DRAFT DELIBERATIVE: FOR INTERAGENCY REVIEW ONLY.
DO NOT DISTRIBUTE OUTSIDE YOUR AGENCY.
EPA/635/R-14/302
Interagency Review Draft
www.epa.gov/iris
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX)
(CASRN 121-82-4)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
September 2014
NOTICE
This document is an Interagency Review draft. This information is distributed solely for the
purpose of pre-dissemination peer review under applicable information quality guidelines. It has
not been formally disseminated by EPA. It does not represent and should not be construed to
represent any Agency determination or policy. It is being circulated for review of its technical
accuracy and science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
vvEPA
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS viii
PREFACE x
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xiv
EXECUTIVE SUMMARY ES-1
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION LS-1
1. HAZARD IDENTIFICATION 1-1
1.1. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-1
1.1.1. Nervous System Effects 1-1
1.1.2. Kidney and Other Urogenital System Effects 1-3
1.1.3. Reproductive and Developmental Effects 1-19
1.1.4. Liver Effects 1-30
1.1.5. Carcinogenicity 1-41
1.1.6. Other Toxicological Effects 1-49
1.2. INTEGRATION AND EVALUATION 1-68
1.2.1. Effects Other Than Cancer 1-68
1.2.2. Carcinogenicity 1-70
1.2.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 1-71
2. DOSE-RESPONSE ANALYSIS 2-1
2.1.ORAL REFERENCE DOSE FOR EFFECTS OTHERTHAN CANCER 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis 2-1
2.1.2. Methods of Analysis 2-3
2.1.3. Derivation of Candidate Values 2-7
2.1.4. Derivation of Organ/System-Specific Reference Doses 2-11
2.1.5. Selection of the Proposed Overall Reference Dose 2-12
2.1.6. Uncertainties in the Derivation of Reference Dose 2-12
2.1.7. Confidence Statement 2-13
2.1.8. Previous IRIS Assessment 2-13
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER 2-13
2.2.1. Previous IRIS Assessment 2-14
This document is a draft for review purposes only and does not constitute Agency policy.
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2.3. ORAL SLOPE FACTOR FOR CANCER 2-14
2.3.1. Analysis of Carcinogenicity Data 2-14
2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods 2-16
2.3.3. Derivation of the Oral Slope Factor 2-18
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 2-19
2.3.5. Previous IRIS Assessment: Oral Slope Factor 2-20
2.4. INHALATION UNIT RISK FOR CANCER 2-21
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 2-21
REFERENCES R-l
This document is a draft for review purposes only and does not constitute Agency policy.
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TABLES
Table ES-1. Organ/system-specific RfDs and proposed overall RfD for RDX ES-2
Table ES-2. Summary of reference dose (RfD) derivation ES-3
Table LS-1. Overview of the search strategy employed for RDX LS-2
Table LS-2. Studies determined not to be informative because of significant issues with design,
conduct, or reporting LS-5
Table LS-3. Experimental animal studies considered less informative because of certain study
design, conduct, or reporting limitations LS-11
Table 1-1. Evidence pertaining to nervous system effects in humans 1-5
Table 1-2. Evidence pertaining to nervous system effects in animals 1-6
Table 1-3. Evidence pertaining to kidney effects in humans 1-6
Table 1-4. Evidence pertaining to kidney and other urogenital system effects in animals 1-7
Table 1-5. Six-, 12-, and 24-month incidence of kidney endpoints in male F344 rats reported for
statistical evaluation in Levine et al. (1983) 1-12
Table 1-6. Six-, 12-, and 24-month incidence of urinary bladder endpoints in male F344 rats
reported for statistical evaluation in Levine et al. (1983) 1-13
Table 1-7. Six-, 12-, and 24-month incidence of prostate endpoints in male F344 rats reported
for statistical evaluation in Levine et al. (1983) 1-14
Table 1-8. Evidence pertaining to reproductive and developmental effects in animals 1-21
Table 1-9. Evidence pertaining to male reproductive effects in animals 1-26
Table 1-10. Evidence pertaining to liver effects in humans 1-33
Table 1-11. Evidence pertaining to liver effects in animals 1-34
Table 1-12. Liver tumors observed in chronic animal bioassays 1-43
Table 1-13. Lung tumors observed in chronic animal bioassays 1-46
Table 1-14. Evidence pertaining to systemic effects (hematological) in humans 1-52
Table 1-15. Evidence pertaining to systemic effects in animals 1-54
Table 2-1. Summary of derivation of PODs following oral exposure to RDX 2-5
Table 2-2. Effects and corresponding derivation of candidate values 2-9
Table 2-3. Organ/system-specific RfDs and proposed overall RfD for RDX 2-11
Table 2-4. Incidence of hepatocellular and alveolar/bronchiolar tumors in female B6C3Fi mice
administered RDX for 2 years in diet 2-16
Table 2-5. Model predictions and oral slope factors for hepatocellular and alveolar/bronchiolar
adenomas or carcinomas in female B6C3Fi mice administered RDX in the diet
for 2 years (Lish et al., 1984a) 2-18
Table 2-6. Summary of uncertainty in the derivation of the cancer risk value for RDX 2-19
FIGURES
Figure LS-1. Summary of literature search and screening process for RDX LS-4
Figure 1-1. Exposure response array of nervous system effects following oral exposure 1-12
Figure 1-2. Exposure-response array of kidney and urogenital system effects 1-16
Figure 1-3. Exposure response array of reproductive and developmental effects following oral
exposure 1-25
Figure 1-4. Exposure response array of male reproductive effects following oral exposure 1-29
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Figure 1-5. Exposure response array of liver effects following oral exposure 1-40
Figure 2-1. Approach for dose-response analysis 2-3
Figure 2-2. Candidate values with corresponding POD and composite UF 2-10
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ABBREVIATIONS
AAP Army ammunition plants NCEA
ACGIH American Conference of Governmental
Industrial Hygienists NHANES
AChE acetylcholinesterase
ADAF age-dependent adjustment factor NICNAS
ALP alkaline phosphatase
ALT alanine aminotransferase NIOSH
AST aspartate aminotransferase
atm atmosphere NOAEL
ATSDR Agency for Toxic Substances and NPL
Disease Registry NRC
AUC area under the curve NTP
BDNF brain-derived neurotrophic factor OR
BMC benchmark concentration ORD
BMCL benchmark concentration lower OSF
confidence limit OSHA
BMD benchmark dose
BMDL benchmark dose lower confidence limit PBPK
BMDS Benchmark Dose Software POD
BMR benchmark response POD[adj]
BUN blood urea nitrogen PWG
BW body weight RBC
CASRN Chemical Abstracts Service Registry RfC
Number RfD
CCL Contaminant Candidate List RNA
CI confidence interval SD
CNS central nervous system SDMS
CYP450 cytochrome P450
DAF dosimetric adjustment factor SDWA
DMSO dimethylsulfoxide SGOT
DNA deoxyribonucleic acid
DTIC Defense Technical Information Center SGPT
EPA Environmental Protection Agency
ER extra risk SLE
FDA Food and Drug Administration SS
FOB functional observational battery TNT
GABA gamma amino butyric acid TSCATS
GD gestational day
GLP good laboratory practices TWA
HEC human equivalent concentration U.S.
HED human equivalent dose UCL
HERO Health and Environmental Research UCM
Online UF
IARC International Agency for Research on UFa
Cancer UFd
IOM Institute of Medicine UFh
IRIS Integrated Risk Information System UFl
LDH lactate dehydrogenase UFs
LOAEL lowest-observed-adverse-effect level
LOD limit of detection WBC
miRNA microRNA WOS
MOA mode of action
National Center for Environmental
Assessment
National Health and Nutrition
Examination Survey
National Industrial Chemicals
Notification and Assessment Scheme
National Institute for Occupational
Safety and Health
no-observed-adverse-effect level
National Priorities List
Nuclear Regulatory Commission
National Toxicology Program
odds ratio
Office of Research and Development
oral slope factor
Occupational Safety and Health
Administration
physiologically based pharmacokinetic
point of departure
duration-adjusted POD
Pathology Working Group
red blood cell
inhalation reference concentration
oral reference dose
ribonucleic acid
Sprague-Dawley
spontaneous death or moribund
sacrifice
Safe Drinking Water Act
glutamic oxaloacetic transaminase, also
known as AST
glutamic pyruvic transaminase, also
known as ALT
systemic lupus erythematosus
scheduled sacrifice
trinitrotoluene
Toxic Substances Control Act Test
Submissions
time-weighted average
United States of America
upper confidence limit
Unregulated Contaminant Monitoring
uncertainty factor
animal-to-human uncertainty factor
database deficiencies uncertainty factor
human variation uncertainty factor
LOAEL-to-NOAEL uncertain factor
subchronic-to-chronic uncertainty
factor
white blood cells
Web of Science
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Louis D'Amico, Ph.D. (Assessment Manager) U.S. EPA/ORD/NCEA
Todd Blessinger, Ph.D. Washington, DC
Ravi Subramaniam, Ph.D.
Christopher Brinkerhoff, Ph.D. ORISE Postdoctoral Fellow at U.S.
EPA/ORD/NCEA
Currently at U.S. EPA/Office of Chemical Safety
and Pollution Prevention
Washington, DC
Contributors
Rob DeWoskin, Ph.D.
Karen Hogan, MS
Anne Loccisano, Ph.D.
Jordan Trecki, Ph.D.
Belinda Hawkins, Ph.D.
Scott Wesselkamper, Ph.D.
Production Team
Maureen Johnson
U.S. EPA/ORD/NCEA
Washington, DC
U.S. EPA/ORD/NCEA
Cincinnati, OH
U.S. EPA/ORD/NCEA
Washington, DC
Contractor Support
Heather Carlson-Lynch, S.M., DABT SRC, Inc, North Syracuse, NY
Julie Melia, Ph.D., DABT
Megan Riccardi, M.S.
Pam Ross, M.S. ICF International, Fairfax, VA
Robin Blain, Ph.D.
Executive Direction
Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director) U.S. EPA/ORD/NCEA
John Vandenberg, Ph.D, (National Program Director, HHRA) Washington, DC
Lynn Flowers, Ph.D., DABT (Associate Director for Health)
Vincent Cogliano, Ph.D. (IRIS Program Director—acting)
Samantha Jones, Ph.D. (IRIS Associate Director for Science)
Susan Rieth, MPH (Quantitative Modeling Branch Chief)
Internal Review Team
General Toxicity/Cancer/Immunotoxicity Workgroup U.S. EPA/ORD/NCEA
Neurotoxicity Workgroup Washington, DC
Pharmacokinetics Workgroup
Reproductive and Developmental Toxicity Workgroup
Statistics Workgroup
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicity Pathways Workgroup
Reviewers
This assessment was provided for review to scientist in EPA's program and regional offices.
Comments were submitted by:
Office of Children's Health Protection, Washington, DC
Office of Chemical Safety and Pollution Prevention Programs/Office of Pesticide Programs,
Washington, DC
Office of Solid Waste and Emergency Response, Washington, DC
Office of Water, Washington, DC
Region 2, New York, NY
Region 8, Denver, CO
Federal Facilities Forum
1
This document is a draft for review purposes only and does not constitute Agency policy.
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PREFACE
This Toxicological Review critically reviews the publicly available studies on hexahydro-
l,3,5-trinitro-l,3,5-triazine (RDX) in order to identify its adverse health effects and to characterize
exposure-response relationships. It was prepared under the auspices of EPA's Integrated Risk
Information System (IRIS) program. This assessment updates a previous IRIS assessment of RDX
that included an oral reference dose (RfD) for effects other than cancer (posted in 1988), a
determination on the carcinogenicity of RDX, as well as derivation of an oral slope factor to quantify
the cancer risk associated with RDX exposure (posted in 1990). New information has become
available and this assessment reviews information on all health effects by all exposure routes.
Organ/system-specific RfDs are calculated based on data for applicable hazards, e.g., nervous
system toxicity. These reference values may be useful for cumulative risk assessments that
consider the combined effect of multiple agents acting on the same biological system.
This assessment was conducted in accordance with EPA guidance, which is cited and
summarized in the Preamble to IRIS Toxicological Reviews. The findings of this assessment and
related documents produced during its development are available on the IRIS web site
fhttp://www.epa.gov/irisI Appendices for assessments by other health agencies, chemical and
physical properties, toxicokinetic information, and summaries of supporting toxicity information
are provided as Supplemental Information to this assessment (See Appendices A to C).
A public meeting was held in December 2013 to obtain input on preliminary materials for
RDX, including draft literature searches and associated search strategies, evidence tables, and
exposure-response arrays prior to the development of the IRIS assessment. All public comments
provided were taken into consideration in developing the draft assessment The complete set of
public comments are available on the docket at http: //www.regulations.gov (Docket ID No. EPA-
HQ-0RD-2 013-0430).
In April 2011, the National Research Council (NRC) released its Review of the Environmental
Protection Agency's Draft IRIS Assessment of Formaldehyde. In addition to offering comments
specifically about EPA's draft formaldehyde assessment, the NRC made several recommendations
to EPA for improving the development of IRIS assessments. EPA agreed with the recommendations
and is implementing them consistent with the Panel's "Roadmap for Revision," which viewed the
full implementation of their recommendations by the IRIS Program as a multi-year process.
In response to the NRC's 2011 recommendations, the IRIS Program has made changes to
streamline the assessment development process, improve transparency, and create efficiencies in
the Program. The NRC has acknowledged EPA's successes in this area. In May 2014, the NRC
released their report Review of EPA's Integrated Risk Information System Process reviewing the IRIS
This document is a draft for review purposes only and does not constitute Agency policy.
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assessment development process and found that EPA has made substantial improvements to the
IRIS Program in a short amount of time.
The draft RDX assessment represents a significant advancement in implementing the NRC
recommendations. This assessment is streamlined, and uses tables, figures, and appendices to
increase transparency and clarity. It is structured to have distinct sections for the literature search
and screening strategy, study selection and evaluation, hazard identification, and dose-response
assessment The assessment includes a comprehensive, systematic, and documented literature
search and screening approach, provides the database search strategy in a table (databases,
keywords), visually represents the inclusion and exclusion of studies in a flow diagram, and all of
the references are integrated within the Health and Environmental Research Online (HERO)
database. A study evaluation section provides a systematic review of methodological aspects of
epidemiology and experimental animal studies, including study design, conduct, and reporting, that
was subsequently taken into consideration in the evaluation and synthesis of data from these
studies. The evidence is presented in standardized evidence tables, and exposure-response arrays.
The hazard identification and dose-response sections include subsections based on organ/system-
specific effects in which the evidence is synthesized within and integrated across all evidence for
each target organ/systems.
In the draft RDX assessment, the IRIS Program has attempted to transparently and
uniformly identify strengths and limitations that would affect interpretation of results. All human
and animal studies of RDX that were considered to be of acceptable quality, whether yielding
positive, negative, or null results, were considered in assessing the evidence for health effects
associated with chronic exposure to RDX. These studies were evaluated for aspects of design,
conduct, and reporting that could affect the interpretation of results and the overall contribution to
the synthesis of evidence for determination of human hazard potential using the study quality
considerations outlined in the Preamble. A brief summary of the evaluation is included in the
section on methods for study selection and evaluation. Information on study features related to this
evaluation is reported in evidence tables and documented in the synthesis of evidence. Discussion
of study strengths and limitations (that ultimately supported preferences for the studies and data
relied upon) were included in the text where relevant.
In this assessment, the IRIS Program is using existing guidelines to systematically approach
the integration of noncancer human, animal, and mechanistic evidence. In conducting this analysis
and developing the synthesis, the IRIS Program evaluates the data for the: strength of the
relationship between the exposure and response and the presence of a dose-response relationship;
specificity of the response to chemical exposure and whether the exposure precedes the effect;
consistency of the association between the chemical exposure and response; and biological
plausibility of the response or effect and its relevance to humans. The IRIS Program uses this
weight-of-evidence approach to identify the potential human hazards associated with chemical
exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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The IRIS RDX assessment provides a streamlined presentation of information, integrated
hazard identification of all toxic effects, and derivation of organ/system-specific reference values.
Additionally, consistent with the goal that assessments should provide a scientifically sound and
transparent evaluation of the relevant scientific literature and presentation of the analyses
performed, this assessment contains an expanded discussion of study selection and evaluation, as
well as increased documentation of key assessment decisions.
For additional information about this assessment or for general questions regarding IRIS,
please contact EPA's IRIS Hotline at 202-566-1676 (phone), 202-566-1749 (fax), or
hotline.iris@epa.gov.
Chemical Properties
RDX is a white, crystalline solid member of the nitramine class of organic nitrate explosives
(Boileau etal.. 2005: Bingham etal.. 2001). It is a synthetic chemical not found naturally in the
environment. The solubility of RDX in water is poor, having been reported as 59.7 mg/L at 25°C
(Yalkowskv and He. 2003). The Henry's law constant for RDX is approximately 2 x 10"11 atm-
m3/mole at 25°C, suggesting slow volatilization from water or moist soil (ATSDR. 2012). The
normalized soil organic carbon/water partition coefficient (Koc) values for RDX range from 42 to
167, indicating a potential for RDX to be mobile in soil fSpanggord etal.. 19801. The vapor pressure
of 4.10 x 10"9 mm Hg at 20°C suggests that it will exist as particulate matter in air and be removed
by both wet and dry deposition. RDX degrades in the environment, and can be subject to both
photolysis (Sikkaetal.. 1980: Spanggord etal.. 1980) andbiodegradation (Funketal.. 1993:
Mccormick etal.. 1981). Further information on the physical and chemical properties of RDX are
provided in Appendix C, Section C.l.
Uses and Environmental Occurrence
RDX is used primarily as a military explosive. In the United States, RDX is produced at Army
ammunition plants (AAP) and is not produced commercially. RDX production peaked in the 1960s;
180 million pounds per year were produced from 1969 to 1971. Yearly total production dropped
to 16 million pounds in 1984 (ATSDR. 2012). According to the U.S. EPA Inventory Update
Reporting program, the aggregated national production volume in 2006 was between 1 and
10 million pounds.
RDX releases have been reported into the air, water, or soil fATSDR. 2012.1999.1993.
1992). RDX is mobile in soil; leaching into groundwater has been reported in samples from military
facilities (Best etal.. 1999a: Godeiohann etal.. 1998: Bart etal.. 1997: Steuckartetal.. 1994:
Spanggord etal.. 1980). RDX transport in soil is generally through dissolution by precipitation and
subsequent downward movement, including migration to groundwater aquifers, and not much via
surface runoff (U.S. EPA. 2012b). An extensive discussion of RDX properties and fate and transport
is available in U.S. EPA f2012bl. Detectable levels of RDX have been observed in plants irrigated or
grown with RDX-contaminated water fBestetal.. 1999b: Simini and Checkai. 1996: Harvey etal..
This document is a draft for review purposes only and does not constitute Agency policy.
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19911. RDX has also been detected in indoor air samples from military facilities where RDX is
produced (Bishop etal.. 19881.
Exposures to RDX among the general population are likely to be confined to individuals in
or around military facilities where RDX is or was produced, stored, or used. Oral, inhalation, and
dermal routes of exposure may be relevant
RDX has been detected in surface water, groundwater, sediment, or soil at 34 current U.S.
EPA National Priorities List (NPL) sites. The NPL serves as a list of sites with known releases or
threatened releases of hazardous substances, pollutants, or contaminants throughout the United
States and its territories. The NPL list aids the Agency in identifying the most serious sites that may
warrant cleanup. The majority of the NPL sites where RDX was listed are associated with military
facilities, although the total number of sites where RDX is present is unknown.
RDX is not regulated under the Safe Drinking Water Act (SDWA), although it was included
as a contaminant to be monitored under the Unregulated Contaminant Monitoring (UCM) Rule by
EPA's Office of Water from 2007 to 2011. Contaminants included in the UCM program are
suspected of being present in drinking water, but do not have existing health-based standards set
under the SDWA. RDX has also been included the Office of Water's Drinking Water Contaminant
Candidate Lists (CCL) since the initial listing was published in 1998. The presence of a chemical on
the list suggests that it is known or anticipated to occur in public water systems.
Assessments by Other National and International Health Agencies
Toxicity values for RDX have been established by the Agency for Toxic Substances and
Disease Registry (ATSDR), the American Conference of Governmental Industrial Hygienists
(ACGIH), the Australian National Industrial Chemicals Notification and Assessment Scheme
(NICNAS), the National Institute for Occupational Safety and Health (NIOSH), and the Occupational
Safety and Health Administration (OSHA). These toxicity values and their basis are presented in
Appendix A. It is important to recognize that the assessments performed by other health agencies
may have been prepared for different purposes and may utilize different methods, and that newer
studies may be included in the IRIS assessment.
This document is a draft for review purposes only and does not constitute Agency policy.
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PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
1. Scope of the IRIS Program
Soon after the EPA was established in
1970, it was at the forefront of developing risk
assessment as a science and applying it in
decisions to protect human health and the
environment. The Clean Air Act, for example,
mandates that the EPA provide "an ample
margin of safety to protect public health"; the
Safe Drinking Water Act, that "no adverse
effects on the health of persons may
reasonably be anticipated to occur, allowing
an adequate margin of safety." Accordingly,
the EPA uses information on the adverse
effects of chemicals and on exposure levels
below which these effects are not anticipated
to occur.
IRIS assessments critically review the
publicly available studies to identify adverse
health effects from exposure to chemicals and
to characterize exposure-response
relationships. In terms set forth by the
National Research Council (NRC, 1983), IRIS
assessments cover the hazard identification
and dose-response assessment steps of risk
assessment, not the exposure assessment or
risk characterization steps that are conducted
by the EPA's program and regional offices and
by other federal, state, and local health
agencies that evaluate risk in specific
populations and exposure scenarios. IRIS
assessments are distinct from and do not
address political, economic, and technical
considerations that influence the design and
selection of risk management alternatives.
An IRIS assessment may cover a single
chemical, a group of structurally or
toxicologically related chemicals, or a complex
mixture. These agents may be found in air,
water, soil, or sediment. Exceptions are
chemicals currently used exclusively as
pesticides, ionizing and non-ionizing
41 radiation, and criteria air pollutants listed
42 under Section 108 of the Clean Air Act (carbon
43 monoxide, lead, nitrogen oxides, ozone,
44 particulate matter, and sulfur oxides).
45 Periodically, the IRIS Program asks other
46 EPA programs and regions, other federal
47 agencies, state health agencies, and the
48 general public to nominate chemicals and
49 mixtures for future assessment or
50 reassessment. Agents may be considered for
51 reassessment as significant new studies are
52 published. Selection is based on program and
53 regional office priorities and on availability of
54 adequate information to evaluate the potential
55 for adverse effects. Other agents may also be
56 assessed in response to an urgent public
57 health need.
2. Process for developing and peer-
reviewing IRIS assessments
58 The process for developing IRIS
59 assessments (revised in May 2009 and
60 enhanced in July 2013) involves critical
61 analysis of the pertinent studies, opportunities
62 for public input, and multiple levels of
63 scientific review. The EPA revises draft
64 assessments after each review, and external
65 drafts and comments become part of the
66 public record (U.S. EPA. 2009).
67 Before beginning an assessment, the IRIS
68 Program discusses the scope with other EPA
69 programs and regions to ensure that the
70 assessment will meet their needs. Then a
71 public meeting on problem formulation
72 invites discussion of the key issues and the
73 studies and analytical approaches that might
74 contribute to their resolution.
75 Step 1. Development of a draft
76 Toxicological Review. The draft
77 assessment considers all pertinent
78 publicly available studies and applies
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consistent criteria to evaluate study
quality, identify health effects, identify
mechanistic events and pathways,
integrate the evidence of causation for
each effect, and derive toxicity values. A
public meeting prior to the integration of
evidence and derivation of toxicity values
promotes public discussion of the
literature search, evidence, and key issues.
Step 2. Internal review by scientists in EPA
programs and regions. The draft
assessment is revised to address the
comments from within the EPA.
Step 3. Interagency science consultation
with other federal agencies and the
Executive Offices of the President. The
draft assessment is revised to address the
interagency comments. The science
consultation draft, interagency comments,
and the EPA's response to major
comments become part of the public
record.
Step 4. Public review and comment,
followed by external peer review. The
EPA releases the draft assessment for
public review and comment. A public
meeting provides an opportunity to
discuss the assessment prior to peer
review. Then the EPA releases a draft for
external peer review. The peer review
meeting is open to the public and includes
time for oral public comments. The peer
reviewers assess whether the evidence
has been assembled and evaluated
according to guidelines and whether the
conclusions are justified by the evidence.
The peer review draft, written public
comments, and peer review report
become part of the public record.
Step 5. Revision of draft Toxicological
Review and development of draft IRIS
summary. The draft assessment is
revised to reflect the peer review
comments, public comments, and newly
published studies that are critical to the
conclusions of the assessment. The
disposition of peer review comments and
public comments becomes part of the
public record.
Step 6. Final EPA review and interagency
science discussion with other federal
agencies and the Executive Offices of
the President The draft assessment and
summary are revised to address the EPA
and interagency comments. The science
discussion draft, written interagency
comments, and EPA's response to major
comments become part of the public
record.
Step 7. Completion and posting. The
Toxicological Review and IRIS summary
are posted on the IRIS website
(http://www.epa.gOv/iris/l.
The remainder of this Preamble addresses
step 1, the development of a draft
Toxicological Review. IRIS assessments follow
standard practices of evidence evaluation and
peer review, many of which are discussed in
EPA guidelines fIJ.S. EPA. 2005a. b, 2000b.
1998. 1996. 1991. 1986a. b) and other
methods (U.S. EPA, 2012a, b, 2011, 2006a, b,
2002, 1994). Transparent application of
scientific judgment is of paramount
importance. To provide a harmonized
approach across IRIS assessments, this
Preamble summarizes concepts from these
guidelines and emphasizes principles of
general applicability.
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3. Identifying and selecting
pertinent studies
3.1. Identifying studies
Before beginning an assessment, the EPA
conducts a comprehensive search of the
primary scientific literature. The literature
search follows standard practices and includes
the PubMed and ToxNet databases of the
National Library of Medicine, Web of Science,
and other databases listed in the EPA's HERO
system (Health and Environmental Research
Online, http://her0.epa.g0v/l. Searches for
information on mechanisms of toxicity are
inherently specialized and may include
studies on other agents that act through
related mechanisms.
Each assessment specifies the search
strategies, keywords, and cut-off dates of its
literature searches. The EPA posts the results
of the literature search on the IRIS web site
and requests information from the public on
additional studies and ongoing research.
The EPA also considers studies received
through the IRIS Submission Desk and studies
(typically unpublished) submitted under the
Toxic Substances Control Act or the Federal
Insecticide, Fungicide, and Rodenticide Act
Material submitted as Confidential Business
Information is considered only if it includes
health and safety data that can be publicly
released. If a study that may be critical to the
conclusions of the assessment has not been
peer-reviewed, the EPA will have it peer-
reviewed.
The EPA also examines the toxicokinetics
of the agent to identify other chemicals (for
example, major metabolites of the agent) to
include in the assessment if adequate
information is available, in order to more fully
explain the toxicity of the agent and to suggest
dose metrics for subsequent modeling.
In assessments of chemical mixtures,
mixture studies are preferred for their ability
to reflect interactions among components.
The literature search seeks, in decreasing
order of preference (U.S. EPA. 2000b. §2.2:
1986b. §2.1Y|:
Studies of the mixture being assessed.
Studies of a sufficiently similar
mixture. In evaluating similarity, the
assessment considers the alteration of
mixtures in the environment through
partitioning and transformation.
Studies of individual chemical
components of the mixture, if there are
not adequate studies of sufficiently
similar mixtures.
3.2. Selecting pertinent epidemiologic
studies
Study design is the key consideration for
selecting pertinent epidemiologic studies from
the results of the literature search.
Cohort studies, case-control studies,
and some population-based surveys
(for example, NHANES) provide the
strongest epidemiologic evidence,
especially if they collect information
about individual exposures and
effects.
Ecological studies (geographic
correlation studies) relate exposures
and effects by geographic area. They
can provide strong evidence if there
are large exposure contrasts between
geographic areas, relatively little
exposure variation within study areas,
and population migration is limited.
Case reports of high or accidental
exposure lack definition of the
population at risk and the expected
number of cases. They can provide
information about a rare effect or
about the relevance of analogous
results in animals.
The assessment briefly reviews ecological
studies and case reports but reports details
only if they suggest effects not identified by
other studies.
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3.3. Selecting pertinent experimental
studies
Exposure route is a key design
consideration for selecting pertinent
experimental animal studies or human clinical
studies.
Studies of oral, inhalation, or dermal
exposure involve passage through an
absorption barrier and are considered
most pertinent to human
environmental exposure.
Injection or implantation studies are
often considered less pertinent but
may provide valuable toxicokinetic or
mechanistic information. They also
may be useful for identifying effects in
animals if deposition or absorption is
problematic (for example, for particles
and fibers).
Exposure duration is also a key design
consideration for selecting pertinent
experimental animal studies.
Studies of effects from chronic
exposure are most pertinent to
lifetime human exposure.
Studies of effects from less-than-
chronic exposure are pertinent but
less preferred for identifying effects
from lifetime human exposure. Such
studies may be indicative of effects
from less-than-lifetime human
exposure.
Short-duration studies involving animals
or humans may provide toxicokinetic or
mechanistic information.
For developmental toxicity and
reproductive toxicity, irreversible effects may
result from a brief exposure during a critical
period of development. Accordingly,
specialized study designs are used for these
effects flJ.S. EPA. 2006b. 1998.1996. 19911.
4. Evaluating the quality of
individual studies
40 After the subsets of pertinent
41 epidemiologic and experimental studies have
42 been selected from the literature searches, the
43 assessment evaluates the quality of each
44 individual study. This evaluation considers
45 the design, methods, conduct, and
46 documentation of each study, but not whether
47 the results are positive, negative, or null. The
48 objective is to identify the stronger, more
49 informative studies based on a uniform
50 evaluation of quality characteristics across
51 studies of similar design.
4.1. Evaluating the quality of
epidemiologic studies
52 The assessment evaluates design and
53 methodological aspects that can increase or
54 decrease the weight given to each
55 epidemiologic study in the overall evaluation
56 flJ.S. EPA. 2005a. 1998. 1996.1994.19911:
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Documentation of study design,
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methods, population characteristics,
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and results.
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Definition and selection of the study
61
group and comparison group.
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- Ascertainment of exposure to the
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chemical or mixture.
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- Ascertainment of disease or health
65
effect
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Duration of exposure and follow-up
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and adequacy for assessing the
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occurrence of effects.
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Characterization of exposure during
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critical periods.
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Sample size and statistical power to
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detect anticipated effects.
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Participation rates and potential for
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selection bias as a result of the
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achieved participation rates.
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Measurement error (can lead to
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misclassification of exposure, health
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1 outcomes, and other factors) and other
2 types of information bias.
3 - Potential confounding and other
4 sources of bias addressed in the study
5 design or in the analysis of results. The
6 basis for consideration of confounding
7 is a reasonable expectation that the
8 confounder is related to both exposure
9 and outcome and is sufficiently
10 prevalent to result in bias.
11 For developmental toxicity, reproductive
12 toxicity, neurotoxicity, and cancer there is
13 further guidance on the nuances of evaluating
14 epidemiologic studies of these effects fU.S.
15 EPA. 2005a. 1998.1996.19911.
4.2. Evaluating the quality of
experimental studies
16 The assessment evaluates design and
17 methodological aspects that can increase or
18 decrease the weight given to each
19 experimental animal study, in-vitro study, or
20 human clinical study fU.S. EPA. 2005a. 1998.
21 1996. 19911. Research involving human
22 subjects is considered only if conducted
23 according to ethical principles.
24 - Documentation of study design,
25 animals or study population, methods,
26 basic data, and results.
27 - Nature of the assay and validity for its
28 intended purpose.
29 - Characterization of the nature and
30 extent of impurities and contaminants
31 of the administered chemical or
32 mixture.
33 - Characterization of dose and dosing
34 regimen (including age at exposure)
35 and their adequacy to elicit adverse
36 effects, including latent effects.
37 - Sample sizes and statistical power to
38 detect dose-related differences or
39 trends.
40 - Ascertainment of survival, vital signs,
41 disease or effects, and cause of death.
42 - Control of other variables that could
43 influence the occurrence of effects.
44 The assessment uses statistical tests to
45 evaluate whether the observations may be due
46 to chance. The standard for determining
47 statistical significance of a response is a trend
48 test or comparison of outcomes in the exposed
49 groups against those of concurrent controls.
50 In some situations, examination of historical
51 control data from the same laboratory within
52 a few years of the study may improve the
53 analysis. For an uncommon effect that is not
54 statistically significant compared with
55 concurrent controls, historical controls may
56 show that the effect is unlikely to be due to
57 chance. For a response that appears
58 significant against a concurrent control
59 response that is unusual, historical controls
60 may offer a different interpretation fU.S. EPA.
61 2005a. §2.2.2.1.31.
62 For developmental toxicity, reproductive
63 toxicity, neurotoxicity, and cancer there is
64 further guidance on the nuances of evaluating
65 experimental studies of these effects fU.S. EPA.
66 2005a. 1998. 1996. 19911. In multi-
67 generation studies, agents that produce
68 developmental effects at doses that are not
69 toxic to the maternal animal are of special
70 concern. Effects that occur at doses associated
71 with mild maternal toxicity are not assumed to
72 result only from maternal toxicity. Moreover,
73 maternal effects may be reversible, while
74 effects on the offspring may be permanent
75 CU.S. EPA. 1998. §3.1.2.4.5.4: 1991. §3.1.1.41..
4.3. Reporting study results
76 The assessment uses evidence tables to
77 present the design and key results of pertinent
78 studies. There may be separate tables for each
79 site of toxicity or type of study.
80 If a large number of studies observe the
81 same effect, the assessment considers the
82 study quality characteristics in this section to
83 identify the strongest studies or types of study.
84 The tables present details from these studies,
85 and the assessment explains the reasons for
86 not reporting details of other studies or
87 groups of studies that do not add new
88 information. Supplemental information
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provides references to all studies considered,
including those not summarized in the tables.
The assessment discusses strengths and
limitations that affect the interpretation of
each study. If the interpretation of a study in
the assessment differs from that of the study
authors, the assessment discusses the basis for
the difference.
As a check on the selection and evaluation
of pertinent studies, the EPA asks peer
reviewers to identify studies that were not
adequately considered.
5. Evaluating the overall evidence of
each effect
5.1. Concepts of causal inference
For each health effect, the assessment
evaluates the evidence as a whole to
determine whether it is reasonable to infer a
causal association between exposure to the
agent and the occurrence of the effect This
inference is based on information from
pertinent human studies, animal studies, and
mechanistic studies of adequate quality.
Positive, negative, and null results are given
weight according to study quality.
Causal inference involves scientific
judgment, and the considerations are nuanced
and complex. Several health agencies have
developed frameworks for causal inference,
among them the U.S. Surgeon General (CDC,
2004; HEW, 1964), the International Agency
for Research on Cancer (IARC. 20061. the
Institute of Medicine (IOM, 2008), and the EPA
(2010, §1.6; 2005a, §2.5). Although developed
for different purposes, the frameworks are
similar in nature and provide an established
structure and language for causal inference.
Each considers aspects of an association that
suggest causation, discussed by Hill (1965)
and elaborated by Rothman and Greenland
(1998), and U.S. EPA f2005a. §2.2.1.7:
1994. Appendix C).
Strength of association: The finding of a large
relative risk with narrow confidence
intervals strongly suggests that an
association is not due to chance, bias, or
44 other factors. Modest relative risks,
45 however, may reflect a small range of
46 exposures, an agent of low potency, an
47 increase in an effect that is common,
48 exposure misclassification, or other
49 sources of bias.
50 Consistency of association: An inference of
51 causation is strengthened if elevated risks
52 are observed in independent studies of
53 different populations and exposure
54 scenarios. Reproducibility of findings
55 constitutes one of the strongest arguments
56 for causation. Discordant results
57 sometimes reflect differences in study
58 design, exposure, or confounding factors.
59 Specificity of association: As originally
60 intended, this refers to one cause
61 associated with one effect Current
62 understanding that many agents cause
63 multiple effects and many effects have
64 multiple causes make this a less
65 informative aspect of causation, unless the
66 effect is rare or unlikely to have multiple
67 causes.
68 Temporal relationship: A causal
69 interpretation requires that exposure
70 precede development of the effect.
71 Biologic gradient (exposure-response
72 relationship): Exposure-response
73 relationships strongly suggest causation.
74 A monotonic increase is not the only
75 pattern consistent with causation. The
76 presence of an exposure-response
77 gradient also weighs against bias and
78 confounding as the source of an
79 association.
80 Biologic plausibility: An inference of
81 causation is strengthened by data
82 demonstrating plausible biologic
83 mechanisms, if available. Plausibility may
84 reflect subjective prior beliefs if there is
85 insufficient understanding of the biologic
86 process involved.
87 Coherence: An inference of causation is
88 strengthened by supportive results from
89 animal experiments, toxicokinetic studies,
90 and short-term tests. Coherence may also
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be found in other lines of evidence, such as
changing disease patterns in the
population.
"Natural experiments": A change in exposure
that brings about a change in disease
frequency provides strong evidence, as it
tests the hypothesis of causation. An
example would be an intervention to
reduce exposure in the workplace or
environment that is followed by a
reduction of an adverse effect.
Analogy: Information on structural analogues
or on chemicals that induce similar
mechanistic events can provide insight
into causation.
These considerations are consistent with
guidelines for systematic reviews that
evaluate the quality and weight of evidence.
Confidence is increased if the magnitude of
effect is large, if there is evidence of an
exposure-response relationship, or if an
association was observed and the plausible
biases would tend to decrease the magnitude
of the reported effect. Confidence is decreased
for study limitations, inconsistency of results,
indirectness of evidence, imprecision, or
reporting bias (Guvatt et al.. 2008b: Guvatt et
al.. 2008a],
5.2. Evaluating evidence in humans
For each effect, the assessment evaluates
the evidence from the epidemiologic studies as
a whole. The objective is to determine
whether a credible association has been
observed and, if so, whether that association is
consistent with causation. In doing this, the
assessment explores alternative explanations
(such as chance, bias, and confounding) and
draws a conclusion about whether these
alternatives can satisfactorily explain any
observed association.
To make clear how much the
epidemiologic evidence contributes to the
overall weight of the evidence, the assessment
may select a standard descriptor to
characterize the epidemiologic evidence of
association between exposure to the agent and
occurrence of a health effect
47 Sufficient epidemiologic evidence of an
48 association consistent with causation:
49 The evidence establishes a causal
50 association for which alternative
51 explanations such as chance, bias, and
52 confounding can be ruled out with
53 reasonable confidence.
