c EDA
WtrM
EPA/635/R-18/065a
Final Agency/Interagency Draft
www.epa.gov/iris
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX)
(CASRN 121-82-4]
May 2018
NOTICE
This document is a Final Agency Review/Interagency Science Discussion 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.
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
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 ix
PREFACE xiii
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xvi
EXECUTIVE SUMMARY xxiv
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION xxix
1. HAZARD IDENTIFICATION 1-1
1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS 1-1
1.1.1. Chemical Properties 1-1
1.1.2. Toxicokinetics 1-3
1.1.3. Description of Toxicokinetic Models 1-3
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-4
1.2.1. Nervous System Effects 1-4
1.2.2. Urinary System (Kidney and Bladder) Effects 1-24
1.2.3. Prostate Effects 1-40
1.2.4. Developmental Effects 1-47
1.2.5. Liver Effects 1-54
1.2.6. Other Noncancer Effects 1-65
1.2.7. Carcinogenicity 1-68
1.3. INTEGRATION AND EVALUATION 1-76
1.3.1. Effects Other Than Cancer 1-76
1.3.2. Carcinogenicity 1-80
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 1-82
2. DOSE-RESPONSE ANALYSIS 2-1
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER 2-1
2.1.1. Identification of Studies for Dose-Response Analysis of Selected Effects 2-1
2.1.2. Methods of Analysis 2-5
2.1.3. Derivation of Candidate Values 2-12
2.1.4. Derivation of Organ/System-Specific Reference Doses 2-18
2.1.5. Selection of the Overall Reference Dose 2-20
This document is a draft for review purposes only and does not constitute Agency policy.
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2.1.6. Comparison with Mortality LDoiS 2-21
2.1.7. Uncertainties in the Derivation of the Reference Dose 2-25
2.1.8. Confidence Statement 2-26
2.1.9. Previous IRIS Assessment 2-27
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER 2-27
2.2.1. Previous IRIS Assessment 2-28
2.3. ORAL SLOPE FACTOR FOR CANCER 2-28
2.3.1. Analysis of Carcinogenicity Data 2-28
2.3.2. Dose-Response Analysis—Adjustments and Extrapolation Methods 2-30
2.3.3. Derivation of the Oral Slope Factor 2-32
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 2-33
2.3.5. Previous IRIS Assessment: Oral Slope Factor 2-36
2.4. INHALATION UNIT RISK FOR CANCER 2-36
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 2-36
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 overall RfDfor RDX xxv
Table ES-2. Summary of reference dose (RfD) derivation xxvi
Table LS-1. Inclusion-exclusion criteria for health effect studies xxxiv
Table LS-2. Studies determined not to be informative because of significant issues with design,
conduct, or reporting xxxvi
Table LS-3. Considerations and relevant experimental information for evaluation of
experimental animal studies xxxviii
Table LR-4. Summary of experimental animal database xl
Table LS-5. Experimental animal studies considered less informative because of certain study
design, conduct, or reporting limitations xlv
Table 1-1. Chemical identity and physicochemical properties of RDX from EPA's Chemistry
Dashboard 1-2
Table 1-2. Evidence pertaining to nervous system effects in humans 1-11
Table 1-3. Evidence pertaining to nervous system effects in animals 1-12
Table 1-4. Evidence pertaining to kidney effects in humans 1-27
Table 1-5. Evidence pertaining to urinary system (kidney and bladder) effects in animals 1-27
Table 1-6. Six-, 12-, and 24-month incidence of kidney endpoints in male F344 rats reported for
statistical evaluation in (Levine et al., 1983) 1-34
Table 1-7. Six-, 12-, and 24-month incidence of urinary bladder endpoints in male F344 rats
reported for statistical evaluation in (Levine et al., 1983) 1-37
Table 1-8. Two-year prostate inflammation incidence in male F344 rats (Levine et al., 1983) 1-42
Table 1-9. Evidence pertaining to prostate effects in animals 1-44
Table 1-10. Evidence pertaining to developmental effects in animals 1-49
Table 1-11. Evidence pertaining to liver effects in humans 1-57
Table 1-12. Evidence pertaining to liver effects in animals 1-58
Table 1-13. Liver tumors observed in chronic animal bioassays 1-71
Table 1-14. Lung tumors observed in chronic animal bioassays 1-73
Table 2-1. Information considered for evaluation of studies that examined convulsions 2-3
Table 2-2. Summary of derivation of PODs following oral exposure to RDX 2-8
Table 2-3. Effects and corresponding derivation of candidate values 2-16
Table 2-4. Organ/system-specific RfDs and overall RfD for RDX 2-18
Table 2-5. Comparison of dose levels associated with mortality and convulsions in selected
studies 2-22
Table 2-6. Summary of dose-response evaluation for mortality following oral exposure to RDX 2-23
Table 2-7. Model predictions and OSFs for hepatocellular and alveolar/bronchiolar adenomas or
carcinomas in female B6C3Fi mice administered RDX in the diet for 2 years (Lish
et al., 1984) 2-32
Table 2-8. Summary of uncertainty in the derivation of the cancer risk value for RDX 2-34
This document is a draft for review purposes only and does not constitute Agency policy.
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FIGURES
Figure LS-1. Summary of literature search and screening process for RDX xxxiii
Figure 1-1. Exposure response array of nervous system effects following oral exposure 1-18
Figure 1-2. Exposure-response array of urinary system (kidney and bladder) effects 1-38
Figure 1-3. Exposure-response array of prostate effects 1-45
Figure 1-4. Exposure response array of developmental effects following oral exposure 1-53
Figure 1-5. Exposure response array of liver effects following oral exposure 1-64
Figure 2-1. Conceptual approach to dose-response modeling for oral exposure 2-6
Figure 2-2. Candidate values with corresponding POD and composite UF 2-17
This document is a draft for review purposes only and does not constitute Agency policy.
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ABBREVIATIONS
AAP Army ammunition plant FUDS
ACGIH American Conference of Governmental GAB A
Industrial Hygienists GD
AChE acetylcholinesterase GI
ADAF age-dependent adjustment factor GLP
AIC Akaike's information criterion HED
ALP alkaline phosphatase HERO
ALT alanine aminotransferase
AOP adverse outcome pathway HGPRT
AST aspartate aminotransferase
atm atmosphere HMX
ATSDR Agency for Toxic Substances and
Disease Registry IARC
AUC area under the curve
BDNF brain-derived neurotrophic factor i.p.
BHC beta-hexachlorocyclohexane IPCS
BMC benchmark concentration
BMCL benchmark concentration lower IRIS
confidence limit IUR
BMD benchmark dose i.v.
BMDL benchmark dose lower confidence limit LDH
BMDS Benchmark Dose Software LOAEL
BMDU benchmark dose upper bound LOD
BMR benchmark response miRNA
BUN blood urea nitrogen MNX
BW body weight
CAAC Chemical Assessment Advisory MOA
Committee MRL
CASRN Chemical Abstracts Service Registry NAPDH
Number
CCL Contaminant Candidate List NAS
CI confidence interval NCE
CICAD Concise International Chemical NCEA
Assessment Document
CNS central nervous system NCI
CSF cerebrospinal fluid NCTR
CYP450 cytochrome P450
DAF dosimetric adjustment factor NHANES
DDT dichlorodiphenyltrichloroethane
d.f. degrees of freedom NICNAS
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid NIEHS
DNX l-nitro-3,5-dinitroso-
1,3,5-triazacyclohexane NIOSH
DTIC Defense Technical Information Center
EEG electroencephalogram NOAEL
EHC Environmental Health Criteria NOEL
EPA Environmental Protection Agency NPL
ER extra risk NRC
FDA Food and Drug Administration NSCEP
FOB functional observational battery
Formerly Used Defense Sites
gamma-amino butyric acid
gestational day
gastrointestinal
good laboratory practices
human equivalent dose
Health and Environmental Research
Online
hyp oxan thine-guanine
phosphoribosyltransferase
octahydro-l,3,5,7-tetranitro-
1,3,5,7-tetrazocine
International Agency for Research on
Cancer
intraperitoneal
International Programme on Chemical
Safety
Integrated Risk Information System
inhalation unit risk
intravenous
lactate dehydrogenase
lowest-observed-adverse-effect level
limit of detection
microRNA
hexahydro-l-nitroso-3,5-dinitro-
1,3,5-triazine
mode of action
Minimal Risk Level
nicotinamide adenine dinucleotide
phosphate
National Academy of Science
normochromatic erythrocyte
National Center for Environmental
Assessment
National Cancer Institute
National Center for Toxicological
Research
National Health and Nutrition
Examination Survey
National Industrial Chemicals
Notification and Assessment Scheme
National Institute of Environmental
Health Sciences
National Institute for Occupational
Safety and Health
no-observed-adverse-effect level
no-observed-effect level
National Priorities List
Nuclear Regulatory Commission
National Service Center for
Environmental Publications
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
NTP
National Toxicology Program
SGOT
glutamic oxaloacetic transaminase, also
NZW
New Zealand White
known as AST
OR
odds ratio
SGPT
glutamic pyruvic transaminase, also
ORD
Office of Research and Development
known as ALT
OSF
oral slope factor
SLE
systemic lupus erythematosus
OSHA
Occupational Safety and Health
SS
scheduled sacrifice
Administration
TLV
Threshold Limit Value
PBPK
physiologically based pharmacokinetic
TNT
trinitrotoluene
PCB
polychlorinated biphenyl
TNX
hexahydro-l,3,5-trinitroso-
PCE
polychromatic erythrocyte
1,3,5-triazine
PEL
Permissible Exposure Limit
TSCATS
Toxic Substances Control Act Test
PND
postnatal day
Submissions
POD
point of departure
TWA
time-weighted average
PWG
Pathology Working Group
U.S.
United States of America
RBC
red blood cell
UCM
Unregulated Contaminant Monitoring
RDX
Royal Demolition eXplosive
UF
uncertainty factor
(hexahydro-l,3,5-trinitro-
UFa
animal-to-human uncertainty factor
1,3,5-triazine]
UFd
database deficiencies uncertainty factor
REL
Recommended Exposure Limit
UFh
human variation uncertainty factor
RfC
inhalation reference concentration
UFl
LOAEL-to-NOAEL uncertain factor
RfD
oral reference dose
UFs
subchronic-to-chronic uncertainty
SDMS
spontaneous death or moribund
factor
sacrifice
WBC
white blood cell
SDWA
Safe Drinking Water Act
WHO
World Health Organization
This document is a draft for review purposes only and does not constitute Agency policy.
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AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Louis D'Amico, Ph.D. (Assessment Manager) U.S. EPA/Office of Research and Development/National
Todd Blessinger, Ph.D. Center for Environmental Assessment
Ravi Subramaniam, Ph.D.
Christopher Brinkerhoff, Ph.D. Former ORISE Postdoctoral Fellow at U.S.
EPA/ORD/NCEA
Currently at U.S. EPA/Office of Chemical Safety and
Pollution Prevention
Contributors
Rob DeWoskin, Ph.D. (retired) U.S. EPA/ORD/NCEA
Belinda Hawkins, Ph.D.
Karen Hogan, MS
Andrew Kraft, Ph.D.
Jordan Trecki, Ph.D. (formerly with EPA)
Scott Wesselkamper, Ph.D.
Tammy Stoker, Ph.D.
Charles Wood, DVM, PhD, DACVP
U.S. EPA/ORD/National Health and Environmental
Research Laboratory (NHEERL)
Anne Loccisano, Ph.D.
Former ORISE Postdoctoral Fellow at U.S.
EPA/ORD/NCEA
Kelly Garcia
Carolyn Gigot
EPA Environmental Health Assessment Support
Associate (ORAU Student Services Contractor)
Production Team
Maureen Johnson U.S. EPA/ORD/NCEA
Vicki Soto
Dahnish Shams
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
Tina Bahadori, Sc.D. (Center Director, National Program U.S. EPA/ORD/NCEA
Director, HHRA)
Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director - Retired)
Michael Slimak, Ph.D. (Acting Center Director)
This document is a draft for review purposes only and does not constitute Agency policy.
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John Vandenberg, Ph.D, (Former National Program Director,
HHRA)
Lynn Flowers, Ph.D., DABT (Former Associate Director for
Health)
Kristina Thayer, Ph.D. (IRIS Division Director)
Vincent Cogliano, Ph.D. (Former IRIS Division Director)
James Avery, Ph.D. (IRIS Division Deputy Director)
Gina Perovich, M.S. (Former IRIS Division Deputy Director)
Samantha Jones, Ph.D. (IRIS Associate Director for Science)
Susan Rieth, MPH (Quantitative Modeling Branch Chief)
Internal Review Team
General Toxicity/Immunotoxicity/Cancer Workgroup U.S. EPA/ORD/NCEA
Epidemiology Workgroup
Neurotoxicity Workgroup
Pharmacokinetics Workgroup
Reproductive and Developmental Toxicity Workgroup
Scoping and Problem Formulation Workgroup
Statistics Workgroup
Toxicity Pathways Workgroup
Reviewers
This assessment was provided for review to scientists in EPA's program and regional offices.
Comments were submitted by:
Office of the Administrator/Office of Children's Health Protection
Office of Chemical Safety and Pollution Prevention/Office of Pesticide Programs
Office of Land and Emergency Management
Office of Land and Emergency Management/Federal Facilities Forum
Office of Water
Region 2, New York City
Region 8, Denver
This assessment was provided for review to other federal agencies and the Executive Office of the
President (EOP). A summary and EPA's disposition of major comments from the other federal
agencies and the EOP is available on the IRIS website. Comments were submitted by:
Department of Defense
Department of Energy
Department of Health and Human Services/Agency for Toxic Substances and Disease Registry
Department of Health and Human Services/National Institute of Environmental Health Sciences/National
Toxicology Program
Department of Health and Human Services/National Institute for Occupational Safety and Health
National Aeronautics and Space Administration
Executive Office of the President/Council on Environmental Quality
Executive Office of the President/Office of Management and Budget
This assessment was released for public comment on March 10,2016 and comments were due on
May 9, 2016. The public comments are available on Regulations.gov. A summary and EPA's
disposition of the comments from the public is included in the external review draft assessment on
the IRIS website. Comments were received from the following entities:
This document is a draft for review purposes only and does not constitute Agency policy.
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Nancy Beck, American Chemistry Council (currently Deputy Assistant Administrator, Office of Chemical
Safety and Pollution Prevention, U.S. EPA)
Andy Nong, Health Canada
Johns Hopkins Bloomberg School of Public Health, Special Studies in Risk Assessment Class as
submitted by Mary Fox
Ron Melnick
U.S. Army Public Health Command and Uniformed Services University of the Health Sciences
Larry Williams
Anonymous member of the public
A public science meeting was held on May 10, 2016 to obtain public input on the IRIS Toxicological
Review of RDX (Public Comment Draft). Public commenters, stakeholders, and members of the
scientific community were joined by independent experts identified by the National Academies'
National Research Council (NRC) (identified by * below) in a discussion of key science topics.
Discussants and public commenters were:
Desmond Bannon
Nancy Beck
Maria Braga*
Marion Ehrich*
Kendall Frazier*
Andy Nong*
Karen Regan*
Evelyn Tiffany-Castiglioni*
Larry Williams
U.S. Army Public Health Center
American Chemistry Council (currently Deputy Assistant Administrator,
Office of Chemical Safety and Pollution Prevention, U.S. EPA)
Uniformed Services University of the Health Sciences
Virginia-Maryland College of Veterinary Medicine (Virginia Tech)
GlaxoSmithKline
Health Canada
Research Pathology Associates
Texas A&M University
This assessment was peer reviewed by independent, expert scientists external to EPA convened by
EPA's Science Advisory Board (SAB), the SAB Chemical Assessment Advisory Committee (CAAC)
Augmented for Review of the Draft IRIS RDX Assessment A peer-review meeting was held on
December 12-14, 2016. The report of the SAB's review of the EPA's draft Toxicological Review of
RDX, dated September 27, 2017, is available on the IRIS website. A summary and EPA's disposition
of the comments received from the SAB is included in Appendix E.
Dr. Kenneth Ramos (Chair)
Dr. Hugh A. Barton
Dr. Maarten C. Bosland
Dr. Mary Boudreau
Dr. James V. Bruckner
Dr. George Cobb
Dr. David Eastmond
Dr. Joanne English
Dr. Alan Hoberman
Dr. Jacqueline Hughes-Oliver
Dr. Susan Laffan
Associate Vice-President of Precision Health Sciences and Professor of
Medicine, University of Arizona Health Sciences, Tucson, AZ
Associate Research Fellow, Pharmacokinetics, Dynamics, and Metabolism,
Pfizer Inc., Groton, CT
Professor of Pathology, College of Medicine, University of Illinois at Chicago,
Chicago, IL
Research Toxicologist, Division of Biochemical Toxicology, National Center
for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR
Professor, Department of Pharmacology & Toxicology, College of Pharmacy,
University of Georgia, Athens, GA
Professor, Environmental Science, College of Arts and Sciences, Baylor
University, Waco, TX
Professor and Chair, Department of Cell Biology and Neuroscience,
Toxicology Graduate Program, University of California at Riverside,
Riverside, CA
Independent Consultant, Menlo Park, CA
Toxicologist, Research, Charles River Laboratories, Inc., Horsham, PA
Professor, Statistics Department, North Carolina State University, Raleigh,
NC
Safety Assessment, GlaxoSmithKline, King of Prussia, PA
This document is a draft for review purposes only and does not constitute Agency policy.
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Dr. Lawrence Lash
Dr. Stephen Lasley
Dr. Melanie Marty
Dr. Marvin Meistrich
Dr. Marilyn Morris
Dr. Victoria Persky
Dr. Isaac Pessah
Dr. Kenneth M. Portier
Dr. Samba Reddy
Dr. Stephen M. Roberts
Dr. Thomas Rosol
Dr. Alan Stern
Dr. Robert Turesky
Professor, Department of Pharmacology, Wayne State University School of
Medicine, Wayne State University, Detroit, MI
Professor of Pharmacology and Assistant Head, Cancer Biology &
Pharmacology, College of Medicine, University of Illinois at Chicago, Peoria,
IL
Adjunct Professor, Environmental Toxicology, University of California at
Davis, Davis, CA
Professor, Experimental Radiation Oncology, M.D. Anderson Cancer Center,
University of Texas, Houston, TX
Professor of Pharmaceutical Sciences, School of Pharmacy and
Pharmaceutical Sciences, University at Buffalo, State University of New York,
Buffalo, NY
Professor, Epidemiology & Biostatistics Program, School of Public Health,
University of Illinois at Chicago, Chicago, IL
Professor, Molecular Biosciences, School of Veterinary Medicine, University
of California at Davis, Davis, CA
Independent Consultant, Athens, GA
Professor, Neuroscience and Experimental Therapeutics, College of
Medicine, Texas A&M University, Bryan, TX
Professor, Center for Environmental and Human Toxicology, University of
Florida, Gainesville, FL
Professor, Veterinary Biosciences, College of Veterinary Medicine, Ohio State
University, Columbus, OH
Chief, Bureau for Risk Analysis, Division of Science, Research and
Environmental Health, New Jersey Department of Environmental Protection,
Trenton, NJ
Professor, Masonic Cancer Center and Department of Medicinal Chemistry,
College of Pharmacy, University of Minnesota, Minneapolis, MN
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
PREFACE
This Toxicological Review critically reviews the publicly available studies on hexahydro-
l,3,5-trinitro-l,3,5-triazine (RDX, Royal Demolition eXplosive, or cyclonite) in order to identify its
adverse health effects and to characterize exposure-response relationships. It was prepared under
the auspices of the U.S. Environmental Protection Agency (EPA) 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, and derivation of an oral slope factor (OSF) 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.
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 on the preliminary materials were taken into consideration in developing the draft
assessment. A second public meeting was held in May 2016 to discuss key science topics on the
public comment draft assessment. These topics included: (1) suppurative prostatitis as a marker
for hazard to the urogenital system following RDX exposure; (2) evaluation and use of RDX
physiologically-based pharmacokinetic (PBPK) models; (3) neurotoxicity observed with RDX and
consideration of dose and duration of exposure and the potential relationship to mortality; and
(4) other science topics in the RDX assessment. Independent experts identified by the National
Academies' National Research Council (NRC) joined members of the scientific community,
stakeholders, and the general public in the discussion of these science topics. The complete set of
public comments submitted in connection with the December 2013 and May 2016 public meetings
are available on the docket at https://www.regulations.gov fDocket ID No. EPA-HQ-ORD-2013-
0430).
Organ/system-specific reference values are calculated based on nervous system, urinary
system (kidney and bladder), and prostate. 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 summarized in
the Preamble to IRIS Toxicological Reviews and cited at appropriate places in this assessment. The
findings of this assessment and related documents produced during its development are available
on the IRIS website fhttps: //www.epa.gov/irisI Appendices containing information on
assessments by other health agencies, details of the literature search strategy, toxicokinetic
This document is a draft for review purposes only and does not constitute Agency policy.
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information, summaries of supplementary toxicity information, and dose-response modeling are
provided as Supplemental Information to this assessment (see Appendices A to D).
The IRIS Program released preliminary assessment materials for RDX in December 2013
and the draft assessment for public comment in March 2016, during the period of development and
implementation of systematic review methods by the IRIS Program. The approach to
implementation is to use procedures and tools available at the time, without holding assessments
until new methods become available. Accordingly, the IRIS Program conducted literature searches
and evaluated studies using tools and documentation standards then available. Updated problem
formulation materials and systematic review protocol development began with assessments
started in 2015, after this assessment was well into assessment development. Implementation of
systematic review is a process of continuous improvement and this assessment represents a step in
the evolution of the IRIS Program.
Uses and Environmental Occurrence
RDX is a military munitions explosive with limited civilian commercial uses (Gadagbui etal..
20121. In the United States, RDX is produced at Army ammunition plants (AAPs) and is not
manufactured 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
fATSDR. 20121. According to the U.S. EPA ChemViewTool (https://chemview.epa.gov/chemview),
the aggregate national production volume in 2015 was between 1 million and 10 million pounds.
RDX can be released into environmental media (air, water, soil) as a result of waste
generated during manufacture, packing, or disposal of the pure product, or use and disposal of RDX-
containingmunitions fATSDR. 2012: Gadagbui etal.. 2012: ATSDR. 1999.1993.19921. 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 et al..
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). Discussion of RDX properties and fate and transport is available in U.S.
EPA (2012b) and on the EPA's Chemistry Dashboard at https: //comptox.epa.gov/dashboard/.
Detectable levels of RDX have been observed in plants irrigated or grown with RDX-contaminated
water (Best etal.. 1999b: Simini and Checkai. 1996: Harvey etal.. 1991). RDX has also been
detected in indoor air samples from military facilities where RDX is produced fBishop etal.. 19881.
Exposures to RDX among the general population are likely to be confined to individuals in
or around active or formerly-used military facilities where RDX is or was produced, stored, or used.
Oral, inhalation, and dermal routes of exposure may be relevant.
As of 2018, RDX was detected in surface water, groundwater, sediment, or soil at 32 active
U.S. EPA National Priorities List (NPL) sites. The NPL serves as a list of sites with known or
threatened releases of hazardous substances, pollutants, or contaminants throughout the United
States and its territories. The NPL aids the Agency in identifying the most serious sites that may
This document is a draft for review purposes only and does not constitute Agency policy.
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warrant cleanup. The majority of the NPL sites where RDX was listed are associated with military
facilities. Based on Department of Defense records, Gadagbui etal. T20121 reported that RDX
contamination is present on 76 active military sites, 9 closed sites, and 15 sites under the Formerly
Used Defense Sites (FUDS) program. Not all sites under the FUDS program have been sampled, and
additional sites with RDX contamination in this program could be identified.
As of 2018, RDX was 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 on the Office of Water's Drinking Water
Contaminant Candidate List (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 information on RDX has been evaluated by the Agency for Toxic Substances and
Disease Registry (ATSDR), National Institute for Occupational Safety and Health (NIOSH),
Occupational Safety and Health Administration (OSHA), and Australian National Industrial
Chemicals Notification and Assessment Scheme (NICNAS). The results of these assessments (as of
2018) are presented in Appendix A of the Supplemental Information. 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. In addition, newer studies may be included in the IRIS
assessment.
For additional information about this assessment or for general questions regarding IRIS,
please contact EPA's IRIS Hotline at 202-566-1676 (phone), or hotline.iris@epa.gov.
This document is a draft for review purposes only and does not constitute Agency policy.
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The summarizes the objectives and scope of the IRIS program, general principles and
systematic review procedures used in developing IRIS assessments¦, and the overall
development process and document structure.
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
Objectives and Scope of the IRIS
Program
Soon after EPA was established in 1970, it
was at the forefront of developing risk
assessment as a science and applying it in
support of actions to protect human health and
the environment EPA's IRIS program1
contributes to this endeavor by reviewing
epidemiologic and experimental studies of
chemicals in the environment to identify adverse
health effects and characterize exposure-
response relationships. Health agencies
worldwide use IRIS assessments, which are also
a scientific resource for researchers and the
public.
IRIS assessments cover the hazard
identification and dose-response steps of risk
assessment. Exposure assessment and risk
characterization are outside the scope of IRIS
assessments, as are political, economic, and
technical aspects of risk management. An IRIS
assessment may cover one chemical, a group of
structurally or toxicologically related chemicals,
or a chemical mixture. Exceptions outside the
scope of the IRIS program are radionuclides,
chemicals used only as pesticides, and the
"criteria air pollutants" (particulate matter,
ground-level ozone, carbon monoxide, sulfur
oxides, nitrogen oxides, and lead).
Enhancements to the IRIS program are
improving its science, transparency, and
productivity. To improve the science, the IRIS
program is adapting and implementing
principles of systematic review (i.e., using
explicit methods to identify, evaluate, and
synthesize study findings). To increase
transparency, the IRIS program discusses key
science issues with the scientific community and
the public as it begins an assessment. External
peer review, independently managed and in
public, improves both science and transparency.
Increased productivity requires that
assessments be concise, focused on EPA's needs,
and completed without undue delay.
IRIS assessments follow EPA guidance2 and
standardized practices of systematic review.
This Preamble summarizes and does not change
IRIS operating procedures or EPA guidance.
Periodically, the IRIS program asks for
nomination of agents for future assessment or
reassessment. Selection depends on EPA's
priorities, relevance to public health, and
availability of pertinent studies. The IRIS
multiyear agenda3 lists upcoming assessments.
The IRIS program may also assess other agents
in anticipation of public health needs.
Planning an Assessment: Scoping,
Problem Formulation, and
Protocols
Early attention to planning ensures that IRIS
assessments meet their objectives and properly
frame science issues.
Scoping refers to the first step of planning
where the IRIS program consults with EPA's
program and regional offices to ascertain their
MRIS program website: http: IIwww.epa.gov /iris /.
2EPA guidance documents: http://www.epa.gov/iris/basic-information-about-integrated-risk-information-
svstem#guidance/.
3IRIS multiyear agenda: https: //www.epa.gov/iris/iris-agenda.
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needs. Scoping specifies the agents an
assessment will address, routes and durations of
exposure, susceptible populations and lifestages,
and other topics of interest
Problem formulation refers to the science
issues an assessment will address and includes
input from the scientific community and the
public. A preliminary literature survey,
beginning with secondary sources (e.g.,
assessments by national and international health
agencies and comprehensive review articles),
identifies potential health outcomes and science
issues. It also identifies related chemicals (e.g.,
toxicologically active metabolites and
compounds that metabolize to the chemical of
interest).
Each IRIS assessment comprises multiple
systematic reviews for multiple health
outcomes. It also evaluates hypothesized
mechanistic pathways and characterizes
exposure-response relationships. An
assessment may focus on important health
outcomes and analyses rather than expand
beyond what is necessary to meet its objectives.
Protocols refer to the systematic review
procedures planned for use in an assessment.
They include strategies for literature searches,
criteria for study inclusion or exclusion,
considerations for evaluating study methods and
quality, and approaches to extracting data.
Protocols may evolve as an assessment
progresses and new agent-specific insights and
issues emerge.
Identifying and Selecting Pertinent
Studies
IRIS assessments conduct systematic
literature searches with criteria for inclusion and
exclusion. The objective is to retrieve the
pertinent primary studies (i.e., studies with
original data on health outcomes or their
mechanisms). PECO statements (Populations,
Exposures, Comparisons, Outcomes) govern the
literature searches and screening criteria.
"Populations" and animal species generally have
no restrictions. "Exposures" refers to the agent
and related chemicals identified during scoping
and problem formulation and may consider
route, duration, or timing of exposure.
"Comparisons" means studies that allow
comparison of effects across different levels of
exposure. "Outcomes" may become more specific
(e.g., from "toxicity" to "developmental toxicity"
to "hypospadias") as an assessment progresses.
For studies of absorption, distribution,
metabolism, and elimination, the first objective
is to create an inventory of pertinent studies.
Subsequent sorting and analysis facilitates
characterization and quantification of these
processes.
Studies on mechanistic events can be
numerous and diverse. Here, too, the objective is
to create an inventory of studies for later sorting
to support analyses of related data. The
inventory also facilitates generation and
evaluation of hypothesized mechanistic
pathways.
The IRIS program posts initial protocols for
literature searches on its website and adds
search results to EPA's HERO database.4 Then
the IRIS program takes extra steps to ensure
identification of pertinent studies: by
encouraging the scientific community and the
public to identify additional studies and ongoing
research; by searching for data submitted under
the Toxic Substances Control Act or the Federal
Insecticide, Fungicide, and Rodenticide Act; and
by considering late-breaking studies that would
impact the credibility of the conclusions, even
during the review process.5
Evaluating Study Methods and Quality
IRIS assessments evaluate study methods
and quality, using uniform approaches for each
group of similar studies. The objective is that
subsequent syntheses can weigh study results on
their merits. Key concerns are potential bias
(factors that affect the magnitude or direction of
an effect) and insensitivity (factors that limit the
ability of a study to detect a true effect).
4Health and Environmental Research Online: https: //hero.epa.gov/hero/.
5IRIS "stopping rules": https: //www.epa.gov/sites/production/files/2014-06/documents/
iris stoppingrules.pdf.
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For human and animal studies, the
evaluation of study methods and quality
considers study design, exposure measures,
outcome measures, data analysis, selective
reporting, and study sensitivity. For human
studies, this evaluation also considers selection
of participant and referent groups and potential
confounding. Emphasis is on discerning bias that
could substantively change an effect estimate,
considering also the expected direction of the
bias. Low sensitivity is a bias towards the null.
Study-evaluation considerations are specific
to each study design, health effect, and agent.
Subject-matter experts evaluate each group of
studies to identify characteristics that bear on
the informativeness of the results. For
carcinogenicity, neurotoxicity, reproductive
toxicity, and developmental toxicity, there is EPA
guidance for study evaluation (U.S. EPA. 2005a.
1998. 1996. 19911. As subject-matter experts
examine a group of studies, additional agent-
specific knowledge or methodologic concerns
may emerge and a second pass become
necessary.
Assessments use evidence tables to
summarize the design and results of pertinent
studies. If tables become too numerous or
unwieldy, they may focus on effects that are
more important or studies that are more
informative.
The IRIS program posts initial protocols for
study evaluation on its website, then considers
public input as it completes this step.
Integrating the Evidence of Causation
for Each Health Outcome
Synthesis within lines of evidence. For
each health outcome, IRIS assessments
synthesize the human evidence and the animal
evidence, augmenting each with informative
subsets of mechanistic data. Each synthesis
considers aspects of an association that may
suggest causation: consistency, exposure-
response relationship, strength of association,
temporal relationship, biological plausibility,
coherence, and "natural experiments" in humans
flLS. EPA. 1994. §2.1.3) flJ.S. EPA. 2005a. §2.5).
Each synthesis seeks to reconcile ostensible
inconsistencies between studies, taking into
account differences in study methods and
quality. This leads to a distinction between
conflicting evidence (unexplained positive and
negative results in similarly exposed human
populations or in similar animal models) and
differing results (mixed results attributable to
differences between human populations, animal
models, or exposure conditions) (U.S. EPA. 2005a.
§2.5).
Each synthesis of human evidence explores
alternative explanations (e.g., chance, bias, or
confounding) and determines whether they may
satisfactorily explain the results. Each synthesis
of animal evidence explores the potential for
analogous results in humans. Coherent results
across multiple species increase confidence that
the animal results are relevant to humans.
Mechanistic data are useful to augment the
human or animal evidence with information on
precursor events, to evaluate the human
relevance of animal results, or to identify
susceptible populations and lifestages. An agent
may operate through multiple mechanistic
pathways, even if one hypothesis dominates the
literature fU.S. EPA. 2005a. §2.4.3.3).
Integration across lines of evidence. For
each health outcome, IRIS assessments integrate
the human, animal, and mechanistic evidence to
answer the question: What is the nature of the
association between exposure to the agent and the
health outcome?
For cancer, EPA includes a standardized
hazard descriptor in characterizing the strength
of the evidence of causation. The objective is to
promote clarity and consistency of conclusions
across assessments (U.S. EPA. 2005a. §2.5).
Carcinogenic to humans: convincing
epidemiologic evidence of a causal
association; or strong human evidence of
cancer or its key precursors, extensive
animal evidence, identification of mode-of-
action and its key precursors in animals, and
strong evidence that they are anticipated in
humans.
Likely to be carcinogenic to humans: evidence
that demonstrates a potential hazard to
humans. Examples include a plausible
association in humans with supporting
experimental evidence, multiple positive
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results in animals, a rare animal response, or
a positive study strengthened by other lines
of evidence.
Suggestive evidence of carcinogenic potential:
evidence that raises a concern for humans.
Examples include a positive result in the only
study, or a single positive result in an
extensive database.
Inadequate information to assess carcinogenic
potential: no other descriptors apply.
Examples include little or no pertinent
information, conflicting evidence, or negative
results not sufficiently robust for not likely.
Not likely to be carcinogenic to humans: robust
evidence to conclude that there is no basis
for concern. Examples include no effects in
well-conducted studies in both sexes of
multiple animal species, extensive evidence
showing that effects in animals arise through
modes-of-action that do not operate in
humans, or convincing evidence that effects
are not likely by a particular exposure route
or below a defined dose.
If there is credible evidence of
carcinogenicity, there is an evaluation of
mutagenicity, because this influences the
approach to dose-response assessment and
subsequent application of adjustment factors for
exposures early in life fU.S. EPA. 2005a. §3.3.1,
§3.5), fU.S. EPA. 2005b. §5).
Selecting Studies for Derivation of
Toxicity Values
The purpose of toxicity values (slope factors,
unit risks, reference doses, reference
concentrations; see section 7) is to estimate
exposure levels likely to be without appreciable
risk of adverse health effects. EPA uses these
values to support its actions to protect human
health.
The health outcomes considered for
derivation of toxicity values may depend on the
hazard descriptors. For example, IRIS
assessments generally derive cancer values for
agents that are carcinogenic or likely to be
carcinogenic, and sometimes for agents with
suggestive evidence (U.S. EPA. 2005a. §3).
Derivation of toxicity values begins with a
new evaluation of studies, as some studies used
qualitatively for hazard identification may not be
useful quantitatively for exposure-response
assessment. Quantitative analyses require
quantitative measures of exposure and response.
An assessment weighs the merits of the human
and animal studies, of various animal models,
and of different routes and durations of exposure
fU.S. EPA. 1994. §2.1). Study selection is not
reducible to a formula, and each assessment
explains its approach.
Other biological determinants of study
quality include appropriate measures of
exposure and response, investigation of early
effects that precede overt toxicity, and
appropriate reporting of related effects (e.g.,
combining effects that comprise a syndrome, or
benign and malignant tumors in a specific
tissue).
Statistical determinants of study quality
include multiple levels of exposure (to
characterize the shape of the exposure-response
curve) and adequate exposure range and sample
sizes (to minimize extrapolation and maximize
precision) (U.S. EPA. 2012. §2.1).
Studies of low sensitivity may be less useful
if they fail to detect a true effect or yield toxicity
values with wide confidence limits.
Deriving Toxicity Values
General approach. EPA guidance describes
a two-step approach to dose-response
assessment: analysis in the range of observation,
then extrapolation to lower levels. Each toxicity
value pertains to a route (e.g., oral, inhalation,
dermal) and duration or timing of exposure (e.g.,
chronic, subchronic, gestational) (U.S. EPA. 2002.
§4).
IRIS assessments derive a candidate value
from each suitable data set. Consideration of
candidate values yields a toxicity value for each
organ or system. Consideration of the organ/
system-specific values results in the selection of
an overall toxicity value to cover all health
outcomes. The organ/system-specific values are
useful for subsequent cumulative risk
assessments that consider the combined effect of
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multiple agents acting at a common anatomical
site.
Analysis in the range of observation.
Within the observed range, the preferred
approach is modeling to incorporate a wide
range of data. Toxicokinetic modeling has
become increasingly common for its ability to
support target-dose estimation, cross-species
adjustment, or exposure-route conversion. If
data are too limited to support toxicokinetic
modeling, there are standardized approaches to
estimate daily exposures and scale them from
animals to humans (U.S. EPA. 1994. §3), (U.S. EPA.
2005a. §3.1), ru.S. EPA. 2011. 20061.
For human studies, an assessment may
develop exposure-response models that reflect
the structure of the available data fU.S. EPA.
2005a. §3.2.1). For animal studies, EPA has
developed a set of empirical ("curve-fitting")
models6 that can fit typical data sets (U.S. EPA.
2005a. §3.2.2). Such modeling yields a point of
departure, defined as a dose near the lower end
of the observed range, without significant
extrapolation to lower levels (e.g., the estimated
dose associated with an extra risk of 10% for
animal data or 1% for human data, or their 95%
lower confidence limits)(U.S. EPA. 2005a. §3.2.4),
flLS. EPA. 2012. §2.2.11.
When justified by the scope of the
assessment, toxicodynamic ("biologically
based") modeling is possible if data are sufficient
to ascertain the key events of a mode-of-action
and to estimate their parameters. Analysis of
model uncertainty can determine the range of
lower doses where data support further use of
the model fU.S. EPA. 2005a. §3.2.2, §3.3.2).
For a group of agents that act at a common
site or through common mechanisms, an
assessment may derive relative potency factors
based on relative toxicity, rates of absorption or
metabolism, quantitative structure-activity
relationships, or receptor-binding
characteristics fU.S. EPA. 2005a. §3.2.6).
