EPA/635/R-16/208a External Review Draft www.epa.gov/iris Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (CASRN 121-82-4) September 2016 NOTICE This document is an External Review draft. This information is distributed solely for the purpose of pre-dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. It is being circulated for review of its technical accuracy and science policy implications. National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Washington, DC Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-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. ii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine CONTENTS AUTHORS CONTRIBUTORS REVIEWERS ..................................................................................... ix PREFACE ..........................................................................................................................................xii PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS................................................................................xv EXECUTIVE SUMMARY ................................................................................................................. xxiii LITERATURE SEARCH STRATEGY STUDY SELECTION AND EVALUATION ................................. xxviii 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 ....................................................................................................... 1-2 1.1.3. Description of Toxicokinetic Models .................................................................... 1-3 1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM ............................... 1-3 1.2.1. Nervous System Effects ........................................................................................ 1-4 1.2.2. Kidney and Other Urogenital System Effects ..................................................... 1-21 1.2.3. Reproductive and Developmental Effects .......................................................... 1-39 1.2.4. Liver Effects ........................................................................................................ 1-50 1.2.5. Carcinogenicity ................................................................................................... 1-61 1.2.6. Other Noncancer Effects .................................................................................... 1-70 1.3. INTEGRATION AND EVALUATION .................................................................................. 1-70 1.3.1. Effects Other Than Cancer.................................................................................. 1-70 1.3.2. Carcinogenicity ................................................................................................... 1-73 1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes . 1-75 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 ............................................................................................. 2-4 2.1.3. Derivation of Candidate Values .......................................................................... 2-10 2.1.4. Derivation of Organ/System-Specific Reference Doses ..................................... 2-16 2.1.5. Selection of the Overall Reference Dose ............................................................ 2-17 2.1.6. Comparison with Mortality LD01s ....................................................................... 2-18 This document is a draft for review purposes only and does not constitute Agency policy. iii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 2.1.7. Uncertainties in the Derivation of the Reference Dose ..................................... 2-22 2.1.8. Confidence Statement ........................................................................................ 2-23 2.1.9. Previous IRIS Assessment ................................................................................... 2-24 2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER........ 2-24 2.2.1. Previous IRIS Assessment ................................................................................... 2-25 2.3. ORAL SLOPE FACTOR FOR CANCER ................................................................................ 2-25 2.3.1. Analysis of Carcinogenicity Data ........................................................................ 2-25 2.3.2. Dose-Response Analysis—Adjustments and Extrapolation Methods ................ 2-26 2.3.3. Derivation of the Oral Slope Factor .................................................................... 2-27 2.3.4. Uncertainties in the Derivation of the Oral Slope Factor ................................... 2-29 2.3.5. Previous IRIS Assessment: Oral Slope Factor ..................................................... 2-31 2.4. INHALATION UNIT RISK FOR CANCER ............................................................................ 2-31 2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS ......................................... 2-31 REFERENCES .................................................................................................................................. R-1 This document is a draft for review purposes only and does not constitute Agency policy. iv DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine TABLES Table ES-1. Table ES-2. Table LS-1. Table LS-2. Organ/system-specific RfDs and overall RfD for RDX............................................... xxiv Summary of reference dose (RfD) derivation ........................................................... xxv Inclusion-exclusion criteria for health effect studies .............................................. xxxii Studies determined not to be informative because of significant issues with design, conduct, or reporting ............................................................................................. xxxiv Table LS-3. Considerations and relevant experimental information for evaluation of experimental animal studies ................................................................................... xxxv Table LS-4. Summary of experimental animal database.......................................................... xxxvii Table LS-5. Experimental animal studies considered less informative because of certain study design, conduct, or reporting limitations ....................................................................xli Table 1-1. Chemical identity and physicochemical properties of RDX ........................................ 1-1 Table 1-2. Evidence pertaining to nervous system effects in humans ........................................ 1-9 Table 1-3. Evidence pertaining to nervous system effects in animals ....................................... 1-10 Table 1-4. Evidence pertaining to kidney effects in humans ..................................................... 1-25 Table 1-5. Evidence pertaining to kidney and other urogenital system effects in animals ....... 1-25 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) ............................................................ 1-31 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) ....................................... 1-33 Table 1-8. Six-, 12-, and 24-month incidence of prostate endpoints in male F344 rats reported for statistical evaluation in Levine et al. (1983) ...................................................... 1-34 Table 1-9. Evidence pertaining to male reproductive effects in animals .................................. 1-42 Table 1-10. Evidence pertaining to reproductive and developmental effects in animals ......... 1-46 Table 1-11. Evidence pertaining to liver effects in humans ....................................................... 1-53 Table 1-12. Evidence pertaining to liver effects in animals ....................................................... 1-54 Table 1-13. Liver tumors observed in chronic animal bioassays ............................................... 1-64 Table 1-14. Lung tumors observed in chronic animal bioassays................................................ 1-67 Table 2-1. Information considered for evaluation of studies that examined convulsions .......... 2-2 Table 2-2. Summary of derivation of PODs following oral exposure to RDX ............................... 2-7 Table 2-3. Effects and corresponding derivation of candidate values ....................................... 2-14 Table 2-4. Organ/system-specific RfDs and overall RfD for RDX ............................................... 2-16 Table 2-5. Comparison of dose levels associated with mortality and convulsions in selected studies ..................................................................................................................... 2-19 Table 2-6. Summary of dose-response evaluation for mortality following oral exposure to RDX 220 Table 2-7. Model predictions and OSFs for hepatocellular and alveolar/bronchiolar adenomas or carcinomas in female B6C3F1 mice administered RDX in the diet for 2 years (Lish et al., 1984) .................................................................................................................. 2-28 Table 2-8. Summary of uncertainty in the derivation of the cancer risk value for RDX ............ 2-29 This document is a draft for review purposes only and does not constitute Agency policy. v DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine FIGURES Figure LS-1. Summary of literature search and screening process for RDX................................ xxxi Figure 1-1. Exposure response array of nervous system effects following oral exposure. ....... 1-16 Figure 1-2. Exposure-response array of kidney and other urogenital system effects. .............. 1-36 Figure 1-3. Exposure response array of male reproductive effects following oral exposure. ... 1-45 Figure 1-4. Exposure response array of reproductive and developmental effects following oral exposure. ................................................................................................................. 1-49 Figure 1-5. Exposure response array of liver effects following oral exposure........................... 1-60 Figure 2-1. Conceptual approach to dose-response modeling for oral exposure. ...................... 2-5 Figure 2-2. Candidate values with corresponding POD and composite UF. .............................. 2-15 This document is a draft for review purposes only and does not constitute Agency policy. vi DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine ABBREVIATIONS AAP ACGIH AChE ADAF AIC ALP ALT AOP AST atm ATSDR AUC BDNF BHC BMC BMCL BMD BMDL BMDS BMDU BMR BUN BW CAAC CASRN CCL CI CICAD CNS CSF CYP450 DAF DDT d.f. DMSO DNA DNX DTIC EEG EHC EPA ER FDA FOB Army ammunition plant American Conference of Governmental Industrial Hygienists acetylcholinesterase age-dependent adjustment factor Akaike’s information criterion alkaline phosphatase alanine aminotransferase adverse outcome pathway aspartate aminotransferase atmosphere Agency for Toxic Substances and Disease Registry area under the curve brain-derived neurotrophic factor beta-hexachlorocyclohexane benchmark concentration benchmark concentration lower confidence limit benchmark dose benchmark dose lower confidence limit Benchmark Dose Software benchmark dose upper bound benchmark response blood urea nitrogen body weight Chemical Assessment Advisory Committee Chemical Abstracts Service Registry Number Contaminant Candidate List confidence interval Concise International Chemical Assessment Document central nervous system cerebrospinal fluid cytochrome P450 dosimetric adjustment factor dichlorodiphenyltrichloroethane degrees of freedom dimethylsulfoxide deoxyribonucleic acid 1-nitro-3,5-dinitroso1,3,5-triazacyclohexane Defense Technical Information Center electroencephalogram Environmental Health Criteria Environmental Protection Agency extra risk Food and Drug Administration functional observational battery FUDS GABA GD GI GLP HED HERO Formerly Used Defense Sites gamma-amino butyric acid gestational day gastrointestinal good laboratory practices human equivalent dose Health and Environmental Research Online HGPRT hypoxanthine-guanine phosphoribosyltransferase HMX octahydro-1,3,5,7-tetranitro1,3,5,7-tetrazocine IARC International Agency for Research on Cancer i.p. intraperitoneal IPCS International Programme on Chemical Safety IRIS Integrated Risk Information System IUR inhalation unit risk i.v. intravenous LDH lactate dehydrogenase LOAEL lowest-observed-adverse-effect level LOD limit of detection miRNA microRNA MNX hexahydro-1-nitroso-3,5-dinitro1,3,5-triazine MOA mode of action MRL Minimal Risk Level NAPDH nicotinamide adenine dinucleotide phosphate NAS National Academy of Science NCE normochromatic erythrocyte NCEA National Center for Environmental Assessment NCI National Cancer Institute NCTR National Center for Toxicological Research NHANES National Health and Nutrition Examination Survey NICNAS National Industrial Chemicals Notification and Assessment Scheme NIEHS National Institute of Environmental Health Sciences NIOSH National Institute for Occupational Safety and Health NOAEL no-observed-adverse-effect level NOEL no-observed-effect level NPL National Priorities List NRC Nuclear Regulatory Commission NSCEP National Service Center for Environmental Publications This document is a draft for review purposes only and does not constitute Agency policy. vii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine NTP NZW OR ORD OSF OSHA PBPK PCB PCE PEL PND POD PWG RBC RDX REL RfC RfD SDMS SDWA National Toxicology Program New Zealand White odds ratio Office of Research and Development oral slope factor Occupational Safety and Health Administration physiologically based pharmacokinetic polychlorinated biphenyl polychromatic erythrocyte Permissible Exposure Limit postnatal day point of departure Pathology Working Group red blood cell Royal Demolition eXplosive (hexahydro-1,3,5-trinitro1,3,5-triazine) Recommended Exposure Limit inhalation reference concentration oral reference dose spontaneous death or moribund sacrifice Safe Drinking Water Act SGOT SGPT SLE SS TLV TNT TNX TSCATS TWA U.S. UCM UF UFA UFD UFH UFL UFS WBC WHO glutamic oxaloacetic transaminase, also known as AST glutamic pyruvic transaminase, also known as ALT systemic lupus erythematosus scheduled sacrifice Threshold Limit Value