54 Suggestive epidemiologic evidence of an
55 association consistent with causation:
56 The evidence suggests a causal association
57 but chance, bias, or confounding cannot be
58 ruled out as explaining the association.
59 Inadequate epidemiologic evidence to infer
60 a causal association: The available
61 studies do not permit a conclusion
62 regarding the presence or absence of an
63 association.
64 Epidemiologic evidence consistent with no
65 causal association: Several adequate
66 studies covering the full range of human
67 exposures and considering susceptible
68 populations, and for which alternative
69 explanations such as bias and confounding
70 can be ruled out, are mutually consistent
71 in not finding an association.
5.3. Evaluating evidence in animals
72 For each effect, the assessment evaluates
73 the evidence from the animal experiments as a
74 whole to determine the extent to which they
75 indicate a potential for effects in humans.
76 Consistent results across various species and
77 strains increase confidence that similar results
78 would occur in humans. Several concepts
79 discussed by Hill (1965) are pertinent to the
80 weight of experimental results: consistency of
81 response, dose-response relationships,
82 strength of response, biologic plausibility, and
83 coherence fU.S. EPA. 2005a. §2.2.1.7:
84 1994. Appendix C).
85 In weighing evidence from multiple
86 experiments, U.S. EPA (2005a. §2.5)
87 distinguishes:
88 Conflicting evidence (that is, mixed positive
89 and negative results in the same sex and
90 strain using a similar study protocol) from
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Differing results (that is, positive results and
negative results are in different sexes or
strains or use different study protocols).
Negative or null results do not invalidate
positive results in a different experimental
system. The EPA regards all as valid
observations and looks to explain differing
results using mechanistic information (for
example, physiologic or metabolic differences
across test systems) or methodological
differences (for example, relative sensitivity of
the tests, differences in dose levels,
insufficient sample size, or timing of dosing or
data collection).
It is well established that there are critical
periods for some developmental and
reproductive effects (U.S. EPA. 2006b. 2005a.
b, 1998. 1996. 1991). Accordingly, the
assessment determines whether critical
periods have been adequately investigated.
Similarly, the assessment determines whether
the database is adequate to evaluate other
critical sites and effects.
In evaluating evidence of genetic toxicity:
Demonstration of gene mutations,
chromosome aberrations, or
aneuploidy in humans or experimental
mammals [in vivo) provides the
strongest evidence.
- This is followed by positive results in
lower organisms or in cultured cells
[in vitro) or for other genetic events.
Negative results carry less weight,
partly because they cannot exclude the
possibility of effects in other tissues
fTARC. 20061.
For germ-cell mutagenicity, The EPA has
defined categories of evidence, ranging from
positive results of human germ-cell
mutagenicity to negative results for all effects
of concern fU.S. EPA. 1986a. §2.31.
5.4. Evaluating mechanistic data
Mechanistic data can be useful in
answering several questions.
- The biologic plausibility of a causal
interpretation of human studies.
- The generalizability of animal studies
to humans.
- The susceptibility of particular
populations or lifestages.
The focus of the analysis is to describe, if
possible, mechanistic pathways that lead to a
health effect. These pathways encompass:
Toxicokinetic processes of absorption,
distribution, metabolism, and
elimination that lead to the formation
of an active agent and its presence at
the site of initial biologic interaction.
Toxicodynamic processes that lead to a
health effect at this or another site
(also known as a mode of action).
For each effect, the assessment discusses
the available information on its modes of
action and associated key events [key events
being empirically observable, necessary
precursor steps or biologic markers of such
steps; mode of action being a series of key
events involving interaction with cells,
operational and anatomic changes, and
resulting in disease). Pertinent information
may also come from studies of metabolites or
of compounds that are structurally similar or
that act through similar mechanisms.
Information on mode of action is not required
for a conclusion that the agent is causally
related to an effect fU.S. EPA. 2005a. §2.51.
The assessment addresses several
questions about each hypothesized mode of
action fU.S. EPA. 2005a. §2.4.3.41.
1) Is the hypothesized mode of action
sufficiently supported in test animals?
Strong support for a key event being
necessary to a mode of action can come
from experimental challenge to the
hypothesized mode of action, in which
studies that suppress a key event observe
suppression of the effect Support for a
mode of action is meaningfully
strengthened by consistent results in
different experimental models, much
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more so than by replicate experiments in
the same model. The assessment may
consider various aspects of causation in
addressing this question.
2) Is the hypothesized mode of action
relevant to humans? The assessment
reviews the key events to identify critical
similarities and differences between the
test animals and humans. Site
concordance is not assumed between
animals and humans, though it may hold
for certain effects or modes of action.
Information suggesting quantitative
differences in doses where effects would
occur in animals or humans is considered
in the dose-response analysis. Current
levels of human exposure are not used to
rule out human relevance, as IRIS
assessments may be used in evaluating
new or unforeseen circumstances that
may entail higher exposures.
3) Which populations or lifestages can be
particularly susceptible to the
hypothesized mode of action? The
assessment reviews the key events to
identify populations and lifestages that
might be susceptible to their occurrence.
Quantitative differences may result in
separate toxicity values for susceptible
populations or lifestages.
The assessment discusses the likelihood
that an agent operates through multiple
modes of action. An uneven level of support
for different modes of action can reflect
disproportionate resources spent
investigating them fU.S. EPA. 2005a. §2.4.3.31.
It should be noted that in clinical reviews, the
credibility of a series of studies is reduced if
evidence is limited to studies funded by one
interested sector fGuvattetal.. 2008al.
For cancer, the assessment evaluates
evidence of a mutagenic mode of action to
guide extrapolation to lower doses and
consideration of susceptible lifestages. Key
data include the ability of the agent or a
metabolite to react with or bind to DNA,
positive results in multiple test systems, or
similar properties and structure-activity
relationships to mutagenic carcinogens fU.S.
EPA. 2005a .§2.3.51.
5.5. Characterizing the overall weight
of the evidence
After evaluating the human, animal, and
mechanistic evidence pertinent to an effect,
the assessment answers the question: Does
the agent cause the adverse effect? (NRC,
2009, 1983). In doing this, the assessment
develops a narrative that integrates the
evidence pertinent to causation. To provide
clarity and consistency, the narrative includes
a standard hazard descriptor. For example,
the following standard descriptors combine
epidemiologic, experimental, and mechanistic
evidence of carcinogenicity fU.S. EPA. 2005a.
§2.51.
Carcinogenic to humans: There is convincing
epidemiologic evidence of a causal
association (that is, there is reasonable
confidence that the association cannot be
fully explained by chance, bias, or
confounding); or there is strong human
evidence of cancer or its precursors,
extensive animal evidence, identification
of key precursor events in animals, and
strong evidence that they are anticipated
to occur in humans.
Likely to be carcinogenic to humans: The
evidence demonstrates a potential hazard
to humans but does not meet the criteria
for carcinogenic. There may be a plausible
association in humans, multiple positive
results in animals, or a combination of
human, animal, or other experimental
evidence.
Suggestive evidence of carcinogenic
potential: The evidence raises concern for
effects in humans but is not sufficient for a
stronger conclusion. This descriptor
covers a range of evidence, from a positive
result in the only available study to a single
positive result in an extensive database
that includes negative results in other
species.
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Inadequate information to assess
carcinogenic potential: No other
descriptors apply. Conflicting evidence can
be classified as inadequate information if
all positive results are opposed by
negative studies of equal quality in the
same sex and strain. Differing results,
however, can be classified as suggestive
evidence or as likely to be carcinogenic.
Not likely to be carcinogenic to humans:
There is robust evidence for concluding
that there is no basis for concern. There
may be no effects in both sexes of at least
two appropriate animal species; positive
animal results and strong, consistent
evidence that each mode of action in
animals does not operate in humans; or
convincing evidence that effects are not
likely by a particular exposure route or
below a defined dose.
Multiple descriptors may be used if there
is evidence that carcinogenic effects differ by
dose range or exposure route (U.S. EPA. 2005a.
§2.51
Another example of standard descriptors
comes from the EPA's Integrated Science
Assessments, which evaluate causation for the
effects of the criteria pollutants in ambient air
(U.S. EPA, 2010, §1.6).
Causal relationship: Sufficient evidence to
conclude that there is a causal
relationship. Observational studies
cannot be explained by plausible
alternatives, or they are supported by
other lines of evidence, for example,
animal studies or mechanistic
information.
Likely to be a causal relationship: Sufficient
evidence that a causal relationship is
likely, but important uncertainties remain.
For example, observational studies show
an association but co-exposures are
difficult to address or other lines of
evidence are limited or inconsistent; or
multiple animal studies from different
laboratories demonstrate effects and
there are limited or no human data.
Suggestive of a causal relationship: At least
one high-quality epidemiologic study
shows an association but other studies are
inconsistent
Inadequate to infer a causal relationship:
The studies do not permit a conclusion
regarding the presence or absence of an
association.
Not likely to be a causal relationship: Several
adequate studies, covering the full range of
human exposure and considering
susceptible populations, are mutually
consistent in not showing an effect at any
level of exposure.
The EPA is investigating and may on a trial
basis use these or other standard descriptors
to characterize the overall weight of the
evidence for effects other than cancer.
6. Selecting studies for derivation of
toxicity values
For each effect where there is credible
evidence of an association with the agent, the
assessment derives toxicity values if there are
suitable epidemiologic or experimental data.
The decision to derive toxicity values may be
linked to the hazard descriptor.
Dose-response analysis requires
quantitative measures of dose and response.
Then, other factors being equal:
Epidemiologic studies are preferred
over animal studies, if quantitative
measures of exposure are available
and effects can be attributed to the
agent
- Among experimental animal models,
those that respond most like humans
are preferred, if the comparability of
response can be determined.
Studies by a route of human
environmental exposure are
preferred, although a validated
toxicokinetic model can be used to
extrapolate across exposure routes.
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Studies of longer exposure duration
and follow-up are preferred, to
minimize uncertainty about whether
effects are representative of lifetime
exposure.
Studies with multiple exposure levels
are preferred for their ability to
provide information about the shape
of the exposure-response curve.
Studies with adequate power to detect
effects at lower exposure levels are
preferred, to minimize the extent of
extrapolation to levels found in the
environment
Studies with non-monotonic exposure-
response relationships are not necessarily
excluded from the analysis. A diminished
effect at higher exposure levels may be
satisfactorily explained by factors such as
competing toxicity, saturation of absorption or
metabolism, exposure misclassification, or
selection bias.
If a large number of studies are suitable for
dose-response analysis, the assessment
considers the study characteristics in this
section to focus on the most informative data.
The assessment explains the reasons for not
analyzing other groups of studies. As a check
on the selection of studies for dose-response
analysis, the EPA asks peer reviewers to
identify studies that were not adequately
considered.
7. Deriving toxicity values
7.1. General framework for dose-
response analysis
The EPA uses a two-step approach that
distinguishes analysis of the observed dose-
response data from inferences about lower
doses flJ.S. EPA. 2005a. §31
Within the observed range, the preferred
approach is to use modeling to incorporate a
wide range of data into the analysis. The
modeling yields a point of departure (an
exposure level near the lower end of the
observed range, without significant
extrapolation to lower doses) (Sections 7.2-
7.3).
Extrapolation to lower doses considers
what is known about the modes of action for
each effect (Sections 7.4-7.5). If response
estimates at lower doses are not required, an
alternative is to derive reference values, which
are calculated by applying factors to the point
of departure in order to account for sources of
uncertainty and variability (Section 7.6).
For a group of agents that induce an effect
through a common mode of action, the dose-
response analysis may derive a relative
potency factor for each agent. A full dose-
response analysis is conducted for one well-
studied index chemical in the group, then the
potencies of other members are expressed in
relative terms based on relative toxic effects,
relative absorption or metabolic rates,
quantitative structure-activity relationships,
or receptor binding characteristics fU.S. EPA.
2005a. §3.2.6: 2000b. §4.41.
Increasingly, the EPA is basing toxicity
values on combined analyses of multiple data
sets or multiple responses. The EPA also
considers multiple dose-response approaches
if they can be supported by robust data.
7.2. Modeling dose to sites of biologic
effects
The preferred approach for analysis of
dose is toxicokinetic modeling because of its
ability to incorporate a wide range of data.
The preferred dose metric would refer to the
active agent at the site of its biologic effect or
to a close, reliable surrogate measure. The
active agent may be the administered chemical
or a metabolite. Confidence in the use of a
toxicokinetic model depends on the
robustness of its validation process and on the
results of sensitivity analyses (U.S. EPA.
2006a: 2005a. §3.1: 1994. §4.31.
Because toxicokinetic modeling can
require many parameters and more data than
are typically available, the EPA has developed
standard approaches that can be applied to
typical data sets. These standard approaches
also facilitate comparison across exposure
patterns and species.
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Intermittent study exposures are
standardized to a daily average over
the duration of exposure. For chronic
effects, daily exposures are averaged
over the lifespan. Exposures during a
critical period, however, are not
averaged over a longer duration fU.S.
EPA. 2005a. 33.1.1: 1991. §3.21.
Doses are standardized to equivalent
human terms to facilitate comparison
of results from different species.
Oral doses are scaled allometrically
using mg/kg3/4-day as the equivalent
dose metric across species. Allometric
scaling pertains to equivalence across
species, not across lifestages, and is
not used to scale doses from adult
humans or mature animals to infants
or children (U.S. EPA. 2011:
2005a. §3.1.31.
Inhalation exposures are scaled using
dosimetry models that apply species-
specific physiologic and anatomic
factors and consider whether the
effect occurs at the site of first contact
or after systemic circulation (U.S. EPA.
2012a: 1994. §31.
It can be informative to convert doses
across exposure routes. If this is done, the
assessment describes the underlying data,
algorithms, and assumptions (U.S. EPA.
2005a. §3.1.41.
In the absence of study-specific data on,
for example, intake rates or body weight, the
EPA has developed recommended values for
use in dose-response analysis fU.S. EPA.
19881.
7.3. Modeling response in the range of
observation
Toxicodynamic ("biologically based")
modeling can incorporate data on biologic
processes leading to an effect. Such models
require sufficient data to ascertain a mode of
action and to quantitatively support model
parameters associated with its key events.
Because different models may provide
equivalent fits to the observed data but
diverge substantially at lower doses, critical
biologic parameters should be measured from
laboratory studies, not by model fitting.
Confidence in the use of a toxicodynamic
model depends on the robustness of its
validation process and on the results of
sensitivity analyses. Peer review of the
scientific basis and performance of a model is
essential fU.S. EPA. 2005a. §3.2.21.
Because toxicodynamic modeling can
require many parameters and more
knowledge and data than are typically
available, the EPA has developed a standard
set of empirical ("curve-fitting") models
(http://www.epa.gOv/ncea/bmds/l that can
be applied to typical data sets, including those
that are nonlinear. The EPA has also
developed guidance on modeling dose-
response data, assessing model fit, selecting
suitable models, and reporting modeling
results fU.S. EPA. 2012al. Additional
judgment or alternative analyses are used if
the procedure fails to yield reliable results, for
example, if the fit is poor, modeling may be
restricted to the lower doses, especially if
there is competing toxicity at higher doses
fU.S. EPA. 2005a. §3.2.31.
Modeling is used to derive a point of
departure fU.S. EPA. 2012a: 2005a. §3.2.41.
(See Section 7.6 for alternatives if a point of
departure cannot be derived by modeling.):
If linear extrapolation is used,
selection of a response level
corresponding to the point of
departure is not highly influential, so
standard values near the low end of
the observable range are generally
used (for example, 10% extra risk for
cancer bioassay data, 1% for
epidemiologic data, lower for rare
cancers).
For nonlinear approaches, both
statistical and biologic considerations
are taken into account.
For dichotomous data, a response level
of 10% extra risk is generally used for
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minimally adverse effects, 5% or
lower for more severe effects.
For continuous data, a response level
is ideally based on an established
definition of biologic significance. In
the absence of such definition, one
control standard deviation from the
control mean is often used for
minimally adverse effects, one-half
standard deviation for more severe
effects.
The point of departure is the 95% lower
bound on the dose associated with the
selected response level.
7.4. Extrapolating to lower doses and
response levels
The purpose of extrapolating to lower
doses is to estimate responses at exposures
below the observed data. Low-dose
extrapolation, typically used for cancer data,
considers what is known about modes of
action fU.S. EPA. 2005a. §3.3.1 and §3.3.21.
1) If a biologically based model has been
developed and validated for the agent,
extrapolation may use the fitted model
below the observed range if significant
model uncertainty can be ruled out with
reasonable confidence.
2) Linear extrapolation is used if the dose-
response curve is expected to have a
linear component below the point of
departure. This includes:
- Agents or their metabolites that are
DNA-reactive and have direct
mutagenic activity.
- Agents or their metabolites for which
human exposures or body burdens are
near doses associated with key events
leading to an effect
Linear extrapolation is also used when
data are insufficient to establish mode of
action and when scientifically plausible.
The result of linear extrapolation is
described by an oral slope factor or an
inhalation unit risk, which is the slope of
the dose-response curve at lower doses or
concentrations, respectively.
3) Nonlinear models are used for
extrapolation if there are sufficient data to
ascertain the mode of action and to
conclude that it is not linear at lower
doses, and the agent does not demonstrate
mutagenic or other activity consistent
with linearity at lower doses. Nonlinear
approaches generally should not be used
in cases where mode of action has not
ascertained. If nonlinear extrapolation is
appropriate but no model is developed, an
alternative is to calculate reference values.
4) Both linear and nonlinear approaches may
be used if there a multiple modes of action.
For example, modeling to a low response
level can be useful for estimating the
response at doses where a high-dose mode
of action would be less important.
If linear extrapolation is used, the
assessment develops a candidate slope factor
or unit risk for each suitable data set. These
results are arrayed, using common dose
metrics, to show the distribution of relative
potency across various effects and
experimental systems. The assessment then
derives or selects an overall slope factor and
an overall unit risk for the agent, considering
the various dose-response analyses, the study
preferences discussed in Section 6, and the
possibility of basing a more robust result on
multiple data sets.
7.5. Considering susceptible
populations and lifestages
The assessment analyzes the available
information on populations and lifestages that
may be particularly susceptible to each effect.
A tiered approach is used fU.S. EPA.
2005a. §3.51.
1) If an epidemiologic or experimental study
reports quantitative results for a
susceptible population or lifestage, these
data are analyzed to derive separate
toxicity values for susceptible individuals.
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2) If data on risk-related parameters allow
comparison of the general population and
susceptible individuals, these data are
used to adjust the general-population
toxicity values for application to
susceptible individuals.
3) In the absence of chemical-specific data,
the EPA has developed age-dependent
adjustment factors for early-life exposure
to potential carcinogens that have a
mutagenic mode of action. There is
evidence of early-life susceptibility to
various carcinogenic agents, but most
epidemiologic studies and cancer
bioassays do not include early-life
exposure. To address the potential for
early-life susceptibility, the EPA
recommends fU.S. EPA. 2005b. §51:
10-fold adjustment for exposures
before age 2 years.
3-fold adjustment for exposures
between ages 2 and 16 years.
7.6. Reference values and uncertainty
factors
An oral reference dose or an inhalation
reference concentration is an estimate of an
exposure (including in susceptible subgroups)
that is likely to be without an appreciable risk
of adverse health effects over a lifetime fU.S.
EPA. 2002. §4.21. Reference values are
typically calculated for effects other than
cancer and for suspected carcinogens if a well
characterized mode of action indicates that a
necessary key event does not occur below a
specific dose. Reference values provide no
information about risks at higher exposure
levels.
The assessment characterizes effects that
form the basis for reference values as adverse,
considered to be adverse, or a precursor to an
adverse effect. For developmental toxicity,
reproductive toxicity, and neurotoxicity there
is guidance on adverse effects and their
biologic markers fU.S. EPA. 1998.1996.19911.
To account for uncertainty and variability
in the derivation of a lifetime human exposure
where adverse effects are not anticipated to
occur, reference values are calculated by
applying a series of uncertainty factors to the
point of departure. If a point of departure
cannot be derived by modeling, a no-
observed-adverse-effect level or a lowest-
observed-adverse-effect level is used instead.
The assessment discusses scientific
considerations involving several areas of
variability or uncertainty.
Human variation. The assessment accounts
for variation in susceptibility across the
human population and the possibility that
the available data may not be
representative of individuals who are
most susceptible to the effect. A factor of
10 is generally used to account for this
variation. This factor is reduced only if the
point of departure is derived or adjusted
specifically for susceptible individuals
(not for a general population that includes
both susceptible and non-susceptible
individuals) fU.S. EPA. 2002. §4.4.5:
1998. §4.2: 1996. §4: 1994. §4.3.9.1:
1991. §3.41.
Animal-to-human extrapolation. If animal
results are used to make inferences about
humans, the assessment adjusts for cross-
species differences. These may arise from
differences in toxicokinetics or
toxicodynamics. Accordingly, if the point
of departure is standardized to equivalent
human terms or is based on toxicokinetic
or dosimetry modeling, a factor of 101/2
(rounded to 3) is applied to account for the
remaining uncertainty involving
toxicokinetic and toxicodynamic
differences. If a biologically based model
adjusts fully for toxicokinetic and
toxicodynamic differences across species,
this factor is not used. In most other cases,
a factor of 10 is applied fU.S. EPA. 2011:
2002. §4.4.5: 1998. §4.2: 1996. §4:
1994. §4.3.9.1: 1991. §3.41.
Adverse-effect level to no-observed-
adverse-effect level. If a point of
departure is based on a lowest-observed-
adverse-effect level, the assessment must
infer a dose where such effects are not
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expected. This can be a matter of great
uncertainty, especially if there is no
evidence available at lower doses. A factor
of 10 is applied to account for the
uncertainty in making this inference. A
factor other than 10 may be used,
depending on the magnitude and nature of
the response and the shape of the dose-
response curve (U.S. EPA. 2002. §4.4.5:
1998. §4.2: 1996. §4: 1994. §4.3.9.1:
1991. §3.41.
Subchronic-to-chronic exposure. If a point
of departure is based on subchronic
studies, the assessment considers whether
lifetime exposure could have effects at
lower levels of exposure. A factor of 10 is
applied to account for the uncertainty in
using subchronic studies to make
inferences about lifetime exposure. This
factor may also be applied for
developmental or reproductive effects if
exposure covered less than the full critical
period. A factor other than 10 may be
used, depending on the duration of the
studies and the nature of the response
riJ.S. EPA. 2002. §4.4.5: 1998. §4.2: 1994.
§4.3.9.11.
Incomplete database. If an incomplete
database raises concern that further
studies might identify a more sensitive
effect, organ system, or lifestage, the
assessment may apply a database
uncertainty factor (U.S. EPA. 2002. §4.4.5:
1998. §4.2: 1996. §4: 1994. §4.3.9.1:
1991. §3.41. The size of the factor depends
on the nature of the database deficiency.
For example, the EPA typically follows the
suggestion that a factor of 10 be applied if
both a prenatal toxicity study and a two-
generation reproduction study are
missing and a factor of 101/2 if either is
missing fU.S. EPA. 2002. §4.4.51.
In this way, the assessment derives
candidate values for each suitable data set and
effect that is credibly associated with the
agent. These results are arrayed, using
common dose metrics, to show where effects
occur across a range of exposures (U.S. EPA.
1994. §4.3.91.
The assessment derives or selects an
organ- or system-specific reference value for
each organ or system affected by the agent.
The assessment explains the rationale for each
organ/system-specific reference value (based
on, for example, the highest quality studies,
the most sensitive outcome, or a clustering of
values). By providing these organ/system-
specific reference values, IRIS assessments
facilitate subsequent cumulative risk
assessments that consider the combined effect
of multiple agents acting at a common site or
through common mechanisms (NRC. 20091.
The assessment then selects an overall
reference dose and an overall reference
concentration for the agent to represent
lifetime human exposure levels where effects
are not anticipated to occur. This is generally
the most sensitive organ/system-specific
reference value, though consideration of study
quality and confidence in each value may lead
to a different selection.
7.7. Confidence and uncertainty in the
reference values
The assessment selects a standard
descriptor to characterize the level of
confidence in each reference value, based on
the likelihood that the value would change
with further testing. Confidence in reference
values is based on quality of the studies used
and completeness of the database, with more
weight given to the latter. The level of
confidence is increased for reference values
based on human data supported by animal
data (TJ.S. EPA. 1994. §4.3.9.21.
High confidence: The reference value is not
likely to change with further testing,
except for mechanistic studies that might
affect the interpretation of prior test
results.
Medium confidence: This is a matter of
judgment, between high and low
confidence.
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Low confidence: The reference value is
especially vulnerable to change with
further testing.
These criteria are consistent with
guidelines for systematic reviews that
evaluate the quality of evidence. These also
focus on whether further research would be
likely to change confidence in the estimate of
effect (Guvatt etal.. 2008b).
All assessments discuss the significant
uncertainties encountered in the analysis. The
EPA provides guidance on characterization of
uncertainty (U.S. EPA. 2005a. §3.61. For
example, the discussion distinguishes model
uncertainty (lack of knowledge about the most
appropriate experimental or analytic model)
and parameter uncertainty (lack of knowledge
about the parameters of a model).
Assessments also discuss human variation
(interpersonal differences in biologic
susceptibility or in exposures that modify the
effects of the agent).
August 2013
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EXECUTIVE SUMMARY
Occurrence and Health Effects
Hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) is a synthetic chemical used
primarily as a military explosive. RDX releases have been reported in air, water, and
soil. Exposure to RDX 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 ingestion of crops irrigated with
contaminated water. Inhalation or dermal exposures are more likely in occupational
settings.
Epidemiological studies provide only limited information on occupational
populations exposed to RDX; several case reports describe effects primarily in the
nervous system following acute exposure to RDX. Animal studies demonstrate
toxicity, including nervous system effects, kidney and other urogenital effects, and
male reproductive effects.
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.
Effects Other Than Cancer Observed Following Oral Exposure
EPA identified nervous system effects as a human hazard of RDX exposure. Several human
case reports and animal studies provide consistent evidence of associations between RDX exposure
and effects on the nervous system, including seizures or convulsions. Increased mortality was
generally observed at RDX doses that induced nervous system effects, and several studies
documented that deaths in most cases were preceded by tremors and convulsions. Although
mechanistic data are insufficient to establish a mode of action (MOA) for RDX-induced convulsions,
the available information suggests that nervous system effects are mediated by RDX binding to the
picrotoxin convulsant site of the GABAa channel, resulting in disinhibition that leads to the onset of
seizures.
EPA identified kidney and other urogenital effects as a potential human hazard of RDX
exposure based on observations in 2-year studies of increased relative kidney weights in male and
female mice and histopathological changes in the urogenital system of male rats exposed to RDX.
An increased incidence of suppurative prostatitis was identified, and is considered a marker for
RDX-related urogenital effects. There is no established MOA for RDX-related effects on the
urogenital system.
Based on the finding of testicular degeneration in male mice exposed to RDX in diet for 2
years, in the only mouse study conducted of that duration, EPA identified suggestive evidence of
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male reproductive effects as a potential human hazard of RDX exposure. There is no known MOA
for male reproductive effects of RDX exposure.
Evidence for effects on other organs/systems, including the liver and developmental effects,
was more limited than for the endpoints summarized above. EPA concluded that the evidence does
not support effects on other organs/systems, including liver and developmental effects, as a
potential human hazard of RDX exposure.
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 RfDs and proposed overall RfD for RDX
Effect
Basis
RfD (mg/kg-day)
Study exposure
description
Confidence
Nervous system
Convulsions
9 x 10"4
Subchronic
Medium
Kidney/urogenital
Suppurative prostatitis
2 x 10"3
Chronic
Low
Male reproductive
Testicular degeneration
2 x 10"2
Chronic
Low
Proposed overall RfD
Nervous system effects
9 x 10"4
Subchronic
Medium
The overall RfD (see Table ES-2) is derived to be protective of all types of hazards
associated with RDX exposure. 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 it represents the most sensitive human hazard of RDX
exposure. Incidence of seizures or convulsions as observed in a subchronic gavage study (Crouse et
al.. 2006] was selected for derivation of the overall RfD as the study was well-conducted, utilized a
more pure form of test material than other studies, and had five closely-spaced dose groups that
allowed characterization of the dose-response curve. Benchmark dose (BMD) modeling was
utilized to derive the point of departure (POD) for RfD derivation (expressed as the BMDLoi). A 1%
response level was chosen because of the severity of the endpoint; this is supported by the
observation in Crouse etal. (2006) that for all the dose groups where unscheduled deaths were
recorded, mortality was strongly associated with convulsions. A physiologically-based
pharmacokinetic (PBPK) model was used to extrapolate the BMDLoi to a human equivalent dose
(HED) based on RDX arterial blood concentration, which was then used for RfD derivation.
The proposed overall RfD was calculated by dividing the BMDLoi-hed for nervous system
effects by a composite uncertainty factor of 300 to account for extrapolation from animals to
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1 humans (3), inter individual differences in human susceptibility (10), extrapolation of results from a
2 subchronic study to a chronic study (3), and deficiencies in the toxicity database (3).
Table ES-2. Summary of reference dose (RfD) derivation
Critical effect
Point of departure*
UF
Chronic RfD
Nervous system effects (convulsions)
90-d F344 rat study
Crouse et al. (2006)
BMDLoi-hed: 1.3 mg/kg-d
300
9 x 10"4 mg/kg-d
3
4 *A benchmark response (BMR) of 1% was used to derive the BMD and BMDL given the severity of the endpoint.
5 The resulting POD was converted to a BMDLoi-hed using a PBPK model based on modeled arterial blood
6 concentration. The concentration was derived from the area under the curve (AUC) of modeled RDX
7 concentration in arterial blood, which reflects the average blood RDX concentration for the exposure duration
8 normalized to 24 hours.
9
Effects Other Than Cancer Observed Following Inhalation Exposure
10 No studies were identified that provided useful information on effects observed following
11 inhalation exposure to RDX. Of the available human epidemiological studies of RDX, none provided
12 data that could be used for dose-response analysis of inhalation exposures. The single
13 experimental animal study involving inhalation exposure is not publicly available, and was
14 excluded from consideration due to significant study limitations, including small numbers of
15 animals tested, lack of controls, and incomplete reporting of exposure levels. Therefore, the
16 available health effects literature does not support the identification of hazards following inhalation
17 exposure to RDX.
Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
18 An RfC for RDX could not be derived based on the available health effects data. Additionally,
19 a PBPK model for inhaled RDX is not available to support route-to-route extrapolation from the RfD.
Evidence for Human Carcinogenicity
20 Under EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al. the database for
21 RDX provides "suggestive evidence of carcinogenic potential" based on the finding of statistically
22 significant trends for hepatocellular adenomas or carcinomas and alveolar/bronchiolar adenomas
23 or carcinomas in female, but not male, B6C3Fi mice fLish etal.. 19841. This is further supported by
24 the finding of a statistically significant trend for hepatocellular carcinomas in male, but not female,
25 F344 rats fLevine etal.. 19831 exposed to RDX in the diet for two years. On the other hand, there
26 was no evidence of carcinogenicity in Sprague-Dawley rats in a 2-year dietary study of RDX (Hart.
27 19761. No human studies are available to assess the carcinogenic potential of RDX. TheMOAfor
28 liver and lung tumors in experimental animals is not known. Available in vitro and in vivo
29 genotoxicity assays were largely negative for RDX, suggesting that parent RDX does not interact
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directly with DNA. /V-nitroso metabolites of RDX generated anaerobically have tested positive in
some genotoxicity assays; their contribution to the overall carcinogenic potential of RDX is not
known.
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 (Lish etal..
19841. This two-year dietary study was generally well conducted, with four dose groups and
adequate numbers of animals per dose group (85/sex/group, with interim sacrifices of
10/sex/group at 6 and 12 months), and included 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.
Although EPA concluded that there is "suggestive evidence of carcinogenic potential" for
RDX, the Agency determined that quantitative analysis of the mouse tumor data may be useful for
providing a sense of the magnitude of potential carcinogenic risk.
EPA calculated a single oral slope factor (OSF) that considered the combination of tumors.
Point of departure (i.e., BMD and BMDL) estimates that corresponded to a specific risk of incidence
of either of the tumors (liver or lung) were calculated. The single BMDLio so derived from the
mouse tumors was extrapolated to the HED using BW3/4 scaling, and an OSF was derived by linear
extrapolation from the BMDLio hed- The OSF is 4 x 10"2 per mg/kg-day, based on the liver and lung
tumor response in female mice (Lish etal.. 1984).
Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure
The carcinogenic potential of RDX by inhalation has not been investigated. A PBPK model to
support route-to-route extrapolation of an inhalation unit risk based on oral carcinogenicity data
was not available.
Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
Little information is available on populations that may be especially vulnerable to the toxic
effects of RDX. Lifestage, and in particular childhood susceptibility, has not been observed 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. Data to suggest males may be more
susceptible than females to noncancer toxicity associated with RDX exposure are limited.
Specifically, urogenital effects have been noted at lower doses than in females. Some evidence
suggests 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 may represent another group that may be susceptible to RDX exposure; however,
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there is no information to indicate how genetic polymorphisms may affect susceptibility to RDX.
Key Issues Addressed in Assessment
In most instances, the spectrum of effects associated with chemical exposure will range in
severity, with relatively less severe effects generally occurring at doses lower than those associated
with more severe or "frank" toxicity. Convulsions in rats were selected as the basis for derivation of
the RDX RfD; less severe nervous system effects were generally not observed at lower doses. U.S.
EPA f2012al emphasizes that when modeling a dose-response relationship from a given set of data,
statistical and biological characteristics of the dataset must be considered, including consideration
of the severity of the effect. For convulsions, because of the severity of the effect itself and the
strong association with mortality, a benchmark response (BMR) level of 1% was selected for
modeling, balancing the quantitative limitations of the available animal bioassays and the severity
of the effect. Use of a BMR of 1% extra risk of convulsions resulted in extrapolation below the range
of experimental data and could potentially increase uncertainty in the BMD and BMDL values.
The candidate RfD for kidney and other urogenital effects is based on suppurative
prostatitis. This organ/system-specific RfD is based on a dose-related increase in suppurative
prostatitis as reported in a 2-year feeding study in male F344 rats (Levine etal.. 19831. the only
2-year study in rats that examined the prostate. Some reports have hypothesized that the observed
suppurative prostatitis was secondary to a bacterial infection unrelated to RDX toxicity fATSDR.
2012: Sweeney et al.. 2012a: Crouse etal.. 20061. In reviewing the findings in Levine etal. (19831.
EPA concluded that while an opportunistic bacterial infection may have been the proximal cause of
the suppurative prostatitis, the infection was secondary to urogenital effects associated with RDX
exposure. Histopathological findings for the bladder are not definitive because the design of the
principal study called for histopathological examination of the bladder only if gross abnormalities
were observed. Although the pathogenesis of kidney and urogenital effects is unclear, suppurative
prostatitis was considered to be a marker for the broader array of kidney and other urogenital
effects observed by Levine etal. (19831.
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LITERATURE SEARCH STRATEGY |
STUDY SELECTION AND EVALUATION
Literature Search and Screening Strategy
A literature search and screening strategy was applied to identify literature related to
characterizing the health effects of RDX. This strategy consisted of a search of online scientific
databases and other sources, casting a wide net in order to identify all potentially pertinent studies.
In subsequent steps, references were screened to exclude papers not pertinent to an assessment of
the health effects of RDX, and remaining references were sorted into categories for further
evaluation.
The literature search for RDX was conducted through January 2014 using the databases and
general keywords listed in Table LS-1 (see Appendix B for further details of the literature search
strategy). More specifically, the literature search for RDX was conducted in four online scientific
databases—Pubmed, Toxline, Toxcenter, and TSCATS. The detailed search approach for these
databases, including the search strings and number of citations identified per database, is provided
in Appendix B, Table B-l. Given the military applications of RDX, the Defense Technical Information
Center (DTIC) database, a central online repository of defense-related scientific and technical
information within the Department of Defense, was also searched. A separate strategy was applied
in searching DTIC because of limitations in the classification and distribution of materials in DTIC;
the detailed search strategy is described in Appendix B, Table B-2. This search of the five online
databases identified 995 citations (after electronically eliminating duplicates). The computerized
database searches were supplemented by review of online regulatory sources, "forward" and
"backward" searches of Web of Science (Appendix B, Table B-3), as well as additional references
added during development of the toxicological review (including guidance documents and other
references that provide context for evaluating RDX health effects); 113 citations were obtained
using these additional search strategies. In total, 1108 citations were identified using online
scientific databases and additional search strategies.
EPA requested public submissions of additional information in 2010 (75 FR 76982;
December 10, 2010); no submissions were received in response to this call for data. Additionally,
EPA issued a request to the public for additional information in a Federal Register Notice in 2013
(78 FR 48674; August 9, 2013), and established a docket for public comment (EPA-HQ-ORD-2013-
0430; available at www.regulations.gov) maintained through the development of the assessment.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table LS-1. Overview of the search strategy employed for RDX
Database
Keywords
Pubmed
Toxline
TSCATS1
Toxcenter
DTIC
WOS (forward and backward
search only)
Chemical CASRN: 121-82-4
Synonyms: Cyclonite OR RDX OR Cyclotrimethylenetrinitramine OR
"cyclotrimethylene trinitramine" OR "Hexahydro-l,3,5-trinitro-l,3,5-triazine"
OR "Hexahydro-l,3,5-trinitro-s-triazine" OR Hexogen OR "l,3,5-trinitro-l,3,5-
triazine" OR "l,3,5-Triaza-l,3,5-trinitrocyclohexane" OR "l,3,5-Trinitro-l,3,5-
triazacyclohexane" OR "l,3,5-Trinitrohexahydro-l,3,5-triazine" OR "1,3,5-
Trinitrohexahydro-s-triazine" OR "l,3,5-Trinitroperhydro-l,3,5-triazine" OR
"Esaidro-l,3,5-trinitro-l,3,5-triazina" OR "Hexahydro-l,3,5-trinitro-l,3,5-
triazin" OR "Perhydro-l,3,5-trinitro-l,3,5-triazine" OR
Cyclotrimethylenenitramine ORTrimethylenetrinitramine OR "Trimethylene
trinitramine" ORTrimethyleentrinitramine OR "Trinitrocyclotrimethylene
triamine" OR Trinitrotrimethylenetriamine OR "CX 84A" OR Cyklonit OR
Geksogen OR Heksogen OR Hexogeen OR Hexolite OR "KHP 281" OR "PBX (af)
108" OR "PBXW 108(E)" OR "Pbx(AF) 108"
Synonym and CASRN search for all databases; Toxcenter, Pubmed, and WOS
limited using toxicity-related keywords
Toxicity-related terms (see Appendix B for specific keywords)
Toxicity (including duration, effects to children and occupational exposure);
development; reproduction; teratogenicity; exposure routes;
pharmacokinetics; toxicokinetics; metabolism; body fluids; endocrinology;
carcinogenicity; genotoxicity; antagonists; inhibitors
ChemID
TSCATS 2 & 8e submissions
Searched by CASRN
1 The citations identified using the search strategy described above were screened using the
2 title, abstract, and in limited instances, full text for pertinence to examining the health effects of
3 RDX exposure. The process for screening the literature is described below and is shown graphically
4 in Figure LS-1.1
• 21 references were identified as potential sources of health effects data and were
considered for data extraction to evidence tables and exposure-response arrays.