Extrapolation: slope factors and unit
risks. An oral slope factor or an inhalation unit
risk facilitates subsequent estimation of human
cancer risks. Extrapolation proceeds linearly
(i.e., risk proportional to dose) from the point of
benchmark Dose Software: http: IIwww.epa.gov/bmds/.
departure to the levels of interest. This is
appropriate for agents with direct mutagenic
activity. It is also the default if there is no
established mode-of-action (U.S. EPA. 2005a.
§3.3.1, §3.3.3).
Differences in susceptibility may warrant
derivation of multiple slope factors or unit risks.
For early-life exposure to carcinogens with a
mutagenic mode-of-action, EPA has developed
default age-dependent adjustment factors for
agents without chemical-specific susceptibility
data fU.S. EPA. 2005a. §3.5), fU.S. EPA. 2005b. §5).
If data are sufficient to ascertain the mode-
of-action and to conclude that it is not linear at
low levels, extrapolation may use the reference-
value approach (U.S. EPA. 2005a. §3.3.4).
Extrapolation: reference values. An oral
reference dose or an inhalation reference
concentration is an estimate of human exposure
(including in susceptible populations) likely to
be without appreciable risk of adverse health
effects over a lifetime fU.S. EPA. 2002. §4.2).
Reference values generally cover effects other
than cancer. They are also appropriate for
carcinogens with a nonlinear mode-of-action.
Calculation of reference values involves
dividing the point of departure by a set of
uncertainty factors (each typically 1, 3, or 10,
unless there are adequate chemical-specific
data) to account for different sources of
uncertainty and variability (U.S. EPA. 2002.
§4.4.5), fU.S. EPA. 20141.
Human variation: An uncertainty factor covers
susceptible populations and lifestages that
may respond at lower levels, unless the data
originate from a susceptible study
population.
Animal-to-human extrapolation: For reference
values based on animal results, an
uncertainty factor reflects cross-species
differences, which may cause humans to
respond at lower levels.
Subchronic-to-chronic exposure: For chronic
reference values based on subchronic
studies, an uncertainty factor reflects the
likelihood that a lower level over a longer
duration may induce a similar response. This
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factor may not be necessary for reference
values of shorter duration.
Adverse-effect level to no-observed-adverse-effect
level: For reference values based on a lowest-
observed-adverse-effect level, an
uncertainty factor reflects a level judged to
have no observable adverse effects.
Database deficiencies: If there is concern that
future studies may identify a more sensitive
effect, target organ, population, or lifestage, a
database uncertainty factor reflects the
nature of the database deficiency.
Process for Developing and Peer-
Reviewing IRIS Assessments
The IRIS process (revised in 2009 and
enhanced in 2013) involves extensive public
engagement and multiple levels of scientific
review and comment IRIS program scientists
consider all comments. Materials released,
comments received from outside EPA, and
disposition of major comments (steps 3, 4, and 6
below) become part of the public record.
Step 1: Draft development. As outlined in
section 2 of this Preamble, IRIS program
scientists specify the scope of an assessment
and formulate science issues for discussion
with the scientific community and the public.
Next, they release initial protocols for the
systematic review procedures planned for
use in the assessment IRIS program
scientists then develop a first draft, using
structured approaches to identify pertinent
studies, evaluate study methods and quality,
integrate the evidence of causation for each
health outcome, select studies for derivation
of toxicity values, and derive toxicity values,
as outlined in Preamble sections 3-7.
Step 2: Agency review. Health scientists across
EPA review the draft assessment.
Step 3: Interagency science consultation.
Other federal agencies and the Executive
Office of the President review the draft
assessment
Step 4: Public comment, followed by external
peer review. The public reviews the draft
assessment IRIS program scientists release
a revised draft for independent external peer
review. The peer reviewers consider
whether the draft assessment assembled and
evaluated the evidence according to EPA
guidance and whether the evidence justifies
the conclusions.
Step 5: Revise assessment. IRIS program
scientists revise the assessment to address
the comments from the peer review.
Step 6: Final agency review and interagency
science discussion. The IRIS program
discusses the revised assessment with EPA's
program and regional offices and with other
federal agencies and the Executive Office of
the President
Step 7: Post final assessment. The IRIS
program posts the completed assessment
and a summary on its website.
General Structure of IRIS Assessments
Main text. IRIS assessments generally
comprise two major sections: (1) Hazard
Identification and (2) Dose-Response
Assessment. Section 1.1 briefly reviews chemical
properties and toxicokinetics to describe the
disposition of the agent in the body. This section
identifies related chemicals and summarizes
their health outcomes, citing authoritative
reviews. If an assessment covers a chemical
mixture, this section discusses environmental
processes that alter the mixtures humans
encounter and compares them to mixtures
studied experimentally.
Section 1.2 includes a subsection for each
major health outcome. Each subsection
discusses the respective literature searches and
study considerations, as outlined in Preamble
sections 3 and 4, unless covered in the front
matter. Each subsection concludes with evidence
synthesis and integration, as outlined in
Preamble section 5.
Section 1.3 links health hazard information
to dose-response analyses for each health
outcome. One subsection identifies susceptible
populations and lifestages, as observed in human
or animal studies or inferred from mechanistic
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data. These may warrant further analysis to
quantify differences in susceptibility. Another
subsection identifies biological considerations
for selecting health outcomes, studies, or data
sets, as outlined in Preamble section 6.
Section 2 includes a subsection for each
toxicity value. Each subsection discusses study
selection, methods of analysis, and derivation of
a toxicity value, as outlined in Preamble sections
6 and 7.
Front matter. The Executive Summary
provides information historically included in
IRIS summaries on the IRIS program website. Its
structure reflects the needs and expectations of
EPA's program and regional offices.
A section on systematic review methods
summarizes key elements of the protocols,
including methods to identify and evaluate
pertinent studies. The final protocols appear as
an appendix.
The Preface specifies the scope of an
assessment and its relation to prior assessments.
It discusses issues that arose during assessment
development and emerging areas of concern.
This Preamble summarizes general
procedures for assessments begun after the date
below. The Preface identifies assessment-
specific approaches that differ from these
general procedures.
August 2016
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References
U.S. EPA. (1991). Guidelines for developmental toxicity risk assessment (pp. 1-83). (EPA/600/FR-
91/001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=23162
U.S. EPA. (1994). Methods for derivation of inhalation reference concentrations and application of
inhalation dosimetry [EPA Report] (pp. 1-409). (EPA/600/8-90/066F). Research Triangle
Park, NC: U.S. Environmental Protection Agency, Office of Research and Development, Office
of Health and Environmental Assessment, Environmental Criteria and Assessment Office.
https://cfpub.epa. gov/ncea/risk/recordisplay.cfm?deid=71993&.CFID=51174829&.CFTOKE
N=25006317
U.S. EPA. (1996). Guidelines for reproductive toxicity risk assessment (pp. 1-143). (EPA/630/R-
96/009). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
U.S. EPA. (1998). Guidelines for neurotoxicity risk assessment Fed Reg 63: 26926-26954.
U.S. EPA. (2002). A review of the reference dose and reference concentration processes (pp. 1-192).
(EPA/630/P-02/002F). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, http: //www.epa.gov/osa/review-reference-dose-and-reference-
concentration-processes
U.S. EPA. (2005a). Guidelines for carcinogen risk assessment [EPA Report] (pp. 1-166).
(EPA/630/P-03/001F). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, http: //www2.epa.gov/osa/guidelines-carcinogen-risk-assessment
U.S. EPA. (2005b). Supplemental guidance for assessing susceptibility from early-life exposure to
carcinogens (pp. 1-125). (EPA/630/R-03/003F). Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum.
U.S. EPA. (2006). Approaches for the application of physiologically based pharmacokinetic (PBPK)
models and supporting data in risk assessment (Final Report) [EPA Report] (pp. 1-123).
(EPA/600/R-05/043F). Washington, DC: U.S. Environmental Protection Agency, Office of
Research and Development, National Center for Environmental Assessment.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=l 57668
U.S. EPA. (2011). Recommended use of body weight 3/4 as the default method in derivation of the
oral reference dose (pp. 1-50). (EPA/100/R11/0001). Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum, Office of the Science Advisor.
https://www.epa.gov/risk/recommended-use-body-weight-34-default-method-derivation-
oral-reference-dose
U.S. EPA. (2012). Benchmark dose technical guidance (pp. 1-99). (EPA/100/R-12/001).
Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
U.S. EPA. (2014). Guidance for applying quantitative data to develop data-derived extrapolation
factors for interspecies and intraspecies extrapolation. (EPA/100/R-14/002F). Washington,
DC: Risk Assessment Forum, Office of the Science Advisor.
http://www.epa.gov/raf/DDEF/pdf/ddef-final.pdf
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1 EXECUTIVE SUMMARY
2
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5
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16
17 ES.l EVIDENCE FOR HAZARDS OTHER THAN CANCER: ORAL EXPOSURE
18 Nervous system effects are a human hazard of RDX exposure. Several human case reports
19 and animal studies provide consistent evidence of an association between RDX exposure and effects
20 on the nervous system, including findings related to increased seizure induction, including
21 abnormal electrical activity, convulsions, tremors, and reducing the threshold for seizure induction
22 by other stimuli; behavioral effects that may be related to seizures such as hyperirritability, hyper-
23 reactivity, and other behavioral changes. Mechanistic data support the hypothesis that RDX-
24 induced seizures and related behavioral effects likely result from inhibition of GABAergic signaling
25 in the limbic system. Some investigators reported that unscheduled deaths in experimental animals
26 exposed to RDX were frequently preceded by convulsions or seizures.
27 Urinary system effects are a potential human hazard of RDX exposure based largely on
28 observations of histopathological changes in the kidney and urinary bladder of male rats exposed to
29 RDX at doses higher than those associated with nervous system effects. The available evidence
30 indicates that male rats are more sensitive than females, and rats are more sensitive than mice to
31 RDX-related urinary system toxicity. There is suggestive evidence of male prostate effects
32 associated with RDX exposure based on an increased incidence of suppurative prostatitis in male
33 rats exposed to RDX in the diet for 2 years, in one of the few studies that evaluated the prostate.
34 There is no known mode of action (MOA) for effects of RDX exposure on the urinary system or
35 prostate, although there are studies indicating GABA helps regulate urinary system and prostate
Summary of 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 ingesting crops irrigated with contaminated water.
Inhalation or dermal exposures are more likely in occupational settings.
Epidemiological studies provide only limited information on worker populations
exposed to RDX; several case reports describe effects primarily in the nervous system
following acute exposure to RDX. Animal studies of ingested RDX demonstrate
toxicity, including effects on the nervous system, urinary system (kidney and
bladder), and prostate.
Results from animal studies provide suggestive evidence of carcinogenic potential for
RDX based on evidence of positive trends in liver and lung tumor incidence in
experimental animals. There are no data on the carcinogenicity of RDX in humans.
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1 function. Evidence for effects on other organs/systems, or developmental effects, was more limited
2 than for the endpoints summarized above.
3 E.S.1.1 Oral Reference Dose (RfD) for Effects Other Than Cancer
4 Organ-specific RfDs were derived for hazards associated with RDX exposure (see
5 Table ES-1). These organ- or system-specific reference values may be useful for subsequent
6 cumulative risk assessments that consider the combined effect of multiple agents acting at a
7 common site.
Table ES-1. Organ/system-specific RfDs and overall RfD for RDX
Effect
Basis
RfD (mg/kg-d)
Study exposure
description
Confidence
Nervous system
Convulsions
4 x 10"3
Subchronic
Medium
Urinary system
Kidney medullary
papillary necrosis
1 x 10"2
Chronic
Medium
Prostate
Suppurative
prostatitis
8 x 10"4
Chronic
Low
Overall RfD
Nervous system
effects
4 x 10"3
Subchronic
Medium
8
9 The overall RfD (see Table ES-2) is derived to be protective of all types of hazards
10 associated with RDX exposure. Although the RfD for prostate effects results in a smaller value, it
11 was not selected as the overall RfD due to uncertainties in the evaluation of this endpoint ("low
12 confidence"). The effect of RDX on the nervous system was chosen as the basis for the overall RfD
13 because nervous system effects were observed most consistently across studies, species, and
14 exposure durations, and because they represent a sensitive human hazard of RDX exposure.
15 Evidence for effects of RDX on the urinary system and prostate is more limited relative to the
16 effects of RDX on the nervous system. Incidence of seizures or convulsions as reported in a
17 subchronic gavage study (Crouse etal.. 20061 was selected for derivation of the overall RfD as this
18 endpoint was measured in a study that was well-conducted, utilized a test material of high purity
19 (99.99%), and had five closely-spaced dose groups that supported characterization of the dose-
20 response curve. In contrast, most other studies used a technical grade with ~10% or more
21 impurities. Benchmark dose (BMD) modeling was utilized to derive the point of departure (POD)
22 for RfD derivation (expressed as the BMDLos). A 5% response level was chosen because of the
23 severity of the endpoint
24 A physiologically-based pharmacokinetic (PBPK) model was used to extrapolate the BMDLos
25 derived from a rat study to a human equivalent dose (HED) based on RDX arterial blood
26 concentration, which was then used for RfD derivation.
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The overall RfD was calculated by dividing the BMDLos-hed for nervous system effects by a
composite uncertainty factor (UF) of 300 to account for extrapolation from animals to humans (3),
interindividual differences in human susceptibility (10), and uncertainty in the database (10).
The overall confidence in the RfD is medium based on high confidence in the principal study
fCrouse etal.. 20061 and medium to low confidence in the database. Confidence in the database is
reduced largely because of (1) differences in test material used across studies (i.e., differences in
formulation and particle size that may have affected RDX absorption and subsequent toxicity),
(2) uncertainties in the influence of oral dosing methods (in particular, based on evidence that
bolus dosing of RDX resulting from gavage administration induces neurotoxicity at doses lower
than administration in the diet), and (3) significant limitations in the available studies to fully
characterize subconvulsive neurological effects as well as developmental neurotoxicity.
Table ES-2. Summary of reference dose (RfD) derivation
Critical effect
Point of departure3
UF
Chronic RfD
Confidence
Nervous system effects (convulsions)
90-d F344 rat study
Crouse et al. (2006)
BMDLos-hed: 1.3 mg/kg-d
300
4 x 10"3 mg/kg-d
Medium
aA benchmark response (BMR) of 5% was used to derive the BMD and BMDL The resulting POD was converted to
a BMDLos-hed using a PBPK model based on modeled arterial blood concentration. The concentration was derived
from the area under the curve (AUC) of modeled RDX concentration in arterial blood, which reflects the average
blood RDX concentration for the exposure duration normalized to 24 hours.
ES.2 EVIDENCE FOR HAZARDS OTHER THAN CANCER: INHALATION EXPOSURE
No studies were identified that provided useful information on the effects observed
following inhalation exposure to RDX. Of the available human epidemiological studies of RDX, none
provided data that could be used for dose-response analysis of inhalation exposures. The single
experimental animal study involving inhalation exposure is not publicly available, and was
excluded from consideration due to significant study limitations, including small numbers of
animals tested, lack of controls, and incomplete reporting of exposure levels. Therefore, the
available health effects literature does not support the identification of hazards following inhalation
exposure to RDX nor the derivation of an RfC.
While inhalation absorption of RDX particulates is a plausible route of exposure, there are
no toxicokinetic studies of RDX inhalation absorption to support development of an inhalation
model. Therefore, a PBPK model for inhaled RDX was not developed to support route-to-route
extrapolation of an RfC from the RfD.
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ES.3 EVIDENCE FOR HUMAN CARCINOGENICITY
Under EPA's cancer guidelines fU.S. EPA. 2005al. there is suggestive evidence of carcinogenic
potential for RDX. RDX induced benign and malignant tumors in the liver and lungs of mice (Parker
etal.. 2006: Lish etal.. 19841 or rats fLevine etal.. 19831 following long-term administration in the
diet The potential for carcinogenicity applies to all routes of human exposure.
ES.4 QUANTITATIVE ESTIMATE OF CARCINOGENIC RISK FROM ORAL EXPOSURE
A quantitative estimate of carcinogenic risk from oral exposure to RDX was based on the
increased incidence of hepatocellular adenomas or carcinomas and alveolar/bronchiolar adenomas
or carcinomas in female B6C3Fi mice observed in the carcinogenicity bioassay in mice fLish etal..
19841. This 2-year dietary study included four dose groups and a control group, adequate numbers
of animals per dose group (85/sex/group, with interim sacrifices of 10/sex/group at 6 and
12 months), and detailed reporting of methods and results (including individual animal data). The
initial high dose (175 mg/kg-day) was reduced to 100 mg/kg-day at week 11 due to high mortality.
When there is suggestive evidence of carcinogenicity to humans, EPA generally would not
conduct a dose-response assessment and derive a cancer value. However, when the evidence
includes a well-conducted study (as is the case with RDX), quantitative analyses may be useful for
some purposes, for example, providing a sense of the magnitude and uncertainty of potential risks,
ranking potential hazards, or setting research priorities (U.S. EPA. 2005a).
An oral slope factor (OSF) was derived that considered the combination of female mouse
liver and lung tumors. In modeling these data sets, the highest dose group was excluded because of
the initial high mortality (loss of almost half the mice in that dose group). BMD and BMDL
estimates were calculated that correspond to a 10% extra risk (ER) of either tumor. The BMDLio so
derived was extrapolated to the HED using BW3/4 scaling, and an OSF was derived by linear
extrapolation from the BMDLio-hed. The OSF is 0.08 per mg/kg-day, based on the liver and lung
tumor response in female mice (Lish etal.. 1984).
ES.5 QUANTITATIVE ESTIMATE OF CARCINOGENIC RISK FROM INHALATION EXPOSURE
An inhalation unit risk (IUR) value was not calculated because inhalation carcinogenicity
data for RDX are not available. While inhalation absorption of RDX particulates is a plausible route
of exposure, there are no toxicokinetic studies of RDX inhalation absorption to support an
inhalation model. Therefore, a PBPK model for inhaled RDX was not developed to support route-to-
route extrapolation of an IUR from the OSF. Thus, a quantitative cancer assessment was not
conducted.
ES.6 SUSCEPTIBLE POPULATIONS AND LIFESTAGES
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 well-studied in
human or animal studies of RDX toxicity. In rats, transfer of RDX from the dam to the fetus during
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gestation and to pups via maternal milk has been reported; however, reproductive and
developmental toxicity studies did not identify effects in offspring at doses below those that also
caused maternal toxicity. Yet, based on the primary mode-of-action for RDX exposure-induced
nervous system effects (GABA receptor antagonism), and the fact that GABAergic signaling plays a
prominent role in nervous system development, a significant concern is raised regarding the
potential for developmental neurotoxicity. In addition, data on the incidence of convulsions and
mortality provide some indication that pregnant animals may be a susceptible population, although
the evidence is inconclusive. Data to suggest that males may be more susceptible than females to
noncancer toxicity associated with RDX are limited. Some evidence suggests that cytochrome P450
(CYP450) enzymes may be involved in the metabolism of RDX, indicating a potential for genetic
polymorphisms in these metabolic enzymes to affect susceptibility to RDX. Similarly, individuals
with epilepsy or other seizure syndromes that have their basis in genetic mutation to GABAa
receptors may represent another group that may be susceptible to RDX exposure; however, there is
no information to indicate how genetic polymorphisms may affect susceptibility to RDX.
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LITERATURE SEARCH STRATEGY |
STUDY SELECTION AND EVALUATION
SR.1 LITERATURE SEARCH AND SCREENING STRATEGY
A literature search and screening strategy was applied to identify literature related to
characterizing the health effects of hexadydro-l,3,5-trinitro-l,3,5-triazine (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 chronic health effects of RDX, and the remaining
references were sorted into categories for further evaluation.
The literature search for RDX was conducted in four online scientific databases—PubMed,
Toxline, Toxcenter, and Toxic Substances Control Act Test Submissions (TSCATS). The initial
search was performed in April 2012, and literature search updates were conducted in February
2013, January 2014, January 2015, and May 2016. Searches of TSCATS were performed in February
2013, January 2015, and May 2016 only. In addition, a post-peer review literature search was
conducted in November 2017 (described below). The detailed pre-peer review search approach for
these databases, including the query strings, and the numbers of citations identified per database
are provided in Appendix B, Table B-l. The Department of Defense has conducted several
unpublished toxicological studies on RDX; to ensure that all such studies were located, 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.
Searches of the five online databases identified 1,247 citations (after electronically eliminating
duplicates). The computerized database searches were supplemented by reviewing online
regulatory sources, performing "forward" and "backward" searches of Web of Science (see
Appendix B, Table B-3), and adding additional references that were identified during the
development of the Toxicological Review (including submissions from the Department of Defense);
34 citations were obtained using these additional search strategies. In total, 1,281 citations were
identified using online scientific databases and additional search strategies.
The U.S. Environmental Protection Agency (EPA) requested public submissions of
additional information in 2010 (75 FR 76982; December 10, 2010). No submissions were received
in response to these calls for data. EPA also 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
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docket for public comment (EPA-HQ-ORD-2013-0430; available at www.regulations.govl
maintained through the development of the assessment
The citations identified using the search strategy described above were screened based on
title and abstract, and when needed, full text for pertinence to examining the health effects of
chronic RDX exposure. The process for screening the literature is described below and is shown
graphically in Figure LS-1 and on the RDX project page on EPA's Health and Environmental
Research Online (HERO) website at:
https://hero.epa.gov/index.cfm/project/page/project id/2216.7 The objective of this manual
screen was to identify sources of primary human health effects data (i.e., human data and pertinent
data from in vivo animal models) and other sources of primary data that inform the assessment of
RDX health effects (i.e., genotoxicity and other mechanistic studies and toxicokinetic studies).
These data sources are represented by the bottom three boxes in Figure LS-1. Inclusion and
exclusion criteria used to manually screen the references in order to identify health effect studies
(i.e., the green boxes with dashed boarders in Figure LS-1) are provided in Table LS-1.
All studies that provided data on adsorption, distribution, metabolism, or elimination,
physiologically-based pharmacokinetic (PBPK) models, or relevant RDX mode of action (MOA)
were tracked and considered in the assessment.
Reviews and other sources of RDX information (e.g., studies with exposure level
information) that did not meet the inclusion criteria for primary health effect studies in Table LS-1
were tracked as "Secondary Literature and Sources of Other RDX Information," and were
considered as appropriate during development of this assessment. Studies identified as
"Excluded/Not on Topic" (see exclusion criteria in Table LS-1) were not further considered in this
assessment.
The results of this literature screening are described below and graphically in Figure LS-1:
• 21 references (including both human and animal studies) were identified as sources of
health effects data and were considered for data extraction to evidence tables and
exposure-response arrays.
7HERO is a database of scientific studies and other references used to develop EPA's assessments aimed at
understanding the health and environmental effects of pollutants and chemicals. It is developed and
managed in EPA's Office of Research and Development (ORD) by the National Center for Environmental
Assessment (NCEA). The database includes more than two million scientific references, including articles
from the peer-reviewed literature. New studies are added continuously to HERO.
Studies were assigned (or "tagged") to a given category in HERO that best reflected the primary content of the
study. In general, 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 et al. (1986). a case report of accidental ingestion of RDX by a child,
was tagged to the human case reports under Supplementary 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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• 25 references were identified as sources of supplementary health effects data, including
experimental animal studies involving acute or short-term exposures or dermal
exposure, and human case reports. Studies investigating the effects of acute/short-term
and dermal exposures and case reports are generally less pertinent for characterizing
health hazards associated with chronic oral and inhalation exposure. Therefore,
information from these studies was not extracted into evidence tables. Nevertheless,
these studies were still considered as possible sources of supplementary health effects
information.
• 91 references provided information on nonmammalian species (tagged as ecosystem
studies) that can inform the hazard evaluation or potential MOA, and specifically in the
case of RDX, the conservation of neurotoxic response across phylogenetically diverse
organisms. Information from these studies was not extracted into evidence tables;
however, these studies were tracked as supplementary health effects information.
• 47 references were identified as sources of mechanistic and toxicokinetic data; these
included 19 studies describing PBPK models and other toxicokinetic information,
11 studies providing genotoxicity information, and 17 studies pertaining to other
mechanistic information. Information from these studies was not extracted into
evidence tables; however, these studies supplemented the assessment of RDX health
effects. Specifically, mechanistic studies were used in the evaluation of potential MOAs
and to develop the mechanistic evidence stream that was considered in the overall
integration of evidence for assessing hazard. Toxicokinetic data were used to inform
extrapolation of experimental animal findings to humans.
• 190 references were identified as secondary literature (e.g., reviews and other agency
assessments), peer review reports of primary (unpublished) health effect studies, or
contextual information (e.g., studies with RDX exposure information). These references
were kept as additional resources for development of the Toxicological Review.
• 907 references were identified as not being pertinent (or not on topic) to an evaluation
of the chronic health effects of RDX and were excluded from further consideration (see
Figure LS-1 and Table LS-1 for exclusion criteria).
SR.1.1. Post-peer Review Literature Search Update
A post-peer review literature search update was conducted in PubMed, Toxline, TSCATS,
and DTIC for the period May 2016 to November 2017 using a search strategy consistent with
previous literature searches (see Appendix B, Tables B-l and B-2). Toxcenter, used in previous
searches, was not searched in the November update. Toxcenter is a proprietary, fee-based database
produced by Chemical Abstract Service. Evaluation of the references retrieved using Toxcenter in
searches conducted through May 2016 revealed that this database did not locate pertinent
references not already identified by other online databases. Results of the November 2017
literature search update are summarized in Appendix B, Tables B-l and B-2.
Consistent with the IRIS Stopping Rules
(https://www.epa.gov/sites/production/files/2014-06/documents/iris stoppingrules.pdf).
manual screening of the literature search update focused on identifying new studies that might
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1 change a major conclusion of the assessment No potentially pertinent references were identified in
2 the post-peer review literature search.
3
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Supplementary Studies
Combined Dataset
n=l,281
Sources of Health Effects Data
(n=21)
4 Human health effects studies
17 Animal toxicology studies
Additional Search Strategies
(see Table B-3 for methods and results)
n=34
Manual Screening For Pertinence
(Title/Abstract/Full Text)
Secondary Literature and Sources of
Other RDX Information (n=190)
113 Exposure levels
7 Mixtures only studies (applies to animal
studies only)
16 Regulatory documents
51 Reviews; risk assessments; editorials
3 Other
Excluded/Not on Topic (n=907)
32 Abstract only; inadequate reporting in
abstract; no abstract
244 Treatment/remediation
154 Chemical, physical or explosive
properties
258 Laboratory methods
182 Not chemical specific
37 Other
Other Sources of Supplementary
Health Effects Data (n=116)
9 Acute/short-term animal studies
16 Human case reports
91 Studies in ecological and other
nonmammalian species
Sources of Mechanistic and
Toxicokinetic Data (n=47)
11 Genotoxicity studies
17 Other mechanistic studies
19 Toxicokinetic studies
Pubmed
n=698
n=l,247 (After duplicates removed electronically)
Toxline
n=433
Database Searches (through May 2016)
(see Tables B-l and B-2 for keywords and limits)
Toxcenter
n=32
DTIC
n=85
TSCATS l/2/8e
n=2
Figure LS-1. Summary of literature search and screening process for RDX.
The numbers on this figure match the HERO project page as of 5/1/2018. See text for search strategy and results
of an updated literature search conducted in November 2017 (post peer review).
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Table LS-1. Inclusion-exclusion criteria for health effect studies3
Inclusion criteria
Exclusion criteria
Population
• Humans
• Standard mammalian animal models,
including rat, mouse, rabbit, guinea pig,
monkey, dog
• In vitro studies -- tracked as
supplementary information
• Ecological and nonmammalian species-
tracked as supplementary information
Exposure
• Exposure is to RDX
• Exposure is measured in an
environmental medium (e.g., air, water,
diet)
• Exposure via oral or inhalation routes
• Study population is not exposed to RDX
• Exposure to a mixture only (applied to animal studies
only)
• Exposure via injection (e.g., intravenous [i.v.])b
Outcome
• Study includes a measure of one or
more health effect endpoints, including
effects on the nervous, urinary,
musculoskeletal, cardiovascular,
immune, and gastrointestinal systems,
reproduction, development, liver, eyes,
and cancer
• Mechanistic and toxicokinetic studies -
tracked as supplementary information
Other
• Reviews, regulatory documents (i.e., not primary
sources of health effect data)b
• Exposure levels'5
• Not on topic, including:
o Abstract only, inadequately reported abstract, or
no abstract, and not considered further because
study was not potentially relevant
o Bioremediation, biodegradation, or chemical or
physical treatment of RDX and other munitions,
including evaluation of wastewater treatment
technologies and methods for remediation of
contaminated water and soil
o Chemical, physical, or explosive properties,
including studies of RDX crystal quality, energetics
characteristics, sublimation kinetics, isotope ratios,
and thermal decomposition and other explosive
properties
o Analytical methods for measuring/detecting/
remotely sensing RDX in environmental media, and
use in sample preparations and assays
o Not chemical specific (studies that do not involve
testing of RDX)
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Inclusion criteria
Exclusion criteria
o Other studies not informative for evaluating RDX
health effects and not captured by other exclusion
criteria, including:
-- Superfund site records of decision that describe
remedial action plans for waste sites
-- characterization of waste sites contaminated by
explosives
-- foreign language studies where translation was
not warranted because, based on title or abstract,
the added value to the evaluation of RDX health
effects was considered small (e.g., Chinese paper
of case reports of RDX poisonings)
-- duplicate studies not previously identified during
electronic screening
alnclusion/exclusion criteria were designed to identify sources of primary human health effects data (i.e., human
data and pertinent data from in vivo animal models).
bStudies that met this exclusion criterion were not considered a primary source of health effects or supplementary
health effects data; however, these studies were tracked and considered as other sources of information
potentially useful in assessing the health effects of RDX, including potential MOAs.
The documentation and results for the literature search and screen, including the specific
references identified using each search strategy and tags assigned to each reference based on the
manual screen, can be found on the HERO website on the RDX project page at:
fhttps://hero.epa.gov/index.cfm/proiect/page/project id/22161.
SR.2 STUDY EVALUATION AND SELECTION OF CRITICAL STUDIES
SR.2.1. Selection of Critical Studies
In order to systematically summarize the important information from the primary health
effects studies in the RDX database, evidence tables were constructed in a standardized tabular
format as recommended by the NRC (2011). Of the studies that were retained after the literature
search and screen, 21 were categorized as "Sources of Health Effects Data" (see Figure LS-1, Table
LS-1) and were considered for extraction into evidence tables for hazard identification in Chapter 1.
A study was not subject to a more thorough review of study quality and was not presented
in evidence tables if flaws in its design, conduct, or reporting were so great that the results would
not be considered credible (e.g., studies where concurrent control information is lacking). Such
study design flaws are discussed in a number of EPA's guidelines (see
https: //www.epa.gov/iris /backgrd.html and Section 4 of the Preamble). For RDX, four studies
were considered uninformative and were removed from further consideration in the assessment
because of fundamental issues with study design, conduct, or reporting. The specific studies and
basis for considering the studies to be uninformative are summarized in Table LS-2.
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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);
repeat dose studies in dogs and rats
Inadequate reporting of study design (e.g., limited exposure
information, breed of dog was not reported) and results; sections of
document were illegible. Deficiencies in experimental design of
dog study (e.g., investigation of only blood pressure in 3 dogs
exposed for 2,14, or 16 weeks; no separate control). Rat study
included only 10 rats treated 41 times with RDX over an unspecified
exposure duration; only body weight and survival findings were
reported.
von Oettingen et al. (1949);
10-wk oral study in rats
No control group; strain of rat was not reported.
Note: other studies included in the paper bv von Oettingen et al.
(1949) were retained; results of these studies are included in
evidence tables.
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.
Unpublished report (dated 1944) 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, incomplete information on
exposure levels, and inadequate reporting of results. [Because this
report is classified as a limited distribution document in the DTIC
database, it was not added to the HERO project page for RDX.]
1 The health effects literature for RDX is not extensive. With the exception of the studies
2 listed in Table LS-2 (i.e., those determined to be uninformative), all human and experimental animal
3 studies of RDX involving repeated exposure were considered in assessing the evidence for health
4 effects associated with chronic exposure to RDX.
5 Studies that contain pertinent information for the toxicological review and augment hazard
6 identification conclusions, such as genotoxicity and other mechanistic studies, studies describing
7 the toxicokinetics of RDX, human case reports, and experimental animal studies involving
8 exposures of acute/short-term duration or routes of exposure other than oral and inhalation, were
9 not included in evidence tables. Nevertheless, these studies were considered, where relevant, in the
10 evaluation of RDX health hazards.
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SR.2.2. Study Evaluation
For this assessment, primary sources of health effects data consisted of three human
studies8 and 16 reports9 presenting results of experimental animal studies. These studies were
evaluated using the study quality considerations described below that addressed aspects of design,
conduct, or reporting that could affect the interpretation of results, overall contribution to the
synthesis of evidence, and determination of hazard potential as noted in various EPA guidance
documents (U.S. EPA. 2005a. 2002.19941. The objective was to identify the stronger, more
informative studies based on a uniform evaluation of quality characteristics across studies of
similar design.
Additionally, a number of general questions, presented in Table LS-3, were considered in
evaluating the animal studies. Much of the key information for conducting this evaluation can be
determined based on study methods and how the study results were reported. Importantly, the
evaluation at this stage does not consider the direction or magnitude of any reported effects.
8Two reports with human data were determined not to be informative; see Table LS-2. The study by ATS PR
(19961 was included in HERO and in Figure LS-1. The unpublished report from the DTIC database was not
included in either HERO or Figure -1 because this report is classified as a limited distribution document in
DTIC. This accounts for the three human studies being reviewed for study evaluation rather than the four
identified in the literature search (see Figure LS-1],
90ne of 17 animal toxicity studies identified in Figure 1 fHaskell Laboratories f~19421 was determined to be
uninformative; see Table LS-2. This study was included in HERO and Figure LS-1, but was not considered a
primary source of health effects data and was not carried forward for further review.
Also, it should be noted that the number of reports of experimental animal studies does not equal the number
of studies for several reasons. The results of some studies were documented in multiple reports (e.g., a 2-
year study in F344 rats by Levine etal. (19831 was published in three volumes]. The Cholakis et al. (1980]
study included, in a single report, subchronic studies in rats and mice, a 2-generation reproductive toxicity
study in rats, and developmental toxicity studies in rats and rabbits. A 13-week toxicity study of RDX in rats
was reported initially as a laboratory report study (Levine et al.. 1981a], and results were subsequently
included in two published papers. A Pathology Working Group review of the female mouse liver tumor data
in the Lish et al. (1984] 2-year bioassay was provided as a study report and subsequently as a published
paper.
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Table LS-3. Considerations and relevant experimental information for
evaluation of experimental animal studies
Methodological
feature
Considerations
(relevant information extracted into evidence tables)
Test animal
Suitability of the species, strain, sex, and source of the test animals
Experimental design
Suitability of animal age/lifestage at exposure and endpoint testing; periodicity and
duration of exposure (e.g., hrs/d, d/wk); timing of endpoint evaluations; and sample size
and experimental unit (e.g., animals, dams, litters)
Exposure
Characterization of test article source, composition, purity, and stability; suitability of the
control (e.g., vehicle control); documentation of exposure techniques (e.g., route,
chamber type, gavage volume); verification of exposure levels (e.g., consideration of
homogeneity, stability, analytical methods)
Endpoint evaluation
Suitability of specific methods for assessing the endpoint(s) of interest
Results presentation
Data presentation for endpoint(s) of interest (including measures of variability) and for
other relevant endpoints needed for results interpretation (e.g., maternal toxicity,
decrements in body weight in relation to organ weight)
Information on study features related to this evaluation is reported in evidence tables and
was considered 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 If EPA's interpretation of a study differed from that of the study authors, the
assessment discusses the basis for the difference.
The general findings of this evaluation are presented in the remainder of this section and
discussed in the relevant health effect sections in Section 1.2.
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) fWest and Stafford. 1997: Ma and Li.
1993: Hathaway and Buck. 1977). To varying degrees, these epidemiology studies are limited in
their ability to assess the relationship between RDX exposure and the incidence of human health
effects. Some studies lacked information related to study design, such as a clear definition of the
study population, while others did not include a comprehensive exposure assessment or details
regarding potential confounders. All three studies had small sample sizes (60-69 exposed workers
in the cross-sectional studies and 32 cases in the case-control study), which limits their statistical
power when comparing exposed workers or cases and unexposed or control participants.
The study by Ma and Li (1993) of Chinese industrial workers provided limited information
on participant recruitment, selection, and participation rate; the available information was not
adequate to evaluate the potential for selection bias. Also, no information on adjustment for co-
exposure to trinitrotoluene (TNT) or other neurological risk factors (e.g., alcohol consumption) was
provided. The study by Hathaway and Buck (1977) included details on exposure assessment, but
did not provide information on length of employment or other metrics that could be used to
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ascertain duration of exposure. In the case-control study by West and Stafford (1997). RDX was
identified as one of the many chemicals that workers may have been exposed to in the ordnance
factory. Thus, there is a potential for co-exposure to other chemicals that may elicit the observed
effects. 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 1.2).
In addition to the three epidemiological studies, the human health effects literature includes
16 case reports that describe effects following acute exposure to RDX. Case reports can suggest
organ systems and health outcomes that might be related to RDX exposure but are often anecdotal,
and typically describe unusual or extreme exposure situations; thus, they provide little information
that would be useful for characterizing chronic health effects or deriving toxicity values. Therefore,
RDX case reports were only briefly reviewed; a critical evaluation was not undertaken. A summary
of these case reports is provided in Appendix C, Section C.2.
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 shorter-term studies in dogs and rats, one
two-generation reproductive toxicity study in the rat, four developmental toxicity studies in rats
and rabbits, and a single-exposure study of audiogenic seizures in rats (Table LS-4).
