of
the Panel reflects the wide knowledge and experience
that is judged to be necessary for the evaluation of these
materials via experts in biochemistry, metabolism and
pharmacokinetics, natural product chemistry, clinical
medicine, pathology and toxicology.
In other safety evaluation programs, the Joint FAO/
WHO Expert Committee on Food Additives (JECFA)
has regularly evaluated individual flavoring agents
beginning in 1967. During the next 30 years approximately one substance was evaluated annually by JECFA
and an acceptable daily intake was assigned. In 1995,
JECFA adopted a novel safety evaluation program for
flavoring substances. Using a group assessment approach, JECFA has approved the use of more than
1300 substances for use as flavoring agents under current conditions of intake. In the case of Europe, the
Council of Europe (CoE) published its so-called ‘‘Blue
Book’’ in 1973 which provides a list of ‘‘permitted flavor
substances’’ the majority of which appear to have been
adopted from the FEMA GRAS Lists. From 1993 to
1995, the European Scientific Committee for Food
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(SCF) convened a Flavor Working Group to evaluate
the safety of chemically-defined flavoring substances.
After a short hiatus the Flavor Working Group was
reconvened in 2000 to continue the evaluation process.
Since then the SCF and its Working Group has been
succeeded by a new Scientific Panel, under the auspices
of the European Food Safety Authority (EFSA), which
will assess the safety of flavors as part of its ongoing
responsibilities. Under EFSA, a database (FLAVIS)
has been developed to collect and organize scientific
data on ‘‘European only’’ flavors. These data are to be
further evaluated by the EFSA Scientific Panel on Food
Additives, Flavourings, Processing Aids and Materials
in Contact with Food.
What has emerged over the years with the evaluation
of so many materials is the recognition, by Governmental and non-Governmental Agencies that they cannot,
for many reasons, be evaluated by traditional toxicology
and that novel approaches are required. In recent years
there has developed an encouraging convergence of approaches adopted by the various Agencies and these
place emphasis on exposure, structural group approach,
metabolic fate and toxicology. In particular these approaches take advantage of the fact that the vast majority of single chemical entity flavors (1700) belong to
relatively few well-defined structural groups (40) where
there exists a reasonable homology in terms of likely
toxicity and metabolic fate.
1.2. Scope of flavoring substances reviewed by the
expert panel
Two types of flavor ingredients exist, chemically identified (e.g., ethyl butyrate and l-carvone) and naturallyoccurring flavor mixtures (e.g., essential oils, oleoresins).
The vast majority of chemically identified flavoring substances (1700) exists naturally in foods or are formed
during the preparation of foods via heating or mixing.
However, with few exceptions, the naturally-occurring
individual substances used in commerce are not isolated
from nature, but are synthesized chemically in order to
ensure the purity and identity of the product intended
for commerce. The availability of technologically advanced methods of synthesis, separation and identification of flavoring substances has provided the
opportunity to produce and maintain a quality of product not available by isolation of the same substance
from natural sources. The remaining chemically identified flavoring substances (350) that are produced by
synthesis but not found in nature are, almost without
exception, structurally related to naturally-occurring flavoring substances. For example, cinnamyl acetate and
butyrate are reported to occur in nature while cinnamyl
propionate and valerate are not (Nijssen et al., 2003).
Regardless of their origin, essentially all chemically iden-
tified flavoring substances belong to well-defined chemical groups (e.g., pyrazines, aliphatic lactones, cinnamyl
derivatives). The ability to organize the large number
of substances into a limited number of chemical
groups provides an efficient and effective approach to
evaluate chemically identified flavor ingredients. In this
framework, each flavor ingredient can be evaluated
individually and within the context of its chemical
group.
The second type of flavor ingredient is the naturallyoccurring flavor mixtures isolated primarily from plants.
Historically, they have been recognized as essential oils,
extracts, oleoresins, tinctures, and in some cases foods
(e.g., cardamom and black pepper). Approximately
300 mixtures are now collectively recognized as natural
flavor complexes (NFC). The volatile components of
most NFCs are mixtures containing as many as 250
chemically identified constituents with likely more to
be discovered. Advances in analytical technology will
doubtless yield more constituents. A limited number of
these constituents are responsible for the technical flavor
effect of the NFC itself. As the flavor industry has developed over time, most of these important flavoring constituents were separated, identified, and synthesized.
They now constitute many of the important chemically
identified flavor ingredients (e.g., citral and limonene)
used by the flavor industry worldwide.
Being of plant origin, NFCs form their chemical constituents by well-recognized biochemical pathways (e.g.,
isoprene and shikimic acid pathways). Constituents
formed by these pathways exhibit similar skeletal structures and possess a limited number of functional groups.
In fact, NFCs of different plant origin contain many of
the same constituents. The vast majority of constituents
fall into one of a few well-defined chemical groups (e.g.,
terpene hydrocarbons and alicyclic terpene secondary
alcohols and ketones). Although the safety evaluation
of chemical mixtures involves significant complexity,
the safety evaluation of NFC mixtures can be reduced
to an analysis of relatively few chemical groups each
containing one or a number of constituents. As is evident from the above discussion the use of the chemical
group approach provides a powerful tool for the safety
evaluation of both chemically identified and NFC flavor
ingredients.
Although these two groups constitute what is commonly known as flavor ingredients, flavors added to
food are a mixture of flavor ingredients. Commercial flavors added to food normally contain 50 or more substances, both chemically identified and NFCs, and
accompany other ingredients that perform non-flavor
functions such as preservatives (BHA), solvents (ethyl
alcohol), modifiers (neohespiridin dihydrochalcone),
and emulsifiers (guar gum). Substances that do not impart flavor may serve several functions and have significant uses in the food supply. In these instances, the
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Panel evaluates the substance for GRAS status based
only on its intended use as a component of a food flavor.
This criterion is entirely consistent with Section 201(s) of
the Federal Food, Drug, and Cosmetic Act (21 CFR
172.515 a and b) which states that in order for a substance to be considered GRAS it must be ‘‘safe under
conditions of intended use.’’ It is also consistent with
past Panel decisions in which substances having nonflavor functions (solvents, modifiers, antioxidants, etc.)
have been recognized as GRAS for their intended use
in a compounded flavor.
2. GRAS, GRASa, and GRASr evaluation
2.1. GRAS process
The GRAS assessment performed by the Expert Panel includes a thorough evaluation of all the available
data on the candidate flavor ingredient and on structurally related substances in the same chemical group. The
analyses include a comprehensive evaluation of the potential exposure to the flavor ingredient through food,
compared with the available toxicologic, metabolic,
and pharmacokinetic characteristics of the substance
and its structural relatives. Available information on
the absorption, distribution, excretion, and metabolic
options on the flavoring ingredient and structurally related substances provide the basis for understanding
the biochemical fate of the substance. Data from acute,
subchronic and long-term oral studies provide the fundamental basis to understand the toxic potential of the
substance. In addition, in vitro and in vivo genotoxicity
data for the substances, principal metabolites, and members of the respective chemical group are evaluated to
screen for potential genotoxic effects. Behavioral, neurotoxicity, immunotoxicity, and reproductive effects are
evaluated, if available. The combined expert judgment
of the Panel is then applied to the above data and any
other data deemed to be relevant to the safety evaluation. In order to reach a GRAS decision, there must
be (1) an understanding of the known or anticipated biochemical fate of the substance and its potential to produce toxicity, and (2) establishment of the safety of
low levels of use of the substances in the context of its
toxic potential. The criteria for the final decision of
the Panel are described below. For the vast majority
of flavoring substances, very low levels are required to
create the intended flavor effect and low volumes are
added to the food supply annually. For many of these
substances, little or no toxicology data is available. This,
however, does not preclude evaluation of the substance
for its intended use as a flavoring substance. If the structure of the substance (e.g., benzyl butyrate) is such that
it can be assigned to a well defined chemical group (e.g.,
benzyl alcohol, benzaldehyde, benzoic acid and related
1145
esters) for which extensive safety data exists the safety
evaluation can proceed. For instance, benzyl butyrate
participates in the same biochemical pathways and
exhibits similar toxicologic potential as the well-studied
substances, benzyl acetate and benzyl propionate. Provided the substance is (1) metabolized by well-recognized detoxication pathways, (2) anticipated exposure
is orders of magnitude less than no effect levels reported
in studies on structurally related substances, (3) The
group of structurally related substances exhibit no potential for genotoxicity effects at anticipated levels of
use as flavoring substances and (4) the substance presents no unique structural features that warrant special
considerations, the substance is considered safe under
intended conditions of use.
Beginning with the publication of GRAS 3 in 1965
the Expert Panel has published nineteen (19) GRAS
articles identifying more than 2000 substances as GRAS
for their intended use as flavor ingredients. GRAS 20
(Smith et al., 2001) represented a landmark in that it
contained the 2000th substance (FEMA No. 4000) to
be recognized as ‘‘GRAS’’ by the FEMA Expert Panel.
In addition to a complete listing of new GRAS flavor
ingredients, the Expert Panel regularly presents critical
interpretations of recent scientific studies on flavor
ingredients with the goal to ensure that the results of
these studies are consistent with their current GRAS status. Flavor issues, for example, the evaluation of methods for the calculation of human dietary exposure to
flavoring substances and a novel method for the safety
evaluation of NFCÕs are also discussed in GRAS
articles.
The application for GRAS recognition is submitted
by FEMA member companies. This Association represents flavor companies accounting for at least 90% of
all flavor business conducted in the US. The GRAS
application contains physical and chemical data to identify the structure of the candidate flavoring substance
and verify its purity. Results of literature searches and
company generated safety data on the substance and
structurally related substances are included in the application to support the safety for the substance under conditions of use. Information on the anticipated pattern of
use as a flavor (use levels in food categories), technical
flavor function and the results of sensory panel evaluations are also provided. Upon completion of the evaluation of these data and other information the Panel
deems appropriate, the Panel has three options. They
may conclude that the substance is GRAS for its intended use as a flavoring substance; they may place
the substance on HOLD requesting that the applicant
provide additional data; or notify the applicant that
the substance in not GRAS, in which case the applicant
usually withdraws the application. Over the last four
decades, more than 150 applications have been withdrawn. Far more applications have been placed on
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HOLD and additional data is later provided to complete
the GRAS evaluation. Normally, the applicant performs
a cost benefit analysis prior to submitting a GRAS
application. However, in some cases, the request for
additional studies alters the results of the analysis such
that completing GRAS submission is economically
unfeasible. Under these circumstances, the application
is withdrawn.
During its review of the scientific data related to flavors, the Panel identifies new data that may affect prior
GRAS decisions. In these cases, the Panel reevaluates
the safety of the flavor in the context all relevant safety
data. Upon completion of the review they may conclude
the substance is no longer GRAS and remove it from the
GRAS list of flavors (deGRASed). In some instances,
they request additional studies be performed by industry
members or the industry as a whole. If sufficient interest
exists in the continued use of the substance as a flavor,
the studies are performed and the reevaluation of the
GRAS status of the substance is completed. In the absence of industry interest and data, the Panel completes
the revaluation based on the data available. Over the
last four decades, this review process has lead to the
deGRASing of 10 substances (e.g. o-vinylanisole,
2-methyl 5-vinylpyrazine,), mainly due to the lack of
interest in the continued use of the substance as a flavor.
2.2. GRASa and GRASr
As advancements are made in science, new information becomes available on existing FEMA GRAS flavoring substances. The dynamic FEMA GRAS assessment
program incorporates this new information into the program by way of systematic reviews of all GRAS flavor
ingredients.
Between 1965 and 1985, the first comprehensive and
systematic scientific literature reviews (SLRs) of flavoring substances were completed by FEMA under contract by the US Food and Drug Administration. These
SLRs served as the basis for a comprehensive review
of substances already designated as FEMA GRAS. This
GRAS status reassessment program was known as
‘‘GRAS affirmation’’ or ‘‘GRASa’’ and was completed
in 1985.
In 1994, the Expert Panel initiated a second comprehensive reassessment program known as ‘‘GRAS reaffirmation’’ or ‘‘GRASr’’. It is anticipated that this
reaffirmation program will be completed in 2005. As
part of the GRASr program, the Expert Panel regularly
publishes interpretations of key scientific data on structurally related groups of flavoring substances on which
GRAS decisions are based. Since 1995, FEMA GRAS
assessments of alicyclic substances, pyrazines, cinnamyl
derivatives, furfural, lactones, and trans-anethole,
allylalkoxybenzene derivatives (methyl eugenol and
estragole) have been published as part of the GRASr
program (Adams et al., 1996; Adams et al., 1997;
Adams et al., 1998; Adams et al., 2004; Newberne
et al., 1999; Smith et al., 2002a; Smith et al., 2002b).
Regular publication of at least 20 other structurally related groups of flavoring substances are scheduled.
2.3. GRASr group assessments
In its initial years of operation, the Panel used its
combined background of expertise and judgment to deal
with the often limited amount of safety data available on
a flavoring substance. As the effort progressed, the Panel
was exposed to a broad array of simple chemical substances, most of which could be assigned to specific
chemical classes (e.g., benzyl alcohol, benzaldehyde,
benzoic acid and related esters and acetals) based on
their close structural relationship, common pathways
of metabolism and consistent pattern of toxicologic potential. The Panel came to use these well-established patterns, not as a substitute for data-driven judgments, but
to assure that those judgments were applied consistently
across increasingly larger amount of data for growing
numbers of structurally related substances. These patterns in the form of group assessments are regularly
published in the peer-reviewed literature (Adams et al.,
1996; Adams et al., 1997; Adams et al., 1998; Adams
et al., 2004; Newberne et al., 1999; Smith et al., 2002a;
Smith et al., 2002b).