trinitrotoluene hexahydro-1,3,5-trinitroso1,3,5-triazine Toxic Substances Control Act Test Submissions time-weighted average United States of America Unregulated Contaminant Monitoring uncertainty factor animal-to-human uncertainty factor database deficiencies uncertainty factor human variation uncertainty factor LOAEL-to-NOAEL uncertain factor subchronic-to-chronic uncertainty factor white blood cell World Health Organization This document is a draft for review purposes only and does not constitute Agency policy. viii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine AUTHORS CONTRIBUTORS REVIEWERS Assessment Team Louis D’Amico, Ph.D. (Assessment Manager) Todd Blessinger, Ph.D. Ravi Subramaniam, Ph.D. Christopher Brinkerhoff, Ph.D. Contributors U.S. EPA/ORD/NCEA Washington, DC ORISE Postdoctoral Fellow at U.S. EPA/ORD/NCEA Currently at U.S. EPA/Office of Chemical Safety and Pollution Prevention Washington, DC Rob DeWoskin, Ph.D. Karen Hogan, MS Jordan Trecki, Ph.D. U.S. EPA/ORD/NCEA Washington, DC Tammy Stoker, Ph.D. Charles Wood, DVM, PhD, DACVP U.S. EPA/ORD/National Health and Environmental Research Laboratory (NHEERL) Research Triangle Park, NC Belinda Hawkins, Ph.D. Scott Wesselkamper, Ph.D. U.S. EPA/ORD/NCEA Cincinnati, OH Anne Loccisano, Ph.D. ORISE Postdoctoral Fellow at U.S. EPA/ORD/NCEA Washington, DC Production Team Maureen Johnson Vicki Soto Dahnish Shams U.S. EPA/ORD/NCEA Washington, DC Heather Carlson-Lynch, S.M., DABT Julie Melia, Ph.D., DABT Megan Riccardi, M.S. SRC, Inc., North Syracuse, NY Contractor Support Pam Ross, M.S. Robin Blain, Ph.D. Executive Direction ICF International, Fairfax, VA Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director - Retired) Michael Slimak, Ph.D. (Acting Center Director) John Vandenberg, Ph.D, (National Program Director, HHRA) Lynn Flowers, Ph.D., DABT (Associate Director for Health, currently with the Office of Science Policy) Vincent Cogliano, Ph.D. (IRIS Program Director) Gina Perovich, M.S. (IRIS Program Deputy Director) Samantha Jones, Ph.D. (IRIS Associate Director for Science) Susan Rieth, MPH (Quantitative Modeling Branch Chief) U.S. EPA/ORD/NCEA Washington, DC This document is a draft for review purposes only and does not constitute Agency policy. ix DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Internal Review Team General Toxicity/Immunotoxicity/Cancer Workgroup Epidemiology Workgroup Neurotoxicity Workgroup Pharmacokinetics Workgroup Reproductive and Developmental Toxicity Workgroup Scoping and Problem Formulation Workgroup Statistics Workgroup Toxicity Pathways Workgroup U.S. EPA/ORD/NCEA 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: Nancy Beck, American Chemistry Council 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 This document is a draft for review purposes only and does not constitute Agency policy. x DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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* U.S. Army Public Health Center American Chemistry Council 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* Marion Ehrich* Kendall Frazier* Andy Nong* Karen Regan* Evelyn Tiffany-Castiglioni* Larry Williams This document is a draft for review purposes only and does not constitute Agency policy. xi DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine PREFACE This Toxicological Review critically reviews the publicly available studies on hexahydro- 1,3,5-trinitro-1,3,5-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 a 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 http://www.regulations.gov (Docket ID No. EPA-HQ-ORD-20130430). Organ/system-specific reference values are calculated based on nervous system, kidney/ urogenital system, and male reproductive toxicity data. 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 (http://www.epa.gov/iris). Appendices containing information on assessments by other health agencies, details of the literature search strategy, toxicokinetic information, This document is a draft for review purposes only and does not constitute Agency policy. xii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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. Problem formulation materials and 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 uses (Gadagbui et al., 2012). 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 (ATSDR, 2012). According to the U.S. EPA ChemView Tool (https://java.epa.gov/chemview), the aggregate national production volume in 2011 was approximately 6.3 million pounds per year. 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 RDXcontaining munitions (ATSDR, 2012; Gadagbui et al., 2012; ATSDR, 1999, 1993, 1992). RDX is mobile in soil; leaching into groundwater has been reported in samples from military facilities (Best et al., 1999a; Godejohann et al., 1998; Bart et al., 1997; Steuckart et al., 1994; Spanggord et al., 1980a). 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, 2012d). An extensive discussion of RDX properties and fate and transport is available in U.S. EPA (2012d). Detectable levels of RDX have been observed in plants irrigated or grown with RDX-contaminated water (Best et al., 1999b; Simini and Checkai, 1996; Harvey et al., 1991). RDX has also been detected in indoor air samples from military facilities where RDX is produced (Bishop et al., 1988). 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 2015, RDX was detected in surface water, groundwater, sediment, or soil at 34 current U.S. EPA National Priorities List (NPL) sites. The NPL serves as a list of sites with known 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. xiii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine warrant cleanup. The majority of the NPL sites where RDX was listed are associated with military facilities. Based on Department of Defense records, Gadagbui et al. (2012) 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 2015, 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 2015) 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. xiv DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS The Preamble 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. 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 guidance 2 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 agenda 3 lists upcoming assessments. The IRIS program may also assess other agents in anticipation of public health needs. 1. 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 program 1 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 2. 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 needs. Scoping specifies the agents an 1IRIS program website: http://www.epa.gov/iris/. guidance documents: http://www.epa.gov/iris/basic-information-about-integrated-risk-informationsystem#guidance/. 3IRIS multiyear agenda: https://www.epa.gov/iris/iris-agenda. 2EPA This document is a draft for review purposes only and does not constitute Agency policy. xv DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 3. Identifying and Selecting Pertinent Studies 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 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 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). 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. 4. Evaluating Study Methods and Quality 4Health and Environmental Research Online: https://hero.epa.gov/hero/. “stopping rules”: https://www.epa.gov/sites/production/files/2014-06/documents/ iris_stoppingrules.pdf. 5IRIS This document is a draft for review purposes only and does not constitute Agency policy. xvi DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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, 1991). As subject-matter experts examine a group of studies, additional agentspecific 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. 5. 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 (U.S. EPA, 1994, §2.1.3) (U.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 (U.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-ofaction 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 This document is a draft for review purposes only and does not constitute Agency policy. xvii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 (U.S. EPA, 2005a, §3.3.1, §3.5), (U.S. EPA, 2005b, §5). 6. 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 (U.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. 7. 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 This document is a draft for review purposes only and does not constitute Agency policy. xviii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 (U.S. EPA, 2005a, §3.5), (U.S. EPA, 2005b, §5). If data are sufficient to ascertain the modeof-action and to conclude that it is not linear at low levels, extrapolation may use the referencevalue 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 (U.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), (U.S. EPA, 2014). 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), (U.S. EPA, 2011, 2006). For human studies, an assessment may develop exposure–response models that reflect the structure of the available data (U.S. EPA, 2005a, §3.2.1). For animal studies, EPA has developed a set of empirical (“curve-fitting”) models 6 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), (U.S. EPA, 2012, §2.2.1). 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 (U.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 (U.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 6Benchmark 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 Dose Software: http://www.epa.gov/bmds/. This document is a draft for review purposes only and does not constitute Agency policy. xix DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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. 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 lowestobserved-adverse-effect level, an uncertainty factor reflects a level judged to have no observable adverse effects. Step 5: Revise assessment. IRIS program scientists revise the assessment to address the comments from the peer review. 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. 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. 8. Process for Developing and PeerReviewing 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 7: Post final assessment. The IRIS program posts the completed assessment and a summary on its website. 9. 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 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 This document is a draft for review purposes only and does not constitute Agency policy. xx DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 assessmentspecific approaches that differ from these general procedures. or animal studies or inferred from mechanistic 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. August 2016 This document is a draft for review purposes only and does not constitute Agency policy. xxi DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine References U.S. EPA. (1991). Guidelines for developmental toxicity risk assessment (pp. 1-83). (EPA/600/FR91/001). 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/R96/009). 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-referenceconcentration-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=157668 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-derivationoral-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 This document is a draft for review purposes only and does not constitute Agency policy. xxii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine EXECUTIVE SUMMARY Summary of Occurrence and Health Effects 1 Hexahydro-1,3,5-trinitro-1,3,5-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 nervous system effects, kidney and other urogenital effects, and male reproductive effects. Results from animal studies provide suggestive evidence of carcinogenic potential for RDX based on evidence of positive trends in liver and lung tumor incidence in experimental animals. There are no data on the carcinogenicity of RDX in humans. Effects Other Than Cancer Observed Following Oral Exposure 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Nervous system effects are a human hazard of RDX exposure. Several human case reports and animal studies provide consistent evidence of an association between RDX exposure and effects on the nervous system, including seizures or convulsions, tremors, hyperirritability, hyper- reactivity, and behavioral changes. Mechanistic data support the hypothesis that RDX-induced hyperactivity and seizures likely result from inhibition of GABAergic signaling in the limbic system. Kidney and other urogenital effects are a potential human hazard of RDX exposure based on observations in 2-year oral toxicity studies of increased relative kidney weights in male and female mice and histopathological changes in the urogenital system of male rats exposed to RDX. An increased incidence of suppurative prostatitis was identified, and is considered a surrogate marker for RDX-related urogenital effects. There is no established mode of action (MOA) for RDX-related effects on the urogenital system. There is suggestive evidence of male reproductive effects associated with RDX exposure based on the finding of testicular degeneration in male mice exposed to RDX in the diet for 2 years, in the only mouse study conducted of that duration. There is no known MOA for male reproductive effects of RDX exposure. Evidence for effects on other organs/systems, including the liver and developmental effects, was more limited than for the endpoints summarized above. This document is a draft for review purposes only and does not constitute Agency policy. xxiii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Oral Reference Dose (RfD) for Effects Other Than Cancer 1 2 3 4 5 Organ-specific RfDs were derived for hazards associated with RDX exposure (see Table ES-1). These organ- or system-specific reference values may be useful for subsequent cumulative risk assessments that consider the combined effect of multiple agents acting at a common site. Table ES-1. Organ/system-specific RfDs and overall RfD for RDX Effect Nervous system 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Basis Study exposure description RfD (mg/kg-d) 3 × 10−3 