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Table LR-4. Summary of experimental animal database
Study category
Study duration, species/strain, and oral administration method
Chronic
2-Yr study in B6C3Fi mice (diet) (Lish et al., 1984)
2-Yr studv in Sprague-Dawlev rats (diet) (Hart, 1976)
2-Yr studv in F344 rats (diet) (Levine et al., 1983)
Subchronic
13-Wk studv in B6C3Fi mice, experiment 1 (diet) (Cholakis et al., 1980)
13-Wk studv in B6C3Fi mice, experiment 2 (diet) (Cholakis et al., 1980)
13-Wk studv in F344 rats (diet) (Cholakis et al., 1980)
13-Wk studv in F344 rats (diet) (Levine et al., 1990; Levine et al., 1981a, b)
13-Wk studv in F344 rats (gavage) (Crouse et al., 2006)
13-Wk studv in rats, strain not specified (diet) (von Oettingen et al., 1949)
13-Wk studv in beagle dogs (diet) (Hart, 1974)
13-Wk studv in monkevs (gavage) (Martin and Hart, 1974)
6-Wk studv in dogs, breed not specified (diet) (von Oettingen et al., 1949)
30-D studv in Sprague-Dawlev rats (gavage) (MacPhail et al., 1985)
Reproductive
2-Generation reproductive toxicity studv in CD rats (diet) (Cholakis et al., 1980)
Developmental
Developmental studv (gestational davs fGDsl 6-19) in F344 rats (gavage) (Cholakis et al., 1980)
Developmental studv (GDs 6-15) in Sprague-Dawlev rats, range-finding (gavage) (Angerhofer et
al., 1986)
Developmental studv (GDs 6-15) in Sprague-Dawlev rats (gavage) (Angerhofer et al., 1986)
Developmental studv (GDs 7-29) in New Zealand White (NZW) rabbits (gavage) (Cholakis et al.,
1980)
Nervous system
8-Hr studv of audiogenic seizures in Long Evans rats (gavage) (Burdette et al., 1988)a
Acute EEG and in vitro studies of RDX evoked seizure activity in Sprague-Dawley male rats
(Williams et al., 2011)
aAs an 8-hour study, Burdette et al. (1988) was tagged in Figure LS-1 and the HERO database as "Other Sources of
Supplementary Health Effects Data," but was nevertheless included in the evidence table for nervous system
effects of RDX as the only study to examine potential effects of RDX on seizure threshold.
With the exception of two studies (Levine etal.. 1990: von Oettingen et al.. 1949). these
toxicity studies are available only as unpublished contract laboratory reports. Peer reviews of four
unpublished studies identified as most informative to the assessment of the health effects of RDX—
the 2-year bioassays by Levine etal. T19831 and Lish etal. fl9841. the subchronic toxicity study by
Crouse etal. f2006I and the collection of repeat-dose studies reported in fCholakis etal.. 19801—
were conducted by Versar, Inc. or Eastern Research Group, Inc. for EPA. The reports of the peer
reviews (U.S. EPA. 2017. 2012c) are available at https://epa.gov/hero. The peer reviewers
generally concluded that the 2-year bioassay reports provided useful information on the toxicity of
RDX, noting that there were limitations in interpretation due to aspects of the histopathological
analysis and the statistical approaches employed. The peer reviewers similarly determined that the
report by Crouse etal. f20061 provided useful information on RDX toxicity, including an array of
endpoints for neurotoxicity and immunotoxicity, although the assessment of neurotoxicity in the
study could have been improved with more histological evaluation as well as additional behavioral
assessment. (U.S. EPA. 2012c). The peer review report of the repeat-dose studies in Cholakis et al.
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(1980) found that the studies were generally appropriate and adequate for evaluating the toxicity
of RDX, and conducted consistent with standards in place at the time the experiments were
conducted fU.S. EPA. 20171.
Only one unpublished inhalation study of RDX (dated 1944) was identified. This inhalation
study was considered uninformative and was 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, see Appendix B and Table
LS-2). 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.
Test animal
The RDX database consists of health effect studies conducted in multiple strains of rats
(F344, Sprague-Dawley, CD), mice (B6C3Fi), dogs (beagle), and monkeys. 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. The
species, strain, and sex of the animals used are 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 (tagged under Supplementary Studies; see Figure LS-1) are
not included in evidence tables, but are discussed where relevant in the assessment.
Experimental design
General aspects of experimental design were evaluated for all studies that included health
effects data to determine if they were appropriate for evaluation of specific endpoints. Key features
of the experimental design, 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, as studies with a small number of animals can still inform the
consistency of effects observed for a specific endpoint. Nevertheless, the informativeness of studies
with small sample sizes, e.g., three animals/sex/group in the case of Hart (1974) and Martin and
Hart (1974). was reduced. Elements of the experimental setup that could influence interpretation
of study findings are discussed in the relevant hazard identification sections of the assessment
Exposure
Studies were evaluated with respect to the reliability of the reported exposure to RDX,
focusing on considerations related to properties of the test material and confirmation of the
administered dose.
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Two properties of the RDX test materials that varied across experimental animal studies
and that were taken into consideration in evaluating the evidence for RDX hazards are the particle
size and purity of the test material. The purity of RDX used in health effects studies varied from 84
to 99.99%. The major contaminant was octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine (HMX),
which is produced during manufacturing. The majority of studies used RDX with ~10% impurities;
only Crouse etal. (2006) used 99.99% pure RDX as a test material in their study. The toxicity of
HMX was assessed by the Integrated Risk Information System (IRIS) Program in 1988
(https://cfpub.epa.gov/ncea/iris2 /chemicalLanding.cfm7substance nmbr=311): histopathological
changes in the 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 >20 mg/kg-day. It should be noted
that the test materials employed in these studies (i.e., with ~10% impurities) are consistent with
the purity 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 test material with a relatively
fine particle size (majority of particles <66 [im in size), while others used a 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 response 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. The lack of
characterization of the test material in the studies by Hart (1974). Hart (1976). and Martin and Hart
(1974) was considered a deficiency.
Only four studies assayed dose preparations to determine how close the actual RDX
concentrations were to target (nominal) concentrations (Crouse etal.. 2006: Lish etal.. 1984:
Levine etal.. 1983: Cholakis etal.. 19801. Cholakis etal. f!9801 described the largest difference
between target and actual dose concentrations; assays of the suspensions prepared for the oral
(gavage) developmental toxicity study showed RDX dosing suspensions ranging from 36 to 501% of
the target concentrations (Appendix I of Cholakis etal. (1980)). Assays of RDX-treated feed used in
the 90-day studies in rats and mice and the two-generation reproductive toxicity study in rats
showed RDX concentrations that were 78 to 209% of target concentrations (Appendix I of Cholakis
etal. (1980)). The authors stated, "maintaining uniform suspensions was not always easy." In the
90-day oral (gavage) toxicity study in rats fCrouse etal.. 20061. fresh dose suspensions were
prepared monthly, mixed with a magnetic stir bar until a uniform suspension was obtained, and
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remixed each day during the dosing procedure; each dose suspension was analyzed prior to use.
RDX concentrations varied from 83 to 114%; the 114% suspension was adjusted to 100% before
administration fCrouse etal.. 20061. In 30 assays performed over the course of a 24-month
bioassay in mice, Lish etal. f19 841 determined dietary concentrations of RDX to be 73 to 103% of
target concentrations. In 32 assays performed over a 24-month bioassay in rats, Levine et al.
(1983) reported that dietary concentrations ofRDXwere 67% to 122% of target concentrations. In
the remaining studies, failure to analyze or report actual concentrations of RDX in the dosing
suspension or test diet is 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 1.2.
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 the number of animals with
seizures as part of clinical observations that, in general, were recorded only once daily. This
frequency of observations could have missed neurobehavioral events or identify subtler
subconvulsive behaviors. While these studies can provide qualitative evidence of neurotoxicity,
they may have underestimated the true incidence of seizures or convulsive behaviors because they
were not designed to systematically evaluate neurotoxic outcomes.
Results presentation
In evaluating studies, consideration was given to whether data were reported for all
endpoints specified in the methods section and for all 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 by individual fetus). Study results have been extracted and
presented in evidence tables.
Notable features of the RDX database
Three 2-year toxicity bioassays of RDX are available as unpublished laboratory studies (Lish
etal.. 1984: Levine etal.. 1983: Hart. 1976). The bioassays by Levine etal. (1983) in the rat and by
Lish etal. (1984) 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 (including brain, gonads, heart, liver, kidneys, spleen, and spinal cord in
both species, and lungs and tissue masses in the mouse), limiting the ability to identify dose-related
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trends for tissues with incomplete histopathology. Additionally, in the mouse bioassay by Lish et al.
fl9841. the initial high dose (175 mg/kg-day) was reduced to 100 mg/kg-day at week 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-5).
An earlier unpublished 2-year study in rats by Hart f!9761 used a dose range that was
lower than the Levine etal. (1983) and Lish etal. (1984) bioassays. Histopathology findings were
limited by the lack of pathology examinations in the mid-dose groups and 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 in the study (see Table LS-5).
Experimental animal toxicity studies of RDX involving less-than-lifetime exposure (Crouse
etal.. 2006: Angerhofer etal.. 1986: MacPhail etal.. 1985: Levine etal.. 1981a: Cholakis etal.. 1980:
Hart. 1974: Martin and Hart. 1974: von Oettingen et al.. 1949) were published or reported between
the years 1949 and 2006, and differences in robustness of study design, conduct, and reporting
reflect that time span. All but two of the eight short-term and subchronic toxicity studies of RDX
are available as unpublished laboratory studies; published studies include von Oettingen et al.
f!9491 and Levine etal. fl981al. a laboratory report of a 13-week study of RDX in F344 rats with
subsets of the data subsequently published as Levine etal. f!981bl and Levine etal. f 19901 The
majority of studies conducted histopathological examinations on only some of the experimental
groups (e.g., control and high dose).
Some of the more important limitations in study design, conduct, and reporting of
experimental animal toxicity studies of RDX are summarized in Table LS-5. Limitations of these
studies as well as the study evaluation consideration described in this section were taken into
consideration in evaluating and synthesizing the evidence for each of the health effects in
Section 1.2.
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Table LS-5. 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)
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. Interpretation of the
histopathology findings was limited by the lack of pathology examinations
in the mid-dose groups and lack of individual time of death. Test material
poorly characterized; purity was not reported.
Cholakis et al. (1980)
13-wk mouse study (Experiment 1)
The dose range was too low to produce effects in mice. Assays of RDX-
treated feed showed RDX concentrations between 123% and 209% of
target concentrations. Histopathological examinations were not
performed.
Cholakis et al. (1980)
13-wk mouse study (Experiment 2)
Nonstandard dosing regimen followed: 0,40, 60, or 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.
Cholakis et al. (1980)
Developmental study in rats
Large differences were reported between target and actual dose
concentrations in the suspensions prepared for oral (gavage)
administration; actual RDX concentrations in dosing suspensions ranged
from 36 to 501% of the target concentrations.
Levine et al. (1981a)
13-wk rat study
Analysis of one lot of rodent feed showed measurable levels of
contaminants, including chlorinated pesticides (dieldrin, heptachlor
epoxide, beta-hexachlorocyclohexane [BHC], and
dichlorodiphenyltrichloroethane [DDT]), polychlorinated biphenyls (PCBs),
and organophosphates (methyl parathion, carbophenothion, and
disulfeton).
Martin and Hart (1974)
13-wk monkey study
The 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). Test material poorly characterized; purity was
not reported.
von Oettingen et al. (1949)
12-wk rat study
The strain of rat was not reported. Only gross observations were made at
autopsy.
von Oettingen et al. (1949)
6-wk dog study
The breed of dog was not reported. Only gross observations were made
at autopsy.
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1.HAZARD IDENTIFICATION
1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS
1.1.1. Chemical Properties
Hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) is a member of the nitramine class of organic
nitrate explosives fBoileau et al.. 2003: Bingham etal.. 20011 and is not found naturally in the
environment. RDX exists as a white, crystalline solid Bingham etal. f20011. It has low solubility in
water (Yalkowskv and He. 2003) and slowly volatilizes from water or moist soil (ATSDR. 2012).
The normalized soil organic carbon/water partition coefficient (K0c) values for RDX indicate a
potential for RDX to be mobile in soil fSpanggord et al.. 1980). The vapor pressure suggests that
RDX will exist as particulate matter in air and be removed by both wet and dry deposition
fSpanggord etal.. 19801. Information on physiochemical properties for RDX is available at EPA's
Chemistry Dashboard (https://comptox.epa.gov/dashboard/) and is summarized in Table 1-1.
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Table 1-1. Chemical identity and physicochemical properties of RDX from
EPA's Chemistry Dashboard
Characteristic or property
Value
Chemical structure
cu -Cr
N
I
^ N -
r i
~0. , N „ „ N _ ^ G
N N
II 1
0 0-
CASRN
121-82-4
Synonyms
l,3,5-triaza-l,3,5-trinitrocyclohexane; 1,3,5-triazine, hexahydro-l,3,5-trinitro-;
l,3,5-trinitro-l,3,5-triazacyclohexane; l,3,5-trinitro-l,3,5-triazinane; 1,3,5-
trinitrohexahydro-l,3,5-triazine; 1,3,5-trinitrohexahydro-s-triazine; 1,3,5-
trinitroperhydro- 1,3,5-triazine; cyclonite; cyclotrimethylenenitramine;
cyclotrimethylenetrinitramine; hexahydro-l,3,5-trinitro-l,3,5-s-triazine;
hexahydro-1,3,5-trinitro-1,3,5-triazine; hexahydro-l,3,5-trinitro-s-triazine;
hexogen; perhydro-1,3,5-trinitro-1,3,5-triazine; RDX; Research Development
Explosive; Royal Demolition explosive; sym-trimethylene trinitramine; s-
triazine, hexahydro-1,3,5-trinitro-; trimethylenetrinitramine;
trinitrocyclotrimethylene triamine; trinitrotrimethylenetriamine (see
https://comptox.epa.gov/dashboard for additional svnonvms)
Molecular formula
c3h6n6o6
Molecular weight
222.117
Average experimental value3
Average predicated value3
Flash point (°C)
-
388
Boiling point (°C)
-
407
Melting point (°C)
205
162
Log Kow
0.87
-0.425
Water solubility (mol/L)
2.69 x 10"4
8.37 x 10"3
Density (g/cm3)
-
1.84
Henry's law constant
(atm-m3/mole)
-
2.53 x 10"6
Vapor pressure (mm Hg at 20°C)
4.10 x 10"9
3.76 x 10"9
aMedian values and ranges for physical chemical properties of RDX are also provided on the Chemistry Dashboard
at https://comptox.epa.gov/dashboard/.
1 RDX degrades in the environment and can be subject to both photolysis (Sikka etal.. 1980:
2 Spanggord etal.. 19801 and biodegradation (Funk etal.. 1993: McCormick etal.. 19811. RDX is
3 metabolized by microbial nitroreductases to form the N-nitroso derivatives hexahydro-l-nitroso-
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3,5-dinitro-l,3,5- triazine (MNX), hexahydro-l,3-dinitroso-5-nitro-l,3,5-triazine (DNX), and
hexahydro-l,3,5-trinitroso-l,3,5-triazine (TNX) flaligama etal.. 2013: Halasz etal.. 2012: Smith et
al.. 2006: Meyer etal.. 2005: Beller andTiemeier. 20021. 4-Nitro-2,4-diazabutanal (NDAB) and
methylenedinitramine (MEDINA) have also been detected as microbial metabolites of RDX (Halasz
etal.. 2012: Fuller etal.. 20101.
1.1.2. Toxicokinetics
RDX is absorbed following exposure by oral and inhalation routes (see Appendix C,
Section C.l.l). Studies in experimental animals indicate that oral absorption rates can range from
approximately 50 to 90% (Krishnanetal.. 2009: Guo etal.. 1985: Schneider etal.. 1978.19771. with
the rate and extent of absorption dependent on the physical form of RDX (i.e., the increased surface
area associated with finely powdered RDX allows for increased absorption) and the dosing
preparation or matrix (Bannon etal.. 2009a: Krishnan et al.. 2009: Crouse etal.. 2008: Bannon.
2006: Guo etal.. 1985: MacPhail etal.. 1985: Schneider et al.. 19771. Dermal absorption of RDX has
been demonstrated in in vitro studies using human and pig skin (Reddv etal.. 2008: Reifenrath et
al.. 20081.
RDX is systemically distributed, including to the brain (i.e., RDX can cross the blood:brain
barrier), heart, kidney, liver, and fat fMusick etal.. 2010: Bannon etal.. 2006: MacPhail etal.. 1985:
Schneider etal.. 19771. In rats, RDX can be transferred from dam to fetus across the placental:blood
barrier, and has been identified in maternal milk (Hess-Ruth et al.. 2007).
The metabolism of RDX in humans has not been investigated. Studies in experimental
animals indicate that metabolism of RDX is extensive and includes denitration, ring cleavage, and
generation of C02 possibly through cytochrome P450 (CYP450) fMusick etal.. 2010: Maior etal..
2007: Fellows etal.. 2006: B hush an etal.. 2003: Schneider et al.. 1978.1977).
RDX and metabolites are eliminated primarily via urinary excretion and exhalation of C02
(Sweeney etal.. 2012a: Musick etal.. 2010: Krishnan et al.. 2009: Major etal.. 2007: Schneider etal..
1977). Estimated elimination half-lives (ti/2; estimated ti/2 values based on RDX concentrations in
blood) indicate that RDX is more rapidly metabolized in mice than in rats and humans; estimated
ti/2 values were 1.2 hours for mice, 5-10 hours for rats, and 15-29 hours for humans (Sweeney et
al.. 2012b: Krishnan et al.. 2009: Ozhan etal.. 2003: Woody etal.. 1986: Schneider et al.. 19771.
A more detailed summary of RDX toxicokinetics is provided in Appendix C, Section C.l.
1.1.3. Description of Toxicokinetic Models
A physiologically based pharmacokinetic (PBPK) model to simulate the pharmacokinetics of
RDX in rats was first developed by Krishnan et al. (2009) and revised to extend the model to
humans and mice (Sweeney etal.. 2012a: Sweeney etal.. 2012b). The Sweeney etal. (2012a) model
consists of six main compartments: blood, brain, fat, liver, and lumped compartments for rapidly
perfused tissues and slowly perfused tissues, and can simulate RDX exposures via the intravenous
(i.v.) or oral route. This model assumes that the distribution of RDX to tissues is flow-limited, and
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represents oral absorption as first-order uptake from the gastrointestinal (GI) tract into the liver,
with 100% of the dose absorbed. RDX is assumed to be cleared by first-order metabolism in the
liver. The model does not represent the kinetics of any RDX metabolites. The Sweeney et al.
f2012al and Sweeney et al. f2012bl PBPK models were evaluated and subsequently modified by
the U.S. Environmental Protection Agency (EPA) for use in dose-response modeling in this
assessment (see Appendix C, Section C. 1.5).
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM
In experimental animal studies, RDX test material administered in toxicology studies
included formulations that ranged in purity (from 84 to 99.99%) and in particle size (from <66 to
~200 [im particle size). Differences in test material purity and particle size were taken into
consideration while evaluating RDX toxicity findings; this is discussed in the literature search
section and incorporated in the synthesis of evidence.
Mortality has been reported in the animal toxicology studies conducted for RDX. Due to the
serious nature associated with a frank effect such as mortality, EPA specifically evaluated the
database with respect to mortality (see Appendix C, Section C.3.1). In brief, mortality was observed
following exposure to a range of doses in chronic-duration studies, in studies up to 6 months in
duration, and during gestation fLish etal.. 1984: Levine etal.. 1983: Levine etal.. 1981a: Cholakis et
al.. 1980: von Oettingen et al.. 1949). In further analyzing the available evidence, mortality
occurred at lower doses in rats compared with mice and following gavage administration compared
with dietary administration. Additionally, mortality occurred to a greater extent with
administration of RDX in the form of relatively finer particle sizes, likely due to faster dissolution of
RDX leading to higher blood concentrations. Some investigators attributed the mortality to RDX-
related cancer or noncancer effects (e.g., kidney or nervous system effects); others identified no
cause for the animal deaths. Typically, evidence related to various hazards is presented and
synthesized in distinct organ- or system-specific sections. However, in this case, the assessment
does not present mortality in a hazard section by itself due to the likelihood that events leading to
mortality fall under other specific hazards. Mortality evidence is considered in discussions of the
evidence for organ/system-specific hazards where applicable.
1.2.1. Nervous System Effects
In humans, 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 cross-sectional 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. No developmental neurotoxicity studies were available and minimal information was
available to evaluate potential cognitive or behavioral effects associated with RDX exposure. A
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summary of nervous system effects associated with RDX exposure is presented in Tables 1-2 and
1-3 and Figure 1-1. Experimental animal studies are ordered in the evidence table and exposure-
response array by duration of exposure and then species.
Observational Studies in Humans
In a cross-sectional study by Ma and Li (1993). neurobehavioral effects were evaluated in
Chinese workers occupationally exposed to RDX. Memory retention and block design scores10 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, and letter cancellation test) (see Table 1-2). This study did not
consider potential confounders such as alcohol consumption or co-exposure to trinitrotoluene
(TNT), and there was limited information characterizing exposure to RDX.
Case reports suggest an association between RDX exposure (via ingestion, inhalation, and
possibly dermal exposure) and neurological effects (see Appendix C, Section C.2). Severe
neurological disturbances include tonic-clonic seizures (formerly known as grand mal seizures) in
factory workers (Testud etal.. 1996a: Testud etal.. 1996b: Kaplan etal.. 1965: Barsotti and Crotti.
19491. seizures and convulsions in exposed soldiers serving in Vietnam fKetel and Hughes. 1972:
Knepshield and Stone. 1972: Hollander and Colbach. 1969: Stone etal.. 1969: Merrill. 19681.
seizures, dizziness, headache, and nausea following nonwartime/nonoccupational exposures
(Kasuske etal.. 2009: Davies etal.. 2007: Kuctikardalietal.. 2003: Hettand Fichtner. 2002: Harrell-
Bruder and Hutchins. 1995: Goldberg et al.. 19921. and seizures in a child following ingestion of
plasticized RDX from the mother's clothing fWoodvetal.. 19861.
Studies in Experimental Animals
Nervous system effects in experimental animals include an array of behavioral changes
consistent with the induction of seizures by RDX exposure, and have been observed in the majority
of chronic, subchronic, and developmental studies examining oral exposure to RDX (see Table 1-3
and Figure 1-1). Although study authors interchangeably used the terms seizures and convulsions,
seizures, which result from abnormal electrical activity in the brain, can outwardly manifest in a
variety of ways, including as convulsions. However, seizures can also manifest as facial twitches or
tremors, or more subtly as increased irritability or aggression, absence of response to external
stimuli, or they may go unnoticed. While behavioral methods exist to capture a spectrum of
responses known to occur as a result of this aberrant neuronal activity, the most reliable detection
10The 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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methods are electrophysiological (Racine. 19721. Only one acute exposure study, testing a single,
high dose of RDX, included electrophysiologic recordings fWilliams etal.. 20111.
Convulsions (a sudden and irregular movement of a limb or of the body) have been
reported in studies with different animal species and experimental designs. In every study that
reported convulsions, the incidence of convulsions increased with dose. In 2-year dietary studies in
rats (F344 and Sprague-Dawley) and mice (B6C3Fi), convulsions were observed beginning at doses
of 35-40 mg/kg-day, butnotatlower doses (Lish etal.. 1984: Levine etal.. 1983: Hart. 19761.11
Subchronic dietary exposure to RDX 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 fCrouse etal..
2006: von Oettingen et al.. 19491. In three other studies of non-pregnant, adult rats involving
exposure durations of 30-90 days, no evidence of seizures, convulsions, or tremors was reported at
doses ranging from 1 to 50 mg/kg-day (MacPhail etal.. 1985: Cholakis etal.. 1980) (both
unpublished technical reports).12 Levine etal. (1990) reported convulsions in rats following
subchronic exposure only at a dose of 600 mg/kg-day (a dose associated with 100% mortality);
however, the unpublished technical report of this study f Levine etal.. 1981al reported convulsions
at 600 and >30 mg/kg-day, thereby reducing confidence in the identification of the dose level at
which nervous system effects were observed in this study. 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 (Angerhofer etal.. 1986: Cholakis etal.. 1980) (unpublished
technical reports), 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 for 6 weeks fvon Oettingen et al.. 19491. but not 10 mg/kg-day for 13 weeks fHart.
19741 (unpublished technical report); however, five of six monkeys exhibited convulsions following
a gavage dose of 10 mg/kg-day for 13 weeks fMartin and Hart. 19741 (unpublished technical
report). Linkage of these convulsions to seizure activity was most directly demonstrated by
Williams etal. (2011). who observed abnormal electroencephalogram (EEG) activity consistent
with seizure activity that coincided with physical manifestations ranging from subtle convulsive
1 'The 2-year dietary studies in F344 rats bv Levine et al. f19831 and B6C3Fi mice bv Lish et al. f19841 were
available only as a laboratory reports. An external peer review was sought by EPA in July 2012 to evaluate
the accuracy of experimental procedures, results, and interpretation and discussion of the findings presented.
A report of this peer review organized by Versar, Inc. is available on the Health and Environmental Research
Online (HERO) database (U.S. EPA. 2012c). The 2-year dietary study in Sprague-Dawley rats by Hart (19761
is available as an unpublished technical report.
12The series of nine toxicology studies reported in Cholakis et al. f!9801 were available only as a laboratory
reports. An external peer review was sought by EPA in 2017 to evaluate the accuracy of experimental
procedures, results, and interpretation and discussion of the findings presented in six of the nine studies (90-
day toxicity study in rats, initial 90-day toxicity study in mice, supplemental 90-day toxicity study in mice,
teratology study in rats, teratology study in rabbits, and two-generation reproductive toxicity study in rats).
A report of this peer review organized by Eastern Research Group, Inc. is available on the HERO database
fU.S. EPA. 20171.
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behaviors (e.g., twitches) to tonic-clonic seizures in rats acutely exposed to 75 mg/kg-day RDX via
gavage.
In the only study addressing susceptibility to seizures (chemicals that may alter seizure
frequency, severity, duration, or threshold), Burdette etal. f!9881 found that seizure occurrence
was more frequent 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 (Note: some
uncertainty exists regarding the administered dose as neither the purity nor the specific particle
size of the RDX used in the experiments by Burdette etal. (1988) was reported.) 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, which could suggest that the
blockade of GABAergic signaling by RDX (see Mechanistic Evidence section) needs to be sustained
for some minimal duration to induce these types of effects. In a complementary experiment, Long
Evans rats treated daily with 6 mg/kg-day RDX for up to 18 days required fewer stimulation trials
compared to animals not treated with RDX to exhibit amygdaloid kindled seizures compared to
controls. These findings provide evidence that RDX exposure can reduce the seizure threshold for
other pro-convulsant stimuli, an adverse effect fU.S. EPA. 19981.
The majority of animal studies reported convulsions and/or seizures as clinical
observations; thus, interpretation of these observations is limited because the nature and severity
of convulsions and seizures were not more fully characterized. The 90-day study by Crouse etal.
(2006)13 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 1-3). 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 convulsion duration and
onset after the start of dosing was not reported fCrouse et al.. 20061.
In general, gavage dosing induced convulsions at lower doses than did dietary
administration. For example, in the subchronic gavage study by Crouse etal. (2006) and the
developmental gavage study by Cholakis et al. (1980). convulsions were observed in 1-3 F344
rats/group at doses of 2-8 mg/kg-day; at doses of 15-20 mg/kg-day, convulsions were observed in
approximately 60-70% of the animals. Consistent with this pattern, even an acute (single dosing)
gavage study reported seizures in 2/10 rats shortly after exposure to 12.5 mg/kg-day, and
approximately 80% of rats developed spontaneous seizures shortly after exposure to 25-50
mg/kg-day (Burdette etal.. 1988): the longevity of the seizure behaviors was also highly dose-
dependent. In contrast, in a 2-year dietary study by Levine etal. (1983). convulsions were reported
13The 13-week gavage study in F344 rats by Crouse etal. (20061 was available only as a laboratory report. An
external peer review was organized by Versar, Inc. in July 2012 to evaluate the accuracy of experimental
procedures, results, and interpretation and discussion of the findings presented. The U.S. EPA (2012cl report
of this peer review is available on the HERO database.
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only at a dose of 40 mg/kg-day; no convulsions were observed at lower doses (<8 mg/kg-day). The
difference in response between gavage and dietary administration may be due to the bolus dosing
resulting from gavage administration and the comparatively faster absorption and higher peak
blood concentrations of RDX.
Several experimental animal studies documented that unscheduled deaths were frequently
preceded by convulsions or seizures. In a 2-year study in rats, Levine etal. (1983) noted that
tremors and/or convulsions were often seen in high-dose animals prior to their death. In a rat
developmental toxicity 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 around the mouth and nose) in other dams that
died during the study. Convulsions preceding death were also observed in pregnant Sprague-
Dawley rats exposed to RDX during gestation fAngerhofer etal.. 19861. fBurdette etal.. 19881
reported that 9/28 rats died during spontaneous seizure within 8 hours of administration (by
gavage) of a single dose of 50 or 60 mg/kg RDX.
The 90-day Crouse etal. (2006) study provides the most detailed information on the
relationship between convulsions and mortality (see Appendix C, Table C-10 for additional
information on evidence of mortality associated with RDX exposure). Convulsions (3/20) and pre-
term deaths (2/20)14 were observed in male and female rats exposed to 8 mg/kg-day RDX; the
incidences of both convulsions and pre-term deaths were higher in dose groups with greater
exposures. Investigators stated that nearly all observed pre-term deaths in rats exposed to the
three higher doses (10,12, and 15 mg/kg-day RDX) for 90 days were preceded by neurotoxic signs
such as rearing behavior, tremors and convulsions; however, pre-term death did not occur in all
animals that convulsed. Convulsions were not typically observed during a functional observational
battery (FOB) test conducted after exposure, possibly due to the time needed to complete
exposures prior to beginning behavioral testing (convulsions typically occurred shortly after
dosing). Of the 100 RDX-treated rats in the Crouse etal. (2006) study, convulsions were
documented in 34 male and female rats across the five dose groups (with convulsions initially
observed anywhere from day 7 to 87); based on additional information provided as a memorandum
by study investigators (Tohnson. 2015a). 26 of these 34 rats (76%) survived to the end of the 90-
day study. In general, higher doses of RDX were associated with fewer days of exposure before the
first convulsion was observed. Of the eight rats that exhibited convulsions prior to pre-term death,
convulsions were documented anywhere from the same day that the animal died to 8 weeks prior
to death. Of the 26 rats that seized and survived to day 90, the first seizures were observed as early
as day 10 and as late as day 87. Thus, while an increase in mortality was observed in the Crouse et
al. (2006) study at the same dose as convulsions, the additional information provided by Tohnson
(2015a) does not show as clear a correspondence between convulsions (and other neurotoxic
14At the 8 mg/kg-day dose level, the three rats that convulsed survived to the end of the study; no convulsions
were observed in the two rats that died before study termination.
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signs) and mortality. Analysis of these data is limited to the extent that convulsions may have
occurred at times when animals were not observed and therefore may be undercounted in the
individual animal data; however, Tohnson f2015al noted that it is unlikely that seizure observations
were missed, since seizures generally occurred soon after dosing.
A few studies reported mortality that was not specifically associated with neurological
effects (see Appendix C, Table C-10) (Angerhofer etal.. 1986: Levine etal.. 1981a: von Oettingen et
al.. 19491: however, in these studies, animals may not have been monitored for clinical observations
or monitored with sufficient frequency to have observed convulsive activity prior to death. There
were no reports of mortality subsequent to convulsions in case reports of nervous system effects in
workers exposed to RDX during manufacture and in individuals exposed acutely as a result of
accidental or intentional ingestion (see Appendix C, Section C.2).
Additional neurobehavioral effects associated with RDX exposure in rats included increased
hyperactivity, hyper-reactivity to approach, fighting, and irritability at doses similar to those that
induced tremors, convulsions, and seizures (20-100 mg/kg-day) f Levine etal.. 1990: Angerhofer et
al.. 1986: Levine etal.. 1983: Levine etal.. 1981a. b; von Oettingen et al.. 19491. Hyperactivity and
nervousness were also reported in male mice that received a subchronic exposure to 320 mg/kg-
day RDX fCholakis 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 at relatively
low dose levels (<10 mg/kg-day), although changes in acoustic startle response in acute exposures
at higher doses (12.5-50 mg/kg) were noted (MacPhail etal.. 1985). No significant changes in
behavioral or neuromuscular activity were observed in rats following exposure to <15 mg/kg-day
for 90 days (Crouse etal.. 2006). Crouse etal. (2006) observed 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.
Changes in absolute and relative brain weight were mixed across studies, and no studies
included histopathologic evaluation of neuronal damage. Elevated absolute brain weights were
reported in subchronic assays in B6C3Fi mice and F344 rats (Crouse etal.. 2006: Levine etal..
1990: Levine etal.. 1981a. b; Cholakis etal.. 1980): 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: Cholakis etal..
19801. but these changes were likely a result of changes in body weight in the study, and were not
as useful a measure of effects of RDX on brain weights as absolute brain weight In 2-year 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 (Lish etal.. 1984). whereas an increase in absolute brain weight
(2% relative to control) was observed in F344 rats at a dose of 40 mg/kg-day (Levine et al.. 1983).
Less emphasis is placed on evidence of organ weight changes from chronic (2-year) studies because
normal physiological changes associated with aging and intercurrent disease may contribute to
inter-animal variability that could confound organ weight interpretation fSellers etal.. 20071.
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
In some studies, seizures appeared soon after dosing, suggesting that seizure induction was
more strongly correlated with dose level than with duration of exposure. Consistent with this
observation are the findings of Williams etal. f2011I who demonstrated that RDX is rapidly
absorbed and crosses the blood:brain barrier following oral administration in rats, and that
distribution of RDX (8 |ig/g wet weight) to the brain correlated with seizure onset. However, the
incomplete or slow reversibility of the blockade of GABA receptor signaling after removal of RDX in
the in vitro study by Williams etal. (20111 suggests that some effects might persist without the
continued presence of RDX in the brain, which could permit cumulative effects.
While a dose-response relationship was observed consistently within studies, a dose that
induced convulsions in animals in one study did not necessarily induce convulsions at the same
dose in another study. This lack of consistency may be attributed, at least in part, to differences in
the purity or particle size of the test material across studies. Assuming that increased particle size
(and the corresponding reduction in available surface area compared with smaller particle sizes)
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 et al.
fl9801 used a relatively large RDX particle size (200 [im) compared to the rat study by Levine et al.
f!9831 that used a smaller (<66 [im) particle size. This may explain why the Cholakis etal. f 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 protocols for observation for clinical signs (e.g., observations
performed once daily in the morning in Levine etal. (1983)) may not have been sufficiently
frequent to accurately measure the incidence of seizures or other nervous system effects.
The lack of developmental neurotoxicity studies was identified as a data gap within the
available studies on RDX. A pilot study in rats did not directly investigate potential RDX nervous
system effects but did find RDX in the brains of offspring rats as well as milk from dams treated
with RDX during gestation (Hess-Ruth etal.. 2007). Studies on chemicals with similar modes of
action to RDX (e.g., bicuculline), combined with demonstrated transfer of RDX to perinatal rodent
brains, suggest a potential for RDX to be harmful during brain development, possibly at a lower
dose than required for neurotoxicity in adults. Further discussion on the potential developmental
neurotoxicity of RDX can be found in Susceptible Populations and Lifestages for Cancer and
Noncancer Outcomes (see Section 1.3.3).
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
Table 1-2. Evidence pertaining to nervous system effects in humans
Reference and study design
Results
Ma and Li (1993) (China)
Cross-sectional study, 60 workers from
Neurobehavioral function tests, scaled scores (mean, standard
deviation)
the same plant 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; two groups of workers
exposed to the following mean RDX
concentrations in air (basis for dividing
workers into two exposure groups was
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)
not provided).
Concentration (mg/m3) (mean
± standard deviation):
Group A 0.407 (± 0.332)
Group B 0.672 (± 0.556)
*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)
Effect measures3: Five neurobehavioral
function tests and five additional
memory subtests.
Analysis: Variance (F-test); unadjusted
linear regression, multiple regression,
and correlation analysis.
Subtest
Control
Group A
Group B
Directional memory*
Associative learning*
Image free recall*
23.5 (3.6)
24.9 (5.1)
24.1 (3.8)
17.2 (4.9)
20.0 (4.3)
20.9 (4.1)
18.1 (5.7)
18.5 (4.6)
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).
aSymptom data were not included in evidence table because of incomplete reporting.
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
Table 1-3. Evidence pertaining to nervous system effects in animals
Reference and study design
Results
Convulsions and neurobehavioral effects
Lish 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
2 yrs
One male in the 35 mg/kg-d dose group and one female 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 nervous system effects, as evidenced by clinical signs or changes
in appearance or behavior, were reported.
Levine et al. (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
2 yrs
Tremors, convulsions, and hyper-responsiveness to stimuli were
noted in males and females 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)3
Diet
13 wks
Hyperactivity and/or nervousness observed in 50% of the high-dose
males; no signs observed in females'5; no incidence data were
reported.
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 nervous system effects, as evidenced by clinical signs or changes
in appearance or behavior, were reported.
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
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
FO exposure: 13 wks pre-mating, and
during mating, gestation, and lactation of
Fl; Fl exposure: 13 wks after weaning,
and during mating, gestation, and
lactation of F2; F2 exposure: until
weaning
No nervous system 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
13 wks
Doses
0 4 8b 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. (1990); Levine et al. (1981a);
Levine et al. (1981b)c
Rats, F344,10/sex/group; 30/sexfor
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 rats (sex not specified)
receiving >100 mg/kg-d; no incidence data were reported.
Tremors and convulsions were observed prior to death in one female
and two male rats receiving 600 mg/kg-d.d (600 mg/kg-d was lethal to
all rats.)
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
13 wks
Hyperirritability and convulsions were observed in the 25 and
50 mg/kg-d groups'5; no incidence data were reported.
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
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; purity
and particle size not specified
0,0.1,1, or 10 mg/kg-d
Diet
13 wks
No nervous system effects, as evidenced by clinical signs or changes
in appearance or behavior, were reported.