During its 40 year-plus history, the Expert Panel has
evaluated and reevaluated chemically identified flavor
ingredients within the context of their respective chemical group (e.g., benzyl derivatives). This experience has
provided the basis for understanding the relationship
between the biochemical fate of members of a chemical
group and their toxicologic potential. Within this framework, the Panel regularly assesses new scientific data for
GRAS candidates, GRAS substances, and structurally
related non-GRAS materials. The objective is to continuously build a more complete understanding of the
absorption, distribution, metabolism and excretion of
members of the chemical group and their potential to
cause systemic or target-organ toxicity. Against this
background, exposure to each chemically identified flavor ingredient is evaluated for safety (see example of
geranyl butyrate, Fig. 1).
One of the principal objectives of the GRASr program is to publish the PanelÕs chemical group safety
assessments in the peer-reviewed scientific literature
(see above for listing). These assessments contain all
the key scientific data relevant to the safety of chemical
groups of flavoring substances. Upon completion of the
GRASr program, approximately 35 chemical group
assessments covering approximately 1600 flavor ingredients will be published. The chemical groups of flavor
ingredients currently being evaluated by the Expert
Panel are listed in Table 1.
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
1147
Database for Congeneric Groups and Metabolites
Exposure
Metabolism/Toxicity
Intake from added
flavor use:
Daily per capita intake
("eaters only')=0.003
mg/person/day
Subchronic toxicity, chronic bioassay
(NTP, 1987) and genotoxicity data for
geranyl acetate and group of 26 terpenoid
esters
O
O
geranyl butyrate
rat oral LD50=10,600 mg/kg
(Jenner et al., 1964)
Hyrolysis data on 3 esters formed from
terpenoid primary alcohols and simple
aliphatic carboxylic acids
hydrolysis
Annual Volume of Use:
Added flavor; 130 kg
As component of food;
21,252 kg
OH
geraniol
Metabolized by w-, w-1, and functional group oxidation
(Chadha and Madyastha, 1984; Licht and Corsia, 1978;
Diliberto et al., 1990)
90-day NOEL=500 mg/kg; 28-week NOEL=50 mg/kg
(Hagan et al., 1967)
Ratio
+
food intake/flavor intake:
O
>160 (food predominant)
OH
Metabolic, subchronic, chronic and
genotoxicity data on a groups of 28 terpenoid
primary alcohols and structurally related
substances
Metabolism, toxicity, and genotoxicity data on
15 linear aliphatic carboxylic acids
butyric acid
endogenous in humans
1-yr NOEL=500 mg/kg
(Mori, 1953)
Fig. 1. Safety evaluation of a flavor ingredient-geranyl butyrate.
During the last three years, the Panel has expanded
the application of the group assessment approach to
the safety evaluation of NFCs. As noted above, NFCs
are mixtures of constituents, most of which have been
chemically identified, that belong to a limited number
of chemical groups. For instance, greater than 98% of
the composition of coriander oil is accounted for by
approximately 30 constituents that belong to three distinct chemical groups, aliphatic terpene hydrocarbons,
terpene tertiary alcohols and esters, and alicyclic terpene
secondary alcohols and ketones (Lawrence and Shu,
1993; FEMA, 2003,2 Heck et al., 1989). Studies on the
biochemical fate and toxicologic potential along with intake data for members of each of these chemical groups
provide the scientific basis to perform a comprehensive
safety evaluation of the NFC. Because of the complexities involved in the evaluation of a mixture, the Panel
has developed a scheme for the systematic evaluation
of the chemical composition of an NFC. The principles,
details and examples of this novel approach to safety
evaluation are the subject of recent publications (Newberne et al., 1998; Smith et al., 2004; Smith et al., 2005).
The types and numbers of chemical groups in the
safety evaluation program are, by no means, static. As
new scientific data and information become available,
some chemical groups are combined while others are
subdivided. This has been the case for the group of alicyclic secondary alcohols and ketones that were the sub-
2
FEMA (2003) Private communication.
ject of a comprehensive scientific literature review (SLR)
in 1978 (FEMA, 1978). Over the last two decades, experimental data has become available indicating that a few
members of this group exhibit biochemical fate and toxicologic potential inconsistent with that for other members of the same group. These inconsistencies, almost
without exception, arise at high dose levels that are irrelevant to the safety evaluation of low levels of exposure
to flavor use of the substance. However, given the
importance of the chemical group in the safety assessment program, it is critical to resolve these inconsistencies. Often the Expert Panel has requested additional
metabolic and toxicologic studies to distinguish the factors that determine these differences. In many cases, the
effect of dose and a unique structural feature results in
utilization of another metabolic pathway not utilized
by other members of a chemical class.
For instance, within the class of alicyclic ketones
mentioned above, (+)-isomenthone and (+)-pulegone
differ only in that the former contains an isopropyl substituent adjacent to the ketone function whereas the latter contains an isopropylidene substituent at the same
position. The presence of an exocyclic isopropylidene
group induces increased allylic hydroxylation (Moorthy
et al., 1989a,b; Thomassen et al., 1988, 1990, 1991; Madyastha and Raj, 1990, 1991, 1992, 1993) eventually leading to a reactive cis-2-ene-1,4-dicarbonyl metabolite that
readily forms protein adducts in the liver (McClanahan
et al., 1989; Thomassen et al., 1992) and induces hepatotoxicity (Moorthy and Madyastha, 1989; Madyastha
and Raj, 1993) at dose levels >75 mg/kg per day in mice
1148
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Table 1
Chemical groups of flavor materials
1
2
3
4
5
6
7
8
9
10
11
12
13
13
14
15
16
17
18
20
21
22
23
24
25
26
27
28
30
31
32
33
34
35
36
Saturated aliphatic, acyclic, linear primary alcohols, aldehydes, carboxylic acids and related esters
Saturated aliphatic, acyclic, branched-chain primary alcohols, aldehydes, carboxylic acids and related esters
Aliphatic linear and branched-chain a,b-unsaturated aldehydes and related alcohols, acids and esters
Aliphatic allyl esters
Unsaturated linear and branched-chain aliphatic, non-conjugated aldehydes, related primary alcohols, carboxylic acids and esters
Aliphatic primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated functional groups
Saturated alicyclic primary alcohols, aldehydes, acids, and related esters
Saturated and unsaturated aliphatic acyclic secondary alcohols, ketones and related esters
Aliphatic acyclic and alicyclic a-diketones and related a-hydroxyketones
Alicyclic ketones, secondary alcohols and related esters
Pulegone and structurally- and metabollically-related substances
Aliphatic and aromatic tertiary alcohols and related esters
Aliphatic, alicyclic, alicyclic-fused and aromatic-fused ring lactones
Aliphatic and aromatic hydrocarbons
Benzyl derivatives
Hydroxy- and alkoxy-substituted benzyl derivatives
Cinnamyl alcohol, cinnamaldehyde, cinnamic acid, and related esters
Phenyl-substituted primary alcohols, aldehydes, carboxylic acids, and related esters
Phenyl-substituted secondary alcohols, ketones, and related esters
Phenol derivatives
Hydroxyallylbenzene and hydroxypropenylbenzenes derivatives
Phenethyl alcohol, phenylacetaldehyde and related acetals and esters
Aliphatic and aromatic ethers
Furfuryl alcohol, furfural, and related substances
Furan derivatives
Aliphatic and aromatic sulfides and thiols
Sulfur-substituted furan derivatives
Sulfur-containing heterocyclic and heteroaromatic derivatives
Aliphatic and aromatic amines and related amides
Nitrogen-containing heterocyclic and heteroaromatic substances
Pyrazine derivatives
Anthranilate derivatives
Amino acids
Maltol derivatives
Epoxide derivatives
(NTP, 2002a) and >10 mg/kg per day in rats (NTP,
2002b). Conversely, the menthone derivative cannot undergo exocyclic allylic hydroxylation because it contains
no allyl group. As expected, the menthone derivative
and other structurally related alicyclic ketones, do not
exhibit hepatoxicity at dose levels at or above those
causing hepatotoxicity with pulegone (Adams et al.,
1996). Thus, the pulegone derivatives are now considered as a unique chemical group separate from other alicyclic ketones.
2.4. Formation, function, and composition of the
FEMA expert panel
GRAS decisions rely on a broad range of knowledge,
experience, and judgment. Biological events are the result of a network of chemical reactions involving macromolecules (proteins, enzymes, nucleic acids) with
micromolecules (e.g., flavoring substances) that may, given sufficient concentration, produce physiologic and/or
toxicologic effects. The interpretation of these effects in
animals, including humans, requires expert scientific
judgment that is dependent upon the training, experi-
ence, and expertise of the individual. Given the number
of different disciplines involved in understanding these
biological events, a multidisciplinary approach provides
the comprehensive analysis required to address issues
regarding human health. Since its inception, the composition of the Panel has been determined by the scientific
expertise required to perform an exhaustive assessment
of the safety of flavor ingredients added to food. Over
time, the PanelÕs expertise has broadened as new disciplines have developed (i.e. molecular toxicology and genetic toxicology). Currently the areas of expertise among
the members of the Panel include toxicology, molecular
toxicology, metabolism and disposition, biochemistry,
pathology, nutrition, organic chemistry, medicinal
chemistry and some related disciplines. Ad hoc consultants provide special expertise not available through regular members of the Panel.
The Panel acts as an independent body of experts and
has set its own agenda for more than 40 years. Although
Panel decisions affect the flavor industry marketplace, a
clear separation exists between the safety activities of the
Panel and the commercial interests of the flavor industry. The industry has long recognized the critical part
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
the Panel plays in self-regulatory aspects of GRAS. It is
continuously aware of the need to maintain the unquestioned credibility of the Panel to perform it safety evaluations in a completely independent manner.
3. Criteria for the GRAS evaluation of flavoring
substances
The criteria used by the Panel for the determination
of GRAS for a flavor ingredient are (1) intake of the flavor ingredient intentionally added to food based on (a)
recommended levels of use in specific food categories
(e.g., baked goods, chewing gum) and (b) the total annual amount of flavor ingredient sold into the marketplace by the flavor manufacturers of the USA; (2)
natural occurrence, purity and specifications; (3) chemical structure and interaction with biologically important
macromolecules; (4) metabolic and pharmacokinetic
characteristics; and (5) toxicity testing including that
for general toxicology, carcinogenicity, genotoxicity
screening, and developmental and reproductive toxicology, immunotoxicity, and neurotoxicity. The Panel applies the above criteria simultaneously in the
evaluation of individual defined chemical substances
and NFCs for their intended use as flavor ingredients.
All criteria must be mutually supportive and the data
being reviewed must be consistent with similar data
for the chemical group or groups to which those substances or mixture of substance belong. All criteria must
be unanimously agreed upon by the Panel for it to conclude that a substance can be used safely as a flavor
ingredient. The Panel decision is a subjective scientific
judgment based on a consideration of all relevant factors, which of course, reflects the scientific training,
experience, and expertise of its individual members.
The criteria discussed below provide the basis for the
PanelÕs safety evaluation program for flavoring substances. In some cases, the discussion of individual criteria is supplemented with examples to illustrate how the
criteria are applied to individual flavoring substances.
3.1. Estimate of intake of flavor ingredients
The intake of flavor ingredient is defined as the
amount of substance ingested and is essential to assessing the safety of food ingredients. Quantifying intake of
flavor ingredients is an intimidating task and challenged
by many technical and economic difficulties. There are
greater than 20,000 different food products and more
than 2000 flavor ingredients available for consumption
in the western diet (FMI, 1998; Woods and Doull,
1991). Food products are occasionally consumed by a
large heterogeneous population, which makes it difficult
to determine any one individualÕs intake of a food constituent. Added to this is the difficulty and expense of
1149
obtaining accurate intake data from a detailed dietary
analysis of a large enough population to obtain statistically significant results for the diverse group of eaters.
Over its 40 year-plus history, the Expert Panel has
used various methods of estimating exposure to flavor
ingredients in food. In the absence of any accurate intake data, the Panel in the 1960Õs estimated daily intake
using a method called ‘‘the possible average daily intake’’ (PADI) which is based on the level of flavor ingredient added to various food categories and the amounts
of those foods consumed (see below). In Europe, an approach similar to PADI is currently used. The theoretical added maximum daily intake (TAMDI) paradigm is
based on the same assumptions as the PADI estimate.
However, as actual intake data became available, it
became apparent that the assumptions inherent in the
PADI approach produced vastly exaggerated overestimates of intake. The PADI method estimates intake
by (1) multiplying usual use levels of the substance in
each of 33 food categories (e.g., baked goods and meat
products) times the average amount of that food category consumed daily, and (2) summing the intake over
all 33 food categories (USDA, 1972). The PADI calculation assumes that all foods in a food category always
contain that substance and that the food category is
consumed daily (Oser and Hall, 1977). An example of
how these assumptions can be problematic is illustrated
with the flavoring ingredient, ethyl methylphenylglycidate, which is added to impart strawberry flavor to hard
candy. The PADI method assumes all hard candy,
including peppermints, cherry-flavored lollipops, and
butterscotch, contains ethyl methylphenylglycidate. In
addition, the approach assumes that the amount added
to the food is actually the amount ingested. This is often
not the case in that it fails to account for significant
quantities of flavor ingredient lost during food processing (heat processing, baking, etc.). For example, the
majority of allyl disulfide, a volatile disulfide, added to
garlic breads is lost during the baking process. Since
more than 98% of the chemically identified flavor ingredients are low molecular weight substances (MW < 300
Da), processing (heating) will lead to substantial loss
and concomitant lower levels of intake. Based on results
of numerous industry surveys (NAS, 1970, 1975, 1982,
1987; Lucas et al., 1999; IOFI, 19953) of the annual volumes of use of flavor ingredients, it is evident that, for
the vast majority of flavor ingredients that have low reported annual volumes of use, the PADI method, as currently determined, provides a gross exaggeration of the
average daily intake. However, the PADI method could
be vastly improved if more accurate data were available
on (1) the concentration, not added levels, of flavoring
3
International Organization of the Flavor Industry (IOFI) (1995)
European inquiry on volume use. Private communication to the Flavor
and Extract Manufacturers Association (FEMA).
1150
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
substances in specific foods and (2) the pattern of intake
(e.g., number of servings consumed per week) of the
foods. The PADI calculation would then provide a
meaningful estimate of intake of flavoring substances.