Convulsions Confidence Subchronic Medium −3 Chronic Low Kidney/urogenital Suppurative prostatitis 2 × 10 Male reproductive Testicular degeneration 2 × 10−2 Chronic Low Overall RfD Nervous system effects 3 × 10−3 Subchronic Medium The overall RfD (see Table ES-2) is derived to be protective of all types of hazards associated with RDX exposure. The effect of RDX on the nervous system was chosen as the basis for the overall RfD because nervous system effects were observed most consistently across studies, species, and exposure durations, and because they represent a sensitive human hazard of RDX exposure. Evidence for effects of RDX on the kidney/urogenital system and male reproductive system is limited relative to the effects of RDX on the nervous system. Incidence of seizures or convulsions as reported in a subchronic gavage study (Crouse et al., 2006) was selected for derivation of the overall RfD as this endpoint was measured in a study that was well-conducted, utilized a test material of higher purity than other studies, and had five closely-spaced dose groups that allowed characterization of the dose-response curve. Benchmark dose (BMD) modeling was utilized to derive the point of departure (POD) for RfD derivation (expressed as the BMDL01). A 1% response level was chosen because of the severity of the endpoint. A physiologically-based pharmacokinetic (PBPK) model was used to extrapolate the BMDL01 derived from a rat study to a human equivalent dose (HED) based on RDX arterial blood concentration, which was then used for RfD derivation. The overall RfD was calculated by dividing the BMDL01-HED for nervous system effects by a composite uncertainty factor (UF) of 100 to account for extrapolation from animals to humans (3), interindividual differences in human susceptibility (10), and uncertainty in the database (3). This document is a draft for review purposes only and does not constitute Agency policy. xxiv DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Table ES-2. Summary of reference dose (RfD) derivation Critical effect Nervous system effects (convulsions) 90-d F344 rat study Crouse et al. (2006) 2 3 4 5 6 7 a Point of departurea BMDL01-HED: 0.28 mg/kg-d UF Chronic RfD 100 3 × 10−3 mg/kg-d A benchmark response (BMR) of 1% was used to derive the BMD and BMDL given the severity of the endpoint. The resulting POD was converted to a BMDL01-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. Effects Other Than Cancer Observed Following Inhalation Exposure 8 9 10 11 12 13 14 15 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. Inhalation Reference Concentration (RfC) for Effects Other Than Cancer 16 17 18 19 inhalation absorption of RDX particulates is a plausible route of exposure, there are no toxicokinetic 20 Evidence for Human Carcinogenicity An RfC for RDX could not be derived based on the available health effects data. While 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 RfC from the RfD. 21 22 23 24 potential for RDX. RDX induced benign and malignant tumors in the liver and lungs of mice (Parker 25 Quantitative Estimate of Carcinogenic Risk from Oral Exposure 26 27 28 29 30 Under EPA’s cancer guidelines (U.S. EPA, 2005a), there is suggestive evidence of carcinogenic et al., 2006; Lish et al., 1984) or rats (Levine et al., 1983) following long-term administration in the diet. The potential for carcinogenicity applies to all routes of human 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 B6C3F1 mice observed in the carcinogenicity bioassay in mice (Lish et al., 1984). 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 This document is a draft for review purposes only and does not constitute Agency policy. xxv DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 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. Considering these data along with the uncertainty associated with the suggestive nature of the weight of the evidence for RDX carcinogenicity, quantitative analysis of the mouse tumor data may be useful for providing a sense of the magnitude of potential carcinogenic risk. An oral slope factor (OSF) that considered the combination of female mouse liver and lung tumors was derived from BMD and BMDL estimates that correspond to a 10% extra risk (ER) of either tumor. The BMDL10 so derived was extrapolated to the HED using BW3/4 scaling, and an OSF was derived by linear extrapolation from the BMDL10-HED. The OSF is 0.04 per mg/kg-day, based on the liver and lung tumor response in female mice (Lish et al., 1984). Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure 11 12 13 14 15 16 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-toroute extrapolation of an IUR from the OSF. Thus, a quantitative cancer assessment was not conducted. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Little information is available on populations that may be especially vulnerable to the toxic effects of RDX. Lifestage, and in particular childhood, susceptibility has not been observed in human or animal studies of RDX toxicity. In rats, transfer of RDX from the dam to the fetus during gestation and to pups via maternal milk has been reported; however, reproductive and developmental toxicity studies did not identify effects in offspring at doses below those that also caused maternal toxicity. Data to suggest that males may be more susceptible than females to noncancer toxicity associated with RDX exposure are limited. Specifically, urogenital effects have been noted at lower doses in males than in females. Data on the incidence of convulsions and mortality provide some indication that pregnant animals may be a susceptible population, although the evidence is unclear. 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. Key Issues Addressed in Assessment 32 33 Selection of a 1% benchmark response (BMR) for convulsions. In most instances, the spectrum of effects associated with chemical exposure will range in severity, with relatively less This document is a draft for review purposes only and does not constitute Agency policy. xxvi DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 severe effects generally occurring at doses lower than those associated with more severe or “frank” toxicity. Convulsions in rats were selected as the basis for derivation of the RDX RfD; less severe nervous system effects were generally not observed at lower doses. U.S. EPA (2012b) recommends considering the statistical and biological characteristics of the dataset when selecting a BMR, including the severity of the effect. For convulsions, a BMR level of 1% ER was selected for modeling, balancing the quantitative limitations of the available animal bioassays and the severity of this effect. Modeling convulsion incidence from Crouse et al. (2006) using this BMR resulted in a moderate extrapolation of the BMD (3.0 mg/kg-day) below the range of experimental data (dose range from Crouse et al. (2006): 4−15 mg/kg-day). Influence of the method of oral dosing (diet and gavage). Some uncertainty in the RfD is also associated with the influence of the method of oral dosing on the magnitude of dose required to induce nervous system effects. Findings from animal studies suggest that gavage administration generally induced convulsions in experimental animals at lower doses than did dietary administration, possibly due to the bolus dose received from gavage administration resulting in a comparatively faster absorption and higher peak blood concentrations of RDX (see Section 1.2.1). The difference in neurotoxic response associated with gavage versus dietary administration is in part reflected in the 14-fold difference in the candidate PODHED values derived from the Crouse et al. (2006) (gavage administration) and Levine et al. (1983) (dietary administration) studies (see Table 2-2). A more rigorous examination of the effect of oral dosing method cannot be performed because of the differences across studies in test materials and experimental designs (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) that could also have contributed to differences in response. As dietary administration is more representative of potential human exposures to RDX, the use of toxicity data from a gavage (bolus dosing) study may introduce uncertainty in the RfD. Suppurative prostatitis as a surrogate marker for kidney and other urogenital effects. The candidate RfD for kidney and other urogenital effects is based on a dose-related increase in the incidence of suppurative prostatitis from a 2-year feeding study in male F344 rats (Levine et al., 1983). This study is the only 2-year study in rats that examined the prostate. Some reports have hypothesized that the observed suppurative prostatitis is a secondary effect from a bacterial infection unrelated to RDX toxicity (ATSDR, 2012; Sweeney et al., 2012a; Crouse et al., 2006). While an opportunistic bacterial infection may have been the proximal cause of the suppurative prostatitis, the infection could have been secondary to urogenital effects associated with RDX exposure. Histopathological findings for the bladder are not definitive because the design of the principal study called for histopathological examination of the bladder only if gross abnormalities were observed. Although the pathogenesis of kidney and urogenital effects is unclear, suppurative prostatitis was considered to be a surrogate marker for the broader array of kidney and other urogenital effects observed by Levine et al. (1983). This document is a draft for review purposes only and does not constitute Agency policy. xxvii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine LITERATURE SEARCH STRATEGY STUDY SELECTION AND EVALUATION Literature Search and Screening Strategy 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 A literature search and screening strategy was applied to identify literature related to characterizing the health effects of hexadydro-1,3,5-trinitro-1,3,5-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. The detailed search approach for these databases, including the query strings, and the numbers of citations identified per database are provided in Appendix B, Table B-1. 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 1,248 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); 33 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). 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 docket for public comment (EPA-HQ-ORD-2013-0430; available at www.regulations.gov) maintained through the development of the assessment. No submissions were received in response to these calls for data. This document is a draft for review purposes only and does not constitute Agency policy. xxviii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 The citations identified using the search strategy described above were screened using the title, abstract, and in limited instances, 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. 7 The objective of this manual screen was to identify sources of primary human health effects data and sources of primary data that inform the assessment of RDX health effects (i.e., 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 in Figure LS-1) are provided in Table LS-1. Specific inclusion criteria were not applied in identifying sources of mechanistic and toxicokinetic data. The number of such studies for RDX is not large, and therefore, all studies that provided data on adsorption, distribution, metabolism, or elimination, physiologically-based pharmacokinetic (PBPK) models, or relevant RDX mode of action (MOA) were considered. Studies that met one or more of the exclusion criteria in Table LS-1 were binned as “Excluded/Not on Topic” and were not further considered in this assessment. A final group of studies consisted of reviews and other sources of RDX information (e.g., exposure, ecosystem effects) that did not meet the inclusion criteria in Table LS-1. These studies were binned into a category called “Secondary Literature and Sources of Other RDX Information,” and were considered as appropriate during development of this assessment. The results of this literature screening are described below and graphically in Figure LS-1: • • • 22 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. 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. 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 7Studies were assigned (or “tagged”) to a given category in Health and Environmental Research Online (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. This document is a draft for review purposes only and does not constitute Agency policy. xxix DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine • • 1 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. 279 references were identified as secondary literature (e.g., reviews and other agency assessments) or as studies providing potentially useful information on RDX (e.g., studies providing information on exposure levels or effects on nonmammalian species); these references were kept as additional resources for development of the Toxicological Review. 