Martin and Hart (1974)
Monkeys, Cynomolgus or Rhesus6,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
13 wks
Doses
_Q
o
1
1
1
o
o
CNS effects characterized as depression, trembling, shaking, jerking,
or convulsions (incidence)
M
F
0/3 0/3 0/3 2/3
0/3 0/3 0/3 3/3
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 reflexesb; no incidence data were reported.
MacPhailetal. (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 (incidence)
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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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Angerhofer et al. (1986)
Rats, Sprague-Dawley, 39-51 mated
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
Convulsions and hyperactivity15 were observed at 20 mg/kg-d;
no incidence data were reported.
Burdette et al. (1988)
Rats, Long Evans, 10-21 males/group
Exp 1: 0,10, 20, or 60 mg/kg-d
Exp 2: 0,12.5, 25, or 50 mg/kg-d
Experiments 1 and 2 conducted using the
same study design, each with a control
group
Gavage (single exposure)
8-hr after exposure, rats placed in
observation chamber; 0-64 kHz, 95 dB
ultrasonic cleaner turned on for 1 min or
until seizure initiated with uncontrolled
running (whichever occurred first)
Doses
0 10 12.5 20 25 50 60
Number of spontaneous seizures during 8-hr interval between
dosing and audiogenic seizure testing (mean)
M
0 -- 0.17 ± -- 1.4 ± 4.5 ±
0.2 0.2* 0.6*
Note: first seizures in all 3 treatment groups observed
within first 2 hrs after RDX exposure.
Prevalence of audiogenic seizures (%p
M
0/31 1/10 0/10 3/10 4/10 10/12* 13/16*
Values estimated from graph using Grab It! Software and numbers of
animals from Figure 2 of the paper. Statistical significance indicated
by study authors; spontaneous seizures - p < 0.012; audiogenic
seizures - p< 0.017.
Brain weight
Lish 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
2 yrs
Doses
0 1.5 7 35 175/100
Absolute brain weight (percent change compared to control)
M
F
0% -0.2% 0.61% 0.81% -1%
0% -2% -2% -4%* -3%*
Relative brain weight (percent change compared to control)
M
F
0% 4% 2% 2% 5%
0% -4% -1% -3% 18%*
Levine et al. (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
2 yrs
Doses
o
00
LO
m
o
o
Absolute brain weight (percent change compared to control)
M
F
0% 2% -1% 2% 2%
0% -0.3% -0.4% 1% 2%*
Relative brain weight (percent change compared to control)
M
F
0% 0% 8% 2% 22%*
0% -1% 3% 4% 20%*
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
Reference and study design
Results
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
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
13 wks
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 10 14 20 28 40
Absolute brain weight (percent change compared to control)
M
F
0% - - - 2% 2%
0% - 4% 2%
Relative brain weight (percent change compared to control)
M
F
0% - 6% 2%
0% - - - 0% 3%
Doses
0 80 160 320
Absolute brain weight (percent change compared to control)
M
F
0% 0% 2% 10%
0% 0% 4% 2%
Relative brain weight (percent change compared to control)
M
F
0% -3% 1% 8%
0% 0% 3% -4%
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 brain weight (percent change compared to control)
M
F
0% - - - 3% 0%
0% - - - 0% 0%
Relative brain weight (percent change compared to control)
M
F
0% - 7%* 10%*
0% - - 5% 6%
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0,4, 8,10,12, or 15 mg/kg-d
Gavage
13 wks
Doses
0 4 8 10 12 15
Absolute brain weight (percent change compared to control)
M
F
0% -1% -0.3% 2% 5%* 7%*
0% -2% 6% 1% 4% 6%
Relative brain weight (percent change compared to control)
M
F
0% 6% 10% 5% 3% 4%
0% -2% -2% -12%* -12%* -15%*
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
Reference and study design
Results
Levine et al. (1990); Levine et al. (1981a);
Levine et al. (1981b)c
Rats, F344,10/sex/group; 30/sexfor
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
Absolute brain weight (percent change compared to control)
M
F
0% 1% 0.53% -6%
0% -1% 1% 2%
Relative brain weight (percent change compared to control)
M
F
0% 4% 7% 14%
0% 0.3% 2% 5%
^Statistically significant (p < 0.05) based on analysis by study authors.
aDoses were calculated by the study authors.
bMortality was reported in some RDX-treated groups in this study.
cLevine 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.
discrepancies in the doses at which convulsions occurred were identified in the technical report. The nervous
system effects reported in this table and in the corresponding exposure-response array are those provided in
the results section of the technical report (Levine et al., 1981a) and in the published paper (Levine et al., 1990).
In other sections of the technical report, the authors reported that hyperactivity to approach and convulsions
were observed in rats receiving >30 mg/kg-day (abstract and executive summary), or that mortality was
observed in rats receiving 100 mg/kg-day and that hyperactivity to approach, tremors, and convulsions were
observed in animals exposed to lethal doses (discussion).
eThe species of monkey used in this study was inconsistently reported in the study as either Cynomolgus (in the
methods section) or Rhesus (in the summary).
CNS = central nervous system; GD = gestational day; HMX = octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine;
TWA = time-weighted average
Note: A dash ("-") indicates that the study authors did not measure or report a value for that dose group.
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
1000 q
100 -
• signifies ntly changed
O not significantly changed
-3 10
tu)
E
o
Q
1 :
6 6 o o
0.1
JU U
ra —
Chronic
15 «
"a —
OJ —
Subchronic
Convulsions and/or Seizures
Gestational (dams)
— rq
"qj Si
Absolute Brain Weight
Figure 1-1. Exposure response array of nervous system effects following oral exposure.3
aBecause convulsions and seizures are rare in experimental animals, any occurrence in an RDX-exposed group was considered treatment-related. Given the
severity of this endpoint, a response in treated groups was determined to be significant (filled circles) in the array where there was any occurrence of
convulsions and/or seizures reported in the study, whether or not the incidence was statistically significantly elevated over the control.
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Mechanistic Evidence
Studies that have explored the mode of action (MOA) of RDX on the central nervous system
(CNS) have focused on the potential impacts on neurotransmission. These studies implicate a MOA
for RDX-induced seizures involving distribution of RDX from the blood to the brain (across the
blood:brain barrier) and subsequent effects on neurotransmission, specifically gamma-amino
butyric acid (GABA)-mediated signaling in limbic regions of the brain. There is significant evidence
from the scientific literature to suggest that RDX neurotoxicity results from interactions of RDX
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. A large literature
base exists to support the relationship between blockade of GABAAergic neurotransmission and
seizure induction, and GABAAergic pharmaceuticals are routinely used to suppress seizures in the
treatment of epilepsy and other disorders (perhaps most recognizably, drugs in the benzodiazepine
family).
In research conducted by the U.S. Army Center for Health Promotion and Preventative
Medicine, Williams etal. f20111 and Bannon et al. f2009al 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 (Williams etal.. 2011). 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.. 2009a).
In receptor binding studies, RDX showed significant affinity for GABAa receptors (Williams
etal.. 2011: Williams and Bannon. 2009). Specifically, RDX showed an affinity for the picrotoxin
convulsant site of the GABA channel, with nearly 100-fold less potency than picrotoxin itself.
Consistent with the observations of abnormal electrical activity after in vivo RDX exposure (see
discussion in previous section), in vitro RDX treatment of brain slices from the basolateral
amygdala inhibited GABAA-mediated inhibitory postsynaptic currents and initiated seizure-like
electrical activity. Thus, RDX exposure appears to reduce the inhibitory effects of GABAergic
neurons, resulting in a loss of inhibitory tone and enhanced excitability that can eventually lead to
seizures (Williams etal.. 2011: Williams and Bannon. 2009). 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 (Teffervs etal.. 2012: Gilbert. 1994).
Consistent with the in vitro observations by Williams etal. (2011). Burdette etal. (1988) also
implicated the limbic system in seizures caused by RDX exposure. Burdette etal. (1988) reported
that the pattern of the seizure behaviors manifest in response to RDX exposure mimicked the
sequence of behavioral stages observed following repeated electrical stimulation of temporal lobe
structures by Racine f 19721. In addition, amygdaloid kindled rats (rats subjected to patterns of
electrical stimulation to this limbic region, which promotes the development of seizures) exhibited
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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
1 pro-convulsant activity at a dose that was approximately half of the dose necessary for RDX to
2 induce spontaneous seizures (rats treated with RDX also required fewer electrical stimulations to
3 trigger kindled seizures) fBurdette etal.. 19881. As these latter findings occurred at lower doses
4 than RDX-induced increases in audiogenic seizures fBurdette etal.. 19881. this further suggests a
5 primary role for limbic regions (brain structures involved in sound-induced seizures may be
6 indirectly affected). Potential limbic system involvement is also suggested given its role in
7 integrating emotional and behavioral responses (including aggression) and the anecdotal
8 observations of hyperactivity, hyper-responsiveness to approach, and irritability noted across
9 several studies of RDX toxicity fLevine etal.. 1990: Levine etal.. 1983: Levine etal.. 1981a. b;
10 Cholakis etal.. 1980: von Oettingen etal.. 19491.
11 It is possible to construct a hypothetical MOA for RDX-induced seizure activity based on the
12 evidence summarized above. These steps are consistent with ongoing efforts to identify an adverse
13 outcome pathway (AOP) for ionotropic GABA receptor antagonism, reviewed in Gong etal. (2015)
14 and Collier etal. (2016) and described in greater detail in the draft AOP available at
15 https://aopwiki.org/. Following distribution of RDX to the brain:
16 1) Parent RDX acts as a GABAa receptor antagonist (supported by Schneider etal. f19771 and
17 Williams etal. f201111. binding noncompetitively to the picrotoxin convulsant site of the
18 GABAa receptor (supported by Williams and Bannon (2009) and Williams etal. (2011)).
19 2) RDX binding to the GABAa receptor at the picrotoxin site blocks the conduction of chloride
20 through the ion channel.
21 3) Reduced chloride conduction results in reduced GABA-mediated inhibition of neuronal
22 signaling, often manifesting as a reduction in spontaneous inhibitory postsynaptic currents
23 (sIPSCs). Williams etal. (2011) observed a reduction in the amplitude and frequency of
24 sIPSCs in whole-cell in vitro recordings of neurons in brain slices from the rat basolateral
25 amygdala after exposure to RDX. In addition, RDX treatment of slices inhibited GABA-
26 induced currents.
27 4) Reduced inhibitory tone (e.g., reduced sIPSCs) increases the likelihood of action potentials
28 by decreasing the resting potential of neuronal membranes (depolarization).
29 5) As a group of neurons begins firing abnormally and excessively (e.g., due to the reduced
30 inhibitory tone, which typically would hyperpolarize, or reset, the membrane after firing),
31 they can begin firing in a synchronized manner and initiate a wave of depolarization; these
32 events can be detected electrophysiologically. Williams etal. f20111 observed a pattern of
33 seizure-like neuronal discharges after in vivo RDX exposure and in vitro from slices of the
34 basolateral amygdala in rats after adding RDX (the in vitro effects were not reversible after
35 40 minutes of washout).
36 The steps above provide a biologically plausible sequence of mechanistic events that result
37 in the generation of seizure-like neuronal activity. Reduction of the inhibitory GABAergic signaling
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is common to many convulsants, as summarized in Kalueff (2007). Some organochlorine
insecticides, including alpha-endosulfan, dieldrin, and lindane, also exert neurotoxic effects through
interaction with the GABAa receptor, and can produce a range of hyperexcitability effects (including
convulsions) in mammals fVale etal.. 2003: Bloomquist. 1992: Sunol etal.. 19891. The interaction
of RDX with the GABAa receptor is directly supported by receptor-binding assays fWilliams etal..
2011). Although these binding assays were performed on rat receptors, it is plausible that the
results are relevant to human neurotoxicity. Seizures have been observed in many species,
including humans, rats, mice, dogs, lizards, and birds at varying dosages and durations of exposure
fOuinnetal.. 2013: Mcfarland etal.. 2009: Tohnson etal.. 2007: Bruchim etal.. 2005: Kuctikardali et
al.. 2003: Woody etal.. 1986: Lish etal.. 1984: Berry etal.. 1983: Levine etal.. 19831. A more recent
meta-analysis of toxicogenomic data across a phylogenetically diverse set of organisms (rat, quail,
fathead minnow, earthworm, and coral) demonstrated that neurotoxic responses are conserved in
more highly-related species and that binding to the GABAa receptor is a common molecular
initiating event (Garcia-Revero etal.. 2011). While these lines of evidence do not preclude a role of
other receptors as yet unscreened for RDX binding affinity, they support a primary role for the
GABAergic pathway described above in the development of RDX neurotoxicity.
As mentioned previously, the GABAa receptor is also a target of many anticonvulsant
therapies (e.g., benzodiazepines, propofol, barbiturates) fMeldrum and Rogawski. 2007: Mohler.
2006). Additional support for the involvement of GABAergic signaling in the neurotoxicity of RDX
comes from human case reports. In multiple case reports, medical intervention included treatment
with benzodiazepines (commonly diazepam or lorazepam) to treat seizing patients (Kasuske etal..
2009: Davies etal.. 2007: Kuctikardali etal.. 2003: Hettand Fichtner. 2002: Woody etal.. 1986).
Benzodiazepines act in large part by enhancing the effects of GABA at the GABAa receptor by
increasing chloride conductance, resulting in anticonvulsant and relaxant effects f Goodman etal..
19961.
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. 1997). 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 contributing mechanism because:
(1) blood and brain levels of AChE are unaffected by RDX fWilliams etal.. 2011: Williams and
Bannon. 2009): and (2) in vitro neurotransmitter receptor binding studies do not reveal any affinity
of RDX for acetylcholine receptors (Williams etal.. 2011: Williams and Bannon. 2009). Additionally,
common AChE-induced symptoms (salivation and lacrimation) have not routinely been observed
fWilliams etal.. 2011). 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 fWilliams etal.. 2011: Williams and Bannon. 20091.
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In a microarray experiment, Bannon etal. (2009a) 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 action for 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 RDX/kg diet (estimated dose: 0.75-
1.5 mg/kg-day; Bannon et al. f2009all 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) (Zhang 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.
Deng etal. (2014) conducted miRNA and mRNA profiling in rats to identify targets up or
downregulated after 48-hour exposure to RDX, finding that many of the gene targets of these
miRNAs were associated with nervous system function, and may contribute to the neurotoxicity of
RDX. However, while effects of RDX on BDNF expression or other downstream targets may play a
role in RDX neurotoxicity, the utility of miRNAs as predictors of toxicity has not been demonstrated
and downstream targets of miRNA require verification (Bannon et al.. 2009b). Despite this
uncertainty, the potential for RDX exposure to modulate the expression or function of BDNF and
other factors crucial to normal brain development raises concern for the possibility of neurotoxic
effects with developmental exposure. Overall, the contribution, if any, of aberrant expression of a
suite of miRNAs to the MOA for RDX neurotoxicity is unknown.
Some uncertainty remains regarding how the mechanistic understanding of RDX
neurotoxicity may inform longer-term or cumulative exposures. To some extent, RDX binding at
the picrotoxin convulsant site of the GABA channel may inform the relationship between exposure
to the chemical and the time when a seizure is observed. Many of the available studies reported
that seizures or convulsions were typically observed shortly after exposure, and several studies
associated seizures with blood (and, correspondingly, brain) levels of RDX, indicating that a major
contributing factor to the seizurogenic effects of RDX exposure appears to be the transient presence
of RDX at target sites in the brain. Observations by Crouse etal. f20061. clarified in Tohnson
(2015a). showed that the median time to seizure after dosing in F344 rats is 55 minutes (range of
20-85 minutes); peak brain concentrations of RDX in F344 rats after single oral doses occurred
within the first 3-4 hours after dosing (Bannon etal.. 2009a). In addition, seizure intensity and the
longevity of seizure-related behaviors was directly related to RDX dose, even with acute exposure
fBurdette etal.. 19881. These observations are all consistent with the presumed primary MOA. In
general, across the RDX database, neurotoxicity, including induction of convulsions and seizures,
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appears to be more strongly correlated with dose than duration of exposure. Crouse etal. (2006)
reported that 80-90% of rats exposed to 12 and 15 mg/kg-day exhibited signs of neurotoxicity
beginning on day 0 of the study, which continued for the study duration. However, some
uncertainty remains. Gerkin etal. f20101 demonstrated that young C57/B16 mice injected
intraperitoneally (i.p.) with picrotoxin to induce seizures had a significantly increased frequency of
elevated neuronal activity ("Up state"), and firing rates were significantly increased in neocortical
neurons up to 24 hours after exposure, despite the rapid clearance (within a few hours) of
picrotoxin (Soto-Otero etal.. 1989). It is possible that this extended period of elevated neuronal
activity could increase the likelihood that a subsequent stimulus could trigger a seizure. While the
study authors did not look at longer durations post exposure, these observations with picrotoxin
may be consistent with the lack of complete reversibility of GABAergic signaling inhibition after
removal of RDX in Williams etal. f2011I Thus, there remains the possibility that, in a chronic
exposure scenario with repeated exposure to RDX and binding at the same site as picrotoxin, a
generalized increase in elevated neuronal activity could increase the likelihood of seizures
developing over time, or have other longer-term effects on normal brain function. While duration
of exposure alone generally did not appear to be predictive of seizures (e.g., in Crouse etal. f20061.
of the rats that survived the 90-day study, the range of time to onset of first observed convulsion
after gavage exposure to 10 mg/kg-day RDX was as early as day 7 and as late as day 87), exposure
to higher doses of RDX was associated with fewer days of exposure before the first convulsion was
observed. The variation in time between the start of the experiment and the onset to first seizure
with increasing dose could simply reflect the increased probability of action potentials with greater
decreases in inhibitory tone at higher doses; however, it may also indicate a cumulative component
of RDX neurotoxicity not accounted for by currently available mechanistic understanding.
Recent research in experimental animals has provided greater insight to inform a
mechanistic basis of RDX neurotoxicity. While other possible MOA(s) may contribute to the overall
neurotoxicity of RDX, the demonstrated affinity of RDX for the GABAa receptor, evidence of
supportive electrophysiological changes in vivo or with direct application of RDX, and toxicokinetic
evidence of distribution of RDX to the brain provide a mechanistic basis for the association of
seizures with exposure to RDX. This MOA is similar to other well-studied convulsants and relevant
to humans. The available information supports that RDX-induced seizures and related behavioral
effects likely result from inhibition of GABAergic signaling within limbic regions of the brain.
Integration 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
in certain neurobehavioral tests in workers exposed to RDX compared to controls (Ma and Li.
19931. and human case reports provide other evidence of an association between acute RDX
exposure and neurological effects. There was consistent evidence of neurotoxicity associated with
exposure to RDX; 11 of 16 repeat-dose animal studies (of varying design) reported neurological
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effects (some severe), 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 et al.. 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 fMacPhail etal.. 1985: Cholakis etal.. 1980: Hart.
1976.1974). differences in dosing, particle size, and purity of the RDX administered potentially
account for the lack of effect. Seizures resulting from RDX exposure likely result from inhibition of
GABAergic signaling due to the interaction of RDX with the GABAa receptor. The pro-convulsant
effects of RDX exposure are specific to CNS toxicity, as supported by observations of aberrant brain
electrical activity corresponding with physical seizure behaviors Williams etal. (20111. as well as
evidence of decreases in the seizure threshold for other centrally acting convulsants, including
amygdaloid kindling and audiogenic stimuli fBurdette etal.. 19881.
Together, toxicological information in animals and humans, supported by toxicokinetic and
mechanistic information, provides a coherent identification of nervous system effects as a human
hazard of RDX exposure.
1.2.2. Urinary System (Kidney and Bladder) Effects
The association between RDX exposure and effects on clinical measures of kidney function
was examined in one occupational epidemiology study. Case reports, involving accidental exposure
to ingested or inhaled RDX, offer some information on the potential for acute exposure 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 urinary system (kidney and bladder)
effects. A summary of these effects associated with RDX exposure is presented in Tables 1-4 to 1-7
and Figure 1-2. Experimental animal studies are ordered in the evidence table and exposure-
response array by duration of exposure and then by species.
Human case reports of individuals accidently exposed to unknown amounts of RDX by
ingestion or inhalation provide some evidence that RDX affects the kidney. Reported symptoms
included decreased urine output (Ketel 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. 1968). proteinuria (Kasuske etal.. 2009: Kuctikardali et al..
2003: Ketel and Hughes. 1972: Hollander and Colbach. 1969: Merrill. 19681. glucosuria
(Kuctikardali etal.. 2003). elevated blood urea nitrogen (BUN) levels (Hollander and Colbach. 1969:
Merrill. 1968). and one case of acute renal failure requiring hemodialysis following accidental
inhalation of RDX (Ketel and Hughes. 1972). 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 f 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 exposed to RDX and octahydro-l,3,5,7-tetranitro-
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1,3,5,7-tetrazocine (HMX); RDX exposure range: undetectable [<0.01 mg/m3] to 1.6 mg/m3), no
statistically significant differences in BUN or total serum protein between nonexposed and RDX-
exposed groups were observed fHathawav and Buck. 19771 (Table 1-4). As it is a cross-sectional
study, no information was provided on the length of employment or other proxies that could be
used to indicate exposure duration or cumulative exposure.
Studies in experimental animals provide some evidence that RDX exposure is associated
with urinary system effects (see Table 1-5 and Figure 1-2). The strongest evidence of effects on this
organ system is the collection of histopathological changes, including increased incidences of
kidney medullary papillary necrosis and pyelitis, uremic mineralization, and bladder distention
and/or cystitis, observed in male F344 rats exposed to 40 mg/kg-day RDX in the diet for 12 months
or longer fLevine etal.. 19831. The incidences of urinary system changes were higher at 2 years
than at 12 months, but the response at both time points was robust (e.g., incidence of medullary
papillary necrosis in male 40-mg/kg-day rats: 15/29 at 12 months, 18/31 at 2 years)15. Renal
effects were considered the principal cause of treatment-related morbidity and mortality in these
high-dose males. Similar kidney lesions were not observed in male rats in any dose group at the 6-
month interim sacrifice (see Tables 1-6 and 1-7). Histopathological changes reported in some male
rats in the lower-dose groups (0.3,1.5, and 8 mg/kg-day) after 2 years on study were not dose-
related, few in number, and consistent with background changes seen in aged rats.
Results from Levine etal. (1983) demonstrate a marked sex difference in response to RDX
urinary system toxicity; no kidney or urinary bladder changes were associated with RDX exposure
in female rats. In addition, mice appear to be less sensitive to the urinary system effects of RDX
than rats; the incidences of kidney histopathological changes in male and female B6C3Fi mice
exposed to RDX in the diet for 2 years at concentrations as high as 100 mg/kg-day were similar to
controls fLishetal.. 19841.
Histopathological findings in the urinary system from other experimental animal studies
are largely consistent with the 2-year findings from Levine et al. (1983) and (Lish etal.. 1984). i.e.,
that kidney and urinary bladder system effects are generally observed after RDX exposures longer
than 6 months in duration and at high doses (e.g., >40 mg/kg-day). Specifically, no pattern of
histopathological changes in the kidney were reported in rats exposed to RDX for 13 weeks (Crouse
etal.. 2006: Levine etal.. 1990: Levine etal.. 1981a. b; Cholakis etal.. 19801. in a 2-year study in
Sprague-Dawley rats or 13-week study in beagle dogs thatused a maximum dose of 10 mg/kg-day
fHart. 1976.19741. or in rabbits exposed dermally to a cumulative dose of 165 mg/kg RDX in
dimethylsulfoxide (DMSO) received over a 4-week period (5 days/week) (McNamara et al.. 1974).
In fact, in the 13-week study in F344 rats (Levine etal.. 1981a) conducted by the same investigators
that conducted the 2-year study in the same strain (Levine et al.. 1983). chronic nephropathy was
15Denominator represents scheduled sacrifice animals plus spontaneous deaths and moribund sacrifice
animals.
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observed in both control and treated animals with no evidence of a dose-related increase in
incidence.
Evidence of kidney histopathological changes in RDX-exposed animals following an
exposure duration of less than 6 months is limited to an increased incidence of tubular nephrosis
observed in B6C3Fi mice exposed for 13 weeks to 320 mg/kg-day RDX fCholakis etal.. 19801. a
dose eightfold higher than the dose that produced kidney and urinary bladder pathology in rats
after 2 years of exposure. 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. 19741. but the study authors did not identify this as treatment related. Finally, in a 2-
generation study, Cholakis etal. f 19801 reported an increased incidence of renal tubular epithelial-
lined cysts in the kidney cortex in F2-generation rats exposed to RDX at doses up to 16 mg/kg-day.
Because F2 animals were exposed for a relatively short duration (during gestation and through
weaning only), and because no histopathology was performed for the parental and F1 generations,
the kidney findings from this 2-generation study are difficult to interpret
Other kidney endpoints—serum chemistry parameters that may indicate changes in renal
function and kidney weights—did not provide consistent evidence of treatment-related changes.
Measurement of serum chemistry parameters (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. b; 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. Kidney
weights in subchronic oral toxicity studies in rats, dogs, and monkeys did not show a clear pattern
of change associated with RDX exposure. Kidney weight changes were either not dose-related or
were inconsistently increased or decreased across studies (see Table 1-5). Less emphasis is placed
on evidence of organ weight changes from chronic (2-year) studies fLish etal.. 1984: Hart. 19761
because normal physiological changes associated with aging and intercurrent disease may
contribute to inter-animal variability that could confound organ weight interpretation (Sellers et al..
20071.
Exposure to HMX, the major contaminant in many of the available RDX studies, was
associated with histopathological changes in the kidney and alterations in renal function in female,
but not male, rats fed doses >450 mg/kg-day HMX for 13 weeks (see the Integrated Risk
Information System [IRIS] assessmentofoctahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine [HMX] at
https: //www.epa.gov/irisl. No effects were observed at doses <115 mg/kg-day. Given the dose
levels where HMX appears to exhibit toxicity and the percentage of HMX (up to 10%) present as an
impurity in technical grade RDX that would result in HMX exposures <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. Further, differences in the pattern of toxicity (i.e., kidney effects observed
only in RDX-exposed males and HMX-exposed females) also suggest that HMX contaminants were
not responsible for kidney effects in rats exposed to RDX.
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Table 1-4. Evidence pertaining to kidney effects in humans
Reference and study design
Results
Hathawav and Buck (1977)
Renal function tests: mean (standard deviation not reported)
Cross-sectional study, 2,022 workers,
RDX exposed males*
1,491 participated (74% response rate).
Referent
Undetected (0.01 mg/m3
Analysis group: limited to whites;
Test
(n = 237)
(n = 22) (n = 45)
69 workers exposed to RDX alone and
BUN
15.5
15.6 16.4
24 workers exposed to RDX and HMX,
Total protein
7.2
7.2 7.3
compared to 338 workers not exposed to
RDX exposed females*
RDX, HMX, or TNT.
Exposure measures: Exposure
Referent
(n = 101)
Undetected (0.01 mg/m3
(n = 1) (n = 25)
determination based on job title and
industrial hygiene evaluation; exposed
BUN
Total protein
13.2
7.3
8 12.6
7.6 7.2
subjects assigned to two groups:
undetected (0.01 mg/m3
(mean for employees with exposures
includes both workers exposed to RDX alone and RDX and HMX.
No differences were statistically significant in men or women.
>LOD: 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).
LOD = limit of detection
Table 1-5. Evidence pertaining to urinary system (kidney and bladder) effects
in animals
Reference and study design
Results
Histopathological lesions
Lish 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
2 yrs
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 controls as males treated with
RDX. There was no increase in incidence of this lesion in females at
any time point.
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Reference and study design
Results
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)
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
Data for male rats sacrificed on schedule (SS) and those that died
spontaneously or were sacrificed moribund (SDMS) (summarized
below) were analyzed separately. There were no treatment-related
changes in incidence of kidney or urinary bladder lesions in females.
contaminant; 83-89% of particles <66 urn
0,0.3,1.5, 8.0, or 40 mg/kg-d
Diet
Doses
0
0.3
1.5
8.0
40
Kidney, medullary papillary necrosis; 24 mo (incidence)
2 yrs
Note: More detailed histopathological
results, including interim sacrifice data at
6 and 12 mo, are provided in Tables 1-6 to
1-8.
(SS)
(SDMS)
(Sum)
0/38
0/17
0/55
0/36
1/19
1/55
0/25
0/27
0/52
0/29
0/26
0/55
0/4
18/27*
18/31*
Kidney, suppurative pyelitis; 24 mo (incidence)
(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*
This document is a draft for review purposes only and does not constitute Agency policy.
1-28 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
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, 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
Tubular nephrosis (incidence)
M
F
0/10 - - 4/9*
0/11 - - 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.
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
F0 exposure: 13 wks pre-mating, and
during mating, gestation, and lactation of
Fl; Fl exposure: 13 wks after weaning,
and during mating, gestation, and
lactation of F2; F2 exposure: until weaning
Data were reported only for F2 generation controls and 5 and
16 mg/kg-d groups.
Doses
0 5 16 50
Renal tubule cysts, cortex (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
13 wks
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.
This document is a draft for review purposes only and does not constitute Agency policy.
1-29 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Levine et al. (1990); Levine et al. (1981a);
Levine et al. (1981b)b
Rats, F344,10/sex/group; 30/sexfor
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
Nephropathy, chronic, unilateral (incidence)
M
F
7/30 0/10 2/10 1/10
4/30 0/10 0/10 1/10
Nephropathy, chronic, bilateral (incidence)
M
F
22/30 8/10 7/10 1/10
13/30 2/10 5/10 1/10
Microcretions, focal, unilateral (incidence)
M
F
0/30 0/10 0/10 0/10
4/30 5/10 0/10 1/10
Microcretions, focal, bilateral (incidence)
M
F
0/30 0/10 0/10 0/10
21/30 4/10 8/10 6/10
Note: Incidence data not presented for 300 and 600 mg/kg-day dose
groups since all rats died by week 3 at these doses.
Hart (1974)
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow containing
20 mg RDX/g-chow, 60 g dog food; purity
and particle size not specified
0, 0.1,1, or 10 mg/kg-d
Diet
13 wks
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 Rhesus0,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
13 wks
Doses
0 0.1 1 10
Medulla; mineralization, minimal to mild (incidence)
M + F
0/6 1/6 0/6 4/6
Dilated tubules, mild to moderate (incidence)
M + F
4/6 3/6 6/6 3/6
Multinucleated cells, tubules, minimal to moderate (incidence)
M + F
5/6 0/6 3/6 6/6
Eosinophilic inclusions, minimal to moderate (incidence)
M + F
2/6 0/6 0/6 3/6
This document is a draft for review purposes only and does not constitute Agency policy.
1-30 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Kidney weightd
Lish 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
2 yrs
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)
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
2 yrs
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%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-31 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
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
Experiment 1: 0,10,14, 20, 28, or
40 mg/kg-d
Diet
13 wks
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 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%
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, 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% - -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, or
50 mg/kg-d
Diet
F0 exposure: 13 wks pre-mating, and
during mating, gestation, and lactation of
Fl; Fl exposure: 13 wks after weaning,
and during mating, gestation, and
lactation of F2; F2 exposure: until weaning
Doses
0 5 16 50
Absolute kidney weight (percent change compared to control)
M
F
0% 6% -12%
0% -4% -21%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-32 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0,4, 8,10,12, or 15 mg/kg-d
Gavage
13 wks
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. (1990); Levine et al. (1981a);
Levine et al. (1981b)b
Rats, F344,10/sex/group; 30/sexfor
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
Absolute kidney weight (percent change compared to control)
M
F
0% 1% 1% -9%
0% 1% 3% -1%
Relative kidney weight (percent change compared to control)
M
F
0% 5% 7% 10%
0% 3% 5% 2%
Hart (1974)e
Dogs, Beagle, 3/sex/group
Pre-mix with ground dog chow containing
20 mg RDX/g-chow, 60 g dog food; purity
and particle size not specified
0, 0.1,1, or 10 mg/kg-d
Diet
13 wks
Doses
o
1
1
1
o
o
Absolute kidney weight (percent change compared to control)
M
F
0% - - 38%
0% - - -18%
This document is a draft for review purposes only and does not constitute Agency policy.
1-33 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Reference and study design
Results
Martin and Hart(1974)e
Monkeys, Cynomolgus or Rhesus6,
3/sex/group
Purity of test material not specified
0,0.1,1, or 10 mg/kg-d
Gavage
13 wks
Doses
o
1
1
1
o
o
Absolute kidney weight (percent change compared to control)
M + F
0% -2% -3% 4%
^Statistically significant (p < 0.05) based on analysis by study authors.
aDoses were calculated by the study authors.
bLevine 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.
cThe species of monkey used in this study was inconsistently reported in the study as either Cynomolgus (in the
methods section) or Rhesus (in the summary).
dAn analysis by Craig et al. (2014) found a statistically significant correlation between absolute, but not relative,
kidney weights and renal histopathology. Therefore, only absolute kidney weight data from RDX studies are
presented in Figure 1-2.
eKidney weight data from the Hart (1974) and Martin and Hart (1974) studies were considered less informative
than other studies. Hart (1974) reported organ weight data for high-dose dogs (3/sex/group) only, and the kidney
weights from Martin and Hart (1974) were highly variable across monkeys (e.g., kidney weights for the control
animals ranged from 4.9 to 13.1 g). Therefore, kidney weight data from these two studies were not presented in
the exposure-response array for urinary system effects (Figure 1-2).
Note: A dash ("-") indicates that the study authors did not measure or report a value for that dose group.
SDMS =spontaneous death or moribund sacrifice; SS = scheduled sacrifice.
Table 1-6. Six-, 12-, and 24-month incidence of kidney endpoints in male F344
rats reported for statistical evaluation in Levine etal. (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*
This document is a draft for review purposes only and does not constitute Agency policy.
1-34 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Doses (mg/kg-d)
0
0.3
1.5
8.0
40
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
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
This document is a draft for review purposes only and does not constitute Agency policy.
1-35 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Doses (mg/kg-d)
0
0.3
1.5
8.0
40
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
^Statistically significant (p < 0.05) based on analysis by study authors.
Note: A dash indicates that the study authors did not measure or report a value for that dose group.
SDMS = spontaneous death or moribund sacrifice; SS = scheduled sacrifice.
Source: Levine et al. (1983).
This document is a draft for review purposes only and does not constitute Agency policy.
1-36 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Table 1-7. Six-, 12-, and 24-month incidence of urinary bladder endpoints in
male F344 rats reported for statistical evaluation in Levine etal. (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
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*
^Statistically significant (p < 0.05) based on analysis by study authors.
Note: A dash indicates that the study authors did not measure or report a value for that dose group.
SDMS = spontaneous death or moribund sacrifice; SS = scheduled sacrifice.
Source: Levine et al. (1983).
This document is a draft for review purposes only and does not constitute Agency policy.
1-37 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
1000
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Figure 1-2. Exposure-response array of urinary system (kidney and bladder) effects.
This document is a draft for review purposes only and does not constitute Agency policy.
1-38 DRAFT—DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Mechanistic Evidence
No MOA information is available for RDX-induced urinary system effects. 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 urinary
system.
GABA and GABA receptors have been identified in a number of peripheral tissues (Erdo et
al.. 1991: Ong and Kerr. 1990: Erdo. 19851. Brar etal. (20141 demonstrated that pretreatment with
picrotoxin reduced the renoprotective effects of sodium valproate (which acts on both GABAa and
GABAb receptors) in a rat model of ischemia-induced acute kidney injury, suggesting that GABAa
receptors may be important in renal function. GABA is believed to play a role in the regulation of
urination and bladder capacity (reviewed in Fowler et al. (2008) and Yoshimura and de Groat
(1997)1. In rats, injection of a GABAa receptor agonist inhibits the urination reflex (Igawa etal..
1993: Kontani etal.. 1987). 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 f Stone etal.. 20111. RDX would be expected to act
like an antagonist and increase bladder activity, 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, suggests a possible MOA for the kidney and urinary bladder lesions observed in
particular by Levine etal. (1983): however, there does not appear to be any direct evidence (basic
science or RDX-specific) to help discern the role of GABAa receptor in mediating these lesion types.
In summary, there are no studies available that inform mechanistically how RDX might lead
to urinary system effects. There is evidence that RDX binds to GABAa receptors in neuronal tissues
(Williams etal.. 2011: Williams and Bannon. 2009). and it is biologically plausible that binding to
the GABA receptor could occur in other tissues as well, contributing to the observed kidney and
urinary bladder effects. However, the way(s) by which GABAa receptors may work in non-neuronal
tissues and organs is not well understood, and the MOA by which RDX induces urinary system
effects is not established.
Integration of Urinary System (Kidney and Bladder) Effects
Evidence for kidney effects resulting from RDX exposure consists of human case reports and
findings of 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
fHathawav and Buck. 19771.
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The 2-year Levine etal. (1983) study in F344 rats reported histopathological changes
(papillary necrosis, pyelitis, luminal distension, and cystitis) in the kidney and urinary bladder in
approximately 50% of male rats exposed to 40 mg/kg-day (the highest dose tested in this study),
but only following exposure to RDX for longer than 6 months. Histopathological findings from
other studies in rats, mice, and dogs fCrouse etal.. 2006: Levine etal.. 1990: Levine etal.. 1981a. b;
Cholakis etal.. 1980: Hart. 1976.1974) are largely consistent with the 2-year findings from Levine
etal. (1983). i.e., that kidney and urinary bladder effects are generally observed after RDX
exposures longer than 6 months in duration and at high doses (e.g., >40 mg/kg-day). Other
measures of kidney effects (kidney weights and serum chemistry parameters) did not provide
consistent evidence of dose-related changes associated with RDX exposure.
Histopathologic findings from 2-year studies in F344 rats fLevine etal.. 19831 and B6C3Fi
mice fLish etal.. 19841 provide evidence of sex and species differences in response to RDX. In
contrast to the substantial urinary system toxicity observed in high-dose F344 male rats that was
considered the primary cause of RDX-related morbidity and mortality (Levine etal.. 1983). no
kidney toxicity was associated with RDX in similarly-exposed female rats. Additionally, mice
appear to be less sensitive than rats, based on an absence of RDX-related kidney histopathological
changes in male and female B6C3Fi mice exposed to RDX in the diet for 2 years at doses more than
twofold greater than doses that produced substantial urinary system toxicity in male rats fLish et
al.. 19841.