Over the last three decades, two comprehensive studies of flavor intake have been undertaken. One involved
a detailed dietary analysis (DDA) of a panel of 12,000
consumers. The other is based on a robust, stochastic
model (FSM). The results of the data intensive DDA
method and the model-based FSM support the use of
annual volume of use data in the determination of daily
intake.
The DDA method is based on the detailed reporting
of dietary intake of all foods by a panel of consumers
over the period of 14 days spread over a year to accommodate seasonal variations in diet (Hall, 1976; Hall and
Ford, 1999). The Market Research Corporation of
America (MRCA) enlisted a diverse panel of 12,000
consumers that came from urban, suburban and rural
communities and ranged in age from infant to over 65
years. The results were then statistically analyzed and
sorted by age groups and consumption patterns. The
participants had more than 4000 descriptors to choose
from over all food eaten. Food categories were narrow
and highly specific. For example, the baked goods category was divided into 500 subcategories allowing for
garlic bread to not be combined with cinnamon-flavored
coffee cake.
This study assessed the amount of each specific food
eaten, the frequency of consumption of each food, the
amount of flavoring agent in each food and the classification of consumer by age, weight or other pertinent
characteristics. The amount of flavoring agent in foods
was difficult to determine since flavor formulas were
proprietary. These levels were assured by a panel of food
chemists and flavorists familiar with the flavor substance
levels in particular food products. Once all of the data
were compiled, it was taken through 8 steps of analysis
to produce average intake levels for both eaters-only
groups and non-eaters. Eaters can be defined as participants who consumed foods containing specific flavoring
substances; and non-eaters are consumers with zero
reported intake for a particular flavoring substance.
Although this data intense method is accurate and
reliable, it is expensive and time consuming. Conservative estimates derived from this data are obtained from
using the 99th percentile intake levels. In the vast majority of cases, estimates of intake are orders of magnitude
less than those obtained from PADI calculations. Based
on the results of the DDA study, it was determined that
intake could be reasonably predicted by using data from
the industry-wide annual poundage surveys. The
method is known as the per capita intake (PCI) · 10
method (Rulis et al., 1984; Woods and Doull, 1991).
The method assumes that only 10% of the population consumes the total annual reported volume of use of a flavor
ingredient. This approximation provides a practical and
cost effective approach to the estimation of intake for flavoring substances. The annual volumes of flavoring
agents are relatively easy to obtain by industry-wide
surveys, which can be performed on a regular basis to account for changes in food trends and flavor consumption.
The 1995 poundage survey of US flavor producers was
published by FEMA in 1999 (Lucas et al., 1999).
This method can be evaluated by comparison to the
data obtained by the DDA method discussed above.
Since the dietary analyses were completed in 1970, it is
necessary to use poundage information from that time
(NAS, 1970). To correct for possible incompleteness in
the 1970 poundage survey, these data are assumed to
be 60% of total annual volume of the flavoring agents
(0.6 correction factor in the equation below) actually
used. The per capita daily intakes (PCI) are then calculated from the annual volume, in kg, for the US population in 1970 (i.e., 210,000,000) by the following
equation:
PCIðmg=dayÞ ¼ kg 106 =210; 000; 000 0:6 365:
ð1Þ
The calculated PCI is then multiplied by ten to obtain
a reasonably conservative estimate for intake by the eaters of the ingredient. The data obtained from PCI · 10 is
more conservative than that obtained from the DDA
method.
For 10 of the substances studied in the panel survey,
the PADI is a gross over-estimation of the DDA intake.
For two high volume substances, b-ionone and methyl
salycilate, PADI is comparable to PCI · 10. This demonstrates that PADI is a reasonable model to follow
for intake estimation of high volume substances that
are used in many food categories. However, for low volume substances (i.e., allyl disulfide), it gives an estimation three or four (4) orders of magnitude higher than
DDA and three (3) orders of magnitude higher than
PCI · 10.
A second method that has been developed to improve
upon our understanding and estimation of exposure to
flavoring agents is based upon the theoretical full stochastic model (FSM) (Lambe et al., 2002). It was developed to assist the European Union in their goal to
evaluate flavor ingredients. Previously, European intake
estimates were based on a TAMDI approach. TAMDI
estimates intake based on maximum levels of incorporation to 31 different categories of food or beverages. Dietary intake data were collected from British males aged
16–24 years in the 1988 Dietary and Nutritional Survey
of British Adults. This data provided the maximum concentrations, distributions of concentrations and the
maximum probability of encountering each substance
in a flavored food or beverage in any one of the 31 different categories. TAMDI produces intake estimates,
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
which are on the same order of magnitude as PADI. If
the model is refined and full stochastic treatment of
the data is performed, the FSM data are lower than
TAMDI estimates by three orders of magnitude for
the twelve substances studied.
The FSM allows for the complete randomization of
conventional intake data and assumes that only a small
portion of the population consumes a given flavoring
substance at its maximum level on a daily basis. This
application of probability to dietary intake provides a
more realistic estimation of intake in that it eliminates
the exaggeration that the maximum level of added flavoring is consumed daily in each food category. To
some extent even the FSM method overestimates intake
in that it does not account for loss due to processing
(cooking) or manufacturing waste and the market share
of flavored foods in that food category, which has the
potential to skew the data depending on the concentration of substance reported in that food. An example of
this would be a soft drink containing a maximum concentration of isoamyl acetate which has market dominance at the time of data collection. Therefore, the
concentration of isoamyl acetate assumed for all similar
soft drinks will be higher in all cases, even though only
one brand contains the maximum concentration. If that
brand dominance fades, then the familiar scenario of
higher estimated intake than manufacture of flavoring
agent would occur.
Upon comparison of the FSM and TAMDI methods
to the PCI · 10 method it is revealed again that
TAMDI, like PADI, is an over estimation of exposure
to flavoring ingredients through food consumption.
The PCI · 10 method is a reasonably conservative estimation for safety analysis when compared to the levels
of exposure calculated by the FSM. The FSM estimates
are comparable to those obtained for 10 different substances by the DDA method with respect to order of
magnitude.
The FSM study points out that the PCI · 10 estimates are a close match to the FSM data, which is lower
by at least one order of magnitude than TAMDI estimates, in most cases. The probability (pFSM>) of
FSM methods overestimating intake determined by
either TAMDI or PCI · 10 is very small This analysis
affirms that FSM estimates are in good agreement with
PCI · 10 estimates.
An advantage of using PCI · 10 estimations is that
the common problem shared by other methods of a
decreasing supply of flavoring substance being eclipsed
and surpassed by intake estimates based on food categories cannot occur. The exposure to flavoring agents is
strictly limited to the volume distributed for the use in
food. Industry poundage surveys are regularly updated
as are estimations of the population through census. Improved intake determinations can be obtained provided
accurate concentrations of flavoring agents in food and
1151
comprehensive food intake data become available. Certainly for particular types of flavoring substances, such
as ‘‘niche’’ flavoring substances, it may be useful to collect concentration and intake data. Flavorings used in
niche food products consumed by a limited population
in a specific geographic region would cause significantly
higher exposure even for flavorings that have a low annual volume of reported use. Similar to the DDA study,
it seems reasonable to collect appropriate data for selected low volume flavoring substances (Arcella and
Leclerq, 2004).
It can be concluded that the DDA and FSM approaches offer a more realistic assessment of intake of
flavoring substances through consumption of food.
The drawback to the DDA method is the cost and time
needed to evaluate the data on a fairly regular basis. The
FSM approach requires a fairly extensive food intake
survey as well, which is economically challenging. The
PCI · 10 method offers a simple calculation based on
easily obtained data and its results are consistent with
those provided by the DDA and FSM methods. Therefore PCI · 10 offers conservative intake estimates and
would be easy to implement on a national and global basis. The Expert Panel uses the PCI · 10 method as a satisfactory means of assessing exposure to flavoring
substances.
3.2. The relationship of intake to threshold of
toxicologic concern
Based on the results of the most recent annual poundage survey (Lucas et al., 1999) and application of the
PCI x10 method, the estimated daily per capita intake
(‘‘eaters only’’) of approximately 85% of all flavoring
substances is less than 1 lg/kg/day in the USA. Less
than 5 chemically identified substances have a daily
per capita intake (‘‘eaters only’’) greater than 1 mg/kg/
day. However, exposure to these few substances occurs
principally from their consumption as naturally occurring components of food and not as intentionally added
flavoring substances. At these low levels of exposure, it
is reasonable to expect that substances used as flavors
would not exhibit any significant toxic effects. Compared to structures of other chemicals used as pesticides,
herbicides, pharmaceuticals, and industrial chemicals,
flavoring agents present no significant structural features that would raise safety concerns, especially at such
low levels of use. Based primarily on these two factors,
the concept of threshold for toxicologic concern was
born, that is, the quantitative levels of exposure below
which there is no significant concern for toxic effects.
Early on, the ability to associate structure and exposure to toxic potential was limited to the effect of an
atom, functional group (e.g., sulfur atom, aldehyde,
amino, carbamoyl, carboxyl, cyano, ester, hydroxyl, nitro, nitrile, and phosphoryl groups), or carbon skeletal
1152
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Table 2
Structural class definitions and their human intake thresholds
Class
Description
Fifth percentile noel
(mg/kg/day)
Human exposure
thresholda (lg/day)
I
Structure and related data suggest a low order of toxicity
If combined with low human exposure, they should enjoy an
extremely low priority for investigation. The criteria for
adequate evidence of safety would also be minimal. Greater
exposures would require proportionately higher
priority for more exhaustive study
Intermediate substances. They are less clearly innocuous
than those of Class I, but do not offer the basis either of the
positive indication of toxicity or of the lack of knowledge
characteristic of those in Class III
Permit no strong initial presumptions of safety, or that may
even suggest significant toxicity. They thus deserve the highest
priority for investigation. Particularly when per capita intake
is high of a significant subsection of the population has a
high intake, the implied hazard would then require the
most extensive evidence for safety-in-use
3.0
1800
0.91
540
0.15
90
II
III
a
The human exposure threshold was calculated by multiplying the fifth percentile NOEL by 60 (assuming an individual weighs 60 kg) and dividing
by a safety factor of 100.
structure (aliphatic or aromatic compounds). The first
attempt to formalize the relationship of chemical structure and the level of use (and importance) to the estimation of toxic hazard was the Cramer et al. decision tree
(Cramer et al., 1978). The ‘‘decision tree’’ assigns substances into three classes, Class I being those chemicals
with simple structures and related data suggesting a
low order of oral toxicity. Class II substances were those
not clearly as harmless as the first class but which did
not show positive indications of toxicity or the structural complexity of Class III. The last class comprised
‘‘those substances that permit no strong initial presumption of safety, or that may even suggest significant toxicity’’. Class I contains several chemical groups of
substances consisting of more than 80% of chemically
identified flavor ingredients that are predictably substrates in high capacity detoxication pathways and that
are without any adverse biochemical or pharmacologic
effect. The chemicals in Class III (i.e., approximately
200 flavor ingredients) that exhibit the highest toxic potential at the lowest levels of exposure are of the greatest
interest. In hindsight, it is now apparent that the decision tree was a conservative, but valid approach to
screening a large number of chemicals for their toxic potential. However, when evaluating individual chemicals,
the structural requirements for assignment to Class III
are, in most cases, too generalized and result in the
inclusion of many relatively innocuous substances.
The toxic potential of each of the three structural
classes has been quantified (Munro et al., 1996). An
extensive toxicity database has been compiled for substances in each structural class. The database covers a
wide range of chemical structures, including food additives, naturally-occurring substances, pesticides, drugs,
antioxidants, industrial chemicals, flavors and fra-
grances. Conservative no observable effect levels (fifth
percentile NOELs) have been determined for each class.
These 5th percentile NOELs in each structural class are
converted to human exposure threshold levels in lg/day
by applying a 100-fold safety factor and correcting for
mean bodyweight (60/100). The 5th percentile NOELs
and human exposure thresholds are recorded for each
structural class (see Table 2). With regards to flavoring
substances, these thresholds are even more conservative,
given that the vast majority of NOELs for flavoring substances are above the 90th percentile. These conservative
exposure thresholds have since been adopted by JECFA
(1997) and Commission of the European Communities
(EC, 2000) for use in the evaluation of chemically identified flavoring agents.
The Panel has recently adopted these thresholds for
their application to the safety evaluation of natural flavor complexes. With the caveat that expert judgment is
used at every step in the procedure, these thresholds provide an efficient method to organize and prioritize the
significant amount of data on a relatively large number
of chemical constituents and chemical groups in an NFC.
In a series of recent papers, Waddell (2002,
2003a,b,c,d) has proposed that the high dose carcinogenicity studies in rodents show evidence of a threshold
dose for carcinogenicity. This conclusion was based on
fundamental principles of chemistry and the observation
that the animal studies show a linear increase in the incidence of tumors in the animals over the range of 2–99%
tumors. The analysis of data is based on a dose-response
relationship in which dose is plotted as the logarithm of
dose in units of molecules/kg bw/day with the scale
continuing down to one molecule/kg/day. The dose is
typically plotted as the incidence of tumors at each dose
level. This scale was first proposed by Rozman et al.
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
(1996) and the log-linear plot is consistent with the traditional analysis of quantal responses in pharmacology
and toxicology. The Panel continues to monitor the progress of the research into carcinogenic thresholds.
3.3. Natural occurrence
The objective of a flavor chemist is not to replace nature, but to duplicate it. Therefore, as analytical methodology has become increasingly sophisticated, many
additional flavors and other ingredients have been found
in raw and processed food. Literally thousands of flavor
ingredients are present in food from natural occurrence
alone before and/or after processing for final consumption. Many of the major and minor constituents of natural and/or processed foods, flavors, and otherwise have
been identified as discrete chemical structures. If that
naturally-occurring chemical is then synthesized it is
considered to be identical to the ‘‘natural’’ product.
Such substances are recognized as natural-identical.