• 65 references were identified as supporting studies; these included 16 studies describing
physiologically-based pharmacokinetic (PBPK) models and other toxicokinetic information,
25 studies providing genotoxicity and other mechanistic information, 7 acute toxicity
studies, and 17 human case reports. Studies investigating the effects of acute exposures and
1 Studies were assigned (or "tagged") to a given category in HERO that best reflected the primary content of
the study. Studies were not assigned multiple tags in order to simplify the tracking of references.
Nevertheless, the inclusion of a citation in a given category (or tag) did not preclude its use in one or more
other categories. For example, Woody etal. (1986). a case report of accidental ingestion of RDX by a child,
was tagged to the human case reports under Supporting Studies in Figure LS-1. This case report also
provides pharmacokinetic data and was a pertinent source of information on RDX toxicokinetics, but was not
assigned a second tag for toxicokinetics.
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case reports are generally less pertinent for characterizing health hazards associated with
chronic oral and inhalation exposure. Therefore, information from these studies was not
considered for extraction into evidence tables. Nevertheless, these studies were still
evaluated as possible sources of supporting health effects information.
• 277 references were identified as secondary sources of health effects information (e.g.,
reviews and other agency assessments) or as studies providing potentially useful contextual
information (e.g., studies providing information on exposure levels); these references were
kept as additional resources for development of the Toxicological Review.
• 745 references were identified as not being pertinent to an evaluation of the health effects
of RDX and were excluded from further consideration (see Figure LS-1 for exclusion
categories).
1 The documentation and results for the literature search and screen can be found on the
2 Health and Environmental Research Online (HERO) website
3 (http://hero.epa.gov/index.cfm/project/page/project_id/2216).
4
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Combined Dataset
n=1109
Additional Search Strategies
(see Table B-3 for methods and results)
Plus guidance documents/contextual references
added during assm development (n=95)
n=114
Manual Screening For Pertinence
{Title/Abstract/Full Text)
Supporting Studies
(n=65)
16 Toxicokinetics
10 Genotoxicity
15 Other mechanistic
studies
7 Acute/short-term
animal studies
17 Human case reports
Sources of Health Effects
Data(n=21)
3 Human health effects
studies
18 Animal toxicology studies
Secondary Sources of Health
Effects Information (n=278)*
10 Regulatory documents
13 Reviews
2 Editorials
11 Risk assessments
46 Ecosystem effects
98 Exposure levels
3 Mixtures only
95 Guidance/contextual references
added during assm development
includes other sources that provided context
for eva I u ati ng RDX hea 1th effects.
Excluded/Not Pertinent (n=745)
19 Abstract only
9 No abstract
4 Inadequate reporting in abstract
141 Not chemical specific
14 Use in sample prep or assay
154 Bioremediation or biodegradation
113 Chemical/physical properties or
explosive properties
63 Chemical or physical treatment
206 Measurement methods
2 Foreign language and not informative
18 Miscellaneous
2 Excluded afterfurther evaluation
Pubmed
n=531
Toxcenter
n=29
Database Searches
(see Tables B-l and B-2 for keywords and limits)
(After duplicates removed electronically)
n=995
Toxline (incl. TSCATS)
n=425
TSCATS 2
n=2
DTIC
n=8
Figure LS-1. Summary of literature search and screening process for RDX.
This document is a draft for review purposes only and does not constitute Agency policy,
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Selection of Critical Studies for Inclusion in Evidence Tables
Selection of Critical Studies
The 21 studies retained after the literature search and screen (Figure LS-1) were evaluated
for aspects of its design or conduct that could affect the interpretation of results and the overall
contribution to the synthesis of evidence for determination of hazard potential. Much of the key
information for conducting this evaluation can generally be found in the study's methods section
and in how the study results are reported. Importantly, the evaluation at this stage does not
consider study results, or more specifically, the direction or magnitude of any reported effects.
To facilitate this evaluation, evidence tables were constructed that systematically
summarize the important information from each study in a standardized tabular format as
recommended by the NRC f2011I The studies selected for inclusion in evidence tables are critical
for assessing the health effects of RDX. The evidence tables include all studies that inform the
overall synthesis of evidence for hazard potential; in general, the goal in developing evidence tables
is to be inclusive.
Studies were excluded from evidence tables if flaws in its design, conduct, or reporting are
so great that the results would not be considered credible (e.g., studies where concurrent or
essential historical control information is lacking). Such study design flaws are discussed in a
number of EPA's guidelines (see http: //www.epa.gov/iris/backgrd.htmll or summarized in the
Preamble. For RDX, four studies were considered uninformative and removed from further
consideration in the assessment because of fundamental issues with study design, conduct, or
reporting. The specific studies and basis for exclusion are summarized in Table LS-2.
Table LS-2. Studies determined not to be informative because of significant
issues with design, conduct, or reporting
Reference
Rationale for exclusion
Haskell Laboratories (1942);
14-wk study in dogs
Incomplete information on exposure levels; low numbers of
animals; breed of dog not reported; inadequate reporting of
results; sections of document illegible.
Von Oettingen et al. (1949);
10-wk oral study in rats
No control group; strain of rat was not reported.
ATSDR (1996);
Disease prevalence study in residential
population
Study of a population residing in two neighborhoods where RDX
had been detected in well water. The study was conducted 7 yrs
after residents were provided the opportunity to connect to a
municipal water supply. Only one target-area household reported
using private well water for bathing and cooking at the time of the
health study. The study was not considered informative because
the design was not able to adequately define the exposed
population.
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Reference
Rationale for exclusion
Unpublished report from the DTIC
database;
Human and animal data
One section of the report describes a human case series with no
referent group. Issues with the inhalation experimental animal
studies included lack of control groups, low numbers of animals
tested, incomplete information on exposure levels, and inadequate
reporting of results.
The health effects literature for RDX is not extensive. All human and experimental animal
studies of RDX involving repeated exposure that were not identified as uninformative because of
fundamental issues with study design, conduct, or reporting were considered in assessing the
evidence for health effects associated with chronic exposure to RDX. These studies are considered
the "critical" studies for which study methods and results are presented in evidence tables and
exposure-response arrays.
Other health effect studies of RDX, including human case reports and experimental animal
studies involving exposures of short-term duration or routes of exposure other than oral and
inhalation, were not included in evidence tables. Nevertheless, these studies were considered,
where relevant, in the evaluation of RDX health hazards.
Study Evaluation
In evaluating the evidence to determine whether RDX exposure may pose a hazard for each
of the health effects considered in this assessment, methodological aspects of a study's design,
conduct, and reporting were considered in the overall evaluation and synthesis of the pertinent
data. In general the relevance and informativeness of the available studies were evaluated as
outlined in the Preamble and in EPA guidance (e.g., A Review of the Reference Dose and Reference
Concentration Processes fU.S. EPA. 20021 and Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhaled Dosimetry fU.S. EPA. 199411. In addition, in 2012, EPA
obtained external peer reviews of two 2-year bioassays (Lish etal.. 1984: Levine etal.. 19831 and
one 90-day study (Crouse etal.. 20061 that were available only as laboratory reports. The report of
the peer reviews is available at www.epa.gov/hero (search for HERO ID 2519581).
The general findings of this evaluation are presented in the remainder of this section. Study
evaluation considerations that are outcome specific are discussed in the relevant health effect
sections in Section 1.1.
Human Studies
The body of literature on RDX includes three studies of populations occupationally exposed
to RDX (one case-control and two cross-sectional studies) (West and Stafford. 1997: Ma and Li.
1992: Hathaway and Buck. 19771.
To varying degrees, these epidemiology studies of RDX are limited by their study design,
uncertainty in estimates of exposure, inadequate reporting, and/or failure to account for potential
confounding exposures. All three studies were based on a relatively small number of participants
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(60-69 exposed workers in the cross-sectional studies and 32 cases in the case-control study). The
study by Ma and Li (19921 of Chinese industrial workers provided limited information on
participant recruitment, selection, and participation rate; information was not adequate to evaluate
the potential for selection bias.
Of the three epidemiological studies, more detailed exposure information was collected by
Hathaway and Buck f 19771. Atmospheric and paired breathing zone sampling was performed;
however, the paper included limited reporting of RDX concentrations in workplace air. Ma and Li
(19921 reported mean RDX exposure concentrations (with standard deviations) for two exposure
groups, but provided no information on the source of these concentrations or how monitoring was
performed. In the case-control study by West and Stafford f!9971. semi-quantitative exposure
estimates (low, moderate, or high) were based on interviews with employees.
Ma and Li T19921 did not adjust for any potential risk factors, e.g., alcohol consumption. In
the study by Hathaway and Buck (19771 that included evaluations of liver, renal, and hematology
endpoints, workers with trinitrotoluene (TNT) exposure were appropriately excluded from the
exposed groups, since TNT is another explosive that is associated with liver and hematological
system toxicity. The case-control study by West and Stafford (19971. which examined hematology
outcomes, did not perform statistical analyses to adjust for other risk factors or occupational
exposures (including TNT). Further, the impact of age or gender could not be assessed as the cases
and controls were not matched.
The methodological limitations in these three studies were considered in the synthesis of
evidence for each of the health effects and in reaching determinations of hazard (see Section 1.1).
In addition to these three occupational epidemiology studies, the human health effects
literature includes 16 case reports that describe effects following acute exposure to RDX. Case
reports are often anecdotal and typically describe unusual or extreme exposure situations,
providing little information that would be useful for characterizing chronic health effects.
Therefore, RDX case reports were only briefly reviewed; a critical evaluation of these studies was
not undertaken. A summary of these case reports is provided in Appendix C, Section C.3.
Experimental Animal Studies
The oral toxicity database for RDX includes three chronic studies in rats and mice, eight
subchronic studies in rats, mice, dogs, and monkeys, two short-term studies, and four
reproductive/developmental toxicity studies in rats and rabbits (including a two-generation
reproductive study). Only one inhalation study of RDX was identified. As discussed in Appendix B
and Table LS-2, this inhalation study was considered uninformative and excluded from
consideration in the development of the Toxicological Review because of study design issues
(including lack of a control group, incomplete information on exposure levels, and inadequate
reporting). Therefore, evaluation of the experimental animal database for RDX is limited to studies
of oral toxicity. An evaluation of the oral toxicity literature, organized by general methodological
features, is provided in the remainder of this section.
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Test animal
The RDX database consists of health effect studies conducted in multiple strains of rats
(F344, Sprague-Dawley, CD), mice (B6C3Fi), dog (beagle), and monkey. The species and strains of
animals used are consistent with those typically used in laboratory studies. All of these species or
strains were considered relevant to assessing the potential human health effects of RDX. Several
studies in the RDX database provided inadequate information on test animals. The strain of
monkey (rhesus or cynomolgus) used in the study by Martin and Hart f 1974-1 was not clearly
specified. In one study, the breed of dog and strain of rat were unreported (Von Oettingen et al..
19491. The species, strain, and sex of the animals used is recorded in the evidence tables.
Other studies of RDX were identified that used nonstandard species, including deer mice
[Peromyscus maniculatus), western fence lizards (Sceloporus occidentalis), prairie voles (Microtus
ochrogaster), and northern bobwhite quail (Colinus virginianus). These studies provide information
relevant to RDX toxicokinetics and mechanism of action on the nervous system, but not health
effects data. Therefore, these studies are not included in evidence tables, but are discussed where
relevant in the assessment
Experimental setup
General aspects of study design and experimental setup were evaluated for all studies that
included health effect data to determine if they were appropriate for evaluation of specific
endpoints. Key features of the experimental setup, including the periodicity and duration of
exposure, timing of exposure (e.g., gestational days for developmental studies), experimental group
sample sizes, and interim sacrifices are summarized in the evidence tables. Note that sample size
was not a basis for excluding a study from consideration. For example, the informativeness of Hart
f!9741 and Martin and Hart f 19741 was reduced in light of the small sample sizes in each study
(3/sex/group), but the studies would still inform the consistency of effects observed for a specific
endpoint across species (dog and monkey). Elements of the experimental setup that could
influence interpretation of study findings are discussed in the relevant hazard identification
sections of the assessment.
Exposure
Properties of the test material were also considered in determining whether the exposures
were sufficiently specific to the compound of interest Two properties of the RDX test materials
that varied across experimental animal studies and that were taken into consideration in evaluating
RDX hazard are the particle size and purity of the test material. The purity of RDX used in health
effects studies varied from 84-99.99%. The major contaminants were octahydro-1,3,5,7-tetranitro-
1,3,5,7-tetrazocine (HMX) and water, which are the primary contaminants of RDX produced
through the Bachmann process. The majority of studies used RDX with ~10% impurities; only
Crouse etal. f20061 used 99.99% pure RDX as a test material in their study. The toxicity of HMX
was reviewed by the IRIS Program in 1988 (www.epa.gov/iris); histopathological changes in the
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liver in male F344 rats and in the kidney in female rats were reported in a 13-week feeding study.
No chronic studies were available to evaluate the carcinogenicity of HMX. The presence of the
impurities introduces some uncertainty in attribution of toxicity to RDX. However, consistency in
the doses at which some toxic effects were seen across studies suggests that the uncertainty
associated with the use of less pure test materials may be relatively small. Evidence of neurotoxic
effects in the study with 99.99% pure RDX occurred at doses of 8-15 mg/kg-day; studies with less
pure RDX reported similar symptoms at doses of >20 mg/kg-day. It should be noted that the test
materials employed in these studies are considered representative of RDX that would be released
into the environment.
Differences in milling procedures used to generate the test material resulted in the use of
RDX of varying particle sizes across studies. Some studies utilized a relatively fine particle size
(majority of particles were <66 [im in size) while others used test material with comparatively
coarse particle size (~200 [im particle size). Differences in particle size across studies could result
in different rates of absorption of RDX into the blood stream, which could account for differences in
some of the toxicities observed across studies, including neurotoxicity. Information on test
material purity and particle size as provided by study authors is reported in the evidence tables,
and was considered in evaluating the toxicity of RDX. Lack of characterization of the test material
in the studies by Hart (1974). Hart (1976). and Martin and Hart (1974) was considered a deficiency.
Endpoint evaluation procedures
Some methodological considerations used to evaluate studies of RDX toxicity are outcome
specific—in particular effects on the nervous system and development. Outcome-specific
methodological considerations are discussed in the relevant health effect sections in Section 1.1.
For example, many of the studies that noted neurotoxicity in the form of seizures or convulsions
were not designed to assess that specific endpoint and reported number animals with seizures
anecdotally. While these studies can provide qualitative evidence of neurotoxicity, they may have
underestimated the true incidence of seizures or convulsions because they were not designed to
systematically evaluate neurotoxic outcomes.
Outcomes and data reporting
In evaluating studies, consideration was given to whether data were reported for all pre-
specified endpoints and study groups, and whether any data were excluded from presentation or
analysis. For example, it was noted where histopathological analysis was limited to control and
high-dose groups, a study reporting feature that limited the ability to identify dose-related trends.
In limited cases, EPA performed additional statistical analysis to identify trends or refine analyses
consistent with EPA guidance (e.g., analyzing developmental data sets on a per litter basis rather
than individual fetus). Data from studies have been extracted and presented in evidence tables.
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Notable features of the RDX database
Three two-year toxicity bioassays of RDX are available as unpublished laboratory studies.
The bioassays by Levine etal. T19831 in the rat and by Lish etal. f19841 in the mouse were
conducted in accordance with Food and Drug Administration (FDA) Good Laboratory Practices
(GLPs) in place at the time of the studies. Both studies included interim sacrifices (at 6 and
12 months). Complete histopathological examinations were performed on all animals in the control
and high-dose groups; however, only a subset of tissues was examined in the mid-dose groups,
limiting the ability to identify dose-related trends for tissues with incomplete histopathology. In
the mouse bioassay by Lish etal. f!9841. the initial high dose (175 mg/kg-day) was reduced to
100 mg/kg-day atweek 11 because of high mortality, thereby reducing the number of high-dose
animals on study for the full 2 years of dosing (see Table LS-3). Because they were available only as
laboratory reports, peer reviews of the Levine etal. T19831 and Lish etal. T19841 studies were
conducted by EPA in 2012. The peer reviewers generally concluded that the reports provided
useful information on the toxicity of RDX, noting that there were limitations in interpretation due to
the histopathological analysis and the statistical approaches employed in the reports. An earlier
two-year study in the rat by Hart T19761 used a dose range that was lower than the subsequent
studies (high dose of 10 mg/kg-day), and that may not have been sufficient to elicit some effects in
treated animals. Histopathology findings were limited by the lack of pathology examinations in the
mid-dose groups and the lack of individual time of death, which impacts the ability to interpret the
histopathology data. In addition, a heating system malfunction on days 75-76 of the study resulted
in the death of 59 rats from the control and treatment groups, thereby reducing the number of
animals on study (see Table LS-3).
Short-term and subchronic toxicity studies of RDX were published or reported between the
years 1949 and 2006, and differences in robustness of study design, conduct, and reporting reflects
that range. All but one of the eight short-term and subchronic toxicity studies of RDX are available
as unpublished laboratory studies; only Von Oettingen et al. (1949) was published. The majority of
studies conducted histopathological examinations on only some of the experimental groups (e.g.,
control and high dose). One subchronic study Crouse etal. (2006) was peer-reviewed by EPA in
2012. The peer reviewers determined that the report provided useful information on the toxicity of
RDX, including an array of endpoints for neurotoxicity and immunotoxicity. Limitations in the
study were based on an incomplete understanding of the neurotoxicity that may have been
resolved with more histological evaluation as well additional behavioral assessment
Some of the more important limitations in study design, conduct, and reporting of
experimental animal toxicity studies of RDX are summarized in Table LS-3. Limitations of these
studies were taken into consideration in evaluating and synthesizing the evidence for each of the
health effects in Section 1.1.
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Table LS-3. Experimental animal studies considered less informative because
of certain study design, conduct, or reporting limitations
References
Study design, conduct, and reporting limitations
Lish et al. (1984); Levine et al. (1984)
2-yr mouse study
The initial high dose (175 mg/kg-d) was reduced to 100 mg/kg-d at wk 11
due to high mortality. Mortality of surviving mice was similar to controls
after dose reduction.
Hart (1976)
2-yr rat study
A heating system malfunction on d 75-76 of the study resulted in the
deaths of 59 rats from the control and treatment groups. Dead animals
were subsequently eliminated from the analysis. There were still more
than 80 rats/sex/group after the overheating incident, and >50
rats/sex/group at termination.
Histopathology findings were limited by the lack of pathology
examinations in the mid-dose groups and the lack of individual time of
death, which impacts the ability to interpret the histopathology data.
Cholakis et al. (1980)
13-wk mouse study (Experiment 1)
Dose range was too low to produce effects in mice. Histopathological
examinations were not performed.
Cholakis et al. (1980)
13-wk mouse study (Experiment 2)
Nonstandard dosing regimen followed: 0, 40, 60, 80 mg/kg-d for 2 wks.
For the next 11 wks, the dosing was inverted, so that the 40 mg/kg-d
group received 320 mg/kg-d, the 60 mg/kg-d group received 160 mg/kg-d,
and the 80 mg/kg-d group continued to receive the same dose. The
rationale for this dosing regimen was not provided in the study report.
Von Oettingen et al. (1949)
12-wk rat study
The strain of rat was not reported. Only gross observations made at
autopsy.
Von Oettingen et al. (1949)
6-wk dog study
The breed of dog was not reported. Only gross observations made at
autopsy.
Martin and Hart (1974)
90-d monkey study
Species of monkey is unclear (either Cynomolgus or Rhesus). Some test
subjects may have had variable dosing due to emesis. Small sample size
per dose group (n=3).
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1.HAZARD IDENTIFICATION
1.1. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM
1.1.1. Nervous System Effects
Nervous system effects following RDX exposure have been observed in multiple case
reports, and the association between RDX exposure and neurobehavioral effects has been examined
in a single occupational epidemiology study. Information relevant to an examination of the
association between RDX exposure and nervous system effects also comes from experimental
animal studies involving chronic, subchronic, and gestational exposure to ingested RDX. A
summary of nervous system effects associated with RDX exposure is presented in Tables 1-1 and
1-2 and Figure 1-1.
In a cross-sectional study by Ma and Li f!9921. neurobehavioral effects were evaluated in
Chinese workers occupationally exposed to RDX. Memoiy retention and block design scores2 were
significantly lower among exposed workers (mean concentrations of RDX in two exposed groups:
0.407 and 0.672 mg/m3) compared to unexposed workers from the same plant However, no
significant differences were observed between the groups on other neurobehavioral tests (e.g.,
simple and choice reaction times, block design, and letter cancellation test) (Table 1-1). This study
did not consider potential confounders such as alcohol consumption or co-exposure to TNT.
Case reports support an association between RDX exposure and neurological effects (see
Appendix C, Section C.3). Severe neurological disturbances include tonic-clonic seizures (formerly
known as grand mal seizures) in factory workers (Testud etal.. 1996b: Testud etal.. 1996a: Kaplan
etal.. 1965: Barsotti and Crotti. 1949). seizures and convulsions in exposed soldiers serving in
Vietnam (Ketel and Hughes. 1972: Knepshield and Stone. 1972: Hollander and Colbach. 1969: Stone
etal.. 1969: Merrill. 1968). seizures, dizziness, headache and nausea followingnon-wartime/non-
occupational exposures (Kasuske etal.. 2009: Davies etal.. 2007: Kuctikardali etal.. 2003: Hettand
Fichtner. 2002: Harrell-Bruder and Hutchins. 1995: Goldberg etal.. 19921. and seizures in a child
following ingestion of plasticized RDX from the mother's clothing fWoodvetal.. 19861.
Nervous system effects in experimental animals, including seizures and convulsions (used
interchangeably by study authors), tremors, behavioral changes, irritability, and hyperactivity, have
been observed in the majority of chronic, subchronic, and developmental studies following oral
exposure to RDX (see Table 1-2 and Figure 1-1). In a 2-year dietary study in F344 rats,
2The memory quotient index measured short-term hearing memory, visual memory, combined hearing and
visual memory, and learning ability. The block design index measured visual perception and design
replication, and the ability to analyze spatial relationships.
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administration of 40 mg/kg-day RDX resulted in convulsions (Levine etal.. 19831: convulsions were
not observed at lower doses in the same study (<8 mg/kg-day) (Levine etal.. 19831 or in Sprague-
Dawley rats following chronic dietary administration of 10 mg/kg-day, the highest dose tested
(Hart. 19761. Convulsions were observed in B6C3Fi mice exposed to RDX for 2 years at doses
similar to or higher than those inducing convulsions in the rat f Levine etal.. 1984: Lish etal.. 19841.
Subchronic dietary exposure was also associated with convulsions in the rat, although doses
reported to increase convulsive activity were inconsistent across studies. Convulsions were
reported in RDX-exposed rats at subchronic doses as low as 8 and 25 mg/kg-day (Crouse etal..
2006: Von Oettingen et al.. 19491. In contrast, Levine etal. (19901 reported convulsions in rats
following subchronic exposure only at a dose of 600 mg/kg-day; however, the unpublished
technical report of this study (Levine etal.. 198 lal inconsistently reported convulsions at 600
mg/kg-day and >30 mg/kg-day, thereby reducing confidence in the identification of the dose level
at which nervous system effects are observed in this study. No evidence of seizures, convulsions or
tremors was reported in three subchronic rat studies that used relatively lower doses of RDX
(highest administered doses: 10-50 mg/kg-day) (MacPhail etal.. 1985: Cholakis etal.. 19801. RDX
exposure (by gavage) during gestation in the rat was associated with induction of seizures or
convulsions in the dams at doses ranging from 2 to 40 mg/kg-day fAngerhofer et al.. 1986: Cholakis
etal.. 19801—demonstrating that effects on the nervous system can be observed following
exposure durations as short as 10-14 days. Convulsions were also reported in dogs exposed to 50
mg/kg-day RDX fVon Oettingen et al.. 19491. but not 10 mg/kg-day fHart. 19741. and in two of
three monkeys of both sexes following a gavage dose of 10 mg/kg-day (Martin and Hart. 19741.
In the only study addressing susceptibility to seizures, Burdette etal. (19881 found that
seizure occurrence was greater in Long Evans rats exposed to a single dose of 50 or 60 mg/kg RDX
by gavage when challenged with an audiogenic stimulus 8 and 16 hours after treatment. However,
no audiogenic seizures were observed at the earlier 2- and 4-hour post-dosing test periods even
though RDX plasma concentrations were elevated throughout the testing period. In a
complementary experiment, Long Evans rats treated daily with 6 mg/kg-day RDX for up to 18 days
required fewer stimulation trials to exhibit amygdaloid kindled seizures compared to controls.
Neither the purity nor the specific particle size of the RDX used in these experiments were reported.
The majority of animal studies reported convulsions and/or seizures as clinical
observations; interpretation of these observations is limited to some extent because the nature and
severity of convulsions and seizures were not more fully characterized. The 90-day study by
Crouse etal. f20061 was one of the few studies that collected and reported incidence data for
convulsions and tremors, and demonstrated a clear dose-related increase in convulsions and
tremors in male and female F344 rats associated with RDX exposure via gavage (see Table 1-2).
Tremors were reported following administration of >12 mg/kg-day, persisting throughout the
90-day study. Convulsions were observed at >8 mg/kg-day in male and female rats; information on
duration and onset was not reported (Crouse etal.. 20061. In general, gavage dosing (Crouse etal..
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2006: Cholakis etal.. 19801 induced convulsions at lower doses than did dietary administration,
possibly due to the bolus dosing resulting from gavage administration and the comparatively faster
peak absorption of RDX.
Several experimental animal studies documented that unscheduled deaths were frequently
preceded by convulsions or seizures. Crouse etal. f20061 stated that nearly all observed pre-term
deaths in rats exposed to RDX for 90 days were preceded by neurotoxic signs such as tremors and
convulsions. In a 2-year study in rats, Levine et al. (1983) observed that tremors and/or
convulsions were often seen in high-dose animals prior to their death. Further, in a rat
developmental study (Cholakis etal.. 19801. investigators concluded that early deaths in dams were
preceded by convulsions based on the observation of convulsions in one rat prior to death, and a
similar appearance (e.g., dried blood round the mouth and nose) in other dams that died during the
study. A few studies reported mortality that was not specifically or directly associated with
neurological effects (Angerhofer etal.. 1986: Levine etal.. 1981a: Von Oettingen et al.. 19491:
however, in these studies, animals may not have been monitored for clinical observations with
sufficient frequency to have observed convulsive activity prior to death.
Additional neurobehavioral effects associated with RDX exposure in rats included increased
hyperactivity, hyper-reactivity, fighting, and irritability at doses similar to those that induced
tremors, convulsions, and seizures (10-100 mg/kg-day) (Levine etal.. 1990: Angerhofer etal..
1986: Levine etal.. 1983: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Von
Oettingen etal.. 19491. Hyperactivity and nervousness were also reported in male mice that
received a subchronic exposure to 320 mg/kg-day RDX (Cholakis etal.. 19801. No changes in motor
activity, flavor aversion, scheduled-controlled behavior, or acoustic startle response were observed
in a 30-day gavage study in rats, but doses were relatively low (<10 mg/kg-day) (MacPhail etal..
19851. and no significant changes in behavioral or neuromuscular activity were observed in rats
following exposure to <15 mg/kg-day for 90 days (Crouse etal.. 20061. Crouse etal. (20061
concluded that stained haircoats and increased barbering in female F344 rats receiving 15 mg/kg-
day may have been caused by the oral dosing procedure (gavage) alone.
Observations of changes in absolute and relative brain weight were mixed across studies.
Among chronic oral studies, a decrease in absolute brain weight of female B6C3Fi mice (3-4%
relative to control) was reported at doses >35 mg/kg-day (Levine etal.. 1984: Lish etal.. 19841.
Conversely, an increase in absolute brain weight of 2% relative to control was observed in F344
rats at 40 mg/kg-day in another two-year oral bioassay (Levine etal.. 1983: Thompson. 19831.
Similarly elevated absolute brain weights were reported in subchronic assays in B6C3Fi mice and
F344 rats (Crouse etal.. 2006: Levine etal.. 1990: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis
etal.. 19801: however, the changes were not consistently observed across studies. Relative brain
weights in some studies showed correspondingly greater increases compared to absolute brain
weight fCrouse etal.. 2006: Levine etal.. 1983: Thompson. 1983: Cholakis etal.. 19801. but these
changes were likely a result of changes in body weight in the study, and were not a useful measure
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of effects of RDX on brain weights. Based on an evaluation of the relationship between organ
weight and body/brain weight to determine which endpoint (organ weight, organ-to-body weight
ratio, or organ-to-brain weight ratio) is likely to more accurately detect target organ toxicity, Bailey
etal. (20041 concluded that brain weights are not modeled well by any of the choices, and that
alternative analysis methods should be utilized.
Across the studies summarized in Table 1-2, nervous system responses to RDX did not show
a predicted relationship with duration of exposure. For example, seizures or convulsions were
observed in F344 rats in some subchronic studies at doses lower than in studies of chronic
duration, and at even lower doses in dams exposed for approximately 2 weeks during gestation. In
some studies, seizures appeared soon after dosing, suggesting that seizure induction was more
strongly correlated with dose level rather than with duration of exposure. Williams etal. (20111
demonstrated that RDX is rapidly absorbed and crosses the blood-brain barrier following oral
administration in rats, and that distribution of low levels of RDX (8 |ig/g ww) to the brain
correlated with seizure onset
Similarly, nervous system effects across studies did not show a consistent relationship with
dose. This lack of consistency may, at least in part, be attributed to differences in the purity or
particle size of the test material across studies. Assuming that increased particle size results in
slowed absorption and distribution to the brain, studies that used a larger particle size may be
expected to produce less neurotoxicity in test animals. The mouse study by Cholakis etal. T19801
used a relatively large RDX particle size (200 [im) compared to the rat study by Levine etal. (19831
that used a smaller (<66 [im) particle size. This could contribute to why the Cholakis etal. (19801
subchronic dietary study in the mouse (doses up to 320 mg/kg-day RDX) and rat (doses up to
40 mg/kg-day) failed to report seizures or convulsions. Finally, differences in study design may
have contributed to differences in reported neurological responses in subchronic and chronic
duration studies; in particular, the number of daily observations for clinical signs may not have
been sufficiently frequent to provide an accurate measure of the incidence of seizures or other
nervous system effects.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table 1-1. Evidence pertaining to nervous system effects in humans
Reference and study design
Results
Ma and Li (1992) (China)
Cross-sectional study, 60 workers
Neurobehavioral function tests, scaled scores (mean, standard
deviation)
exposed to RDX (30 in Group A [26
males; 4 females]; 30 in Group B [24
males; 6 females]), compared to
32 workers with similar age, education
level, and length of employment from
same plant with no exposure to RDX (27
males; 5 females).
Exposure measures: Details of
exposure measurement were not
provided; exposed workers were
divided into two groups based on RDX
concentration in the air:
Concentration (mg/m3)
Test
Control
Group A
Group B
Memory retention*
Simple reaction time
(milliseconds)
Choice reaction time
(milliseconds)
Block design*
(elapsed time)
Letter cancellation
(quality per unit time)
111.3 (9.3)
493 (199)
763 (180)
18.0 (5.4)
1,487 (343)
96.9 (9.6)
539 (183)
775 (161)
16.0 (4.3)
1,449 (331)
91.1 (10.3)
578 (280)
770 (193)
13.5(6.7)
1,484 (443)
Group A 0.407 (± 0.332)
Group B 0.672 (± 0.556)
Effect measures3: Five neurobehavioral
function tests and five additional
memory subtests.
*p < 0.01 (overall F-test); no statistically significant differences between
Group A and Group B.
Lower score indicates worse performance.
Memory retention subtests, scaled scores (mean, standard deviation)
Analysis: Variance (F-test); unadjusted
linear regression, multiple regression,
and correlation analysis.
Subtest
Control
Group A
Group B
Directional memory*
23.5 (3.6)
17.2 (4.9)
18.1 (5.7)
Associative learning*
24.9 (5.1)
20.0 (4.3)
18.5 (4.6)
Image free recall*
24.1 (3.8)
20.9 (4.1)
20.4 (3.3)
Recognition of
nonsense pictures*
26.3 (3.6)
23.2 (4.9)
21.6 (4.3)
Associative recall of
portrait characteristics*
26.3 (3.3)
20.3 (4.4)
18.5 (4.3)
*p < 0.01 (overall F-test); no statistically significant differences between
Group A and Group B.
Lower score indicates worse performance.
Total behavioral score negatively correlated with exposure index (high
exposure correlated with poor performance).
1
2 aSymptom data were not included in evidence table because of incomplete reporting.
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Table 1-2. Evidence pertaining to nervous system effects in animals
Reference and study design
Results
Convulsions and neurobehavioral effects
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11 due
to excessive mortality)
Diet
24 mo
One male mouse in the 35 mg/kg-d dose group and one female
mouse in the 175/100 mg/kg-d group convulsed near the end of the
study.
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
No neurological effects, as evidenced by clinical signs or changes in
appearance or behavior, were reported.
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Tremors, convulsions, and hyper-responsiveness to stimuli were
noted at 40 mg/kg-d; no incidence data were reported.
Cholakis et al. (1980)
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants
~200 nm particle size
0, 40, 60, or 80 mg/kg-d for 2 wks
followed by 0, 320,160, or 80 mg/kg-d
(TWA doses of 0, 79.6, 147.8, or 256.7
mg/kg-d for males and 0, 82.4,136.3, or
276.4 mg/kg-d for females)b
Diet
13 wks
Hyperactivity and/or nervousness observed in 50% of the high-dose
males; no signs observed in females3; no incidence data were
reported.
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Reference and study design
Results
Cholakis et al. (1980)
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 urn particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
No neurological effects, as evidenced by clinical signs or changes in
appearance or behavior, were reported.
Cholakis et al. (1980)
Rats, CD, two-generation study;
FO: 22/sex/group; Fl: 26/sex/group;
F2: 10/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 urn particle size
FO and Fl parental animals: 0, 5,16, or
50 mg/kg-d
Diet
13 wks
No neurological effects were reported.
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Doses
0 4 8a 10 12 15
Convulsions (incidence)
M
F
0/10 0/10 1/10 3/10 8/10 7/10
0/10 0/10 2/10 3/10 5/10 5/10
Tremors (incidence)
M
F
0/10 0/10 0/10 0/10 2/10 3/10
0/10 0/10 0/10 0/10 0/10 1/10
Levine et al. (1981a); Levine et al. (1990);
Levine et al. (1981b)d
Rats, F344,10/sex/group; 30/sex for
control
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 nm, ~90% of particles
< 66 nm
0,10, 30,100, 300, or 600 mg/kg-d
Diet
13 wks
Hyper-reactivity to approach was observed in groups receiving
>100 mg/kg-d; no incidence data were reported.
Tremors and convulsions were observed prior to death in some
animals receiving 600 mg/kg-d; no incidence data were reported.0
Von Oettingen et al. (1949)
Rats, sex/strain not specified, 20/group
90-97% pure, with 3-10% HMX; particle
size not specified
0,15, 25, or 50 mg/kg-d
Diet
3 mo
Hyperirritability and convulsions were observed in the 25 and
50 mg/kg-d groups3; no incidence data were reported.
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Reference and study design
Results
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow containing
20 mg RDX/g-chow, 60 g dog food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
No neurological effects, as evidenced by clinical signs or changes in
appearance or behavior, were reported.
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Doses
(O
o
1
1
1
o
o
CNS effects characterized as trembling, shaking, jerking, or
convulsions (incidence)
M
F
0/3 0/3 0/3 2/3
0/3 0/3 0/3 2/3
Von Oettingen et al. (1949)
Dogs, breed not specified,
5 females/group (control);
7 females/group (exposed)
90-97% pure, with 3-10% HMX; particle
size not specified
0 or 50 mg/kg-d
Diet
6 d/wk for 6 wks
Treated dogs exhibited convulsions, excitability, ataxia, and
hyperactive reflexes3; no incidence data were reported.
MacPhail etal. (1985)
Rats, Sprague-Dawley derived CD,
8-10 males or females/group
Purity 84 ± 4.7%; <66 nm particle size
0,1, 3, or 10 mg/kg-d
Gavage
30 d
No changes in motor activity, flavor aversion, scheduled-controlled
response, or acoustic startle-response were reported.
Cholakis et al. (1980)
Rats, F344, 24-25 females/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants
0, 0.2, 2.0, or 20 mg/kg-d
Gavage
GDs 6-19
Doses
0 0.2 2.0 20
Convulsions
F
0/24 0/24 1/24 18/25
Angerhofer et al. (1986) (range-finding
study)
Rats, Sprague-Dawley, 6 pregnant
females/group
Purity 90%; 10% HMX and 0.3% acetic acid
occurred as contaminants
0,10, 20, 40, 80, or 120 mg/kg-d
Gavage
GDs 6-15
Convulsions preceding death were observed at >40 mg/kg-d;
no incidence data were reported.
This document is a draft for review purposes only and does not constitute Agency policy.