In light of the dose-related increase in histopathological changes in the kidney and urinary
bladder in male rats in the Levine etal. (1983) study, and in particular the robust response in the
high-dose animals, urinary system effects are a potential human hazard of RDX exposure.
1.2.3. Prostate Effects
No human studies were identified that evaluate the potential of RDX to cause effects on the
prostate. There was limited information to evaluate prostate effects in animal studies, including
two-year dietary studies in rats and mice (Lish etal.. 1984: Levine etal.. 1983). and one 90-day
gavage study fCrouse etal.. 2006). A summary of the prostate effects associated with RDX exposure
in animals is presented in Tables 1-8 and 1-9 and Figure 1-3. Studies are ordered in the evidence
tables and exposure-response arrays by duration of exposure and then by species.
The majority of animal studies available did not specifically evaluate whether there were
prostate effects associated with RDX exposure. 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 2 years (Levine etal.. 1983). Neither suppurative prostatitis nor any other
treatment-related prostate effects were observed in a 2-year dietary study in mice (Lish etal..
1984). Suppurative prostatitis was not observed in 90-day studies in the rat involving oral (dietary
or gavage) exposure to RDX fCrouse etal.. 2006: Levine etal.. 1990: Levine etal.. 1981a. b). In the
90-day gavage study fCrouse etal.. 20061. mild subacute inflammation was observed in the prostate
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of one of the rats at terminal sacrifice in the high dose group (15 mg/kg-day)16. The study authors
considered the single observation to be consistent with expected background incidence and not
treatment related. Excluding inflammation, there was little additional information to identify
prostate effects associated with RDX exposure. Histopathological analysis identified a statistically
significant increase in the incidence of spermatic granuloma of the prostate in rats fed 40 mg/kg-
day RDX for up to 6 months. No gross abnormalities of the prostate were observed to accompany
this finding, nor was this endpoint observed in 12- or 24-month dietary exposures to RDX (Levine
etal.. 19831.
Suppurative prostatitis is part of a continuum of inflammation. Further, suppurative
prostatitis and non-suppurative prostatitis are not mutually exclusive; one form can evolve into
another. Levine etal. f 19831 also reported the incidence of non-suppurative (chronic-active)
inflammation as well as subacute inflammation in male rats (see Table 1-8).
16A reporting discrepancy exists in Crouse et al. (20061 between the results section and the summary of
histopathological findings in males in the appendix. The results section reports that mild subacute
inflammation of the prostate was present in 1/7 males in the 15 mg/kg-day dose group at terminal sacrifice.
The summary of histopathological findings (Appendix U) reports an incidence of 1/8 at 15 mg/kg-day.
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Table 1-8. Two-year prostate inflammation incidence in male F344 rats
Levine etal. (1983)
Doses (mg/kg-d)
0
0.3
1.5
8.0
40
Inflammation, subacute
TS
1/38
0/36
0/25
0/29
0/4
SDMS
0/16
0/19
0/27
0/26
0/27
Sum
1/54
0/55
0/52
0/55
0/31
Inflammation, chronic-active
TS
15/38
13/36
6/25
6/29
1/4
SDMS
5/16
5/19
7/27
5/26
1/27
Sum
20/54
18/55
13/52
11/55
2/31
Inflammation, suppurative
TS
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*
All inflammation
Sum
23/54
22/55
21/52
23/55
21/31
^Statistically significant (p < 0.05) based on analysis by study authors.
SDMS = spontaneous death or moribund sacrifice; TS = terminal sacrifice.
Source: Levine et al. (1983)
As noted by the SAB in their review of the external review draft of the RDX assessment
fSAB. 20171. the incidences of all observations of inflammation at 24 months in the Levine et al.
f!9831 study were similar in all dose group (approximately 40%) except for the high-dose group
(68%). The incidence rate in the control and three lowest dose groups is lower than the
background incidence of inflammation (70.4%) in a retrospective analysis of background lesions in
male accessory sex organs of F344 rats reported by Suwaetal. (2001). The lower incidence in
F344 rats reported in Levine etal. f 19831 suggests there may have been differences in
histopathological practices between those employed by Levine etal. f 19831 and more recent
diagnostic criteria. For example, inflammation incidence varies across lobes of the prostate and the
methods reporting in Levine etal. (1983) does not provide sufficient information to determine how
the prostates were evaluated for inflammation. Finally, male rats in the high-dose group (40
mg/kg-day) were moved from group to individual housing between weeks 30 to 40 during the
study, due to a high incidence of fighting. The increased incidence of fighting may have contributed
to conditions that lead to urogenital infections in male rats f Creasy etal.. 20121.
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The severity of inflammation differs in Levine etal. (1983) compared to that reported in
Suwa etal. f20011. In reviewing the background incidence of all inflammation in the prostate in
1,768 F344 rats, Suwa etal. f20011 identified an average severity grade of "mild." In Levine et al.
f!9831. there is an increased incidence of suppurative prostatitis, which is more severe
(characterized by the formation of pus and a high concentration of neutrophils). There was also a
shift from chronic inflammation to suppurative inflammation with increasing dose of RDX starting
at 1.5 mg/kg-day (see Table 1-8). At the highest dose, the shift from chronic to suppurative
inflammation is clear, with only two animals exhibiting chronic inflammation and 19 identified as
having suppurative inflammation.
Some reports have hypothesized that the observation of prostate inflammation in Levine et
al. f!9831 is secondary to a bacterial infection unrelated to RDX toxicity fATSDR. 2012: Sweeney et
al.. 2012a: Crouse etal.. 20061. For example, in describing the results from the 2-year dietary study
in rats, Crouse etal. (2006) observed 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. 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 were combined.
Additionally, the dose-related nature of the increased incidence suggests that the primary cause
(potentially leading to bacterial infection) was treatment-related, as 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. f 19831 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 et al.. 20061. In general, causes of
prostatitis other than infection exist, including stress, endocrine effects (i.e., changing prolactin
levels), and autoimmune dysfunction (see, for example. Bosland. 1992: Gatebeck etal.. 1987: Parker
and Grabau. 1987).
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Table 1-9. Evidence pertaining to prostate effects in animals
Reference and study design
Results
Levine et al. (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 urn
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
2 yrs
Data for male rats sacrificed on schedule (SS) and those that died
spontaneously or were sacrificed moribund (SDMS) (summarized
below) were analyzed separately.
0
0.3
1.5
8.0
40
Prostate, suppurative inflammation (prostatitis); 24 mo (incidence)
SS
SDMS
(Sum)
0/38
1/36
2/25*
4/29*
0/4
2/16
3/19
7/27*
8/26
19/27
2/54
4/55
9/52*
12/55*
19/31
Spermatic granuloma of the prostate; 6 mo (incidence)
SS
SDMS
(Sum)
0/10
0/10
2/10
2/10
2/10
2/10
1/10
1/10
6/10*
2/5
8/15*
Lish et al. (1984)
Mice, B6C3F1, 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
2 yrs
0
1.5
7.0
35 175/100
Prostate, chronic inflammation; 24 mo (incidence)0
M
1/62
1/3
0/1
1/1
0/27
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
13 wks
Doses
0
8
10
12
Prostate, mild subacute inflammation (incidence)
M
0/10
15
1/8
*Statistically significant (p < 0.05) based on analysis by study authors.
Examination only required by protocol in the control and high-dose groups.
Note: A dash ("-") indicates that the study authors did not measure or report a value for that dose group.
SDMS = spontaneous death or moribund sacrifice; SS = scheduled sacrifice.
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Mechanistic Evidence
No MOA information is available for RDX-induced prostate effects. 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, and provides
some potential MOA hypotheses for the effects reported in Levine etal. (1983) that do not require
bacterial infection.
One 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 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
(lohnson. 2015b). No evidence was found to support this potential pathway leading to prostate
inflammation.
Another possibility is that 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 et al.. 1999b: Stoker etal.. 1999a: Tangbanluekal and
Robinette. 1993: Robinette. 19881. Typically, the inflammation seen is chronic and does not reverse
over time (Robinette. 19881. Functional GABAa receptors have been identified in the anterior
pituitary (Zemkova et al.. 2008: Maverhofer etal.. 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 et al.
f!9831 could have been produced by disruption of pituitary prolactin or another hormonal signal
via interference with normal regulatory GABA-related hormonal control. However, no direct
evidence for this hypothesized MOA is available. Levine etal. (1983) did not evaluate serum
endocrine measures or pituitary weights, and pituitary adenomas that could account for higher
prolactin levels were not observed.
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 et al.. 2007: Tian etal.. 20041. and GABAa receptor agonists have decreased
cytotoxic immune responses and hypersensitivity reactions (Tian etal.. 1999: Bergeretetal.. 1998).
In a mouse autoimmune model of multiple sclerosis, Bhatetal. (2010) 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, as it binds to the
same site) was able to reduce this effect. However, picrotoxin on its own did not significantly alter
cytokine production, suggesting that the effects are limited to reversal of agonist-induced
GABAergic activity flohnson. 2015bl. If an autoimmune mechanism was contributing to the effects
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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
fCrouse et al.. 20061.
In summary, there are no studies available that inform mechanistically how RDX exposure
might lead to prostate effects. There is evidence that RDX binds to GABAa receptors in neuronal
tissues (Williams etal.. 2011: Williams and Bannon. 2009). and it is biologically plausible that
binding to the GABA receptor could occur in other tissues as well. Among the mechanistic
information presented above, MOAs that require direct action on the prostate appear less likely;
however, the ways that GABAa receptors work in non-neuronal tissues and organs is still not well
understood, and the MOA by which RDX may induce prostate effects is unknown.
Integration of Prostate Effects
Suppurative prostatitis was reported in male F344 rats chronically exposed to RDX in the
diet for 24 months (Levine etal.. 19831. No other studies of equivalent duration were performed in
rats to determine the consistency of this effect Spermatic granuloma of the prostate was identified
in F344 rats exposed to RDX for up to 6 months, but not at 12 or 24 months in the study; therefore,
the biological significance of the 6-month finding is uncertain. A 24-month study in mice (Lish et
al.. 19841 did not report prostate effects associated with RDX exposure. No other animal studies of
shorter duration identified prostate effects associated with RDX exposure. In light of the dose-
related, statistically significant increase in suppurative prostatitis, there is suggestive evidence that
prostate effects are a potential human hazard of RDX exposure.
1.2.4. Developmental Effects
No human studies were identified that evaluate the potential of RDX to cause
developmental effects. Information relevant to an examination of the association between RDX
exposure and developmental effects comes from a 2-generation reproductive toxicity study in rats
and developmental studies in rats and rabbits involving oral administration of RDX during
gestation. A summary of the developmental effects associated with RDX exposure is presented in
Table 1-10 and Figure 1-4. Studies are ordered in the evidence tables and exposure-response
arrays by duration of exposure and then by species.
Animal studies have reported decreases in offspring survival following administration of
RDX. Pup survival rates in the F0 and F1 generations (including both stillborn pups and postnatal
deaths through the age of weaning) were statistically significantly decreased in RDX-exposed CD
rats compared to controls in the only available two-generation reproductive toxicity study of RDX
(Cholakis etal.. 1980). This observation was noted only at the highest dose tested (50 mg/kg-day)
that also produced toxicity in adults (mortality [18%], reduced body weights [8-14%], and reduced
food consumption [10-17%]). Decreased fetal viability was observed at the highest dose tested, 20
mg/kg-day, in a developmental toxicity study in F344 rats fCholakis etal.. 19801. although no effect
on live fetuses was observed in a developmental toxicity study in Sprague-Dawley rats at the same
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dose (Angerhofer et al.. 19861: both of these studies reported significant mortality (29-31%) in
dams at 20 mg/kg-day. Increased resorptions were similarly limited to the highest dose tested (20
mg/kg-day) fCholakis etal.. 19801. Both studies started treatment with RDX on gestational day
(GD) 6, which may contribute to the incidence of resorptions observed in the control and treated
groups. As noted in EPA's Guidelines for Developmental Toxicity Risk Assessment fU.S. EPA. 19911.
treatment beginning around the time of implantation may result in an increase in implantation loss
that reflects variability that is not treatment related. There was no evidence of maternal toxicity,
embryotoxicity, or decreased fetal viability in a teratology study of pregnant New Zealand White
(NZW) rabbits administered RDX by gavage from GD 7 to 29 at doses up to 20 mg/kg-day fCholakis
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 administered RDX by gavage from GD 6 to 15 f Angerhofer etal..
1986).17 Decreased fetal body weight (9%) and body length (5%), with statistically significant
trends, were observed at 20 mg/kg-day, a dose that produced significant (31%) 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 29% maternal mortality
fCholakis etal.. 19801. Dose-related reductions in fetal body weight were not observed in NZW
rabbits at doses up to 20 mg/kg-day fCholakis etal.. 19801.
No treatment-related effects on morphological development have been reported in rats
exposed to a dose as high as 20 mg/kg-day RDX, a dose that resulted in 29-31% maternal mortality
(Angerhofer etal.. 1986: Cholakis etal.. 1980). Examination of rabbits administered RDX at doses
up to 20 mg/kg-day from GD 7 to 29 also provided no evidence of treatment-related developmental
anomalies fCholakis etal.. 19801. Although increased incidences of enlarged frontal fontanel and
unossified sternebrae were observed in fetuses of all groups of NZW rabbits administered RDX
fCholakis etal.. 19801. these developmental anomalies did not exhibit a dose-related increase in the
number of either fetuses or litters affected, and were thus interpreted as not being treatment-
related by the study authors (Cholakis etal.. 1980). Neither individual litter data nor historical
control data from the performing laboratory were available to assist in the interpretation of these
findings. The author's interpretation is supported by the following additional considerations. A
report of historical control incidences of fetal skeletal observations in NZW rabbits for 224 prenatal
developmental toxicology studies conducted in 8 contract research laboratories during the period
of 1988-1992 fMTA. 19921 included findings from 26,166 fetuses of 3,635 litters. Background
control incidences of enlarged anterior fontanel were observed in 8 fetuses (0.031%) of 7 litters
(0.193%), while sternebrae agenesis (which may not be entirely comparable to the finding of
17The 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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unossified sternebrae in Cholakis etal. (1980) was found in 10 fetuses (0.038%) of 5 litters
(0.138%). Although the use of concurrent control data is preferable for the interpretation of
developmental toxicity data, this historical information supports the low control incidences of these
findings in the Cholakis etal. f 19801 study as being within typical historical parameters. It is also
noted that the non-dose-related pattern of increased enlarged fontanel and unossified sternebrae
across treated groups in Cholakis etal. (1980) was similar to the pattern of decreases in fetal body
weight in the same study, suggesting a possible link between these particular sternebral and
fontanel anomalies with fetal growth status. Given the lack of dose-related increases in the
incidences of these anomalies, and patterns that mirrored fetal body weight decreases (which were
also not dose-related), the findings of enlarged frontal fontanel and unossified sternebrae were not
considered treatment-related. Gestational administration of RDX to NZW rabbits did not result in
any other dose- and treatment-related skeletal abnormalities.
Table 1-10. Evidence pertaining to developmental effects in animals
Reference and study design
Results
Prenatal mortality/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
F0 exposure: 13 wks pre-mating, and
during mating, gestation, and lactation of
Fl; Fl exposure: 13 wks after weaning,
and during mating, gestation, and
lactation of F2; F2 exposure: until weaning
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%*
Survival at weaning (percent ofliveborn pups)
Fl
F2
87% 96% 90% 8%
79% 86% 79% 0%
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
surviving six died during subsequent treatment.
Note: results on a per litter basis were not provided.
Cholakis et al. (1980)
Rabbits, NZW, 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 20
Early resorptions (mean percent per dam)
6% 5% 4% 1%
Late resorptions (mean percent per dam)
8% 5% 3% 3%
Viable fetuses (mean percent per dam)
85% 82% 11% 94%
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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
Doses
0 0.2 2.0 20
Early resorptions (mean percent per dam)
6.0% 2.5% 4.8% 15.3%
Late resorptions (mean percent per dam)
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)
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
Resorptions (percent of total implantations)
4.8% 6.1% 5.9% 6.4%
Early resorptions (percent of total implantations)
4.8% 6.1% 5.9% 6.2%
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.
Percent resorptions and live fetuses based on number of surviving
females at time of necropsy.
Offspring growth
Cholakis et al. (1980)
Rabbits, NZW, 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
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.
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
Reference and study design
Results
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)
(O
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, NZW, 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
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
Unossified sternebrae (incidence)
Fetuses
Litters
4/49 12/53 8/50 12/58
4/11 7/11 4/11 6/12
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
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%
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
Reference and study design
Results
0, 2, 6, or 20 mg/kg-d
Gavage
GDs 6-15
Significant maternal mortality (16/51) occurred at 20 mg/kg-d.
^Statistically significant (p < 0.05) based on analysis by study authors.
statistically significant dose-related trend (p < 0.05) by linear trend test, performed for this assessment. Average
fetal weights or lengths for each litter comprised the sample data for this test.
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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
• slgnficantly changed
O not signifcantly changed
o
oo
en
o
_c
O
4/ Offspring survival
o
oo
en
o
_c
O
Offpsring growth
Note: Filled circle indicates that response was statistically significantly different from the control.
(1) Statistically signficant dose-related trend (p <= 0.05) by linear trend test, performed for this assessment.
o
oo
cn
o
_c
O
Morphological
development
Figure 1-4. Exposure response array of developmental effects following oral exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Integration of Developmental Effects
Developmental studies in rats fAngerhofer et al.. 1986: Cholakis etal.. 19801 demonstrated
effects on offspring survival, growth, and morphological development only at doses associated with
severe maternal toxicity and mortality. No dose-related developmental effects were observed in
rabbits (Cholakis etal.. 1980). As noted in EPA's Guidelines for Developmental Toxicity Risk
Assessment (U.S. EPA. 1991). 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. At this
time, the available data do not support developmental effects as a human hazard of RDX exposure.
1.2.5. Liver Effects
One occupational epidemiology study examined the association between RDX exposure and
changes in serum liver enzymes. 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 1-11 and 1-12 and Figure 1-5.
Experimental animal studies are ordered in the evidence table and exposure-response array by
duration of exposure and then by species.
Reports in humans provide inconsistent evidence of liver toxicity associated with acute
exposure to RDX. Elevated serum levels of aspartate aminotransferase (AST) and/or alanine
aminotransferase (ALT) were reported in several case reports of individuals who ingested
unknown amounts of RDX fKiicukardali etal.. 2003: Woody etal.. 1986: Knepshield and Stone.
1972: Hollander and Colbach. 1969: Stone etal.. 1969: Merrill. 1968) (see Appendix C, Section C.2).
Liver biopsies did not reveal any abnormal observations (Stone etal.. 1969). In other case reports,
no significant changes in serum levels of liver enzymes were observed (Testud etal.. 1996a: 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; RDX exposure range:
undetectable (<0.01 mg/m3) to 1.6 mg/m3) f Hath away and Buck. 19771. serum chemistry analysis
(including the serum liver enzymes AST, ALT, and alkaline phosphatase [ALP]) revealed no
statistically significant differences between exposed and unexposed workers (see Table 1-11).
In experimental animals, some, but not all, subchronic studies reported increased liver
weight associated with RDX exposure (see Table 1-12 and Figure 1-5). Dose-related increases in
This document is a draft for review purposes only and does not constitute Agency policy.
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relative liver weight18 (11-25% in high-dose groups) were observed in male and female B6C3F1
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 fLevine etal.. 1990: Levine etal.. 1981a. b; Cholakis etal..
19801: however, relative liver weights were not increased in female F344 rats in another 90-day
gavage study fCrouse etal.. 20061. Male F344 rats exhibited an increase in relative liver weight
only in one of these subchronic studies (Levine etal.. 1990: Levine etal.. 1981a. b). In subchronic
studies in other species, absolute liver weights were increased in male and female monkeys
(6-16% relative to control at 1 and 10 mg/kg-day) fMartin and Hart. 19741 and in male, but not
female, beagle dogs (53% relative to control in male dogs at 10 mg/kg-day) (Hart. 19741.
Chronic RDX exposures in B6C3F1 mice and F344 or Sprague-Dawley rats showed a less
consistent pattern of liver weight increases. Interpretation of liver weight increases in the 2-year
mouse study 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 (Levine etal.. 1983). relative liver weights were increased in high-dose
(40 mg/kg-day) males and females (by 11 and 18% compared to controls, respectively), likely
reflecting the depressed weight gain in the high-dose rats (2-30% in males and 10-15% in females).
In evaluating organ weight data across studies of all durations, less emphasis is placed on evidence
of organ weight changes from chronic (2-year) studies because normal physiological changes
associated with aging and intercurrent disease contributes to inter-animal variability that could
confound organ weight interpretation (Sellers etal.. 2007). as is true of the mouse liver weight data
for RDX.
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. b; Hart. 1976.1974: Martin and Hart.
19741. including 2-year oral studies in mice at doses up to 100 mg/kg-day fLish etal.. 19841 and in
rats at doses up to 40 mg/kg-day fLevine etal.. 19831. 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 (Lish etal..
1984: Levine etal.. 1983). For example, the incidence of liver portal inflammation was increased in
female rats, but not male rats, exposed to 40 mg/kg-day in the diet for 90 days fCholakis etal..
1980). There was an increase in the incidence of mild liver microgranulomas in female mice only
fCholakis etal.. 19801 and karyomegaly of hepatocytes in male mice only exposed to
18Based 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 evaluation of the effects of a test
chemical on liver weight are optimally analyzed using organ-to-body weight ratios. Therefore, the analysis of
liver weight here focuses on relative weight data where study authors reported both relative and absolute
weights, although both relative and absolute data are summarized in the evidence table (see Table 1-12).
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320 mg/kg-day RDX in the diet for 90 days (Cholakis etal.. 19801. Because both the rat and mouse
studies by Cholakis et al. f 19801 used relatively small group sizes (n = 10/sex/group) and provided
histopathologic findings for the control and high-dose groups only, less emphasis is placed on these
findings than on those from the 2-year bioassays. 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. However, 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..
1984): 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% at the
high doses) in a subchronic oral (dietary) study (Levine etal.. 1990: Levine etal.. 1981a. b). 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% at the 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 (Crouse etal.. 2006). and in monkeys exposed to RDX in the diet for 90 days (Martin
and Hart. 19741 were relatively small (within 38% of control mean) and were not dose related.
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Toxicological Review of Hexahydro-l,3,5-trinitro-l,3,5-triazine
Table 1-11. Evidence pertaining to liver effects in humans
Reference and study design
Results
Hathaway and Buck (1977) (United States)
Mean laboratory values of liver enzymes in men (mean; standard
Cross-sectional study, 2,022 workers,
deviation not reported)
1,491 participated (74% response rate).
Analysis group: limited to whites;
RDX exposed*
69 exposed to RDX alone and 24 exposed
Referent
Undetected (0.01 mg/m3
to RDX and HMX; 338 not exposed to RDX,
Test
(n = 237)
(n = 22)
(n = 45)
HMX, or TNT.
Exposure measures: Exposure
LDH
173
191
174
determination based on job title and
ALP
82
78
80
industrial hygiene evaluation. Exposed
subjects assigned to two groups: 0.01 mg/m3 (mean for employees with
AST (SGPT)
21
26
18
exposures >LOD: 0.28 mg/m3).
Effect measures: Liver function tests.
Bilirubin
0.5
0.4
0.4
Analysis: Types of statistical tests were not
includes both workers exposed to RDX alone and RDX and HMX.
reported (assumed to be t-tests for
No differences were statistically significant as reported by study
comparison of means and x2 tests for
authors. Similar results in women.
comparison of proportions).
Liver function tests in men (prevalence of abnormally elevated
values)
Test
RDX exposed*
(abnormal
range)
Referent
Undetected (0.01 mg/m3
LDH (>250)
2/237
1/22
0/45
ALP (>1.5)
34/237
1/22
6/45
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)
includes both workers exposed to RDX alone and RDX and HMX.
No differences were statistically significant as reported by study
authors. Similar results in women.
LDH = lactate dehydrogenase; SGOT = glutamic oxaloacetic transaminase; SGPT = glutamic pyruvic transaminase
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
Table 1-12. Evidence pertaining to liver effects in animals
Reference and study design
Results
Liver weight
Lish 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
2 yrs
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, suggesting no real effect on liver weight.
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)
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
2 yrs
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%
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
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 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
F0 exposure: 13 wks pre-mating, and
during mating, gestation, and lactation
of Fl; Fl exposure: 13 wks after
weaning, and during mating, gestation,
and lactation of F2; F2 exposure: until
weaning
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
13 wks
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%
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
Reference and study design
Results
Levine et al. (1990); Levine et al.
(1981a); Levine etal. (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 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
Absolute liver weight (percent change compared to control)
M
F
0% 5% -1% -2%
0% 2% 4% 16%*
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 g
dog food; purity and particle size not
specified
0, 0.1,1, or 10 mg/kg-d
Diet
13 wks
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 Rhesusd,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
13 wks
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)
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
2 yrs
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.
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Reference and study design
Results
Levine et al. (1983)
Rats, F344, 3-4 wks old; 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 urn
0, 0.3,1.5, 8.0, or 40 mg/kg-d
Diet
2 yrs
Doses
o
o
00
LO
m
o
o
Microgranulomas (incidence)
M
F
0/38 0/36 0/25 0/29 0/4
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 nm
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 (incidence)
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 (incidence)
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
13 wks
Histopathology examination of the 15 mg/kg-d group showed one male
with mild liver congestion and one female with a moderate-sized focus of
basophilic cytoplasmic alteration; neither finding was attributed by study
authors to RDX treatment.
Levine et al. (1990); Levine et al.
(1981a); Levine etal. (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. No
histopathology findings available for the 300 or 600 mg/kg-d dose groups
because all rats in these groups died before the 13-wk necropsy.
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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; purity and particle size not
specified
0, 0.1,1, or 10 mg/kg-d
Diet
13 wks
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 Rhesusd,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
13 wks
An increase in the amount of iron-positive material in liver cord cytoplasm
was reported in monkeys treated with 10 mg/kg-d RDX, which the study
authors considered to be of uncertain toxicological significance. Because
iron-positive stain was present in controls and no further characterization
of the staining was provided in the study report, the toxicological
significance of this finding could not be determined.
Serum chemistry
Lish 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
2 yrs
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)
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
2 yrs
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%*
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Reference and study design
Results
Crouse et al. (2006)
Rats, F344,10/sex/group
99.99% pure
0, 4, 8,10,12, or 15 mg/kg-d
Gavage
13 wks
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%
Levine et al. (1990); Levine et al.
(1981a); Levine etal. (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
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 Rhesusd,
3/sex/group
Purity of test material not specified
0, 0.1,1, or 10 mg/kg-d
Gavage
13 wks
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%
^Statistically significant (p < 0.05) based on analysis by study authors.
aDoses were calculated by the study authors.
bLevine 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.
cLiver weight data from the Hart (1974) and Martin and Hart (1974) studies were considered less informative than
other studies. Hart (1974) reported organ weight data for high-dose dogs (3/sex/group) only, and the liver
weights from Martin and Hart (1974) were highly variable across monkeys (e.g., liver weights for the control
animals ranged from 46 to 141 g). Therefore, liver weight data from these two studies were not presented in the
exposure-response array for liver effects (Figure 1-5).
dThe species of monkey used in this study was inconsistently reported in the study as either Cynomolgus (in the
methods section) or Rhesus (in the summary).
Note: A dash ("-") indicates that the study authors did not measure or report a value for that dose group.
1
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1000
100 ,
lie
£
O 1
a 1
o.i
1 significantly changed o not significantly changed x not determined
4 o a
a o
CD —
fD
X
on
00
01
rfi
oo
IT.
Chronic
o
oo
CTl
O
JZ
o
3
c
E
o
00
cn
o
.c
o
o
00
c
_c
u
o
en
ffr
0/
c
o
cn
en
Subchronic
1s Relative liver weight
Chronic
3
O
E
o
00
CTi
O
-C
u
3
D
E
o
00
cr.
^ ^ tj
g tf
® s
a/ oi
S
o p
X
"D
Subchronic
Histopathology
S!
3
O
E
Chronic
lO
8
(N
I
¦o
Subchronic
4, Cholesterol
- ^
o E
£
Chronic
o
L-.
Subchr
•I Trigylcerides
Serum biochemistry changes
Note: Filled circle indicates that response was statistically significantly different from the control.
X - Not considered due to confounding caused by presence of tumors.
Figure 1-5. Exposure response array of liver effects following oral exposure.
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Integration of Liver Effects
There is limited evidence from human studies and from studies in experimental animals
that RDX may affect the liver. The observation of transient elevations of serum liver enzymes in
several human case reports of individuals who ingested unknown amounts of RDX suggests that
RDX might target the liver; however, serum liver enzymes were not elevated in a small cross-
sectional study of munition plant workers exposed to RDX. In experimental animals, dose-related
increases in liver weight were observed in some studies following subchronic oral exposure, but
liver weight changes were not consistent across sexes within a study or across different studies.
Changes in serum chemistry 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. At this time, the
available data do not support liver effects as a human hazard of RDX exposure.
1.2.6. Other Noncancer Effects
There are some reports that RDX may induce effects on the eyes, on the cardiovascular,
musculoskeletal, immune, gastrointestinal (GI), hematological, and male reproductive systems, and
on body weight. However, there is less evidence for these effects compared to organ systems
described earlier in Section 1.2. Generally, human evidence for effects in these organ systems is
limited to human case reports. Evidence of effects in experimental animals is generally inconsistent
across studies of similar duration in the same species, or lacks consistent, dose-related patterns of
increasing or decreasing effect. A summary of the evidence for an association between these other
noncancer effects and RDX exposure is provided below; a more detailed discussion is provided in
Appendix C.3.2. As discussed below, the information to assess the association between RDX
exposure and toxicity for the organ systems presented below is considered inadequate.
Ocular Effects
There is no human evidence of ocular effects following exposure to RDX. In animals, the
incidence of cataracts was significantly increased in high-dose female rats (73%) relative to
controls (32%) in one chronic oral study fLevine etal.. 19831. This finding was not observed in
males in the same chronic study or in other chronic or subchronic studies in rats, mice, or monkeys
fCrouse etal.. 2006: Lish etal.. 1984: Cholakis etal.. 1980: Martin and Hart. 19741. There is
insufficient information to assess ocular toxicity following exposure to RDX.
Cardiovascular Effects
Human evidence of cardiovascular effects consists of case reports of transient arterial
hypertension, sinus tachycardia, and premature ventricular beats in male workers or men who
accidentally ingested RDX fKuctikardali etal.. 2003: Barsotti and Crotti. 19491. In animals, evidence
is limited to inconsistent findings of changes in heart weight and a report of increased incidence of
minimal histopathological changes in a 90-day rat study at a dose that also produced 40% mortality
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(Cholakis etal.. 19801. There is insufficient information to assess cardiovascular effects following
exposure to RDX.
Musculoskeletal Effects
Evidence for musculoskeletal effects in humans is limited to case reports that described
muscle twitches, soreness, and muscle injury as indicated by elevated levels of aspartate
aminotransferase (AST), creatine phosphokinase, and myoglobinuria (Testud etal.. 2006:
Kiiciikardali et al.. 2003: Hettand Fichtner. 2002: Hollander and Colbach. 1969: Stone etal.. 1969:
Merrill. 19681. In animal studies, evaluations of muscle and skeletal tissues did not reveal any
histopathological alterations in rats or mice following chronic exposure or in mice, rats, or dogs
following subchronic exposure. There is insufficient information to assess musculoskeletal effects
following exposure to RDX.
Immune System Effects
Increased white blood cell (WBC) counts were reported in several case reports of humans
acutely exposed to RDX (Knepshield and Stone. 1972: Hollander and Colbach. 1969: Stone etal..
1969: Merrill. 19681. In animals, there were no consistent patterns of change in WBC count or
spleen weight across the RDX database. No dose-related immune effects were observed in a 90-day
study in F344 rats that evaluated structural measures of immunotoxicity (including red blood cell
[RBC] and WBC 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.. 20061. None of the available studies included evaluation of more sensitive measures
of functional immune system changes. Therefore, there is insufficient information to assess
immunotoxicity following exposure to RDX.
Gastrointestinal Effects
Nausea, vomiting, and erosive gastroduodenitis were identified in human case reports of
RDX poisonings, generally concurrent with severe neurotoxicity (Kasuske etal.. 2009: Davie s etal..
2007: Kiiciikardali 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. 19491. There have been similar reports of vomiting in swine, dogs, and
monkeys fMusick etal.. 2010: Hart. 1974: Martin and Hart. 19741. Generally, histopathological
changes of the GI tract were not observed in RDX-exposed animals. There is insufficient
information to assess gastrointestinal toxicity following exposure to RDX.
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Hematological Effects
Temporary hematological alterations, including anemia, decreased hematocrit, hematuria,
and methemoglobinemia were observed in some human case reports following acute exposure
fKasuske etal.. 2009: Kiiciikardali et al.. 2003: Knepshield and Stone. 1972: Hollander and Colbach.
1969: Stone etal.. 1969: Merrill. 1968). Observations of anemia in case reports may reflect co-
exposure to 2,4,6-trinitrotoluene (TNT). Levine and colleagues identified that anemia resulted
from exposure to TNT in F344 rats, but not RDX fLevine etal.. 1990: Levine etal.. 1981a. b).
Hematological findings in a case-control and cross-sectional occupational study were inconsistent
fWestand Stafford. 1997: Hathaway and Buck. 19771: both studies used small sample sizes and
were considered low confidence studies. In general, subchronic and chronic animal studies showed
no consistent dose-related patterns of change in hematological parameters. There is insufficient
information to assess hematological toxicity following exposure to RDX.
Reproductive Effects
Investigation of the potential effects of RDX on reproductive function is limited to a two-
generation study in rats by Cholakis etal. f!9801 that also included a dominant lethal mutation
study. A reduction in number of pregnancies was reported only at a dose that also resulted in
decreased food consumption, decreased body weight gain, and increased mortality. The limited
investigation of reproductive function in RDX-exposed rats by a single investigator provides
insufficient information to assess female reproductive toxicity following exposure to RDX.
Evidence of male reproductive toxicity comes largely from the finding of increased
incidence of testicular degeneration in male B6C3Fi mice exposed to >35 mg/kg-day RDX for 2
years in the diet compared to concurrent controls fLish etal.. 19841. The biological significance of
this finding is unclear because no similar histopathological changes were observed in this study at 6
or 12 months, durations longer than the 1.4-month duration of spermatogenesis in mice, and
because of the loss of testicular function that occurs in aging rodents. The evidence for testicular
degeneration in mice suggested by Lish etal. (1984) was generally not supported by
histopathological findings in male reproductive organs in other studies, and changes in testes
weight across the RDX database were generally small, not dose-related, and directionally
inconsistent There is insufficient information to assess male reproductive toxicity following
exposure to RDX.
Body Weight Effects
Changes in body weight gain were reported in experimental animal studies involving
chronic and subchronic exposure to ingested RDX, but generally at high doses that were also
associated with elevated mortality or with severe kidney and urinary bladder toxicity in male rats
in the case of the Levine etal. (1983) study. For the most part, at lower doses, there were no
apparent patterns of treatment-related body weight changes across dose groups or sexes within a
study or across studies. Thus, available studies of RDX provide evidence that RDX exposure causes
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decreases in body weight gain in mice and rats, but these effects appear to be secondary to effects
on other primary targets of RDX toxicity.
1.2.7. Carcinogenicity
The relationship between exposure to RDX and cancer has not been investigated in human
populations. The carcinogenicity of RDX has been examined in one oral chronic/carcinogenicity
bioassay in mice (Lish etal.. 1984) and two bioassays in rats (Levine etal.. 1983: Hart. 1976). The
2-year studies by Lish etal. (1984) and Levine etal. (1983) included comprehensive
histopathological examination of major organs, multiple dose groups and a control, and
>50 animals/dose group (plus additional interim sacrifice groups). In both studies, the maximum
tolerated dose was reached or exceeded in high-dose animals (based on decreased terminal body
weight in high-dose male and female mice of 5 and 19%, respectively, and decreased survival in
male and female rats by approximately 50 and 25%, respectively, compared to the control).19 The
earlier Hart (1976) study is largely limited by the lack of characterization of the test material and
histopathologic examination in control and high-dose groups only. 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 >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 peer review of
histopathological evaluations by the study pathologist was performed only for female mouse liver
tissues from the Lish etal. (1984) study (see discussion of the Pathology Working Group (PWG)
below). A summary of the evidence for liver and lung tumors in experimental animals from these
three bioassays is provided in Tables 1-13 and 1-14.
Liver Tumors
An increased incidence of liver tumors was observed in one chronic mouse study fLish etal..
1984) and one of two chronic rat studies (Levine etal.. 1983). Incidences of hepatocellular tumors
are presented in Table 1-13 and discussed in further detail below.
The incidence of hepatocellular carcinomas and the combined incidence of hepatocellular
adenomas or carcinomas showed a statistically significant positive trend with RDX dose in female,
but not male, B6C3Fi mice as compared to concurrent controls in a 2-year dietary study fLish etal..
19841. In female B6C3Fi mice, Lish etal. f19841 observed that the liver tumor incidence in the
concurrent female control mice was relatively low (1/65), and significantly lower than the
19In high-dose mice in the Lish et al. (19841 study, reduced survival due to acute RDX toxicity occurred during
the first 11 weeks on study at a dietary dose of 175 mg/kg-day; survival in high-dose animals was similar to
controls after 11 weeks when the dose was reduced to 100 mg/kg-day. By contrast, in high-dose rats in the
Levine et al. (19831 study, elevated mortality, particularly in males, occurred gradually over the entire period
of the study beginning after 6 months, and was attributable in large part to kidney toxicity.
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incidence from historical controls (historical incidence data not provided by study authors). The
study authors also compared liver tumor incidence in RDX-exposed female mice to mean historical
control incidence for female mice of the same strain from National Toxicology Program (NTP)
studies conducted during the same time period (147/1,781 or 8%; range: 0-20%) fHaseman etal..
19851.20 The combined incidence of hepatocellular adenomas or carcinomas in female mice at RDX
doses >35 mg/kg-day (19% at both doses) was statistically significantly elevated when statistical
analysis was performed using NTP historical control data; limitations associated with comparisons
to historical control data originating from a different laboratory are acknowledged given cross-
study differences in diet, laboratory, pathological evaluation, and animal provider.
