ACUTE TOXICITY OF RDX NITROSO PRODUCTS 427
JOURNAL OF APPLIED TOXICOLOGY
J. Appl. Toxicol. 2005; 25: 427–434
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1090
Up-and-down procedure (UDP) determinations of
acute oral toxicity of nitroso degradation products
of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
Sharon A. Meyer,1,* Adam J. Marchand,1 Jennifer L. Hight,1 George H. Roberts,2 Lynn B. Escalon,3
Laura S. Inouye4 and Denise K. MacMillan5
1
2
3
4
5
Department of Toxicology, University of Louisiana at Monroe, Monroe, LA 71209, USA
Department of Clinical Laboratory Science, University of Louisiana at Monroe, Monroe, LA 71209, USA
Analytical Services, Inc., Huntsville, AL, 35806, USA
U.S. Army Engineer Research and Development Center, Environmental Laboratory, Environmental Risk Assessment Branch,
Vicksburg, MS 39180, USA
U.S. Army Engineer Research and Development Center, Environmental Laboratory, Environmental Chemistry Branch, Omaha,
NE 68102, USA
Received 10 January 2005; Revised 11 April 2005; Accepted 12 April 2005
ABSTRACT: Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a widely used military explosive and soil and ground
water contaminant of munitions manufacturing and artillery training sites, undergoes microbial nitroreductase metabolism to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and
hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Human occupational and accidental exposures to RDX, as well as acute
oral exposures in rats, result in seizures, but little is known about the toxicity of the RDX degradation products. The main
objective of the present study was to determine the oral LD50 of the most potent RDX N-nitroso product in female SpragueDawley rats using the recently validated up-and-down procedure (UDP). With only 26 rats, MNX was identified as the
most potent metabolite and a maximum likelihood estimate of 187 mg kg−1 (95% confidence interval 118–491 mg kg−1) for
its LD50 was established and found equivalent to that of RDX determined with the same protocol. CNS toxicity, manifested
as forelimb clonic seizures progressing to generalized clonic-tonic seizures, was the critical adverse effect. Further, confirmation of the UDP LD50 for MNX with a fixed-dose design enabled identification of 94 mg kg−1 as the highest nonlethal
dose. An ED50 of 57 mg kg−1 was determined for neurotoxicity, while splenic hemosiderosis and decreased blood hematocrit
and hemoglobin concentration occurred with a threshold at 94 mg kg−1 in 14-day survivors. These studies, while providing new toxicity data necessary for the management of RDX-contaminated sites, illustrate the efficiency of the UDP for
comparative acute toxicity determinations and its value in guiding further characterization of dose dependency of identified adverse effects. Copyright © 2005 John Wiley & Sons, Ltd.
KEY WORDS: N-nitroso toxicity; toxicity testing; neurotoxicity; anemia
Introduction
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a highly
energetic chemical used in conventional, biological,
chemical and nuclear weapons (National Academy of
Sciences, 2000). The need for site remediation of
munitions manufacturing and artillery training sites with
known RDX-contaminated soil and ground water, some
of which are entries on the U.S. Environmental Protection Agency (USEPA) National Priorities List (Agency
for Toxic Substances and Disease Registry [ATSDR],
1995), has necessitated the evaluation of the health
* Correspondence to: Dr. S. A. Meyer, Department of Toxicology, College
of Health Sciences, University of Louisiana at Monroe, Monroe, LA 712090470, USA.
E-mail: meyer@ulm.edu
Contract/grant sponsor: Environmental Quality and Technology Program,
Engineer Research and Development Center, U.S. Army Corps of Engineers;
contract/grant number: BS04 008D5P; BAA-02-3435.
Copyright © 2005 John Wiley & Sons, Ltd.
hazards of RDX. RDX is also being evaluated for future
regulatory action under provisions of the Safe Drinking
Water Act (USEPA, 2005).