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Reference and study design
Results
Angerhofer et al. (1986)
Convulsions and hyperactivity3 were observed at 20 mg/kg-day;
Rats, Sprague-Dawley, 39-51 mated
no incidence data were reported.
females/group
Purity 90%; 10% HMX and 0.3% acetic acid
occurred as contaminants
0, 2, 6, or 20 mg/kg-d
Gavage
GDs 6-15
Brain weight
Lish et al. (1984); Levine et al. (1984)
Doses
0
1.5
7
35
175/100
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
Absolute brain weight
M
0%
-0.2%
0.61%
0.81%
-1%
contaminant; 83-89% of particles <66 nm
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11 due
F
0%
-2%
-2%
-4%*
-3%*
Relative brain weight
to excessive mortality)
Diet
M
0%
4%
2%
2%
5%
24 mo
F
0%
-4%
-1%
-3%
18%*
Levine et al. (1983); Thompson (1983)
Doses
0
0.3
1.5
8
40
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
Absolute brain weight
M
0%
2%
-1%
2%
2%
contaminant; 83-89% of particles <66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
F
0%
-0.3%
-0.4%
1%
2%*
Diet
Relative brain weight
24 mo
M
0%
0%
8%
2%
22%*
F
0%
-1%
3%
4%
20%*
Cholakis et al. (1980)
Doses
0
10
14 20
28
40
Mice, B6C3F1,10-12/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
Absolute brain weight
M
0%
-
-
2%
2%
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
F
0%
-
-
4%
2%
Relative brain weight
13 wks
M
0%
-
-
6%
2%
F
0%
-
-
0%
3%
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Reference and study design
Results
Experiment 2: 0, 40, 60, or 80 mg/kg-d for
Doses
0
80
160
320
2 wks followed by 0, 320,160, or
80 mg/kg-d (TWA doses of 0, 79.6,147.8,
or 256.7 mg/kg-d for males and 0, 82.4,
Absolute brain weight
M
0%
0%
2%
10%
136.3, or 276.4 mg/kg-d for females)b
Diet
F
0%
0%
4%
2%
13 wks
Relative brain weight
M
0%
-3%
1%
8%
F
0%
0%
3%
-4%
Cholakis et al. (1980)
Doses
0
10
14
20
28
40
Rats, F344,10/sex/group
Absolute brain weight
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
M
0%
-
-
-
3%
0%
0,10,14, 20, 28, or 40 mg/kg-d
Diet
F
0%
-
-
-
0%
0%
13 wks
Relative brain weight
M
0%
-
-
-
7%*
10%*
F
0%
-
-
-
5%
6%
Crouse et al. (2006)
Doses
0
4
8
10
12
15
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Absolute brain weight
M
0%
-1%
-0.3%
2%
5%*
7%*
Gavage
90 d
F
0%
-2%
6%
1%
4%
6%
Relative brain weight
M
0%
6%
10%
5%
3%
4%
F
0%
-2%
-2%
-12%*
-12%*
-15%*
Levine et al. (1981a); Levine et al. (1990);
Doses
0
10
30
100
300
600
Levine et al. (1981b)d
Rats, F344,10/sex/group; 30/sex for
control
Absolute brain weight
M
0%
1%
0.53%
-6%
-
-
84.7 ± 4.7% purity, ~10% HMX, median
0%
-1%
1%
2%
particle diameter 20 nm, ~90% of particles
<66 nm
r
Relative brain weight
0,10, 30,100, 300, or 600 mg/kg-d
Diet
M
0%
4%
7%
14%
-
-
13 wks
F
0%
0.3%
2%
5%
-
-
1
2 aMortality was reported in some RDX-treated groups in this study.
3 bDoses were calculated by the study authors.
4 discrepancies in the doses at which convulsions occurred were identified in the technical report. The nervous
5 system effects reported in this table and in the corresponding exposure-response array are those provided in the
6 results section of the technical report (Levine et al., 1981a) and in the published paper (Levine et al., 1990). In
7 other sections of the technical report, the authors reported that hyperactivity to approach and convulsions were
8 observed in rats receiving >30 mg/kg-day (abstract and executive summary), or that mortality was observed in
This document is a draft for review purposes only and does not constitute Agency policy.
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1 rats receiving 100 mg/kg-d and that hyperactivity to approach, tremors, and convulsions were observed in
2 animals exposed to lethal doses (discussion).
3 dLevine et al. (1981a) is a laboratory report of a 13-week study of RDX in F344 rats; two subsequently published
4 papers (Levine et al., 1990; Levine et al., 1981b) present subsets of the data provided in the full laboratory report.
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1000
100
10
W>
£ 1
o
Q
• significantly changed
O not significantly changed
© M
-M-
M
0 M • M
+ M
• ° • °
O M
M
M
HVh
M
M •
OIV
C) o
o o
M
o iyi
M O M
o o
O O O M o M
-o i>
0.1
a;
Chronic
03
Subchronic
Convulsions and/or Seizures
-ir a;
Gestational (dams)
— ro
Absolute Brain Weight
M- Mortality observed at this dose and above
Figure 1-1. Exposure response array of nervous system effects following oral exposure.3
3Due to the severity of the endpoint for convulsions and/or seizures, a response in treated groups was determined to be significant (filled circles) in the
exposure-response array where there was an observation of convulsions and/or seizures reported in the study.
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Mechanistic Evidence
The few studies that have explored the MOA of RDX on the central nervous system have
focused on potential impacts on neurotransmission. These studies suggest that the MOA for RDX-
induced seizures and convulsions involves distribution to the brain (across the blood-brain barrier)
and subsequent effects on neurotransmitters, including gamma amino butyric acid (GABA) and
glutamate. The strongest mechanistic information for RDX neurotoxicity comes from documented
interactions with the GABAa receptor. GABA is a major inhibitory neurotransmitter in the brain,
and the GABAa receptor has been implicated in susceptibility to seizures (Galanopoulou. 20081. It
is also a target of many anticonvulsant therapies (e.g., benzodiazepines, propofol, barbiturates)
fMeldrum and Rogawski. 2007: Mohler. 20061. The affinity of RDX for the GABAa receptor provides
biological plausibility for the association of seizures with exposure to RDX in both human case
reports and experimental animal studies.
In research conducted by the U.S. Army Center for Health Promotion and Preventative
Medicine, Williams etal. (20111 and Bannonetal. (20091 showed a correlation between blood and
brain concentrations of RDX in rats that received a single oral dose of RDX (>98-99.5% purity) by
gavage, which closely correlated with the time of seizure onset. RDX (75 mg/kg) was distributed to
the brain in direct proportion to levels found in the blood, while time to seizure onset was reduced
as RDX brain levels increased fWilliams etal.. 20111. Similarly, oral exposure to RDX (via a gel
capsule: 3 or 18 mg/kg) resulted in quick absorption followed by transport to the brain and
subsequent alterations in neurotransmission (Bannon et al.. 20091.
Some other pro-convulsant agents with minimal direct toxicity to nerve cells, such as sarin
and some organophosphate pesticides, are known to act through inhibition of acetylcholinesterase
(AChE) activity (Mcdonough and Shih. 19971. Some of the clinical signs observed following RDX
exposure are similar to the clinical signs associated with organophosphate pesticides and nerve
agents fCrouse etal.. 2006: Burdette etal.. 1988: Barsotti and Crotti. 19491. However, the limited
data available for RDX do not support AChE inhibition as a primary mechanism because:
1) common AChE-induced symptoms such as salivation and lacrimation have not always been
observed (Williams et al.. 2 0111: 2) blood and brain levels of AChE are unaffected by RDX (Williams
etal.. 2011: Williams and Bannon. 20091: and 3) in vitro neurotransmitter receptor binding studies
do not reveal any affinity of RDX for acetylcholine receptors (Williams etal.. 2011: Williams and
Bannon. 20091. RDX showed no affinity for other receptors that are known targets of convulsants,
including the glutamate family of receptors, nicotinic receptors, glycine receptors, and several
monoamine receptors (Williams et al.. 2011: Williams and Bannon. 20091.
As noted above, in receptor binding assays RDX only showed affinity for GABAa receptors
(Williams etal.. 2011: Williams and Bannon. 20091. Specifically, RDX showed a significant affinity
for the picrotoxin convulsant site of the GABA channel. The authors demonstrated that RDX
treatment in brain slices from the basolateral amygdala inhibit GABAA-mediated inhibitory
postsynaptic currents and initiated seizure-like neuronal discharges. RDX exposure may reduce the
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inhibitory effects of GABAergic neurons, resulting in enhanced excitability that could lead to
seizures (Williams etal.. 2011: Williams and Bannon. 20091. although additional studies are
necessary to substantiate this observation and to clarify the potential cellular and regional targets
of RDX-induced neurotoxicity.
The limbic system, and the amygdala and hippocampus in particular, are known to be
critical to the development of seizures in various human conditions (e.g., epilepsy) and animal
models (e.g., kindling) (Teffervs etal.. 2012: Gilbert. 1994). Burdette etal. (1988) hypothesized that
the limbic system was involved in seizures caused by RDX exposure, given than rats exhibited pro-
convulsant activity in response to amygdaloid kindling at a dose that was approximately half the
dose necessary for RDX to induce spontaneous seizures. Potential limbic system involvement is
also suggested given its role in integrating emotional and behavioral responses (including
aggression) and the anecdotal observations of hyperactivity, hyper-responsiveness, and irritability
noted across several studies of RDX toxicity (Levine etal.. 1990: Levine etal.. 1983: Thompson.
1983: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Von Oettingenetal.. 1949).
In a microarray experiment, Bannon et al. (2009) found that RDX caused a down regulation
of an abundance of genes in the cerebral cortex related to neurotransmission, including those
encoding proteins involved in synaptic transmission and vesicle transport Genes encoding
proteins involved in the glutamate pathway were also underexpressed, indicating a possible
mechanism of RDX via excessive glutamate stimulation. The authors speculated that this
depression of the major excitatory neurotransmitter system could be a negative response to the
increase in seizure likelihood from RDX influx into the brain. Molecular changes in response to RDX
have been described by Zhang and Pan (2009b). who observed significant changes in micro-RNA
(miRNA) expression in the brains of B6C3Fi mice fed 5 mg/kg-day for 28 days. One miRNA, miR-
206, was upregulated 26-fold in RDX-exposed brains; brain-derived neurotrophic factor (BDNF)
was identified as a downstream gene target of this miRNA, along with two other miRNAs that were
upregulated in RDX-exposed brains (miR-30a and miR-195) fZhang and Pan. 2009a. b). BDNF is a
member of the neurotrophin family of growth factors, and promotes the survival and differentiation
of existing and new neurons. Effects of RDX on BDNF expression may play a role in RDX
neurotoxicity, but the utility of miRNAs as predictors of toxicity has not been established, and the
contribution, if any, of aberrant expression of a suite of miRNAs to the MOA for RDX neurotoxicity is
unknown.
Information from a small number of studies suggests that inhibition of GABAergic signaling
in the limbic system could represent a likely mechanism for RDX-induced hyperactivity and
seizures. However, the available data are insufficient to identify any specific mode(s) of action for
the nervous system effects observed following RDX exposure.
Summary of Nervous System Effects
Evidence for nervous system effects associated with exposure to RDX comes from studies in
both humans and animals. One occupational study reported memory impairment and decrements
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in certain neurobehavioral tests in workers exposed to RDX compared to controls (Ma and Li.
19921. and human case reports provide other evidence of an association between acute RDX
exposure and neurological effects. Eleven of 16 repeat-dose animal studies reported neurological
effects, including seizures, convulsions, tremors, hyperirritability, hyper-reactivity and behavioral
changes, associated with RDX exposure fCrouse etal.. 2006: Angerhofer etal.. 1986: Levine etal..
1983: Levine etal.. 1981b: Cholakis etal.. 1980: Von Oettingen et al.. 19491. In most of these
studies, the occurrence of neurological effects was dose related. In those studies that found no
evidence of RDX-associated neurotoxicity (MacPhail etal.. 1985: Cholakis etal.. 1980: Hart. 1976.
19741. differences in particle size and purity of the RDX administered could possibly account for the
lack of effect. Although the specific mode(s) of action for RDX-induced nervous system effects
remains unknown, evidence that RDX exposures may lead to seizures through binding to the GABAa
receptor provides biological support for this association. EPA identified nervous system effects as a
human hazard of RDX exposure.
1.1.2. Kidney and Other Urogenital System Effects
The association between RDX exposure and effects on clinical measures of kidney function
was examined in a single occupational epidemiology study. Case reports involving accidental
exposure to ingested or inhaled RDX provide some information on the potential for acute exposures
to RDX to affect the kidney in humans. Organ weight and histopathology findings from
experimental animal studies involving subchronic and chronic exposure to ingested RDX also
provide data relevant to an examination of the association between RDX exposure and kidney and
other urogenital system effects. A summary of kidney and other urogenital effects associated with
RDX exposure is presented in Tables 1-3 to 1-7 and Figure 1-2.
Human case reports of individuals accidently exposed to unknown amounts of RDX by
ingestion or inhalation provide some evidence that RDX may affect the kidney and urogenital
system. Reported symptoms included decreased urine output fKetel and Hughes. 1972: Knepshield
and Stone. 1972: Hollander and Colbach. 1969: Merrill. 19681. blood in urine fKasuske etal.. 2009:
Knepshield and Stone. 1972: Hollander and Colbach. 1969: Merrill. 19681. proteinuria fKasuske et
al.. 2009: Kuctikardali etal.. 2003: Ketel and Hughes. 1972: Hollander and Colbach. 1969: Merrill.
19681. glucosuria f Kuctikardali etal.. 20031. elevated blood urea nitrogen (BUN) levels (Hollander
and Colbach. 1969: Merrill. 19681. and one case of acute renal failure requiring hemodialysis
following accidental inhalation of RDX fKetel and Hughes. 19721. In many of these case reports,
renal parameters returned to normal within a few days following exposure. No changes in renal
parameters were reported in other individuals exposed to unknown amounts of RDX (Stone etal..
1969: Kaplan etal.. 19651. In a cross-sectional epidemiologic study of workers from five U.S. Army
munitions plants (69 exposed to RDX alone and 24 to RDX and HMX; average exposure of up to
1.5 mg/m3), no statistically significant differences in BUN or total serum protein between
nonexposed and RDX-exposed groups were observed (Hathaway and Buck. 19771 (Table 1-3).
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Studies in experimental animals provide some evidence that RDX exposure is associated
with kidney and other urogenital effects (Table 1-4 and Figure 1-2). Dose-related increases in
absolute and relative kidney weights (19-27% compared to control) were observed in male B6C3Fi
mice exposed to RDX in the diet for 2 years (Lish etal.. 1984) and a dose-related increase in relative
kidney weights (up to 19%) was observed in female mice. Relative, but not absolute, kidney
weights were increased (20-21% compared to control) in male and female F344 rats exposed to
40 mg/kg-day RDX in the diet for 2 years (Levine etal.. 1983). Changes in kidney weights in other
subchronic oral toxicity studies in rats, dogs, and monkeys did not show a clear pattern of increase
or decrease associated with RDX exposure; kidney weight changes were either not dose-related or
were inconsistent across sexes when absolute and relative weights were compared (Crouse etal..
2008: Levine etal.. 1990: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Hart. 1974:
Martin and Hart. 1974). Based on an evaluation of the relationship between organ weight and
body/brain weight to determine which endpoint (organ weight, organ-to-body weight ratio, or
organ-to-brain weight ratio) is likely to more accurately detect target organ toxicity, Bailey et al.
f20041 concluded that kidney weights are not modeled well by any of the choices, and that
alternative analysis methods should be utilized.
Histopathological changes in the urogenital system associated with exposure to RDX were
observed in a 2-year bioassay in which increased incidences of kidney medullary papillary necrosis
and pyelitis, uremic mineralization, bladder distention and/or cystitis, and suppurative prostatitis
were observed in high-dose (40 mg/kg-day) male rats that died spontaneously or were sacrificed in
moribund condition (Levine etal.. 1983). Similar kidney lesions were not observed in female rats
in this study. An increased incidence of tubular nephrosis was observed in male B6C3Fi mice
exposed to 320 mg/kg-day RDX in feed for 90 days, but not in female mice in this study fCholakis et
al.. 1980). In other chronic and subchronic oral studies in rats and mice, no histopathological
changes in the kidney were associated with RDX exposure (Crouse etal.. 2006: Levine etal.. 1990:
Lish etal.. 1984: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Hart. 1976).
Increased incidence of minimal to mild mineralization of the medulla was observed in male and
female monkeys exposed to 10 mg/kg-day RDX for 90 days by gavage (Martin and Hart. 1974). but
the study authors did not identify this as treatment related. No dose-related histopathological
changes were reported in a subchronic study in dogs fHart. 19741. and no histological alterations
were noted in the kidneys of rabbits exposed dermally to 165 mg/kg RDX in DMSO for 4 weeks
(McNamara et al.. 1974). Measurement of serum chemistry parameters that may indicate effects on
renal function, including BUN and uric acid, in studies of RDX in mice, rats, dogs, and monkeys
fCrouse etal.. 2008: Levine etal.. 1990: Lish etal.. 1984: Levine etal.. 1981a: Levine etal.. 1981b:
Cholakis etal.. 1980: Hart. 1976.1974: Martin and Hart. 1974) revealed variations (increases or
decreases) from the respective control groups that were not dose-related.
Exposure to the major contaminant in many of the available RDX studies, HMX, was
associated with histopathological changes in the kidney and alterations in renal function in female
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rats fed doses >450 mg/kg-day HMX for 13 weeks. No effects were observed at doses <115 mg/kg-
day. Because the percentage of HMX as an impurity ranged from 3-10% resulting in HMX
exposures of <60 mg/kg-day in the studies of RDX toxicity, the contribution of HMX to the observed
kidney toxicity in studies of RDX is expected to be negligible.
A significant, dose-related increase in the total incidence of suppurative prostatitis was
reported in male F344 rats exposed to >1.5 mg/kg-day RDX in the diet for two years fLevine etal..
19831. The Levine etal. (19831 report is the only 2-year study that reported examination of the
prostate in rats. Suppurative prostatitis was not observed in 90-day studies in the rat involving
oral (dietary or gavage) exposure to RDX (Crouse etal.. 2006: Levine etal.. 1990: Levine etal..
1981a: Levine etal.. 1981bl. Similarly, prostate effects were not observed in a 2-year dietary study
in mice (Lish etal.. 19841. Some reports have hypothesized that the observation of prostate
inflammation in Levine etal. f 19831 is secondary to a bacterial infection unrelated to RDX toxicity
(ATSDR. 2012: Sweeney etal.. 2012a: Crouse etal.. 20061. For example, Crouse etal. (20061
concluded that the inflammation reflects a common condition in rodents, noting that since 85% of
the incidence occurred in rats found at spontaneous death or moribund sacrifice (SDMS), it was
most likely that the condition was a result of an incidental bacterial infection. However, Levine et
al. T19831 distinguish between nonsuppurative and suppurative inflammation (the latter being
characterized by the formation of pus and a high concentration of neutrophils). Although the
proportion of suppurative prostatitis was higher in SDMS rats, there was an increasing trend with
dose in both the scheduled sacrifice (SS) and SDMS groups; the incidence of suppurative prostatitis
in the control group was 4% when the SS and SDMS groups are combined. Additionally, the dose-
related nature of the increased incidence suggests that the primary cause (potentially leading to
bacterial infection) was treatment-related since a more uniform distribution of rats with
suppurative prostatitis would be expected with a spontaneous or age-related lesion. The dose-
responsiveness could be explained if the infections were secondary to treatment-related
immunotoxicity, but there is no evidence from Levine etal. T19831 to support this possibility; a
more thorough analysis of immune endpoints in a 90-day gavage exposure of F344 rats did not
identify any immunotoxic effects associated with RDX fCrouse etal.. 20061.
Levine etal. (19831 document an array of kidney and other urogenital lesions in their
2-year dietary exposure of F344 rats to RDX. However, the sequence by which those effects may
have occurred is unclear. Renal medullary necrosis, bladder distension and cystitis were observed
mainly in the male rats exposed to 40 mg/kg-day RDX for 24 months, although one rat in the
0.3 mg/kg-day dose group also exhibited these lesions. Treatment-related effects on the kidney
(necrosis) and bladder (distension/obstruction and hemorrhagic cystitis) were also identified in
the 12-month pathology report (see Tables 1-5 to 1-7). The absence of these observations in the
6-month interim pathology report suggests that an exposure duration of greater than 6 months
may be required before RDX-induced effects on the urogenital system are observed. Suppurative
prostatitis was observed with increasing incidence in each dose group in the study at 24 months.
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Considered as a group, treatment-related kidney and urogenital lesions may have led to a blockage
that resulted in urinary stasis. Reduced urinary flow and/or retrograde flow may have contributed
to an environment that allowed bacterial infection of the prostate. Thus while an opportunistic
bacterial infection could be the proximal cause of the suppurative prostatitis, it may have been
secondary to the effects of RDX on the urogenital system. This hypothesis is consistent with the
observed dose-related increase in incidence of the suppurative prostatitis fATSDR. 2012: Sweeney
etal.. 2012a: Crouse etal.. 20061.
Although the ultimate sequence of effects in the urogenital system is unclear, even from
review of the scheduled sacrifices at 6 or 12 months on study, it is plausible that the observations of
suppurative prostatitis would arise after other kidney or bladder lesions that resulted in the initial
blockage and urinary stasis. The incidence of suppurative prostatitis reported in Levine et al.
(19831 was increased at doses lower than the doses associated with an increased incidence of other
urogenital lesions. However, the incidence of bladder lesions may have been underreported, since
the bladders were only examined following observation of a gross abnormality. Bladder distension
was reported sporadically among the lower dose groups (0.3,1.5, or 8.0 mg/kg-day), but the
bladder was not routinely examined in these dose groups (Levine etal.. 1983: Thompson. 19831.
Although the pathogenesis of kidney and urogenital effects cannot be established, the available
evidence is consistent with suppurative prostatitis as an indirect effect of RDX exposure and as a
marker for the broader array of kidney and urogenital effects observed by Levine etal. f!9831.
Table 1-3. Evidence pertaining to kidney effects in humans
Reference and study design
Results
Hathawav and Buck (1977)
Renal function tests in men: mean (standard deviation not
Cross-sectional study, 2,022 workers,
reported)
1,491 participated (74% response rate).
RDX exposed
Analysis group: limited to whites;
Referent Undetected >0.01 mg/m3
69 workers exposed to RDX alone and
Test
(n = 237) (n = 22) (n =45)
24 workers exposed to RDX and HMX,
BUN
15.5 15.6 16.4
compared to 338 workers not exposed to
Total protein
7.2 7.2 7.3
RDX, HMX, or TNT.
Exposure measures: Exposure
No differences were statistically significant. Similar results in
women.
determination based on job title and
industrial hygiene evaluation; exposed
subjects assigned to two groups:
undetected (0.01 mg/m3
(mean 0.28 mg/m3).
Effect measures: Renal function tests
(blood)
Analysis: Types of statistical tests were
not reported (assumed to be t-tests for
comparison of means and x2 tests for
comparison of proportions).
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Table 1-4. Evidence pertaining to kidney and other urogenital system effects
in animals
Reference and study design
Results
Kidney weight
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
Doses
0 1.5 7.0 35 175/100
Absolute kidney weight at 104 wks (percent change compared to
control)
M
F
0% -1% 4% 9%* 19%*
0% 3% 1% 1% -2%
Relative kidney weight at 104 wks (percent change compared to
control)
M
F
0% 3% 6% 11%* 27%*
0% 1% 1% 2% 19%*
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Doses
o
1
1
rn
o
O
Absolute kidney weight (percent change compared to control)
M
F
0% -3% -7% 2%
0% 14% -4% 8%
Relative kidney weight (percent change compared to control)
M
F
0% -1% -4% 4%
0% 22% 3% 18%
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Doses
o
o
00
LO
m
o
o
Absolute kidney weight at 105 wks (percent change compared to
control)
M
F
0% 2% -7% 1% 0%
0% 3% 3% 2% 2%
Relative kidney weight at 105 wks (percent change compared to
control)
M
F
0% 1% 0% 2% 20%*
0% 3% 6% 5% 21%*
Cholakis et al. (1980)
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Absolute kidney weight (percent change compared to control)
M
F
0% - 18% 2%
0% - - - -8% -5%
Relative kidney weight (percent change compared to control)
M
F
0% - 29% 0%
0% - - - -8% -3%
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Reference and study design
Results
Experiment 2: 0, 40, 60, or 80 mg/kg-d for
2 wks followed by 0, 320,160, or
80 mg/kg-d (TWA doses of 0, 79.6,147.8,
or 256.7 mg/kg-d for males and 0, 82.4,
136.3, or 276.4 mg/kg-d for females)3
Diet
13 wks
Doses
0 80 160 320
Absolute kidney weight (percent change compared to control)
M
F
0% 8% 11% 13%
0% -5% -3% 0%
Relative kidney weight (percent change compared to control)
M
F
0% 5% 9% 10%
0% -5% -4% -5%
Cholakis et al. (1980)
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
0,10,14, 20, 28, 40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Absolute kidney weight (percent change compared to control)
M
F
0% - -2% -5%
0% - - - 1% 0%
Relative kidney weight (percent change compared to control)
M
F
0% - 1% 5%
0% - - - 6% 6%
Cholakis et al. (1980)
Rats, CD, two-generation study;
F0: 22/sex/group; Fl: 26/sex/group;
F2: 10/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
F0 and Fl parental animals: 0, 5,16, 50
mg/kg-d
Diet
13 wks
Doses
0 5 16 50
Absolute kidney weight (percent change compared to control)
M
F
0% 6% -12%
0% -4% -21%*
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Doses
0 4 8 10 12 15
Absolute kidney weight (percent change compared to control)
M
F
0% -3% -4% -1% 3% 5%
0% 2% 5% 13%* 10% 15%*
Relative kidney weight (percent change compared to control)
M
F
0% 3% 6% 2% 1% 3%
0% 1% -3% -1% -6% -7%*
Levine et al. (1981a);Levine et al. (1990);
Levine et al. (1981b)b
Rats, F344,10/sex/group; 30/sex for
control
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 nm, ~90% of particles
< 66 nm
0,10, 30,100, 300, or 600 mg/kg-d
Data were not reported for rats in the 300 or 600 mg/kg-d groups
because all of the rats died before the 13-wk necropsy.
Doses
0 10 30 100 300 600
Absolute kidney weight (percent change compared to control)
M
F
0% 1% 1% -9%
0% 1% 3% -1%
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Reference and study design
Results
Diet
13 wks
Relative kidney weight (percent change compared to control)
M
F
0% 5% 7% 10%
0% 3% 5% 2%
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow containing
20 mg RDX/g-chow, 60 grams of dog food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
Numerical values given only for control and 10 mg/kg-d groups.
Doses
o
1
1
1
o
o
Absolute kidney weight (percent change compared to control)
M
F
0% - - 38%
0% - - -18%
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Doses 0 0.1 1 10
Absolute kidney weight (percent change compared to control)
M + F
0% -2% -3% 4%
Histopathological lesions
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
The incidence of cytoplasmic vacuolization of renal tubules was
greater for RDX-treated males than the control group males after
6 mo of treatment. However, at 12 and 24 mo of treatment, this
lesion was observed as frequently in control animals as animals
treated with RDX.
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Histopathological examination of kidney did not reveal any significant
differences compared to controls; lesions observed were not
attributed to RDX treatment; incidence data were reported only for
control and 10 mg/kg-d groups.
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Note: More detailed histopathological
results, including interim sacrifice data at
6 and 12 mo, are provided in Tables 1-5 to
1-7.
Data were analyzed separately for animals sacrificed on schedule (SS)
and those that died spontaneously or were sacrificed moribund
(SDMS); incidence data were not reported for females.
Doses
0
0.3
1.5
8.0
40
Kidney, medullary papillary necrosis; 24 mo (incidence)
(SS)
(SDMS)
(Sum)
0/38
0/36
0/25
0/29
0/4
0/17
1/19
0/27
0/26
18/27
0/55
1/55
0/52
0/55
18/31
Kidney, suppurative pyelitis; 24 mo (incidence)
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Results
(SS)
0/38
0/36
0/25
0/29
0/4
(SDMS)
0/17
1/19
0/27
1/26
5/27*
(Sum)
0/55
1/55
0/52
1/55
5/31*
Kidney, uremic mineralization; 24 mo (incidence)
(SS)
1/38
0/36
0/25
0/29
0/4
(SDMS)
0/17
1/19
2/27
0/26
13/27
(Sum)
1/55
1/55
2/52
0/55
13/31
Urinary bladder, luminal distention; 24 mo (incidence)
(SS)
0/38
0/36
0/25
0/29
1/4*
(SDMS)
0/16
2/19
1/27
3/22
24/28*
(Sum)
0/54
2/55
1/52
3/51
25/32*
Urinary bladder, cystitis hemorrhagic/suppurative; 24 mo
(incidence)
(SS)
0/38
0/36
0/25
1/29
0/4
(SDMS)
0/16
2/19
1/27
0/22
18/27*
(Sum)
0/54
2/55
1/52
1/51
18/31*
Prostate, suppurative inflammation (prostatitis); 24 mo (incidence)
SS
0/38
1/36
2/25*
4/29*
0/4
SDMS
2/16
3/19
7/27*
8/26
19/27*
(Sum)
2/54
4/55
9/52*
12/55*
19/31*
Cholakis et al. (1980)
Incidence data reported only for controls and the 320 mg/kg-d group.
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 urn particle size
Doses
0
80
160
320
Tubular nephrosis (incidence)
0, 80, 60, 40 mg/kg-d for 2 wks followed
by 0, 80,160, or 320 mg/kg-d (TWA doses
of 0, 79.6,147.8, or 256.7 mg/kg-d for
males and 0, 82.4,136.3, or
276.4 mg/kg-d for females)3
Diet
13 wks
M
F
0/10
0/11
-
-
4/9*
1/11
Cholakis et al. (1980)
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
Histopathological examination of kidney did not reveal any significant
differences compared to controls; incidence data were reported only
for control and 40 mg/kg-d groups.
This document is a draft for review purposes only and does not constitute Agency policy.
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Reference and study design
Results
Cholakis et al. (1980)
Rats, CD, two-generation study; FO:
22/sex/group; Fl: 26/sex/group; F2:
10/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 urn particle size
FO and Fl parental animals: 0, 5,16, or
50 mg/kg-d
Diet
13 wks
Data were reported only for F2 generation controls and 5 and
16 mg/kg-d groups.
Doses
0 5 16 50
Cortical cysts (incidence)
M
F
4/10 4/10 8/10
3/10 4/10 8/10
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Doses
0 4 8 10 12 15
Prostate, mild subacute inflammation (incidence)
M
0/10 1/8
Histopathological examination of kidney did not reveal any significant
differences compared to controls; incidence data were reported only
for control and 15 mg/kg-d groups.
Levine et al. (1981a);Levine et al. (1990);
Levine et al. (1981b)b
Rats, F344,10/sex/group; 30/sex for
control
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 nm, ~90% of particles
< 66 nm
0,10, 30,100, 300, or 600 mg/kg-d
Diet
13 wks
Histopathological examination of kidney did not reveal any significant
differences compared to controls.
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow containing
20 mg RDX/g-chow, 60 grams of dog food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
Histopathological examination of kidney did not reveal any significant
differences compared to controls; incidences were reported only for
control and 10 mg/kg-d groups.
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Doses
o
1
1
1
o
o
Medulla; mineralization, minimal to mild (incidence)
M + F
0/6 1/6 0/6 4/6
1
2 ^Statistically significant (p < 0.05) based on analysis by study authors.
3 aDoses were calculated by the study authors.
4 bLevine et al. (1981a) is a laboratory report of a 13-week study of RDX in F344 rats; two subsequently published
5 papers (Levine et al., 1990; Levine et al., 1981b) present subsets of the data provided in the full laboratory report.
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Table 1-5. Six-, 12-, and 24-month incidence of kidney endpoints in male F344
rats reported for statistical evaluation in Levine et al. (1983)
Doses (mg/kg-d)
0
0.3
1.5
8.0
40
Medullary papillary necrosis (incidence)
6 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
-
-
0/5
Sum
0/10
0/10
0/10
0/10
0/15
12 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
0/3
-
15/19*
Sum
0/10
0/10
0/13
0/10
15/29*
24 mo
SS
0/38
0/36
0/25
0/29
0/4
SDMS
0/17
1/19
0/27
0/26
18/27*
Sum
0/55
1/55
0/52
0/55
18/31*
Pyelitis (incidence)
6 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
-
-
0/5
Sum
0/10
0/10
0/10
0/10
0/15
12 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
0/3
-
1/19
Sum
0/10
0/10
0/13
0/10
1/29
24 mo
SS
0/38
0/36
0/25
0/29
0/4
SDMS
0/17
1/19
0/27
1/26
5/27*
Sum
0/55
1/55
0/52
1/55
5/31*
Pyelonephritis (incidence)
6 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
-
-
0/5
Sum
0/10
0/10
0/10
0/10
0/15
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Doses (mg/kg-d)
0
0.3
1.5
8.0
40
12 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
0/3
-
1/19
Sum
0/10
0/10
0/13
0/10
1/29
24 mo
SS
0/38
0/36
0/25
1/29
0/4
SDMS
0/17
0/19
2/27
1/26
1/27
Sum
0/55
0/55
2/52
2/55
1/31
1 ^Statistically significant (p < 0.05) based on analysis by study authors.
2
3 Source: Levine et al. (1983).
4
Table 1-6. Six-, 12-, and 24-month incidence of urinary bladder endpoints in
male F344 rats reported for statistical evaluation in Levine et al. (1983)
Doses (mg/kg-d)
0
0.3
1.5
8.0
40
Luminal distention (incidence)
6 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
-
-
0/5
Sum
0/10
0/10
0/10
0/10
0/15
12 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
0/3
-
18/19*
Sum
0/10
0/10
0/13
0/10
18/29
24 mo
SS
0/38
0/36
0/25
0/29
1/4*
SDMS
0/16
2/19
1/27
3/22
24/28*
Sum
0/54
2/55
1/52
3/51
25/32*
Cystitis, hemorrhagic/suppurative (incidence)
6 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
-
-
0/5
Sum
0/10
0/10
0/10
0/10
0/15
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Doses (mg/kg-d)
0
0.3
1.5
8.0
40
12 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
0/3
-
17/19*
Sum
0/10
0/10
0/13
0/10
17/29
24 mo
SS
0/38
0/36
0/25
1/29
0/4
SDMS
0/16
2/19
1/27
0/22
18/27*
Sum
0/54
2/55
1/52
1/51
18/31*
1
2 ^Statistically significant (p < 0.05) based on analysis by study authors.
3
4 Source: Levine et al. (1983).
Table 1-7. Six-, 12-, and 24-month incidence of prostate endpoints in male
F344 rats reported for statistical evaluation in Levine et al. (1983)
Doses (mg/kg-d)
0
0.3
1.5
8.0
40
Spermatic granuloma (incidence)
6 mo
SS
0/10
2/10
2/10
1/10
6/10*
SDMS
-
-
-
-
2/5
Sum
0/10
2/10
2/10
1/10
8/15*
12 mo
SS
0/10
0/10
1/10
1/10
0/10
SDMS
-
-
0/3
-
0/19
Sum
0/10
0/10
1/13
1/10
0/29
24 mo
SS
0/38
0/36
0/25
0/29
0/4
SDMS
0/16
0/19
0/27
0/26
0/27
Sum
0/54
0/55
0/52
0/55
0/31
Suppurative inflammation (incidence)
6 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
-
-
0/5
Sum
0/10
0/10
0/10
0/10
0/15
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Doses (mg/kg-d)
0
0.3
1.5
8.0
40
12 mo
SS
0/10
0/10
0/10
0/10
0/10
SDMS
-
-
0/3
-
0/19
Sum
0/10
0/10
0/13
0/10
0/29
24 mo
SS
0/38
1/36
2/25*
4/29*
0/4
SDMS
2/16
3/19
7/27*
8/26
19/27*
Sum
2/54
4/55
9/52*
12/55*
19/31*
1
2 ^Statistically significant (p < 0.05) based on analysis by study authors.
3
4 Source: Levine et al. (1983).
This document is a draft for review purposes only and does not constitute Agency policy.
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ToxicologicalReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
1000
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Chronic
Subchronic
Histopathological lesions
"T" Relative kidney
weight
The following studies were excluded from array because absolute kidney weight was reported: Cholakis, 1980 (2-gen rat); Hart, 1974; Martin and Hart, 1974
M - Mortality observed at this dose and above
1 statistical significance determined from incidence at time of of scheduled sacrfice
2 statistical significance determined from incidence at spontaneous death.
Figure 1-2. Exposure-response array of kidney and urogenital system effects.
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Mechanistic Evidence
No MOA information is available for RDX-induced kidney and other urogenital effects,
including suppurative prostatitis. However, mechanistic information underlying the neurotoxicity
observed with RDX exposure, and the specific affinity of RDX to the GABAa receptor-convulsant site
fWilliams etal.. 2011: Williams and Bannon. 20091. suggests a biologically plausible role for the
GABAa receptor in RDX-related effects on the urogenital system and provides some potential modes
of action for the effects reported in Levine etal. (19831.
Alterations in hormonal signaling or circulating levels of estrogen or prolactin may lead to
prostatitis. Prostate inflammation has been associated with endocrine disruptors in the
environment fCowin etal.. 20101. and increased prolactin has been shown to cause lateral lobe
prostatitis (Stoker etal.. 1999b: Stoker etal.. 1999a: Tangbanluekal and Robinette. 1993: Robinette.
19881. Typically the inflammation seen is chronic and does not reverse over time fRobinette.
19881. Functional GABAa receptors have been identified in the anterior pituitary (Zemkova etal..
2008: Maverhofer. 20011. which also serves as the primary source of prolactin. Thus, the prostate
inflammation observed in the rat in the 2-year study by Levine etal. (19831 could have been
produced by disruption of pituitary prolactin or other hormonal signal via interference with normal
regulatory GABA-related hormonal control. However, no direct evidence for this hypothesized
MOA is available. Levine etal. (19831 did not evaluate serum endocrine measures or pituitary
weights, and pituitary adenomas that could account for higher prolactin levels were not observed.
A MOA based on pituitary-mediated alterations in endocrine signaling also does not explain the
other urogenital lesions observed by Levine etal. (19831.
Another hypothesis is that the prostate effects could be mediated through an autoimmune
inflammatory response. GABAa receptor transcripts have been identified in immune cells of mouse
models f Reyes-Garcia etal.. 2007: Tian etal.. 20041. and GABAa receptor agonists have decreased
cytotoxic immune responses and hypersensitivity reactions (Tian etal.. 1999: Bergeretetal.. 19981.
In a murine autoimmune model of multiple sclerosis, Bhatetal. (20101 found that treatment of
macrophages challenged with lipopolysaccharide with various GABA agonists decreased cytokine
production; addition of picrotoxin (which may have effects similar to those of RDX, since they bind
to the same site) was able to reduce this effect However, picrotoxin on its own did not significantly
alter cytokine production, suggesting the effects are limited to reversal of agonist-induced
GABAergic activity. If an autoimmune mechanism was contributing to the effects observed with
RDX exposure, it is unclear why inflammation would be limited to the prostate. RDX has also tested
negative in the only battery of immunotoxicity tests to which it was subjected (Crouse etal.. 20061.