A PWG reviewed the slides of female mouse liver lesions from the Lish etal. f19841 study
fParker etal.. 2006: Parker. 20011. Some malignant tumors were downgraded to benign status, and
several lesions initially characterized as adenomas were changed to non-neoplastic lesions based
on more recent diagnostic criteria used by the PWG (Harada etal.. 1999). There remained a
statistically significant positive trend in the combined incidence of hepatocellular adenomas or
carcinomas, consistent with the original findings of Lish etal. (1984). 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. f20061 were considered the
more appropriate measure of liver tumor response in female mice from the Lish etal. T19841
bioassay. The PWG also offered observations about the histopathology methods used by Lish et al.
(1984) that raised some concerns about the uniformity of histologic processing (Parker etal.. 2006:
Parker. 2001). These included variation in size and shape of sections, suggesting that liver sections
were not uniformly taken from the same area of the liver of all animals; only one liver section
present from most animals (two sections are commonly examined in current carcinogenicity
bioassays); and more than one section prepared for 20 mice across different groups, raising some
concern of sample bias but likely reflecting sections taken from visible lesions at gross necropsy.
In male mice from the Lish etal. (1984) study, the incidences of hepatocellular carcinomas
in treated groups were higher than in the control, and the combined incidences of hepatocellular
adenomas or carcinomas of male mice were higher in three of four treated groups than in the
control; however, there were no statistically significant trends in either case. The incidences of
liver carcinoma in control (21%) and treated groups of male mice (22-33%) were generally within
20Comparison of control incidences of hepatocellular adenomas or carcinomas between Lish et al. (19841 and
Haseman et al. (19851 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. (19841 study, and it is not clear if the diet used in the Lish et al.
(19841 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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the range for the same mouse strain reported by NTP (8-32%) (Haseman et al.. 19851. Similarly,
the combined incidences of liver adenoma or carcinoma in control (32%) and treated groups
(27-48%) were within the range for the same mouse strain reported by NTP (14-58%) (Haseman
etal.. 19851.21 The PWG did not re-analyze liver tumor slides from male mice; the SAB fSAB. 20171
noted this as unusual since typically sections from both male and female animals are reevaluated to
ensure that findings in both sexes are reliable.
In the two-year bioassay in F344 rats (Levine etal.. 19831. RDX was not associated with
dose-related increases in the incidence of nonmalignant liver tumors (neoplastic nodules) or
combined incidence of liver neoplastic nodules or carcinomas.22 However, a statistically significant
positive trend with dose was observed in the incidence of hepatocellular carcinomas in male, but
not female, F344 rats fLevine etal.. 19831. In the Levine etal. T19831 study, there were only a few
tumors observed in the exposed groups of male rats (0/55, 0/52, 2/55, 2/31) relative to the control
(1/55), and inferences made from such a sparse response are uncertain. Because hepatocellular
carcinomas are rare tumors in the rat23, some perspective is obtained by considering historical
control data. In a paper published concurrently with the Levine etal. (19831 study, NTP reported
an incidence of liver carcinomas in untreated control male F344 rats of 0.7% (12/1,719; range:
0-2%) fHaseman et al.. 19851. In Levine etal. T19831. the incidence of liver carcinomas in control
male rats (1/55 or 1.8%) was at the upper end of this NTP range, and the incidence in RDX-treated
male F344 rats in the highest two dose groups (3.6 and 6.4%) exceeded the NTP historical control
range. Using incidence data from NTP historical controls, the trend for carcinoma in the RDX-
treated F344 rats was statistically significant (p-value = 0.003; one-sided exact Cochrane-Armitage
trend test). It should be noted that although the NTP historical controls (Haseman et al.. 19851 are
comparable with Levine etal. T19831 in terms of the time period, they may not be directly
comparable in terms of diet, laboratory, pathological evaluation, and animal provider. However,
other historical control datasets from male F344 rats, both recent and of the time period of the
Levine study, indicate similar low incidences of liver carcinomas (0.36%, (NTP. 2009): 0.31%,
(Maita et al.. 1987)). In the Levine etal. (1983) study, mortality in the highest dose group was
substantially higher than in the other dose groups during the second year leading to uncertainty in
the true cancer incidence in the high dose group. It was not possible to estimate mortality-adjusted
incidences because no time-to-death information was available.
In a second 2-year dietary study using a different rat strain (Sprague-Dawley), the
combined incidence of hepatocellular adenomas or carcinomas was not increased with dose in rats
^Considerations listed in footnote 20 apply to the comparison of combined liver adenoma and carcinoma
incidence to historical controls as well.
22The incidence of neoplastic nodules of 7.3% in control male rats in Levine et al. (19831 was consistent with
the NTP historical control range of 0-12% (mean: 3.5% or 61/1,719) (Haseman et al.. 19851.
23NTP historical control data for hepatocellular carcinomas F344 rats as reported in Haseman et al. (19851:
12/1,719 (0.7%) in males; 3/1,766 (0.17%) in females. Historical control data for Charles River Sprague-
Dawley rats as reported in Chandra et al. (19921: 6/1,340 (0.45%) in males; 1/1,329 (0.08%) in females.
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of either sex at doses up to 10 mg/kg-day (Hart. 19761. However, interpretation of results from this
study is limited by the comparatively lower doses employed in the study, and the recording of
effects only at the control and high dose groups.
Table 1-13. Liver tumors observed in chronic animal bioassays
Reference and study design
Results3
Lish et al. (1984)
Doses
o
LO
o
35
175/100b
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 (incidence)c
M
8/63 6/60 1/62*
7/59
7/27
contaminant; 83-89% of particles <66 urn
(12.7) (10.0) (1.6)
(11.9)
(25.9)
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
F
1/65 1/62 6/64
(1.5) (1.6) (9.4)
6/64
(9.4)
3/31
(9.7)
Hepatocellular carcinomas (incidence)c
2 yrs
M
13/63 20/60 16/62
18/59
6/27
(20.6) (33.3) (25.8)
(30.5)
(22.2)
F
0/65 4/62 3/64
6/64
3/3 ld
(0.0) (6.5) (4.7)
(9.4)
(9.7)
Hepatocellular adenoma or carcinoma combined (incidence)c
M
20/63 26/60 17/62
25/59
13/27
(31.7) (43.3) (27.4)
(42.4)
(48.1)
F
1/65 5/62 9/64*
12/64*
6/31*d
(1.5) (8.1) (14.1)
(18.8)
(19.4)
PWG reanalysis of liver lesion slides from female mice (Parker et al.,
2006; Parker, 2001).e
Doses
o
LO
o
35
175/100b
Hepatocellular adenomas (incidence)c
F
1/67 3/62 2/63
8/64
2/31
(1.5) (4.8) (3.2)
(12.5)
(6.5)
Hepatocellular carcinomas (incidence)c
F
0/67 1/62 3/63
2/64
2/31
(0.0) (1.6) (4.8)
(3.1)
(6.5)
Hepatocellular adenoma or carcinoma combined (incidence)c
F
1/67 4/62 5/63
10/64
4/3 ld
(1.5) (6.5) (7.9)
(15.6)
(12.9)
Hart (1976)
Doses
o
O
3.1
10
Rats, Sprague-Dawley, 100/sex/group
Purity and particle size not specified
0,1.0, 3.1, or 10 mg/kg-d
Neoplastic nodules (incidence)c
M
0/82
-
3/77
Diet
2 yrs
F
1/72
-
1/81
Hepatocellular carcinomas (incidence)c
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M
1/82
-
-
1/77
F
1/72
-
-
1
00
1
Neoplastic nodules or hepatocellular carcinomas combined
(incidence)c
M
1/82
-
-
4/77
F
2/72
-
-
2/81
Levine et al. (1983)
Doses
0
0.3
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
contaminant; 83-89% of particles <66 nm
0,0.3,1.5, 8.0, or 40 mg/kg-d
Diet
2 yrs
Neoplastic nodules (incidence)c
M
F
4/55
(7.3)
3/53
(5.6)
3/55
(5.5)
1/55
(1.8)
0/52
(0.0)
1/54
(1.9)
2/55
(3.6)
0/55
(0.0)
1/31
(3.2)
4/48
(8.3)
Hepatocellular carcinomas (incidence)c
M
1/55
(1.8)
0/55
(0.0)
0/52
(0.0)
2/55
(3.6)
2/3 ld
(6.5)
F
0/53
(0.0)
1/55
(1.8)
0/54
(0.0)
0/55
(0.0)
0/48
(0.0)
Neoplastic nodules or hepatocellular carcinomas combined
(incidence)c
M
5/55
(9.1)
3/55
(5.5)
0/52
(0.0)
4/55
(7.3)
3/31
(9.7)
F
3/53
(5.6)
2/55
(3.6)
1/54
(1.9)
0/55
(0.0)
4/48
(8.3)
^Statistically significant difference compared to the control group (p < 0.05), identified by the authors.
aSelected percent incidences are provided in parentheses below the incidences to help illustrate patterns in the
responses.
bThe lower dose of 100 mg/kg-day was started in week 11, resulting in a duration-weighted average dose of
107 mg/kg-day.
cThe incidences reflect the animals surviving to month 12.
Statistically significant trend (p < 0.05) was identified using a one-sided Cochran-Armitage trend tests performed
by EPA.
eThe numbers of animals at risk (i.e., the denominators) in the control group (n = 67) and 7 mg/kg-day dose group
(n = 63) as reported in the PWG reanalysis (Parker et al., 2006; Parker, 2001) differed from the numbers reported
in the original study by Lish et al. (1984) (n = 65 and 64, respectively). Further investigation of these differences
by the U.S. Army (sponsor of the mouse bioassay and subsequent PWG reevaluation) was unable to resolve the
discrepancy (email to Louis D'Amico, U.S. EPA, from Mark Johnson, U.S. Army Public Health Command, February
13, 2015).
fHart (1976) distinguishes the single high-dose carcinoma in the liver from a hepatocellular carcinoma; the
incidence of hepatocellular carcinomas in this dose group is shown as 0/81 (p. 119 of the publication).
Note: A dash ("-") indicates that the study authors did not measure or report a value for that dose group.
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Lung Tumors
Lung tumors were observed in female and male B6C3Fi mice exposed to RDX in the diet for
2 years fLish etal.. 19841 (see Table 1-14). Incidence of alveolar/bronchiolar carcinomas and the
combined incidence of alveolar/bronchiolar adenomas or carcinomas showed a statistically
significant positive trend (one-sided p-values of 0.016 and 0.009, respectively, for the Cochran-
Armitage trend test) in female mice. Incidence of alveolar/bronchiolar carcinomas in male mice
showed a statistically significant positive trend (p-value = 0.015; one-sided Cochran-Armitage trend
test). However, the combined incidence of adenomas and carcinomas was not elevated in male
mice. In such a case, NTP policy recommends analyzing the tumors both separately and in
combination fMcConnell etal.. 19861. This recommendation arose out of concern that combining
benign and malignant neoplasms can result in a false negative if the chemical shows a statistically
significant increase in malignant tumors without an increase in the combined incidence. 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. (19841 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, but regarded these neoplasms as
random and not biologically significant (rationale for this conclusion not provided).
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. 19761 or in F344 rats of either sex exposed chronically to RDX at doses up to
40 mg/kg-day (Levine et al.. 19831. Alveolar/bronchiolar carcinomas are rare tumors in both
species of rats, male or female f Chandra etal.. 1992: Haseman etal.. 19851.
Table 1-14. Lung tumors observed in chronic animal bioassays
Reference and study design
Results3
Lish et al. (1984)
Doses
0
1.5
7.0
35
175/100b
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 (incidence)c
M
6/63
5/60
5/62
7/59
1/27
contaminant; 83-89% of particles
(9.5)
(8.3)
(8.1)
(11.9)
(3.7)
<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)
F
4/65
(6.2)
2/62
(3.2)
5/64
(7.8)
9/64
(14.1)
3/31
(9.7)
Alveolar/bronchiolar carcinomas (incidence)c
Diet
M
3/63
6/60
3/62
7/59
5/27d
2 yrs
(4.8)
(10.0)
(4.8)
(11.9)
(18.5)
F
3/65
1/62
3/64
3/64
4/3 ld
(4.6)
(1.6)
(4.7)
(4.7)
(12.9)
Alveolar/bronchiolar adenoma or carcinoma combined (incidencef
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Results3
M
9/63 11/60 8/62
14/59
6/27
(14.3) (18.3) (12.9)
(23.7)
(22.2)
F
7/65 3/62 8/64
12/64
7/3 ld
(10.8) (4.8) (12.5)
(18.8)
(22.6)
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
No alveolar/bronchiolar carcinomas reported by study authors.
Levine et al. (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 (incidence)c
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 (incidence)c
Diet
2 yrs
M
- - -
-
-
F
0/53 0/7 1/8
0/10
0/48
Alveolar/bronchiolar adenoma or carcinoma combined (incidencef
M
F
3/53 0/7 1/8
1/10
0/48
1
2 aSelected percent incidences are provided in parentheses below the incidences to help illustrate patterns in the
3 responses.
4 bThe lower dose of 100 mg/kg-day was started in week 11, resulting in a duration-weighted average dose of
5 107 mg/kg-day.
6 cThe incidences reflect the animals surviving to month 12.
7 Statistically significant trend (p < 0.05) was identified using a one-sided Cochran-Armitage trend test performed by
8 EPA.
9
10 Note: A dash indicates that the study authors did not measure or report a value for that dose group.
11
12 Mechanistic Evidence
13 There are few mechanistic data to inform a MOA determination for either liver or lung
14 tumors induced by exposure to RDX.
15 Largely negative findings in in vitro and in vivo genotoxicity assay for parent RDX or its
16 oxidative metabolites (see Appendix C, Section C.3.2) suggest that parent RDX or its oxidative
17 metabolites do not interact directly with deoxyribonucleic acid (DNA). In contrast, there are some
18 positive genotoxicity results for the N-nitroso metabolites of RDX, specifically hexahydro-l-nitroso-
19 3,5-dinitro-l,3,5-triazine (MNX) andhexahydro-l,3,5-trinitroso-l,3,5-triazine (TNX). Trace
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amounts of MNX and TNX metabolites were identified in minipigs orally exposed to 14C-RDX in an
ADME study; minipigs were chosen as the animal model for investigation of RDX metabolism
because the GI tract of pigs more closely resembles that of humans fMusick etal.. 2010: Mai or 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 (Snodgrass. 19841. MNX was not mutagenic in Salmonella typhimurium (strains TA98, TA100,
TA1535, TA1537, and TA1538), with or without the addition of the S9 metabolic activating mixture
fPan etal.. 2007b: Snodgrass. 19841. WhenS. typhimurium strains TA97a andTA102, 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. N-nitroso
metabolites, including MNX and TNX, are generated anaerobically and are likely a result of bacterial
transformation of parent RDX in the GI tract to various N-nitroso derivatives (Pan etal.. 2007b).
Exposure to potentially mutagenic N-nitroso metabolites of RDX generated in the GI tract of mice
may occur in the liver (and subsequently in the systemic circulation) via enterohepatic circulation.
However, as noted earlier 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-diazabutanal and 4-nitro-
2,4-diaza-butanamide fMaior etal.. 20071. Thus, the contribution of the N-nitroso metabolites to
the overall carcinogenic potential of RDX is unclear.
Aberrant expression of miRNAs was observed in the brains and livers of female B6C3Fi
mice fed 5 mgRDX/kg in the diet for 28 days (Zhang and Pan. 2009b) (dose of 0.75-1.5 mg/kg-day
estimated by Bannon etal. (2009bl). with several oncogenic miRNAs being upregulated, while
several tumor-suppressing miRNAs were downregulated. 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) f Sweeney etal..
2012b). miRNAs have been associated with several cancers (Wiemer. 2007: Zhang etal.. 2007). but
the utility of miRNAs as predictive of carcinogenesis has not been demonstrated (Bannon etal..
2009b). Further, it is unknown whether or not aberrant expression of a specific miRNA (or suite of
miRNAs) plays a role in the MOA of RDX carcinogenicity. Microarray analysis of gene expression in
male Sprague-Dawley rats after exposure to a single oral (capsule) dose of RDX revealed a general
upregulation in gene expression (predominantly genes involved in metabolism) in liver tissues
fBannon et al.. 2009al: however, the relevance of this finding to the carcinogenicity of RDX is
unclear.
Sweeney etal. (2012b) hypothesized a set of MOAs for the liver tumors:
• Genotoxicity mediated by either: (1) RDX; (2) tissue-generated oxidative metabolites; or
(3) N-nitroso metabolites generated anaerobically in the GI tract. The key events in this
hypothesized MOA are: production of DNA damage, gene mutation, formation of neoplastic
lesions, and promotion/progression of tumors. The largely negative results for genotoxicity
led Sweeney etal. f2012bl to conclude that this MOA is not plausible for RDX or its
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oxidative metabolites. Although there are some positive results for the N-nitroso
metabolites, the limited evidence to support systemic uptake and distribution of
metabolites to the liver led Sweeney etal. f2012bl to conclude that this MOA is not
sufficiently plausible.
• Cell proliferation. The key events in this hypothesized MOA are Gl-tract generation of
N-nitroso metabolites, absorption, distribution to the liver, cytotoxicity (optional), and
enhanced cell proliferation, leading to preneoplastic foci that progress to hepatocellular
adenomas and carcinomas. Sweeney etal. (2012b) cited evidence of increased liver weights
in mice as consistent with cell proliferation, but noted that increased liver weights were
also observed in rats without proceeding to liver tumors. They considered this MOA
"plausible, but not particularly well supported."
In addition to the inconsistencies in the evidence identified by Sweeney etal. f2012bl. EPA
notes the following evidence (or lack of evidence) that fails to support this hypothesized MOA.
(1) The absence of significant liver histopathology in mice after subchronic or chronic exposure to
RDX at doses that induced liver tumors (Lish etal.. 1984: Cholakis et al.. 1980) suggests that cellular
toxicity is not a precursor to these tumors. (2) As discussed in Section 1.2.4, changes in liver weight
showed no consistent pattern across studies or sexes, and did not correlate with tumor response.
(3) No studies were available that directly measured RDX-induced cell proliferation rates. (4) No
information was available to rule out non-precancerous causes of liver weight increase.
In summary, the available evidence indicates that RDX is likely not mutagenic (see
Appendix C, Section C.3.2), although anaerobically-derived N-nitroso metabolites have
demonstrated some genotoxic potential. While these metabolites have been measured in the
mouse (Pan etal.. 2007b) and identified in minipigs (Musick etal.. 2010: Major etal.. 2007). 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 nor a
mutagenic N-nitroso metabolite MOA is supported. Thus, the MOA leading to the increased
incidence of liver and lungs tumors is not known.
1.3. INTEGRATION AND EVALUATION
1.3.1. Effects Other Than Cancer
The majority of evidence for the health effects of RDX comes from oral toxicity studies in
animals. The three epidemiology studies that document possible inhalation exposure are limited by
various study design deficiencies, including inability to distinguish exposure to TNT (associated
with liver and hematological system toxicity), inability to adequately characterize exposure levels,
small sample sizes, and inadequate reporting. The single animal inhalation study identified in the
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literature search had deficiencies (e.g., lack of a control and incomplete exposure information) that
precluded its inclusion in this assessment (see literature search section).
The strongest evidence for a human health hazard following exposure to RDX is for nervous
system effects. Toxicity studies in multiple animal species involving chronic, subchronic, and
gestational exposures provide consistent evidence of nervous system effects following oral
exposure. Effects included dose-related increases in seizures and convulsions, as well as
observations of tremors, hyperirritability, hyper-reactivity, and other behavioral changes (Crouse
etal.. 2006: Angerhofer etal.. 1986: Levine etal.. 1983: Levine etal.. 1981a. b; Cholakis etal.. 1980:
von Oettingen etal.. 19491.
Human studies provide supporting evidence for RDX as a neurotoxicant and provide
support for the assumption that the nervous system effects observed in experimental animals are
relevant to humans. In particular, several case reports provide evidence of associations between
exposure to RDX (via ingestion, inhalation, and possibly dermal exposure) and seizures and
convulsions (Kasuske etal.. 2009: Kuctikardali etal.. 2003: Testudetal.. 1996a: Testud etal..
1996b: Woody etal.. 1986 and others, see Appendix C.2). Other nervous system effects identified in
human case reports include dizziness, headache, confusion, and hyperirritability. A cross-sectional
study described memory impairment and visual-spatial decrements in RDX-exposed workers fMa
and Li. 19931. although confidence in these findings is relatively low because of issues with design
and reporting.
Additional support for an association between RDX exposure and nervous system effects
comes from consistent evidence of neurotoxicity across taxa, including humans, laboratory animal
species, birds, lizards, fathead minnows, and earthworms (Ouinn etal.. 2013: Garcia-Revero etal..
2011: Mcfarland et al.. 2009: Gogal etal.. 20031. Studies in rats demonstrate a correlation between
blood and brain concentrations of RDX and the time of seizure onset fWilliams etal.. 2011: Bannon
etal.. 2009al. 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.
Induction of convulsions and seizures appears to be more strongly correlated with dose
than with duration of exposure. However, there is mechanistic information that suggests repeated
binding to the receptor convulsant site of GABAa may promote a state of increased neuronal activity
that increases the likelihood of subsequent neurological effects fGerkin etal.. 20101. As a result,
some uncertainty remains as to whether the available mechanistic information adequately
addresses potential neurotoxicity after longer-duration exposure to RDX. It is unclear if nervous
system effects progressed in severity (e.g., from subtle behavioral changes or nonconvulsive
seizures to tonic-clonic seizures) with increasing dose, as 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.
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The nervous system effects following oral exposure to RDX were observed in humans
acutely exposed to RDX and in multiple experimental animal studies in rats, mice, monkeys, and
dogs following exposures ranging from 10 days to 2 years in duration. Notably, despite the
potential for effects on the developing nervous system based on the presumed MOA for RDX
neurotoxicity (discussed in Section 1.2.3), no studies included a thorough evaluation of potential
developmental neurotoxicity. Across the database, behavioral manifestations of seizure activity
were the most consistently observed nervous system effect associated with RDX exposure. This
most commonly included evidence of increased convulsions, as well as other related effects such as
tremors, shaking, hyperactivity, or nervousness, which were generally observed at doses that were
the same as or higher than doses that induced convulsions. Nervous system effects are a human
hazard of RDX exposure and are carried forward for consideration for dose-response analysis.
Convulsions, considered a severe adverse effect, were selected as a consistent and sensitive
endpoint representative of nervous system effects.
Evidence for urinary system toxicity is more limited than evidence for neurotoxicity. In
humans, kidney effects (including decreased urine output, blood in urine, and proteinuria) were
observed only in individuals with acute accidental exposure (ingestion and inhalation) to unknown
amounts of RDX. In experimental animal studies, histopathological changes in the kidney and
urinary bladder (medullary papillary necrosis, suppurative pyelitis, and uremic mineralization of
the kidney; luminal distention and cystitis of the urinary bladder) were reported in male rats
exposed to RDX in the diet following exposure durations of 1 year or longer (Levine etal.. 1983).
but not in similarly exposed female rats. Evidence for milder renal effects reported in subchronic
studies of RDX in mice, rats, and monkeys was limited and inconsistent Mice appeared to be less
sensitive than rats. Other measures of kidney effects (kidney weights and serum chemistry
parameters) did not provide consistent evidence of dose-related changes associated with RDX
exposure. In light of the dose-related increase in histopathological changes in the kidney and
urinary bladder in male rats in the Levine etal. (1983) study, and in particular the robust response
in the high-dose animals, urinary system effects are a potential human hazard of RDX exposure.
Medullary papillary necrosis was selected as an endpoint representative of kidney effects.
This histopathologic lesion was observed at higher incidence than other kidney histopathologic
lesions, was present at both the 12-month interim and 2-year final sacrifices (Table 1-6), and
represents a severe measure of toxicity. Renal toxicity was, in fact, considered the principal cause
of RDX-related mortality and morbidity in male rats in the Levine etal. T19831 2-year bioassay.
Hemorrhagic/suppurative cystitis was selected as an endpoint representative of urinary bladder
effects. Like medullary papillary necrosis of the kidney, urinary bladder cystitis is a clearly adverse
effect and was observed at both the 12-month interim and 2-year final sacrifices (Table 1-7). A
dose-related increased incidence of luminal distention was also observed in male rats, but was not
selected as representative of urinary bladder toxicity because it is a less specific diagnosis than
cystitis and can be caused by various factors, including partial obstruction of the bladder.
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Evidence for prostate toxicity is also more limited than evidence for neurotoxicity. A dose-
related, increase in the incidence of suppurative prostatitis was observed in male rats exposed to
RDX in the diet for 2 years fLevine etal.. 19831. There was also a concomitant shift from chronic
inflammation to suppurative inflammation with increasing dose of RDX starting at 1.5 mg/kg-day.
Similar types and patterns of inflammation were not observed in mice, and no other rat studies of
equivalent duration that examined the prostate were available. RDX and its interaction with GABAa
receptors, which have also been identified on the prostate (Napoleone et al.. 19901. increases
biological plausibility by providing a potential mechanism by which RDX could have effects directly
on the prostate. In their evaluation of the external review draft assessment, the SAB determined
the weight-of-evidence to be sufficient for identifying prostate effects as a hazard of RDX exposure
fSAB. 20171. Consistent with this determination, the incidence of suppurative prostatitis was
selected as the endpoint most representative of prostate effects.
Evidence for developmental toxicity and liver toxicity was more limited than that for the
endpoints discussed above. In animal studies, developmental effects, including offspring survival,
growth, and morphological development, were observed only at doses associated with maternal
mortality fAngerhofer etal.. 1986: Cholakis etal.. 19801. Evidence for potential hepatic effects
comes from observations of increases (generally dose-related) in liver weight in some subchronic
oral animal studies fLish etal.. 1984: Levine etal.. 1983: Levine etal.. 1981a. b; Cholakis etal.. 1980:
Hart. 1976). However, these elevations in liver weight were not consistently observed across
studies nor were they accompanied by RDX-related histopathological changes in the liver or
increases in serum liver enzymes. In addition, the interpretation of liver weight changes in the
mouse bioassay by Lish et al. (1984) is complicated by the relatively high incidence of liver tumors
in this study. At this time, the available data do not support liver and developmental toxicity as
human hazards of RDX exposure; these effects were not considered further for dose-response
analysis and derivation of reference values.
Evidence that RDX may induce effects in other organs, including the eyes, and the
cardiovascular, musculoskeletal, immune, GI, hematological, and reproductive systems, is generally
limited to human case reports or to findings in experimental animals that were inconsistent across
studies or lacked dose-related patterns of response. Therefore, information to assess toxicity in
these organs following exposure to RDX was insufficient Treatment-related changes in body
weight or body weight gain were generally observed at high doses in association with elevated
mortality or with severe kidney and urinary bladder toxicity, and thus appeared to be secondary to
effects on other primary targets of RDX toxicity. Effects on body weight and on these other organs
were not considered further for dose-response analysis and derivation of reference values.
In a number of the animal studies reporting nervous system effects, unscheduled deaths
occurred at RDX doses as low as those that induced nervous system effects (Crouse etal.. 2006:
Angerhofer et al.. 1986: Levine etal.. 1983: Levine etal.. 1981a: Cholakis etal.. 1980: von Oettingen
etal.. 19491. In a 90-day study that recorded nervous system effects and survival more thoroughly
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than earlier studies, Crouse etal. (2006) reported that nearly all pre-term deaths were preceded by
neurotoxic signs such as tremors and convulsions. Convulsions did not, however, necessarily lead
to early mortality; of the animals observed to have convulsed in the Crouse etal. f20061 study,
approximately 75% survived to the end of the 90-day study. Most of the earlier studies provide a
limited understanding of the association between mortality and nervous system effects because the
frequency of clinical observations was likely insufficient to observe convulsions prior to death. In
humans, mortality has not been reported in case reports involving workers with symptoms of
neurotoxicity exposed to RDX during manufacture or in individuals exposed acutely as a result of
accidental or intentional ingestion. Survival has not been specifically evaluated in studies of worker
populations exposed chronically to RDX. Ultimately, the convulsion findings, without consideration
of mortality, are sufficient to identify neurotoxic effects associated with RDX exposure as severe
and adverse.
Regarding mortality, the preference is not to use a frank health effect as severe as mortality
as the basis for a reference value. As noted in U.S. EPA (20021. a chemical may cause a variety of
effects ranging from severe—such as death—to more subtle biochemical, physiological, or
pathological changes; primary attention in assessing health risk should be given to those effects in
the lower exposure range and/or the effects most biologically appropriate for a human health risk
assessment Where mortality occurs as a consequence of a chemical's effects on a specific
organ/system (e.g., in the case of RDX, evidence suggests some relationship between mortality and
effects on the nervous system and kidney), the preference would be to develop a quantitative
assessment based on the initial hazard and not on death. Because unscheduled deaths were
observed with some consistency across studies and, in some studies, at doses as low as those
associated with convulsions, two additional analyses of mortality data are presented in Chapter 2.
In the first analysis, BMDs derived using mortality data sets are compared to the BMD used to
derive the RfC (Section 2.1.6). As discussed in Section 1.2.1, the relationship between convulsions
and mortality is not clear and raises concerns for the potential underreporting of convulsions. An
analysis, described in Section 2.1.7, addresses the possibility that the analyses of convulsions
brought forward for dose-response analysis resulted in an underestimate of the toxicity for RDX.
1.3.2. Carcinogenicity
As presented in Section 1.2.7, dietary administration of RDX induced dose-related increases
in the incidence of hepatocellular adenomas or carcinomas in male and female B6C3Fi mice (Parker
etal.. 2006: Lish etal.. 1984). In the same study, RDX also induced dose-related increases in the
incidence of alveolar/bronchiolar adenomas or carcinomas in both sexes. Some of these trends in
liver and lung were statistically significant. In Fischer 344 rats, dietary administration of RDX
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yielded a statistically significant trend in the incidence of hepatocellular carcinomas24 in males, but
not in females fLevine etal.. 19831. A 2-year dietary study in Sprague-Dawley rats was negative in
both sexes Hart f19761. although the highest dose in this study, and the only dosed group for which
pathology was examined, was somewhat lower (no increase in carcinomas at doses up to
10 mg/kg-day in Hart f!9761. versus hepatocellular carcinomas in male rats at 8 and 40 mg/kg-day
in the Levine etal. (1983) study). The human studies are not informative.
This evidence leads to consideration of two hazard descriptors under the EPA's Guidelines
for Carcinogen Risk Assessment (U.S. EPA. 2005a). The descriptor likely to be carcinogenic to humans
is appropriate when the evidence is "adequate to demonstrate carcinogenic potential to humans"
but does not support the descriptor carcinogenic to humans. One example from the cancer
guidelines 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." RDX
matches the conditions of this example, having induced dose-related increases in tumors in two
species (mouse and rat), in both sexes, and at two sites (liver and lung). Liver carcinomas,
increased in male F344 rats in the Levine etal. (1983) study, are considered rare in that species.
Alternatively, the descriptor suggestive evidence of carcinogenic potential is appropriate
when the evidence raises "a concern for potential carcinogenic effects in humans" but is not
sufficient for a stronger conclusion. The incidences of alveolar/bronchiolar tumors showed a
positive trend in male and female B6C3Fi mice. Evidence of carcinogenicity in the liver from rodent
bioassays is less clear. The hepatocellular carcinoma result in male F344 rats is based on a small
number of tumors (1/55, 0/55, 0/52, 2/55, and 2/31, respectively, at 0, 0.3,1.5, 8.0, and
40 mg/kg-day) that is not matched by an increase in hepatocellular neoplasms overall (5/55, 3/55,
0/52, 4/55, and 3/31, respectively), and RDX did not increase the incidence of carcinomas at any
other site in F344 or Sprague-Dawley rats of either sex. The incidence of liver tumors in female
B6C3Fi mice showed a statistically significant positive trend fLish etal.. 19841. although the
authors noted the relatively low tumor incidence in concurrent female control mice (1/65). The
PWG that reviewed the slides from this study (Parker etal.. 2006: Parker. 2001) confirmed the
positive trend in female mouse liver tumors, but also raised some concerns related to
histopathological methods and the absence of necropsy and histopathology processing records that
limited their evaluation. In male mice from this study fLish etal.. 19841. the incidences of liver
tumors in some treated groups were higher than in the control, but trend tests were not statistically
significant. Interpretation of male mouse liver tumor incidence is complicated by the high and
variable background incidence of this tumor in the male mouse.
^Hepatocellular carcinoma may be regarded as a rare tumor in male F344 rats. Although there is no
compilation of historical control data for the Levine laboratory, Haseman et al. (19841 reported that in NTP
studies during 1980-1983,18/2306 (0.8%) of male F344 rats developed hepatocellular carcinomas and
78/2306 (3.4%) developed neoplastic nodules.
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As discussed in Section 1.2.7, few mechanistic studies are available to inform the mode of
action by which RDX induces liver and lung tumors in rodents. The available evidence indicates
that RDX is likely not mutagenic. Anaerobically-derived N-nitroso metabolites have demonstrated
some genotoxic potential. These metabolites have not been identified in humans, and their
contribution to any genotoxic potential of RDX is unknown. Precursor events leading to the
observed liver and lung tumor response in RDX-exposed rodents have not been identified.
Although characterization of the cancer MOA is not needed to determine a chemical's cancer
hazard, understanding the MOA can contribute to a cancer hazard determination. In the case of
RDX, mechanistic information is not helpful in guiding selection of a cancer descriptor.
As noted in the EPA's cancer guidelines fU.S. EPA. 2005al. choosing a hazard descriptor
cannot be reduced to a formula, as descriptors may be applicable to a variety of potential data sets
and represent points along a continuum of evidence. In the case of RDX, there are plausible
scientific arguments for more than one hazard descriptor. Overall, the considerations discussed
above, interpreted in light of the cancer guidelines, lead to the conclusion that there is suggestive
evidence of carcinogenic potential for RDX. Although the evidence includes dose-related tumor
increases in two species, two sexes, and two sites, the evidence of carcinogenicity outside the
B6C3Fi mouse is not robust, and this factor was decisive in choosing a hazard descriptor. Within
the spectrum of results covered by the descriptor suggestive evidence, the evidence for RDX is
strong. There are well-conducted studies that tested large numbers of animals at multiple dose
levels, making the cancer response suitable for dose-response analysis (Section 2).
The descriptor suggestive evidence of carcinogenic potential applies to all routes of human
exposure. Dietary administration of RDX to mice and rats induced tumors of the liver or lung, sites
beyond the point of initial contact, and human case reports have demonstrated absorption and
distribution of inhaled RDX into systemic circulation. Under the cancer guidelines, this information
provides sufficient basis to apply the cancer descriptor developed from oral studies to other
exposure routes.
1.3.3. 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 RDX toxicokinetics
or differences in RDX toxicodynamics between susceptible and other populations. Little
information is available on populations that may be especially vulnerable to the toxic effects of RDX.
Reproductive and developmental toxicity studies generally did not identify effects in offspring at
doses below those that also caused severe maternal toxicity (Angerhofer et al.. 1986: Cholakis etal..
19801. However, the developmental importance of GABAergic systems fKirmse etal.. 2018: Ben-
Ari. 2014: Williams et al.. 2011: Williams and Bannon. 2009: Galanopoulou. 20081 and
developmental neurotoxicity of chemicals with similar modes of action fSalari and Amani. 2017:
Marty et al.. 20001 suggest RDX may be harmful during the period of brain development Further
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raising cause for concern, seizures and seizure disorders such as epilepsy occur more frequently in
infants and children than in any other age group (many are caused by early-life insults such as fever
or trauma), and research suggests that early life seizures (i.e., before the brain has fully matured)
can lead to long-lasting neurological consequences fRonnie. 2003: Volpe. 2001: Tensen and Baram.
2000: Moshe. 2000.19871. A pilot study in rats demonstrated transfer of RDX from dam to fetus
during gestation, found RDX in milk from treated dams, and recommended further study (Hess-
Ruth etal.. 2007). Given the understanding of RDX toxicokinetics (see Section 1.1.2), it is expected
that RDX reaching the fetus or infant through either the blood or ingested milk would be readily
distributed to the brain, although specific studies have not been conducted. For these reasons, and
as noted in Section 1.2.1, the lack of developmental neurotoxicity studies was identified as a
significant data gap in understanding the nervous system effects of RDX exposure.
The primary MOA for the neurotoxic effects of RDX exposure involves RDX binding to
GABAa receptors, specifically the picrotoxin convulsant site of the GABA channel, and blocking
inhibitory GABAergic transmission, that eventually leads to the development of seizures and
related behavioral changes (see Section 1.2.1, Mechanistic Evidence). In addition to its role as the
major inhibitory neurotransmitter system in many regions of the adult brain, GABAergic signaling
plays a key role in brain development, where it contributes to a delicate equilibrium with other
signaling processes (e.g., glutamatergic) to help establish the appropriate functional connectivity of
the mature brain (Kirmse etal.. 2018: Ben-Ari. 2014). While GABAergic signaling and the overall
balance between excitation and inhibition is essential throughout brain development, which
continues through sexual maturation, a number of critical developmental processes occur
simultaneously during the perinatal period, and these coincide with prominent shifts in GABAergic
function. As a result, the perinatal period may represent a vulnerable lifestage for the neurotoxic
effects of RDX exposure through GABAergic inhibition.
In the perinatal mammalian brain, GABA activity is primarily depolarizing and excitatory
(as compared to hyperpolarizing and inhibitory in the adult brain), which is presumably necessary
for its specific functions at this stage of brain development. In animals, expression of chloride co-
transporters NKCC1 and KCC2 around or shortly after birth reduces intracellular CI- and mediates a
switch in GABA activity to primarily hyperpolarizing and inhibitory (Ben-Ari. 2014: Rivera etal..
19991. For GABAAergic signaling, the switch from depolarizing to the hyperpolarizing phenotype in
adults occurs by the end of the first postnatal month in rats, although this differs by brain region
and sex fGalanopoulou. 20081. In addition, the composition of GABAa receptors is also subject to
developmental regulation, with some subunits varying in their pattern of expression during
development as compared to adulthood (Lujan etal.. 2005: Fritschv etal.. 1994: Laurie etal.. 1992).