908 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). Retrieving a large number of references that are not on topic is a consequence of applying an initial search strategy designed to cast a wide net and to minimize the possibility of missing potentially relevant health effects data. This document is a draft for review purposes only and does not constitute Agency policy. xxx DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 The numbers on this figure match the HERO project page as of 5/20/2016; subsequent changes may not be reflected. A limited number of references were assigned more than one tag; therefore, the sum of the references in boxes below “Manual Screening for Pertinence” does not match exactly the total number of references in the “Combined Dataset.” Figure LS-1. Summary of literature search and screening process for RDX. This document is a draft for review purposes only and does not constitute Agency policy. xxxi DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Table LS-1. Inclusion-exclusion criteria for health effect studies Inclusion criteria Exclusion criteria Population • Humans • Standard mammalian animal models, including rat, mouse, rabbit, guinea pig, monkey, dog • Ecological speciesa • Nonmammalian speciesa 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 • Exposure via injection (e.g., intravenous [i.v.]) Outcome • Study includes a measure of one or more health effect endpoints, including effects on the nervous, kidney/urogenital, musculoskeletal, cardiovascular, immune, and gastrointestinal systems, reproduction, development, liver, eyes, and cancer Other Not on topic, including: • Abstract only, inadequately reported abstract, or no abstract, and not considered further because study was not potentially relevant • 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 • Chemical, physical, or explosive properties, including studies of RDX crystal quality, energetics characteristics, sublimation kinetics, isotope ratios, and thermal decomposition and other explosive properties • Analytical methods for measuring/detecting/ remotely sensing RDX in environmental media, and use in sample preparations and assays • Not chemical specific (studies that do not involve testing of RDX) • 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 This document is a draft for review purposes only and does not constitute Agency policy. xxxii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Inclusion criteria Exclusion criteria was considered small (e.g., Chinese paper of case reports of RDX poisonings) -- duplicate studies not previously identified 1 2 3 4 5 6 7 8 a Studies that met this exclusion criterion were not considered a source of health effects or supplementary health effects data, but were considered as other sources of information potentially useful in assessing the health effects of RDX. The documentation and results for the literature search and screen can be found on the Health and Environmental Research Online (HERO) website on the RDX project page at: (http://hero.epa.gov/index.cfm/project/page/project_id/2216). Selection of Critical Studies for Presentation in Evidence Tables Selection of Critical Studies 9 10 11 12 13 14 15 16 17 18 19 20 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, 25 were categorized as “Sources of Health Effects Data” (Figure LS-1, Table LS-1) and were considered for extraction into evidence tables for hazard identification in Chapter 1. A study was not presented in the 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 http://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. This document is a draft for review purposes only and does not constitute Agency policy. xxxiii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 Table LS-2. Studies determined not to be informative because of significant issues with design, conduct, or reporting Reference 3 4 5 6 7 8 9 10 11 12 Rationale for exclusion Haskell Laboratories (1942); 14-wk study in dogs Incomplete information on exposure levels; breed of dog was not reported; inadequate reporting of results; sections of document were illegible. von Oettingen et al. (1949); 10-wk oral study in rats No control group; strain of rat was not reported. ATSDR (1996); Disease prevalence study in residential population Study of a population residing in two neighborhoods where RDX had been detected in well water. The study was conducted 7 yrs after residents were provided the opportunity to connect to a municipal water supply. Only one target-area household reported using private well water for bathing and cooking at the time of the health study. The study was not considered informative because the design was not able to adequately define the exposed population. 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.] The health effects literature for RDX is not extensive. With the exception of the studies listed in Table LS-2 (i.e., those determined to be uninformative), all human and experimental animal studies of RDX involving repeated exposure were considered in assessing the evidence for health effects associated with chronic exposure to RDX. Studies that contain pertinent information for the toxicological review and augment hazard identification conclusions, such as genotoxicity and other mechanistic studies, studies describing the toxicokinetics of RDX, human case reports, and experimental animal studies involving exposures of acute/short-term duration or routes of exposure other than oral and inhalation, were not included in evidence tables. Nevertheless, these studies were considered, where relevant, in the evaluation of RDX health hazards. This document is a draft for review purposes only and does not constitute Agency policy. xxxiv DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Study Evaluation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For this assessment, primary sources of health effects data consisted of three human studies 8 and 21 reports 9 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, 1994). 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. Table LS-3. Considerations and relevant experimental information for evaluation of experimental animal studies Methodological feature 15 16 17 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 8Two reports with human data were determined not to be informative; see Table LS-2. The study by ATSDR (1996) was included in HERO and in Figure LS-1. The unpublished report from the DTIC database was not included in either HERO or Figure LS-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). 9The number of reports of experimental animal studies does not equal the number of studies. The results of some studies were documented in multiple reports (e.g., a 2-year study in F344 rats by Levine et al. (1983) 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. This document is a draft for review purposes only and does not constitute Agency policy. xxxv DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 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. Study evaluation considerations that are outcome specific are discussed in the relevant health effect sections in Section 1.2. Human Studies 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 The body of literature on RDX includes three studies of populations occupationally exposed to RDX (one case-control and two cross-sectional studies) (West and Stafford, 1997; 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 precise 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 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 aforementioned 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. 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 34 35 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 This document is a draft for review purposes only and does not constitute Agency policy. xxxvi DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 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). Table LS-4. Summary of experimental animal database Study category 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Study duration, species/strain, and oral administration method Chronic 2-Yr study in B6C3F1 mice (diet) (Lish et al., 1984) 2-Yr study in Sprague-Dawley rats (diet) (Hart, 1976) 2-Yr study in F344 rats (diet) (Levine et al., 1983) Subchronic 13-Wk study in B6C3F1 mice, experiment 1 (diet) (Cholakis et al., 1980) 13-Wk study in B6C3F1 mice, experiment 2 (diet) (Cholakis et al., 1980) 13-Wk study in F344 rats (diet) (Cholakis et al., 1980) 13-Wk study in F344 rats (diet) (Levine et al., 1990; Levine et al., 1981a, b) 13-Wk study in F344 rats (gavage) (Crouse et al., 2006) 13-Wk study in rats, strain not specified (diet) (von Oettingen et al., 1949) 13-Wk study in beagle dogs (diet) (Hart, 1974) 13-Wk study in monkeys (gavage) (Martin and Hart, 1974) 6-Wk study in dogs, breed not specified (diet) (von Oettingen et al., 1949) 30-D study in Sprague-Dawley rats (gavage) (MacPhail et al., 1985) Reproductive 2-Generation reproductive toxicity study in CD rats (diet) (Cholakis et al., 1980) Developmental Developmental study (gestational days [GDs] 6−19) in F344 rats (gavage) (Cholakis et al., 1980) Developmental study (GDs 6−15) in Sprague-Dawley rats, range-finding (gavage) (Angerhofer et al., 1986) Developmental study (GDs 6−15) in Sprague-Dawley rats (gavage) (Angerhofer et al., 1986) Developmental study (GDs 7−29) in New Zealand White (NZW) rabbits (gavage) (Cholakis et al., 1980) Nervous system 8-Hr study of audiogenic seizures in Long Evans rats (gavage) (Burdette et al., 1988) With the exception of two studies (Levine et al., 1990; von Oettingen et al., 1949), these toxicity studies are available only as unpublished contract laboratory reports. Peer reviews of three unpublished studies identified as most informative to the assessment of the health effects of RDX—the 2-year bioassays by Levine et al. (1983) and Lish et al. (1984) the subchronic toxicity study by Crouse et al. (2006)—were conducted by Versar, Inc. for EPA in 2012. The report of the peer reviews (U.S. EPA, 2012e) is 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 et al. (2006) provided useful information on RDX toxicity, including an array of endpoints for neurotoxicity and immunotoxicity (U.S. EPA, 2012e). Only one unpublished inhalation study of RDX (dated 1944) was identified. As discussed in Appendix B and Table LS-2, 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). Therefore, evaluation of the experimental animal database for RDX is limited to studies This document is a draft for review purposes only and does not constitute Agency policy. xxxvii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 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 3 4 5 6 7 8 9 10 11 12 13 14 15 16 The RDX database consists of health effect studies conducted in multiple strains of rats (F344, Sprague-Dawley, CD), mice (B6C3F1), 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. Several studies in the RDX database provided inadequate information on test animals. The strain of monkey (Rhesus or Cynomolgus) used in the study by Martin and Hart (1974) was not clearly specified. In one study, the breed of dog and strain of rat were unreported (von Oettingen et al., 1949). 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 are not included in evidence tables, but are discussed where relevant in the assessment. Experimental setup 17 18 19 20 21 22 23 24 25 26 27 General aspects of study design and experimental setup 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 setup, including the periodicity and duration of exposure, timing of exposure (e.g., gestational days for developmental studies), experimental group sample sizes, and interim sacrifices are summarized in the evidence tables. Note that sample size was not a basis for excluding a study from consideration, 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 28 29 30 31 32 33 34 Properties of the test material were also considered in determining whether the exposures were sufficiently specific to the compound of interest. Two properties of the RDX test materials that varied across experimental animal studies and that were taken into consideration in evaluating 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 contaminants were octahydro- 1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and water, which are the primary contaminants of RDX produced during manufacturing. The majority of studies used RDX with ~10% impurities; only This document is a draft for review purposes only and does not constitute Agency policy. xxxviii DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Crouse et al. (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 (http://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_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 µm in size), while others used a test material with comparatively coarse particle size (~200 µm 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. Endpoint evaluation procedures 23 24 25 26 27 28 29 30 31 32 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. While these studies can provide qualitative evidence of neurotoxicity, they may have underestimated the true incidence of seizures or convulsions because they were not designed to systematically evaluate neurotoxic outcomes. Outcomes and data reporting 33 34 35 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 This document is a draft for review purposes only and does not constitute Agency policy. xxxix DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 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 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 Three 2-year toxicity bioassays of RDX are available as unpublished laboratory studies (Lish et al., 1984; Levine et al., 1983; Hart, 1976). The bioassays by Levine et al. (1983) in the rat and by Lish et al. (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, limiting the ability to identify dose-related trends for tissues with incomplete histopathology. Additionally, in the mouse bioassay by Lish et al. (1984), 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 (1976) used a dose range that was lower than the Levine et al. (1983) and Lish et al. (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 et al., 2006; Angerhofer et al., 1986; MacPhail et al., 1985; Levine et al., 1981a; Cholakis et al., 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. (1949) and Levine et al. (1981a), a laboratory report of a 13-week study of RDX in F344 rats with subsets of the data subsequently published as Levine et al. (1981b) and Levine et al. (1990). The majority of studies conducted histopathological examinations on only some of the experimental groups (e.g., control and high dose). One subchronic study Crouse et al. (2006) was peer-reviewed by Versar, Inc. for EPA in 2012. The peer reviewers determined that the report provided useful information on the toxicity of RDX, including an array of endpoints for neurotoxicity and immunotoxicity (U.S. EPA, 2012e). The assessment of neurotoxicity in the study could have been improved with more histological evaluation as well as additional behavioral assessment. This document is a draft for review purposes only and does not constitute Agency policy. xl DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 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. 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. 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. 