Knowledge of the toxicity of RDX environmental
degradation products also is important for site remediation. Anaerobic microbial metabolism of RDX results
successively in nitroreductase-catalysed formation of
hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX),
hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) and
hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) (Fig. 1).
All three nitroso degradation products have been detected
in ground water at RDX contaminated sites (Beller and
Tiemeier, 2002) and as intermediates of bioremediation
processes (Hawari et al., 2000).
Acute occupational and accidental exposures to high
doses of RDX have identified the central nervous system
(CNS) as the critical target tissue as manifested by reversible seizure activity (Woody et al., 1986; Goldberg
et al., 1992; Harrel-Bruder and Hutchins, 1995; Testud
J. Appl. Toxicol. 2005; 25: 427–434
428
S. A. MEYER ET AL.
acceptance of the up-and-down procedure (UDP) for
determination of acute toxicity was in response to this
goal. Since categorical data generated by the UDP are
statistically analysed to yield maximum likelihood estimations of potency, the UDP was employed for these
comparative studies. Further, the UDP LD50 for the most
potent metabolite MNX was confirmed with a traditional,
fixed-dose design, thus enabling dose-response determinations of MNX toxic effects qualitatively identified in the
UDP study.
Materials and Methods
Figure 1. Structures of parent compound hexahydro1,3,5-trinitro-1,3,5-triazine
(RDX)
and
products
hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX),
hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) and
hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX)
et al., 1996; Küçükardalı et al., 2003). Seizures are also
induced by acute oral exposure of rats to RDX and are
causal to lethality, for which an LD50 of ~100 mg kg−1
has been determined (Cholakis et al., 1980; cited in
ATSDR, 1995). Transient deposition of radioactivity
from [14C]RDX within the brain occurs within hours of
acute oral exposure, followed by accumulation in the
liver and kidneys. Disposition in liver is due, in part,
to retention of an unidentified metabolite(s) (Schneider
et al., 1977).
Nothing is known of the activities of the nitroso products relative to defined RDX toxic effects, while the
recent demonstration of the weak mutagenicity of TNX
(George et al., 2001) illustrates the potential for unique
toxic effects contributed by the nitroso toxicophore.
Effects typical of other N-nitroso compounds include
cytotoxicity and genotoxicity from alkylating metabolites
and effects resulting from nitroso redox activity, such as
methemoglobinemia with consequential splenic hemosiderosis and anemia. Certain N-nitroso compounds are
also sources of nitric oxide (Ohwada et al., 2001) and
thus associated with the many toxic effects attributed to
reactive nitrogen species. Clearly, research into the potential for MNX, DNX and TNX to elicit these toxic effects
will provide information essential for remediation of sites
contaminated by these military-unique compounds. In
addition, comparative assessment of toxic effects of these
compounds with parent RDX provides an opportunity to
evaluate the relative activity of N-nitro vs N-nitroso
functional groups in eliciting these effects. The objectives
of the present study were to identify the most potent of
the RDX N-nitroso products and compare its LD50 with
that of its parent. A current priority of chemical hazard
identification has been the implementation of validated
procedures that reduce experimental animal usage. Recent
Copyright © 2005 John Wiley & Sons, Ltd.
Chemicals
MNX, DNX and TNX were obtained from Dr Ron
Spanggord (SRI Intl., Menlo Park, CA). Purities, determined by HPLC with UV detection, were >99.9% for
TNX, 98.4% for MNX with ~1.2% RDX, and 66.4% for
DNX with ~25.3% MNX and 7.1% TNX. Compounds
were used without additional purification. Because of the
highly energetic property of RDX, nitrosamine derivatives were also handled as potential explosives. RDX
(>99%) was obtained from Stan Caulder (Naval Surface
Warfare Center, Indianhead, MD) and was stored under
absolute ethanol. Stored quantities were limited to <1 g
and sonication was avoided in the preparation of corn
oil-based dosing solutions.
