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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX)
Executive Summary
(CASRN 121-82-4]
August 2018
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC

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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
EXECUTIVE SUMMARY
Summary of Occurrence and Health Effects
RDX is a synthetic chemical used primarily as a military explosive. RDX releases have
been reported in air, water, and soil, and exposure is likely limited to individuals in
or around military facilities where RDX is or was produced, used, or stored. Oral
exposure may occur from drinking contaminated groundwater or ingesting crops
irrigated with contaminated water. Inhalation or dermal exposures are more likely
in occupational settings.
Epidemiological studies provide only limited information on worker populations
exposed to RDX; several case reports describe effects primarily in the nervous system
following acute exposure to RDX. Animal studies of ingested RDX demonstrate
toxicity, including effects on the nervous system, urinary system (kidney and
bladder], and prostate.
Results from animal studies provide suggestive evidence of carcinogenic potential for
RDX based on evidence of positive trends in liver and lung tumor incidence in
experimental animals. There are no data on the carcinogenicity of RDX in humans.
ES.1. EVIDENCE FOR HAZARDS OTHER THAN CANCER: ORAL EXPOSURE
Nervous system effects are a human hazard of RDX exposure. Several human case reports
and animal studies provide consistent evidence of an association between RDX exposure and effects
on the nervous system, including findings related to the induction of seizures, abnormal electrical
activity, convulsions, tremors, and a reduced threshold for seizure induction by other stimuli;
behavioral effects that may be related to seizures such as hyperirritability, hyper-reactivity, and
other behavioral changes. Mechanistic data support the hypothesis that RDX-induced seizures and
related behavioral effects likely result from inhibition of gamma-aminobutyric acid (GABA)ergic
signaling in the limbic system. Some investigators reported that unscheduled deaths in
experimental animals exposed to RDX were frequently preceded by convulsions or seizures.
Urinary system effects are a potential human hazard of RDX exposure based largely on
observations of histopathological changes in the kidney and urinary bladder of male rats exposed to
RDX at doses higher than those associated with nervous system effects. The available evidence
indicates that male rats are more sensitive than females, and rats are more sensitive than mice to
RDX-related urinary system toxicity. There is suggestive evidence of male prostate effects
associated with RDX exposure based on an increased incidence of suppurative prostatitis in male
rats exposed to RDX in the diet for 2 years, in one of the few studies that evaluated the prostate.
There is no known mode of action (MOA) for effects of RDX exposure on the urinary system or
prostate, although there are studies indicating GABA helps regulate urinary system and prostate
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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
function. Evidence for effects on other organs/systems, or developmental effects, was more limited
than for the endpoints summarized above.
ES.1.1. Oral Reference Dose (RfD) for Effects Other Than Cancer
Organ-specific RfDs were derived for hazards associated with RDX exposure (see
Table ES-1). These organ- or system-specific reference values may be useful for subsequent
cumulative risk assessments that consider the combined effect of multiple agents acting at a
common site.
Table ES-1. Organ/system-specific reference doses (RfDs) and overall RfD for
hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX)
Effect
Basis
RfD (mg/kg-day)
Study exposure
description
Confidence
Nervous system
Convulsions
4 x 10"3
Subchronic
Medium
Urinary system
Kidney medullary papillary necrosis
1 x 10"2
Chronic
Medium
Prostate
Suppurative prostatitis
8 x 10"4
Chronic
Low
Overall RfD
Nervous system effects
4 x 10"3
Subchronic
Medium
The overall RfD (see Table ES-2) is derived to be protective of all types of hazards
associated with RDX exposure. Although the RfD for prostate effects results in a smaller value, it
was not selected as the overall RfD due to uncertainties in the evaluation of this endpoint ("low
confidence"). The effect of RDX on the nervous system was chosen as the basis for the overall RfD
because nervous system effects were observed most consistently across studies, species, and
exposure durations, and because they represent a sensitive human hazard of RDX exposure.
Evidence for effects of RDX on the urinary system and prostate is more limited relative to the
effects of RDX on the nervous system. Incidence of seizures or convulsions as reported in a
subchronic gavage study fCrouse etal.. 20061 was selected for deriving the overall RfD because this
endpoint was measured in a study that was well conducted, used a test material of high purity
(99.99%), and had five closely spaced dose groups that supported characterization of the
dose-response curve. In contrast, most other studies used a technical grade with ~10% or more
impurities. Benchmark dose (BMD) modeling was used to derive the point of departure (POD) for
RfD derivation (expressed as the lower confidence limit on the benchmark dose [BMDL05]). A 5%
response level was chosen because of the severity of the endpoint.