For the evaluation of any synthetic flavor ingredient,
the Panel requires documentation of identity, purity,
and the method of chemical synthesis. The latter information allows the Panel to evaluate potential toxic intermediates or by-products.
3.4. Chemical structure and interaction with biologically
important macromolecules
Key to the safety evaluation process is understanding
the relationships between structure and inherent toxicity, and between dose and the onset of toxicity. Without
exception, every chemical will become toxic provided a
threshold is reached. Although the current state of the
science does not permit the exact prediction of the toxic
effect of a new chemical, recent studies of organic molecules possessing a range of heteroatoms, functional
groups, skeletal structures, and substituents have provided new insight on the interaction of these molecules
with bio-functional macromolecules such as protein,
glycoprotein, lipoprotein, and nucleic acids. Reaction
of these low molecular weight molecules with macromolecules that serve as enzymes, receptors, genes, or
permeability barriers is the primary event that eventually determines the biological response to a substance.
A reciprocal relationship exists between the macromolecule and the low molecular weight substance. The beneficial or toxic biological response to any substance, a
drug, flavoring agent, nutrient or pesticide, is determined by the specific conformation of the target macromolecule. On the other hand, the three-dimensional
structure of the substance determines the action on the
macromolecule (receptor, enzyme, etc.). Flavor substances are selective for the olfactory and gustatory
receptors. Although these functional effects are not of
direct concern to the Panel, flavoring substances must
1153
be evaluated for their potential to interact with other
macromolecules.
The presence of specific atoms, functional groups and
substituents greatly influence the nature and extent of
interaction of a chemical substance with a macromolecule. The interaction may be direct or indirect, in that
the substance may be metabolized prior to interaction
with the macromolecule. For chemically identified flavor
ingredients used at low levels, the substance undergoes
short-lived binding with an enzyme and in the process
is converted to an innocuous product that is less reactive
with macromolecules and is easily eliminated from the
animal. Alternately, the substance may be a substrate
for high capacity enzymes involved in catabolism resulting in the complete breakdown of the substance to carbon dioxide and water. Provided levels of intake are
low, these macromolecular interactions are either beneficial in that the resulting product is of lower toxicity
and, in most cases, more easily excreted that the parent
substance. However, as levels of exposure increase, primary enzymatic pathways may become saturated creating conditions for the involvement of other pathways,
one or more of which may produce reactive metabolites
of greater toxic potential than the parent substance.
Reactive metabolites may bind covalently to proteins,
membrane components and even nucleic acids.
In the last two decades, research in flavor safety has
focused on those few flavor ingredients possessing structural features and concomitant reactivity with macromolecules producing toxic effects at low dose levels in
animal studies. It should be emphasized that, even in
these cases, the dose levels causing deleterious reactions
with macromolecules and subsequent toxicity are orders
of magnitude greater than the average daily intake of
these substances used as flavor ingredients. The following discussion is intended to demonstrate the type of
data on the interaction of flavoring substances with
macromolecules currently being considered by the Expert Panel during the safety evaluation of important flavoring substances.
Formation of protein and DNA adduct of p-allylalkoxybenzene derivatives
Based on the metabolic and biochemical evidence,
the hepatotoxicity and hepatocarcinogenicity of allylalkoxybenzene derivatives (i.e., estragole and methyl
eugenol) in rodents have been related to the
dose-dependent formation of the corresponding 1 0 hydroxy metabolite (Miller et al., 1985). In repeated
oral dose studies in rats, low doses (10 or 30 mg/kg/
d for 5 days) of methyl eugenol have been shown to
produce a single 44 kDa microsomal protein adduct
which is likely formed from the reaction of the electrophilic 1 0 -hydroxylation metabolite (carbonium
1154
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
ion) with a peripheral membrane protein (Gardner
et al., 1996). It is also the major adduct at higher hepatotoxic dose levels (100 and 300 mg/kg/d) which
have been shown to produce as many as 20 other protein adducts. The formation of protein adducts has
been directly related to the formation of the 1 0 -hydroxy metabolite (Borchert et al., 1973; Gardner
et al., 1995) in a dose-related manner (Gardner et al.,
1995). A similar pattern of adduct formation occurs
in vitro when the 1Õ-hydroxy metabolite is incubated
with rat hepatocytes (Gardner et al., 1995, 1996).
In studies beginning two decades ago, methyl eugenol and estragole, the 1 0 -hydroxy metabolites of
methyl eugenol and estragole, and the corresponding
sulfate esters of the 1 0 -hydroxy metabolites were
shown to form DNA adducts in vivo and in vitro.
Adult female CD-1 mice (mean weight 35 g) were
given 12 umol/mouse (58 mg/kg) of [2 0 ,3 0 -3H]-1 0 hydroxyestragole by intraperitoneal injection in trioctanoin, and DNA adduct formation was monitored
over 20 days post exposure. Similarly, 9-day old male
or female B6C3F1 mice (mean weight, 6g) were given
intraperitoneal injections of 0.5 lmol (14 mg/kg) of labeled estragole and sacrificed after 23 h. Three adducts were formed by the reaction of 1 0 or 3 0
positions (cis or trans isomers) of estragole with the
exocyclic amino group (N2) of deoxyguanosine. An
additional adduct was formed by the reaction of the
3 0 position of estragole and the N6 position of deoxyadenosine. Unlike adducts of aromatic amines (e.g.,
N-acetyl-2-aminofluorene) which persist at near maximum levels of binding for several weeks, the three adducts of estragole-deoxyribonucleoside were removed
rapidly from mouse liver DNA. Timed measurement
of DNA adducts revealed a biphasic loss indicated
by a sharp decline in one of the two major 1 0 -hydroxyestragole adducts followed by relatively constant levels of liver DNA adducts from days 3 to 20. This
suggests that at least one of the adducts undergoes
excision repair. Dose levels of the 1 0 -hydroxyestragole
in the adult female and pre-weanling male and female
mice were approximately 58 mg/kg bw and 14 mg/kg
bw, respectively (Phillips et al., 1981).
In 32P-post-labelling experiments with adult
female CD-1 mice (mean weight, 25 g) (Randerath
et al., 1984), a 2 or 10 mg dose of estragole, methyl
eugenol, safrole or other alkenylbenzene derivative
was given by intraperitoneal injection and liver
DNA samples were collected 24 h later. The dose levels in this study were equivalent to 100 or 500 mg/kg
bw of each test substances. Safrole, methyl eugenol,
and estragole show binding activities higher than
allylbenzene, anethole, and other allyl substituted
benzene derivatives. Similar to the previous experi-
ment, a rapid drop in total adduct formation occurred within 7 days after dosing and was followed
by a relatively constant level over the next 140 days.
The authors noted that the significant decrease in
DNA adduct levels was probably related to DNA
repair processes.
In a related 32P-post-labelling experiment (Phillips
et al., 1984), newborn male B6C3F1 mice were given
0.25, 0.5, 1.0, and 3.0 umol of the same series of allylbenzene derivatives by intraperitoneal injection on
day 1, 8, 15, and 22, respectively, after birth. Dose
levels on days 1 and 22 were estimated to be approximately 27 and 35 mg/kg bw, 1Õ-hydroxyestragole and
1 0 -hydroxysafrole, respectively. Mice were terminated on days 23, 29, and 43 and their liver DNA
was isolated and analysed. Highest DNA adduct levels were measured for methyl eugenol, estragole, and
safrole. A significant (p < 0.05) amount of adduct
was detected at 43 days. Based on the results of a
study of carcinogenic activity of these substances in
the same species and strain (Miller et al., 1983), the
authors concluded that threshold adduct levels of at
least 15 pmoles/mg of DNA at 23 days were required
for statistically significant tumour formation (Phillips
et al., 1984). The authors also noted that, compared
to adults, newborn mice showed greater sensitivity
to alkenylbenzene carcinogenicity.
Based on the protein and DNA adduct studies the
Expert Panel concludes that methyl eugenol and
estragole can form covalently-bound protein and
DNA adducts. It is anticipated that the 1 0 -hydroxy
derivative is the reactive intermediary metabolite that
forms protein and DNA adducts, since the 1 0 hydroxy metabolite forms these same adducts but
at lower dose levels than for the parent substance.
Studies performed at relatively high dose levels (15
mg/kg bw) demonstrate that the 1 0 -hydroxy metabolite reacts with an exocyclic amine function to form a
single covalent bond to the deoxyribonucleoside.
Clearly, at high dose levels, DNA adduct formation
has been directly related to administration of the
parent allylalkoxybenzene derivative or its principal
hepatotoxic metabolite. Currently there is no information on the formation of DNA adducts at dose
levels <10 mg/kg bw; there is some evidence that liver
tumors are not induced at detectable levels at a low
dose level (1.5 mg/kg bw) of the 1 0 -hydroxy metabolite (Wiseman et al., 1987).
3.5. Metabolism and disposition
Upon completion of the Scientific Literature Reviews
(1974–1979), the Expert Panel concluded that data on
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
dose-dependent metabolism was critical to understanding the quantitative link between the biochemical fate
of a substance and the dose resulting in toxicity. Beginning in 1980, a series of dose-dependent metabolism
studies were undertaken on key flavor ingredients (benzyl acetate, cinnamaldehyde, furfural, anethole, estragole, 2-ethyl-1-hexanol) for which extensive toxicity and
carcinogenicity data were available. The objective of
the studies was to evaluate the effect of dose on the metabolic options available to a substance and correlate
those data with dose levels required for the onset of
toxicity.
Fundamentally, the metabolic options available to a
substance depend on the chemical structure, that is,
the functional group or groups, carbon skeleton, position and type of substituents, and the electronic and
steric forces present. Ordinarily, the molecule is enzymatically altered to increase its hydrophilic properties
to facilitate excretion via the kidney or entero-hepatic
system. In this case, the toxicity of the substance is decreased by metabolism (detoxication). Often there are
multiple metabolic options available in an animal for
the detoxication of a substance. This is not unexpected,
given redundancy in the evolutionary process. However,
in many cases, as dose increases, other metabolic pathways may emerge due to saturation of high affinity
metabolic pathways (e.g., sulfation) or xenobiotic induction of expression of other metabolic pathways (e.g.,
cytochromes P450). Detoxication and metabolic activation pathways compete and the predominance of a particular pathway is governed by a threshold dose of
substance required to induce a particular pathway and
the capacity of each pathway to react to a given quantity
of substance.
Typically, as one or more detoxication pathways become saturated due mainly to an increase in dose, metabolic activation may emerge. This is particularly true
for conjugating enzymes that scavenge reactive intermediates. Since toxicity studies are routinely performed at
high dose levels (i.e., those required to induce toxicity),
it is critical to evaluate the metabolic events occurring
at anticipated low levels of exposure from use as a flavor
ingredient. The change in metabolic disposition that occurs with increasing dose provides a basis for evaluating
the safe use of a flavor ingredient at low levels compared
to animal toxicity observed at high dose levels.
In addition to dose, the threshold and the capacity of
a metabolic pathway are dependent on the species and
sex of the animal studied. Since animal models (e.g. rats,
mice, and dogs) must be relied on to evaluate the toxic
potential of a substance in man, a properly designed
study of metabolism should be performed over a broad
range of dose levels (including toxic dose levels) in different species.
The evaluation of dose-, sex-, and species-dependent
metabolic studies has become an integral part of the Pa-
1155
nelÕs safety evaluation program. The metabolic fate of
anethole described below demonstrates the PanelÕs integration of metabolic data with observed toxicological
effects.
Metabolism of anethole
trans-Anethole (FEMA No. 2086) is 4-methoxypropenylbenzene. It is the major volatile component
in sweet and bitter fennel, and anise. Based on the reported annual volume in 1987 of 17,100 kg (NAS,
1987), the estimated daily per capita intake (‘‘eaters
only’’) is 0.054 mg/kg bw/d from use of anethole as
an added flavor ingredient. It is calculated as follows:
lg/kg bw = (Annual volume, kg) (1 · 109 lg/kg) · (1/
260 · 106 people in the US) · (1/365 days) · (1/60 kg
bw) · (1/0.6) · 10 (eaters only) and is based on the
assumptions that (1) only 10% of the population,
the ‘‘eaters only’’, consumed the entire reported annual volume of a flavoring substance (NAS, 1987)
and, (2) only 60% of the flavor volume was reported
by flavor manufacturers in the 1987 annual survey
(NAS, 1987).
In 1989, it was reported that chronic intake of
high dose levels of trans-anethole in the female rat
was associated with hepatotoxicity and a low incidence of liver tumors (Truhau et al., 1989; Newberne
et al., 1989). Subsequent studies on the toxicity, pharmacokinetics, and metabolism of trans-anethole in
laboratory animals and humans have been performed
and were used in a mechanism-based safety
evaluation to interpret the observed hepatotoxicity
and related tumorigenic effects (Newberne et al.,
1999). Changes reported in the liver of laboratory rodents exposed to trans-anethole in the diet for periods
more than 2 years (i.e., 833 days) may be further
understood in terms of the hepatic intoxication pathway in which trans-anethole is metabolized to the
ultimate hepatotoxic agent, anethole epoxide.
In a 2-year study, Sprague–Dawley rats exhibited
evidence of hepatotoxicity at dietary levels of 200
and 400 mg/kg bw/d for males and 250 and 550 mg/
kg bw/d for females based on a statistically significant
increased incidence of focal and nodular hyperplasia,
sinusoidal dilatation, and distended bile ducts (Truhau et al., 1989). An independent histopathological
evaluation confirmed the presence of hepatic injury
at these dose levels (Newberne et al., 1989). Additionally, a statistically significant increase in hepatocellular carcinoma was reported in female rats at the
highest dose level (i.e., 550 mg/kg bw/d) (Truhau
et al., 1989; Newberne et al., 1989). The neoplasms
occurred primarily in livers with significant nonneoplastic lesions indicative of hepatotoxicity and
1156
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
necrosis (Newberne et al., 1989). They were found to
be of late onset, after week 98, and had no effect on
the longevity of the animals. Based on the evidence
of hepatotoxicity in males at P200 mg/kg bw and females at P250 mg/kg bw, the NOAEL for the study
was concluded to be 100 mg/kg bw/d for males and
120 mg/kg/bw/d for females (Truhau et al., 1989;
Newberne et al., 1989). In a more recent 90-day dietary study, no significant hepatotoxicity was observed
in male or female rats at dose levels up to 300 mg
trans-anethole/kg bw/d (55–60 mg AE/kg bw/d)
for 90 days (Minnema, 1997).