Thus, RDX exposure during the perinatal period in humans could be impactful.
During this potentially sensitive period, excitatory GABAAergic signaling helps to regulate
the proliferation, migration, survival, and differentiation of new neurons, as well as synaptogenesis
and the development of mature neural networks fDeidda etal.. 2014: Galanopoulou. 20081.
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Modulation of GABAAergic signaling at this lifestage is presumably tightly controlled, as it serves to
orchestrate these processes in a region-specific manner for specific glial and neuronal subsets,
often stimulating these processes (e.g., increasing neuronal migration or survival) in some regions
while simultaneously inhibiting the same processes (e.g., decreasing neuronal migration or
survival) in other regions fCreelev. 2016: Deiddaetal.. 2014: Galanopoulou. 2008: Ikonomidou et
al.. 2000). Additional concern for susceptibility during this lifestage may be raised due to the
prominent role for BDNF during this time, with high expression during the first two postnatal
weeks (in rodents) before declining to adult levels, and whose role as a neurotrophic factor
includes the regulation of neuronal excitation and its sequelae fAguado etal.. 20031. As discussed
in Section 1.2.1, molecular evidence suggests that RDX exposure in adults may impact the
expression or function of BDNF and related factors in the brain fZhang and Pan. 2009bl: the lack of
data on brain BDNF after developmental RDX exposure remains a data gap.
Alterations of GABA activity have been linked to developmental brain disorders (Kirmse et
al.. 2018) and genetic mutations causing aberrant GABAergic signaling lead to a number of seizure
disorders in infants and children (Galanopoulou. 2008). although GABAergic signaling in the
immature brain may be required for epileptogenesis fKhalilov etal.. 20031. Exposure of early
postnatal rodent hippocampus to the GABAa receptor antagonist, bicuculline, which has a similar
mode of action to RDX, increased the density of inhibitory but not excitatory synapses f Marty etal..
2000). White etal. (2008) reported that a 2.7 mg/kg subcutaneous dose of bicuculline provoked
seizures in 97% adult mice, but Salari and Amani (2017) found developmental and behavioral
impairment after a 0.3 mg/kg subcutaneous dose to neonatal mice, suggesting developmental
neurotoxicity from bicuculline is evident as low as 1/10 the convulsive dose. Findings from studies
of bicuculline provide suggestive evidence of perinatal susceptibility to the neurotoxicity elicited by
compounds that alter GABAergic signaling. The lack of data on how RDX exposure might impact the
critical role of GABAergic signaling during the perinatal period (and at later stages of brain
development and maturation) represents an important uncertainty.
Limited data suggest that male laboratory animals may be more susceptible to noncancer
toxicity associated with RDX exposure. In general, male animals were more sensitive to RDX
neurotoxicity than females (i.e., more convulsions; more hyperactive; greater brain weight
changes). In the two-year study in F344 rats fLevine etal.. 19831. RDX exposure induced severe
toxicity of the kidney and urinary bladder in males, but no similar effects in females, suggesting a
sex-based difference in susceptibility to RDX urinary system toxicity.
Data on the incidence of convulsions and mortality from gavage studies of RDX in the rat
provide some indication that pregnant animals may be a susceptible population. In the
developmental toxicity study by Cholakis etal. (1980). deaths were observed in pregnant F344 rats
only at a dose of 20 mg/kg-day, but convulsions were reported in a single rat at 2 mg/kg-day. In a
range-finding developmental toxicity study fAngerhofer etal.. 19861. mortality and convulsions
were reported in pregnant.Sprague-Dawley rats at a dose of >40 mg/kg-day, but not at <20 mg/kg-
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day, although the relatively small group sizes in this study should be noted. In the main study by
these investigators, convulsions were reported in pregnant rats only at 20 mg/kg-day, but one
death (in dose groups of 40 rats) was reported at both 2 and 6 mg/kg-day fAngerhofer etal.. 19861.
In comparison, increased mortality and convulsions were reported at >8 mg/kg-day in a 90-day
gavage study in F344 rats fCrouse et al.. 20061. The instances of one convulsion and two deaths in
pregnant rats in the Cholakis etal. (1980) and Angerhofer et al. (1986) studies at doses of 2 or
6 mg/kg-day raise the possibility that pregnant animals may be more susceptible to the effects of
RDX; however, direct comparison between the available gavage studies in pregnant and
nonpregnant rats is uncertain because of differences in study design, including numbers of animals
tested per group, test material characteristics, and rat strain. Overall, the available information is
not considered sufficient to conclude that pregnant animals are a susceptible population.
There is limited evidence that CYP450 or similar enzymes are involved in the metabolism of
RDX (Bhushan et al.. 2003). 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 or the presence of seizure
syndromes may affect susceptibility to RDX exposure.
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2.DOSE-RESPONSE ANALYSIS
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER
The oral reference dose (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 for Dose-Response Analysis of Selected Effects
As discussed in Section 1.3.1, based on findings from oral studies in experimental animals,
nervous system effects are a human hazard of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX)
exposure, and urinary system (kidney and bladder) effects are a potential human hazard of RDX
exposure. There is suggestive evidence of prostate effects associated with RDX exposure. Although
animal mortality has been reported in a number of the toxicology studies conducted for RDX, it was
not considered a hazard by itself or as the basis for the derivation of a reference value (see Sections
2.1.6 and 2.1.7 for further discussion).
The effects selected to best represent each of the hazards, identified in Section 1.3.1, are
carried forward in the sections below. In order to identify the stronger studies for dose-response
analysis, several attributes of the studies reporting the endpoints selected for each hazard were
reviewed (i.e., study size and design, relevance of the exposure paradigm, and measurement of the
endpoints of interest). In considering the study size and design, preference was given to studies
using designs reasonably expected to have power to detect responses of suitable magnitude.
Exposure paradigms including a route of human environmental exposure (i.e., oral and inhalation)
are preferred. When developing a chronic reference value, chronic or subchronic studies are
preferred over studies of acute exposure durations. Studies with a broad exposure range and
multiple exposure levels are preferred to the extent that they can provide information about the
shape of the exposure-response relationship. Additionally, with respect to measurement of the
endpoint, studies that can reliably distinguish the presence or absence (or degree of severity) of the
effect are preferred.
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
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to RDX examined the relationship between certain health endpoints and inhalation exposure;
however, no epidemiological studies of ingested RDX are available. Multiple case reports support
the identification of hazards associated with RDX exposure but are inadequate for dose-response
analysis because they do not yield incidence estimates, exposure durations are short, and
quantitative exposure information is lacking. Therefore, human studies could not be used for oral
dose-response analysis or to serve as the basis for the RfD. In the absence of human data, the
animal studies were considered for dose-response analysis.
Experimental animal studies considered for each health effect were evaluated using general
study quality considerations discussed in Section 4 of the Preamble and in the literature search
section, and the attributes described above. The rationales for selecting the strongest studies that
reported effects on the nervous system, urinary system, and prostate are summarized below.
Nervous System Effects
Convulsions, a severe adverse effect, were selected for dose-response analysis as a
consistent endpoint of nervous system effects (see Section 1.3.1 for discussion). This endpointwas
reported in seven studies fCrouse etal.. 2006: Lish etal.. 1984: Levine etal.. 1983: Levine etal..
1981a: Cholakis etal.. 1980: Martin and Hart. 1974: von Oettingen et al.. 19491. Table 2-1 provides
an overview of the information considered in the studies reporting nervous system effects (i.e.,
convulsions) evaluated for dose-response analysis.
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Table 2-1. Information considered for evaluation of studies that examined
convulsions
Study
reference
Study design and size
Exposure paradigm
Measurement
of endpoint
Design
ft of
animals
Route
Duration
# of dose
groups3
Levels
(mg/kg-d)
Purity
(%)
Analytical
conc?b
Incidence data
reported
Crouse et
al. (2006)
Toxicity study
10 rats/
sex/
group
Gavage
13-wk
5
4-15
99.99
Yes
Yes
Cholakis et
al. (1980)
Developmental
study
24-25
female
rats/group
Gavage
14-d
3
0.2-20
89
Yes
Yes
Martin and
Hart (1974)
Toxicity study
3
monkeys/
sex/group
Gavage
13-wk
3
0.1-10
Not
specified
Not
specified
Yes
Levine et
al. (1983)
Toxicity and
carcinogenicity
bioassay
75 rats/
sex/group
Diet
2-yr
4
0.3-40
89-99
Yes
No
Lish et al.
(1984)
Toxicity and
carcinogenicity
bioassay
85 mice/
sex/group
Diet
2-yr
4
1.5-175
89-99
Yes
No
Levine et
al. (1981a)
Toxicity study
10 rats/
sex/group
Diet
13-wk
5
10-600
85
Yes
No
von
Oettinsen
et al.
(1949)
Toxicity study
20 rats/
group
Diet
13-wk
3
15-50
90-97
Not
specified
No
Excluding the control group.
indicates whether authors performed analysis to confirm the concentration of RDX in the suspension or diet
administered to the animals (e.g., to determine percentage of target concentration).
Incidence of convulsions was reported in three studies of RDX—all involving gavage
administration: Crouse etal. (20061. Cholakis etal. (19801 (developmental toxicity study), and
Martin and Hart (19741. Qualitative findings of nervous system effects were reported in other
chronic and subchronic studies—all involving dietary administration: Lish et al. f19841. Levine et
al. f!9831. Levine etal. f!981al. and von Oettingen et al. f!9491. Incidence data on neurotoxic
effects of RDX were not collected in any of the dietary studies. For example, Levine etal. f 19831
reported only that convulsions and other nervous system effects were noted in rats exposed to RDX
for 2 years at the highest dose (40 mg/kg-day) tested. The studies that included incidence data (i.e.,
the gavage studies) were preferred over those studies only reporting qualitative results (i.e., the
dietary studies).
The three gavage studies reporting incidence data were further considered. Crouse etal.
f20061 reported a dose-related increase in convulsions and tremors in both male and female F344
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rats following a 90-day oral (gavage) exposure to RDX. This study used a test material of high
purity and six dose groups (including the control) that provided good resolution of the dose-
response curve. Cholakis etal. f!9801 reported a dose-related increase in convulsions in a
developmental toxicity study in F344 rats, following a 14-day exposure to RDX on gestational days
(GDs) 6-19. Although this study was designed as a standard developmental toxicity study (i.e., not
specifically to examine nervous system effects), it reported information on the identity of the test
material and used three dose groups that adequately characterized the dose-response curve.
Further, this study provided evidence of nervous system effects at a relatively low dose. The study
in monkeys by Martin and Hart f!9741 provides supporting evidence of nervous system effects
(trembling, shaking, ataxia, hyperactive reflexes, and convulsions); however, this study was not
selected for dose-response analysis because of small group sizes (n = 3/sex) and uncertainty in
measures of exposures (e.g., purity of the test material was not specified, and reported emesis in
some animals likely influenced the delivered dose).
Although the gavage studies reporting incidence data were preferred over four dietary
studies (Lish etal.. 1984: Levine etal.. 1983: Levine etal.. 1981a: von Oettingen et al.. 1949) that did
not provide incidence data, it is important to note that the reported neurotoxic effects in the dietary
studies were observed at dose levels higher than the doses at which effects were observed in the
gavage studies fCrouse etal.. 2006: Cholakis et al.. 1980: Martin and Hart. 19741. Given this
potential difference based on dosing method, the dietary studies were also considered for
quantitative analysis, despite the lack of incidence data, to evaluate the influence of oral dosing
method on candidate values. In the 2-year study by Levine etal. (1983). a LOAEL for nervous
system effects (convulsions, tremors, and hyper-irritability) of 40 mg/kg-day and a NOAEL of 8
mg/kg-day were identified. Other studies identified higher effect levels (i.e., 100 mg/kg-day in the
2-year mouse study by Lish etal. f!9841 and 50 mg/kg-day in the 3-month rat study by von
Oettingen et al. f194911. and, with the exception of Lish etal. T19841. used shorter exposure
durations. The unusual dosing regimen in the Cholakis etal. (1980) 13-week mouse study
precluded identification of a NOAEL and LOAEL, and the single-dose design of the 6-week dog study
by von Oettingen et al. (1949) did not allow identification of a NOAEL. As discussed in Section 1.2.1
and Table 1-3, the technical report of the 13-week study by Levine etal. (1981a) inconsistently
identified the dose level at which convulsions occurred; therefore, a reliable NOAEL and LOAEL
from this study could not be identified.
Therefore, two gavage studies, Crouse etal. f20061 and Cholakis etal. f!9801. and one
dietary study, Levine etal. (1983). were selected for dose-response analysis.
Urinary System (Kidney and Bladder) Effects
Medullary papillary necrosis and hemorrhagic/suppurative cystitis were selected for dose-
response analysis as biologically significant measures of kidney and urinary bladder effects,
respectively (see Section 1.3.1 for discussion). These histopathologic lesions of the urinary system
were primarily observed in the 2-year study by Levine etal. f!9831. Levine etal. T19831 included
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histopathologic examination of kidney and bladder tissues at 6-, 12-, and 24-month time points;
included four dose groups and a control group, and adequate numbers of animals per dose group
(75/sex/group, with interim sacrifice groups of 10/sex/group at 6 and 12 months); and reported
individual animal data. Other studies in rats using subchronic exposure durations or lower dose
levels did not observe similar effects on the urinary system as did Levine etal. T1983I and studies
in mice suggest that this species is less sensitive to RDX toxicity on the urinary system. Therefore,
incidence data from the 2-year dietary study by Levine et al. (19831 were selected for dose-
response analysis.
Prostate Effects
Suppurative prostatitis as reported in male rats in the Levine etal. f 19831 study was
selected for dose-response analysis as a biologically significant measure of prostate effects (see
Section 1.3.1 for discussion). This study included histopathologic examination of the prostate at 6-,
12-, and 24-month time points; included four dose groups and a control group, and adequate
numbers of animals per dose group (75/sex/group, with interim sacrifice groups of 10/sex/group
at 6 and 12 months); and reported individual animal data. Levine etal. f!9831. the only study to
identify an increased incidence of suppurative prostatitis associated with RDX exposure, was
selected for dose-response analysis.
2.1.2. Methods of Analysis
No biologically based dose-response models are available for RDX. In this situation, the U.S.
Environmental Protection Agency (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 BMDL using a
benchmark response (BMR) selected for each effect. A conceptual model of the analysis approach
used for RDX is provided in Figure 2-1. In this assessment, points of departure (PODs) are
identified through BMD modeling (preferred) or identification of a NOAEL, and followed by animal-
to-human extrapolation using physiologically based pharmacokinetic (PBPK) models or the
application of a dosimetric adjustment factor, depending on the data available.
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BMD modeling
(orNOAEL)
Animal PBPK
model
Dosimetric
adjustment
factor (DAF)
Human PBPK
model
POO
Animal internal dose
Animal administered
(external) dose
Human equivalent
(external) dose (HED)
Human equivalent
(externa!) dose (HED)
PBPK MODELING BW3'4 SCALING
Figure 2-1. Conceptual approach to dose-response modeling for oral
exposure.
Nervous System Effects
Incidence data for convulsions from Crouse etal. f20061 and Cholakis etal. fl9801 were
amenable to BMD modeling. For Crouse etal. f20061. statistical analysis conducted by EPA
indicated no significant difference in convulsion rates of male and female rats (exact Wald-type chi-
square test, accounting for dose; see Table 2-2); thus, combined incidence data from male and
female rats were used for modeling convulsion data from this study.
In general, there is a strong preference to use a less severe endpoint as the basis for a
noncancer toxicity value. As discussed in the evaluation of nervous system effects (Section 1.2.1),
evidence from other seizurogenic compounds with modes of action similar to RDX suggests that
other generally subclinical cognitive and behavioral neurological effects are likely to occur at lower
RDX doses, although limited investigation has been conducted to establish such subclinical effects.
EPA guidelines indicate that a BMR of 5% or lower may be warranted for frank effects (such
as convulsions) (U.S. EPA. 2012a). EPA considered BMRs of 1 and 5% extra risk (ER) for
convulsions. A BMR of 1% ER was considered appropriate to address the severity of convulsions, a
frank effect The use of a 1% ER BMR for convulsions in Crouse etal. (2006) resulted in
extrapolation below the range of the experimental doses. More specifically, the BMD of 3.02
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mg/kg-day with a 1% BMR was below the LOAEL of 8 mg/kg-day (with a 15% response rate for
convulsions), although the BMD was not far below the dose range of 4-15 mg/kg-day used in the
study.
EPA considered the trade-off between (1) a BMR of 1% ER that addresses the severity of
convulsions, but results in extrapolation outside the experimental range, and (2) a 5% ER BMR that
may be inadequate for addressing the severity of these specific outcomes, but is more consistent
with the available data. EPA selected a BMR of 5% ER, addressing the lack of incidence data for less
severe endpoints through application of the database uncertainty factor (i.e., reflecting insufficient
investigation of less severe, subclinical, nervous system effects for RDX). See Section 2.1.3,
Derivation of Candidate Values, for further discussion of the database uncertainty factor.
Because incidence data for convulsions were not provided by Levine etal. fl9831. a NOAEL
was used as the POD for this dataset rather than a BMDL.
Table 2-2 summarizes the PODs derived for each data set More detailed BMD modeling
information is available in Appendix D; BMD and BMDL estimates for 1 and 10% ER for the selected
model (see Appendix D, Section D.1.2, Tables D-3 and D-4) are provided for comparative purposes.
Urinary System (Kidney and Bladder) Effects
Incidence data for medullary papillary necrosis in the kidney (as reported by Levine et al.
fl98311 was considered unsuitable for modeling. Aside from the lowest positive dose (which had
incidence of 1/55), only the highest dose group had a positive response (18/31 or 58%), which was
higher than a level of change considered to be minimally biologically significant (e.g., 10% ER). In
this case, because there is insufficient information to estimate the BMD (U.S. EPA. 2012a). these
data were not modeled. In the absence of sufficient information to conduct BMD modeling, a
NOAEL of 8 mg/kg-day was used as the POD for this dataset (see Table 2-2).
Incidence data for hemorrhagic/suppurative cystitis in the urinary bladder as reported by
Levine etal. (1983) were amenable to BMD modeling. The BMDS models were fit to these data
using a BMR of 10% ER, under the assumption that it represents a minimally biologically significant
level of change. Table 2-2 summarizes the POD derived using data on the incidence of
hemorrhagic/suppurative cystitis. More detailed BMD modeling information is available in
Appendix D, Section D.1.2, Table D-6.
Prostate Effects
Incidence data on suppurative prostatitis as reported by Levine etal. (1983) were amenable
to BMD modeling. A BMR of 10% ER was applied under the assumption that it represents a
minimally biologically significant level of change. Table 2-2 summarizes the POD derived using
data on the incidence of suppurative prostatitis. More detailed BMD modeling information is
available in Appendix D, Section D.1.2, Table D-7.
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Table 2-2. 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)
Admin-
istered
doseb
RDX
AUCC
RDX
Cmax"
Nervous system
Incidence of
convulsions
Crouse et al. (2006)
(90-d/gavage)
Male and
female F344
rat, combined6
Multistage 3°
5% ER
5.19
2.66
0.64
1.3
1.7
Incidence of
convulsions
Cholakis et al. (1980)
(GDs 6-19/gavage)
Female F344
rat
Quantal-
linear
5% ER
0.915
0.628
0.15
0.31
0.41
Incidence of
convulsions
Levine et al. (1983)
(2-yr/diet)
Male and
female F344
rat
LOAEL = 40 mg/kg-d; NOAEL = 8 mg/kg-df
1.9
3.9
4.3
Urinary system (kidney and bladder)
Kidney: medullary
papillary necrosis
Levine et al. (1983)
(2-yr/diet)
Male F344 rat
LOAEL = 40 mg/kg-d; NOAEL = 8 mg/kg-d8
1.9
3.9
4.3
Urinary bladder:
hemorrhagic/
suppurative cystitis
Levine et al. (1983)
(2-yr/diet)
Male F344 rat
Multistage 3°
10% ER
20.0
11.6
2.8
5.6
6.3
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Endpoint and
reference
(exposure
duration/route)
Species/sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODhed (mg/kg-d)
Admin-
istered
doseb
RDX
AUC
RDX
Cmax"
Prostate
Incidence of
suppurative
prostatitis
Levine et al. (1983)
(2-yr/diet)
Male F344 rat
LogProbit
10% ER
1.67
0.47
0.11
0.23
0.25
aFor modeling details, see Appendix D.
bPOD was converted to an HED using a standard DAF based on BW3/4. See Section 2.1.2, Methods of Analysis/
Extrapolation using BW3/4 scaling for DAFs.
cPOD was converted to an HED based on the equivalence of internal RDX dose (expressed as AUC for RDX
concentration in arterial blood) derived using PBPK models.
dPOD was converted to an HED based on the equivalence of internal RDX dose (expressed as peak RDX
concentration in arterial blood, Cmax) derived using PBPK models.
eExact Wald-type chi-square exact test for differences in convulsion incidence across sexes yielded p-value >0.05.
'Nervous system effects for male and female rats reported qualitatively; incidence of convulsions and other
nervous system effects was not reported. Therefore, available data do not support BMD modeling.
gBMD modeling was not supported for this data set; see discussion in text.
AUC = area under the curve; BW = body weight; DAF = dosimetric adjustment factor; ER = extra risk; HED = human
equivalent dose
Human Extrapolation
EPA guidance fU.S. EPA. 20111 describes a hierarchy of approaches for deriving human
equivalent doses (HEDs) from data in laboratory animals, with the preferred approach being PBPK
modeling. Other approaches can include using chemical-specific information in the absence of a
complete PBPK 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.
Candidate PODs for endpoints selected from rat and mouse bioassays were expressed as
HEDs. HEDs were derived using both PBPK modeling (with alternative measures of internal dose),
and a BW3/4 scaling approach. These approaches are outlined in Figure 2-1, and the resulting
PODhed values are presented in Table 2-2.
Extrapolation Using PBPK Modeling.
PBPK models for RDX in rats, humans, and mice have been published (Sweeney etal..
2012a: Sweeney etal.. 2012b: Krishnan et al.. 2009) based on RDX-specific data. EPA evaluated and
further developed these models for extrapolating doses from animals to humans (see Appendix C,
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Section C.1.5). In general, appropriately chosen internal dose metrics are expected to correlate
more closely with toxic responses than external doses for effects that are not occurring at the point
of contact fMcLanahan etal.. 20121. 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 two dose metrics: the area under the curve (AUC) and the peak concentration (Cmax) for
RDX concentration in arterial blood. The AUC represents the average blood RDX concentration for
the exposure duration normalized to 24 hours and the Cmax represents the maximum RDX
concentration for the exposure duration.
Ideally, use of RDX concentrations in the brain would serve as the internal dose metric for
analyzing convulsion data. However, 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 predictions
of RDX blood concentrations, since the model is flow-limited and no metabolism is assumed in that
organ. Greater confidence was placed in model estimates of blood AUC than peak blood
concentrations because, as discussed in Appendix C, Section C.1.5, the rate constant for oral
absorption (KAS) is uncertain, and peak concentrations are more sensitive to variations in this
parameter than average values. RDX-induction of convulsions and seizures appears to be more
strongly correlated with dose than exposure duration, which might argue for use of peak blood
concentration as an appropriate dose metric; however, biological support for blood AUC, rather
than peak blood concentration, comes from: (1) mechanistic information on RDX binding at the
picrotoxin convulsant site of the gamma-amino butyric acid (GABA) channel; and (2) observations
from animals studies of convulsions occurring only after repeated exposures. There is evidence
from examination of picrotoxin binding to GABAAthat a resulting period of elevated neuronal
activity post-exposure could result in increased likelihood of seizures developing over time or other
longer-term effects on normal brain function (see Section 1.2.1 for further discussion). Also, as
discussed in Section 1.2.1, the range of time to onset of the first observed convulsion in the Crouse
etal. (20061 90-day study was as early as day 10 to as late as day 87, indicating a possible
cumulative component of RDX neurotoxicity not accounted for in the currently available
mechanistic studies. Therefore, the AUC for RDX concentration in arterial blood was selected as the
internal dose metric for analyzing dose-response data for convulsions. Tissue-specific dose metrics
for kidney, bladder, and prostate were not available in the PBPK model. Because effects in these
organs were observed only after subchronic or chronic exposure to RDX (i.e., there is no evidence
that effects are associated with peak exposure) and because greater confidence was placed in
model estimates of blood AUC, AUC for RDX concentration in arterial blood was also selected as the
internal dose metric for analyzing dose-response data for the kidney, urinary bladder, and prostate.
PODhed values based on both blood AUC and peak blood concentration (Cmax) are presented in Table
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2-2 for completeness. As demonstrated in Table 2-2, the PODhedS derived using administered dose,
AUC, and Cmax do not differ greatly; thus, the selection of AUC is not a major determinant of the POD.
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 (internal) 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.
Because the sequence of the calculation does not influence the results, applying the PBPK model
after BMD modeling is more efficient—BMD modeling would not have to be redone if there were
changes to the PBPK model, and it is easier to evaluate and show two dose metrics (as discussed
above). Because of relatively high confidence in the PBPK models developed for the rat and human,
these models were used to derive reliable internal dose metrics for extrapolation. For datasets
selected from the rat bioassays, the candidate oral values were calculated assuming cross-species
toxicological equivalence of the AUC of RDX blood concentration derived from PBPK modeling.
Extrapolation Using BW3/4 Scaling.
HEDs were also calculated using a BW3/4 scaling approach consistent with EPA guidance
fU.S. EPA. 20111. PODs (BMDLs or NOAELs) based on the RDX dose administered in the
experimental animal study were adjusted by a standard dosimetric adjustment factor (DAF)
derived as follows:
DAF = (BWaVVBWh1/4),
where
BWa = animal body weight
BWh = human body weight
Using BWa values of 0.25 kg for rats and 0.036 kg for mice and a BWh of 70 kg for humans
(U.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 2-2):
PODhed = laboratory animal dose (mg/kg-day) x DAF
Further details of the BMDL modeling, BMDS outputs, and graphical results for the best fit
model for each dataset included in Table 2-2 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.1.5. Table 2-2 summarizes the results of the BMD modeling and the PODhed for each data
set discussed above.
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2.1.3. Derivation of Candidate Values
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 when determining the application of UFs to the PODs presented in
Table 2-2. 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. The available
human pharmacokinetic data are not sufficient to inform human kinetic variability and derive a
chemical-specific UF for intraspecies uncertainty.
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. 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 external doses in
humans (see rationale in Section 2.1.2—Human Extrapolation). 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, of 1 was applied to all PODs. Where a POD
was based on a 2-year bioassay (i.e., for urinary system [kidney/urinary bladder] and prostate), a
UFs was not necessary because the RfD was based on effects associated with chronic exposure.
Where a POD was based on studies of subchronic exposure to RDX (i.e., for nervous system effects),
EPA considered the application of either a UFs of 3 or 1. Although EPA guidance recommends a
default UFs of 10 on the assumption that effects in a subchronic study would occur at an
approximately 10-fold higher concentration than in a corresponding (but absent) chronic study
(U.S. EPA. 20021. the RDX database does not support a UFS of 10. This determination is based on
the MOA for nervous system effects and the support across studies that nervous system effects are
more strongly driven by dose than duration of exposure (see Section 1.2.1). The argument for the
application of a UFs of 3 is based on some remaining uncertainty regarding the potential for effects
to accumulate over time. While the MOA strongly suggests that the convulsive effects of RDX are
driven by the transient binding of RDX to target (GABA) receptors in the brain, the lack of complete
reversibility of the inhibited GABAergic signaling after removal of RDX in vitro by Williams et al.
(20111. as well as observations from related chemicals suggesting that prolonged decreases in
inhibitory tone might predispose nervous system tissues to future seizurogenic events (see Section
1.2.1), introduce the possibility that mechanisms leading to cumulative effects over time have not
been adequately investigated. The application of a UFs of 1 is supported by the findings across most
studies that convulsions occurred shortly after dosing (minutes to hours) and generally did not
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appear to be appreciably influenced by duration of exposure. For example, convulsive effects were
observed after a single RDX dose of 12.5 mg/kg-day (the lowest dose tested in this experiment),
and several animals exhibited reduced seizure thresholds to other convulsants at 10 mg/kg-day
(again, the lowest dose tested) fBurdette et al.. 19881. These doses are comparable to the LOAEL of
8 mg/kg-day from the 90-day study by Crouse etal. f20061. Similarly, at 12 and 15 mg/kg-day in
the 90-day study by Crouse etal. (2006). neurotoxic signs were present in >80% of animals
beginning on day 0 and continuing for the duration of the experiment. Convulsions in Crouse et al.
(2006) were not observed until 7 to 15 days of exposure at doses of 10-15 mg/kg-day, and only
after 48 days of exposure in rats receiving 8 mg/kg-day RDX flohnson. 2015al. In a 14-day range-
finding study in 6 animals/group, Crouse etal. f20061 reported that neuromuscular signs (tremors,
convulsions) were observed at 17 mg/kg-day and above (the next lower dose was 8.5 mg/kg-day).
Thus, studies with comparable dosing methods reported seizure-related effects within the narrow
range of 8-17 mg/kg-day, regardless of exposure duration (acute to subchronic). Data from
chronic rodent studies (Lish etal.. 1984: Levine etal.. 1983) leads to identification of a higher
effective dose range (>35 mg/kg-day) for convulsions than these acute and subchronic exposure
studies (and would result in the identification of a higher POD). However, because of cross-study
differences in methods of outcome measurement, peak internal dose from gavage administration
versus dietary administration, physical form of RDX (e.g., particle size), and dose matrix in the
dietary versus gavage preparations that could have influenced absorption rate and internal (e.g.,
peak) RDX dose, direct comparison of the effective convulsive doses from the available subchronic
and chronic studies is not appropriate. Overall, evaluation of the available evidence leaves open the
possibility for a small influence of chronic (as compared to subchronic) RDX exposure duration on
the manifestation of neurotoxicity; however, current data suggest that any such influence
specifically on convulsions would be small and as such a UFs of 3 would not be warranted. Based on
the strong evidence supporting a negligible-to-minimal impact of exposure duration on the effective
dose for convulsions, a UFs of 1 was applied to PODs for neurotoxic effects derived from studies of
less-than-chronic duration.
A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied to all POD values because every
POD was a BMDL or a NOAEL. When the POD is a BMDL, the current approach is to address this
factor as one of the considerations in selecting a BMR for BMD 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 10 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. As discussed
below, some uncertainty is associated with characterization of the RDX neurotoxicity.
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EPA prefers to identify reference values based on upstream (less severe) effects that would
precede frank effects like convulsions, and uncertainty remains in the understanding of RDX-
induced neurotoxicity. In part, this is due to limitations in study design to assess neurotoxicity
across the RDX database; the frequency of animal observations in the available studies raises
concerns that there may be underreporting of the true incidence of convulsions, and in general the
reporting of this effect does not include a measure of the severity at the time of observation. No
follow-up studies were identified that employed more sensitive assays to assess more subtle
neurotoxicity, and the database lacks a chronic duration study that could inform residual
uncertainty regarding the potential for chronic exposure to magnify effects (as compared to
subchronic exposure). As noted by the SAB, the convulsion endpoint in rodents does not capture
the breadth of potential human hazard, and the lack of information on more sensitive endpoints,
including cognitive and behavioral effects, as well as developmental neurotoxicity, is a significant
data gap. Uncertainties in the database for RDX neurotoxicity could be addressed by:
• Analysis of "seizures" using more detailed behavioral scoring methods. In the available
studies, "convulsion" or "seizure (depending on the reporting in the study) might indicate a
range of observable behaviors in response to altered brain activity, ranging from
involuntary limb and facial twitches to tonic-clonic seizures in which animals exhibit a
sustained (seconds to hours) and widespread loss of muscle control sometimes resulting in
respiratory arrest and/or death. As there are studies where convulsions occur at the same
dose as mortality, the convulsive activity in these studies is interpreted as severe. Scoring
methods quantifying the occurrence of different behavioral aspects of the RDX-induced
convulsions, such as the Racine scale fRacine. 19721. employed in Burdette etal. T19881
would provide a much more accurate, complete, and likely more sensitive measure of RDX
neurotoxicity.
• Additional electrophysiological measures of epileptiform activity. Well-established and
sensitive methods for evaluating brain activity exist These measures could not only better
describe the profile of RDX-induced convulsant activity, but could also be used to identify
and quantify sub-convulsive effects of RDX exposure (e.g., EEG spiking).
Electrophysiological characterization of the effects of RDX in vitro and in vivo has already
been demonstrated by Williams etal. (2011). Additional studies building on this work,
looking at the effects of different concentrations of RDX, could potentially identify more
sensitive measures of RDX neurotoxicity.
• A FOB conducted by Crouse etal. f20061 provides some limited information on
neurobehavioral effects associated with RDX exposure, yet the results of that study did not
identify notable effects associated with RDX exposure. While some components of the FOB
testing conducted by Crouse etal. f20061 would be expected to give a screening-level
evaluation of some stimuli-induced behaviors that have the potential to be related to
seizures (e.g., response to handling, touch, click or open field), these observational
descriptions are insensitive and are expected to have missed potential subconvulsive
effects. Additional studies addressing the potential for subconvulsive behaviors resulting
from RDX exposure would be informative. For example, Burdette etal. f!9881 examined
seizure susceptibility in gavaged male Long Evans rats, at doses >10 mg/kg; spontaneous
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seizures were already observed in this study at 12.5 mg/kg-day. Further evaluation of
seizure susceptibility at lower doses and with longer exposure durations, as well as
evaluations of potential effects of subconvulsive doses on subtler behaviors that might be
related to RDX neurotoxicity (e.g., motor, anxiety or social behaviors; learning and memory
tests) may identify additional measures of RDX neurotoxicity.
• Further evaluation of potential developmental neurotoxicity associated with RDX exposure
(see Section 1.3.3 for discussion). Models for examining seizure-related behaviors during
development exist, mainly involving manipulation and analyses in pre-weanling rodents.
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. Examination of specific developmental neurotoxicity
endpoints has not been conducted in studies of RDX toxicity. Well-conducted developmental
neurotoxicity studies could further rule out the possibility that RDX exposure during
development might result in immediate or delayed seizure activity, or predispose animals
to developing seizures as adults, or it could identify other more sensitive indicators of
toxicity.
Overall, while the RDX database adequately covers major systemic effects, including
reproductive and developmental effects, uncertainties in the adequacy of the database were
identified in characterization of the neurotoxicity hazard. There is significant concern that
additional studies described above may lead to identification of a more sensitive endpoint or a
lower POD. Accordingly, a UFd of 10 was applied to all derived PODs.
Table 2-3 is a continuation of Table 2-2 and summarizes the application of UFs to each
PODhed to derive a candidate value for each data set. The candidate values presented in Table 2-3
are preliminary to the derivation of the organ/system-specific reference values. These candidate
values are considered individually in the selection of a representative oral reference value for a
specific hazard and subsequent overall RfD for RDX.
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Table 2-3. 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 (rat)
Incidence of convulsions
Crouse et al. (2006)
1.3
BMDLos
3
10
1
1
10
300
4.3 x 10"3
Incidence of convulsions
Cholakis et al. (1980)
0.31
BMDLos
3
10
1
1
10
300
1.0 x 10"3
Incidence of convulsions
Levine et al. (1983)
3.9
NOAEL
3
10
1
1
10
300
1.3 x 10"2
Urinary system (kidney and bladder) (rat)
Kidney: incidence of
medullary papillary necrosis
Levine et al. (1983)
3.9
NOAEL
3
10
1
1
10
300
1.3 x 10"2
Urinary bladder: incidence
of hemorrhagic/
suppurative cystitis
Levine et al. (1983)
5.6
BMDLio
3
10
1
1
10
300
1.9 x 10"2
Prostate (rat)
Incidence of prostate
suppurative inflammation
Levine et al. (1983)
0.23
BMDLio
3
10
1
1
10
300
7.6 x 10"4
aPODHED values based on data from the rat were derived using PBPK modeling, with the HED based on equivalence
of internal RDX dose expressed as AUC for RDX concentration in arterial blood (see Section 2.1.2 and discussion of
the PBPK models above and in Appendix C, Section C.1.5).
1 Figure 2-2 presents graphically the candidate values, UFs, and PODhed values, with each bar
2 corresponding to one data set described in Tables 2-2 and 2-3.
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Convulsions; Crouse et
al. (2006)
Convulsions; Choiakis et
a I, (1980)
Convulsions; Levine et
al, (1983)
Kidney - medullary
papillary necrosis;
Levine et al, (1983}
Urinary bladder -
hemorrhagic/
suppurative cystitis;
Levine et al. (1983}
Prostate - suppurative
inflammation; Levine et
al. (1983)
~ Candidate value
• 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 2-4 distills the candidate values from Table 2-3 into a single value for each organ or
system. Organ- or system-specific reference values may be useful for cumulative risk assessments
that consider the combined effect of multiple agents acting at a common site. For example,
organ/system-specific reference values can be used to refine the hazard index (HI)25 as described
in EPA's Risk Assessment Guidance for Superfund (U.S. EPA. 1989). As noted by U.S. EPA (1989). one
limitation of the HI approach is the potential to overestimate effects when this approach is applied
to multiple chemicals that induce different types of effects or do not act by the same mode of action.
Availability of organ/system-specific references value allows risk assessors to calculate His for
chemicals acting at a common site (i.e., and thereby more likely to induce similar effects). Further,
derivation of a single reference value for a chemical based on one critical effect only fails to address
other potential health effects caused by exposure to that chemical that may occur at exposures
higher than those associated with the critical effect Derivation of organ/system-specific values for
all health effects with credible evidence addresses this limitation.