Animals and Treatment
Female Sprague-Dawley rats were from the in-house
breeding colony (School of Pharmacy, University of
Louisiana at Monroe [ULM]) and maintained in accordance with the Guide for Use and Care of Animals
(National Research Council, 1996). Breeders were
from Harlan-Sprague Dawley (Madison, WI). Rats were
housed with a 12 h light/dark cycle and free access to tap
water and pelleted rodent chow (no.7012, Harlan/Teklad,
Madison, WI). One week prior to trials, groups of rats
(175–225 g) were housed individually in polycarbonate
cages on hardwood bedding (Sani-chips, Harlan/ Teklad,
Madison, WI). Study protocols had prior approval by the
ULM Animal Care and Use Committee.
Comparative Studies
For comparison of acute toxicity of MNX, DNX and
TNX, a sequential dosing protocol based upon the UDP
was used, but modified to incorporate a smaller dose progression factor (1/3 log unit) to better distinguish between
potencies of related compounds. Also, because chemicals
were available in limited amounts, the progression was
J. Appl. Toxicol. 2005; 25: 427–434
ACUTE TOXICITY OF RDX NITROSO PRODUCTS 429
stopped when a dose was identified with >50% lethality
with one of the N-nitroso products and <50% lethality
with the others and limit dose determinations were not
conducted. Instead, the series was initiated with a starting dose of 400 mg kg−1, a dose approximating that yielding 80% mortality with RDX as determined by Schneider
et al. (1977). Dosing solutions were made in the minimal
amount of dimethylsulfoxide (DMSO, 5% v/v) in corn
oil that allowed preparation of a stable emulsion for
the highest dose of the least soluble compound
(400 mg kg−1 MNX) without sonication. Solutions were
prepared by dissolving compounds in DMSO, and then
mixing with corn oil (Sigma Chemical Co., St Louis,
MO) warmed to 40 °C just before use.
Rats were weighed, then rank ordered based upon
random numbers assigned by weight. Food was withdrawn overnight before administration of starting doses at
10–11 a.m. by gavage to rat 1 (TNX), rat 2 (DNX) and
rat 3 (MNX). Rat 4 received vehicle at 10 ml kg−1, the
volume administered to rats receiving test compounds.
Qualitative assessments of appearance, righting reflex,
forelimb strength and incidence of tremors and/or convulsions were made at 15 min intervals during the first hour
after treatment, hourly over the next 8 h and daily thereafter over the 14 day observation period. Since death was
observed at 400 mg kg−1 MNX within 48 h, rats 5, 6 and
7 received 187 mg kg−1 TNX, DNX or MNX, respectively. Doses for the third group of rats were increased to
400 mg kg−1 or decreased to 87 mg kg−1 depending upon
whether the 187 mg kg−1 rat survived or died, respectively, within 48 h. Doses for subsequent animals were
similarly determined by outcome of the previous rat.
Moribund rats, as judged with criteria of the Organization
for Economic Cooperation and Development (OECD,
2000), and 14-day survivors were euthanized with CO2
and examined for gross pathological effects. Liver,
kidney plus adrenal, stomach plus duodenum and
spleen were fixed in neutral buffered formalin for
histopathology.
UDP Studies and Fixed-dose Acute Toxicity Study
Having established that the most potent RDX N-nitroso
metabolite was MNX, a UDP study was conducted according to the 2002 EPA acute oral toxicity guideline
870.1100 (USEPA, 2002) with a slope factor sigma = 0.33
and a starting dose of 87 mg kg−1. Additional subsequent
dose levels were 187 and 400 mg kg−1. Dose progression
was stopped when one of the three stopping rules of the
AOT425StatPgm program (USEPA, 2003) was satisfied.