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. This document is a draft for review purposes only and does not constitute Agency policy. xli DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1. HAZARD IDENTIFICATION 1.1. Overview of Chemical Properties and Toxicokinetics 1.1.1. Chemical Properties 1 2 3 4 5 6 7 8 9 10 Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a member of the nitramine class of organic nitrate explosives (Boileau et al., 2003; Bingham et al., 2001) and is not found naturally in the environment. It has low solubility in water (Yalkowsky and He, 2003) and slowly volatilizes from water or moist soil (ATSDR, 2012). The normalized soil organic carbon/water partition coefficient (KOC) values for RDX indicate a potential for RDX to be mobile in soil (Spanggord et al., 1980a). The vapor pressure suggests that RDX will exist as particulate matter in air and be removed by both wet and dry deposition (Spanggord et al., 1980a). RDX degrades in the environment and can be subject to both photolysis (Sikka et al., 1980; Spanggord et al., 1980a) and biodegradation (Funk et al., 1993; McCormick et al., 1981) (Table 1-1). Table 1-1. Chemical identity and physicochemical properties of RDX Characteristic or property Value Reference Chemical structure NLM (2011) CASRN 121-82-4 Synonyms Hexahydro-1,3,5-trinitro-s-triazine; 1,3,5-trinitro-1,3,5-triazacyclohexane; 1,3,5-trinitrohexahydro-s-triazine; cyclonite; cyclotrimethylenetrinitramine; hexogen; cyclotrimethylenenitramine; Research Department Explosive; Royal Demolition eXplosive; RDX Color/form White, crystalline solid Bingham et al. (2001) Molecular formula C3H6N6O6 NLM (2011) Molecular weight 222.12 Lide (2005) Density (g/cm3 at 20°C) 1.82 Lide (2005) Boiling point (°C) 276−280 Bingham et al. (2001) This document is a draft for review purposes only and does not constitute Agency policy. 1-1 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Characteristic or property Value Reference Melting point (°C) 205.5 Lide (2005) Heat of formation (kJ/g) −0.277 Ryon et al. (1984) Log KOW 0.87−0.90 Hansch et al. (1995) KOC 42−167 Spanggord et al. (1980b) Henry’s law constant (atm-m3/mole at 25°C) −11 2.0 × 10 ATSDR (2012) Vapor pressure (mm Hg at 20˚C) 4.10 × 10−9 Spanggord et al. (1980a) Solubility in water (mg/L at 25°C) Yalkowsky and He (2003) 59.7 1.1.2. Toxicokinetics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 RDX is absorbed following exposure by oral and inhalation routes (see Appendix C, Section C.1.1). Studies in experimental animals indicate that oral absorption rates can range from approximately 50 to 90% (Krishnan et al., 2009; Guo et al., 1985; Schneider et al., 1978, 1977), 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 et al., 2009a; Krishnan et al., 2009; Crouse et al., 2008; Bannon, 2006; Guo et al., 1985; MacPhail et al., 1985; Schneider et al., 1977). Dermal absorption of RDX has been demonstrated in in vitro studies using human and pig skin (Reddy et al., 2008; Reifenrath et al., 2008). RDX is systemically distributed, including to the brain (i.e., RDX can cross the blood:brain barrier), heart, kidney, liver, and fat (Musick et al., 2010; Bannon et al., 2006; MacPhail et al., 1985; Schneider et al., 1977). 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 CO₂ possibly through cytochrome P450 (CYP450) (Musick et al., 2010; Major et al., 2007; Fellows et al., 2006; Bhushan et al., 2003; Schneider et al., 1978, 1977). RDX and metabolites are eliminated primarily via urinary excretion and exhalation of CO₂ (Sweeney et al., 2012a; Musick et al., 2010; Krishnan et al., 2009; Major et al., 2007; Schneider et al., 1977). Estimated elimination half-lives (t1/2) indicate that RDX is more rapidly metabolized in mice than in rats and humans; estimated t1/2 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; Özhan et al., 2003; Woody et al., 1986; Schneider et al., 1977). A more detailed summary of RDX toxicokinetics is provided in Appendix C, Section C.1. This document is a draft for review purposes only and does not constitute Agency policy. 1-2 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1.1.3. Description of Toxicokinetic Models 1 2 3 4 5 6 7 8 9 10 11 12 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 et al., 2012a; Sweeney et al., 2012b). The Sweeney et al. (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 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. (2012a) and Sweeney et al. (2012b) 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 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 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 µm 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. 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, potentially due to the reduced ability of larger particles of RDX to enter the bloodstream. 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-3 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1.2.1. Nervous System Effects 1 2 3 4 5 6 7 8 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. A 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 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 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 scores 10 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) (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 et al., 1996a; Testud et al., 1996b; Kaplan et al., 1965; Barsotti and Crotti, 1949), seizures and convulsions in exposed soldiers serving in Vietnam (Ketel and Hughes, 1972; Knepshield and Stone, 1972; Hollander and Colbach, 1969; Stone et al., 1969; Merrill, 1968), seizures, dizziness, headache, and nausea following nonwartime/nonoccupational exposures (Kasuske et al., 2009; Davies et al., 2007; Küçükardali et al., 2003; Hett and Fichtner, 2002; HarrellBruder and Hutchins, 1995; Goldberg et al., 1992), and seizures in a child following ingestion of plasticized RDX from the mother’s clothing (Woody et al., 1986). Studies in Experimental Animals Nervous system effects in experimental animals include a wide 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, 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-4 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine seizures, which result from abnormal electrical activity in the brain, can outwardly manifest in a variety of ways. While seizures can be detected in the form of convulsions, they can also manifest as facial twitches, tremors, or increased irritability, 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 methods are electrophysiological (Racine, 1972). Convulsions 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 (B6C3F1), convulsions were observed beginning at doses of 35−40 mg/kg-day, but not at lower doses (Lish et al., 1984; Levine et al., 1983; Hart, 1976). 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 (Crouse et al., 2006; von Oettingen et al., 1949). In three other rat studies 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 et al., 1985; Cholakis et al., 1980) (both unpublished technical reports). Levine et al. (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 (Levine et al., 1981a) inconsistently 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 et al., 1986; Cholakis et al., 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 (von Oettingen et al., 1949), but not 10 mg/kg-day for 1 2 3 4 5 13 weeks (Hart, 1974) (unpublished technical report), and in five of six monkeys following a gavage dose of 10 mg/kg-day (Martin and Hart, 1974) (unpublished technical report). In the only study addressing susceptibility to seizures, Burdette et al. (1988) 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. However, no audiogenic seizures were observed at the earlier 2- and 4-hour post- dosing test periods even though RDX plasma concentrations were elevated throughout the testing 11The 2-year dietary studies in F344 rats by Levine et al. (1983) and B6C3F1 mice by Lish et al. (1984) were available only as a laboratory reports. An external peer review was conducted 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 performed by Versar, Inc. is available through the EPA’s IRIS Hotline at (202) 566-1676 (phone) or hotline.iris@epa.gov (e-mail address), and on the Health and Environmental Research Online (HERO) database (U.S. EPA, 2012e). The 2-year dietary study in Sprague-Dawley rats by Hart (1976) is available as an unpublished technical report. This document is a draft for review purposes only and does not constitute Agency policy. 1-5 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 period. In a complementary experiment, Long Evans rats treated daily with 6 mg/kg-day RDX for up to 18 days required fewer stimulation trials to exhibit amygdaloid kindled seizures compared to controls. Neither the purity nor the specific particle size of the RDX used in the experiments by Burdette et al. (1988) was reported. The majority of animal studies reported convulsions and/or seizures as clinical observations; interpretation of these observations is limited to some extent because the nature and severity of convulsions and seizures were not more fully characterized. The 90-day study by Crouse et al. (2006) 12 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 duration and onset was not reported (Crouse et al., 2006). In general, gavage dosing induced convulsions at lower doses than did dietary administration. For example, in the gavage studies by Crouse et al. (2006) and Cholakis et al. (1980), convulsions were observed in 1−3 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. In contrast, in a 2-year dietary study by Levine et al. (1983), convulsions were reported 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 et al. (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 et al., 1980), 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 SpragueDawley rats exposed to RDX during gestation (Angerhofer et al., 1986). The 90-day Crouse et al. (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- 12The 13-week gavage study in F344 rats by Crouse et al. (2006) was available only as a laboratory report. An external peer review was conducted 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 (2012e) report of this peer review is available through the EPA’s IRIS Hotline at (202) 566-1676 (phone) or hotline.iris@epa.gov (e-mail address), and on the HERO database. This document is a draft for review purposes only and does not constitute Agency policy. 1-6 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 term deaths (2/20) 13 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. Some uncertainty exists in that convulsions were not typically observed during a functional observational battery (FOB) test conducted after exposure, likely due to the time needed to complete exposures prior to beginning behavioral testing. Of the 100 RDX-treated rats in the Crouse et al. (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 (Johnson, 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 Johnson (2015a) do not show as clear a correspondence between convulsions (and other neurotoxic 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, Johnson (2015a) 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 or directly associated with neurological effects (see Appendix C, Table C-10) (Angerhofer et al., 1986; Levine et al., 1981a; von Oettingen et al., 1949); however, in these studies, animals may not have been monitored for clinical observations with sufficient frequency to have observed convulsive activity prior to death. In case reports of convulsions and other nervous system effects in workers exposed to RDX during manufacture and in individuals exposed acutely as a result of accidental or intentional ingestion, there were no reports of mortality subsequent to the convulsions (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) (Levine et al., 1990; Angerhofer et al., 1986; Levine et al., 1983; Levine et al., 1981a, b; von Oettingen et al., 1949). Hyperactivity and nervousness were also reported in male mice that received a subchronic exposure to 320 mg/kg- day RDX (Cholakis et al., 1980). No changes in motor activity, flavor aversion, scheduled-controlled 13At 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-7 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 behavior, or acoustic startle response were observed in a 30-day gavage study in rats (changes in acoustic startle response in acute exposures at higher doses [12.5−50 mg/kg] were noted), but doses were relatively low (≤10 mg/kg-day) (MacPhail et al., 1985), and no significant changes in behavioral or neuromuscular activity were observed in rats following exposure to ≤15 mg/kg-day for 90 days (Crouse et al., 2006). Crouse et al. (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. Elevated absolute brain weights were reported in subchronic assays in B6C3F1 mice and F344 rats (Crouse et al., 2006; Levine et al., 1990; Levine et al., 1981a, b; Cholakis et al., 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 (Crouse et al., 2006; Levine et al., 1983; Cholakis et al., 1980), but these changes were likely a result of changes in body weight in the study, and were not a useful measure of effects of RDX on brain weights. In 2-year oral studies, a decrease in absolute brain weight of female B6C3F1 mice (3−4% relative to control) was reported at doses ≥35 mg/kg-day (Lish et al., 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 weight 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 (Sellers et al., 2007). 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 et al. (2011), who demonstrated that RDX is rapidly absorbed and crosses the blood:brain barrier following oral administration in rats, and that distribution of RDX (8 µg/g wet weight) to the brain correlated with seizure onset. 