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Table ES-2. Summary of reference dose (RfD) derivation
Critical effect
Point of departure3
UF
Chronic RfD
Confidence
Nervous system effects (convulsions)
90-d F344 rat study
Crouse et al. (2006)
BMDLos-hed: 1.3 mg/kg-d
300
4 x 10"3 mg/kg-d
Medium
AUC = area under the curve; BMDL = benchmark dose lower confidence limit.
aA benchmark response (BMR) of 5% was used to derive the BMD and BMDL. The resulting POD was converted to
a BMDLos -hed using a PBPK model based on modeled arterial blood concentration. The concentration was derived
from the AUC of modeled RDX concentration in arterial blood, which reflects the average blood RDX concentration
for the exposure duration normalized to 24 hr.
A PBPK model was used to extrapolate the BMDLos derived from a rat study to a human
equivalent dose (HED) based on RDX arterial blood concentration, which was then used for RfD
derivation.
The overall RfD, 4 x 10"3 mg/kg-day, was calculated by dividing the BMDLos expressed as a
human equivalent dose (BMDLos-hed) for nervous system effects by a composite uncertainty factor
(UF) of 300 to account for extrapolation from animals to humans (3), interindividual differences in
human susceptibility (10), and uncertainty in the database (10).
Because a subchronic-to-chronic uncertainty factor (UFs) of 1 was applied to the POD based
on evidence that nervous system effects (in particular convulsions) are more strongly driven by
dose than duration of exposure, the RfD may be appropriate for assessing health risks of less-than-
lifetime as well as chronic durations of exposure.
The overall confidence in the RfD is medium based on high confidence in the principal study
(Crouse etal.. 2006) and medium to low confidence in the database. Confidence in the database is
reduced largely because of (1) differences in test material used across studies (i.e., differences in
formulation and particle size that may have affected RDX absorption and subsequent toxicity),
(2) uncertainties in the influence of oral dosing methods (in particular, based on evidence that
bolus dosing of RDX resulting from gavage administration induces neurotoxicity at doses lower
than administration in the diet), and (3) significant limitations in the available studies to fully
characterize subconvulsive neurological effects as well as developmental neurotoxicity.
ES.2. EVIDENCE FOR HAZARDS OTHER THAN CANCER: INHALATION EXPOSURE
No studies were identified that provided useful information on the effects observed
following inhalation exposure to RDX. Of the available human epidemiological studies of RDX, none
provided data that could be used for dose-response analysis of inhalation exposures. The single
experimental animal study involving inhalation exposure is not publicly available and was excluded
from consideration due to significant study limitations, including small numbers of animals tested,
lack of controls, and incomplete reporting of exposure levels. Therefore, the available health effects
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literature does not support the identification of hazards following inhalation exposure to RDX nor
the derivation of an inhalation reference concentration (RfC).
While inhalation absorption of RDX particulates is a plausible route of exposure, there are
no toxicokinetic studies of RDX inhalation absorption to support development of an inhalation
model. Therefore, a PBPK model for inhaled RDX was not developed to support route-to-route
extrapolation of an RfC from the RfD.
ES.3. EVIDENCE FOR HUMAN CARCINOGENICITY
Under EPA's cancer guidelines fU.S. EPA. 2005al. there is suggestive evidence of carcinogenic
potential for RDX. RDX induced benign and malignant tumors in the liver and lungs of mice (Parker
etal.. 2006: Lish etal.. 19841 or rats (Levine etal.. 19831 following long-term administration in the
diet. The potential for carcinogenicity applies to all routes of human exposure.
ES.4. QUANTITATIVE ESTIMATE OF CARCINOGENIC RISK FROM ORAL EXPOSURE
A quantitative estimate of carcinogenic risk from oral exposure to RDX was based on the
increased incidence of hepatocellular adenomas or carcinomas and alveolar/bronchiolar adenomas
or carcinomas in female B6C3Fi mice observed in the carcinogenicity bioassay in mice fLish etal..
19841. This 2-year dietary study included four dose groups and a control group, adequate numbers
of animals per dose group (85/sex/group, with interim sacrifices of 10/sex/group at 6 and
12 months), and detailed reporting of methods and results (including individual animal data). The
initial high dose (175 mg/kg-day) was reduced to 100 mg/kg-day at Week 11 due to high mortality.
When there is suggestive evidence of carcinogenicity to humans, EPA generally would not
conduct a dose-response assessment and derive a cancer value. However, when the evidence
includes a well-conducted study (as is the case with RDX), quantitative analyses may be useful for
some purposes, for example, providing a sense of the magnitude and uncertainty of potential risks,
ranking potential hazards, or setting research priorities (U.S. EPA. 2005a).