Subsequent to the 2-year study, biochemical and
metabolic studies were performed that demonstrate
that the hepatotoxicity observed in rats exposed to
trans-anethole is associated with the dose-dependent
metabolic formation of anethole epoxide (AE) (Sangster et al., 1984a,b; Bounds and Caldwell, 1996). At
high dose levels (P100 mg trans-anethole/kg bw), a
metabolic shift to greater epoxidation in rats leads
to increased hepatocellular concentrations of AE.
Epoxidation is more pronounced in rats than mice
(Sangster et al., 1984a,b; Bounds, 1996) and significantly greater than in humans (Sangster et al.,
1987; Caldwell and Sutton, 1988) at low dose levels
(i.e., <12 mg trans-anethole/kg bw/d). Therefore,
the rat is the more sensitive rodent species for evaluating the potential for AE-related hepatotoxicity in
humans exposed to trans-anethole from use as a flavoring substance.
At low doses of trans-anethole in rats, AE is readily detoxified by enzymes such as epoxide hydrolase
(EH; fast-acting enzyme) and glutathione-S-transferase (GST; slower detoxication enzyme) (Marshall
and Caldwell, 1992). With increasing dose levels, hepatic levels of AE increase and these enzymes (especially EH) approach saturation leading to
cytotoxicity. The female rat is probably more sensitive than the male, since it exhibits lower EH activity
that would result in higher hepatocellular levels of
AE (Meijer et al., 1987). Saturation of these detoxication enzymes (i.e., EH and GST) is directly related to
an increase in the cytotoxic effects of trans-anethole.
AE is approximately 10 times more cytotoxic than
trans-anethole in the hepatocytes of rats (Marshall
and Caldwell, 1992, 1996; Caldwell, 1991; Caldwell
et al., 1991; Caldwell et al., 1992). This difference corresponds approximately to the proportion (12–18%)
of trans-anethole that is metabolized to AE in rats
at dose levels (P200 mg trans-anethole/kg bw/d) required to observe hepatotoxicity. Taken together,
these data indicate that cytotoxicity and hepatotoxicity are linked metabolically to the formation of AE
from trans-anethole in the liver.
Based on repeated dose metabolic studies, daily
production of AE is higher in female rats as compared to male rats. At higher dose levels more
trans-anethole is converted to AE that saturates the
‘‘fast-acting’’ epoxide hydrolase detoxication pathway in the liver. This leads to increased hepatocellular AE concentrations that may react with
glutathione or cellular components. At the highest
dose level of anethole (i.e., 400 mg/kg/d in males
and 550 mg/kg bw/d in females) daily hepatic production of AE in females (120 mg/kg bw) was at least
twice that of males (50 mg/kg bw). The NOAEL
of 120 mg trans-anethole/kg bw/d for female rats in
the 2-year study corresponds to production of
approximately 22 mg AE/kg bw/d, which is >10,000
times the level of 0.002 mg AE/kg bw/d produced
by humans from intake of trans-anethole as a flavoring substance.
The panel concludes that daily exposure to significant levels of AE must continue over a long duration
in order to observe the onset of hepatotoxicity in
rats. Cumulative exposure to AE may be directly related to the incidence and severity of the observed
dose-dependent hepatotoxicity. Hepatotoxicity reported in female rats at dose levels P 250 trans-anethole/kg bw/d in the 2-year study corresponds to
cumulative AE production of at least 20,000 mg
AE/kg bw. It is postulated that continuous exposure
to high dose levels of trans-anethole leads to a continuum of biochemical and toxicological events: (1) hepatic metabolism to form AE; (2) saturation of rapid
(EH) detoxication pathways; (3) concomitant increase in hepatocellular concentrations of AE; (4)
cytotoxicity; (5) cell death (necrosis); (6) hepatocyte
proliferation; and ultimately (7) liver tumors in a
few female rats as reported in the 2-year study (Truhau et al., 1989).
The low incidence (6/52) of carcinomas observed
in the severely compromised livers of female rats given 550 mg trans-anethole/kg bw/d in the 2-year
study occurred following an estimated total lifetime
exposure to AE exceeding 100,000 mg/kg bw. The
fact that hepatocellular carcinomas occurred only
in the female rat is a reflection of a higher daily dose
of trans-anethole for the females, increased
conversion to AE compared to the male, and decreased detoxication of AE by the female which
exhibits a lower activity of the AE-detoxication
enzyme EH compared to that of the male (Meijer
et al., 1987).
3.6. Toxicology
As previously described, the Panel considers flavoring
substances in the context of homologous chemical
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
1157
groups. If sufficient biochemical and metabolic information on prototype members of the chemical group are
available and if the substances belong to a well-studied
and toxicologically innocuous group of substances used
at low levels in food, toxicological data on each member
of the group may not be required to reach a GRAS decision. However, under some circumstances, even though
information on safety and metabolic fate is available for
other members of the chemical group, the Panel may
conclude that toxicological data should be obtained
for a specific substance.
In the past when additional data have been needed, a
simple minimal test, referred to as a ‘‘Primary Toxicity
Screen’’ (PTS), developed by the Panel for such needs,
served to replace the standard LD50 test and to provide
a minimal amount of biological data. If the results from
the PTS so indicated, more definitive tests were then
required, on a case-by-case basis, usually sufficient to
establish a no observed adverse effect level (NOAEL).
The 14-day PTS protocol provided important data in
alleviating the scientific and regulatory concerns of the
times. However, advances in molecular biology now
reveal potential methods for enhancement of histological observations.
After carefully considering the OECDÕs ‘‘Guidelines
for Testing Chemicals. No. 407 Repeated Dose Oral Toxicity—Rodent: 28 day study or 14 day study’’ (OECD,
1981), the Panel decided to modify the toxicity testing
guidelines, now referred to as the 4-Week Toxicity Study
for Flavoring Substances, to meet the following objectives: (1) provide a test that meets the special needs of
flavor materials; (2) maintain a flexible test protocol that
is compatible with the ones currently recommended by
national and international regulatory agencies; and (3)
expand the toxicity testing to characterize adverse effects
to the nervous, reproductive and immune systems, if
necessary.
ally related flavor ingredients (e.g., aldehydes, terpene
hydrocarbons) are associated with a single target organ
and tumor type in a specific species and sex using a single mode of administration. Common features among
these substances provide valuable information for the
mechanism of carcinogenic activity.
3.6.1. Carcinogenicity studies on flavoring ingredients
For more than two decades, the Panel has critically
reviewed the results of carcinogenicity studies of more
than 20 flavoring substances or their principal metabolites, the majority of which were sponsored by the National Toxicology Program (NTP). Given that the
studies were hazard determinations, they were normally
performed at dose levels orders of magnitude greater
than the daily intakes as added flavor ingredients or as
naturally occurring constituents of food. Even at these
high intake levels, the majority of flavoring ingredients
show no carcinogenic potential (Table 3). In addition
to dose, the carcinogenic potential of remaining flavor
ingredients are related to several factors including mode
of administration, species and sex of animal model, and
target organ specificity. In the vast majority of studies,
the carcinogenic effect is secondary to pre-existing
chronic organ toxicity. Selected subgroups of structur-
Carcinogenic effects specific to rodents
Kidney neoplasms in male rats chronically exposed
mainly to hydrocarbons, ketones and other
substances.
Administration of high dose levels of hydrocarbons (limonene, camphene, decalin, and JP-4 jet
fuel) and other substances (e.g., isophorone, methyl
isobutyl ketone, and a-methylbenzyl alcohol) in
short-(Hoechst, 1991; Wolf et al., 1956; Webb
et al., 1989; Spindler and Madsen, 1992) and longterm studies (EPA, 1991; or NTP, 1990) exhibit
renal lesions resulting from the accumulation of
aggregates of a2u-globulin (a low molecular-weight
protein synthesized in the male rat liver) and the test
substances or a metabolite in the P2 segment of the
renal proximal tubule. These aggregates prevent
lysosomal degradation which leads to accumulation
Carcinogenic effects related to the mode of
administration
Forestomach neoplasms in rodents chronically
administered irritating reactive substances by gavage.
The appearance of forestomach hyperplasia and
squamous cell papillomas in rodents is a regular
occurrence in bioassay gavage studies in which high
concentrations of an irritant material in corn oil is
delivered daily by dosing tube into the forestomach.
These phenomena are consistently associated with
administration of high concentrations of aldehydes,
e.g., malonaldehyde, furfural, benzaldehyde, and
trans,trans-2,4-hexadienal (NTP, 1988, 1990c,b,
2003b) and other irritating substances, e.g., ethyl
acrylate, benzyl acetate, dihydrocoumarin and coumarin (NTP, 1986a, 1993a,b,c) in corn oil by gavage.
The lack of any evidence of carcinogenicity in bioassays in which aldehydes (e.g. citral and cinnamaldehyde) (NTP, 2003a; NTP, 2004) or reactive
substances (e.g., benzyl acetate) (NTP, 1986b) were
provided in microencapsulated form administered
in the diet illustrates the impact of the mode of
administration on the toxicological sequelae in the
rodent forestomach. Future design of 2-year bioassay
studies with low molecular weight, irritant substances
should avoid the use of gavage as a mode of
administration.
Substance (FEMA No.)
Negative results:
Acrolein
O
Daily per
capita
Intake
(‘‘eaters
only’’),
lg/kg
bw/day
Route of
administration
Dose levels,
mg/kg bw/day
Statistically
significant (P < 0.05)
dose-related
neoplastic effects
in rats (% tumors at
each dose level)
Statistically
significant (P < 0.05)
dose-related
neoplastic effects in
mice (% tumors at
each dose level)
Not
applicable
Oral, drinking
water
Groups of 20m and
20f F344 rats were
given drinking water
containing 100, 250
or 625 mg/L
5 days/week for 124,
124 or 104 weeks,
respectively
None
Not applicable
Lijinsky and
Reuber (1987)
Not applicable
Not
applicable
Gavage, water
Groups of 70m and
70f Sprague–Dawley
rats were
administered 0, 0.05,
0.5 or 2.5 mg/kg bw
7 days/week for
102 weeks
None
Not applicable
Parent et al.
(1992)
Not applicable
Not
applicable
Oral, drinking
water
A group of 20m and
20f F344 rats was
given drinking water
containing 300 mg/L
allyl alcohol
5 days/week for
106 weeks
None
Not applicable
Lijinsky and
Reuber (1987)
Not applicable
100.57
Oral, feeding
study
CD-1 mice (f only)
0% or 0.46% for
12 months
Not applicable
None
Miller et al.
(1983)
Not applicable
NTP (1993a)
Not applicable
O
H
Allyl alcohol
HO
Anethole (2086)
(Z)
Reference (date)
Evaluation of
positive results
O
Benzyl acetate
(2135)
O
O
14.24
Oral, feeding
study
0, 3000, 6000 or
12,000 ppm (R);
0, 330, 1000,
3000 ppm (M)
60 animals/sex/group
None
None
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
H
Acrolein
1158
Table 3
Summary of carcinogenicity studies on FEMA GRAS substances
Benzoin (2132)
Oral, feeding
study
0, 125 or 250 ppm
(mR) 0, 250 or
500 ppm (fR) 0,
2500 or 5000 ppm
(M) 104 weeks
None
None
NCI (1980b)
Not applicable
61.24
Oral, feeding
study
0, 3000 or 6000 ppm
105 weeks (R)
107–108 weeks (M)
None
None
NTP (1979)
Not applicable
61.24
Oral, feeding
study
0%, 0.25% or 1%
WistarR 57m and
57f/group 104 weeks
None
Not applicable
Hirose et al.
(1981)
Not applicable
61.24
Oral, feeding
study
0, 200, 1000 or
5000 ppm (M)
96 weeks
Not applicable
None
Shirai et al.
(1982)
Not applicable
Oral, drinking
water
0%, 0.1% or 0.2%
WistarR 78 weeks
None
Not applicable
Takayama
and Kuwabara
(1982)
Not applicable
Gavage, corn
oil vehicle
0, 375 or
750 (mice only)
Not applicable
None
NTP (1990b)
Not applicable
OH
O
Butylated hydroxytoluene
(BHT) (2184)
OH
Butylated hydroxytoluene
(BHT) (2184)
OH
Butylated hydroxytoluene
(BHT) (2184)
OH
Caffeine (2224)
O
4022.90
N
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
0.35
N
N
N
O
d-Carvone (2247)
OH
0.01
1159
Daily per
capita
Intake
(‘‘eaters
only’’),
lg/kg
bw/day
Route of
administration
Dose levels,
mg/kg bw/day
Statistically
significant (P < 0.05)
dose-related
neoplastic effects
in rats (% tumors at
each dose level)
Statistically
significant (P < 0.05)
dose-related
neoplastic effects in
mice (% tumors at
each dose level)
Reference (date)
Evaluation of
positive results
trans-Cinnamaldehyde (2286)
990.00
Diet
(microencaps)
0, 50, 100 or
200 mg/kg bw in
male and female
F344/N rats and 0,
125, 270 or
550 mg/kg bw in
male and female
B6C3F1 mice
None
None
NTP (2002)
Not applicable
0.19
Oral, feeding
study
0, 30, 80 and 200
to Charles River
CD-COBS rats
through
3 generations and
to F1 generation
for 2 years
None
Not applicable
King et al.
(1979)
Not applicable
0.40
Gavage,
distilled
water with
5 mg
Polyoxyl 35
Caster Oil
(CremophorÒ
EL) per 100 mL vehicle
0, 50, 200, or
750 (M) 78 weeks
0, 50, 150, or
500 (R) 104 weeks
None
None
Astill et al.