A UDP trial with RDX was similarly conducted to provide an oral LD50 generated under the same conditions,
i.e. preparation of corn oil-based solutions by dilution of
compounds dissolved in DMSO. It was anticipated that
prior dissolution in DMSO would control for the wide
Copyright © 2005 John Wiley & Sons, Ltd.
variability in LD50 from other studies that has been attributed to the particle size of RDX solid used to prepare
dosing solutions (Yinon, 1990). Dosing solutions of RDX
were prepared just before use by drying an aliquot of
ethanol slurry and redissolving the weighed, residual
solid in DMSO, then warm corn oil as described above.
To confirm the MNX LD50 and to provide material for
the assessment of dose dependency of endpoints identified in the UDP, a fixed dose protocol was performed
as in the 1998 EPA 870.110 guidelines except that
the number of dose levels was increased from 3 to 5 to
facilitate inclusion of no-adverse-effect- and lowestadverse-effect-levels for outcomes other than lethality.
Five groups of six rats (175–225 g) were weighed and
housed individually, then randomly assigned to dose.
Prior to treatment, food was withdrawn overnight.
Treatments were vehicle or MNX at 47, 94, 187, 280
or 420 mg kg−1. Rats were observed for incidence of
tremors and/or convulsions frequently over the first 8 h
after treatment. Survivors were weighed at 7 and 14 days,
then exsanguinated by cardiac puncture while under CO2
anesthesia. Blood was collected in heparinized syringes
and transferred to EDTA-containing tubes for hemocytology and determination of creatinine and urea nitrogen.
Livers and spleens were weighed and sections taken for
histopathology.
Blood Analyses and Histopathology
Blood urea nitrogen (BUN) and creatinine concentrations
were analysed with a Synchron CX® System (Beckman
Instruments; Brea, CA). In this system, BUN is measured
as the rate of NADH oxidation by a coupled ureaseglutamate dehydrogenase enzymatic reaction. Creatinine
was measured as a complex with picrate upon reaction
with Jaffe’s reagent.
Hemoglobin and red and white blood cell count and
size were determined with a CELL-DYN 3500 System
(Abbott Laboratories; Abbott Park, IL). Hematocrit is
derived from red blood cell size and count. Hemoglobin
was measured as absorbance at 540 nm after leukocyte
lysis and conversion to hemoglobin-hydroxylamine.
Formalin-fixed tissues were embedded in paraffin and
5 µm sections were stained with hematoxylin and eosin.
Qualitative examinations for pathology were made on
all collected tissues. Sections of spleen were also stained
for ferric iron deposits by reacting with potassium
ferrocyanide to form Prussian blue and counterstained
with Nuclear fast red (Vacca, 1985). Quantitative assessment of Fe3+-laden splenic macrophages was done by
counting dark blue staining vesicles of diameter greater
than that of yellow-staining erythrocytes in red pulp of
spleens stained for Prussian blue (Fig. 2). Slides were
coded before observation and at least 20 100× oil immersion fields were counted per animal.
J. Appl. Toxicol. 2005; 25: 427–434
430
S. A. MEYER ET AL.
2003). Estimates of the MNX LD50 and ED50 for convulsions were determined from the fixed-dose acute toxicity
study by log-probit analysis with SAS PROC PROBIT
(SAS, 1989). Effects of MNX on body and organ
weights, hematological parameters, and blood urea nitrogen and creatinine of the 14-day survivors were determined by ANOVA with post-hoc comparisons of
treatment means against vehicle control done with
Dunnett’s test. Count data of large, Prussian blue-stained
splenic vesicles/microscopic field were analysed for dose
dependence using the Cochran-Armitage trend test
(Piegorsch and Bailer, 1997) and comparison of treatment
means against vehicle was done by ANOVA plus
Dunnett’s test on the square root transformation with
3/8 continuity factor.
Results
Comparative Lethality of N-nitroso Products
of RDX
The incidence of lethality was 100%, 67% and 67% at
400 mg kg−1 for MNX, DNX, TNX, respectively (Table 1).