An OSF was derived that considered the combination of female mouse liver and lung
tumors. In modeling these data sets, the highest dose group was excluded because of the initial
high mortality (loss of almost half the mice in that dose group). BMD and benchmark dose lower
confidence limit (BMDL) estimates were calculated that correspond to a 10% extra risk (ER) of
either tumor. The BMDLio so derived was extrapolated to the HED using body-weight scaling to the
% power (BW3/4), and an OSF was derived by linear extrapolation from the BMDLio expressed as an
HED (BMDLio-hed). The OSF is 0.08 per mg/kg-day, based on the liver and lung tumor response in
female mice (Lish etal.. 1984).
ES.5. QUANTITATIVE ESTIMATE OF CARCINOGENIC RISK FROM INHALATION EXPOSURE
An inhalation unit risk (IUR) value was not calculated because inhalation carcinogenicity
data for RDX are not available. While inhalation absorption of RDX particulates is a plausible route
of exposure, there are no toxicokinetic studies of RDX inhalation absorption to support an
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inhalation model. Therefore, a PBPK model for inhaled RDX was not developed to support
route-to-route extrapolation of an IUR from the OSF. Thus, a quantitative cancer assessment was
not conducted for inhalation exposure.
ES.6. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
Little information is available on populations that may be especially vulnerable to the toxic
effects of RDX. Life stage, particularly childhood, susceptibility has not been well-studied in human
or animal studies of RDX toxicity. In rats, transfer of RDX from the dam to the fetus during
gestation and to pups via maternal milk has been reported; however, reproductive and
developmental toxicity studies did not identify effects in offspring at doses below those that also
caused maternal toxicity. Yet, based on the primary mode of action for RDX exposure-induced
nervous system effects (GABA receptor antagonism), and the fact that GABAergic signaling plays a
prominent role in nervous system development, a significant concern is raised regarding the
potential for developmental neurotoxicity. In addition, data on the incidence of convulsions and
mortality provide some indication that pregnant animals may be a susceptible population, although
the evidence is inconclusive. Data to suggest that males may be more susceptible than females to
noncancer toxicity associated with RDX are limited. Some evidence suggests that cytochrome P450
(CYP450) enzymes may be involved in the metabolism of RDX, indicating a potential for genetic
polymorphisms in these metabolic enzymes to affect susceptibility to RDX. Similarly, individuals
with epilepsy or other seizure syndromes that have their basis in genetic mutation to GABAa
receptors (GABA receptors that are ligand-gated ion channels, also known as ionotropic receptors)
may represent another group that may be susceptible to RDX exposure; however, there is no
information to indicate how genetic polymorphisms may affect susceptibility to RDX.
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REFERENCES
Crouse. LCB: Michie. MW: Major. M: Tohnson. MS: Lee. RB: Paulus. HI. (2006). Subchronic oral
toxicity of RDX in rats. (Toxicology Study No. 85-XC-5131-03). Aberdeen Proving Ground,
MD: U.S. Army Center for Health Promotion and Preventive Medicine.
http: / /www, dtic.mil /dtic /tr /fulltext/u2 /10 5 09 0 3 .p df.
Levine. BS: Lish. PM: Furedi. EM: Rac. VS: Sagartz. TM. (1983). Determination of the chronic
mammalian toxicological effects of RDX (twenty-four month chronic
toxicity/carcinogenicity study of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) in the
Fischer 344 rat): Final report-phase V. Chicago, IL: IIT Research Institute.
Lish. PM: Levine. BS: Furedi. EM: Sagartz. TM: Rac. VS. (1984). Determination of the chronic
mammalian toxicological effects of RDX: Twenty-four month chronic
toxicity/carcinogenicity study of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) in the
B6C3F1 hybrid mouse (Volumesl-3). (ADA181766. DAMD17-79-C-9161). FortDetrick,
Parker. GA: Reddv. G: Maior. MA. (2006). Reevaluation of a twenty-four-month chronic
toxicity/carcinogenicity study of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) in the
B6C3F1 hybrid mouse. Int J Toxicol 25: 373-378.
http://dx.doi.Org/10.1080/10915810600846245.
U.S. EPA (U.S. Environmental Protection Agency). (2005a). Guidelines for carcinogen risk
assessment [EPA Report] (pp. 1-166). (EPA/630/P-03/001F). Washington, DC: U.S.
Environmental Protection Agency, Risk Assessment Forum.
http://www2.epa.gov/osa/guidelines-carcinogen-risk-assessment
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