(1996)
Not applicable
56.06
Oral, feeding
study
CD-1 mice (f only)
0% or 0.50% for
12 months
Not applicable
None
Miller et al.
(1983)
Not applicable
56.06
Feeding study
0, 6000 or
12,500 ppm (fR)
0, 3000 or 6000
(mR, M)
None
None
NTP (1983b)
Not applicable
O
H
3-Ethyl-2-hydroxy-2-cyclopenten-1-one
(3152)
OH
O
2-Ethyl-1-hexanol (3151)
HO
Eugenol (2467)
OH
O
Eugenol (2467)
OH
O
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Substance (FEMA No.)
1160
Table 3 (continued )
Geranyl acetate (71%),
Citronellyl acetate (29%) (2509)
8.16
Gavage, corn
oil vehicle
0, 1000, or 2000
(R) 0, 500 or
1000 (M)
None
None
NTP (1987)
Not applicable
1.10
Inhalation
0, 500, 1000 or
2000 ppm
None
None
NTP (1999)
Not applicable
527.76
Feeding study
0, 3750, 7500 (R) 0,
2000, 4000 ppm (M)
None
None
NCI (1978)
Not applicable
600.45
Gavage, corn
oil vehicle
0, 200 or 400 mg/kg
bw in male and
female F344/N rats
and B6C3F1 mice 0,
300 or 600 mg/kg bw
in female B6C3F1
mice for 104 wks
None
Forestomach:
Squamous cell
papillomas:
(females at
300 mg/kg bw,
10%; 600 mg/kg
bw, 12%) lower
incidence in males
NTP (1990c)
Forestomach tumors
associated with chronic
irritant effects (Smith
and Ford, 1993)
0.003
Diet
(microencaps)
0, 22.5, 45, 90 mg/kg
bw in rats and 30, 60,
and 120 mg/kg bw in
B6C3F1mice for
104 weeks
Forestomach:
Squamous cell
carcinoma and
papillomas (mf);
(males at
22.5 mg/kg bw,
7%; 45 mg/kg bw,
25%, 90 mg/kg bw,
67%) lower
incidence in females
Forestomach:
Squamous cell
carcinoma and
papillomas (mf);
(females at 30 mg/kg
bw, 4.3%; 60 mg/kg
bw, 23%, 120 mg/kg
bw, 39%) lower
incidence in males
NTP (2003a)
Forestomach tumors
associated with chronic
irritant effects (Smith
and Ford, 1993)
0.01
Gavage, corn
oil vehicle
0, 100 or 200 mg/kg
bw in Fischer 344
rats and B6C3F1
mice for 104 weeks
Forestomach:
Squamous cell
carcinoma and
papillomas: (males
at 100 mg/kg bw,
36%; 200 mg/kg,
72%); lower
incidence in females
Forestomach:
Squamous cell
carcinoma and
papillomas: (males
at 100 mg/kg bw,
11%; 200 mg/kg
bw, 24%) lower
incidence in females
NTP (1986a)
Forestomach tumors
associated with chronic
irritant effects (NTP,
1986a)
O
O
Isobutyraldehyde (2220)
H
Menthol (2665)
OH
Forestomach effects:
Benzaldehyde (2127)
O
H
2,4-Hexadienal (3429)
(z)
(z)
O
Ethyl acrylate (2418)
o
o
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
O
1161
Substance (FEMA No.)
Liver effects:
Anethole (2086)
Route of
administration
Dose levels,
mg/kg bw/day
Statistically
significant (P < 0.05)
dose-related
neoplastic effects
in rats (% tumors at
each dose level)
Statistically
significant (P < 0.05)
dose-related
neoplastic effects in
mice (% tumors at
each dose level)
Reference (date)
Evaluation of
positive results
100.57
Oral, feeding
study
0%, 0.25%, 0.5%
and 1% (105–1000
mg/kg bw/day)
groups of 312m and
312f
Sprague–Dawley
rats 117–121 weeks
Liver: Hepatocellular
neoplasms (females
at 1000 mg/kg bw,
23%)
Not applicable
Truhaut et al.
(1989)
Although statistically
significant, the increase
hepatocellular carcinoma
remained within the
historical control range
for the strain of rat and
laboratory
14.24
Gavage, corn
oil vehicle
0, 250 or 500 mg/kg
bw day in groups of
50 male and
50 female F344/N
rats 0, 500 or
1000 mg/kg bw day
in groups of 50 male
and female B6C3F1
mice for 103 weeks
Pancreas:
Acinar-cell
adenoma (males
at 250 mg/kg bw,
54%; 500 mg/kg
bw, 76%); testis
mentioned but
data not shown
Liver: Adenomas
or carcinomas
(males at 500 mg/kg
bw, 37%; 1000 mg/kg
bw, 46%;
females-lower
incidence at high
dose only);
Forestomach:
Squamous cell
papilloma or
carcinoma (males
at 500 mg/kg bw,
8%; 1000 mg/kg bw,
22%; females- lower
incidence at high
dose only)
NTP (1986b)
Neoplasms in mice
associated with the
gavage route of
administration and
high spontaneous
incidence of this type
of neoplasm in this
mouse. No tumors
were observed in a
subsequent feeding
study of benzyl
acetate (NTP, 1993a)
Feeding study
Groups of 50
F344/N rats and
50 B6C3F1 mice
were fed 0, 15,000
or 30,000 ppm (0, 750,
1500 mg/kg bw) for
103 weeks
None
Liver: Hepatocellular
carcinomas or
adenomas (combined)
(males at 750 mg/kg
bw, 60%; 1500 mg/kg
bw, 79%) lower
incidence in females
NCI (1980b)
Neoplasms in mice
associated with high
spontaneous incidence
of this type of
neoplasm in this strain
of mouse; the MTD
was exceeded and may
have been a
confounding effect
(Adams et al., 2004)
(Z)
o
Benzyl acetate (2135)
o
o
Cinnamyl anthranilate (2295)
H2N
O
O
(Z)
0
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Daily per
capita
Intake
(‘‘eaters
only’’),
lg/kg
bw/day
1162
Table 3 (continued )
Furfural (2489)
0, 30 or 60 mg/kg
bw in F344/N rats and
0, 50, 100, or
175 mg/kg bw in
B6C3F1 mice for
103 weeks
Liver:
Cholangiocarcinomas
(males at 60 mg/kg
bw, 2%) and bile
duct dysplasia with
fibrosis
Liver: Hepatocellular
carcinomas and
adenomas (males at
50 mg/kg bw, 44%;
100 mg/kg bw, 35%;
175 mg/kg bw, 64%)
lower incidence in
females
NTP (1990c)
1.22
Oral, diet
CD-1 mice (f only)
0, 2300 ppm, or
4600 ppm for
12 months
Not applicable
Hepatomas (females
at 2300 ppm, 58%;
4600 ppm, 71%)
Miller et al.
(1983)
1.22
Gavage
Weanling CD-1 mice
370 mg/kg, twice
weekly for 10 times.
Animals examined
after 14 months
Not applicable
Hepatomas:
(males at 0 mg/kg
bw, 24%; 370 mg/kg
bw, 73%) lower
incidence in females
Miller et al.
(1983)
0.07
Drinking water
8, 17, 36 mg/kg in
male F344/N rats for
104 weeks and 35,
65, or 110 in B6C3F1
mice for 104 (female)
or 105 (male) weeks
None
Hepatocellular
carcinomas or
blastomas in mice:
(males at 250 mg/kg
bw, 94%; 500 mg/kg
bw, 94%, 1000
mg/kg bw, 94%) lower
incidence in females
NTP (1997c)
Gavage, corn
oil vehicle
0, 150, 300 or
600 mg/kg bw in
groups of 60 male
and 60 female
F344/N rats and
0, 200, 400 or
800 in 70 male
and 70 female
B6C3F1 mice (M)
for 105 weeks
Kidney: Renal tubule
hyperplasia (males at
150 mg/kg bw, 5%;
300 mg/kg bw, 6%;
600 mg/kg bw, 8%)
and adenomas (males
at 150 mg/kg bw,
1%; 300 mg/kg bw,
3%; 600 mg/kg bw,
6%) lower incidence
for both in females
Liver:
Hepatocellular
adenomas,
carcinoma, or
hepatoblastoma
(combined): (males
at 200 mg/kg bw,
59%; 400 mg/kg bw,
78%; 800 mg/kg bw,
68%) lower incidence
in females
NTP (1993b)
O
O
H
Estragole (2411)
O
Estragole (2411)
O
Pyridine (2966)
N
Liver and kidney effects:
3,4-Dihydrocoumarin (2381)
O
O
18.52
Rat and mouse liver
tumors were either
insignificant at P < 0.01
(Haseman et al., 1986),
or secondary to chronic
hepatotoxicity (Adams
et al., 1997)
Rat kidney lesions
species/sex specific
(Smith et al., 1996).
Mouse lesions associated
with high incidence of
spontaneous lesions an
an inconsistent dose
response (Smith et al.,
1996)
1163
Gavage, corn
oil vehicle
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
7.68
1164
Table 3 (continued )
Substance (FEMA No.)
Route of
administration
Dose levels,
mg/kg bw/day
Statistically
significant (P < 0.05)
dose-related
neoplastic effects
in rats (% tumors at
each dose level)
Statistically
significant (P < 0.05)
dose-related
neoplastic effects in
mice (% tumors at
each dose level)
Reference (date)
Evaluation of
positive results
0.002
Gavage, corn
oil vehicle
0, 250 or
500 mg/kg bw to
groups of 50
F344/N rats and
50 B6C3 F1 mice
(M) for 103 weeks
Kidney: Renal tubular
cell adenomas (males
at 500 mg/kg bw, 4%)
or adenocarcinomas
(males at 250 mg/kg
bw, 6%); carcinomas
of the preputial gland
(males at 250 mg/kg bw,
10%; 500 mg/kg bw,
10%)
Liver: Hepatocellular
adenomas or
carcinomas
(combined) (males
at 250 mg/kg bw,
36%; 500 mg/kg bw,
58%) and
mesenchymal tumors
in the integumentary
system (males at
250 mg/kg bw,
16%; 500 mg/kg
bw, 28%)
NTP (1986c)
Kidney neoplasms,
a2l-globulin phenomenon;
preputial neoplasms, high
spontaneous incidence in
this rat; Liver neoplasms,
a species-specific effect;
neoplasms of
integumentary system,
high spontaneous
incidence in this rat
(Adams et al., 1996)
Gavage, corn
oil vehicle
0, 75 or 150 mg/kg
bw to groups of 50
F344/N male rats;
0, 300, 600 mg/kg
bw to groups of
50 female F344/N
rats; 0, 250 or
500 mg/kg bw to
groups of 50 male
(mM) 0, 500 or 1000
(fM) for 103 weeks
Kidney: Renal tubular
cell hyperplasia (males
at 75 mg/kg bw, 8%;
150 mg/kg bw, 14%);
adenomas (males at
75 mg/kg bw, 8%;
150 mg/kg bw, 16%);
and adenocarcinomas
(males at 75 mg/kg bw,
8%; 150 mg/kg bw,
6%) no lesions
observed in females
None
NTP (1990d)
Kidney neoplasms
associated with
a2l-globulin, specific to
the male rat (Burdock
et al., 1990; EPA, 1991)
O
(Z)
Kidney effects:
d-Limonene (2633)
(Z)
Line missing
212.10
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Isophorone (3553)
Daily per
capita
Intake
(‘‘eaters
only’’),
lg/kg
bw/day
1.21
a-Methylbenzyl
alcohol (2685)
0, 375 or 750 mg/kg
bw in F344/N rats
and B6C3F1 mice
for 105 weeks
Gavage, corn
oil vehicle
0, 31, or 62 mg/kg
bw to groups of 50
male and 50 female
F344/N rats and
B6C3F1 mice for
105 weeks
Hematopoietic
system: Mononuclear
cell leukemia
(females at 31 mg/kg
bw, 12%; 62 mg/kg
bw, 18%) lower
incidence in males
Hematopoietic
system: Lymphoma
(females at 31 mg/kg
bw, 22%; 62 mg/kg
bw, 36%) lower
incidence in males
NTP (1983c)
Rat and mouse lesions
not statistically significant
(P < 0.01) (Haseman
et al., 1986; Smith et al.,
1996); blood effects
previously observed in
controls at this lab,
associated with Klebsiella
infection
Feed,
microencaps
1000, 2000, or
4000 ppm (50, 100,
or 210 mg/kg bw) to
groups of 50 male and
50 female F344/N
rats for 105 weeks
500, 1000, or
2000 ppm (60, 120,
or 260 mg/kg bw) to
groups of 50 male
and 50 female
B6C3F1 mice for
105 weeks
None
Malignant
lymphoma (all
types): (females at
60 mg/kg bw, 10%;
120 mg/kg bw, 18%;
260 mg/kg bw, 24%)
NTP (2003b)
High incidence in
historical controls
maintained on NIH
diet-07 (NTP, 2001a)
HO
Other effects:
Allyl isovalerate (2045)
0.01
O
O
116.50
Citral (2303)
H
O
(Z)
Kidney: Renal tubular
cell adenomas and
adenomas or
adenocarcinomas
(combined) (males at
375 mg/kg bw, 4%;
750 mg/kg bw, 10%)
None
NTP (1990e)
Inadequate study
(Smith and Ford, 1993)
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Gavage, corn
oil vehicle
1165
1166
NTP (1982)
None
Urinary bladder:
Transitional cell
papillomas (males
at 12 mg/kg bw,
4%; 25 mg/kg bw,
8%)
0, 12 or 25 mg/kg
bw fed to groups of
50 male and 50 female
F344/N rats and
B6C3F1 mice for
103 weeks
N
C
S
2.21
Allyl isothiocyanate
(2034)
O
(E)
O
HO
4-Hydroxy-2,5-dimethyl3(2H)-furanone
87.00
Diet
Gavage, corn
oil vehicle
Kelly and
Bolte (2003)
Not applicable
Pituitary adenomas
of the pars distalis
in 400 mg/kg group
of males after
18 months on test.