Table 2-4. Organ/system-specific RfDs and overall RfD for RDX
Effect
Basis
RfD
(mg/kg-d)
Study exposure
description
Confidence
Nervous system
Incidence of convulsions
(Crouse et al., 2006)
4 x 10"3
Subchronic
Medium
Urinary system
Incidence of kidney medullary
papillary necrosis
(Levine et al., 1983)
1 x 10"2
Chronic
Medium
Prostate
Incidence of suppurative prostatitis
(Levine et al., 1983)
8 x 10"4
Chronic
Low
Overall RfD
Nervous system
4 x 10"3
Subchronic
Medium
Nervous System Effects
The organ/system-specific RfD for nervous system effects was based on the incidence of
convulsions in F344 rats reported in Crouse etal. (2006). 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 studies. Although the candidate value derived from the developmental toxicity study in
F344 rats by Cholakis etal. fl9801 is lower (by approximately fourfold), deficiencies in the Cholakis
etal. (19801 study resulted in a candidate value with less confidence than the value derived from
25The HI is the sum of more than one hazard quotient for multiple substances and/or multiple exposure
pathways, with the hazard quotient derived as the ratio of a single chemical exposure level to an RfD (or RfC)
for that chemical Superfund (U.S. EPA. 1989).
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Crouse etal. (2006). Crouse etal. (2006) was better designed to assess the nervous system effects
of RDX, whereas Cholakis etal. fl9801 was designed as a developmental toxicity study with only
routine monitoring of clinical signs (the methods section states that "Dams were monitored daily
for toxic signs"). Crouse etal. f20061 used five dose groups (plus the control) that provided good
characterization of the dose-response curve for RDX-induced convulsions, whereas Cholakis et al.
(1980) used only three dose group (plus the control) with order of magnitude dose spacing,
resulting in a less well-defined characterization of the dose-response curve for this endpoint Lack
of uniformity/homogeneity of the dosing preparation in Cholakis etal. (1980) raised concerns
about exposure quality and the potential for under- and over-dosing animals. Cholakis etal. f 19801
noted difficulty maintaining uniform dosing suspensions; RDX concentrations in the gavage study
ranged from 36 to 501% of target concentrations. In contrast, Crouse etal. f20061 used methods to
ensure uniform dosing suspensions; actual RDX concentrations varied from 83 to 114% of target
concentrations, and the 114% suspension was adjusted to 100% before administration. In light of
evidence that nervous system effects are more strongly driven by dose than duration of exposure
(see Section 2.1.3), the wide deviations from the target doses in Cholakis etal. (1980) lead to
decreased confidence in the quantitative use of this study. Further, Crouse etal. f20061 used a
higher purity test material than did Cholakis etal. fl9801 (99.99% versus 88.6%, respectively).
Finally, the Crouse etal. f20061 study used a longer exposure duration (90 days) than did the
Cholakis etal. (1980) study (14 days), and is more representative of a chronic exposure duration.
The lower candidate value from the Cholakis etal. (1980) developmental toxicity study could
indicate that pregnant animals are a susceptible population, which could support selection of this
study as the basis for the RfD; however, as discussed in Section 1.3.3, the available studies in
pregnant and nonpregnant rats cannot be directly compared, and the available information is not
considered sufficient to identify pregnant animals as a susceptible population.
As discussed in Section 2.1.1, the 2-year dietary study by Levine et al. T19831 was also
considered for RfD derivation because the available oral studies suggest that bolus doses of RDX
received with gavage administration may induce nervous system effects at doses lower than those
resulting from dietary administration (recognizing that differences in particle size and purity of the
test material may confound direct comparisons between gavage and dietary administration).
Convulsion data from Levine etal. f 19831 yielded a PODhed threefold higher than the PODhed
derived from Crouse etal. f20061. The POD derived from the Levine etal. T19831 study is
considered less certain than that derived from Crouse etal. f20061. Levine etal. T19831 did not
provide information on the incidence of neurotoxic effects, and BMD analysis was thus not
supported (i.e., the POD was based on a NOAEL). As discussed in Section 1.2.1, the frequency of
daily observations in the Levine etal. (1983) study may not have been sufficient to provide an
accurate measure of the occurrence of nervous system effects, potentially leading to
underestimation of convulsions and other nervous system effects. For these reasons, and in light of
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the fact that data from the Levine etal. (1983) study yielded a higher POD, Levine etal. (1983) was
not used as the basis for the organ/system-specific RfD for nervous system effects.
Urinary System (Kidney and Bladder) Effects
Dose-response analysis was conducted for two data sets representing effects on the urinary
system—incidence of medullary papillary necrosis in the kidney and incidence of
hemorrhagic/suppurative cystitis in the urinary bladder, both as reported by Levine et al. (1983).
Both effects were reported primarily in high-dose male rats in this study, and both data sets yielded
similar PODhedS (3.9 and 5.6 mg/kg-day, respectively) and candidate values (1.3 x 10 2 and
1.9 x if)-2 mg/kg-day, respectively). The smaller of the two candidate values (1.3 x 102 mg/kg-day)
was selected as the organ/system-specific RfD for urinary system effects.
Prostate Effects
A single data set for prostate effects, specifically the incidence of suppurative prostatitis in
male F344 rats as reported in a 2-year dietary study by Levine etal. (1983). was brought forward
for quantitative analysis. The organ/system-specific RfD for prostate effects is based on this
dataset
2.1.5. Selection of the Overall Reference Dose
Multiple organ/system-specific reference doses were derived for effects identified as
hazards from RDX exposure, including organ/system-specific reference doses for the nervous
system, urinary system (kidney and bladder), and prostate. There is strong support for RDX as a
nervous system toxicant, with evidence for nervous system effects, and specifically convulsions,
observed in humans and in multiple experimental animal studies, in multiple species, and following
a range of exposure durations.
The organ/system-specific RfD for nervous system effects of 4 x 10"3 mg/kg-day is smaller
than the organ/system-specific RfD for urinary system effects (1 x 10"2 mg/kg-day), suggesting that
the RfD for nervous system effects is protective of effects on both organ systems. This is consistent
with findings from animal bioassays that show RDX-related effects on the kidney and urinary
bladder at exposure levels higher than those associated with convulsions.
The organ/system-specific RfD for nervous system effects is fivefold higher than the
organ/system-specific RfD for prostate effects of 8 x 10"4 mg/kg-day. Although smaller in value,
the RfD for prostate effects was not selected as the overall RfD. Evidence for dose-related effects on
the prostate comes from a single 2-year toxicity study in male rats (Levine etal.. 1983): a second
chronic study in the rat that evaluated prostate histopathology was not available, and the 2-year
study in mice (Lish etal.. 1984) did not identify similar patterns of prostate inflammation. There
are also uncertainties in the diagnosis of suppurative prostatitis. Levine etal. f!9831 do not
provide more extensive detail on the histopathological evaluation of the prostate to account for
potential variation in inflammation inherent to the different lobes of the prostate. Additionally,
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male rats in the high-dose group (40 mg/kg-day) were moved from group to individual housing
during weeks 30-40 on study, due to the high incidence of fighting. As noted by the SAB, Creasy et
al. f20121 reported that fighting may cause urogenital infections in male rats. The fighting
observed by Levine etal. f!9831. along with the change in housing conditions from the other
treatment groups, increases uncertainty in the response of the high-dose group.
Therefore, the organ/system-specific RfD of 4 x 10~3 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 the greater
confidence in the value for nervous system effects compared to urinary system and prostate effects.
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 fU.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. Comparison with Mortality LDoiS
As previously discussed, mortality was considered in discussions of other organ/system-
specific toxicity (and in particular, effects on the nervous system and kidney). EPA did not develop
an RfD for mortality because EPA generally does not develop reference values based on frank
effects such as mortality; rather, reference values are generally based on earlier (less severe)
upstream events, where possible, in order to protect against all adverse outcomes. Nevertheless,
additional analysis of mortality data was undertaken because some studies (see Table 2-5)
identified mortality at the same RDX dose that induced nervous system effects (Crouse etal. (2006):
Angerhofer et al. (1986): Cholakis etal. (1980): von Oettingen et al. (1949)).
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Table 2-5. Comparison of dose levels associated with mortality and
convulsions in selected studies
Study
Doses associated with
mortality
Doses associated with
convulsions
Crouse et al. (2006)
Rats, F344,10/sex/group
0, 4, 8,10,12, or 15 mg/kg-d
13 wks/gavage
>8 mg/kg-d
>8 mg/kg-d
von Oettingen et al. (1949)
Rats, sex/strain not specified, 20/group
0,15, 25, or 50 mg/kg-d
13 wks/diet
>25 mg/kg-day
>25 mg/kg-d
Cholakis et al. (1980)
Rats, F344, 24-25 females/group
0, 0.2, 2.0, or 20 mg/kg-d
GDs 6-19/gavage
20 mg/kg-d
Primarily 20 mg/kg-d; 1
convulsion at 2 mg/kg-d
Angerhofer et al. (1986)
Rats, Sprague-Dawley, 39-51 mated females/group
0, 2, 6, or 20 mg/kg-d
GDs 6-15/gavage
Primarily at 20 mg/kg-d, but
one death each at 2 and
6 mg/kg-d
20 mg/kg-d
A discussion of mortality evidence for RDX is presented in Appendix C, Section C.3.1, and the
relationship between mortality and nervous system effects in Sections 1.2.1 and 1.3.1. Unscheduled
deaths were observed as early as day 8 of a 90-day gavage study (Crouse etal.. 2006) and in
developmental toxicity studies with exposure durations of two weeks (Angerhofer et al. (19861:
Cholakis etal. (198011.
Given the proximity in the dose at which mortality and nervous system effects were
observed in several studies, the dose-response relationships for mortality were compared across
studies with durations similar to those in Table 2-5 by comparing the LDoi (the dose expected to be
lethal to 1% of the animals) or NOAELs derived from each study. A BMR of 1% ER was used for
modeling mortality data in light of the severity of this frank effect. In addition, the LDoi values and
NOAELs for mortality were compared to BMD0i for convulsions.26 For purposes of this analysis, a
BMR of 1% ER was selected for convulsions (rather than 5% ER used in the analysis to derive the
nervous system RfD) to facilitate comparison with the LDoi values for mortality.
Interpretation of mortality data from chronic exposure studies in mice and rats is
complicated by other treatment-related effects and pathology regularly observed in aging animals
26BMDs were compared, as opposed to BMDLs, because, as stated on p. 20 of the BMD Technical Guidance
(U.S. EPA. 2012a). "In general, it is recommended that comparisons across chemicals/studies/endpoints be
based on central estimates; this is in contrast to using lower bounds for PODs for reference values..."
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1 (e.g., kidney pathology, neoplastic lesions), and was not considered in this analysis. Other studies
2 that were less informative and not considered in this analysis are not presented in Table 2-6.27
Table 2-6. Summary of dose-response evaluation for mortality following oral
exposure to RDX
Reference
(exposure duration/route)
Species/sex
Model3
BMR
LDoi
(mg/kg-d)
LDLoi
(mg/kg-d)
Diet studies
Lish et al. (1984)
(11-wk data from 2-yr
study/diet)
Male and female
B6C3Fi mouse
Not amenable to
modeling
NOAEL: 35 mg/kg-d
95% CI for response: 0-4%
Levine et al. (1981a)
(13-wk/diet)
Male and female
F344 rat,
combined
Multistage 4°
1% ER
7.9
2.2
von Oettingen et al. (1949)
(13-wk/diet)
Rats, sex/strain
not specified
Not amenable to
modeling
NOAEL: 15 mg/kg-d
95% CI for response: 0-15%
Cholakis et al. (1980)
(2-generation design/diet)
Female CD rat
Not amenable to
modeling
NOAEL: 16 mg/kg-d
95% CI for response: 0-13%
Levine et al. (1983)
(13-week data from 2-yr
study/diet)
Male and female
F344 rat
NA
(no mortality at
highest dose tested)
NOAEL: 40 mg/kg-d
95% CI for response: 0-4%
Cholakis et al. (1980)
(13-wk/diet)
Male and female
F344 rat
NA
(no mortality at
highest dose tested)
NOAEL: 40 mg/kg-d
95% CI for response: 0-25%
27The following less informative studies were not included in the analysis of early mortality:
13-week dietary study in the mouse by Cholakis et al. (1980). Mortality was observed only in the high-dose
group (257-276 mg/kg-day TWA), and the unusual dosing regimen precluded identification of a NOAEL or
LOAEL.
13-week dietary study in the dog by Hart Q9741 and 13-week study in the monkey by Martin and Hart
("19741 Both studies used small group sizes (3 animals/dose group), and no animals died on study (although
one high-dose monkey was euthanized).
6-week dietary study in the dog from the 1949 publication by von Oettingen et al. (1949). This dog study
included only one treatment group and recorded only one death.
30-day gavage study in the rat by MacPhail et al. (1985). The authors did not identify treatment-related
mortality, but reporting was limited.
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Reference
(exposure duration/route)
Species/sex
Model3
BMR
LDoi
(mg/kg-d)
LDLoi
(mg/kg-d)
Gavage studies
Crouse et al. (2006)
(90-d/gavage)
Male and female
F344 rat,
combined
Multistage 2°
1% ER
2.1
0.46
Cholakis et al. (1980)
(GDs 6-19/gavage)
Female F344 rat
Not amenable to
modeling
NOAEL: 2 mg/kg-day
95% CI for response: 0-12%
Angerhofer et al. (1986)
(GD 6-15/gavage)
Female SD rat
Multistage 3°
1% ER
1.7
0.59
Cholakis et al. (1980)
(GDs 7-29/gavage)
Female New
Zealand white
rabbit
NA
(no mortality at
highest dose tested)
NOAEL: 20 mg/kg-day
95% CI for response: 0-22%
aFor modeling details, see Appendix D, Section D.1.2, Tables D-9 to D-12.
CI = confidence interval; ER = extra risk; LD0i = dose expected to be lethal to 1% of the animals; LDLoi = lower
confidence limit on the LD0i.
Of the studies in Table 2-6, dose-response analysis was conducted for all studies that
showed an increased incidence of unscheduled deaths. LDoi values are provided in Table 2-6, and
detailed modeling results are provided in Appendix D, Section D.1.2. Mortality was observed only
at the highest dose tested at week 11 in the 2-year mouse study by Lish etal. (19841. in the 13-week
rat study by von Oettingen et al. (19491. and in the two-generation reproductive and developmental
toxicity studies by Cholakis etal. f!9801. In these cases, data were not amenable to LDoi estimation,
and a NOAEL (with a confidence interval, CI, on its associated response) was used in this
comparative analysis instead.
LDoi values for mortality in Table 2-6 range from 1.7 mg/kg-day (10-day gavage exposure
in pregnant rats) to 7.9 mg/kg-day (13-week dietary exposure in rats), with the lower values
generally from studies that administered RDX by gavage. These values may be compared to the
BMDoi for convulsions from Crouse etal. f20061 fsee Appendix D, Table D-3). The BMDoi for
convulsions of 3.0 mg/kg-day is in the middle of the distribution of calculated LDoiS, and the lowest
LDoi of 1.7 mg/kg-day is within twofold of the convulsion BMDoi of 3.0 mg/kg-day.
The NOAELs from studies where mortality was observed tend to be higher than the LDoiS.
However, NOAELs are not directly comparable to BMDoiS for several reasons. CIs for the responses
characterize some statistical uncertainty for NOAELs from studies that could not be modeled (note
that the upper bound of a CI is not directly comparable to a lower bound on a benchmark dose).
The CIs suggest that comparable 1% levels for these datasets could be lower than the NOAELs. In
addition, dose-spacing can affect the interpretation of NOAELs, such as that from the Cholakis et al.
f!9801 developmental toxicity study because of the wide (order-of-magnitude) spacing between
doses in that study (i.e., the reported NOAEL of 2 mg/kg-day [see Table 2-6] is 10-fold lower than
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the dose associated with 21% mortality (5/24 dams) at 20 mg/kg-day [see Appendix C, Table C-
10]).
In general, this comparison indicates that PODs derived from mortality data would be
similar to PODs for RDX based on convulsions. The proximity of doses associated with mortality
and nervous system effects should be taken into consideration when assessing health risks from
environmental exposures to RDX.
2.1.7. Uncertainties in the Derivation of the Reference Dose
To derive the RfD, the UF approach fU.S. EPA. 2000.19941 was applied to a PODhed based on
nervous system effects in rats exposed to RDX for a subchronic duration. UFs were applied to the
PODhed values to account for uncertainties in extrapolating from an animal bioassay to human
exposure, the likely existence of a diverse human population of varying susceptibilities, and to
address limitations in the database. For the most part, these extrapolations are carried out with
default approaches given the lack of data to inform individual steps. One exception is the use of
PBPK modeling to perform interspecies (i.e., rat to human) extrapolation. Uncertainties associated
with the PBPK models are considered in Appendix C, Section C.1.5.
Nervous system effects have been documented in multiple studies and animal species and
strains; however, some uncertainty is 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. In the study used to derive the
RfD (Crouse etal.. 2006). Tohnson (2015a) noted that convulsions were observed infrequently
outside the dosing period; more often, seizures were observed during the 2-hour (gavage) dosing
period, typically within 60-90 minutes of dosing. Similar information was not available for other
studies to assess the likelihood that observations of convulsions were missed. However, animals
were not monitored continuously during the Crouse etal. f20061 study, and investigators reported
that nearly all observed pre-term deaths in rats exposed to the three higher doses were preceded
by signs of neurotoxicity. If an animal died during the study as a result of effects on the nervous
system, convulsions preceding death could have been missed, resulting in an underestimation of
the incidence of convulsions. Conversely, attributing all mortality to neurotoxicity (i.e., all deaths
were preceded by convulsions that may not have been observed) could result in an overestimation
of the incidence of convulsions. A dose-response analysis of the combined incidence of seizures and
mortality from Crouse etal. f20061 was conducted to evaluate the impact of these assumptions, as
the true convulsion incidence would likely fall somewhere between the observed convulsion
incidence and the combined incidence of convulsions and mortality. This analysis revealed that the
PODhed of 0.24 mg/kg-day for a combined incidence of convulsions and mortality28 was similar to
28The PODhed values were derived from data in Crouse etal. (2006) using a BMR of 1% ER and PBPK
modeling (see Section 2.1.2 and discussion of the PBPK models in Appendix C, Section C.1.5).
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the PODhed of 0.28 mg/kg-day for convulsions alone (using a BMR of 1% ER for comparability to the
analysis with mortality data), indicating that potential underestimation of convulsion incidence in
the Crouse etal. f20061 study was not likely to impact the RfD.
Some uncertainty is also associated with the influence of the method of oral dosing on the
magnitude of dose required to induce nervous system effects. As noted in Section 1.2.1, gavage
administration generally induced convulsions in experimental animals at lower doses than did
dietary administration, possibly due to the bolus dose resulting from gavage administration that
could lead to comparatively faster absorption and higher peak blood concentrations of RDX. To
some extent, this uncertainty is reflected in the threefold difference in the candidate PODhed values
derived from the Crouse etal. f20061 (gavage administration) and Levine etal. f!9831 (dietary
administration) studies. A more rigorous examination of the effect of oral dosing method cannot be
performed because of the differences in test materials and study designs used in the available
gavage and dietary studies that could also have contributed to differences in response (e.g., test
article purity and particle size, number and spacing of dose groups, exposure duration, frequency of
clinical observations, and thoroughness of the reporting of observations).
Other sources of uncertainty related to the RDX database have already been discussed at
length, namely the lack of more sensitive measures of neurotoxicity than convulsions, the lack of
studies examining the potential for RDX exposure to cause developmental neurotoxicity, and the
possibility for some increase in the incidence of neurotoxic effects with cumulative exposure (see
Sections 1.3.3 and 2.1.3). The use of a BMR of 5% addresses some of the concern for quantification
of a frank effect such as convulsions, while application of a UFd of 10 addresses limitations in the
sensitivity of the neurotoxicity measures as well as the lack of a developmental neurotoxicity study.
2.1.8. 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 (U.S. EPA.
1994). The overall confidence in this RfD is medium. Confidence in the principal study (Crouse et
al.. 2006) 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 in the
dose range of interest. 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 to low. 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
Calculation of PODhed-oi based on incidence of convulsions: BMDLoi = 0.569 mg/kg-day (see Appendix D.1.2,
Table D-3); converted to PODhed-oi based on AUC for RDX in arterial blood = 0.28 mg/kg-day.
Calculation of PODhed-oi based on incidence of convulsions and mortality: BMDLoi = 0.49 mg/kg-day (see
Appendix D.1.2, Table D-5); converted to PODhed-oi based on AUC for RDX in arterial blood = 0.24 mg/kg-day.
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in rats and rabbits (including a two-generation reproductive study). Confidence in the database is
reduced largely because of (1) differences in test material used across studies, (2) uncertainties in
the influence of oral dosing methods, and (3) limitations in the available studies to fully
characterize potential neurological effects and developmental neurotoxicity. As discussed in
Section 2.1.7 and Appendix C, Section C.1.5, differences in test material formulation and particle
size may affect RDX absorption and subsequent toxicity, which in turn could influence the
characterization and integration of toxicity findings across studies. The available evidence also
suggests that bolus dosing of RDX that results from gavage administration induces neurotoxicity at
doses lower than administration in the diet, although a rigorous examination of these differences
cannot be performed with the available database. To the extent that a bolus dose of RDX, with
associated high peak blood concentrations, may not represent likely human exposure, the use of
toxicity data from a gavage (bolus dosing) study may introduce uncertainty in the RfD. Finally, as
noted in Section 1.2.1 and 1.3.3, the convulsion incidence endpointin rodents does not reflect the
spectrum of potential human hazard; the lack of information on developmental neurotoxicity, as
well as more sensitive cognitive and behavioral effects, introduces uncertainty into the derived RfD.
Reflecting high confidence in the principal study and medium to low confidence in the database,
overall confidence in the RfD is medium.
2.1.9. Previous IRIS Assessment
The previous RfD for RDX, posted to the Integrated Risk Information System (IRIS) database
in 1988, was based on a 2-year rat feeding study by Levine etal. (1983). The no-observed-effect
level (NOEL) of 0.3 mg/kg-day based on suppurative inflammation of the prostate 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 inhalation reference concentration (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.
As discussed in Section 1.3.1, the available inhalation literature does not support
characterization of the health hazards specifically associated with chronic inhalation exposure to
RDX, nor do the studies support quantitative dose-response analysis. Of the available human
epidemiological studies of RDX (West and Stafford. 1997: Ma and Li. 1993: Hathaway and Buck.
1977). none provided data that could be used for dose-response analysis. The studies by Ma and Li
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(1993) of neurobehavioral effects in Chinese workers and West and Stafford (1997) 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 T19771 found no evidence of adverse
health effects in munition plant workers (based on evaluation of liver function, renal function, and
hematology), and therefore does not identify a POD at which there would be an effect from which to
derive 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 Literature Search Strategy | Study Selection and Evaluation, a single
experimental animal study involving inhalation exposure was identified in the Defense Technical
Information Center (DTIC) database; the study is not publicly available. However, the study would
not have provided useful data on responses to inhaled RDX, as it was limited by small numbers of
animals tested, 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. While inhalation absorption of RDX particulates is a plausible route of exposure, there are
no toxicokinetic studies of RDX inhalation absorption to support an inhalation model. Therefore, a
PBPK model for inhaled RDX was not developed to support route-to-route extrapolation from the
RfD.
2.2.1. Previous IRIS Assessment
An RfC for RDX was not previously derived under the IRIS Program.
2.3. ORAL SLOPE FACTOR FOR CANCER
The oral slope factor (OSF) is a plausible upper bound on the estimate of risk per
mg/kg-day of oral exposure. The OSF can be multiplied by an estimate of lifetime exposure (in
mg/kg-day) to estimate the lifetime cancer risk.
2.3.1. Analysis of Carcinogenicity Data
As noted in Section 1.3.2, there is "suggestive evidence of carcinogenic potential" for RDX.
The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) state:
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.
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In the case of RDX, there are well-conducted studies that tested large numbers of animals at
multiple dose levels (Lish etal.. 1984: Levine etal.. 19831. making the cancer response suitable for
dose-response analysis. Considering the data from these studies, along with the uncertainty
associated with the suggestive nature of the weight of evidence, 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. (1984)
were selected for quantitative dose-response analysis. The study by Lish etal. (1984): (1) included
comprehensive histopathological examination of major organs; (2) contained four dose groups and
a control; (3) used adequate numbers of animals per dose group (85/sex/group, with interim
sacrifice groups of 10/sex/group at 6 and 12 months) and a sufficient overall exposure duration
(2 years); and (4) 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.. 2006) in order to apply more
up-to-date histopathological criteria established by Harada et al. (1999). The updated liver tumor
incidences from the PWG reanalysis of Lish etal. (1984) 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 (U.S. EPA. 2005a: McConnell etal.. 1986). In addition, the highest dose group
was excluded from the analyses because of the death of almost half the animals in that group from
overdosing. As a group, mice that survived exposure to 175 mg/kg-day RDX for 11 weeks may not
have constituted an unbiased representation of the population of animals exposed to the final high
dose of 100 mg/kg-day from week 11 to study termination at 2 years. These animals may have
been more or less sensitive to RDX than the animals in the general population, and there is no way
to determine to what degree. Therefore, this group was excluded because its tumor rates may not
have been representative of the population tumor rate at this dose. Female mouse liver and lung
tumor incidences from the Lish etal. (1984) study are summarized in Appendix D, Table D-13.
The incidence of hepatocellular carcinomas in male F344 rats from the study by Levine et al.
f!9831 and the incidence of alveolar/bronchiolar carcinomas in male B6C3Fi mice from the study
by Lish etal. (1984) were also considered for quantitative dose-response analysis. Both studies
were well-conducted, using similar study designs (described above). In both instances, the
response was less robust than the response observed in female mice from the Lish etal. (1984)
study. The hepatocellular carcinoma result in male F344 rats is based on a small number of tumors
(1/55, 0/55, 0/52, 2/55, and 2/31, respectively, atO, 0.3,1.5, 8.0, and 40 mg/kg-day), and
inferences made from such a sparse response are uncertain. There was no increased trend in
hepatocellular adenomas and carcinomas combined. The alveolar/bronchiolar carcinomas in male
B6C3Fi mice showed a positive trend; however, a positive trend was not observed when the
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incidence of adenomas and carcinomas was combined. Modeling results are provided in
Appendix D, Section D.2.3 for comparison.
2.3.2. Dose-Response Analysis—Adjustments and Extrapolation Methods
The EPA Guidelines for Carcinogen Risk Assessment fU.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 mode of action (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 (see discussion of Mechanistic Evidence in Section 1.2.7).
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. (19841. the survival curves were determined to be similar across dose groups after
excluding the high-dose group (log-rank test, p-value >0.10); 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% ER was applied to both tumor sites
in the mouse.
Given the finding of an association between RDX exposure in the female mouse and
increased tumor incidence at two tumor sites, basing the OSF 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. 1994). this assumption of independence is not considered likely to produce
substantial error in risk estimates. 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. Additional details on the MS-COMBO procedure are provided in
Appendix D, Section D.2.1.
In addition, a sensitivity analysis was conducted as recommended by the SAB in their
evaluation of the external review draft of the RDX assessment (SAB. 2017). The SAB recommended
this analysis to investigate 1) the fit of the multistage models in the low-dose region; 2) the effect of
dropping the highest dose group; and 3) the impact of low concurrent controls on model selection
and the POD estimate. The sensitivity analysis is provided in Appendix D, Section D.2.4.
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EPA's preferred approach for extrapolating results from animal studies to humans is
toxicokinetic modeling. As described in Appendix C, Section C.1.5, PBPK models for RDX in mice
and humans published by Sweeney etal. f2012bl 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 1.2.7)
point to 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 BW3/4 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. The mouse model was based on fitting both the absorption and
metabolic rate constants to a single set of blood concentration measurements. In this study, the
lowest dose that resulted in a detectable level of RDX in blood was 35 mg/kg, a dose high enough to
manifest some toxicity in the chronic mouse bioassay. At the 4-hour timepoint in this study,
measurement of blood RDX was based on results from only one of six exposed mice (the five other
data points were non-detects, excluded as an outlier, or not collected because of death) (Sweeney et
al.. 2012b). The type of additional data that increased confidence in the rat and human models (e.g.,
in vitro measurements of RDX metabolism and RDX elimination data) are not available for mice.
Consequently, confidence in the mouse model parameter values and in the calibration of the mouse
PBPK model is low. Further, there are no data to enable characterizing the fraction of RDX that is
metabolized in the mouse; this is problematic considering evidence that indicates that the role of
metabolism in RDX toxicity may differ across species (e.g., mice may have more efficient or higher
expression of the cytochrome P450 [CYP450] enzymes). Given the high sensitivity of the model to
the metabolic rate constant, the uncertainty in mouse toxicokinetics significantly decreases
confidence in using the mouse PBPK model for predicting mouse blood RDX concentrations. (See
Summary of Confidence in PBPK Models for RDX in Appendix C, Section C.1.5 for further discussion
of confidence in the mouse model.) In light of insufficient toxicokinetic information to identify a
supported internal dose metric and model uncertainties, the PBPK model developed for the mouse
was not used. Consistent with the EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA.
2005a). the approach used to calculate an HED from the mouse tumors, in the absence of a suitable
PBPK model, was 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 was 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 fU.S. EPA. 19921. where the body
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1 weight for the mouse is 0.036 kg and the reference body weight for humans is 70 kg (U.S. EPA.
2 19881. Details of the BMD modeling can be found in Appendix D, Section D. 2.
3 2.3.3. Derivation of the Oral Slope Factor
4 The lifetime cancer OSF for humans is defined as the slope of the line from the BMR (10%
5 ER) at the BMDL to the estimated control response at zero (OSF = 0.1/BMDLio-hed). This slope, a
6 95% upper confidence limit on the true slope, represents a plausible upper bound on the true slope
7 or risk per unit dose. The PODs estimated for each mouse tumor site are summarized in Table 2-7.
8 Using linear extrapolation from the BMDLio-hed, human equivalent OSFs were derived for each
9 tumor site individually and both sites combined and are listed in Table 2-7.
Table 2-7. Model predictions and OSFs for hepatocellular and
alveolar/bronchiolar adenomas or carcinomas in female B6C3Fi mice
administered RDX in the diet for 2 years (Lish etal.. 19841
Tumor type
Selected
model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
BMDl0-HEDb
(mg/kg-d)
POD =
BMDLio-hedc
(mg/kg-d)
OSFd
(mg/kg-d)"1
Hepatocellular
adenomas or
Multistage 1°
10% ER
25.5
14.2
3.81
2.12
0.047
carcinomas6
Alveolar/
bronchiolar
adenomas or
Multistage 1°
10% ER
29.9
14.9
4.47
2.23
0.045
carcinomas
Liver + lung tumors
Multistage 1°
(MS-COMBO)
10% ER
13.8f
V)
LO
00
2.06
1.28
0.078
aThe highest dose was dropped prior to analysis (see Section 2.3.1).
"BMDio-hed = BMDio x (BWa1/4/BWh1/4), where BWa = 0.036 kg, and BWh = 70 kg.
cBMDLio-hed = BMDLio x (BWa1/4/BWh1/4), where BWa = 0.036 kg, and BWh = 70 kg.
dOSF = BMR/BMDLlo-hed, where BMR = 0.1 (10% ER).
incidences of female mouse liver tumors from Lish et al. (1984) are those reported in the PWG reevaluation
(Parker et al., 2006).
fData for hepatocellular adenomas and carcinomas and for liver and lung tumors combined were remodeled using
the original sample sizes provided in Lish et al. (1984), which were slightly different for two groups than those
reported in Parker et al. (2006). The resulting BMDs and BMDLs from the remodeling were 25.7 and
14.3 mg/kg-day, respectively, for hepatocellular adenomas and carcinomas and 13.8 and 8.56 mg/kg-day,
respectively, for liver and lung tumors combined. See Appendix D, Table D-16 and the subsequent MS-COMBO
results for details.
10 An OSF was derived from the BMDLio-hed based on a significantly increased trend in the
11 incidence of hepatocellular and alveolar/bronchiolar adenomas or carcinomas in female B6C3Fi
12 mice (i.e., the Liver + Lung BMDLio-hed from MS-COMBO). The OSF of 0.08 (mg/kg-day)-1 is
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1 calculated by dividing the BMR (10% ER) by the Liver + Lung BMDLio-hed and represents an upper
2 bound on cancer risk per unit dose associated with a continuous lifetime exposure:
OSF = 0.10 4- (Liver + Lung) BMDLio-hed = 0.10 4 1.28 mg/kg-day
= 7.8 x 10"2 (mg/kg-day)-1
= 8 x 10"2 (mg/kg-day)-1, rounded to one significant figure
3 The slope of the linear extrapolation from the central estimate of exposure associated with
4 10% extra cancer risk (BMDio-hed) from the same data sets is given by:
Slope of the linear extrapolation from the central estimate
= 0.10 4- (Liver + Lung) BMDio-hed = 0.10 4- 2.06 mg/kg-day
= 4.9 x 10"2 (mg/kg-day)-1
= 5 x 10"2 (mg/kg-day)-1 (rounded to one significant figure)
5 The OSF for RDX should not be used with exposures exceeding the POD (1.28 mg/kg-day),
6 because above this level, the fitted dose-response model better characterizes what is known about
7 the carcinogenicity of RDX.
8 2.3.4. Uncertainties in the Derivation of the Oral Slope Factor
9 A number of uncertainties underlie the cancer unit risk for RDX. Table 2-8 summarizes the
10 impact on the assessment of issues such as the use of models and extrapolation approaches
11 particularly those underlying the Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al. the
12 effect of reasonable alternatives, the approach selected, and its justification.
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Table 2-8. 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 female mice/dose
group for all groups besides the highest dose group).
Tumor data from the mouse provided a stronger
basis for estimating the OSF than rat data.
Confidence in the OSF based on rat data was low
because of the small numbers of tumors.
Species/sex
Use of data sets from the male
mouse or male rat would
provide a lower OSF
OSF based on tumors
in female B6C3Fi
mouse
It is assumed that a positive tumor response in
animal cancer studies indicates that the agent can
have carcinogenic potential in humans in the
absence of data indicating that 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 sex would be
most relevant for extrapolating to humans, tumor
data from the most sensitive species and sex were
selected as the basis for the OSF. Other data sets
would provide smaller OSF values, and are not
considered any more or less relevant to humans
than data from the female mouse (i.e., 0.017 per
mg/kg-day based on hepatocellular carcinomas in
male F344 rats, and 0.027 per mg/kg-day based on
alveolar/bronchiolar carcinomas in male B6C3Fi
mice; see Appendix D, Section D.2.3).
Combined tumor types
Human risk would be
underestimated if OSF was
based on analysis using only a
single tumor type
OSF based on liver
and lung tumors in
female B6C3Fi
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 (see
Appendix D, Section D.2.1). Because there is no
known biological dependence between the liver and
lung tumors, independence between the two tumor
sites was assumed. NRC (1994) considered the
assumption of independence in incidence between
tumor types to be reasonable when no evidence
exists to the contrary.
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: administered
dose used
EPA evaluated a published PBPK model in the mouse
(Sweenev et al., 2012b); maior uncertainties
associated with limited toxicokinetic data in the
mouse and unknown differences in metabolism
across species were identified. Although EPA's
preferred approach for extrapolating results from
animal studies to humans is toxicokinetic modeling,
the uncertainties associated with use of the mouse
PBPK model for RDX were considered higher than
use of administered dose.
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Consideration and impact on
cancer risk value
Decision
Justification
Cross-species scaling
Alternatives could 4^ or T* OSF
(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 AUC, 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 not expected over- or underestimate
human equivalent risks.
BMD model uncertainty
Alternative models could 4^ or
-t OSF
Use multistage
model to derive a
BMD and BMDLfor
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, as it allows for the statistical
plausibility of low-dose linearity (see Appendix D,
Section D.2.4), and is the model most consistently
used in EPA cancer assessments (Gehlhaus et al.,
2011). A sensitivity analysis using multistage and
non-multistage models, with the highest dose
dropped, revealed that the multistage models were
the best-fitting or near best-fitting models for both
liver and lung tumors.
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 OSF)
Lower bound is 95% CI on administered exposure at
10% ER of liver and lung tumors.
Sensitive subpopulations
1" OSF to an unknown extent
Considered
qualitatively
There is little information on whether some
subpopulations may be more or less sensitive to the
potential carcinogenicity of RDX (i.e., because of
variability in toxicokinetics or toxicodynamics for
RDX). The mode of carcinogenic action for liver and
lung tumors in experimental animals is unknown,
and little information is available on RDX
metabolites or variation in metabolic rates that
could be used to evaluate human variability in
cancer response to RDX.
Historical control
OSF changes no more than
twofold if mean historical
control tumor rates (from NTP)
used rather than concurrent
control rates
Concurrent control
rate used in BMD
modeling and to
drive OSF
The concurrent control liver tumor rate (1.5%) was
at the low end of the range (0-20%) for historical
controls from NTP studies (Haseman et al., 1985).
Concurrent control is generally preferred to
historical control in BMD modeling, especially where
historical control data come from a different
laboratory. See Appendix D, Section D.2.4, and Table
D-29.
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2.3.5. Previous IRIS Assessment: Oral Slope Factor
The previous cancer assessment for RDX was posted to the IRIS database in 1990. The OSF
in the previous cancer assessment was based on the bioassay by Lish etal. f!9841 and analysis of
data for hepatocellular adenomas or carcinomas in female mice. An OSF of 1.1 x 10"1 (mg/kg-day)"1
was derived using a linearized multistage procedure (extra risk) and scaling by body weight to the
2/3 power for cross-species extrapolation. In addition, the previous assessment dropped the high-
dose group because the dose was reduced at week 11 to address high mortality.
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 (IUR) is a plausible upper bound on the
estimate of risk per |J.g/m3 air breathed.
An IUR value was not calculated because inhalation carcinogenicity data for RDX are not
available. While inhalation absorption of RDX particulates is a plausible route of exposure, there
are no toxicokinetic studies of RDX inhalation absorption to support an inhalation model.
Therefore, a PBPK model for inhaled RDX was not developed to support route-to-route
extrapolation of an IUR 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 fU.S. EPA. 2005bl either default or chemical-specific age-dependent
adjustment factors (ADAFs) are recommended to account for early-life exposure to carcinogens
that act through a mutagenic MOA. Because no chemical-specific data on lifestage susceptibility for
RDX carcinogenicity are available, and because the MOA for RDX carcinogenicity is not known (see
Section 1.2.7), application of ADAFs is not recommended.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
R-13 DRAFT—DO NOT CITE OR QUOTE
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