No rats died upon treatment with DNX or TNX at
187 mg kg−1, the next dose level in the progression, while
two of the three rats treated with MNX died. The most
notable clinical symptom observed was the occurrence
of convulsions. Forelimb clonic seizures were observed
most often and occasionally progressed to severe generalized clonic-tonic seizures requiring euthanasia. Seizures,
which occurred as early as 5 min after dosing, were
often preceded by vacuous chewing movements and a
brown discharge from the mouth. Forelimb strength
and righting reflex appeared unaffected by any of the
chemicals, although performance of these tests would
often precede the onset of convulsions. Seizures occurred
at a 100% incidence for MNX and DNX and at 67% for
TNX at the highest dose, while at 187 mg kg−1, an incidence of 67% with MNX compared with 33% with DNX
or TNX. From these data, it was concluded that MNX
Figure 2. Photomicrographs of Prussian blue-stained
spleens from rats 14 days after treatment with
187 mg kg−1 MNX (B) or vehicle (A). Large iron-laden
lysosomes of macrophages in the red pulp are indicated by arrows
Statistical Analysis
Maximum likelihood estimates of MNX LD50 and the
95% confidence interval from the UDP study were obtained from the AOT425StatPrg, version 1.0 (USEPA,
Table 1. Comparative MNX, DNX and TNX acute toxicitya
Chemical
Dose
(mg kg−1)
Group
1
MNX
DNX
TNX
400
187
87
x|+
400
187
o|+
400
187
o|−
2
3
x|+
4
5
x|+
o|–
x|+
x|+
o|−
x|+
6
7
Lethality
Convulsions
x|+
2/2
2/3
0/2
2/2
2/3
1/2
o|−
2/3
0/3
3/3
1/3
o|+
2/3
0/3
2/3
1/3
o|–
o|+
o|+
o|−
Incidence
x|+
o|−
a
Outcomes are given as lethality|occurrence of convulsions as symbolized by x, death or o, survival and −, absence or +, presence of convulsions. Rats were
administered the indicated dose for each chemical in sequence at 48 h intervals. Rats dosed first were those in group 1, those dosed second in group 2, dosed
third in group 3, etc.
Copyright © 2005 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 2005; 25: 427–434
ACUTE TOXICITY OF RDX NITROSO PRODUCTS 431
Table 2. MNX fixed-dose acute oral toxicity study
Dose
(mg kg−1)
0
47
94
187
280
420
Lethality
Convulsions
Body weight
gain/2 wka (g)
Spleen weight at 14
da (% of body wt)
PrB+ splenic
lysosomesa,b (#/field)
0/5
0/5
0/5
3/5
5/5
4/5
0/5
0/5
4/5
4/5
5/5
5/5
15.8 ± 2.4
17.2 ± 3.7
−8.8 ± 6.3*
17, −18
0.27 ± 0.02
0.26 ± 0.01
0.29 ± 0.02
0.26, 0.23
4.43 ± 0.93
5.20 ± 0.81
7.08 ± 1.21*
13.13, 5.14
0.18
19.11
a
−45
Mean ± SEM for n = 5 for doses 0–94 mg kg . Individual values for survivors of doses >94 mg kg are given. * P < 0.05.
Prussian blue-stained vesicles of diameter greater than that of erythrocytes in 100× oil immersion fields of splenic red pulp. Significant dose-dependent trend,
P < 0.05.
−1
−1
b
was the most potent of the RDX N-nitroso metabolites
with respect to acute oral toxicity.
No obvious pathological lesions were noted either
grossly or histologically for tissues taken from rats
treated with any chemical and euthanized shortly after
treatment. Stomachs were distended and filled with fluid
and yellow granular material, but no histopathological
evidence of tissue damage was observed. For the 14-day
survivors, Prussian blue staining of spleens from rats
treated with 187 mg kg−1 of the chemicals indicated
hemosiderosis for the single MNX survivor and for two
of the three TNX-treated rats. Neither spleen from two
DNX-treated rats exhibited hemosiderosis. Although the
third rat treated with 187 mg kg−1 DNX survived 48 h
after dosing and was thus classified as a survivor for the
UDP determination of lethality (Table 1), death occurred
during night 13 and thus tissue from this animal was not
available for histopathology. No other gross or
histopathological lesions were noted for the 14-day survivors treated with MNX.