Within historical
control data ranges
and within reported
ranges in the
literature for the age
and strain of rat
(McComb et al.,
1984)
0 (control), 100,
200 and 400 mg/kg
bw/day for 24 months
to Sprague–Dawley
rats (60/sex/group)
Pituitary adenomas
common in aging and
aged rats- appear at
between 13 and 24 months
of age. Peto analysis,
which compares tumour
rate and time to tumour
formation, revealed no
statistical difference
between control and
treated males and females
in this study. Adenomas
were concluded to be
common, spontaneous
tumours unrelated to the
administration of DMHF
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
in the cytoplasm of the protein or the protein-chemical complex which leads to single cell necrosis, regeneration and eventually, renal neoplasia in male rats
(Lehman-McKeeman et al., 1990; Hildebrand et al.,
1997; Strasser et al., 1988; Borghoff et al., 1990).
The accumulated evidence indicates that it is the unique interaction of the chemical or metabolites with
a2l-globulin in the male rat kidney, especially the
proximal convoluted tubule, which allows d-limonene to interfere with renal processing of the sex-specific a2l-globulin. Since humans and other species do
not possess a protein comparable to a2l-globulin that
bonds these substances and accumulates in the renal
tubules, this process is not predictive of human carcinogenicity. In a comprehensive review of a2l-globulin nephropathy and associated renal tubule tumors
produced in the male rat exposed to d-limonene and
other simple chemical substances (e.g. isophorone,
decalin and methyl isobutyl ketone), it was concluded
that this mode of action in the male rat is not an
appropriate model for assessing human renal carcinogenic risk (EPA, 1991; IARC, 1989). After careful
review, the Panel concludes that this mode of action
leading to the renal carcinogenic findings in the male
rat are largely known and strongly indicate that the
nephropathy associated with d- limonene has no significance for human risk assessment (Burdock et al.,
1990).
Peroxisome proliferation-liver tumors in mice and
pancreatic acinar cell tumors in rats
Hepatocellular neoplasms in mice.
Neoplastic and non-neoplastic lesions associated
with administration of cinnamyl anthranilate to mice
developed principally in the liver. Treated groups of
male and female mice showed evidence of lipoidosis,
hemosiderosis, hyperplasia of hepatocytes and a statistically significant increase in the incidence of combined hepatocellular adenomas and carcinomas
{control, 14/48; 15,000 ppm or 2250 mg/kg bw, 30/
50 (p = 0.003); 30,000 ppm or 4500 mg/kg bw, 37/
47 (p < 0.001)} in male mice compared to that of
the control group. There was a statistically significant
increase in the incidence of hepatocellular carcinomas {control, 1/50; 15,000 ppm or 2250 mg/kg bw,
8/49 (p = 0.014); 30,000 ppm or 4500 mg/kg bw, 14/
49 (p < 0.001)} and combined adenomas and carcinomas {control, 3/50; 15,000 ppm or 2250 mg/kg bw,
20/49 (p < 0.001); 30,000 ppm or 4500 mg/kg bw,
33/49 (p < 0.001)} in dosed groups of female mice.
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
Four high-dose and two low-dose females were diagnosed as having both adenomas and carcinomas.
The NTP report concluded: ‘‘Based on increased
incidences of hepatocellular adenomas, and hepatocellular adenomas and carcinomas, cinnamyl anthranilate
was considered carcinogenic for male and female
B6C3F1 mice receiving 15,000 or 30,000 ppm cinnamyl anthranilate in the diet’’ (NCI, 1980).
Since performance of the original bioassay (NCI,
1980), additional studies on over 70 substances have
established a direct correlation between the increased
incidence of hepatocarcinogenicity and the induction
of peroxisome proliferation in rodent livers (Ashby
et al., 1994). Studies performed by the European
Centre for Ecotoxicity and Toxicology of Chemicals
(ECETOC) (ECETOC, 1992) show that peroxisome
proliferators form a discrete category of rodent liver
carcinogens, the carcinogenicity of which does not involve direct genotoxic mechanisms.
Histological evidence of peroxisome proliferation
in rodents is reflected by an increased peroxisome/
mitochondrial ratio which is correlated with increases
in target organ weights, total cytochrome P-450 content, and activities in microsomal lauric acid hydroxylation, carnitine acetyl transferase, and cyanide
(CN) insensitive palmitoyl-CoA (Reddy et al.,
1980, 1986; Reddy and Lalwai, 1983; Barber et al.,
1987). Peroxisome proliferation is a transcriptionmediated process involving the peroxisome proliferator-activated receptor (PPARa) in the hepatocyte
nucleus. The role of PPARa in the induction of
hepatocarcinogenicity in the mouse has been clearly
established (Peters et al., 1997). Carcinogenicity studies with mice genetically modified to remove PPAR
show no evidence of either peroxisome proliferation
or carcinogenicity. Given that levels of expression
of PPARa in humans is 1–10% of levels found in
the rat or mouse (Palmer et al., 1994, 1998) and that
the associated response element does not appear for
several of the genes in humans, it is not unexpected
that humans are refractory to peroxisome proliferation following chronic exposure to potent rodent peroxisome proliferators. No significant evidence of
peroxisome proliferation has been observed in human studies with several potent hypolipidemic drugs
that are rodent peroxisome proliferators (reviewed in
Doull et al., 1999; Ashby et al., 1994). Based on these
observations, it is concluded that the hepatocarcinogenic response in rodents is not relevant to the human health assessment of cinnamyl anthranilate.
When the above information is combined with
data on metabolism and enzyme induction, the panel
concludes that hepatic peroxisome proliferation is
both a rodent-specific and dose-dependent phenome-
1167
non induced by the intact ester cinnamyl anthranilate
(Viswalingam et al., 1988; Keyhanfar and Caldwell,
1996; Caldwell, 1992). Specifically, repeated-dose
metabolism studies have shown that above a threshold dose greater than 500 mg/kg bw/d, intact cinnamyl anthranilate given i.p. or in the diet to mice
shows a dose-dependent increase in liver weight, total
cytochrome P-450, microsomal lauric acid hydroxylation and cyanide (CN) insensitive palmitoyl-CoA
activity, and peroxisome/mitochondria ratio in hepatic cells (Caldwell, 1992; Viswalingam et al., 1988).
These markers for peroxisome proliferation correspond to dose levels at which saturation of the hydrolysis pathway leads to the presence of the intact ester
in vivo. Therefore, peroxisome proliferation caused
by cinnamyl anthranilate is a dose-dependent effect.
In addition, the results of chronic studies on the
hydrolysis product, anthranilic acid, a normal metabolite of the amino acid tryptophan, and on the intermediary metabolite cinnamyl alcohol, provide
additional evidence for this mechanism of action.
Pancreatic Acinar-cell neoplasms in male rats
In an NTP bioassay using cinnamyl anthranilate
(NCI, 1980), an increased incidence of pancreatic acinar-cell adenomas (2/45) and carcinomas (1/45) was
reported in the high-dose males (3/45; 7%) compared
to controls (0/42). According to the NTP, although
the difference was not statistically significant, the
incidence of this type of neoplasm in aging F344 control rats is extremely low {historical incidence for
controls in participating NTP laboratories (6/1538;
0.28%)}. Therefore, the NTP considered occurrence
of these neoplasms to be related to administration
of the test material.
Since completion of the 2-year bioassay with cinnamyl anthranilate, other carcinogenicity studies have
established a relationship between peroxisome proliferation and the appearance of pancreatic acinar-cell
neoplasms in the male F344 rat (Critical Reviews in
Toxicology, 11/03). The sex-specific phenomenon
has been observed when F344 male rats were exposed
to high dose levels of other peroxisome proliferators
(e.g. butyl benzyl phthalate and hypolipidemic drugs,
clofibrate and nafenopin) (Malley et al., 1995; NTP,
1997a; Reddy and Qureshi, 1979; Svoboda and Azarnoff, 1979). It appears that the effect on the rat pancreas is secondary to the effect of these substances on
the liver.
The sequence of rat pancreatic acinar cell hypertrophy, hyperplasia, and adenomas in male rats is affected by several factors including steroids, growth
factors such as cholecystokinin (CCK), growth factor
receptors, and diet. Studies show that testosterone
stimulates, and estrogen inhibits, the growth of
1168
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
pancreatic acinar-cell neoplasms in rats (Lhoste
et al., 1987a,b; Sumi et al., 1989; Longnecke, 1987;
Longnecker and Sumi, 1990). Cholecystokinin has
been shown to stimulate adaptive and neoplastic
changes of pancreatic acinar cells (Longnecke,
1987). The impact of diet on stimulation of CCK
and the subsequent appearance of acinar cell neoplasms in male rats has also been reported (Longnecke, 1987; NTP, 1997b). In rat bioassays, the
corn oil vehicle has been shown to increase the incidence of pancreatic acinar call neoplasms (Longnecke, 1987). Also, the incidence of pancreatic
acinar-cell neoplasms induced by benzyl phthalate
was 10/50 for male rats fed ad libitum, but 0/10 for
rats placed on a restricted feed protocol for 2 years.
The latter study clearly demonstrated the effect of excess caloric intake on the incidence of pancreatic acinar cell neoplasms. In summary, the appearance of
these neoplasms is sex, species, dose, and even diet
specific. In addition, relevance of this tumor type to
humans is unlikely since acinar cell proliferations
and tumors in humans are rare.
Apparently, prolonged peroxisome proliferation
in rats inhibits bile flow leading to cholestasis (Lu
et al., 2000; Marrapodi and Chiang, 2000). The cholestasis, in turn, leads to a decrease in trypsin activity
and an increase in monitor protein in the gut lumen
which stimulates cholecystokinin (CCK) (Obourn
et al., 1997a,b). CCK then acts on CCK receptors
on pancreatic acinar cells leading to hyperplasia
and eventually adenomas. This is a high dose phenomenon in rats and is unlikely to occur in humans.
Several human studies of hypolipidemic drugs that
are recognized peroxisome proliferators in rodents
have failed to show any significant difference in cancer deaths between treated patients and placebo-treated group (IARC, 1996). Also, acinar cell neoplasms
are extremely rare in humans. These results are expected, since humans and rodents show quantitative
differences in their response to peroxisome proliferators. Furthermore, increased CCK levels in humans
do not stimulate acinar cell proliferation, because humans possess a relatively small number of CCK
receptors compared to the rat.
Given these more recent data and the lack of any
correspondence between bioassay results and human
studies with peroxisome proliferators, the Panel
believes that the increased incidence of acinar-cell
neoplasms in the F344 male rat are associated with
peroxisomal proliferation induced by high dose levels of cinnamyl anthranilate. This effect is specific
to the male F344 rat and, therefore, is not relevant
to the human health assessment of cinnamyl
anthranilate.
In summary, the FEMA Expert Panel concludes
that the report of pancreatic neoplasms in the male
F344/N rat is related to use of corn oil as a gavage
vehicle and is a secondary response to peroxisome
proliferation. Likewise, hepatic neoplasms in the
B6C3F1 mouse in the NTP bioassay are secondary
responses to peroxisome proliferation. Since peroxisome proliferation is a dose-dependent effect specific
to rodents, the results of the bioassay are not relevant
to the safety of cinnamyl anthranilate in humans at
low levels of intake from its use as a flavoring
substance.
High incidence of background spontaneous
neoplasms
Liver neoplasms in male and female B6C3F1 mice.
High dose levels of groups of flavor ingredients
{i.e., benzyl acetate, furfural dihydrocoumarin,
isophorone, pyridine} have been associated with an
increased incidence of liver neoplasms in primarily
male, but also female mice in NTP gavage studies.
In parallel studies, no liver neoplasms were reported
in male or female rats. The high incidence of hepatocellular adenomas and carcinomas that occurs in
control male and female mice demonstrates the sensitivity of the B6C3F1 mouse liver to neoplastic
changes. A recent analysis of the historical spontaneous incidence of liver neoplasms in control male and
female B6C3F1 mice has revealed background incidences of liver adenoma/carcinomas of 42.2% (range
10–68%) for males and 23.6% (6–56%) for female
B6C3F1 mice (Haseman et al., 1998). Increasing liver
tumor rates are strongly correlated with increasing
mean body weights in control and test mice achieved
when NTP altered its standard protocol from group
to individual housing (Haseman et al., 1994). This
change led to increased body weights in control and
test animals. Analysis of body weight on the incidence of liver neoplasms for control and test mice
in the NTP database revealed that males exhibiting
mean body weights >49 g showed a liver tumour incidence of 54% (test) and 62% (control), respectively.
Test females exhibiting mean body weights >49 g
showed a liver tumour incidence of 35%. In addition,
other factors (Haseman et al., 1998) such as administration of corn oil gavage vehicle and contamination
with Heliobacter hepaticus contribute to the high
background incidence of this type of neoplasm in
both sexes of mice. The profile of neoplastic responses in these studies are consistent with the historically high levels of background hepatocellular
neoplasms in male and female B6C3F1 mice (Maron-
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
pot et al., 1987). Therefore, the appearance of hepatic
neoplasms in NTP mouse bioassays is generally not
relevant to the safety of these flavor ingredients in
humans at low levels of intake from use as flavor
ingredients. This conclusion is based on: the high
incidence of spontaneous hepatocellular neoplasms
(adenomas and carcinomas) in the B6C3F1 mice related to body weight changes, the use of corn oil gavage, the absence of consistent dose-response data,
the lack of hepatocellular neoplastic effects in the
parallel rat studies, low incidences of liver tumors
in other strains of mice that do not have a high spontaneous incidence, and the relatively high dose levels.