If it is assumed that the kidney and other urogenital effects are mediated through localized
interaction with GABAa receptors, another possibility is that effects would result from direct
interactions with GABAa receptors located on the prostate. GABAa receptors have been identified
on the prostate fNapoleone etal.. 19901. providing a potential mechanism by which RDX could
interact directly with the prostate. However, this would require that the prostate is actively
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maintained in a non-inflamed state, mediated by GABA; RDX binding to GABAa receptor-convulsant
sites on the prostate would result in a reduction of the inhibitory effects of the GABA receptor
leading to increased inflammation. No evidence was found to support this potential pathway
leading to prostate inflammation.
Another hypothesis is that the kidney and other urogenital effects of RDX are caused by
interactions with GABAa receptors mediating inputs to the urogenital system. GABA is believed to
play a role in the regulation of urination and bladder capacity (reviewed in Fowler et al. (20081 and
Yoshimura and de Groat (199711. In rats, injection of a GABAa receptor agonist inhibits the
urination reflex (Igawa etal.. 1993: Kontani et al.. 19871. GABAa agonists injected into the
periaqueductal gray area in rats inhibited reflex bladder activity, while injection of an antagonist
reduced bladder capacity and increased the frequency of bladder reflex activity (Stone etal.. 20111.
RDX would be expected to act like an antagonist and increase bladder activity (which would not
result in urinary stasis), although the impact of chronic exposure to RDX acting as a GABAa receptor
antagonist is not known. Evidence of GABAergic signaling regulating bladder function, and the
hypothesized disruption of that regulation by RDX via interaction with GABAa receptors, may
plausibly account for the kidney and other urogenital lesions, including suppurative prostatitis,
observed by Levine etal. f!9831: however, no evidence to support this hypothesized MOA is
available.
In summary, there are no studies available that inform mechanistically how RDX might lead
to kidney and other urogenital effects. There is evidence that RDX binds to GABAa receptors in
neuronal tissues (Williams etal.. 2011: Williams and Bannon. 20091. and it is biologically plausible
that binding to the GABA receptor could occur in other tissues as well, accounting for the observed
kidney and urogenital effects. Among the mechanistic information presented above, modes of
action that require direct action on the prostate are considered less likely, because the available
information suggests the prostatitis is a secondary effect However, the ways GABAa receptors
work in non-neuronal tissues and organs is still not well understood, and the MOA by which RDX
induces kidney and other urogenital effects is unknown.
Summary of Kidney and Other Urogenital System Effects
Evidence for kidney effects resulting from RDX exposure consists of human case reports and
some findings of increased kidney weight and histopathological changes in rodents. In humans,
evidence for kidney effects (including decreased urine output, blood in urine, and proteinuria) is
limited to individuals with acute accidental exposure (ingestion and inhalation) to unknown
amounts of RDX. No RDX-related changes in kidney parameters were found in a small cross-
sectional study of RDX-exposed workers (Hathaway and Buck. 19771. Treatment-related increases
in relative kidney weight were consistently observed in rats and mice of both sexes in two chronic
oral toxicity studies (Lish etal.. 1984: Levine etal.. 19831: however, kidney weights across studies
of subchronic duration generally failed to show a consistent pattern of change. Measurement of
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serum chemistry parameters in multiple animal species did not provide consistent evidence of
dose-related changes associated with RDX exposure.
Histopathological changes in a two-year study in F344 rats, including a dose-related
increase in the incidence of suppurative prostatitis in male rats (Levine etal.. 1983: Thompson.
19831. provides the strongest evidence of RDX-associated kidney and other urogenital effects. As
discussed above, the incidence of suppurative prostatitis is considered to be an indicator for the
broader array of kidney and other urogenital effects seen in this study. A second 2-year study in
Sprague-Dawley rats found no histopathological changes in the kidney or urogenital system (Hart.
19761. but exposure levels used in this study were low compared to Levine etal. (19831. In light of
the dose-related increase in suppurative prostatitis and lack of support for an alternative (i.e., non-
RDX-related) basis for this effect, EPA identified kidney and other urogenital effects as a potential
human hazard of RDX exposure.
1.1.3. Reproductive and Developmental Effects
No human studies were identified that evaluate the potential of RDX to cause reproductive
or developmental effects. Information relevant to an examination of the association between RDX
exposure and reproductive and developmental effects comes from a 2-generation study in rats and
studies in rats and rabbits involving gestational exposure to ingested RDX. In addition, oral
subchronic and chronic studies in experimental animals provide information useful for examining
the association between RDX exposure and effects on the male reproductive system. A summary of
the developmental and reproductive effects associated with RDX exposure is presented in Tables
1-8 and 1-9 and Figures 1-3 and 1-4.
Developmental Effects
Animal studies report effects of RDX on offspring survival. Pup survival rates in the F0 and
F1 generations were statistically significantly decreased in RDX-exposed CD rats compared to
controls in the only available two-generation reproductive toxicity study of RDX (Cholakis et al..
19801. but only at the highest dose tested (5 0 mg/kg-day) that also produced toxicity in adults
(neurotoxicity, mortality, and reduced body weights and food consumption). Decreased fetal
viability was observed at 20 mg/kg-day in F344 rats (Cholakis etal.. 19801. although no effect on
live fetuses was observed in Sprague-Dawley rats at the same dose fAngerhofer etal.. 19861: both
of these studies reported significant mortality in dams at 20 mg/kg-day. Increased resorptions
were similarly limited to the highest dose tested (20 mg/kg-day), i.e., a dose associated with
maternal toxicity (Cholakis etal.. 19801. There was no evidence of maternal toxicity,
embryotoxicity or decreased fetal viability in a teratology study of pregnant rabbits exposed to RDX
by gavage from GD 7 to 29 at doses up to 20 mg/kg-day (Cholakis etal.. 19801. suggesting that
rabbits may be less sensitive to RDX toxicity than rats.
Statistically significant, dose-related reductions in fetal body weight and length were
reported in Sprague-Dawley rats exposed to RDX by gavage from GD 6 to 15 fAngerhofer etal..
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19861.4 Maximum decreases in fetal body weight (9%) and body length (5%) were observed at
20 mg/kg-day, a dose that produced significant mortality in the dams. A similar reduction in fetal
body weight of 7% (not statistically significant) was observed in F344 rats exposed to RDX at 20
mg/kg-day, a dose associated with maternal mortality fCholakis etal.. 19801. The larger Sprague-
Dawley litter sizes and number of fetuses, compared to F344 rats, may account for the greater
statistical power to observe treatment-related effects. Dose-related reductions in fetal body weight
were not observed in rabbits at doses up to 20 mg/kg-day fCholakis etal.. 19801.
No treatment-related teratogenic effects have been reported in rats exposed to a dose as
high as 20 mg/kg-day RDX, a dose that resulted in approximately 30% maternal mortality
fAngerhofer et al.. 1986: Cholakis etal.. 19801. Examination of rabbits administered RDX at doses
up to 20 mg/kg-day from GD 7-29 also provided little evidence of teratogenicity fCholakis etal..
19801. Increased incidences of enlarged front fontanel and unossified sternebrae were observed in
all groups of rabbits exposed to RDX fCholakis etal.. 19801: however, these developmental
anomalies did not exhibit a dose-related increase. Gestational exposure to RDX did not result in any
other skeletal abnormalities.
Reproductive Effects
Evidence of male reproductive toxicity is provided by the finding of testicular degeneration
in male mice (Table 1-9 and Figure 1-4). An increased incidence of testicular degeneration was
observed in male B6C3Fi mice exposed to >35 mg/kg-day RDX for 2 years in the diet (10-11%)
compared to concurrent (0%) and historical (1.5%) controls fLish etal.. 19841. Reductions in
absolute testicular weight were observed, but the magnitude of the effect was small (<6%
compared to controls) and not dose-related. An increased incidence of germ cell degeneration was
observed in rats exposed to 40 mg/kg-day (40%) compared with controls at 12 months (0%); by 24
months all male rats (including controls) had testicular masses and no instances of germ cell
degeneration were identified in control or RDX-treated groups fLevine etal.. 19831. No dose-
related histopathological changes in the testes were identified in other studies in rats (Crouse etal..
2006: Levine etal.. 1990: Levine etal.. 1981a: Levine etal.. 1981b: Hart. 19761 or dogs fHart. 19741.
Changes in testicular weight were inconsistent across studies, with an equivalent number of studies
identifying decreases (Crouse etal.. 2006: Lish etal.. 1984: Cholakis etal.. 19801 or increases
fLevine etal.. 1990: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Hart. 1976.
19741 in testicular weight; in most cases the changes in testicular weight were small (<10% change
compared to control) and not dose-related. Based on an evaluation of the relationship between
organ weight and body/brain weight to determine which endpoint (organ weight, organ-to-body
weight ratio, or organ-to-brain weight ratio) is likely to more accurately detect target organ
4 The statistical analyses presented by the study authors were performed on a per fetus basis; EPA's
Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA. 19911 recommend that fetal data be analyzed
on a per litter (rather than per fetus) basis. In a reanalysis of the Angerhofer et al. (19861 data by EPA on a
per litter basis, fetal body weight and length showed statistically significant decreasing trends.
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toxicity, Bailey etal. (2004) concluded that testes weights are not modeled well by any of the
choices, and that alternative analysis methods should be utilized.
Reproductive function was assessed in two separate studies reported by Cholakis et al.
(19801. In the dominant lethal mutation study, no effects on fertility were observed in male rats
exposed to <16 mg/kg-day RDX. Pregnancy rates were lower in females mated to males exposed to
50 mg/kg-day RDX for 15 weeks prior to mating, although this effect was attributed to decreased
well-being of the males in this high-dose group (Cholakis et al.. 19801. No specific effects on
reproductive function were observed in F0 and F1 rats exposed to <16 mg/kg-day RDX in a two-
generation study. The highest dose tested, 50 mg/kg-day, was associated with reductions in
fertility (specifically a decreased number of pregnancies) in the F0 generation, although these
changes were not statistically significant. The finding of lower fertility rates only at the 50 mg/kg-
day dose, a dose associated with reduced body weight and feed consumption and increased
mortality, suggests that effects on reproductive function were likely due to the general toxicity of
RDX rather than a direct effect of RDX on reproduction.
Table 1-8. Evidence pertaining to reproductive and developmental effects in
animals
Reference and study design
Results
Offspring survival
Cholakis et al. (1980)
Rats, CD, two-generation study;
FO: 22/sex/group; Fl: 26 sex/group;
F2: 10 sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 urn particle size
FO and Fl parental animals: 0, 5,16, or 50
mg/kg-d
Diet
13 wks
Doses
0 5 16 50
Stillborn pups (incidence)
Fl
F2
8/207 6/296 4/259 16/92*
6/288 6/290 2/250 24/46*
Offspring survival at birth (percent of fetuses)
Fl
F2
96% 98% 98% 83%*
98% 98% 99% 48%*
F0 maternal deaths occurred at 50 mg/kg-d. Only six Fl females in
this group survived to serve as parental animals; none of the six died
during subsequent treatment.
Note: results on a per litter basis were not provided.
This document is a draft for review purposes only and does not constitute Agency policy.
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Reference and study design
Results
Cholakis et al. (1980)
Doses
0 0.2
2
20
Rabbits, New Zealand White, 11-12/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 urn particle size
Early resorptions (mean percent per dam)
6% 5%
4%
1%
0, 0.2, 2.0, or 20 mg/kg-d
Gavage
Late resorptions (mean percent per dam)
GDs 7-29
8% 5%
3%
3%
Complete litter resorptions (number of litters)
0 0
0
2
Viable fetuses (mean percent per dam)
85% 82%
11%
94%
Cholakis et al. (1980)
Doses
0 0.2
2.0
20
Rats, F344, 24-25 females/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants.
Early resorptions (mean percent per dam)
6.0% 2.5%
4.8%
15.3%
0, 0.2, 2.0, or 20 mg/kg-d
Gavage
Late resorptions (mean percent per dam)
GDs 6-19
0.5% 0.5%
0.3%
1.6%
Complete litter resorptions (number of litters)
0 0
0
2
Viable fetuses (mean percent per dam)
93.2% 97.6%
94.9%
81.4%
Significant maternal mortality (7/24 dams) occurred at 20 mg/kg-d.
Angerhofer et al. (1986)
Doses
0 2
6
20
Rats, Sprague-Dawley, 39-51 mated
females/group (25-29 pregnant
dams/group)
Resorptions (percent of total implantations)
4.8% 6.1%
5.9%
6.4%
Purity 90%; 10% HMX and 0.3% acetic acid
occurred as contaminants
Early resorptions (percent of total implantations)
0, 2, 6, or 20 mg/kg-d
4.8% 6.1%
5.9%
6.2%
Gavage
GDs 6-15
Late resorptions (percent of total implantations)
0% 0%
0%
0.27%
Live fetuses (mean percent per litter)
100% 100%
100%
100%
Significant maternal mortality (16/51) occurred at 20 mg/kg-d.
This document is a draft for review purposes only and does not constitute Agency policy.
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Reference and study design
Results
Offspring growth
Cholakis et al. (1980)
Rabbits, New Zealand White, 11-12/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 urn particle size
0, 0.2, 2.0, or 20 mg/kg-d
Gavage
GDs 7-29
Doses
0 0.2 2.0 20
Fetal body weight (percent change compared to control)
0% -6.7% -2.3% -9.3%
Cholakis et al. (1980)
Rats, F344, 24-25 females/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants.
0, 0.2, 2.0, or 20 mg/kg-d
Gavage
GDs 6-19
Doses
0 0.2 2.0 20
Fetal body weight (percent change compared to control)
0% 2% 3% -7%
Significant maternal mortality (7/24 dams) occurred at 20 mg/kg-d.
Angerhofer et al. (1986)
Rats, Sprague-Dawley, 39-51 mated
females/group (25-29 pregnant
dams/group)
Purity 90%; 10% HMX and 0.3% acetic acid
occurred as contaminants
0, 2, 6, or 20 mg/kg-d
Gavage
GDs 6-15
Doses
0 2 6 20
Fetal body weight (percent change compared to control)
0% -4% -2% -9%a
Fetal body length (percent change compared to control)
_Q
0s-
LO
1
0s-
1
1
0s-
1
1
0s-
o
Significant maternal mortality (16/51) occurred at 20 mg/kg-d.
Morphological development
Cholakis et al. (1980)
Rabbits, New Zealand White, 11-12/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
0, 0.2, 2.0, or 20 mg/kg-d
Gavage
GDs 7-29
Doses
0 0.2 2.0 20
Spina bifida (incidence)
Fetuses
Litters
0/88 0/99 0/94 3/110
0/11 0/11 0/11 2/12
Misshapen eye bulges (incidence)
Fetuses
Litters
0/88 0/99 0/94 3/110
0/11 0/11 0/11 1/12
Cleft palate (incidence)
Fetuses
Litters
0/39 1/46 2/44 2/52
0/11 1/11 1/11 1/12
Enlarged front fontanel (incidence)
Fetuses
Litters
0/49 5/53 2/50 8/58
0/11 2/11 2/11 2/12
This document is a draft for review purposes only and does not constitute Agency policy.
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Reference and study design
Results
Cholakis et al. (1980)
Rats, F344, 24-25 females/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants.
0, 0.2, 2.0, or 20 mg/kg-d
Gavage
GDs 6-19
No gross or soft-tissue anomalies were seen in any exposure group.
No treatment-related increase in the incidence of litters with skeletal
anomalies was observed.
Significant maternal mortality (7/24 dams) occurred at 20 mg/kg-d.
Angerhofer et al. (1986)
Rats, Sprague-Dawley, 39-51 mated
females/group (25-29 pregnant
dams/group)
Purity 90%; 10% HMX and 0.3% acetic acid
occurred as contaminants
0, 2, 6, or 20 mg/kg-d
Gavage
GDs 6-15
No treatment-related increase in the incidence of anomalies was
observed.
Doses
0 2 6 20
Total malformations (percent of fetuses with malformations)
1% 1% 0% 2%
Significant maternal mortality (16/51) occurred at 20 mg/kg-d.
1
2 ^Statistically significant (p < 0.05) based on analysis by study authors.
3 Statistically significant dose-related trend (p < 0.05) by linear trend test, performed for this assessment. Average
4 fetal weights or lengths for each litter comprised the sample data for this test.
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100
>
TO
-a
M
£
o
Q
10
M
• signficantly changed
O not signifcantly changed
0
0 M
Q M
9
Q M
Q M
0
Q M
0 M
0.1
o
oo
CTl
4_>
ro
ro
ro
_Q
_Q
ro
O
to
ro
O
00
00
00
O
00
cn
i
cn
i
O
00
cn
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cn
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Table 1-9. Evidence pertaining to male reproductive effects in animals
Reference and Study Design
Results
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
Doses
0 1.5 7.0 35 175/100
Testicular degeneration (incidence)
0/63 2/60 2/62 6/59 3/27a
Absolute testes weight; wk 105 (percent change compared to
control)
0% -6% 0% -2% -6%
Relative testes weight; wk 105 (percent change compared to control)
0% -4% 2% -2% -2%
Hart (1976)
Rats, Sprague-Dawley, 100/sex/dose
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Doses
o
1
1
rn
o
O
Absolute testes (with epididymis) weight; wk 104
0% -2% 2% 5%
Relative testes (with epididymis) weight; wk 104
0% -1% 7% 9%
Testes were examined microscopically in control and 10 mg/kg-d
groups; no degeneration or other treatment-related effects were
observed.
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles <66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Doses
o
o
00
LO
m
o
o
Testes, germ cell degeneration; 12 mob (incidence)
SS
SDMS
0/10 0/10 0/10 0/10 4/10*
1/3 - 4/19
Testes, germ cell degeneration; 24 mo (incidence)
SS
SDMS
0/38 0/36 0/25 0/29 0/4
0/16 0/19 0/27 0/26 0/27
Testes weights were not measured at termination due to testicular
masses in nearly all males.
SDMS = spontaneous death or moribund sacrifice;
SS = scheduled sacrifice
Cholakis et al. (1980)
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Absolute testes weight (percent change compared to control)
0s-
1
0s-
1
1
1
1
0s-
o
Relative testes weight (percent change compared to control)
0s-
1
1
0s-
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Reference and Study Design
Results
80 mg/kg-d (TWA doses of 0, 79.6,147.8,
or 256.7 mg/kg-d for males and 0, 82.4,
136.3, or 276.4 mg/kg-d for females)d
Diet
13 wks
0% 4% -4% -8%
Relative testes weight (percent change compared to control)
0% 1% -4% -9%
Testes were examined microscopically in control and 320 mg/kg day
groups; no effects were observed.
Cholakis et al. (1980)
Rats, F344,10/sex/dose
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Absolute testes weight (percent change compared to control)
0% - - - -2% 0%
Relative testes weight (percent change compared to control)
0% - - - 2% 9%
Testes were examined microscopically in control and 40 mg/kg-d
groups; no effects were observed.
Cholakis et al. (1980)
Rats, CD, two-generation study; F0:
22/sex/group; Fl: 26/sex/group; F2:
10/sex/group
88.6% pure, with 9% HMX and 2.2% water
as contaminants; ~200 nm particle size
F0 and Fl parental animals: 0, 5,16, or
50 mg/kg-d
Diet
13 wks
In F2 offspring of 0, 5, and 16 mg/kg-d groups. No high-dose F2
animals available.
Doses
0 5 16 50
Absolute testes weight (percent change compared to control)
0% 3% -31%
Testes were examined microscopically in all F2 groups; no effects
observed.
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Doses
0 4 8 10 12 15
Absolute testes weight (percent change compared to control)
0% -3% -5% -4% -4% -8%
Relative testes weight (percent change compared to control)
0% 4% 5% 0% -6% -10%*
Levine et al. (1981a); Levine et al. (1990);
Levine et al. (1981b)d
Rats, F344,10/sex/group; 30/sex for
control
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 nm, ~90% of particles
< 66 nm
0,10, 30,100, 300, or 600 mg/kg-d
Diet
13 wks
Doses
0 10 30 100 300 600
Testes, germ cell degeneration (incidence)
0/10 0/10 0/10 0/10 1/9 1/10
Absolute testes weight (percent change compared to control)
0% 1% 1% -2%
Relative testes weight (percent change compared to control)
0% 4% 5% 19%*
Hart (1974)
Dogs, Beagle, 3/sex/dose
Pre-mix with ground dog chow containing
20 mg RDX/g-chow, 60 grams of dog food
0, 0.1,1, or 10 mg/kg-d
Doses
0 0.1 1 10
Absolute testes (with epididymis) weight (percent change compared
to control)
0% - - 51%
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Reference and Study Design
Results
Diet
90 d
Testes were not examined microscopically.
^Statistically significant (p < 0.05) based on analysis by study authors.
aAlthough the study authors did not observe a statistically significant increase in the incidence of testicular
degeneration, they determined that the incidences at the 35 and 175/100 mg/kg-day dose groups were "notable"
when compared to concurrent (0%) and historical (1.5%) incidences.
testicular atrophy was observed at 12 months along with a statistically reduced mean testes weight (compared
with controls). By 24 months, all male rats (including controls) had testicular masses; testes weights were not
recorded, and an increased incidence of testicular degeneration was not observed.
cDoses were calculated by the study authors.
dLevine et al. (1981a) is a laboratory report of a 13-week study of RDX in F344 rats; two subsequently published
papers (Levine et al., 1990; Levine et al., 1981b) present subsets of the data provided in the full laboratory report.
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1000
100
• signifcantly changed O not significantly changed
O M
M
O M
Q M
>
re
T3
i
00
o
Q
10
O
o
o
o
o
O M O
O M
O M
0.1
o
.c
(_>
4- Absolute testes weight
Testicular degeneration
o
.c
(_>
1 Increased absolute weight of testes and epididymis
2Although the study authors did not observe a statistically significant increase in the incidence of testicular degeneration, they
determined that the incidences at the 35 and 175/100 mg/kg-day dose groups were "notable" when compared to concurrent (0%) and
historical (1.5%) incidences.
Figure 1-4. Exposure response array of male reproductive effects following oral exposure.
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Summary of Reproductive and Developmental Effects
Developmental studies in rats fAngerhofer etal.. 1986: Cholakis et al.. 19801 and rabbits
fCholakis etal.. 19801 suggest that developmental effects related to offspring survival, growth, and
morphological development were likely associated with severe maternal toxicity. Developmental
effects were observed only at doses that caused maternal mortality. As noted in EPA's Guidelines
for Developmental Toxicity Risk Assessment (U.S. EPA. 19911. where adverse developmental effects
are produced only at doses that cause minimal maternal toxicity, developmental effects should not
be discounted as being secondary to maternal toxicity; however, at doses causing excessive toxicity,
as is the case with RDX, information on developmental effects may be difficult to interpret and of
limited value. Therefore, EPA concluded that the evidence does not support developmental effects
as a potential human hazard of RDX exposure.
Testicular effects were reported in male B6C3Fi mice chronically exposed to RDX in the diet
for 24 months (Lish etal.. 19841. No other studies of equivalent duration were performed in mice
to determine the consistency of this effect Germ cell degeneration was observed in F344 rats at
12 months, but not at 24 months in a 2-year study (Levine etal.. 19841. Other testicular effects
were inconsistent across rat studies. Based on the evidence reported by Lish etal. f!9841. EPA
identified suggestive evidence of male reproductive effects as a potential human hazard of RDX
exposure.
1.1.4. Liver Effects
The association between RDX exposure and changes in serum liver enzymes was examined
in a single occupational epidemiology study. Case reports involving accidental exposure to RDX
provide information on the potential for acute exposure to RDX to affect the liver in humans. In
addition, organ weight, histopathology, and serum chemistry findings from experimental animal
studies involving subchronic and chronic exposure to ingested RDX provide data relevant to an
examination of the association between RDX exposure and liver effects. A summary of the liver
effects associated with RDX exposure is presented in Tables 1-10 and 1-11 and Figure 1-5.
Reports in humans provide limited evidence of liver toxicity associated with acute exposure
to RDX. Elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) were reported in several case reports of individuals who ingested unknown amounts of RDX
(Kuciikardali etal.. 2003: Woody etal.. 1986: Knepshield and Stone. 1972: Hollander and Colbach.
1969: Stone etal.. 1969: Merrill. 19681 (see Appendix C, Section C.3). Liver biopsies did not reveal
any abnormal observations (Stone etal.. 19691. In other case reports, no significant changes in
serum levels of liver enzymes were observed (Testud etal.. 1996b: Ketel and Hughes. 19721. In a
cross-sectional epidemiologic study of workers from five U.S. Army munitions plants (69 exposed to
RDX alone and 24 to RDX and HMX; mean average exposure concentration was 0.28 mg/m3)
fHathawav and Buck. 19771. serum chemistry analysis (including the serum liver enzymes AST,
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ALT, and alkaline phosphatase (ALP)) revealed no statistically significant differences between
exposed and unexposed workers (Table 1-10).
In experimental animals, the most consistent noncancer liver effect associated with RDX
exposure is elevated liver weight in studies ofsubchronic exposure (Table 1-11 and Figure 1-5).
Dose-related increases in absolute and relative liver weight were observed in male and female
B6C3Fi mice given RDX in the diet for 90 days fCholakis etal.. 19801. and in female F344 rats in two
separate 90-day dietary studies of RDX (Levine etal.. 1990: Levine etal.. 1981a: Levine etal..
1981b: Cholakis etal.. 1980). In another 90-day study, only absolute liver weights were increased
in female F344 rats exposed to RDX by gavage (Crouse et al.. 2006). The magnitude of liver weight
increases in B6C3Fi mice and female F344 rats across these studies ranged from 4-29% in the
high-dose groups. Male F344 rats did not exhibit similar increases in liver weight in other
subchronic studies fCrouse etal.. 2006: Levine etal.. 1990: Levine etal.. 1981a: Levine etal.. 1981b:
Cholakis etal.. 19801. In male and female monkeys exposed subchronically to RDX, absolute liver
weights were increased (6-16% relative to control at 1 and 10 mg/kg-day) (Martin and Hart. 1974)
and similarly in male, but not female beagle dogs (53% relative to control in male dogs at
10 mg/kg-day) fHart. 19741. Chronic RDX exposures in B6C3Fi mice and F344 or Sprague Dawley
rats showed a less consistent pattern of liver weight increases. Interpretation of liver weight
increases in 2-year studies is complicated by the incidence of adenomas and carcinomas in each
dose group; the apparent increase in liver weights in male and female mice exposed to RDX in diet
fLish etal.. 19841 was reduced when mice with liver adenomas or carcinomas were removed from
the analysis. In a 2-year rat study, absolute liver weight showed no dose-related changes; however,
relative liver weights were increased in high-dose (40 mg/kg-day) males and females (by 11 and
18% compared to controls, respectively) fLevine etal.. 19831. The changes in relative liver weight
likely reflected the depressed weight gain in the high-dose rats (2-30% in males and 10-15% in
females). Based on an evaluation of the relationship between organ weight and body/brain weight
to determine which endpoint (organ weight, organ-to-body weight ratio, or organ-to-brain weight
ratio) is likely to more accurately detect target organ toxicity, Bailey etal. (2004) concluded that
relative liver weights (expressed as organ to body weight ratios) were better modeled for
quantitative analysis than organ weight alone, or organ-to-brain weight ratios.
Nonneoplastic histopathological changes in the liver were not associated with RDX
exposure in the majority of experimental animal studies (Crouse etal.. 2006: Levine etal.. 1990:
Lish etal.. 1984: Levine etal.. 1983: Levine etal.. 1981a: Levine etal.. 1981b: Hart. 1974: Martin
and Hart. 1974: Von Oettingen etal.. 19491. including 2-year oral studies in mice at doses up to 100
mg/kg-day (Lish etal.. 1984) and in rats at doses up to 40 mg/kg-day (Levine etal.. 1983). The few
findings of liver lesions were reported in studies with more limited histopathological analyses, and
were not confirmed in the studies with more complete histopathologic examination and longer
exposure durations (Levine etal.. 1984: Lish etal.. 1984: Levine etal.. 1983: Thompson. 1983: Von
Oettingen etal.. 1949). For example, the incidence of liver portal inflammation was increased in
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female but not male rats exposed to 40 mg/kg-day in the diet for 90 days (Cholakis etal.. 19801.
There was an increase in the incidence of mild liver microgranulomas in female mice only (Cholakis
etal.. 19801 and karyomegaly of hepatocytes in male mice only exposed to 320 mg/kg-day RDX in
the diet for 90 days (Cholakis etal.. 19801. In both the rat and mouse studies by Cholakis et al.
f!9801. groups sizes were relatively small (n = 10/sex/group) and histopathologic findings were
reported for the control and high-dose groups only. It should be noted that exposure to HMX, the
primary contaminant in several of the RDX studies, was associated with histopathological changes
in the livers of male rats fed doses >450 mg/kg-day for 13 weeks. Similar findings were not
observed in the RDX studies, where the doses of RDX employed in the studies would have resulted
in HMX exposures of <60 mg/kg-day. The contribution of HMX exposure to the overall liver
findings in the studies of RDX toxicity is therefore expected to be negligible.
Clinical chemistry parameters, including serum ALT, AST, and ALP, showed no treatment-
related changes indicative of liver toxicity. Statistically significant changes in these parameters in
some subchronic and chronic toxicity studies in rats and mice were relatively small (generally
<50% of the control mean), were not dose-related in most instances, and showed no consistent
pattern of change between sexes or across studies.
Some subchronic and chronic oral toxicity studies in rats and mice reported dose-related
changes in serum cholesterol and triglyceride levels; however, these changes were not consistently
observed in males and females within the same study, and patterns of changes were not consistent
across studies. Specifically, serum triglyceride levels were elevated (up to 41%) in female B6C3Fi
mice exposed to RDX in the diet for 2 years, although increases were not dose-related (Lish etal..
19841: male mice in the same study did not show a similar increase in triglycerides. In contrast,
serum triglycerides showed dose-related decreases in male and female F344 rats (50-62% atthe
high doses) in a subchronic oral (dietary) study (Levine etal.. 1990: Levine etal.. 1981a: Levine et
al.. 1981b). In a chronic toxicity study by the same investigators (Levine etal.. 1983). serum
triglyceride levels were generally decreased in male and female rats (52 and 51%, respectively, at
the highest dose of 40 mg/kg-day); however, triglyceride levels across the four dose groups in this
study did not show a dose-related response.
Serum cholesterol levels showed a dose-related increase (38% atthe high dose of
100 mg/kg-day) in female B6C3Fi mice exposed to RDX in the diet for 2 years (Lish etal.. 1984):
however, changes in cholesterol in male mice in the same study were not dose related. Changes in
serum cholesterol in male and female F344 rats exposed to RDX in the diet for 2 years at doses up
to 40 mg/kg-day fLevine etal.. 19831. in rats exposed to RDX by gavage for 90 days at doses up to
15 mg/kg-day fCrouse etal.. 20061. and in monkeys exposed to RDX in the diet for 90 days fMartin
and Hart. 1974) were relatively small (within 38% of control mean) and were not dose related.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Table 1-10. Evidence pertaining to liver effects in humans
Reference and study design
Results
Hathawav and Buck (1977) (United States)
Liver function tests in men; mean (standard deviation not reported)
Cross-sectional study, 2,022 workers,
RDX exposed
1,491 participated (74% response rate).
Analysis group: limited to whites;
Referent
Undetected
>0.01 mg/m3
69 exposed to RDX alone and 24 exposed
Test
(n = 237)
(n = 22)
LO
II
C
to RDX and HMX; 338 not exposed to RDX,
HMX, or TNT.
LDH
173
191
174
Exposure measures: Exposure
Alkaline
82
78
80
determination based on job title and
phosphatase
industrial hygiene evaluation. Exposed
subjects assigned to two groups: less than
ALA (SGOT)
22
25
21
the limit of detection (LOD) or
AST (SGPT)
21
26
18
> 0.01 mg/m3 (mean 0.28 mg/m3).
Effect measures: Liver function tests.
Bilirubin
0.5
0.4
0.4
Analysis: Types of statistical tests were not
No differences were statistically significant as reported by study
reported (assumed to be t-tests for
authors. Similar results in women.
comparison of means and x2 tests for
comparison of proportions).
Liver function tests in men: prevalence of abnormal values
Test
RDX exposed
(abnormal
range)
Referent
Undetected
>0.01 mg/m3
LDH (>250)
2/237
1/22
0/45
Alkaline
34/237
1/22
6/45
phosphatase
(>1.5)
AST (SGOT)
20/237
4/22
2/45
(>35)
ALT (SGPT)
15/237
2/22
0/45
(>35)
Bilirubin
5/237
1/22
1/45
(>1.0)
No differences were statistically significant as reported by study
authors. Similar results in women.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Table 1-11. Evidence pertaining to liver effects in animals
Reference and study design
Results
Liver weight
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and
12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d
(high dose reduced to 100 mg/kg-d in
wk 11 due to excessive mortality)
Diet
24 mo
Doses
0 1.5 7.0 35 175/100
Absolute liver weight at 104 wks (percent change compared to control)
M
F
0% 28%* 11% 12% 35%*
0% 7% 7% 15% 18%*
Relative liver weight at 104 wks (percent change compared to control)
M
F
0% 32%* 12% 14% 46%*
0% 6% 8% 18% 45%*
Note: Percent change in liver weights of male and female mice was
reduced in all dose groups when mice with liver tumors were removed
from the analysis.
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Doses
o
1
1
rn
o
O
Absolute liver weight (percent change compared to control)
M
F
0% -6% -6% -6%
0% 7% -11% 1%
Relative liver weight (percent change compared to control)
M
F
0% -5% -2% -3%
0% 17% -2% 13%
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and
12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of
particles <66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Doses
o
o
00
LO
m
o
o
Absolute liver weight at 105 wks (percent change compared to control)
M
F
0% 3% -7% 1% -8%
0% 1% -4% 3% 0%
Relative liver weight at 105 wks (percent change compared to control)
M
F
0% 1% 0% 2% 11%
0% 1% -2% 6% 18%*
Cholakis et al. (1980)
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Absolute liver weight (percent change compared to control)
M
F
0% - - - -6% -5%
0% - - - -4% -1%
Relative liver weight (percent change compared to control)
M
F
0% - - - -4% -4%
0% - -6% 1%
Doses
0 80 160 320
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Experiment 2: 0, 40, 60, or 80 mg/kg-d
for 2 wks followed by 0, 320,160, or 80
mg/kg-d (TWA doses of 0, 79.6,147.8,
or 256.7 mg/kg-d for males and 0, 82.4,
136.3, or 276.4 mg/kg-d for females)3
Diet
13 wks
Absolute liver weight (percent change compared to control)
M
F
0% 2% 12% 26%*
0% 4% 9% 29%*
Relative liver weight (percent change compared to control)
M
F
0% 0% 9% 25%*
0% 4% 4% 22%*
Cholakis et al. (1980)
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Absolute liver weight (percent change compared to control)
M
F
0% - -2% -5%
0% - 6% 4%
Relative liver weight (percent change compared to control)
M
F
0% - 2% 3%
0% - 10% 11%
Cholakis et al. (1980)
Rats, CD, two-generation study; F0:
22/sex/group; Fl: 26/sex/group;
F2: 10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
F0 and Fl parental animals: 0, 5,16, or
50 mg/kg-d
Diet
13 wks
Doses
0 5 16 50
Absolute liver weight (percent change compared to control)
M
F
0% 7% -16%
0% 0% -14%
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Doses
0 4 8 10 12 15
Absolute liver weight (percent change compared to control)
M
F
0% -6% -9% 0% 7% 5%
0% 1% 7% 18%* 15% 28%*
Relative liver weight (percent change compared to control)
M
F
0% 0% -1% 2% 5% 2%
0% 1% -2% 2% -3% 2%
Levine et al. (1981a); Levine et al.
(1990); Levine et al. (1981b)b
Rats, F344, 3-4 wks old; 10/sex/group;
30/sex/group for controls
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 nm, ~90% of
particles <66 nm
0,10, 30,100, 300, or 600 mg/kg-d
Data were not reported for rats in the 300 or 600 mg/kg-d dose groups
because all of the rats died before the 13-wk necropsy.
Doses
0 10 30 100 300 600
Absolute liver weight (percent change compared to control)
M
F
0% 5% -1% -2%
0% 2% 4% 16%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Diet
13 wks
Relative liver weight (percent change compared to control)
M
F
0% 9% 6% 20%
0% 3% 5% 19%*
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow
containing 20 mg RDX/g-chow,
60 grams of dog food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
Doses
o
1
1
1
o
o
Absolute liver weight (percent change compared to control)
M
F
0% - - 53%
0% - - 3%
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Doses
o
1
1
1
o
o
Absolute liver weight (percent change compared to control)
M + F
0% 2% 6% 16%
Histopathological lesions
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and
12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0,1.5, 7.0, 35, or 175/100 mg/kg-d
(high dose reduced to 100 mg/kg-d in
wk 11 due to excessive mortality)
Diet
24 mo
Histopathological lesions in liver other than adenomas and carcinomas
were not significantly different compared to controls, as reported by
study authors.
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Histopathological examination performed only for controls and
10 mg/kg-d rats; no significant differences compared to controls were
reported by study authors.
Levine et al. (1983); Thompson (1983)
Rats, F344, 3-4 wks old; 75/sex/group;
interim sacrifices (10/sex/group) at
6 and 12 mo
Doses
o
o
00
LO
m
o
o
Microgranulomas (incidence)
M
0/38 0/36 0/25 0/29 0/4
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 urn
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
F
10/43 19/45 12/42 17/41 4/28
Cholakis et al. (1980)
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 urn
particle size
0, 80, 60, or 40 mg/kg-d for 2 wks
followed by 0, 80,160, or 320 mg/kg-d
(TWA doses of 0, 79.6, 147.8, or 256.7
mg/kg-d for males and 0, 82.4,136.3,
or 276.4 mg/kg-d for females)3
Diet
13 wks
Doses
0 80 160 320
Liver microgranulomas; mild (incidence)
M
F
2/10 - - 1/9
2/11 - - 7/11*
Increased karyomegaly of hepatocytes
M
F
0/10 - - 5/9*
Cholakis et al. (1980)
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Liver granulomas; mild (incidence)
M
F
0/10 - 1/10
Liver portal inflammation
M
F
2/10 - 3/10
1/10 - 7/10
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Histopathology examination of the 15 mg/kg-d group showed one male
rat with mild liver congestion and one female rat with a moderate-sized
focus of basophilic cytoplasmic alteration; neither finding was attributed
by study authors to RDX treatment.