UDP Determination of MNX LD50 Relative to RDX
Because published values for the LD50 for RDX vary
widely due to difficulties with dispersion of this chemical in a suitable gavage vehicle (Yinon, 1990), UDP
determinations for RDX and MNX were performed under
the same conditions (vehicle, 5% DMSO in corn oil,
starting dose, 87 mg kg−1, sigma = 0.33). Maximum likelihood estimates from these determinations of LD50
(with 95% confidence interval), as estimated with the
AOT425StatPgm program, were 187 mg kg−1 (118–
491 mg kg−1) for MNX and 187 mg kg−1 (71–295 mg kg−1)
for RDX. In both determinations, the 187 mg kg−1 dose
was the only dose which gave a partial response, i.e. two
deaths and one death of three rats treated with MNX and
RDX, respectively. Both rats treated with the high dose
of either MNX or RDX (400 mg kg−1) convulsed within
several minutes, while convulsions occurred with two of
three rats treated with 187 mg kg−1 MNX or RDX. One
of two MNX-treated and both RDX-treated rats receiving
87 mg kg−1 convulsed.
Copyright © 2005 John Wiley & Sons, Ltd.
Table 3. Hematological assessment of MNX acute oral
toxicitya
Dose
(mg kg−1)
0
47
94
187
280
420
Hemoglobin
(g dl−1)
Hematocrit
(%)
WBCb
(×103 µl−1)
14.8 ± 0.5
14.5 ± 0.3
13.1 ± 0.5**
14.8, 15.6
46.0 ± 1.1
45.4 ± 0.7
42.0 ± 1.7*
46.5, 49.9
5.00 ± 0.65
5.06 ± 0.84
6.15 ± 0.88
3.65, 5.36
17.2
54.2
6.39
a
Mean ± SEM for n = 5 for doses 0–94 mg kg−1. Individual values for survivors of doses >94 mg kg−1 are given.
* = P < 0.05, ** = P < 0.01.
b
Leukocyte counts.
MNX Fixed-dose Study and Dose-dependence
of Toxic Effects
The results of acute toxicity of MNX determined with the
fixed-dose design are presented in Tables 2 and 3. The
highest nonlethal dose observed was 94 mg kg−1. The oral
LD50 for MNX determined with a fixed-dose protocol and
regression analysis of probit vs log dose yielded a value
of 186 mg kg−1 with a 95% confidence interval of 103–
268 mg kg−1. Similar analysis of the incidence of convulsions gave an ED50 of 57 mg kg−1 (13–101 mg kg−1).
Further observation of survivors identified nonlethal adverse effects (Table 2). The body weight gain
over the subsequent 14 days was significantly decreased
for rats treated with 94 mg kg−1 MNX compared with
those receiving vehicle. Histopathological examination of hematoxylin/eosin-stained spleens indicated
hemosiderosis of red pulp of MNX-treated rats, although
the relative spleen weight was not significantly increased.
Quantitation of large, iron-laden macrophage lysosomes
in splenic red pulp of Prussian blue-stained sections
(Fig. 2, Table 2) revealed a statistically significant
trend towards greater numbers with increasing dose of
MNX. Relative liver weights of rats treated with 47 and
94 mg kg−1 MNX (mean ± SEM = 3.52 ± 0.13 and 3.61
± 0.15%, respectively) did not differ from that of vehicletreated controls (3.72 ± 0.06%).