Lymphoma in B6C3F1 mice
An increased incidence of malignant lymphoma
was reported in B6C3F1 female mice administered
(1) diets containing 0, 500, 1000 or 2000 ppm of citral
(a mixture of the cis and trans isomers of 3,7-dimethyl-2,6-octadienal) (NTP, 2003a), or (2) 31 or
62 mg/kg bw of allyl isovalerate by gavage daily, five
days per week, for 2 years (NTP, 1983b). A doserelated increase in the incidence of lymphoma was reported that was statistically significant in the high
dose in B6C3F1 female mice given citral (P = 0.011
by Fisher exact test; 12/50 (24%) at 2000 ppm versus
3/50 (6%) in controls) or allyl isovalerate (22% at 31
mg/kg bw and 36% at 62 mg/kg bw). There was no
evidence of increased incidence of malignant lymphoma in either sex of F344/N rats or in male
B6C3F1 mice.
The background incidence of malignant lymphoma in control female B6C3F1 mice maintained
on a NTP-2000 diet is high (98/659), with a historical
incidence of 14.0% (standard deviation ± 7.1%) and
a range of 6–30% (NTP, 2003a). The incidence of
spontaneous malignant lymphoma in female
B6C3F1 mice in all 2-year rodent carcinogenicity
studies carried out by NTP is also high (20.9%)
(Haseman et al., 1998). The historical incidence in
controls maintained on the NIH-07 diet at the same
contract laboratory performing the citral study was
high (167/953) with a historical incidence of 17.5%
(standard deviation, 7.7%) and a range of 6–30%.
Therefore, these tumors occur at a high and variable rate in control animals. It is recommended
(Haseman et al., 1986) that a compound is anticipated to exhibit a carcinogenic potential if the highest
dose is associated with an increased incidence of a
common tumor that is significant at the 1%
(p < 0.01) level, or an increased incidence in a rare tumor at the 5% (p < 0.05) level. Therefore, applying a
significance level of 1% (p < 0.01) and based on pairwise comparisons of the incidence of malignant
lymphoma in the citral study by a Fisher exact test,
1169
the incidence of this commonly observed neoplasm
is not considered to be statistically significant
(p = 0.011) for female mice at the 1% level. Similar
conclusions can be made for the allyl isovalerate gavage study.
Based upon the high frequency of this neoplastic
response in historical controls in NTP studies (Haseman et al., 1998), the fact that toxicity was observed
in female B6C3F1 mice at dose levels producing lymphoma in female mice given citral or allyl isovalerate,
and the observation that the incidences of lymphoma
reported in both NTP studies were not significant at
the 1% level (p < 0.01) (Haseman et al., 1986), the
FEMA Expert Panel concluded that the results of
the bioassays do not provide evidence that these substances present a carcinogenic risk to humans exposed to low levels of citral or allyl isovalerate used
as flavor ingredients added to food.
Carcinogenic effects in multiple species
Liver neoplasms in mice and rats.
High dose levels of allylalkoxybenzene derivatives
(i.e., estragole, methyl eugenol, and safrole) have
been associated with a carcinogenic potential in multiple species and sexes via different routes of administration. Both the qualitative and quantitative aspects
of the molecular disposition of methyl eugenol and
estragole and their associated toxicological sequelae
have been relatively well defined from mammalian
studies. Several studies have clearly established that
the profiles of metabolism, metabolic activation,
and covalent binding are dose-dependent and that
their relative importance diminishes markedly at
low levels of exposure (i.e., these events are not linear
with respect to dose). In particular, rodent studies
show that these events are minimal probably in the
dose range of 1–10 mg/kg bw which is approximately
100–1000 times the anticipated human exposure to
these substances. For these reasons the panel concludes that present exposure to methyl eugenol and
estragole resulting from consumption of food, mainly
spices, and added as such, does not pose a significant
cancer risk. Nevertheless, current toxicologic and
biochemical studies are being performed to confirm
and more thoroughly explore both the nature and
implications of the dose-response curve in rodents
at low levels of exposure to methyl eugenol and
estragole.
3.6.2. Conclusion
3.6.2.1. Conclusions on carcinogenicity. In summary,
the majority of 2-year bioassays on flavor ingredients
either show no evidence of carcinogenicity in rodents at
1170
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
any dose level or exhibit carcinogenic responses that are
not considered relevant to the assessment of risk to
humans. These latter studies show a dose- and toxicitydependent increase in the incidence of neoplasms in a specific rodent species and sex (i.e., a-2u-globulin in male
rats and spontaneous liver neoplasms or lymphoma in
B6C3F1 male and female mice, respectively), or use a
mode of administration (forestomach tumors by gavage)
irrelevant to the intake of flavor ingredients by humans,
or the neoplasms are of a spontaneous background nature specific to a sex, strain, and species of rodent. For
those substances that currently are not the subject of
ongoing research (i.e., allylalkoxybenzene derivatives),
it is clear that flavor ingredients at current levels of intake
provide no significant carcinogenic risk to humans. However, taken together, these assays reveal the importance
of chronic dose-related organ toxicity in the onset of carcinogenic effects in 2-year rodent studies. In this regard, it
is relevant to discuss presence of toxicity thresholds for
the appearance of carcinogenicity in these studies.
3.7. Genotoxicity
The Panel uses data from genotoxicity studies as a
screening tool to evaluate the genotoxic potential of
individual flavoring compounds and groups of structurally related substances. In this respect the Panel has
evaluated the results of thousands of genotoxicity assays. The Panel critically evaluates the results of both
in vitro and in vivo tests for genotoxicity, taking into
consideration metabolic and related toxicity data in
coming to a conclusion as to the biological significance
and relevance of genotoxicity studies in the context of
the use of flavoring substances.
The Panel is often faced with a mix of positive and
negative in vitro studies. In such cases results from well
conducted in vivo studies would typically outweigh positive results obtained in vitro. It is the PanelÕs practice
to provide a clear rationale for its conclusions in resolving divergent data sets.
The original design of standardized in vitro assays was
to provide a basis for understanding the potential of
chemical substances to cause molecular and genetic alterations leading to carcinogenicity. However, the disparity
of positive and negative results observed in the four
widely used in vitro genotoxicity tests (SAL, MLA,
ABS, and SCE) has caused a thorough reevaluation of
the utility of these assays for predicting the carcinogenic
potential of substances in long-term rodent assays.
Based, in part, on the following conclusions by researchers affiliated with the NTP (Haseman et al., 1990; and
Zeiger et al., 1990), the use of ABS, SCE, and MLA assays to prioritize substances for carcinogenicity testing
has been curtailed within the NTP: (1) there appears to
be no evidence of complementariness among the four
genotoxicity assays; (2) the results of a battery of tests
has not been shown to be more conclusive than the use
of the SAL assay alone (Smart, 1994); and (3) the disparity of positive and negative results in selected assays precludes the formation of conclusions on the potential
carcinogenicity of a substance.
The evaluation of genotoxicity assays is also often
complicated by the fact that the results are inconsistent
with other metabolic and toxicologic data on the substance. Thus, the panel considers the results of genotoxicity assays in the broader context of available
information for a given flavoring substance. An example
of such an analysis is provided by consideration of the
panelÕs analysis of the data set available for furanones.
Furanones are flavor ingredients that exhibit uniform
positive responses in both in vitro and in vivo genotoxicity assays. The genotoxic potential of 3-(2H)-furanone
derivatives is consistently demonstrated by the results of
standardized bacterial (Gilroy et al., 1978; Xing et al.,
1988; Hiramoto et al., 1996a,b; Li et al., 1998) and
mammalian assays (Xing et al., 1988; Tian et al., 1992;
Hiramoto et al., 1996b). Yet consideration of metabolic
and toxicological studies along with comparison to data
sets available for chemically-related substances suggests
that furanones added as flavoring agents do not pose a
significant genotoxic risk to humans (see box).
Putative mechanism of genotoxicity of furanone
derivatives
Furanones such as 2,5-dimethyl-2(3H)-furanone
(DMHF) induce DNA damage in vitro by generating
free radicals that induce strand scission. In the presence of metals (e.g., Fe3+) and dissolved oxygen, the
enolic OH of the furanone is oxidized by single electron transfer to yield the corresponding carbon-centered radical and a reduced metal ion (e.g., Fe2+).
The carbon-centered radical can couple to molecular
oxygen to produce a peroxyl radical. Alternately, the
reduced metal ion can autoxidize to form superoxide
radical anion. Superoxide radical then dismutates
into hydrogen peroxide (H2O2). It is well recognized
that reduced metals react with H2O2 to form hydroxyl radical which is a powerful oxidizing agent (see
Fig. 2). Hydrogen peroxide also oxidizes glutathione
leading to decreased GSH/GSSG and an increase in
cellular oxidative stress.
HO
O
OH
O2-.
superoxide
dismutase
O
+ Fe++ + O2-.
+ Fe+++ + O2
H2O2
OH.
hydroxyl radical,
DNA strand breaking
Fig. 2. Mechanism of in vitro furanone derivative oxidation.
R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
The ability of DMHF to induce oxygen radical
formation and DNA strand breaks is reminiscent of
similar activities of Vitamin C, a structurally related
furanone. Vitamin C (ascorbic acid) contains an enediol that is superficially related to the enol of DMHF.
Being both an enol ether and a,b-unsaturated ketone,
DMHF is anticipated to undergo hydrolytic ring
opening to yield an enediol. Like DMHF, Vitamin
C also reduces metal ions and produces superoxide
anion to generate hydroxyl radicals that cleave
DNA. As anticipated, vitamin C exhibits genotoxicity in test systems similar to those in which furanones test positive. In standard Ames assays,
ascorbic acid (vitamin C) induces reverse mutations
in S. typhimurium strains TA104, TA102, TA100,
and TA98 at concentrations 352–1,761 lg/plate
(DÕAgostini et al., 2000; Ichinotsubo et al., 1981).
In the E.coli Mutoxitest, positive results were obtained when 200, 300, or 400 lg/plate of ascorbic acid
in the presence of Cu++ was incubated with E. coli
strain IC203. E.coli IC203 carries a D oxy R30 mutation effectively removing its ability to turn on the biosynthesis of hydrogen peroxide protective proteins
and making the strain sensitive to DNA damage under conditions of oxidative stress (Blanco et al., 1998;
Martinez et al., 2000).
Increased levels of micronuclei were observed
when ascorbic acid (400, 500, or 600 lg/ml) was incubated with Chinese hamster cells (Miller et al., 1995).
An increase in SCE was observed in Chinese hamster
ovary (CHO) cells in the presence of 500 lg/ml of
ascorbic acid without metabolic activation (Tennant
et al., 1987). Ascorbic acid (1 or 2 mg/kg bw) did
not increase the incidence of replicative DNA synthesis in F344 rats or B6C3F1 mice (Miyagawa et al.,
1995). But in a standard mouse micronucleus assay,
1500 mg/kg bw ascorbic acid induced a significant increase in micronuclei in B6C3F1 mice (Shelby et al.,
1993).
Furanones are a class of substances present in
food naturally that are also added as flavoring
agents. The principal furanone used as a flavoring
agent is DMHF. In humans, DMHF is rapidly absorbed in the gastrointestinal tract and conjugated
with glucuronic acid in the liver. Free DMHF is
not detected in the blood of human volunteers to
whom it is administered as a constituent of strawberries; its glucuronic acid conjugate is the principal urinary metabolite (Roscher et al., 1997). Thus, the
potential for chemical reaction of DMHF with
important cellular macromolecules, especially DNA,
appears low.
Despite the fact that DMHF is genotoxic, it does
not produce carcinogenic effects in rodents. Two
1171
studies, one on DMHF and the other on a structurally furanone show no evidence of carcinogenicity
at intake levels orders of magnitude greater than
the intake of furanones added as flavoring agents
(Kelly and Bolte, 2003; Munday and Kirkby, 1973).
Furthermore, Vitamin C, a furanone with a genotoxicity test profile similar to that of DMHF, does not
demonstrate carcinogenicity (NRC, 1996). As a result of the 2-year bioassay of DMHF, a NOAEL of
200 mg/kg bw per day was established in rodents.
This intake level is approximately 2000 times the daily per capita intake (‘‘eaters only’’) of 0.088 mg/kg
bw per day form use of DMHF as a flavoring agent.
Based on the available data, it is highly unlikely that
DMHF or other furanones pose any significant genotoxic risk to humans under conditions of use as flavoring agents.
4. Conclusions
The Panel will continue to evaluate genotoxicity studies for their relevance to the human health assessment of
flavor ingredients. Certainly as a screening tool, the results have been consistent with the metabolic and toxicologic data available for the vast majority of chemical
groups reviewed by the Panel. However, at this time,
the relevance of genotoxicity data must be evaluated in
the context of other data more closely related to actual
human experience, i.e., the results of genotoxicity testing
are not viewed in isolation, these data comprise one
component that is factored into the PanelÕs overall
safety assessment.
5. Summary
This review describes the current status of the GRAS
evaluation of flavoring substances by the Expert Panel
of FEMA. The Panel currently maintains a rotating
10-year program of continuous review of the safety data
related to chemical groups of flavor ingredients. As scientific methodology has evolved, new data relevant to
the safety of individual and groups of structurally related flavoring substances have become available. Studies in dose-dependent metabolism, enzyme induction,
two year bioassays, and in vitro and in vivo genotoxicity
have become a routine part of flavor safety evaluation.
Maintaining its multidisciplinary approach to safety
evaluation, the PanelÕs objective is to integrate relevant
information from these different scientific disciplines to
achieve a truly comprehensive safety evaluation program for flavoring substances. Major emphasis has been
placed on understanding biochemical and biological
events leading to toxicity, the relationship of toxicity
to dose, and comparison of dose to intake of the
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R.L. Smith et al. / Food and Chemical Toxicology 43 (2005) 1141–1177
flavoring substance. The mechanistic approach continues to evolve.
The Panel has now concluded a second comprehensive review of all available GRAS chemically identified
flavoring substances. This second re-evaluation of chemical groups of flavor ingredients is recognized as the
GRAS reaffirmation (GRASr) program The Expert Panel is alert to new methodology that conceivably could
be applied to the evaluation of the safety-in-use of flavor
ingredients. However, the application of such tests
would need to be evaluated within the context of the
very low exposure resulting from the use of flavor ingredients in order to be validated.
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