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. (1980) used a relatively large RDX particle size (200 µm) compared to the rat study by Levine et al. (1983) that used a smaller (<66 µm) particle size. This could contribute to why the Cholakis et al. (1980) 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-8 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 performed once daily in the morning in Levine et al. (1983)) may not have been sufficiently frequent to accurately measure the incidence of seizures or other nervous system effects. Table 1-2. Evidence pertaining to nervous system effects in humans Reference and study design Ma and Li (1993) (China) Cross-sectional study, 60 workers from 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 not provided). Concentration (mg/m3) (mean ± standard deviation): Group A 0.407 (± 0.332) Group B 0.672 (± 0.556) Effect measuresa: Five neurobehavioral function tests and five additional memory subtests. Analysis: Variance (F-test); unadjusted linear regression, multiple regression, and correlation analysis. Results Neurobehavioral function tests, scaled scores (mean, standard deviation) Test Control Group A Group B Memory retention* 111.3 (9.3) 96.9 (9.6) 91.1 (10.3) Simple reaction time 493 (199) 539 (183) 578 (280) Choice reaction time 763 (180) 775 (161) 770 (193) Block design* 18.0 (5.4) 16.0 (4.3) 13.5(6.7) 1,487 (343) 1,449 (331) 1,484 (443) (milliseconds) (milliseconds) (elapsed time) Letter cancellation (quality per unit time) *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) Subtest Control Group A Group B Directional memory* 23.5 (3.6) 17.2 (4.9) 18.1 (5.7) Associative learning* 24.9 (5.1) 20.0 (4.3) 18.5 (4.6) Image free recall* 24.1 (3.8) 20.9 (4.1) 20.4 (3.3) Recognition of nonsense pictures* 26.3 (3.6) 23.2 (4.9) 21.6 (4.3) Associative recall of portrait characteristics* 26.3 (3.3) 20.3 (4.4) 18.5 (4.3) *p < 0.01 (overall F-test); no statistically significant differences between Group A and Group B. Lower score indicates worse performance. Total behavioral score negatively correlated with exposure index (high exposure correlated with poor performance). 4 5 6 a Symptom 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. 1-9 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-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) One male in the 35 mg/kg-d dose group and one female in the Mice, B6C3F1, 85/sex/group; interim 175/100 mg/kg-d group convulsed near the end of the study. 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 µm 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 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) Tremors, convulsions, and hyper-responsiveness to stimuli were Rats, F344, 75/sex/group; interim noted in males and females at 40 mg/kg-d; no incidence data were sacrifices (10/sex/group) at 6 and 12 mo reported. 89.2−98.7% pure, with 3−10% HMX as contaminant; 83−89% of particles <66 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Cholakis et al. (1980) Hyperactivity and/or nervousness observed in 50% of the high-dose Mice, B6C3F1, 10−12/sex/group males; no signs observed in femalesb; no incidence data were 88.6% pure, with 9% HMX and 2.2% water reported. as contaminants; ~200 µm 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)a Diet 13 wks Cholakis et al. (1980) No nervous system effects, as evidenced by clinical signs or changes Rats, F344, 10/sex/group in appearance or behavior, were reported. 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0, 10, 14, 20, 28, or 40 mg/kg-d Diet 13 wks This document is a draft for review purposes only and does not constitute Agency policy. 1-10 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Results Cholakis et al. (1980) No nervous system effects were reported. Rats, CD, two-generation study; F0: 22/sex/group; F1: 26/sex/group; F2: 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size F0 and F1 parental animals: 0, 5, 16, or 50 mg/kg-d Diet F0 exposure: 13 wks pre-mating, and during mating, gestation, and lactation of F1; F1 exposure: 13 wks after weaning, and during mating, gestation, and lactation of F2; F2 exposure: until weaning 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 0/10 0/10 1/10 3/10 8/10 7/10 F 0/10 0/10 2/10 3/10 5/10 5/10 Tremors (incidence) M 0/10 0/10 0/10 0/10 2/10 3/10 F 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/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 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 groupsb; no incidence data were reported. This document is a draft for review purposes only and does not constitute Agency policy. 1-11 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Results Hart (1974) No nervous system effects, as evidenced by clinical signs or changes Dogs, Beagle, 3/sex/group in appearance or behavior, were reported. 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 0 0.1 1 10b Martin and Hart (1974) Monkeys, Cynomolgus or Rhesus e, 3/sex/group Purity of test material not specified 0, 0.1, 1, or 10 mg/kg-d Gavage 13 wks Doses 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. MacPhail et al. (1985) Rats, Sprague-Dawley derived CD, 8−10 males or females/group Purity 84 ± 4.7%; ≤66 μm 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. CNS effects characterized as depression, trembling, shaking, jerking, or convulsions (incidence) M 0/3 0/3 0/3 2/3 F 0/3 0/3 0/3 3/3 Cholakis et al. (1980) Doses 0 Rats, F344, 24−25 females/group Convulsions (incidence) 88.6% pure, with 9% HMX and 2.2% water as contaminants F 0/24 0, 0.2, 2.0, or 20 mg/kg-d Gavage GDs 6−19 0.2 2.0 20 0/24 1/24 18/25 Angerhofer et al. (1986) (range-finding Convulsions preceding death were observed at ≥40 mg/kg-d; study) no incidence data were reported. 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-12 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Results Angerhofer et al. (1986) Convulsions and hyperactivityb were observed at 20 mg/kg-d; Rats, Sprague-Dawley, 39−51 mated no incidence data were reported. females/group Purity 90%; 10% HMX and 0.3% acetic acid occurred as contaminants 0, 2, 6, or 20 mg/kg-d Gavage GDs 6−15 Burdette et al. (1988) Rats, Long Evans, 10−21 males/group 0, 10, 12.5, 20, 25, 50, or 60 mg/kg-d Study conducted as two experiments with the same study design, each with a control group Gavage 8 hrs (single exposure) After an 8-hr 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 Prevalence of audiogenic seizures (%)† M 0 9 0 29 25 50 60 40 82* 78* † Values estimated from graph using Grab It! Software. Statistical significance indicated by study authors; p < 0.017. Brain weight 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 µm 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 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Doses 0 1.5 7 35 175/100 Absolute brain weight (percent change compared to control) M 0% −0.2% 0.61% 0.81% −1% F 0% −2% −2% −4%* −3%* Relative brain weight (percent change compared to control) M 0% 4% 2% 2% 5% F 0% −4% −1% −3% 18%* 0 0.3 1.5 8 40 Absolute brain weight (percent change compared to control) M 0% 2% −1% 2% 2% F 0% −0.3% −0.4% 1% 2%* Relative brain weight (percent change compared to control) M 0% 0% 8% 2% 22%* F 0% −1% 3% 4% 20%* This document is a draft for review purposes only and does not constitute Agency policy. 1-13 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Cholakis et al. (1980) Mice, B6C3F1, 10−12/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm 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)a Diet 13 wks Cholakis et al. (1980) Rats, F344, 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0, 10, 14, 20, 28, or 40 mg/kg-d Diet 13 wks 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 Results Doses 0 10 14 20 28 40 Absolute brain weight (percent change compared to control) M 0% − − − 2% 2% F 0% − − − 4% 2% Relative brain weight (percent change compared to control) M 0% − − − 6% 2% F 0% − − − 0% 3% Doses 0 80 160 320 Absolute brain weight (percent change compared to control) M 0% 0% 2% 10% F 0% 0% 4% 2% Relative brain weight (percent change compared to control) M 0% −3% 1% 8% F 0% 0% 3% −4% Doses 0 10 14 20 28 40 Absolute brain weight (percent change compared to control) M 0% − − − 3% 0% F 0% − − − 0% 0% Relative brain weight (percent change compared to control) M 0% − − − 7%* 10%* F 0% − − − 5% 6% 0 4 8 10 12 15 Doses Absolute brain weight (percent change compared to control) M 0% −1% −0.3% 2% 5%* 7%* F 0% −2% 6% 1% 4% 6% Relative brain weight (percent change compared to control) M 0% 6% 10% 5% 3% 4% F 0% −2% −2% −12%* −12%* −15%* This document is a draft for review purposes only and does not constitute Agency policy. 1-14 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Levine et al. (1990); Levine et al. (1981a); Levine et al. (1981b)c Rats, F344, 10/sex/group; 30/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 0, 10, 30, 100, 300, or 600 mg/kg-d Diet 13 wks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Results Doses 0 10 30 100 300 600 Absolute brain weight (percent change compared to control) M 0% 1% 0.53% −6% − − F 0% −1% 1% 2% − − Relative brain weight (percent change compared to control) M 0% 4% 7% 14% − − F 0% 0.3% 2% 5% − − *Statistically significant (p < 0.05) based on analysis by study authors. a Doses were calculated by the study authors. b Mortality was reported in some RDX-treated groups in this study. c Levine 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. d 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). e The 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-1,3,5,7-tetranitro-1,3,5,7-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. 1-15 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 Figure 1-1. Exposure response array of nervous system effects following oral exposure.1 1Because 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-16 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Mechanistic Evidence 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 37 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 and convulsions involving distribution to the brain (across the blood:brain barrier) and subsequent effects on neurotransmitters, including gamma-amino butyric acid (GABA) and glutamate. 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, 2008). In research conducted by the U.S. Army Center for Health Promotion and Preventative Medicine, Williams et al. (2011) and Bannon et al. (2009a) 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 et al., 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 has only showed affinity for GABAA receptors (Williams et al., 2011; Williams and Bannon, 2009). Specifically, RDX showed a significant affinity for the picrotoxin convulsant site of the GABA channel. RDX treatment in brain slices from the basolateral amygdala inhibited GABAA-mediated inhibitory postsynaptic currents and initiated seizure-like neuronal discharges. RDX exposure may reduce the inhibitory effects of GABAergic neurons, resulting in enhanced excitability that could lead to seizures (Williams et al., 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 (Jefferys et al., 2012; Gilbert, 1994). Burdette et al. (1988) hypothesized that the limbic system was involved in seizures caused by RDX exposure, given that amygdaloid kindled rats (rats subjected to patterns of electrical stimulation to promote the development of seizures) exhibited pro-convulsant activity at a dose that was approximately half of the dose necessary for RDX to induce spontaneous seizures (rats treated with RDX also required fewer electrical stimulations to trigger kindled seizures). Potential limbic system involvement is also suggested given its role in integrating emotional and behavioral responses (including aggression) and the anecdotal observations of hyperactivity, hyper-responsiveness to approach, and irritability noted across several studies of RDX toxicity (Levine et al., 1990; Levine et al., 1983; Levine et al., 1981a, b; Cholakis et al., 1980; von Oettingen et al., 1949). It is possible to construct a hypothetical MOA for RDX-induced seizure activity based on the evidence summarized above. These steps are consistent with ongoing efforts to identify an adverse outcome pathway (AOP) for ionotropic GABA receptor antagonism, reviewed in Gong et al. (2015), This document is a draft for review purposes only and does not constitute Agency policy. 1-17 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 and Collier et al. (2016) and described in greater detail in the draft AOP available at https://aopwiki.org/wiki/index.php/Main_Page. Following absorption and transport to the brain: 1) Parent RDX acts as a receptor antagonist (supported by Schneider et al. (1977) and Williams et al. (2011)), binding noncompetitively to the picrotoxin convulsant site of the GABAA receptor (supported by Williams and Bannon (2009) and Williams et al. (2011)). 2) RDX binding to the GABAA receptor results in decreased conduction of chloride through the ion channel. 3) Reduced chloride conduction results in depolarization of the neuronal membrane, thereby reducing spontaneous inhibitory postsynaptic currents (sIPSCs). Williams et al. (2011) observed a reduction in the amplitude and frequency of sIPSCs in whole-cell in vitro recordings of neurons in brain slices from the rat basolateral amygdala after exposure to RDX. In addition, RDX treatment of slices inhibited GABA-induced currents. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 4) Reduction in sIPSCs results in an overall reduction in inhibitory inputs to the nervous system. Williams et al. (2011) observed a pattern of seizure-like neuronal discharges in vitro from slices of the basolateral amydgala in rats after adding RDX and noted that the effects were not reversible after 40 minutes of washout. The steps above provide a biologically plausible sequence of mechanistic events that result in the generation of seizure-like neuronal activity. Reduction of the inhibitory GABAergic signaling 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 (Vale et al., 2003; Bloomquist, 1992; Suñol et al., 1989). The interaction of RDX with the GABAA receptor is directly supported by receptor-binding assays (Williams et al., 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 (Quinn et al., 2013; McFarland et al., 2009; Johnson et al., 2007; Bruchim et al., 2005; Küçükardali et al., 2003; Woody et al., 1986; Lish et al., 1984; Berry et al., 1983; Levine et al., 1983). 