Levine et al. (1981a);Levine et al.
(1990); Levine et al. (1981b)b
Rats, F344,10/sex/group; 30/sex for
control
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 nm, ~90% of
particles <66 nm
0,10, 30,100, 300, or 600 mg/kg-d
Diet
13 wks
Histopathological examination of liver did not reveal any significant
differences compared to controls, as reported by study authors.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow
containing 20 mg RDX/g-chow, 60
grams of dog food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
Histopathological examination performed only for controls and
10 mg/kg-d dogs; no significant differences compared to controls were
reported.
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
An increase in the amount of iron-positive material in liver cord
cytoplasm was reported in monkeys treated with 10 mg/kg-d RDX;
however, the study authors considered the toxicological significance to
be uncertain.
Serum chemistry
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and
12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0,1.5, 7.0, 35, or 175/100 mg/kg-d
(high dose reduced to 100 mg/kg-d in
wk 11 due to excessive mortality)
Diet
24 mo
Doses
0 1.5 7.0 35 175/100
Serum cholesterol at 105 wks (percent change compared to control)
M
F
0% 11% -11% 5% 39%
0% 5% 15% 25% 38%
Serum triglycerides at 105 wks (percent change compared to control)
M
F
0% 21% -20% 10% -25%
0% 34% 28% 41% 28%
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and
12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Doses
o
o
00
LO
m
o
o
Serum cholesterol at 104 wks (percent change compared to control)
M
F
0% 15% 38% 19% -6%
0% 6% 3% -7% -9%
Serum triglycerides at 104 wks (percent change compared to control)
M
F
0% 14% -15% -12% -52%
0% 18% 5% -42% -51%*
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Doses
0 4 8 10 12 15
Serum cholesterol (percent change compared to control)
M
F
0% -3% -10%* -16%* -18%* -11%*
0% -1% -8% -4% -4% -1%
Serum triglycerides (percent change compared to control)
M
F
0% 1% 1% -7% -2% -19%
0% -16% -21% 7% -37% 18%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Levine et al. (1981a);Levine et al.
(1990); Levine et al. (1981b)b
Rats, F344,10/sex/group; 30/sex for
control
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 urn, ~90% of
particles <66 urn
0,10, 30,100, 300, or 600 mg/kg-d
Diet
13 wks
Data not reported for 300 and 600 mg/kg-d dose groups because all of
the animals died before the 13-wk blood sampling.
Doses
0 10 30 100 300 600
Serum triglyceride levels (percent change compared to control)
M
F
0% -14% -34% -62%*
0% -12% -29% -50%*
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Serum biochemistry analysis revealed scattered deviations, but study
authors indicated they appear to have no toxicological significance.
Doses
o
1
1
1
o
o
Serum cholesterol (percent change compared to control)
M
F
0% -17% -2% -7%
0% 7% 7% 7%
1
2 ^Statistically significant (p < 0.05) based on analysis by study authors.
3 aDoses were calculated by the study authors.
4 bLevine et al. (1981a) is a laboratory report of a 13-week study of RDX in F344 rats; two subsequently published
5 papers (Levine et al., 1990; Levine et al., 1981b) present subsets of the data provided in the full laboratory report.
6
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ToxicologicalReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
1000
• significantly changed O not significantly changed X not determined
• M
• M
100
O M
O M
CM
>
o MO M
o MO
0.1
o
00
00
o
o
00
o
o
o
o
<3-
00
o
00
o
00
<3-
00
o
00
o
00
<3-
00
00
00
00
00
o
o
00
X
X
o
o
00
o
o
00
00
X
00
o
X
X
u
o
u
u
u
u
u
u
u
u
u
Chronic
Subchronic
Chronic
Subchronic
Chronic
Subchronic
Chronic
Sub-
chronic
si/ Cholesterol sb Trigylcerides
^ Relative liver weight
X- not determined due to confounding caused by presence of tumors
These studies were excluded from array because only absolute liver weight was reported: Cholakis, 1980 (2-gen rat); Hart, 1974; Martin and Hart, 1974
Histopathology
Serum biochemistry changes
M - Mortality observed at this dose and above
Figure 1-5. Exposure response array of liver effects following oral exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Summary of Liver Effects
There is limited evidence from reports of human exposure and from studies in experimental
animals that RDX may affect the liver. Several human case reports of short-term elevations of
serum liver enzymes in individuals who ingested unknown amounts of RDX suggest that RDX might
target the liver; however, serum liver enzymes were not elevated in a small prevalence study of
munition plant workers exposed to RDX. In experimental animals, dose-related increases in
relative or absolute liver weight were observed in multiple studies following subchronic oral
exposure, in multiple species (mice, rats, dogs, and monkeys), and in both sexes; however, an
association between RDX exposure and increased liver weight was not similarly supported by
lifetime studies in mice and rats. Changes in serum liver enzymes were not consistent across
studies and the magnitude of change relative to concurrent controls was not indicative of liver
damage. Nonneoplastic histopathologic lesions of the liver were also not consistently associated
with RDX exposure. EPA concluded that the evidence does not support liver effects as a potential
human hazard of RDX exposure.
1.1.5. Carcinogenicity
The relationship between exposure to RDX and cancer in human populations has not been
investigated. The carcinogenicity of RDX has been examined in one oral chronic/carcinogenicity
bioassay in mice fLish etal.. 19841 and two bioassays in rats fLevine etal.. 1983: Hart. 19761. The
2-year studies by Lish etal. (19841 and Levine etal. (19831 were performed in accordance with
FDA Good Laboratory Practice regulations (FDA. 19791 and included comprehensive
histopathological examination of major organs, multiple dose groups and a control, and more than
50 animals/dose group (plus additional interim sacrifice groups). The Hart T19761 study is largely
limited by lack of characterization of the test material and pathology analysis limited to the control
and high-dose groups. A temperature spike in the animal rooms on study day 76 resulted in
significant mortality across all dose groups and control animals; however, there were still more
than 80 rats/sex/group after the overheating incident and >50 rats/sex/group at termination, and
it seems unlikely that the mortality associated with the temperature spike would have affected a
tumor response in the rats. A summary of the evidence for liver and lung tumors in experimental
animals from these three bioassays is provided in Tables 1-12 and 1-13.
Liver tumors
Increased incidence of liver tumors was observed in one chronic mouse study and one of
two chronic rat studies. In the chronic mouse dietary study (Lish etal.. 19841. the combined
incidences of hepatocellular adenomas or carcinomas were increased with increasing RDX doses in
female B6C3Fi mice as compared to concurrent controls, but not in male B6C3Fi mice similarly
exposed to RDX for 2 years. In addition, the incidence of hepatocellular carcinomas showed a cant
positive trend with dose in male, but not female, F344 rats exposed to RDX in the diet for 2 years
fLevine etal.. 19831 (Cochran-Armitage trend test performed for this review, p = 0.032). On the
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other hand, there were no increased incidences of hepatocellular adenomas or carcinomas in
Sprague-Dawley rats of either sex exposed to RDX via diet for two years at doses up to 10 mg/kg-
day fHart. 19761. Incidences of hepatocellular neoplasms are presented in Table 1-12. The tumor
responses are discussed in further detail below.
In the female B6C3Fi mouse study by Lish etal. f!9841. the finding of a statistically
significant increase in hepatocellular tumors may have been influenced by the incidence of
hepatocellular adenomas/carcinomas in the concurrent female control mice, which the study
authors noted was relatively low (1/65). However, as noted by the authors, the incidence of
hepatocellular adenomas or carcinomas at RDX doses >35 mg/kg-day (19% at both doses) was also
statistically significantly elevated when compared to the mean historical control incidence for
female B6C3Fi mice in National Toxicology Program (NTP) studies (147/1781 or 8%; range:
0-20%) fHaseman etal.. 19851.5
A Pathology Working Group (PWG) review of the slides of female mouse liver lesions from
the Lish etal. (1984) study resulted in some changes in lesion diagnosis (Parker etal.. 2006: Parker.
2001). Some malignant tumors were downgraded to benign status and several lesions initially
characterized as tumors were changed to non-tumors based on more recent diagnostic criteria used
by the PWG fHarada et al.. 19991. There was a statistically significant trend in the combined
incidence of hepatocellular adenomas or carcinomas (using a Cochran-Armitage one-sided trend
test performed by EPA), consistent with the original findings of Lish etal. f!9841. Because the PWG
analysis reflects more recent histopathological criteria for the grading of tumors, the incidence of
hepatocellular adenomas or carcinomas as reported by Parker etal. (2006) were considered the
more reliable measure of liver tumor response in female mice from the Lish etal. (1984) bioassav.
As noted above, male F344 rats showed a positive trend with dose in the incidence of
hepatocellular carcinomas in the Levine etal. T19831 bioassay; however, the association with
exposure is not strong, in part reflecting the lower magnitude of response. There were only a few
tumors observed in the exposed groups (0/55, 0/52, 2/55, 2/31) relative to the control (1/55), as
compared with the mice. There is less confidence that the final incidence in the highest-dose group
accurately reflects lifetime cancer incidence because of low survival and no time-to-death
information to estimate mortality-adjusted incidences; the available information may
underestimate lifetime cancer incidence by overestimating the number of rats truly at risk. Some
perspective on the magnitude of response is provided by comparing with incidence rates in
Comparison of control incidences of hepatocellular adenomas or carcinomas between Lish et al. (19841 and
Haseman etal. Q9851 must be interpreted with caution because of cross-study differences in labs, diets, and
sources of animals. Specifically, the labs used by NTP and analyzed by Haseman et al. f19851 did not include
the lab contracted to perform the Lish et al. T19841 study, and it is not clear if the diet used in the Lish et al.
Q9841 study was included in the diets reported in the NTP studies. Further, the NTP studies included three
different suppliers of mice; one supplier was also used in the Lish et al. (19841 study. EPA Guidelines for
Carcinogenic Risk Assessment (U.S. EPA. 2005al also note that, unless the tumor is rare, the standard for
determining statistical significance of tumor incidence is a comparison of dosed animals with the concurrent
controls.
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historical controls, despite the limitation of this comparison due to the historical data originating
from a different laboratory. In a paper published concurrently with the Levine etal. (19831 study,
the NTP reported an incidence of liver carcinomas in untreated control male F344 rats of 0.7%
(12/1,719; range: 0-2%) (Haseman et al.. 19851. The incidence of liver carcinomas in control male
rats in Levine etal. T19831 was at the upper end of the NTP range, and higher than the NTP range in
the highest two dose groups. Nonmalignant liver tumors (neoplastic nodules) in F344 male rats in
the historical controls were reported more frequently than carcinomas, with an average incidence
of 3.5% (61/1,719; range: 0-12%) (Haseman et al.. 19851: Levine et al. (19831 reported a higher
incidence of neoplastic nodules, 7.3%, in their control male rats, with a decline in incidence with
increasing RDX exposure. Although there are several reasons to conclude that the observation of an
association between RDX exposure and liver tumors in rats is not strong, this suggestive site
concordance supports the response in female mice.
Table 1-12. Liver tumors observed in chronic animal bioassays
Reference and study design
Results
Lish et al. (1984); Levine et al. (1984)
Doses
LO
O
7.0
35
175/100
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
Hepatocellular adenomas (incidencef
M
8/63 6/60
1/62*
7/59
7/27
contaminant; 83-89% of particles <66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
F
1/65 1/62
6/64
6/64
3/3 lb
dose reduced to 100 mg/kg-d in wk 11
Hepatocellular carcinomas (incidencef
due to excessive mortality)
Diet
M
13/63 20/60
16/62
18/59
6/27
24 mo
F
0/65 4/62
3/64
6/64
3/3 la
Hepatocellular adenoma or carcinoma combined (incidence)
M
21/63 26/60
17/62
25/59
13/27
F
1/65 5/62
9/64*
12/64*
6/3 l*b
Pathology workgroup reanalysis of liver lesion slides from female mice
(Parker et al., 2006; Parker, 2001)°
Doses
LO
O
7.0
35
175
Hepatocellular adenomas (incidencef
F
1/67 3/62
2/63
8/64
2/3 lb
Hepatocellular carcinomas (incidencef
F
0/67 1/62
3/63
2/64
2/3 lb
Hepatocellular adenoma or carcinoma combined (incidencef
F
1/67° 4/62
5/63°
10/64
4/3 lb
Hart (1976)
Doses
o
O
3.1
10
Rats, Sprague-Dawley, 100/sex/group
Neoplastic nodules (incidence)0
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Reference and study design
Results
Purity and particle size not specified
M
0/82
-
3/77
0,1.0, 3.1, or 10 mg/kg-d
Diet
F
1/72
-
1/81
2 yrs
Hepatocellular carcinomas (incidence)0
M
1/82
-
1/77
F
1/72
-
1/81
Neoplastic nodules or hepatocellular carcinomas combined
(incidence)0
M
1/82
-
4/77
F
2/72
-
2/81
Levine et al. (1983); Thompson (1983)
Doses
m
o
o
1.5
8.0
40
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
Neoplastic nodules (incidencef
M
4/55 3/55
0/52
2/55
1/31
contaminant; 83-89% of particles <66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
F
3/53 1/55
1/54
0/55
4/48
Diet
Hepatocellular carcinomas (incidence)3
24 mo
M
1/55 0/55
0/52
2/55
2/3 lb
F
0/53 1/55
0/54
0/55
0/48
Neoplastic nodules or hepatocellular carcinomas combined
(incidencef
M
5/55 3/55
0/52
4/55
3/31
F
3/53 2/55
1/54
0/55
4/48
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2 ^Statistically significant difference compared to the control group (p < 0.05), identified by the authors.
3 aThe incidences reflect the animals surviving to month 12.
4 Statistically significant trend (p < 0.05) was identified using Cochran-Armitage trend tests performed by EPA.
5 clt is not clear why the numbers of animals at risk in the control group (n = 67) and 7 mg/kg-day dose group (n = 63)
6 differed from the numbers reported in the original study (n = 65 and 64, respectively).
7
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Lung tumors
Cochran-Armitage trend tests (as performed for this review, p = 0.019) found statistically
significant positive trends in the incidences of alveolar/bronchiolar adenomas in female B6C3Fi
mice, alveolar/bronchiolar carcinomas in male mice, and alveolar/bronchiolar adenomas or
carcinomas combined in female mice. The combined incidence in male B6C3Fi mice did not show a
statistically significant trend (see Table 1-13). In an addendum to the study report that included
results of additional examination and sectioning of lung specimens from the mid-dose groups in the
mouse study, Lish etal. (1984) noted an increase in the combined incidences of primary pulmonary
neoplasms in males of all dose groups and in females in the 7.0, 35, and 175/100 mg/kg-day dose
groups. However, the authors regarded these neoplasms as random and not biologically significant
Bioassays in rats provide no evidence of an association between RDX exposure and
induction of lung tumors. The incidence of alveolar/bronchiolar adenomas or carcinomas was not
increased in either sex of Sprague-Dawley rats exposed chronically to RDX at doses up to 10 mg/kg-
day (Hart. 1976) or in F344 rats of either sex exposed chronically to RDX at doses up to 40 mg/kg-
day (Levine etal.. 1983).
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Table 1-13. Lung tumors observed in chronic animal bioassays
Reference and study design
Results
Lish et al. (1984); Levine et al. (1984)
Doses
o
LO
O
35
175/100
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
Alveolar/bronchiolar adenomas (incidencef
M
6/63 5/60 5/62
7/59
1/27
contaminant; 83-89% of particles
<66 nm
F
4/65 2/62 5/64
9/64
3/3 lb
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
Alveolar/bronchiolar carcinomas (incidencef
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
M
3/63 6/60 3/62
7/59
5/27b
Diet
F
3/65 1/62 3/64
3/64
4/31
24 mo
Alveolar/bronchiolar adenoma or carcinoma combined (incidencef
M
9/63 11/60 8/62
14/59
6/27
F
7/65 3/62 8/64
12/64
7/3 lb
Hart (1976)
Doses
1
rn
o
O
10
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Alveolar/bronchiolar adenoma (incidence)
M
2/83
-
1/77
Diet
2 yrs
F
0/73
-
0/82
Alveolar/bronchiolar carcinoma (incidence)
None reported by study authors.
Levine et al. (1983); Thompson (1983)
Doses
LO
m
o
o
8.0
40
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
Alveolar/bronchiolar adenomas (incidencef
M
1/55 0/15 1/17
0/16
1/31
contaminant; 83-89% of particles
<66 nm
F
3/53 0/7 0/8
1/10
0/48
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Alveolar/bronchiolar carcinomas (incidencef
Diet
24 mo
M
- - -
-
-
F
0/53 0/7 1/8
0/10
0/48
Alveolar/bronchiolar adenoma or carcinoma combined (incidence)3
M
F
3/53 0/7 1/8
1/10
0/48
1
2 aThe incidences reflect the animals surviving to month 12.
3 Statistically significant trend (p < 0.05) was identified using Cochran-Armitage trend test performed by EPA.
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Mechanistic Evidence
There are few mechanistic data to support a MOA determination for either liver or lung
tumors induced by exposure to RDX.
The increase in liver weights observed in subchronic studies of RDX in mice (Cholakis etal..
19801 and rats fLevine etal.. 1990: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 19801
and chronic studies in female B6C3Fimice fLish etal.. 1984: Cholakis etal.. 19801 raises the
possibility of RDX-related liver cell proliferation as a precursor to tumorgenicity. Sweeney et al.
(2012bl reviewed hypothesized MOA's for carcinogenicity and concluded that a MOA involving a
proliferative response generated by tissue-derived oxidative metabolites of RDX was the most
plausible of the MOAs considered, but acknowledged that the overall support for this MOA was
limited. The following lines of evidence do not support a metabolite-based proliferative response
as the MOA for RDX carcinogenicity:
• the absence of significant liver histopathology in mice after subchronic or chronic exposure
to RDX at doses that induced liver tumors fLish etal.. 1984: Cholakis etal.. 19801 suggests
that cellular toxicity is not a precursor to these tumors;
• increased liver weight was also observed in rats and male mice where tumors did not occur;
• no studies were available that directly measured RDX-induced cell proliferation rates; and
• no information was available to rule out non-precancerous causes of liver weight increase.
The available in vitro and in vivo genotoxicity assay results are largely negative for parent
RDX (see Appendix C, Section C.4), supporting the hypothesis that parent RDX does not interact
directly with DNA. Sweeney etal. f2012bl proposed that the increased incidence of liver adenomas
and carcinomas in female mice (Parker etal.. 2006: Lish etal.. 19841 may result from liver-
generated metabolites as the most likely agents responsible for liver tumors. Sweeney et al.
(2012bl estimated an approximately 30-fold higher metabolic rate for RDX in mice (which
displayed a more robust liver tumor response to RDX exposure than did rats) compared with rats
based on the results of a PBPK model. These authors hypothesized a non-linear, cell proliferation
MOA in conjunction with the lack of evidence to support a genotoxic/mutagenic MOA for RDX or its
oxidative metabolites. Sweeney etal. f2012bl suggest that RDX is unlikely to be genotoxic because
it does not induce tumors at multiple sites and species. This observation is inconsistent with the
finding in Lish etal. (19841 that showed positive trends in the incidence of both
alveolar/bronchiolar adenomas or carcinomas and liver tumors.
In contrast to the negative results for RDX oxidative metabolites, there are some positive
genotoxicity results for the /V-nitroso metabolites of RDX, specifically hexahydro-l-nitroso-
3,5-dinitro-l,3,5-triazine [MNX) andhexahydro-l,3,5-trinitroso-l,3,5-triazine (TNX). MNXand
TNX have been identified from minipigs; minipigs were chosen as the animal model for the
metabolism of RDX because the gastrointestinal tract of pigs more closely resembles that of humans
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(Musick etal.. 2010: Major etal.. 20071. MNX has tested positive in some in vitro assays, including
unscheduled DNA synthesis in primary rat hepatocytes and the mouse lymphoma forward mutation
assay fSnodgrass. 19841. although MNX tested negative in the only in vivo test performed, a mouse
dominant lethal mutation test fSnodgrass. 19841. MNX was not mutagenic in S. typhimurium
(strains TA98, TA100, TA1535, TA1537, and TA1538), with or withoutthe addition of the S9
metabolic activating mixture fPan etal.. 2007: Snodgrass. 19841. WhenS. typhimurium strains
TA97a and TA102, strains sensitive to frame shift and oxidative DNA damage, were used in
conjunction with elevated concentrations of the metabolizing system (S9), MNX and TNX were
mutagenic. /V-nitroso metabolites, including MNX and TNX, are generated anaerobically and are
likely a result of bacterial transformation of parent RDX in the gastrointestinal tract to various N-
nitroso derivatives (Pan etal.. 20071. Exposure to potentially mutagenic N-nitroso metabolites of
RDX generated in the gastrointestinal tract of mice may occur in the liver (and subsequently in the
systemic circulation) via enterohepatic circulation. However, in pigs the N-nitroso metabolites of
RDX have been identified only in trace amounts in urine compared to the major metabolites, 4-
nitro-2,4-diazbutanal and 4-nitro-2,4-diazbutanamide. Thus, the contribution of the N-nitroso
metabolites to the overall carcinogenic potential of RDX is unclear.
Aberrant expression of microRNAs (miRNAs) was observed in the brains and livers of
female B6C3Fi mice fed 5 mg RDX/kg in the diet for 28 days (Zhang and Pan. 2009b). with several
oncogenic miRNAs being upregulated, while several tumor-suppressing miRNAs were down
regulated. However, the pattern of induction was not always consistent in the livers of RDX-treated
mice (e.g., mIR-92a was downregulated in liver tissue samples when it is typically upregulated in
hepatocellular carcinomas) (Sweeney et al.. 2012b). miRNAs have been associated with several
cancers (Wiemer. 2007: Zhang etal.. 2007): however, the utility of miRNAs as predictive of
carcinogenesis has not been established, and whether or not aberrant expression of a specific
miRNA (or suite of miRNA's) plays a role in the MOA of RDX carcinogenicity is unknown.
Microarray analysis of gene expression in male Sprague-Dawley rats after exposure to a single oral
(capsule) dose of RDX revealed a general up-regulation in gene expression (predominantly genes
involved in metabolism) in liver tissues (Bannon etal.. 2009): however, the relevance of this finding
to the carcinogenicity of RDX is unclear.
In summary, the available evidence indicates that RDX is not mutagenic (see Appendix C,
Section C.4); however, anaerobically-derived N-nitroso metabolites have demonstrated some
genotoxic potential. While these metabolites have been measured in the mouse (Pan etal.. 2007)
and minipig f Musick etal.. 2010: Maior etal.. 20071. they have not been identified in humans, and
may not be the predominant metabolites of RDX. A MOA involving a proliferative response
generated by tissue-derived oxidative metabolites of RDX has been proposed, but is not supported
by the available data. In light of limited information on precursor events leading to the observed
liver and lung tumor response in RDX-exposed rodents and lack of toxicokinetic information on
RDX metabolites, neither a cell proliferative MOA or a mutagenic N-nitroso metabolite MOA is
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supported. Thus, the MOA leading to the increased incidence of liver and lungs tumors is not
known.
1.1.6. Other Toxicological Effects
There is limited evidence that RDX can produce systemic effects in several organs/systems,
including the eyes, and the musculoskeletal, cardiovascular, immune, and gastrointestinal systems.
However, there is less evidence for these effects compared to organ systems described earlier in
Section 1.1. A summary of the evidence for toxicological effects in other organ systems is shown in
Tables 1-14 and 1-15.
Ocular Effects
There are no reports of ocular effects in human case reports or epidemiological studies.
The incidence of cataracts was statistically significantly increased in high-dose female rats in one
chronic oral study; however, this finding was not reproduced in other subchronic and chronic
studies in rats or mice.
The incidence of cataracts was 73% in female F344 rats exposed to 40 mg/kg-day RDX in
the diet for 2 years, compared to 32% in the control group fLevine etal.. 19831. After 76 weeks of
exposure, the incidence of cataracts in female rats at 40 mg/kg-day (23%) was also elevated
compared to controls (6%). The incidence of cataracts was not increased in RDX-exposed male rats
in the same study fLevine etal.. 19831. and other studies have not observed ocular effects
associated with RDX exposure. Only 2 rats (dose groups not reported) were observed to have mild
cataracts in a 90-day study of male and female F344 rats exposed to RDX at doses up to 15 mg/kg-
day by gavage; however, the authors noted that these observations are common in F344 rats at
4 months of age and should not be attributed to treatment fCrouse etal.. 20061. Furthermore,
cataracts were not observed in male or female F344 rats exposed to 40 mg/kg-day RDX by diet for
90 days (Cholakis etal.. 19801. or in male or female B6C3Fi mice exposed to RDX in the diet for
2 years at doses up to approximately 100 mg/kg-day (Lish etal.. 19841. A statistically significant
increase in the incidence of cataracts in male mice was initially noted by Lish etal. (19841. but was
not confirmed when mice used for orbital bleedings were excluded from the analysis, suggesting
the effect was not treatment related.
Cardiovascular Effects
Human evidence for cardiovascular effects is limited to case reports that include
observations of transient arterial hypertension in male Italian workers following inhalation of RDX
during manufacturing (Barsotti and Crotti. 19491. sinus tachycardia, and in one instance premature
ventricular beats in 5 men following accidental ingestion of RDX at 37-250 mg/kg body weight
(Kuctikardali etal.. 20031 (see Appendix C, Section C.3).
Inconsistent observations of cardiovascular effects have been reported in animal studies.
An increase in the relative heart-to-body weight ratio was observed at the highest dose tested in
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B6C3Fi mice (male: 13%; female 17%) and F344 rats (male: 22%; female 15%) following chronic
dietary administration of RDX (Lish etal.. 1984: Levine etal.. 1983): however, this dose also
resulted in reductions in body weight in both males and females. Dose-related decreases in
absolute heart weight were reported following subchronic exposures to RDX in the diet (Levine et
al.. 1990: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 19801. while a subchronic study in
male dogs reported a 31% increase in absolute heart weight at the highest dose tested
(10 mg/kg-day) fHart. 19741.
Evidence for histopathologic changes associated with RDX exposure is limited to findings of
an increased incidence of focal myocardial degeneration in female rats compared to controls (60 vs.
20%, respectively) and male mice (50 vs. 0%, respectively) following exposure to RDX in the diet
for 90 days (Cholakis etal.. 1980). In each study, the finding of myocardial degeneration was
limited to one sex and to the high-dose group only. Other studies in monkeys fMartin and Hart.
1974) and rats (Von Oettingen et al.. 1949) reported no observable cardiovascular effects.
Musculoskeletal Effects
Evidence of musculoskeletal effects in humans consists of case reports that include
observations of muscle twitching, myalgia/muscle soreness, and muscle injury as indicated by
elevated levels of AST or myoglobinuria fKiiciikardali etal.. 2003: Hettand Fichtner. 2002:
Hollander and Colbach. 1969: Stone etal.. 1969: Merrill. 19681 (see Appendix C, Section C.3).
Histological evaluations of musculature or skeletal tissue did not reveal any alterations in
mice (Lish etal.. 1984) or rats (Levine etal.. 1983: Hart. 1976) following chronic oral exposure to
RDX, in mice and rats following subchronic exposure (Cholakis etal.. 1980). or in dogs following a
90-day dietary exposure (Hart. 1974).
Immune Effects
RDX is structurally similar to various drugs known to induce the autoimmune disorder
systemic lupus erythematosus (SLE). Three cases of SLE were initially identified among workers at
one U.S. Army munitions plant; however, upon further investigation of 69 employees at five U.S.
Army munitions plants with potential exposure to RDX, no additional cases of SLE were identified
(Hathaway and Buck. 1977). Increased white blood cell (WBC) counts have been reported in some
case reports of individuals who ingested RDX or C-4 (91% RDX) fKnepshield and Stone. 1972:
Hollander and Colbach. 1969: Stone etal.. 1969: Merrill. 19681.
In animal studies, increased WBC count in female rats following subchronic dietary
exposure to RDX was the only dose-related immune effect reported (Levine etal.. 1990: Levine et
al.. 1981a: Levine etal.. 1981b): WBC counts in male rats were unaffected. Conversely, decreased
WBC counts in were reported in male and female rats in a 2-year study (Hart. 1976). Changes in
spleen weights were observed across studies, but the responses were not consistent and did not
appear to be dose-related. For example, in 90-day studies, Cholakis etal. f 19801 identified a
statistically significant decrease in absolute spleen weight in F344 rats at 40 mg/kg-day, while
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Crouse etal. (2006) observed an increase in spleen weight at 15 mg/kg-day (not statistically
significant). Across studies, there was no significant or dose-dependent pattern of response to
suggest that the WBC changes reflect RDX-induced immunotoxicity. No dose-related immune
effects from oral exposure to RDX were observed in other animal studies, including a 90-day study
in F344 rats specifically designed to evaluate immunotoxicity (parameters included evaluation of
red and white blood cell populations, proportion of cell surface markers, cellularity in proportion to
organ weight, B and T cells in the spleen, and CD4/CD8 antigens of maturing lymphocytes in the
thymus) (Crouse et al.. 2006). Routine clinical and histopathology evaluations of immune-related
organs in a two-generation study in rats (Cholakis et al.. 1980) and chronic studies in rats (Levine et
al.. 19831 and mice fLish etal.. 19841 provide no evidence of immunotoxicity associated with oral
(dietary) exposure to RDX.
In summary, evidence for immunotoxicity associated with RDX exposure is limited to
findings from one study of increased WBC counts in female rats (Levine etal.. 1981a: Levine etal..
1981b). Evidence that RDX is not immunotoxic comes from several animal studies, including other
repeat-dose oral studies in mice and rats (Crouse etal.. 2006: Lish etal.. 1984: Levine etal.. 1983:
Cholakis etal.. 1980).
Gastrointestinal Effects
Clinical signs of nausea and/or vomiting have been frequently identified in case reports of
accidental or intentional RDX poisonings, and generally concurrent with severe neurotoxicity
(Kasuske etal.. 2009: Davies etal.. 2007: Kuctikardali etal.. 2003: Hettand Fichtner. 2002: Ketel
and Hughes. 1972: Knepshield and Stone. 1972: Hollander and Colbach. 1969: Stone etal.. 1969:
Merrill. 1968: Kaplan etal.. 1965: Barsotti and Crotti. 1949) (see Appendix C, Section C.3). In
animal studies, nausea and vomiting have also been observed following oral exposure of swine
fMusick etal.. 20101. dogs fHart. 19741. and monkeys fMartin and Hart. 19741. One subchronic oral
(diet) rat study from the early literature reported congestion of the gastrointestinal tract at doses
also associated with elevated mortality (Von Oettingen et al.. 19491: however, none of the
subsequent subchronic or chronic animal studies reported histological findings of the
gastrointestinal tract related to RDX administered via gavage or the diet (Crouse etal.. 2006: Lish et
al.. 1984: Levine etal.. 1983: Hart. 1974: Martin and Hart. 1974).
Hematological Effects
Elevated prevalence odds ratios (OR) for hematological abnormalities were observed in a
case-control study of males (32 exposed, 322 controls) exposed to RDX in an occupational setting
(West and Stafford. 1997) (see Table 1-14). The prevalence OR for an association between RDX
exposure and hematological abnormalities was 1.7 (95% CI 0.7-4.2) for men with greater than 50
hours of low intensity exposure, while the prevalence OR was 1.2 (95% CI 0.3-5.3) for men with
>50 hours of high intensity exposure. The ORs from this study must be interpreted with caution
given the small sample size and wide confidence intervals. No changes in hematological parameters
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
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13
14
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18
19
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22
23
24
25
26
27
28
Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
(including hemoglobin, hematocrit, and reticulocyte count) were observed in a cross-sectional
epidemiologic study of 69 workers exposed to RDX by inhalation (average of 0.28 mg/m3)
fHathawav and Buck. 19771. Humans who ingested or inhaled unknown amounts of RDX or C-4
(~91% RDX) for an acute duration displayed temporary hematological alterations, including
anemia, decreased hematocrit, hematuria, and methemoglobinemia fKasuske etal.. 2009:
Kuctikardali etal.. 2003: Knepshield and Stone. 1972: Hollander and Colbach. 1969: Stone etal..
1969: Merrill. 1968). In other case reports, normal blood counts were observed in accidentally
exposed individuals (Testud etal.. 1996b: Goldberg etal.. 1992: Woody etal.. 1986: Ketel and
Hughes. 1972: Kaplan etal.. 1965) (see Appendix C, Section C.3).
In animals, hematological alterations were observed following oral exposure in chronic and
subchronic studies in both sexes of rats (F344 or SD) and B6C3Fimice (see Table 1-15). Increases
in platelet count were observed in male and female mice and rats in some subchronic and chronic
studies at doses from 0.3 mg/kg-day to 320 mg/kg-day (Lish etal.. 1984: Levine etal.. 1983:
Cholakis etal.. 1980): however, findings were generally inconsistent across studies and were not
necessarily dose-dependent Similarly, decreased hemoglobin levels/anemia were observed in
some chronic and subchronic studies (Levine etal.. 1983: Cholakis etal.. 1980: Von Oettingen et al..
19491. particularly at doses greater than or equal to 15 mg/kg-day, but trends in hemoglobin levels
across studies did not show a consistent relationship with dose. Other hematological parameters,
including WBC counts, reticulocyte counts, and hematocrit, showed conflicting results between
studies, marginal responses, or inconsistent changes with increasing dose. Other subchronic
studies in rats and dogs (Crouse etal.. 2006: Hart. 1974: Von Oettingen etal.. 1949) did not identify
any changes in hematological parameters.
In summary, evidence for hematological effects associated with RDX exposure in humans
comes from several case reports that found transient fluctuations in hematological endpoints after
acute exposures. Hematological findings from two epidemiological studies were inconsistent and
difficult to interpret because of small sample sizes (Table 1-14). In general, animal studies of
chronic and subchronic durations showed no consistent, dose-related pattern of increase or
decrease in hematological parameters.
Table 1-14. Evidence pertaining to systemic effects (hematological) in
humans
Reference and study design
Results
Hematological effects
West and Stafford (1997) (United Kingdom)
Case-control study, 32 cases with abnormal
and 322 controls with normal hematology test
drawn from 1991 study of 404 workers at
ammunitions plant; participation rate 97% of
Odds ratio (95% CI) [number of exposed cases] of blood
disorder and RDX
Low intensity, 50 hr-duration 1.7 (0.7,4.2) [22]
Medium intensity, 50-hr 1.6 (not reported) [5]
duration
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Hematological effects
cases, 93% of controls. Analysis limited to men
(29 cases, 282 controls).
Exposure measures: Exposure determination
based on employee interviews and job title
analysis; data included frequency (hrs/d, d/yr),
duration (yrs), and intensity (low [1-10 ppm],
moderate [10-100 ppm], and high
[100-1,000 ppm], based on ventilation
considerations).
Effect measures: Hematology tests; blood
disorder defined as neutropenia (2.0 x 109/L),
low platelet count (<150 x 109/L), or
macrocytosis (mean corpuscular volume = 99 fl
or >6% macrocytes).
Analysis: Unadjusted odds ratio.
High intensity, 50-hr duration 1.2 (0.3, 5.3) [2]
Hathawav and Buck (1977) (United States)
Cross-sectional study, 2,022 workers,
1,491 participated (74% response rate).
Analysis limited to whites; 69 exposed to RDX
alone and 24 exposed to RDX and HMX; 338
not exposed to RDX, HMX, or TNT.
Exposure measures: Exposure determination
based on job title and industrial hygiene
evaluation. Exposed subjects assigned to two
groups: 0.01 mg/m3 (mean
0.28 mg/m3).
Effect measures: Hematology tests.
Analysis: Types of statistical tests were not
reported (assumed to be t-tests for
comparison of means and x2 tests for
comparison of proportions).
Hematology tests in men; mean (standard deviation not
reported)
Test
RDX exposed
Referent Undetected >0.01 mg/m3
(n = 237) (n = 22) (n = 45)
Hemoglobin
Hematocrit
Reticulocyte
count
15.2 14.7 15.2
42 45.6 47
0.7 0.9 0.7
No differences were statistically significant. Similar results in
women.
Hematology tests in men: prevalence of abnormal values
Test
(abnormal
range)
RDX exposed
Referent Undetected >0.01 mg/m3
Hemoglobin
(< 14)
Hematocrit
(<40)
Reticulocyte
count (>1.5)
15/237 3/22 4/45
1/237 1/22 1/45
18/237 3/22 2/45
No differences were statistically significant. Similar results in
women.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Table 1-15. Evidence pertaining to systemic effects in animals
Reference and study design
Results
Ocular effects
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
Doses
0 1.5 7.0 35 175/100
Cataracts; 103 wks (incidence)0
M
F
2/47 2/41 0/41 2/37 2/16
2/50 1/37 6/52 0/46 1/26
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Doses
o
o
00
LO
m
o
o
Cataracts; 103 wks (incidence)
M
F
8/40 6/39 6/31 8/35 2/6
14/44 4/48 11/44 8/43 22/30*
Cholakis et al. (1980)
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
No ocular effects were observed (gross examination of eye was
performed in all animals, and microscopic examination in control and
40 mg/kg-d animals).
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
No ocular effects were observed (ophthalmic examinations were
performed in all animals within 1 wk of sacrifice, and microscopic
examination of the eye was performed in control and 15 mg/kg-
danimals).
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
No ocular effects were observed (ophthalmoscopic examination was
performed at the end of exposure).
Cardiovascular effects
Lish et al. (1984); Levine et al. (1984)
Doses
0 1.5 7.0 35 175/100
This document is a draft for review purposes only and does not constitute Agency policy.
1-54 DRAFT—DO NOT CITE OR QUOTE
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
Absolute heart weight; 104 wks (percent change compared to control)
M
F
0% 4% 4% 5% 7%
0% 1% 5% 2% -5%
Relative heart-to-body weight; 104 wks (percent change compared to
control)
M
F
0% 7% 5% 5% 13%*
0% 0% 6% 4% 17%*
Body weight was significantly lower at termination in males and
females exposed to 175/100 mg/kg-d (-5 and -19%, respectively).
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Doses
0 1.0 3.1 10
Myocardial fibrosis (percent incidence; number not reported)
M
F
20% - - 5%
5% - - 1%
Endocardial disease (percent incidence; number not reported)
M
F
1% - - 3%
0% - - 0%
Absolute heart weight; 104 wks (percent change compared to control)
M
F
0% -6% -2% -5%
0% 13% 3% 15%
Relative heart-to-body weight; 104 wks (percent change compared to
control)
M
F
0% -2% 4% 1%
0% 23% 13% 27%
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Doses
0 0.3 1.5 8.0 40
Absolute heart weight; 104 wks (percent change compared to control)
M
F
0% 3% -2% -2% 1%
0% -1% 0% -4% -3%
Relative heart-to-body weight; 104 wks (percent change compared to
control)
M
F
0% 2% 6% 0% 22%
0% -2% 3% -1% 15%
Cholakis et al. (1980)
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
Doses
0 10 14 20 28 40
Absolute heart weight (percent change compared to control)
M
F
0% - 7% 7%
0% - - - 0% 0%
This document is a draft for review purposes only and does not constitute Agency policy.