J. Appl. Toxicol. 2005; 25: 427–434
432
S. A. MEYER ET AL.
Hematology of blood of 14-day survivors detected
statistically significant decreases in hemoglobin and
hematocrit values of rats treated with 94 mg kg−1 MNX
from that of vehicle controls (Table 3). Additional
hematological effects reported for RDX were not evident
in our rodent trials with the nitrosamine product MNX.
Transient leukocytosis has been observed with accidental
human exposure to RDX (ATSDR, 1995; Küçükardalı
et al., 2003), while leukocyte counts were unaffected
by MNX in our studies (Table 3). Further, renal toxicity
observed in rats chronically treated with RDX (U.S.
Army, 1983) was absent in the MNX-treated rats. Blood
urea nitrogen and creatinine concentrations were equivalent for control and MNX-treated rats (data not shown).
Discussion
The primary goal of these studies at the outset was to
compare the relative acute oral toxicity of the three Nnitroso degradation products of RDX. Since all three Nnitroso compounds have been detected in ground water of
RDX-contaminated sites (Beller and Tiemeier, 2002),
knowledge of their relative toxicities is of value for direction of future efforts related to site remediation. Once
identified, a second objective was to begin profiling the
toxicity of this N-nitroso product by determining its oral
LD50 and surveying for target tissue toxicity.
To achieve this goal, the recently validated procedure
for acute oral toxicity, the up-and-down procedure (UDP)
was employed. Acceptance of the UDP by several regulatory agencies has resulted from efforts to minimize
animal numbers used for toxicity testing. The greater
efficiency of the UDP procedure is enabled by a sequential dosing regime in which each successive dose level is
determined by the 48 h outcome of the previous trial.
Another advantage of the UDP is that the singular animal
treatment offers the opportunity to make detailed clinical
observations shortly after dose administration and to
collect preliminary data useful for subsequent hazard
identification, design of dose-response studies and assessment of mechanisms of toxicity.
These features were particularly of value for our
comparative studies with RDX parent and N-nitroso
metabolites since monitoring the incidence and severity
of convulsions, some of which occurred within minutes
after dosing, augmented our conclusion based upon
lethality that MNX was the most potent of the metabolites. The equivalence of the LD50 for MNX and its
parent RDX derived with the UDP, compared with the
lesser incidence of lethality of TNX at that dose, suggests
that a nitroreductive pathway could function in vivo leading to detoxication for this endpoint. Results with DNX
were like those for TNX, even though the DNX preparation contained significant MNX contamination (25%),
and thus supportive of nitroreductive detoxication of
Copyright © 2005 John Wiley & Sons, Ltd.
RDX. An estimated LD50 of 100 mg kg−1 for SpragueDawley rats orally gavaged with a saline slurry of RDX
(Schneider et al., 1977) compares favorably with the
UDP RDX LD50 obtained with female Sprague-Dawley
rats in this study. Both male and female rats were used
by Schneider et al. (1977) and no difference in sensitivity to lethality was indicated. Reduction of the N-nitro
groups of RDX also appeared to reduce seizure incidence, as evidenced by the lowest value for TNX at
187 mg kg−1 and greater incidence for RDX than MNX at
87 mg kg−1. A greater potency of RDX over MNX is also
suggested by the determination of 80% seizure incidence
at 25 mg kg−1 RDX by Burdette et al. (1988) compared
with our estimate of an ED50 of 57 mg kg−1 for MNX.
Archived histological material from the comparative
studies allowed an efficient survey for pathological effects in several selected tissues. The well-known toxicity
of nitrosamines to the stomach justified examination
of this tissue. Exploration for liver and kidney lesions
was suggested by previously observed disposition of
[14C]RDX residues in these tissues of rats shortly after
gavage administration (Schneider et al., 1977) and toxicity to these tissues in chronic dietary trials (U.S. Army,
1983). No overt pathology of these tissues was observed
from MNX-treated rats euthanized early after dosing
or from those that survived for 14 days. However, preliminary evidence for hematological toxicity was obtained
from an obvious hemosiderosis of splenic red pulp of the
14-day surviving rat that had received 187 mg kg−1 MNX.