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-Reyero et al., 2011). While these lines of evidence do not preclude a role of other receptors as yet unscreened for RDX binding affinity, they support involvement of the GABAergic pathway described above in the development of RDX neurotoxicity. The GABAA receptor is also a target of many anticonvulsant therapies (e.g., benzodiazepines, propofol, barbiturates) (Meldrum and Rogawski, 2007; Möhler, 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-18 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 diazepam or lorazepam) to treat seizing patients (Kasuske et al., 2009; Davies et al., 2007; Küçükardali et al., 2003; Hett and Fichtner, 2002; Woody et al., 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 (Goodman et al., 1996). 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 (Crouse et al., 2006; Burdette et al., 1988; Barsotti and Crotti, 1949). However, the limited data available for RDX do not support AChE inhibition as a primary mechanism because: (1) blood and brain levels of AChE are unaffected by RDX (Williams et al., 2011; Williams and Bannon, 2009); and (2) in vitro neurotransmitter receptor binding studies do not reveal any affinity of RDX for acetylcholine receptors (Williams et al., 2011; Williams and Bannon, 2009). Additionally, common AChE-induced symptoms (salivation and lacrimation) have not routinely been observed (Williams et al., 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 (Williams et al., 2011; Williams and Bannon, 2009). In a microarray experiment, Bannon et al. (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 B6C3F1 mice fed 5 mg RDX/kg diet (estimated dose: 0.75‒ 1.5 mg/kg-day; Bannon et al. (2009a)) 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 et al. (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). Thus, the This document is a draft for review purposes only and does not constitute Agency policy. 1-19 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 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. In general across the RDX database, induction of convulsions and seizures appears to be more strongly correlated with dose than duration of exposure. However, Gerkin et al. (2010) demonstrated that young C57/Bl6 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. It is possible that this 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, it is possible in a chronic exposure scenario that repeated exposure to RDX binding at the same site as picrotoxin, through a general increase in brain tissue with elevated neuronal activity, could increase the likelihood of seizures developing over time, or have other longer-term effects on normal brain function. Observations by Crouse et al. (2006), clarified in Johnson (2015a), showed that the median time to seizure after dosing in F344 rats is 55 minutes (range fromof 20‒85 minutes); peak brain concentrations of RDX in F344 rats after single oral doses occured within the first 3‒4 hours after dosing (Bannon et al., 2009a). However, in Crouse et al. (2006), of the rats that survived the 90-day study, the range of time to onset of first observed convulsion was as early as day 10, and as late as day 87, and 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 may indicate a cumulative component of RDX neurotoxicity not accounted for in the currently available mechanistic studies. Recent research 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, and in particular neurotoxicity observed over longer exposure durations, the demonstrated affinity of RDX for the GABAA receptor, evidence of supportive electrophysiological changes 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. The available information supports that RDX-induced hyperactivity and seizures likely result from inhibition of GABAergic signaling in the limbic system. Integration of Nervous System Effects 34 35 36 37 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, 1993), and human case reports provide other evidence of an association between acute RDX This document is a draft for review purposes only and does not constitute Agency policy. 1-20 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 effects (some severe), including seizures, convulsions, tremors, hyperirritability, hyper-reactivity, and behavioral changes, associated with RDX exposure (Crouse et al., 2006; Angerhofer et al., 1986; Levine et al., 1983; Levine et al., 1981b; Cholakis et al., 1980; von Oettingen et al., 1949). In most of these studies, the occurrence of neurological effects was dose-related. In those studies that found no evidence of RDX-associated neurotoxicity (MacPhail et al., 1985; Cholakis et al., 1980; Hart, 1976, 1974), differences in dosing, particle size, and purity of the RDX administered could possibly 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. Convulsant receptor binding leading to a decreased seizure threshold, considered with kindling studies, suggests that the effect is specific to CNS toxicity. 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. Kidney and Other Urogenital System Effects 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 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 kidney and other urogenital system effects. A summary of these effects associated with RDX exposure is presented in Tables 1-4 to 1-8 and Figure 1-2. Experimental animal studies are ordered in the evidence table and exposureresponse 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 may affect the kidney and the urogenital system. Reported symptoms included decreased urine output (Ketel and Hughes, 1972; Knepshield and Stone, 1972; Hollander and Colbach, 1969; Merrill, 1968), blood in urine (Kasuske et al., 2009; Knepshield and Stone, 1972; Hollander and Colbach, 1969; Merrill, 1968), proteinuria (Kasuske et al., 2009; Küçükardali et al., 2003; Ketel and Hughes, 1972; Hollander and Colbach, 1969; Merrill, 1968), glucosuria (Küçükardali et al., 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 (Stone et al., 1969; Kaplan et al., 1965). 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- This document is a draft for review purposes only and does not constitute Agency policy. 1-21 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 1,3,5,7-tetranitro-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 (Hathaway and Buck, 1977) (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 cumulative exposure concentrations. Studies in experimental animals provide some evidence that RDX exposure is associated with kidney and other urogenital system effects (Table 1-5 and Figure 1-2). Histopathological changes in the urogenital system were associated with exposure to RDX in a 2-year bioassay. Specifically, increased incidences of kidney medullary papillary necrosis and pyelitis, uremic mineralization, bladder distention and/or cystitis, and suppurative prostatitis were observed in high-dose (40 mg/kg-day) male rats that died spontaneously or were sacrificed in moribund condition (Levine et al., 1983). These 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 female rats in this study. An increased incidence of tubular nephrosis was observed in male B6C3F1 mice exposed to 320 mg/kg-day RDX in feed for 90 days, but not in female mice in this study (Cholakis et al., 1980). In other chronic and subchronic oral studies in rats and mice, no histopathological changes in the kidney were associated with RDX exposure (Crouse et al., 2006; Levine et al., 1990; Lish et al., 1984; Levine et al., 1981a, b; Cholakis et al., 1980; Hart, 1976). Increased incidence of minimal to mild mineralization of the medulla was observed in male and female monkeys exposed to 10 mg/kg-day RDX for 90 days by gavage (Martin and Hart, 1974), but the study authors did not identify this as treatment related. No dose-related histopathological changes were reported in a subchronic study in dogs (Hart, 1974), and no histological alterations were noted in the kidneys of 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). Measurement of serum chemistry parameters that may indicate effects on renal function, including BUN and uric acid, in studies of RDX in mice, rats, dogs, and monkeys (Crouse et al., 2008; Levine et al., 1990; Lish et al., 1984; Levine et al., 1981a, b; Cholakis et al., 1980; Hart, 1976, 1974; Martin and Hart, 1974) revealed variations (increases or decreases) from the respective control groups that were not dose-related. The findings of suppurative prostatitis provide the strongest evidence of urogenital toxicity. 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 et al., 1983). Suppurative prostatitis was not observed in 90-day studies in the rat involving oral (dietary or gavage) exposure to RDX (Crouse et al., 2006; Levine et al., 1990; Levine et al., 1981a, b). Similarly, prostate effects were not observed in a 2-year dietary study in mice (Lish et al., 1984). The Levine et al. (1983) report is the only 2-year study that reported examination of the prostate in rats. Spontaneous prostatitis in older rats has been used as a model for nonbacterial prostatitis in humans, and aging F344 rats have higher rates of background prostatitis in general (Suwa et al., This document is a draft for review purposes only and does not constitute Agency policy. 1-22 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 2001). Further, suppurative prostatitis and non-suppurative prostatitis are not mutually exclusive; one form can evolve into another. Higher background rates of prostate inflammation in F344 rats along with differences in background inflammation rates in different lobes of the prostate raise some uncertainty in the findings in Levine et al. (1983), which did not provide detailed information on the histopathology methods used to evaluate prostate lesions. Incidences of prostatitis reported by Levine et al. (1983) in the intermediate dose groups could be considered consistent with background incidence rates in F344 rats, and the incidence in the control group may have been unusually low. Some reports have hypothesized that the observation of prostate inflammation in Levine et al. (1983) is secondary to a bacterial infection unrelated to RDX toxicity (ATSDR, 2012; Sweeney et al., 2012a; Crouse et al., 2006). For example, in describing the results from the 2-year dietary study in rats, Crouse et al. (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. However, Levine et al. (1983) distinguished between nonsuppurative and suppurative inflammation (the latter being characterized by the formation of pus and a high concentration of neutrophils). Although the proportion of suppurative prostatitis was higher in SDMS rats, there was an increasing trend with dose in both the scheduled sacrifice (SS) and SDMS groups; the incidence of suppurative prostatitis in the control group was 4% when the SS and SDMS groups 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 et al. (1983) 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 (Crouse et al., 2006). 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 et al., 1987; Parker and Grabau, 1987). As noted above, Levine et al. (1983) documented an array of kidney and other urogenital lesions in their 2-year dietary exposure of F344 rats to RDX. However, the sequence by which those effects may have occurred is unclear. Renal medullary necrosis, bladder distension, and cystitis were observed mainly in the male rats exposed to 40 mg/kg-day RDX for 24 months, although one rat in the 0.3 mg/kg-day dose group also exhibited these lesions. Treatment-related effects on the kidney (necrosis) and bladder (distension/obstruction and hemorrhagic cystitis) were also identified in the 12-month pathology report (see Tables 1-6 to 1-8). The absence of these observations in the 6-month interim pathology report suggests that an exposure duration >6 months may be required before RDX-induced effects on the urogenital system are observed. This document is a draft for review purposes only and does not constitute Agency policy. 1-23 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 Suppurative prostatitis was observed with increasing incidence in each dose group in the study at 24 months. Considered as a group, one hypothesis is that treatment-related kidney and urogenital lesions may have led to a blockage that resulted in urinary stasis. Reduced urinary flow and/or retrograde flow may have contributed to an environment that allowed bacterial infection of the prostate. Thus, while an opportunistic bacterial infection could be the proximal cause of the suppurative prostatitis (ATSDR, 2012; Sweeney et al., 2012a; Crouse et al., 2006), it may have been secondary to the effects of RDX on the urogenital system. This hypothesis would be consistent with the observed dose-related increase in incidence of suppurative prostatitis. Although the ultimate sequence of effects in the urogenital system is unclear, even from review of the scheduled sacrifices at 6 or 12 months on study, it is plausible that suppurative prostatitis would occur after other kidney or bladder lesions that resulted in the initial blockage and urinary stasis. The incidence of suppurative prostatitis reported in Levine et al. (1983) was increased at doses lower than the doses associated with an increased incidence of other urogenital lesions. However, the incidence of bladder lesions may have been underreported, as the bladders were only examined following observation of a gross abnormality. Bladder distension was reported sporadically among the lower dose groups (0.3, 1.5, or 8.0 mg/kg-day), but the bladder was not routinely examined in these groups (Levine et al., 1983). Some urinary tract endpoints (luminal distension, papillary necrosis, pyelitis, pyelonephritis) showed no relationship to either suppurative prostatitis or dose of RDX in the 0.3, 1.5, or 8.0 mg/kg-day dose groups. Although the pathogenesis of kidney and urogenital system effects cannot be established, the available evidence is consistent with suppurative prostatitis as an indirect effect of RDX exposure that may correlate with other kidney/urogentical effects and can serve as a surrogate marker for the broader array of kidney and urogenital system effects observed by Levine et al. (1983). Changes in kidney weights in subchronic oral toxicity studies in rats, dogs, and monkeys did not show a clear pattern of increase or decrease associated with RDX exposure. Kidney weight changes were either not dose-related or were inconsistent across sexes when absolute and relative weights were compared (see Table 1-5). Less weight is placed on evidence of organ weight changes from chronic (2-year) studies (Lish et al., 1984; Hart, 1976) 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., 2007). 