1-55 DRAFT—DO NOT CITE OR QUOTE
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
13 wks
Relative heart weight (percent change compared to control)
M
F
0% - - - 6% 0%
0% - - - -4% 0%
Experiment 2: 0, 40, 60, or 80 mg/kg-d
for 2 wks followed by 0, 320,160, or 80
mg/kg-d (TWA doses of 0, 79.6,147.8,
or 256.7 mg/kg-d for males and 0, 82.4,
136.3, or 276.4 mg/kg-d for females)b
Diet
13 wks
Doses
0 80 160 320
Focal myocardial degeneration (incidence)
M**
p* * *
0/10 - - 5/10*
0/11 - - 2/11
Absolute heart weight (percent change compared to control)
M
F
0% 0% 0% 8%
0% 0% 0% 8%
Relative heart-to-body weight (percent change compared to control)
M
F
0% 0% -2% 6%
0% 0% -2% 2%
**lncludes one affected and three unaffected animals that died
prematurely.
***lncludes one unaffected animal that died prematurely.
Cholakis et al. (1980)
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Focal myocardial degeneration (incidence)
M
F
3/10 - 1/10
2/10 - 6/10
Absolute heart weight (percent change compared to control)
M
F
0% - - - 0% -8%*
0% - -6% -11%*
Relative heart-to-body weight (percent change compared to control)
M
F
0% - - - 3% 0%
0% - - - -3% -8%
Relative heart-to-brain weight (percent change compared to control)
M
F
0% - -4% -10%*
0% - -5% -11%*
Cholakis et al. (1980)
Rats, CD, two-generation study; F0:
22/sex/group; Fl: 26/sex/group; F2:
10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
No cardiac effects were observed (microscopic examination of heart
was performed in randomly selected F2 animals).
Heart weight data were reported only for F2 generation controls, 5 and
16 mg/kg-day groups.
Doses
0 5 16 50
Absolute heart weight (percent change compared to control)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
FO and F1 parental animals: 0, 5,16, or
50 mg/kg-d
Diet
13 wks
F2 M
F2 F
0% 3.2% -6.5%
0% 15% -3.7%
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
Doses
0 4 8 10 12 15
Cardiomyopathy (incidence)
M
F
2/10 - - - - 3/8
0/10 - - - - 1/6
Absolute heart weight (percent change compared to control)
M
F
0% -2% -7% -1% 1% 11%
0% -2% 0% 8% 1% 6%
Relative heart-to-body weight (percent change compared to control)
M
F
0% 4% 2% 1% -1% 8%
0% -2% -7% -6% -9% -16%*
Levine et al. (1981a);Levine et al. (1990);
Levine et al. (1981b)c
Rats, F344,10/sex/group; 30/sex for
control
84.7 ± 4.7% purity, ~10% HMX, median
particle diameter 20 nm, ~90% of
particles <66 nm
0,10, 30,100, 300, or 600 mg/kg-d
Diet
13 wks
All animals in the 300 and 600 mg/kg-d groups died prior to study
termination.
Doses
0 10 30 100 300 600
Chronic focal myocarditis (incidence)
M
F
8/30 8/10 6/10 1/10 1/10 0/10
8/30 3/10 1/10 1/10 1/10 1/9
Absolute heart weight (percent change compared to control)
M
F
0% -2% -10% -15%
0% -3% 0% -5%
Relative heart-to-body weight (percent change compared to control)
M
F
0% 2% -4% 3%
0% -2% 0% -3%
Von Oettingen et al. (1949)
Rats (sex/strain not specified); 20/group
Purity and particle size not specified
0,15, 25, or 50 mg/kg-d
Diet
3 mo
The study authors reported that there were no cardiac effects
(microscopic examination of the heart was performed in all rats; data
were not shown).
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow
containing 20 mg RDX/g-chow, 60 grams
of dog food
Doses
o
1
1
1
o
o
Focal hyalinization of the heart (incidence)
M
F
0/3 - - 0/3
0/3 - - 1/3
This document is a draft for review purposes only and does not constitute Agency policy.
1-57 DRAFT—DO NOT CITE OR QUOTE
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
O, 0.1,1, or 10 mg/kg-d
Absolute heart weight (percent change compared to control)
Diet
90 d
M
F
0% - - 31%
0% - - 5.7%
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Doses
o
1
1
1
o
o
Myocarditis (percent change compared to control)
M
F
1/3 - - 1/3
0/3 - - 0/3
Absolute heart weight (percent change compared to control)
M
F
0% 7% -1% 5%
0% 10% 12% -12%
Immune effects
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
No immune effects were observed with routine hematology, clinical
chemistry, or histopathology evaluations.
Doses
0 1.5 7.0 35 175/100
WBC count; 105 wks (percent change compared to control)
M
F
0% -13% -8% -16% -30%
0% 12% 39%* 28% 0%
Absolute spleen weight; 105 wks (percent change compared to
control)
M
F
0% 24% 31% -10% -28%
0% 4% 15% -17% 16%
Relative spleen weight; 105 wks (percent change compared to control)
M
F
0% 26% 32% -11% -21%
0% 4% 15% -17% 44%
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Doses
o
l->
o
w
h->
o
WBC count; 104 wks (percent change compared to control)
M
F
0% -13% -22%* -34%*
0% 5% -32%* -12%
Absolute spleen weight; 104 wks (percent change compared to
control)
M
F
0% -11% -16% -4%
0% 58% 8% 37%
Relative spleen weight; 104 wks (percent change compared to control)
M
0% -11% -14% 1%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
F
0% 77% 19% 55%
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
No immune effects were observed with routine hematology, clinical
chemistry and histopathology evaluations.
Doses
o
o
00
LO
m
o
o
WBC count; 105 wks (percent change compared to control)
M
F
0% -11% 103%d 184%d 15%
0% 7% 12% 354%d 251%d
Absolute spleen weight; 105 wks (percent change compared to
control)
M
F
0% 5% -10% -32% -49%
0% -28% -44% -35% 17%
Relative spleen weight; 105 wks (percent change compared to control)
M
F
0% 9% 4% -29% -38%
0% -34% -45% -36% 9%
Cholakis et al. (1980)
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
particle size
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
13 wks
Doses
0 10 14 20 28 40
Absolute spleen weight (percent change compared to control)
M
F
0% - 18% 13%
0% - - - -2% -8%
Relative spleen weight (percent change compared to control)
M
F
0% - 24% 14%
0% - - - -3% -5%
Experiment 2: 0, 40, 60, 80 mg/kg-d for
2 wks followed by 0, 320,160, or 80
mg/kg-d (TWA doses of 0, 79.6,147.8,
or 256.7 mg/kg-d for males and 0, 82.4,
136.3, or 276.4 mg/kg-d for females)b
Diet
13 wks
Doses
0 80 160 320
WBC count (percent change compared to control)
M
F
0% -27% -12% 30%
0% -17% 3% -3%
Absolute spleen weight (percent change compared to control)
M
F
0% 17% 0% -17%
0% -22% 0% 0%
Relative spleen weight (percent change compared to control)
M
F
0% 25% 5% 0%
0% -12% 0% -3%
Cholakis et al. (1980)
Rats, F344,10/sex/group
Doses
0 10 14 20 28 40
WBC count (percent change compared to control)
M
0% - -12% 7%
This document is a draft for review purposes only and does not constitute Agency policy.
1-59 DRAFT—DO NOT CITE OR QUOTE
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 urn
particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
13 wks
F
0% - 17% 30%
Absolute spleen weight (percent change compared to control)
M
F
0% - 2% -4%
0% - -10% -12%*
Relative spleen weight (percent change compared to control)
M
F
0% - - - 5% 5%
0% - - - -8% -8%
Cholakis et al. (1980)
Rats, CD, two-generation study; FO:
22/sex/group; Fl: 26/sex/group;
F2: 10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 urn
particle size
FO and Fl parental animals: 0, 5,16, or
50 mg/kg-d
Diet
13 wks
No immune effects were observed upon routine histopathology
evaluation.
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
No effects were observed on thymus or spleen histology, red and white
blood cell populations, or lymphocyte populations.
Doses
0 4 8 10 12 15
WBC count (percent change compared to control)
M
F
0% -5% -12% -7% 1% -3%
0% 22% 45% 12% 52% 29%
Absolute spleen weight (percent change compared to control)
M
F
0% -3% -6% 3% 1% 5%
0% 1% 8% 23%* 17%* 24%*
Relative s
M
F
pleen weight (percent change compared to control)
0% 3% 4% 7% -1% 2%
0% 1% 0% 6% -1% -2%
Absolute thymus weight (percent change compared to control)
M
F
0% -1% 3% -10% -12% -25%
0% -7% 12% 19% 32% 19%
Relative thymus weight (percent change compared to control)
M
F
0% -1% 3% -10% -12% -25%
0% -7% 4% 4% 12% -6%
Levine et al. (1981a);Levine et al. (1990);
Levine et al. (1981b)c
Data were not reported for rats in the 300 or 600 mg/kg dose groups
because all of the rats died before the 13-wk necropsy.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Rats, F344,10/sex/group; 30/sex for
control
84.7 + 4.7% purity, ~10% HMX, median
particle diameter 20 urn, ~90% of
particles < 66 urn
0,10, 30,100, 300, or 600 mg/kg-d
Diet
13 wks
Doses
0 10 30 100 300 600
WBC count (percent change compared to control)
M
F
0% 4% 7% 15%
0% 23%* 24%* 62%*
Absolute spleen weight (percent change compared to control)
M
F
0% -11% -16% -34%
0% 2% 12% 0%
Relative spleen weight (percent change compared to control)
M
F
0% -9% -12% -21%
0% 2% 12% 3%
Von Oettingen et al. (1949)
Rats, sex/strain not specified, 20/group
90-97% pure, with 3-10% HMX; particle
size not specified
0,15, 25, or 50 mg/kg-d
Diet
3 mo
Doses
0 15 25 50
WBC count (percent change compared to control)
M
0% -30% 7% -6%
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow
containing 20 mg RDX/g-chow, 60 grams
of dog food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
Doses
o
1
1
1
o
o
WBC count (percent change compared to control)
M
F
0% 5% 2% -19%
0% -2% 24% 6%
Absolute spleen weight (percent change compared to control)
M
F
0% - - 123%
0% - - -11%
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Doses
o
1
1
1
o
o
WBC count (percent change compared to control)
M
F
0% -32% 0% -3%
0% -38% -1% -41%
Gastrointestinal effects
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Reference and study design
Results
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
No gastrointestinal tract effects were observed as clinical signs or on
gross pathology or histopathology examination.
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
No gastrointestinal tract effects were observed as clinical signs or on
gross pathology or histopathology examination.
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
90 d
No gastrointestinal tract effects were observed on gross pathology or
histopathology examination. Increased salivation and blood stains
around the mouth were noted (affected doses and incidences were not
reported); it is not clear whether these effects occurred in animals also
experiencing convulsions.
Von Oettingen et al. (1949)
Rats (sex/strain not specified); 20/group
90-97% pure, with 3-10% HMX; particle
size not specified
0,15, 25, or 50 mg/kg-d
Diet
3 mo
Congestion of the gastrointestinal tract was observed in 50 and 100
mg/kg-d rats that also exhibited mortality (40%) and severe
neurotoxicity.
Martin and Hart (1974)
Monkeys (Cynomolgus or Rhesus);
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 d
Vomiting was observed more frequently in the 1 and 10 mg/kg-d
groups compared to the control or 0.1 mg/kg-d groups, although some
episodes occurred during the intubation procedure.
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow
containing 20 mg RDX/g-chow, 60 grams
of dog food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
Some nausea and vomiting were reported (incidences and affected
dose groups were not reported).
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Reference and study design
Results
Hematological effects
Lish et al. (1984); Levine et al. (1984)
Mice, B6C3Fi, 85/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 urn
0,1.5, 7.0, 35, or 175/100 mg/kg-d (high
dose reduced to 100 mg/kg-d in wk 11
due to excessive mortality)
Diet
24 mo
Doses
0 1.5 7.0 35 175/100
RBC count; 105 wks (percent change compared to control)
M
F
0% -4% 3% -3% 14%
0% 4% -7% 5% 3%
Hemoglobin; 105 wks (percent change compared to control)
M
F
0% -6% 3% -5% 9%
0% 2% -7% 3% 1%
Hematocrit; 105 wks (percent change compared to control)
M
F
0% -4% 3% -4% 9%
0% 3% -6% 3% 1%
Platelets; 105 wks (percent change compared to control)
M
F
0% 33% 9% 21% 27%
0% -14% -7% 1% 5%
Hart (1976)
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Diet
2 yrs
Doses
o
1
1
rn
o
O
RBC count; 104 wks (percent change compared to control)
M
F
0% 3% 7% -2%
0% -14% 7% 2%
Reticulocyte count; 104 wks (percent change compared to control)
M
F
0% 250%c 500%*c 850%*c
0% 180%*c -40% 20%
Hemoglobin; 104 wks (percent change compared to control)
M
F
0% 3% 4% 0%
0% -1% 1% -2%
Levine et al. (1983); Thompson (1983)
Rats, F344, 75/sex/group; interim
sacrifices (10/sex/group) at 6 and 12 mo
89.2-98.7% pure, with 3-10% HMX as
contaminant; 83-89% of particles
<66 nm
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
24 mo
Doses
o
o
00
LO
m
o
o
Hemoglobin levels; 105 wks (percent change compared to control)
M
F
0% 6% 6% 3% -13%
0% -5% 1% -9% -14%
RBC count; 105 wks (percent change compared to control)
M
F
0% 5% 2% -1% -9%
0% -2% 2% -9% -13%
Platelet count; 105 wks (percent change compared to control)
M
0% 6% -4% -10% -7%
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Reference and study design
Results
F
0% 14% -4% 5%
22%
Hematocrit; 105 wks (percent change compared to control)
M
0% 5% 5% 2%
-7%
F
0s-
00
1
0s-
O
0s-
LO
1
0s-
O
-12%
Cholakis et al. (1980)
Doses
0 80 160
320
Mice, B6C3Fi, 10-12/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 urn
RBC count (percent change compared to control)
M
0% -5% -12%*
-2%
particle size
0, 80, 60, or 40 mg/kg-d for 2 wks
followed by 0, 80,160, or 320 mg/kg-d
F
0% -10% -1%
1%
Reticulocytes (percent change compared to control)
(TWA doses of 0, 79.6, 147.8, or
256.7 mg/kg-d for males and 0, 82.4,
M
0% -36% -13%
15%
136.3, or 276.4 mg/kg-d for females)b
F
0% 21% 25%
-19%
Diet
13 wks
Hematocrit (percent change compared to control)
M
0s-
ID
1
0s-
1
1
0s-
O
0%
F
0% -8% 2%
1%
Hemoglobin (percent change compared to control)
M
0% -2% -7%*
-3%
F
0s-
^1-
0s-
LO
1
0s-
O
1%
Platelets (percent change compared to control)
M
0% 33% 28%
22%
F
0% 3% 9%
39%
Cholakis et al. (1980)
Doses
0 10 14 20
28
40
Rats, F344,10/sex/group
88.6% pure, with 9% HMX and 2.2%
water as contaminants; ~200 nm
RBC count (percent change compared to control)
M
0% -
3%
-1%
particle size
0,10,14, 20, 28, or 40 mg/kg-d
Diet
F
0% -
-1%
-7%
Hemoglobin (percent change compared to control)
13 wks
M
0% -
2%
-1%
F
0% -
-1%
-1%
Platelet (percent change compared to control)
M
0% -
11%
16%*
F
0% -
-23%
-13%
Reticulocytes (percent change compared to control)
M
0% -
26%
76%*
F
0% -
-2%
17%
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Reference and study design
Results
Hematocrit (percent change compared to control)
M
0% -
3%
0%
F
0% -
0%
-2%
Crouse et al. (2006)
Doses
0 4 8 10
12
15
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
RBC count (percent change compared to control)
M
0% 1% -7% -2%
-4%
-5%
Gavage
90 d
F
0s-
1
1
0s-
m
0s-
m
0s-
O
2%
-2%
Hemoglobin (percent change compared to control)
M
0s-
O
0s-
LO
1
0s-
1
1
0s-
O
-1%
-6%
F
0% 2% 4% -1
4%
-4%
Platelet count (percent change compared to control)
M
0% 21% 11% 13%
-8%
34%
F
0% 6% 40% 47%
34%
-36%
Hematocrit (percent change compared to control)
M
0% 2% -5% 0%
-1%
-4%
F
0% 3% 4% 0%
4%
-2%
Levine et al. (1981a);Levine et al. (1990);
Data were not reported for rats in the 300 or 600 mg/kg dose groups
Levine et al. (1981b)c
because all of the rats died before the 13-wk necropsy.
Rats, F344,10/sex/group; 30/sex for
control
Doses
0 10 30 100
300
600
84.7 ± 4.7% purity, ~10% HMX, median
Hematocrit (percent change compared to control)
particle diameter 20 urn, ~90% of
particles < 66 urn
M
0s-
LO
1
0s-
1
1
0s-
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Reference and study design
Results
0,15, 25, or 50 mg/kg-d
Diet
3 mo
Hemoglobin (percent change compared to control)
M + F
0% -25% -7% -11%
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow
containing 20 mg RDX/g-chow, 60 g dog
food
0, 0.1,1, or 10 mg/kg-d
Diet
90 d
Doses
o
1
1
1
o
o
RBC count (percent change compared to control)
M
F
0% -3% 3% 2%
0% 13% 7% 11%
Reticulocyte count (percent change compared to control)
M
F
0% -66% 0% -50%
0% -17% -50% 0%
Hematocrit (percent change compared to control)
M
F
0% -4% 2% 0%
0% 6% 1% 7%
Hemoglobin (percent change compared to control)
M
F
0% 5% -2% 0%
0% 8% -2% 8%
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
90 ds
Histopathological examination revealed increased numbers of
degenerate or necrotic megakaryocytes in all bone marrow sections.
Doses
o
1
1
1
o
o
RBC count (percent change compared to control)
M
F
0% -3% 2% -3%
0% 0% -1% 2%
Reticulocyte count (percent change compared to control)
M
F
0% -33% -50% -50%
0% -18% -36% 45%
Hematocrit (percent change compared to control)
M
F
0% -7% -4% -1%
0% 10% 7% 3%
Hemoglobin (percent change compared to control)
M
F
0% -10% -8% -6%
0% 6% 6% 3%
1
2 ^Statistically significantly different compared to the control, as determined by study authors (p < 0.05).
3 incidence counts exclude individuals from which blood was obtained via the orbital sinus.
4 bDoses were calculated by the study authors.
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1 cLevine et al. (1981a) is a laboratory report of a 13-week study of RDX in F344 rats; two subsequently published
2 papers (Levine et al., 1990; Levine et al., 1981b) present subsets of the data provided in the full laboratory report.
3 Standard deviations accompanying the mean response in a given dose group were high, suggesting uncertainty in
4 the accuracy of the reported percent change compared to control.
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Summary of Other Toxicity Data
Effects on the eyes and the musculoskeletal, cardiovascular, immune, and gastrointestinal
systems have been reported in some studies. EPA concluded that the evidence does not support
these effects as a potential human hazard of RDX exposure.
1.2. INTEGRATION AND EVALUATION
1.2.1. Effects Other Than Cancer
The majority of evidence for the health effects of RDX comes from oral toxicity studies. The
available health effects literature does not support identification of hazards by the inhalation route
of exposure. Three epidemiological studies that document possible inhalation exposure are limited
by various study design features, including inability to distinguish exposure to TNT (associated
with liver and hematological system toxicity), uncertainty in identifying exposure levels, small
sample sizes, and inadequate reporting. The single animal inhalation study identified in the
literature search had deficiencies that precluded its inclusion in this assessment (see Literature
Search Strategy | Study Selection and Evaluation).
The strongest evidence for hazards following exposure to RDX is for nervous system effects.
A human occupational study fMa and Li. 19921 describes memory impairment and visual-spatial
decrements, and several case reports provide additional evidence of associations between exposure
to RDX and seizures and convulsions (Kasuske etal.. 2009: Kuctikardali etal.. 2003: Testud etal..
1996b: Testud etal.. 1996a: Woody et al.. 1986 and others, see Appendix C.3). Other nervous
system effects identified in human case reports include dizziness, headache, confusion, and
hyperirritability. Evidence from toxicity studies in multiple animal species involving chronic,
subchronic and gestational exposures is consistent with the effects seen in humans. Effects
included dose-related increases in seizures and convulsions, as well as observations of tremors,
hyperirritability, hyper-reactivity, and other behavioral changes fCrouse etal.. 2006: Angerhofer et
al.. 1986: Levine etal.. 1983: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Von
Oettingen etal.. 1949). In a number of these studies, death occurred at RDX doses that induced
nervous system effects. Crouse etal. (2006). a study designed to more systematically record
nervous system effects, reported that pre-term deaths occurred earlier in the higher-dose groups
and in almost all cases, deaths were preceded by neurotoxic signs such as tremors and convulsions.
The strength of a direct association between mortality and nervous system effects is less clear in
most of the earlier studies because the frequency of clinical observations may have been
insufficient to observe seizures prior to death.
Induction of convulsions and seizures appears to be more strongly correlated with dose
than with duration of exposure. It is unclear if nervous system effects increased in severity (e.g.,
from behavioral change to seizures and convulsions) with increasing dose because many of the
studies that reported more subtle neurobehavioral changes did not provide detailed dose-response
information, and the majority of studies were not designed to capture this information. Additional
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support for an association between RDX exposure and nervous system effects comes from
consistent evidence of neurotoxicity across taxa, including several species of wildlife (Ouinn etal..
2013: Garcia-Revero etal.. 2011: McFarland etal.. 2009: Gogal etal.. 20031. Although the MOA is
unknown, the association between RDX and neurological effects is biologically plausible, with
studies demonstrating a correlation between blood and brain concentrations of RDX and the time of
seizure onset fWilliams etal.. 2011: Bannon etal.. 20091. Additionally, the affinity of RDX for the
picrotoxin convulsant site of the GABAa channel suggests that the resulting disinhibition could lead
to the onset of seizures (Williams etal.. 20111. EPA identified nervous system effects as a human
hazard of RDX exposure.
Evidence for kidney and other urogenital toxicity is more limited than evidence for
neurotoxicity. Increased relative kidney weight was observed in male and female mice (Lish et al..
19841. and histopathological changes in the urogenital system (including suppurative prostatitis)
were reported in male rats exposed to RDX in the diet for 2 years (Levine etal.. 19831. Similar
histopathological changes of the urogenital system were not observed in mice, and no other rat
studies of similar duration that examined the prostate were available. Among the lesions identified
in the rat, the incidence of suppurative prostatitis is considered a marker for RDX-related
urogenital effects. The plausibility of a MOA that shares a common molecular initiating event
(binding to the GABAa receptor convulsant-site) with the neurotoxic effects of RDX increases
support for an association between RDX exposure and kidney and other urogenital effects. EPA
identified the urogenital system as a potential human hazard of RDX exposure.
Evidence for male reproductive toxicity comes from the finding of testicular degeneration in
male B6C3Fi mice chronically exposed to RDX in the diet (Lish etal.. 19841 in the only mouse study
conducted of that duration (24 months). The effect was noted by study authors at both the
penultimate and high dose tested in the study. However, studies in different rat strains did not
consistently report testicular effects. Although the available data are limited, given the dose-related
findings of mouse testicular degeneration, EPA identified suggestive evidence of male reproductive
effects as a potential human hazard of RDX exposure.
Evidence for developmental toxicity and liver toxicity was more limited than that for the
endpoints discussed above. In animal studies, embryotoxicity and other developmental effects
were observed only at doses associated with maternal mortality (Angerhofer et al.. 1986: Cholakis
etal.. 19801. Evidence for hepatic effects comes from observations of increases (generally dose-
related) in liver weight in some chronic and subchronic oral animal studies (Lish etal.. 1984: Levine
etal.. 1983: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Hart. 19761. However,
these elevations in liver weight were not accompanied by RDX-related histopathological changes in
the liver or increases in serum liver enzymes. In addition, interpretation of liver weight changes in
the mouse bioassay by Lish etal. (19841 is complicated by the relatively high incidence of liver
tumors in this study. EPA concluded that evidence does not support developmental toxicity or liver
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effects as potential human hazards of RDX exposure. Thus, these effects were not considered
further for dose-response analysis and the derivation of reference values.
1.2.2. Carcinogenicity
Under EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al. the database for
RDX provides "suggestive evidence of carcinogenic potential" based on the finding of statistically
significant trends for hepatocellular adenomas or carcinomas and alveolar/bronchiolar adenomas
or carcinomas in female, but not male, B6C3Fi mice (Lish etal.. 19841. This is further supported by
the finding of a statistically significant trend for hepatocellular carcinomas in male, but not female,
F344 rats (Levine etal.. 19831 exposed to RDX in the diet for two years. On the other hand, there
was no evidence of carcinogenicity in Sprague-Dawley rats in a 2-year dietary study of RDX fHart.
19761. No human studies are available to assess the carcinogenic potential of RDX.
EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) emphasizes the
importance of weighing the totality of evidence in reaching conclusions about the human
carcinogenic potential of agents under evaluation. Information taken into consideration in
weighing the evidence for the human carcinogenic potential of RDX includes the magnitude of
response in rats, availability of a PWG reevaluation of tumors, and potential differences in test
material across studies.
The incidence of male rat liver carcinomas as reported by Levine etal. T19831 showed a
positive trend with dose (based on statistical analysis conducted for this review), thus supporting
the positive finding of liver tumors in female mice. However, as discussed in Section 1.1.5, the
association of liver tumors in rats with RDX exposure was judged not to be strong for several
reasons, including the small numbers of carcinomas observed across the study and the low survival
rate in the high-dose group that reduces confidence that the final incidence in that group accurately
reflects lifetime cancer incidence. A PWG reevaluation of rat liver tumors has not been conducted.
The weight of evidence of carcinogenicity also took into consideration the lack of
carcinogenic response in the two-year bioassay in the Sprague-Dawley rat (Hart. 19761. The
incidence of liver tumors in the Hart (19761 study was not increased relative to controls at a dose of
10 mg/kg-day, a dose that fell in the range of doses in the Levine etal. (19831 study that showed a
positive tumor trend.
A cancer descriptor may be applicable to a variety of potential data sets and represent
points along a continuum of evidence fU.S. EPA. 2005al. The available evidence for RDX suggests
that it could be considered a borderline case between two descriptors—"likely to be carcinogenic to
humans" and "suggestive evidence of carcinogenic potential." One of the criteria identified in EPA's
Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) that supports the likely descriptor is
"an agent that has tested positive in animal experiments in more than one species, sex, strain, site,
or exposure route, with or without evidence of carcinogenicity in humans" may qualify as a likely
carcinogen. Bioassay data provide evidence for RDX carcinogenicity in one sex of one species
(female mouse), weaker evidence for carcinogenicity in one sex of a second species (male rat), and
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evidence of tumors in two tissues (liver and lung); this evidence could be considered to meet the
criteria for the "likely to be carcinogenic to humans" descriptor.
The "suggestive evidence of carcinogenic potential" descriptor is appropriate when the
weight of evidence is suggestive of carcinogenicity, and a concern for potential carcinogenic effects
in humans is raised, but the data are judged not sufficient for a stronger conclusion. This descriptor
covers a wide spectrum of evidence associated with varying levels of concern for carcinogenicity,
including a positive cancer result in the only study on an agent, a single positive cancer result in an
extensive database that includes negative studies in other species, or evidence of a positive
response in a study whose power, design, or conduct limits the ability to draw a positive conclusion.
In reviewing the carcinogenicity data for RDX, EPA considered that either descriptor is
plausible, as the evidence for increased trends in tumor incidence in two tissues and possibly a
second species raises a concern for carcinogenic effects in humans. However, in light of the
determination that the association between RDX exposure and liver tumors in rats is not strong,
and the lack of a carcinogenic response in male B6C3Fi mice, female F344 rats, and Sprague-Dawley
rats of both sexes, EPA concluded that there is "suggestive evidence of carcinogenic potential" for
RDX.
U.S. EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al indicate that for
tumors occurring at a site other than the initial point of contact, the weight of evidence for
carcinogenic potential may apply to all routes of exposure that have not been adequately tested at
sufficient doses. An exception occurs when there is convincing toxicokinetic data that absorption
does not occur by other routes. Information available on the carcinogenic effects of RDX via the
oral route demonstrates that tumors occur in tissues remote from the site of absorption.
Information on the carcinogenic effects of RDX via the inhalation and dermal routes in humans or
animals is not available. Based on the observation of systemic tumors following oral exposure, and
in the absence of information to indicate otherwise, it is assumed that an internal dose will be
achieved regardless of the route of exposure. Therefore, there is "suggestive evidence of
carcinogenic potential" following exposure to RDX by all routes of exposure.
1.2.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
Susceptibility refers to factors such as lifestage, genetics, sex, and health status that may
predispose a group of individuals to greater response to an exposure. This greater response could
be achieved either through differences in exposure to the chemical underlying toxicokinetic and
toxicodynamic differences between susceptible and other populations. Little information is
available on populations that may be especially vulnerable to the toxic effects of RDX. Lifestage,
and in particular childhood susceptibility, has not been observed in human or animal studies of
RDX toxicity. Reproductive and developmental toxicity studies did not identify effects in offspring
at doses below those that also caused maternal toxicity fAngerhofer et al.. 1986: Cholakis etal..
19801. Transfer of RDX from dam to the fetus during gestation has been reported, and the presence
of RDX in the milk of dams administered 6 mg/kg-day by gavage has been documented fHess-Ruth
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etal.. 20071. Because RDX is neurotoxic in adult animals, evidence of gestational transfer of RDX to
the developing organism, along with the presence of RDX in milk, suggests that the nervous system
may be a target in the developing organism; however, developmental neurotoxicity studies of RDX
have not been conducted. Limited data suggest that male laboratory animals may be more
susceptible to noncancer toxicity associated with RDX exposure. While no sex-based differences in
neurotoxicity were observed, urogenital effects have been observed in males at lower doses than in
females (Levine etal.. 1983: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 19801.
suggesting a possible sex-based difference in susceptibility to RDX toxicity. There is limited
evidence that CYP450 or similar enzymes are involved in the metabolism of RDX (Bhushan etal..
20031. indicating a potential for genetic polymorphisms in these metabolic enzymes to affect
susceptibility to RDX. This susceptibility may also be influenced by differential expression of these
enzymes during development Individuals with epilepsy or other seizure syndromes, and in
particular those that have their basis in genetic mutation to GABAa receptors, may represent
another group that may be susceptible to RDX exposure. However, there is currently no
information to support predictions of how genetic polymorphisms may affect susceptibility.
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2. DOSE-RESPONSE ANALYSIS
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER
The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty
spanning perhaps an order of magnitude) of a daily exposure to the human population (including
sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a
lifetime. It can be derived from a no-observed-adverse-effect level (NOAEL), lowest-observed-
adverse-effect level (LOAEL), or the 95% lower bound on the benchmark dose (BMDL), with
uncertainty factors (UFs) generally applied to reflect limitations of the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis
Human studies are generally preferred over animal studies as the basis for a reference value
when quantitative measures of exposure are reported and the reported effects are determined to
be associated with exposure. The available epidemiological studies of worker populations exposed
to RDX examined the relationship between certain health endpoints and inhalation exposure; no
epidemiological studies of ingested RDX are available. Therefore, epidemiological studies could not
be used for oral dose-response analysis and as the basis for the RfD. Multiple case reports provide
some evidence of effects in humans associated with acute exposure to RDX; however, while case
reports can support the identification of hazards associated with RDX exposure, data from case
reports are inadequate for dose-response analysis and subsequent derivation of a chronic reference
value because of short exposure durations and incomplete or missing quantitative exposure
information.
As discussed in Section 1.2.1, based on findings from oral studies in experimental animals,
EPA identified nervous system effects as a human hazard of RDX exposure, and effects on the
urogenital system (including the kidney) as a potential human hazard of RDX exposure. EPA also
identified suggestive evidence of male reproductive effects as a potential human hazard of RDX
exposure. Experimental animal studies within each health effect category were evaluated using
general study quality considerations discussed in Section 6 of the Preamble and in the section on
Literature Search Strategy | Study Selection and Evaluation to help inform the selection of studies
from which to derive oral reference values. Rationales for selecting the studies and effects to
represent each of these hazards are summarized below.
Nervous System Effects
Nervous system effects following oral exposure to RDX, including convulsions, seizures, and
hyper-reactivity, were observed in multiple studies in rats, mice, monkeys, and dogs. Only three
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studies reported data on the incidence of nervous system findings—Crouse etal. (2006). Cholakis
etal. (19801. and Martin and Hart (19741. T wo of these—Crouse etal. (20061 and Cholakis et al.
f!9801—were selected for dose-response analysis.
Crouse etal. (20061 reported a dose-related increase in convulsions and tremors in both
male and female F344 rats following a 90-day oral (gavage) exposure to RDX. Additionally, Crouse
etal. f20061 observed that for all the dose groups where unscheduled deaths were recorded,
mortality was strongly associated with seizures or convulsions. This study used a test material of
high purity (99.99% RDX), six dose groups (including the control) that provided good resolution of
the dose-response curve, and relatively low doses that still provided adequate responses. Cholakis
etal. f!9801 reported a dose-related increase in convulsions in a developmental toxicity study, with
convulsions observed at a dose as low as 2 mg/kg-day RDX on GDs 6-19. Because evidence of
nervous system effects was observed in this study at a relatively low dose, this study was also
selected for dose-response analysis.
The study in monkeys by Martin and Hart (1974) was not selected for dose-response
analysis. This study provided supporting evidence of nervous system effects (trembling, shaking,
ataxia, and hyperactive reflexes) with 66% incidence at the high dose of 10 mg/kg-day; however,
this study was not selected for dose-response analysis because it used small group sizes (n = 3/sex)
and the exposures were relatively variable or uncertain (e.g., purity of the test material was not
specified, and reported emesis in some animals likely influenced the amount of dose received).
Other chronic and subchronic studies reported nervous system effects as clinical
observations (Angerhofer etal.. 1986: Lish etal.. 1984: Levine etal.. 1983: Levine etal.. 1981a:
Levine etal.. 1981b: Von Oettingen etal.. 1949). but without incidence data. As discussed in Section
1.1.1, these studies did not systematically monitor or evaluate nervous system effects induced by
RDX, leading to possible underestimates of incidence of such effects. As such, there is some
uncertainty associated with identification of NOAELs and LOAELs for nervous system effects from
these studies. Further, these studies reported convulsions and other indications of nervous system
effects at doses higher than the doses at which effects were observed in Cholakis etal. (1980). i.e.,
>2 mg/kg-day, and Crouse etal. (2006). i.e., >8 mg/kg-day.
Kidney and Other Urogenital Effects
Effects on kidney and other urogenital system endpoints included changes in kidney weight
and histopathological findings in the kidney, bladder, and prostate in experimental animals exposed
orally to RDX. As discussed in Section 1.1.3, kidney weight changes across experimental animal
studies were not consistent and were difficult to interpret; therefore kidney weight data sets were
not selected for quantitative analysis.
Histopathological changes in the urogenital system were reported in a 2-year study in F344
rats by Levine etal. (1983) and in a 13-week study in B6C3Fi mice by Cholakis etal. (1980).
Histopathological changes of the kidney and bladder (medullary papillary necrosis, suppurative
pyelitis, uremic mineralization, and luminal distention and cystitis of the urinary bladder) were
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observed by Levine etal. (1983) in high-dose (40 mg/kg-day) males. The incidence of suppurative
prostatitis, considered to be a marker for the broader range of urogenital effects in these animals,
showed a dose-related trend beginning at doses below 40 mg/kg-day (see Section 1.1.3).
Therefore, suppurative prostatitis was selected for dose-response modeling as a sensitive measure
of RDX effects on the urogenital system.
Cholakis etal. f 19801 examined the kidney for histopathological changes in control and
high-dose (320 mg/kg-day) mice only. Because incidence data from only a single high-dose group
was available, this study was not selected for dose-response analysis.
Male Reproductive Toxicity
Male reproductive effects were identified in mice following chronic administration of RDX
in the diet. Lish etal. f19841 observed an increased incidence of testicular degeneration in mice
given RDX in diet for two years compared to controls. The response was shown to be dose-related
and was selected for dose-response modeling. Changes in other reproductive outcomes were not
dose-related or consistently observed across studies, and therefore were not considered for dose-
response modeling.
2.1.2. Methods of Analysis
Benchmark dose (BMD) modeling and physiologically-based pharmacokinetic (PBPK)
models were used in this assessment to estimate candidate points of departure (PODs) for the
derivation of an RfD for RDX. The general approach for the estimation of PODs is presented in
Figure 2-1 and described further below.
Benchmark
BMDLor
NOAEL
Animal
internal dose
Animal
external dose
Human
external dose
(HED)
Benchmark
dose modeling
Animal PBPK model
Human PBPK model
Figure 2-1. Approach for dose-response analysis.
No biologically based dose-response models are available for RDX. In this situation, EPA
evaluates a range of dose-response models thought to be consistent with underlying biological
processes to determine how best to empirically model the dose-response relationship in the range
of the observed data. Consistent with this approach, EPA evaluated dose-response information
with the models available in EPA's Benchmark Dose Software (BMDS, versions 2.4 and 2.5). EPA
estimated the benchmark dose (BMD) and 95% lower confidence limit on the BMD (BMDL) using a
benchmark response (BMR) selected for each effect A summary of BMD modeling, including
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selection of BMRs, for each of the health effect categories is provided below.
Nervous System Effects
Incidence data from Crouse etal. f20061 and Cholakis etal. f!9801 were amenable to
modeling. For Crouse et al. f20061. statistical analysis (Cochran-Mantel-Haenszel test) conducted
by EPA indicated no significant difference in convulsion rates of male and female rats; thus,
combined incidence data from male and female rats were used for modeling convulsion data from
this study. A BMR of 1% extra risk for convulsions was used to address the relative severity of this
endpoint; across the experimental animal database for RDX, convulsions and seizures were
generally associated with mortality. In general, severe endpoints are not used as the basis of a
noncancer risk value because of relatively high uncertainty in extrapolating to a level of exposure
likely to be without appreciable risk. Less severe nervous system outcomes that precede
convulsions and associated mortality would be preferred, but none were identified for RDX.