Guided by this observation, we were then able to conclusively demonstrate dose-dependent deposition of ferric
iron in spleen of MNX-treated rats using tissue from the
fixed-dose design study.
Pigmentation of spleens from rats chronically treated
with RDX has been previously noted (U.S. Army, 1983),
but no evidence exists for this effect with acute rodent
exposures (ATSDR, 1995). A preliminary survey for
Prussian blue-positive spleens, using material from our
UDP determination of RDX LD50, indicated that hemosiderosis was absent. Since hemosiderosis is conclusively
present with acute MNX treatment and since two of three
spleens from TNX survivors stained heavily for Prussian
blue, these results suggest that the N-nitroso may be the
toxicophore for this endpoint. Thus, splenic hemosiderosis may be an adverse effect acquired upon reduction of parent RDX. Alternately, a recent observation that
phenobarbital-induced rabbit liver microsomes enriched
in CYP2B4 catalyses a high rate of nitrite production
upon RDX denitration (Bhushan et al., 2003) suggests
a mechanism centered on the N-nitro group. Splenic
hemosiderosis is often a sequel to erythrocyte damage
as a consequence of methemoglobin formation. Many
nitrogenous chemicals, such as nitrite (Kohn et al.,
2002), can redox couple with heme ferrous iron to yield
methemoglobin and, if severe, anemia can result from
loss of circulating erythrocytes upon entrapment in the
J. Appl. Toxicol. 2005; 25: 427–434
ACUTE TOXICITY OF RDX NITROSO PRODUCTS 433
red pulp of the spleen. Although anemia was absent
in rats fed RDX in subchronic studies reported by Levine
et al. (1990), human case reports (Stone et al., 1969)
have documented transient mild anemia with up to 4%
methemoglobin in humans within hours of acute oral
exposure to approximately 25 g of the compound C-4
(91% RDX). Transient anemia and methemoglobinemia
was also noted in the case reports of Küçükardalı et al.
(2003). A greater sensitivity of erythrocytes from humans
than rats to products of RDX N-nitro metabolism in
vivo may result from their known lower activity of
methemoglobin reductase (Smith and Beutler, 1966). A
decrease in hematocrit and blood hemoglobin with MNX
observed in our fixed-dose studies (Table 3) is supportive of a mechanism involving methemoglobin for this
RDX degradation product. A more careful assessment
of hemosiderosis in rat spleen with RDX and TNX
treatment than possible with the UDP design is needed
to better determine whether N-nitro or N-nitroso is the
important functional group for this effect.
These studies, along with those of George et al.
(2001), have identified three toxic effects for the reduced
degradation products of the munition RDX; mutagenicity,
neurotoxicity and hematological toxicity. Reduction of
RDX N-nitro groups confers weak mutagenicity (George
et al., 2001), but diminishes lethal neurotoxicity.
Hematological toxicity is associated with acute exposure
to the mono N-nitroso product MNX and further study
is needed to determine whether N-nitro reduction is
conducive to this effect. Related toxicities previously
reported to the nervous and hematological systems of
humans exposed to parent RDX suggest the relevance
of hazards of the RDX degradation products, especially
MNX, identified here in rodents to risk assessment of
RDX contaminated sites.
Acknowledgements—This research was supported by BS04 008D5P
and BAA-02-3435 under the Environmental Quality and Technology
Program, Engineer Research and Development Center, U.S. Army
Corps of Engineers. Permission was granted by the Chief of Engineers
to publish this information. This publication reflects the personal views
of the authors and does not suggest or reflect the policy, practices, programs, or doctrine of the U.S. Army or Government of the United
States. The contents of this report are not to be used for advertising
or promotional purposes. Citation of brand names does not constitute
an official endorsement or approval of the use of such commercial
products.
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