Kidney/Urogenital and Male Reproductive Effects
Incidence data on prostate effects as reported by Levine etal. (19831 and testicular
degeneration as reported by Lish etal. f19841 were amenable to modeling. Cut-offs for the
biological significance of these effects were not identified, and a BMR of 10% was applied under the
assumption that it represents a minimally biologically significant degree of effect Uncertainty in
this characterization should be taken into account in comparisons with PODs from other effects.
Human Extrapolation
EPA guidance (U.S. EPA. 20111 advocates a hierarchy of approaches for deriving human
equivalent doses (HEDs) from data in laboratory animals, with the preferred approach being
physiologically-based toxicokinetic modeling. Other approaches can include using chemical-
specific information in the absence of a complete physiologically-based toxicokinetic model. In lieu
of either reliable chemical-specific models or data to inform the derivation of human equivalent
oral exposures, a body weight scaling to the % power (i.e., BW3/4) approach is generally applied to
extrapolate toxicologically equivalent doses of orally administered agents from adult laboratory
animals to adult humans for the purpose of deriving an oral RfD.
As described below, HEDs for candidate PODs for RDX were derived using PBPK models for
endpoints selected from rat and mouse bioassays, and are compared in Table 2-1 to estimates
derived from administered RDX dose.
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Table 2-1. Summary of derivation of PODs following oral exposure to RDX
Endpoint and
reference
(exposure
duration/route)
Species/sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODhed
(mg/kg-d)
Administered
doseb
RDX
AUC
Nervous system
Convulsions
Crouse et al. (2006)
(90-d/gavage)
Male and
female F344
rat,
combined
Multistage
3°
1% ER
1.53
0.54
0.13
0.27
Convulsions
Cholakis et al.
(1980)
(GDs 6-19/gavage)
Female F344
rat
Quanta 1-
linear
1% ER
0.18
0.12
0.03
0.06
Kidney/urogenital system
Prostate
suppurative
inflammation
Levine et al. (1983)
(2-yr/diet)
Male F344
rat
LogProbit
10% ER
1.67
0.47
0.11
0.23
Male reproductive system
Testicular
degeneration
Lish et al. (1984)
(2-yr/diet)
Male B6C3Fi
mouse
LogProbit
10% ER
56.0
16.3
2.4
0.08
1
2 aFor modeling details, see Appendix D.
3 bPOD was convered to an HED using a standard DAF based on BW3/4.
4 cPOD was converted to an HED based on the equivalence of internal RDX dose (expressed as area under the curve
5 [AUC] for RDX concentration in arterial blood) derived using PBPK models.
6
7 ER = extra risk
8 Physiologically-based pharmacokinetic models for RDX in rats, humans, and mice have been
9 published (Sweeney etal.. 2012a: Sweeney etal.. 2012b: Krishnan et al.. 20091 based on RDX-
10 specific data. EPA evaluated and further developed these models for extrapolating doses from
11 animals to humans (see Appendix C, Section C.2.5). As concluded in the MOA analyses for the
12 various observed noncancer effects associated with RDX exposure, the available data are
13 insufficient to establish any specific mode(s) of action for these effects, and there appears to be no
14 clear evidence linking health effects with RDX-generated metabolites. In general, appropriately
15 chosen internal dose metrics are expected to correlate more closely with toxic responses than
16 external doses, for effects that are not occurring at the point of contact (Mclanahan etal.. 20121.
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Therefore, PBPK model-derived arterial blood concentration of RDX is considered a better dose-
metric for extrapolation of health effects than administered dose when there is adequate
confidence in the estimated value. The PBPK models for RDX were used to estimate the area under
the curve (AUC) for RDX concentration in arterial blood, which represents the average blood RDX
concentration for the exposure duration normalized to 24 hours.
It appears logical to use RDX concentration levels in the brain as the internal dose metric for
analyzing convulsions as the health effect Nevertheless, the blood concentration of RDX was
preferred as the dose metric due to greater confidence in modeling this variable. This is because of
the substantially greater number of measurements of RDX blood levels used in calibrating model
parameters. Additionally, predictions of RDX concentrations in the brain are highly correlated with
RDX blood concentrations because the brain compartment does not have absorption, metabolism,
or elimination of RDX. It may also be noted that there is greater confidence in model estimates of
blood AUC versus peak blood concentrations because, as discussed in Appendix C, Section C.2.5, the
rate constant for oral absorption (KAS) is uncertain, and peak concentrations are more sensitive to
variations in this parameter than average values. Furthermore, a more consistent dose-response
for convulsions is observed in chronic studies than for the higher exposures in subchronic studies.
The rodent PBPK model was applied to the BMDLs generated from BMD modeling to
determine the animal internal dose, expressed as the AUC of RDX blood concentration, and
representing the cross-species toxicologically equivalent dose. The human PBPK model was then
applied to derive the corresponding HEDs (see Figure 2-1). Because the AUC is linear with
exposure level, at least in the exposure range of interest, the value of the HED would be the same
whether the rat or mouse PBPK model is applied before or after BMD modeling is performed (i.e.,
the sequence of this calculation is immaterial for the RDX data).
HEDs were also calculated consistent with EPA guidance fU.S. EPA. 20111 using PODs
(BMDLs or NOAELs) determined from administered RDX doses and employing a standard
dosimetric adjustment factor (DAF) derived as follows:
DAF = (BWa'/'VBWh'/'i),
where
BWa = animal body weight
BWh = human body weight
Using a BWa of 0.25 kg for rats and 0.035 kg for mice and a BWh of 70 kg for humans fU.S.
EPA. 1988). the resulting DAFs for rats and mice are 0.24 and 0.15, respectively. Applying the DAF
to the POD identified for effects in adult rats or mice yields a PODhed as follows (see Table 2-1):
PODhed = Laboratory animal dose (mg/kg-day) x DAF
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Further details of the BMDL modeling, BMDS outputs, and graphical results for the best fit
model for each dataset included in Table 2-1 can be found in Appendix D, Section D.l. Details of the
PBPK model evaluation used for extrapolation from BMDL values can be found in Appendix C,
Section C.2.5. Table 2-1 summarizes the results of the BMD modeling and the PODhed for each data
set discussed above.
2.1.3. Derivation of Candidate Values
Pharmacokinetic models are useful to examine species differences in pharmacokinetic
processing. Because of relatively high confidence in the rat and human PBPK modeling, these
models were used to derive reliable internal dose metrics for extrapolation. For datasets selected
from the rat bioassays, the candidate RfDs were calculated assuming cross-species toxicological
equivalence of the AUC of RDX blood concentration derived from the PBPK modeling. However,
there were major uncertainties identified in the mouse PBPK modeling. Therefore, for endpoints
selected from the mouse bioassay, the preferred approach for determining the candidate RfDs is
that based on the administered dose of RDX extrapolated to humans using allometric BW3/4 scaling.
The evaluation of confidence in the PBPK model results is summarized in Summary of confidence in
PBPK models for RDX in Appendix C, Section C.2.5.
Under EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
20021 (Section 4.4.5), and as described in the Preamble, five possible areas of uncertainty and
variability were considered. An explanation follows.
An intraspecies uncertainty factor, UFh, of 10 was applied to all PODs to account for
potential differences in toxicokinetics and toxicodynamics in the absence of information on the
variability of response in the human population following oral exposure to RDX.
An interspecies uncertainty factor, UFa, of 3 (101/2 = 3.16, rounded to 3) was applied to all
PODs to account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences
between rodents and humans. For the testicular degeneration dataset from the mouse bioassay, a
UFa of 3 was applied because BW3/4 scaling is used to extrapolate oral doses from laboratory
animals to humans. Although BW3/4 scaling addresses some aspects of cross-species extrapolation
of toxicokinetic and toxicodynamic processes, some residual uncertainty remains. In the absence of
chemical-specific data to quantify this uncertainty, EPA's BW3/4 guidance (U.S. EPA. 20111
recommends use of an uncertainty factor of 3. For datasets from the rat bioassays, a PBPK model
was used to convert internal doses in rats to administered doses in humans. This reduces
toxicokinetic uncertainty in extrapolating from the rat to humans, but does not account for
interspecies differences due to toxicodynamics. A UFa of 3 was applied to account for this
remaining toxicodynamic and any residual toxicokinetic uncertainty not accounted for by the PBPK
model.
A subchronic to chronic uncertainty factor, UFS, differs depending on the exposure duration.
An UFS of 1 was applied to the POD values for kidney/urogenital effects and testicular degeneration
derived from the 2-year bioassays in the rat fLevine etal.. 19831 and mouse fLish etal.. 19841. POD
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values for nervous system effects were derived from studies of subchronic duration or gestational
exposure; a UFs of 3 was applied to these PODs. Typically, a UFs of 10 is applied to extrapolate
results from a subchronic duration study in the absence of a chronic study based on the assumption
that effects from a given compound would occur at approximately a 10-fold higher exposure level in
a subchronic study than in a chronic study, if a chronic study were available fU.S. EPA. 20021.
However, the available nervous system effects data for RDX support an UFs of less than 10. As
discussed in Section 1.1.1, seizure induction appears to be more strongly correlated with dose level
than with duration of exposure. In addition, the available empirical evidence from rodent bioassays
provide support for an UFs no greater than 3. Dose levels associated with convulsions in chronic
dietary studies of RDX are >35 mg/kg-day and are higher than doses that induced convulsions in
the 14- and 90-day (gavage) studies that were used to derive candidate PODs for nervous system
effects (i.e., 2 mg/kg-day in Cholakis etal. fl9801 and 8 mg/kg-day in Crouse etal. f200611 (also see
Table 1-2 and Figure 1-1). Thus, the available RDX data for nervous system effects is consistent
with the application of a UFs that is less than the default of 10.
A LOAEL to NOAEL uncertainty factor, UFl, of 1 was applied to all POD values because the
PDO was a BMDL. When the POD is a BMDL, the current approach is to address this factor as one of
the considerations in selecting a BMR for benchmark dose modeling. In this case, the BMR for
modeled endpoints was selected under the assumption that the BMR represents a minimal,
biologically significant change for these effects.
A database uncertainty factor, UFd, of 3 was applied to all POD values. The oral toxicity
database for RDX includes subchronic and chronic toxicity studies in the rat and mouse, a two-
generation reproductive toxicity study in the rat, developmental toxicity studies in the rat and
rabbit, and subchronic studies (with study design limitations) in the dog and monkey. Deficiencies
in the database related to neurobehavioral and neurodevelopmental testing were identified. The
database for neurotoxicity is characterized primarily by observations of frank effects (convulsions).
Additional observations of neurobehavioral effects were reported fLevine etal.. 1990: Angerhofer
etal.. 1986: Levine etal.. 1983: Levine etal.. 1981a: Levine etal.. 1981b: Cholakis etal.. 1980: Von
Oettingen etal.. 1949): however, a FOB conducted by Crouse etal. (2006) did not report any
consistent, treatment-related behavioral effects. Further, Crouse etal. (2006) noted that the ability
of the FOB to identify neurobehavioral effects at doses >8 mg/kg-day was limited due to the timing
of the dosing procedure and timing of the FOB screenings. Given the reports of neurobehavioral
effects in several studies, additional systematic evaluation of neurobehavioral effects would be
informative. Hess-Ruth et al. f20071 reported possible transfer of RDX to offspring during
gestation, as well as the presence of RDX in the milk of dams, indicating a potential for lactational
transfer of RDX to offspring. Given the potential for exposure during gestation and lactation and the
neurotoxic potential of RDX, the lack of a developmental neurotoxicity study was identified as a
data gap. A UFd of 3 was applied to all PODs to account for limitations in neurobehavioral and
neurodevelopmental testing.
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1 Table 2-2 is a continuation of Table 2-1 and summarizes the application of UFs to each
2 PODhed to derive a candidate value for each data set The candidate values presented in the table
3 below are preliminary to the derivation of the organ/system-specific reference values. These
4 candidate values are considered individually in the selection of a representative oral reference
5 value for a specific hazard and subsequent overall RfD for RDX.
Table 2-2. Effects and corresponding derivation of candidate values
Endpoint and reference
PODhed3
POD
type
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Nervous system (rats)
Convulsions
Crouse et al. (2006)
0.27
BMDLoi
3
10
1
3
3
300
8.8 x 10"4
Convulsions
Cholakis et al. (1980)
0.06
BMDLoi
3
10
1
3
3
300
2.0 x 10"4
Kidney/urogenital system (rats)
Prostate suppurative
inflammation
Levine et al. (1983)
0.23
BMDLio
3
10
1
1
3
100
2.3 x 10"3
Male reproductive system (mice)
Testicular degeneration
Lish et al. (1984)
2.4
BMDLio
3
10
1
1
3
100
2.5 x 10"2
6
7 aPODHED values based on data from the rat were derived using PBPK modeling; the HED POD based on data from
8 the mouse was derived using BW3/4 adjustment (see Section 2.1.3 and discussion of the PBPK models in
9 Appendix C, Section C.2.5).
10 Figure 2-2 presents graphically the candidate values, UFs, and PODhedS, with each bar
11 corresponding to one data set described in Tables 2-1 and 2-2.
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Convulsions; Crouse et
al. (2006)
Convulsions; Cholakis et
al. (1980)
Prostate - suppurative
inflammation; Levine et
a I. (1984)
Testicular
degeneration; Lish et al.
(1984)
~ Candidate RfD
• PODhed
Composite UF
0.0001
0.001
0.01 0.1
mg/kg-day
10
Figure 2-2. Candidate values with corresponding POD and composite UF.
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2.1.4. Derivation of Organ/System-Specific Reference Doses
Table 2-3 distills the candidate values from Table 2-2 into a single value for each organ or
system. 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 2-3. Organ/system-specific RfDs and proposed overall RfD for RDX
Effect
Basis
RfD (mg/kg-day)
Study exposure
description
Confidence
Nervous system
Convulsions
9 x 10"4
Subchronic
Medium
Kidney/urogenital
system
Suppurative prostatitis
2 x 10"3
Chronic
Low
Male reproductive
system
Testicular degeneration
2 x 10"2
Chronic
Low
Proposed overall RfD
Nervous system
9 x 10"4
Subchronic
Medium
Nervous System Effects
The organ/system-specific RfD for nervous system effects was based on the incidence of
convulsions in rats reported in Crouse etal. (20061. a well-conducted study that used a 99.99%
pure form of RDX, five closely-spaced dose groups that provided a good characterization of the
dose-response curve for convulsions, and an endpoint (convulsions) that was replicated across
multiple other studies. Although the candidate value derived from Cholakis etal. fl9801 is lower
(by approximately fourfold), there is greater certainty in the value derived from Crouse etal.
(2006) because of the longer exposure duration (90 versus 14 days), more systematic evaluation of
neurobehavioral endpoints, and higher test compound purity.
Kidney/Urogenital Effects
A single data set for incidence of suppurative prostatitis in male B6C3Fi mice as reported by
Lish etal. T19841 was brought forward for quantitative analysis as a sensitive marker for the
broader array of RDX-associated effects observed in the urogenital system. As previously
discussed, the data supporting RDX-related kidney and other urogenital effects are largely limited
to this 2-year study in the mouse. Accordingly, the candidate value for kidney and other urogenital
effects is based on the incidence of suppurative prostatitis in male mice (Lish etal.. 1984).
Male Reproductive Effects
A single dataset for male reproductive effects was brought forward for quantitative
analysis: the incidence of testicular degeneration as reported in male B6C3Fi mice exposed to RDX
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in diet for 24 months (Lish etal.. 19841. The candidate value for male reproductive effects is based
on this dataset.
2.1.5. Selection of the Proposed Overall Reference Dose
Multiple organ/system-specific reference doses were derived for effects identified as
potential hazards from RDX exposure, including nervous system effects, kidney and other
urogenital effects, and male reproductive effects. Evidence for nervous system effects, and
specifically convulsions, was observed in multiple studies, in multiple species, and following a range
of exposure durations. In addition, the organ/system-specific RfD for nervous system effects was
the lowest among the organ/system-specific RfDs derived for RDX. Evidence for dose-related
effects on the urogenital system comes primarily from a single 2-year toxicity study in male rats
fLevine etal.. 19831. and evidence for male reproductive effects comes primarily from a single 2-
year toxicity study in mice (Lish etal.. 19841: neither a second chronic study in the rat that
evaluated prostate histopathology nor a second mouse study was available to validate and replicate
these findings.
The organ/system-specific RfD of 9 x 10~4 mg/kg-day for nervous system effects in the rat
as reported by Crouse etal. (20061 is selected as the overall RfD for RDX given the strength of
evidence for the nervous system as a hazard of RDX exposure, and as the lowest organ/system-
specific RfD. This overall RfD should provide an exposure level below which effects associated with
RDX exposure are not expected to occur.
The overall RfD is derived to be protective of all types of effects for a given duration of
exposure, and is intended to protect the population as a whole, including potentially susceptible
subgroups (U.S. EPA. 20021. Decisions concerning averaging exposures over time for comparison
with the RfD should consider the types of toxicological effects and specific lifestages of concern.
Fluctuations in exposure levels that result in elevated exposures during these lifestages could
potentially lead to an appreciable risk, even if average levels over the full exposure duration were
less than or equal to the RfD. In the case of RDX, no specific lifestages have been identified as a
potentially susceptible subgroup.
2.1.6. Uncertainties in the Derivation of Reference Dose
The following discussion identifies uncertainties associated with the RfD for RDX. To derive
the RfD, the UF approach fU.S. EPA. 2000a. 19941 was applied to a PODhed based on nervous system
effects in rats exposed to RDX for a subchronic duration. UFs were applied to the PODhedS to
account for uncertainties in extrapolating from an animal bioassay to human exposure, the likely
existence of a diverse population of varying susceptibilities, subchronic to chronic duration, and
database deficiencies. These extrapolations are carried out with default approaches given the lack
of data to inform individual steps.
Although the database is adequate for reference value derivation, uncertainty is associated
with the consistency in toxicity results across studies that used RDX test materials that differed in
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purity, formulation, and particle size. There is evidence that differences in test material
formulation and particle size can affect absorption of RDX.
Nervous system effects have been documented in multiple studies and animal species and
strains; however, there is some uncertainty associated with the incidence of reported neurological
effects in studies that employed a study design that did not monitor animals with sufficient
frequency to accurately record neurobehavioral effects, including convulsions.
2.1.7. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfD,
the overall database, and the RfD itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA.
19941. The overall confidence in this RfD is medium. Confidence in the principal study fCrouse et
al.. 20061 is high. The study was well-conducted, utilized 99.99% pure RDX, and had five closely-
spaced dose groups that allowed characterization of dose-response curves for convulsions. One
limitation identified by study authors was the limited ability of the FOB to fully identify
neurobehavioral effects at doses >8 mg/kg-day due to the timing of the dosing procedure and
timing of the FOB screening. Confidence in the database is medium. The database includes three
chronic studies in rats and mice; eight subchronic studies in rats, mice, dogs, and monkeys; two
short-term studies; and four reproductive/developmental toxicity studies in rats and rabbits
(including a two-generation reproductive study). Confidence is reduced largely because of limited
examination of the potential for RDX to induce neurobehavioral and neurodevelopmental effects
and the incomplete understanding of a MOA for convulsions. Reflecting high confidence in the
principal study and medium confidence in the database, overall confidence in the RfD is medium.
2.1.8. Previous IRIS Assessment
The previous RfD for RDX, posted to the IRIS database in 1993, was based on a two-year rat
feeding study by Levine et al. (19831. The no observed effect level (NOEL) of 0.3 mg/kg-day
(LOAEL = 1.5 mg/kg-day) based on suppurative prostate inflammation in male F344 rats from this
study was identified as the POD. An RfD of 3 x 10"3 mg/kg-day was derived following application of
an overall UF of 100 (UFa = 10, UFh = 10).
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER
The RfC (expressed in units of mg/m3) is defined as an estimate (with uncertainty spanning
perhaps an order of magnitude) of a continuous inhalation exposure to the human population
(including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects
during a lifetime. It can be derived from a NOAEL, LOAEL, or the 95% lower bound on the
benchmark concentration (BMCL), with UFs generally applied to reflect limitations of the data used.
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As noted in Section 2.1, human studies are generally preferred over animal studies as the
basis for a reference value when quantitative measures of exposure are reported and the reported
effects are determined to be associated with exposure. Of the available human epidemiological
studies of RDX (West and Stafford. 1997: Ma and Li. 1992: Hathaway and Buck. 19771. none
provided data that could be used for dose-response analysis. The studies by Ma and Li f!9921 of
neurobehavioral effects in Chinese workers and West and Stafford f!9971 of hematological
abnormalities in ordnance factory workers had numerous methodological limitations that preclude
their use for quantitative analysis (see Literature Search Strategy | Study Selection and Evaluation).
The study by Hathaway and Buck (19771 found no evidence of adverse health effects in munition
plant workers, and therefore does not provide a basis for derivation of an RfC. Multiple case
reports provide some evidence of effects in humans associated with acute exposure to RDX;
however, while case reports can support the identification of hazards associated with RDX
exposure, data from case reports are inadequate for dose-response analysis and subsequent
derivation of a chronic reference value because of short exposure durations and incomplete or
missing quantitative exposure information.
As discussed in the Literature Search Strategy | Study Selection and Evaluation, a single
experimental animal study involving inhalation exposure was identified in the DTIC database; the
study is not publicly available. However, the study would not have provided useful data on
responses to inhaled RDX, as the study was limited by small numbers of animals tested, a lack of
controls, and incomplete reporting of exposure levels. Therefore, the available health effects
literature does not support the derivation of an RfC for RDX. Further, a PBPK model for inhaled
RDX is not available to support route-to-route extrapolation from the RfD.
2.2.1. Previous IRIS Assessment
An RfC for RDX was not derived in the previous assessment posted to the IRIS database in
1990.
2.3. ORAL SLOPE FACTOR FOR CANCER
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question, and quantitative estimates of risk from oral and inhalation exposure
may be derived. Quantitative risk estimates may be derived from the application of a low-dose
extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on the estimate
of risk per mg/kg-day of oral exposure.
2.3.1. Analysis of Carcinogenicity Data
As noted in Section 1.2.2, EPA concluded that there is "suggestive evidence of carcinogenic
potential" for RDX. The Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al state:
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When there is suggestive evidence, the Agency generally would not attempt a dose-
response assessment, as the nature of the data generally would not support one;
however, when the evidence includes a well-conducted study, 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.
In the case of RDX, the carcinogenicity of the chemical has been evaluated in one oral
chronic/carcinogenicity bioassay in mice fLish etal.. 19841 and two bioassays in rats fLevine etal..
1983: Hart. 19761. The data in Lish etal. (19841 demonstrated a statistically significant positive
trend with dose6 in the incidence of liver and lung tumors in female, but not male, B6C3Fi mice
associated with dietary administration of RDX. In the study by Levine etal. (19831. the incidence of
liver tumors in male F344 rats showed a statistically significant positive trend with dose7. No
increases in tumors were observed in Sprague-Dawley rats exposed to RDX (Hart. 19761. As
discussed further below, the 2-year studies by Lish etal. T19841 and Levine etal. T19831 were well-
conducted studies that support quantitative analysis. Considering these data along with the
uncertainty associated with the suggestive nature of the weight of evidence, EPA concluded that
quantitative analysis of the tumor data may be useful for providing a sense of the magnitude of
potential carcinogenic risk.
The incidences of liver and lung tumors in female mice from the study by Lish etal. T19841
were selected for quantitative dose-response analysis. The study by Lish etal. (19841 was
performed in accordance with FDA Good Laboratory Practice regulations fFDA. 19791. included
comprehensive histopathological examination of major organs, contained four dose groups and a
control, used adequate numbers of animals per dose group (65/sex/group, plus interim sacrifice
groups of 10/sex/group at 6 and 12 months) and a sufficient overall exposure duration (2 years),
and adequately reported methods and results (including individual animal data). Female mouse
liver tissues from the original unpublished study by Lish etal. f19841 were reevaluated by a
pathology working group (PWG) (Parker et al.. 20061 in order to apply more up-to-date
histopathological criteria established by Harada etal. (19991. The updated liver tumor incidences
from the PWG reanalysis of Lish etal. (19841 were used for quantitative dose-response analysis.
In the case of both liver and lung tumors, benign and malignant tumors (i.e., adenomas and
carcinomas) were combined for dose-response analysis because benign and malignant tumors in
both organs develop from the same cell line and there is evidence for progression from benign to
the malignant stage fU.S. EPA. 2005a: McConnell etal.. 19861.
Female mouse liver and lung tumor incidences from the Lish etal. (19841 study are
summarized in Table 2-4.
6 A two-sided asymptotic Cochran-Armitage test yielded p = 0.041 for liver tumors and p = 0.019 for lung
tumors in female mice.
7 A two-sided exact Cochran-Armitage test yielded p = 0.032 for liver tumors in rat. An exact test was done
because the incidence of tumors was too low for the asymptotic test to be reliable.
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Table 2-4. Incidence of hepatocellular and alveolar/bronchiolar tumors in
female B6C3Fi mice administered RDX for 2 years in diet
Tumor type
Study/Analysis
Dose group
(mg/kg-day)
Control
1.5
7
35
107a
Hepatocellular adenomas or
carcinomas
Parker et al. (2006)
1/67
4/62
5/63
10/64
4/3 lb
Alveolar/bronchiolar adenomas
or carcinomas
Lish et al. (1984)
7/65
3/62
8/64
12/64
7/3 lb
aTWA dose, due to reductions in the highest dose from 175 to 100 mg/kg-day at week 11.
bHistopathology results are based on animals that survived more than 12 month. The smaller number of mice in
the high-dose group reflects the high mortality at a dose of 175 mg/kg-day.
The incidence of liver carcinomas in male F344 rats from the study by Levine etal. T19831
was also considered for quantitative dose-response analysis. Although the study was well
conducted (see Section 1.1.5), EPA considered that the association between RDX exposure and rat
liver tumors is not strong, reflecting the relatively low magnitude of the rat liver carcinoma
response and reduced confidence that the high-dose group accurately reflects lifetime cancer
incidence because, in part, of low survival. A candidate slope factor is provided in Appendix D,
Section D.2. for comparison.
2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods
The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005al recommend that the
method used to characterize and quantify cancer risk from a chemical be determined by what is
known about the MOA of the carcinogen and the shape of the cancer dose-response curve. The
linear approach is recommended when there are MOA data to indicate that the dose-response curve
is expected to have a linear component below the POD or when the weight of evidence evaluation of
all available data are insufficient to establish the MOA for a tumor site fU.S. EPA. 2005al In the case
of RDX, the mode of carcinogenic action for hepatocellular and alveolar/bronchiolar tumors is
unknown. Therefore, a linear low-dose extrapolation approach was used to estimate human
carcinogenic risk associated with RDX exposure.
The survival curves were compared across dose groups in each study to determine whether
time of death should be incorporated in the dose-response analysis of tumors. For female mice in
Lish etal. f!9841. the survival curves were similar across dose groups after the dose was reduced in
the high dose group to 100 mg/kg-day; therefore, a time-to-tumor analysis was not necessary for
this study.
Tumor incidence was modeled using the multistage-cancer models in BMDS (versions 2.4
and 2.5). A standard BMR of 10% extra risk was applied to both tumor sites in the mouse.
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Given the finding of an association between RDX exposure in the female mouse and
increased tumor incidence at two tumor sites, basing the oral slope factor on only one tumor site
could potentially underestimate the carcinogenic potential of RDX. Therefore, an analysis that
combines the results from the mouse liver and lung tumor incidence is preferred. The MS-COMBO
procedure (BMDS, version 2.5), extends the multistage-cancer models to the case with multiple
tumors assuming independence between tumor types. There is no known biological relationship
between liver and lung tumors in RDX-exposed mice, and therefore, as noted by the National
Research Council (NRC. 19941. this assumption of independence is considered not likely to produce
substantial error in risk estimates. MS-COMBO analyzes tumor incidence as present if either organ
(or both) has a tumor and absent otherwise. The procedure derives a maximum likelihood estimate
of the combined risk at a 95% confidence level based on the parameter values obtained for the
individual tumor multistage model fits.
EPA's preferred approach for extrapolating results from animal studies to humans is
toxicokinetic modeling. As described in Appendix C, PBPK models for RDX in mice and humans
published by Sweeney etal. (2012b) were evaluated and further developed by EPA. Consideration
was given to whether the available toxicokinetic information supported using an internal dose
metric derived by PBPK modeling. The available mechanistic data (Section 1.1.5) pointto some
evidence, although not conclusive, that RDX-generated metabolites may be implicated in the
observed tumorigenicity in the female mouse. However, there are no data on the toxicokinetics of
RDX metabolites, and metabolism in the liver is the only route of elimination of RDX in the PBPK
model. In this case, as is to be expected from mass balance principles, the PBPK modeling provides
no further information; the HED obtained from the model-estimated amount of total RDX
metabolites scaled by BW% was equal to that calculated using administered dose scaled by BW3/4
In addition to the lack of data on metabolism, other major uncertainties were identified in the
mouse PBPK modeling; EPA's evaluation of these uncertainties is summarized briefly in Section
2.1.3 and in more detail in Appendix C, Section C.2.5. Therefore, the PBPK model developed for the
mouse was not used, and consistent with the EPA's Guidelines for Carcinogen Risk Assessment (U.S.
EPA. 2005a). the preferred approach for calculating an HED from the mouse tumors is adjustment
of the administered dose by allometric scaling to achieve toxicological equivalence across species.
As discussed in Section 2.1.1, the administered dose in animals is converted to an HED on
the basis of (body weight)3/4 fU.S. EPA. 19921. This was accomplished by multiplying administered
dose by (animal body weight in kg/human body weight in kg)1/4 (U.S. EPA. 1992). where the body
weight for the mouse is 0.035 kg and the reference body weight for humans is 70 kg fU.S. EPA.
1988). It was not necessary to adjust the administered doses to HEDs prior to BMD modeling
because the relationship between the two dose metrics is linear and the same POD would be
produced whether the adjustment was performed before or after modeling. Details of the BMD
modeling can be found in Appendix D, Section D.2.
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2.3.3. Derivation of the Oral Slope Factor
1 The lifetime oral cancer slope factor for humans is defined as the slope of the line from the
2 BMR (10% extra risk) at the BMDL to the estimated control response at zero (slope factor =
3 0.1/BMDLio-hed)- This slope, a 95% upper confidence limit (UCL) on the true slope, represents a
4 plausible upper bound on the true risk. The PODs estimated for each mouse tumor site are
5 summarized in Table 2-5. Using linear extrapolation from the BMDLio-hed, human equivalent oral
6 slope factors (OSFs) were derived for each tumor site individually and both sites combined and are
7 listed in Table 2-5.
Table 2-5. Model predictions and oral slope factors for hepatocellular and
alveolar/bronchiolar adenomas or carcinomas in female B6C3Fi mice
administered RDX in the diet for 2 years (Lish et al., 1984a)
Tumor type
Selected
model
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
POD =
BMDLio-hed3
(mg/kg-d)
OSFb
(mg/kg-d)1
Hepatocellular adenomas or
carcinomas0
Multistage 1°
10% ER
64.2
32.6
4.89
0.020
Alveolar/bronchiolar
adenomas or carcinomas
Multistage 1°
10% ER
52.8
27.7
4.16
0.024
Liver + lung tumors
Multistage 1°
(MS-COMBO)
10% ER
29.0
17.7
2.66
0.038
8
9 aBMDLio-HED = BMDLio x (BWa1/4/BWh1/4), where BWa = 0.035 kg, and BWh = 70 kg.
10 bSlope factor = BMR/BMDLio-hed, where BMR = 0.1 (10% extra risk).
11 incidence of female mouse liver tumors from Lish et al. (1984) are those reported in the PWG reevaluation (Parker
12 et al., 2006).
13 An OSF was derived from the BMDLio-hed based on significantly increased incidence of
14 hepatocellular and alveolar/bronchiolar adenomas or carcinomas in female B6C3Fi mice (i.e., the
15 Liver + Lung BMDLio-hed from MS-COMBO). The OSF of 0.04 (mg/kg-day)-1 is calculated by
16 dividing the BMR (10% extra risk) by the Liver + Lung BMDLio-hed and represents an upper bound
17 on cancer risk associated with a continuous lifetime exposure:
OSF = 0.1 4- (Liver + Lung) BMDLio-hed
= 3.8 x 10"2 (mg/kg-day)-1
= 4 x 10"2 (mg/kg-day)-1, rounded to one significant figure
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2.3.4. Uncertainties in the Derivation of the Oral Slope Factor
1 A number of uncertainties underlie the cancer unit risk for RDX. Table 2-6 summarizes the
2 impact on the assessment of issues such as the use of models and extrapolation approaches
3 (particularly those underlying the Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005all. the
4 effect of reasonable alternatives, the approach selected, and its justification.
Table 2-6. Summary of uncertainty in the derivation of the cancer risk value
for RDX
Consideration and
impact on cancer risk value
Decision
Justification
Selection of study
The cancer bioassay in the rat
(Levine et al.. 1983) would provide a
lower estimate of the OSF
Lish et al. (1984) as
principal oral study to
derive the human
cancer risk estimate
Lish et al. (1984) was a well-conducted studv;
five dose levels (including control) used, with a
sufficient number of animals per dose group (at
terminal sacrifice, n = 62-65/dose group except
highest dose where n = 31). Tumor data from
the mouse provided a stronger basis for
estimating the OSF than rat data, and yielded a
higher (and therefore more health protective)
estimate of risk than data from the rat bioassay.
Species/gender
Use of data sets from the male
mouse would not support
quantitative analysis of carcinogenic
risk
OSF based on tumors in
female mouse
It is assumed that a positive tumor response in
animal cancer studies indicates the agent can
have carcinogenic potential in humans in the
absence of data indicating animal tumors are not
relevant to humans (U.S. EPA, 2005a). As there
are no data to inform whether the response in
any given experimental animal species or gender
would be most relevant for extrapolating to
humans, tumor data from the most sensitive
species and gender were selected as the basis of
the OSF.
Combined tumor types
Human risk would 4, if OSF based on
analysis using only a single tumor
type
OSF based on liver and
lung tumors in female
mouse
Basing the OSF on one tumor site could
potentially underestimate the carcinogenic
potential of RDX, so an analysis that included
data from the two tumor sites was chosen to
calculate the combined risk. Because there is no
known biological dependence between the liver
and lung tumors, independence between the
two tumor sites was assumed. This is not likely
to produce substantial error in the risk estimates
(NRC, 1994).
Selection of dose metric
PBPK models are available for the
rat, mouse and human, and using an
appropriate internal metric can T*
accuracy in human extrapolation.
Mouse liver and lung
tumors: use
administered dose
Lack of sufficient data on RDX metabolism and
major uncertainties identified in the mouse
PBPK model.
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Consideration and
impact on cancer risk value
Decision
Justification
Cross-species scaling
Alternatives could 4^ or T* slope
factor (e.g., 3.5-fold 4^ [scaling by
body weight] or T* 2-fold [scaling by
BW2/3])
BW3/4 scaling (default
approach)
There are no data to support alternatives.
Because the dose metric was not an area under
the curve, BW3/4 scaling was used to calculate
equivalent cumulative exposures for estimating
equivalent human risks. While the true human
correspondence is unknown, this overall
approach is expected neither to over- or
underestimate human equivalent risks.
BMD model uncertainty
Alternative models could 4^ or T*
slope factor
Use multistage model to
derive a BMD and BMDL
for combined tumor
incidence
No biologically-based models for RDX are
available, and there is no a priori basis for
selecting a model other than the multistage.
The multistage model has biological support and
is the model most consistently used in EPA
cancer assessments (Gehlhaus et al., 2011).
Low-dose extrapolation approach
4/ cancer risk would be expected
with the application of nonlinear
extrapolation
Linear extrapolation
from the POD
Where the available information is insufficient to
establish the MOA for tumors at a given site,
linear extrapolation is recommended because
this extrapolation approach is generally
considered to be health-protective (U.S. EPA,
2005a). Because the MOA for RDX-induced liver
and lung tumors has not been established, linear
low-dose extrapolation was applied consistent
with EPA guidance.
Statistical uncertainty at the POD
4/ OSF by 1.6-fold if BMD used as
the POD rather than the BMDL
BMDL (default approach
for calculating plausible
upper bound slope
factor)
Lower bound is 95% CI on administered
exposure at 10% extra risk of liver and lung
tumors.
Sensitive subpopulations
1" OSF to an unknown extent
Considered qualitatively
No data are available to support a range of
human variability/sensitivity in toxicokinetics or
toxicodynamics for RDX, including whether
children are more sensitive than other life
stages.
1
2.3.5. Previous IRIS Assessment: Oral Slope Factor
2 The previous cancer assessment for RDX was posted to the IRIS database in 1990. The oral
3 slope factor in the previous cancer assessment was based on the bioassay by Lish etal. T19841 and
4 analysis of data for hepatocellular adenomas or carcinomas in female mice. A slope factor of
5 1.1 x 10"1 (mg/kg-day)-1 was derived using a linearized multistage procedure (extra risk). This
6 differs from the slope factor for hepatocellular tumors in Table 2-6, because the current OSF is
7 based on the combined incidence of hepatocellular and alveolar/bronchiolar adenomas or
8 carcinomas, PWG reevaluation of female mouse liver tumors, and use of scaling by body weight to
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
the 3/4 power for cross-species extrapolation (whereas the previous assessment scaled by body
weight to the 2/3 power).
2.4. INHALATION UNIT RISK FOR CANCER
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question and quantitative estimates of risk from oral and inhalation exposure
may be derived. Quantitative risk estimates may be derived from the application of a low-dose
extrapolation procedure. If derived, the inhalation unit risk is a plausible upper bound on the
estimate of risk per |J.g/m3 air breathed.
An inhalation unit risk value was not calculated because inhalation carcinogenicity data for
RDX are not available. A PBPK model for inhaled RDX is not available to support route-to-route
extrapolation from the OSF.
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life
Exposure to Carcinogens (U.S. EPA. 2005b). either default or chemical-specific age-dependent
adjustment factors (ADAFs) are applied to account for early-life exposure to carcinogens that act
through a mutagenic MOA. Because no chemical-specific data on life-stage susceptibility for RDX
carcinogenicity are available, and because the MOA for RDX carcinogenicity is not known (see
Section 1.1.5), ADAFs were not applied.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological ReviewofHexahydro-l,3,5-trinitro-l,3,5-triazine
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