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] assessment of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine [HMX] at http://www.epa.gov/iris). No effects were observed at doses ≤115 mg/kg-day. Because the percentage of HMX as an impurity ranged from 3 to 10%, resulting 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-24 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 (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. Table 1-4. Evidence pertaining to kidney effects in humans Reference and study design 4 5 6 7 Hathaway and Buck (1977) Cross-sectional study, 2,022 workers, 1,491 participated (74% response rate). Analysis group: limited to whites; 69 workers exposed to RDX alone and 24 workers exposed to RDX and HMX, compared to 338 workers not exposed to RDX, HMX, or TNT. Exposure measures: Exposure determination based on job title and industrial hygiene evaluation; exposed subjects assigned to two groups: undetected (0.01 mg/m3 Test (n = 237) (n = 22) (n = 45) BUN 15.5 15.6 16.4 Total protein 7.2 7.2 7.3 RDX exposed females* Referent Undetected (0.01 mg/m3 (n = 101) (n = 1) (n = 25) BUN 13.2 8 12.6 Total protein 7.3 7.6 7.2 *Includes both workers exposed to RDX alone and RDX and HMX. No differences were statistically significant in men or women. LOD = limit of detection Table 1-5. Evidence pertaining to kidney and other urogenital system effects in animals Reference and study design Results Histopathological lesions 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 µm 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-25 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 contaminant; 83−89% of particles <66 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 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. Data for male rats sacrificed on schedule (SS) and those that died spontaneously or were sacrificed moribund (SDMS) (summarized below) were analyzed separately; incidence data were not reported for females. Doses 0 0.3 1.5 8.0 40 Kidney, medullary papillary necrosis; 24 mo (incidence) (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* 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* Prostate, suppurative inflammation (prostatitis); 24 mo (incidence) SS 0/38 1/36 2/25* 4/29* 0/4 SDMS 2/16 3/19 7/27* 8/26 19/27* (Sum) 2/54 4/55 9/52* 12/55* 19/31* This document is a draft for review purposes only and does not constitute Agency policy. 1-26 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Cholakis et al. (1980) Mice, B6C3F1, 10−12/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm 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)a Diet 13 wks Results Doses 0 80 160 320 Tubular nephrosis (incidence) M 0/10 − − 4/9* F 0/11 − − 1/11 Cholakis et al. (1980) Histopathological examination of kidney did not reveal any significant Rats, F344, 10/sex/group differences compared to controls; incidence data were reported only 88.6% pure, with 9% HMX and 2.2% water for control and 40 mg/kg-d groups. as contaminants; ~200 µm particle size 0, 10, 14, 20, 28, or 40 mg/kg-d Diet 13 wks Cholakis et al. (1980) Rats, CD, two-generation study; F0: 22/sex/group; F1: 26/sex/group; F2: 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size F0 and F1 parental animals: 0, 5, 16, or 50 mg/kg-d Diet F0 exposure: 13 wks pre-mating, and during mating, gestation, and lactation of F1; F1 exposure: 13 wks after weaning, and during mating, gestation, and lactation of F2; F2 exposure: until weaning 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 Data were reported only for F2 generation controls and 5 and 16 mg/kg-d groups. Doses 0 5 16 50 Cortical cysts (incidence) M 4/10 4/10 8/10 − F 3/10 4/10 8/10 − Doses 0 4 8 10 12 15 − 1/8 Prostate, mild subacute inflammation (incidence) M 0/10 − − − 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. 1-27 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-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/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 0, 10, 30, 100, 300, or 600 mg/kg-d Diet 13 wks Histopathological examination of kidney did not reveal any significant differences compared to controls. 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. Hart (1974) Histopathological examination of kidney did not reveal any significant Dogs, Beagle, 3/sex/group differences compared to controls; incidences were reported only for Pre-mix with ground dog chow containing control and 10 mg/kg-d groups. 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 Martin and Hart (1974) Monkeys, Cynomolgus or Rhesusc, 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 Doses 0 1/6 0/6 4/6 Kidney weightd 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 µm 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 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 1.5 7.0 35 175/100 Absolute kidney weight at 104 wks (percent change compared to control) M 0% −1% 4% 9%* 19%* F 0% 3% 1% 1% −2% Relative kidney weight at 104 wks (percent change compared to control) M 0% 3% 6% 11%* 27%* F 0% 1% 1% 2% 19%* Doses 0 1.0 3.1 10 Absolute kidney weight (percent change compared to control) M 0% −3% −7% 2% F 0% 14% −4% 8% Relative kidney weight (percent change compared to control) M 0% −1% −4% 4% F 0% 22% 3% 18% 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-1,3,5-trinitro-1,3,5-triazine Reference and study design 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Cholakis et al. (1980) Mice, B6C3F1, 10−12/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm 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)a Diet 13 wks Cholakis et al. (1980) Rats, F344, 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0, 10, 14, 20, 28, or 40 mg/kg-d Diet 13 wks Results Doses 0 0.3 1.5 8.0 40 Absolute kidney weight at 105 wks (percent change compared to control) M 0% 2% −7% 1% 0% F 0% 3% 3% 2% 2% Relative kidney weight at 105 wks (percent change compared to control) M 0% 1% 0% 2% 20%* F 0% 3% 6% 5% 21%* Doses 0 10 14 20 28 40 Absolute kidney weight (percent change compared to control) M 0% − − − 18% 2% F 0% − − − −8% −5% Relative kidney weight (percent change compared to control) M 0% − − − 29% 0% F 0% − − − −8% −3% Doses 0 80 160 320 Absolute kidney weight (percent change compared to control) M 0% 8% 11% 13% F 0% −5% −3% 0% Relative kidney weight (percent change compared to control) M 0% 5% 9% 10% F 0% −5% −4% −5% Doses 0 10 14 20 28 40 Absolute kidney weight (percent change compared to control) M 0% − − − −2% −5% F 0% − − − 1% 0% Relative kidney weight (percent change compared to control) M 0% − − − 1% 5% F 0% − − − 6% 6% 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-1,3,5-trinitro-1,3,5-triazine Reference and study design Results Cholakis et al. (1980) Rats, CD, two-generation study; F0: 22/sex/group; F1: 26/sex/group; F2: 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size F0 and F1 parental animals: 0, 5, 16, or 50 mg/kg-d Diet F0 exposure: 13 wks pre-mating, and during mating, gestation, and lactation of F1; F1 exposure: 13 wks after weaning, and during mating, gestation, and lactation of F2; F2 exposure: until weaning Doses 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 5 16 50 Absolute kidney weight (percent change compared to control) M 0% 6% −12% − F 0% −4% −21%* − 0 4 8 10 12 15 Absolute kidney weight (percent change compared to control) M 0% −3% −4% −1% 3% 5% F 0% 2% 5% 13%* 10% 15%* Relative kidney weight (percent change compared to control) M 0% 3% 6% 2% 1% 3% F 0% 1% −3% −1% −6% −7%* 0 10 30 100 300 600 Levine et al. (1990); Levine et al. (1981a); Levine et al. (1981b)b Rats, F344, 10/sex/group; 30/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 0, 10, 30, 100, 300, or 600 mg/kg-d Diet 13 wks Doses 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 Absolute kidney weight (percent change compared to control) M 0% 1% 1% −9% − − F 0% 1% 3% −1% − − Relative kidney weight (percent change compared to control) M 0% 5% 7% 10% − − F 0% 3% 5% 2% − − 0 0.1 1 10 Absolute kidney weight (percent change compared to control) M 0% − − 38% F 0% − − −18% 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-1,3,5-trinitro-1,3,5-triazine Reference and study design Martin and Hart (1974)e Monkeys, Cynomolgus or Rhesuse, 3/sex/group Purity of test material not specified 0, 0.1, 1, or 10 mg/kg-d Gavage 13 wks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Results Doses 0 0.1 1 10 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. a Doses were calculated by the study authors. b Levine 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. c The 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). d An 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. e Kidney 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 kidney and other urogenital 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. 20 21 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) Doses (mg/kg-d) 0 0.3 1.5 8.0 40 0/10 0/10 0/10 0/10 0/10 SDMS − − − − 0/5 Sum 0/10 0/10 0/10 0/10 0/15 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* Medullary papillary necrosis (incidence) 6 mo SS 12 mo SS 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-1,3,5-trinitro-1,3,5-triazine Doses (mg/kg-d) 0 0.3 1.5 8.0 40 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* 0/10 0/10 0/10 0/10 0/10 SDMS − − − − 0/5 Sum 0/10 0/10 0/10 0/10 0/15 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 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* 0/10 0/10 0/10 0/10 0/10 SDMS − − − − 0/5 Sum 0/10 0/10 0/10 0/10 0/15 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 Pyelitis (incidence) 6 mo SS 12 mo SS 24 mo Pyelonephritis (incidence) 6 mo SS 12 mo SS 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-1,3,5-trinitro-1,3,5-triazine Doses (mg/kg-d) 0 0.3 1.5 8.0 40 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 24 mo 1 2 3 4 5 6 7 *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). 8 9 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) Doses (mg/kg-d) 0 0.3 1.5 8.0 40 0/10 0/10 0/10 0/10 0/10 SDMS − − − − 0/5 Sum 0/10 0/10 0/10 0/10 0/15 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 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* Luminal distention (incidence) 6 mo SS 12 mo SS 24 mo 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 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-1,3,5-trinitro-1,3,5-triazine Doses (mg/kg-d) 0 0.3 1.5 8.0 40 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 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* 12 mo SS 24 mo 1 2 3 4 5 6 7 8 *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). 9 10 Table 1-8. Six-, 12-, and 24-month incidence of prostate endpoints in male F344 rats reported for statistical evaluation in Levine et al. (1983) Doses (mg/kg-d) 0 0.3 1.5 8.0 40 0/10 2/10 2/10 1/10 6/10* SDMS − − − − 2/5 Sum 0/10 2/10 2/10 1/10 8/15* 0/10 0/10 1/10 1/10 0/10 SDMS − − 0/3 − 0/19 Sum 0/10 0/10 1/13 1/10 0/29 SS 0/38 0/36 0/25 0/29 0/4 SDMS 0/16 0/19 0/27 0/26 0/27 Sum 0/54 0/55 0/52 0/55 0/31 Spermatic granuloma (incidence) 6 mo SS 12 mo SS 24 mo 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-1,3,5-trinitro-1,3,5-triazine Doses (mg/kg-d) 0 0.3 1.5 8.0 40 0/10 0/10 0/10 0/10 0/10 SDMS − − − − 0/5 Sum 0/10 0/10 0/10 0/10 0/15 0/10 0/10 0/10 0/10 0/10 SDMS − − 0/3 − 0/19 Sum 0/10 0/10 0/13 0/10 0/29 SS 0/38 1/36 2/25* 4/29* 0/4 SDMS 2/16 3/19 7/27* 8/26 19/27* Sum 2/54 4/55 9/52* 12/55* 19/31* Suppurative inflammation (incidence) 6 mo SS 12 mo SS 24 mo 1 2 3 4 5 6 7 8 *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. 1-35 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Figure 1-2. Exposure-response array of kidney and other urogenital system effects. 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-1,3,5-trinitro-1,3,5-triazine Mechanistic Evidence 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 37 No MOA information is available for RDX-induced kidney and other urogenital effects. However, mechanistic information underlying the neurotoxicity observed with RDX exposure, and the specific affinity of RDX to the GABAA receptor-convulsant site (Williams et al., 2011; Williams and Bannon, 2009), suggests a biologically plausible role for the GABAA receptor in RDX-related effects on the urogenital system, and provides some potential MOA hypotheses for the effects reported in Levine et al. (1983) that do not require bacterial infection. One hypothesis is that urogenital effects of RDX are caused by interactions with GABAA receptors mediating inputs to the urogenital system. GABA and GABA receptors have been identified in a number of peripheral tissues (Erdö et al., 1991; Ong and Kerr, 1990; Erdo, 1985). Brar et al. (2014) 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)). In rats, injection of a GABAA receptor agonist inhibits the urination reflex (Igawa et al., 1993; Kontani et al., 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 (Stone et al., 2011). RDX would be expected to act like an antagonist and increase bladder activity (which would not result in urinary stasis), although the impact of chronic exposure to RDX acting as a GABAA receptor antagonist is not known. Evidence of GABAergic signaling regulating bladder function, and the hypothesized disruption of that regulation by RDX via interaction with GABAA receptors, may plausibly account for the kidney and other urogenital lesions, including suppurative prostatitis, observed by Levine et al. (1983); however, no evidence to support this hypothesized MOA is available. Other potential mechanisms by which RDX, through GABAA binding, may lead to kidney and urogenital effects are less apparent. 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 (Cowin et al., 2010), and increased prolactin has been shown to cause lateral lobe prostatitis (Stoker et al., 1999b; Stoker et al., 1999a; Tangbanluekal and Robinette, 1993; Robinette, 1988). Typically, the inflammation seen is chronic and does not reverse over time (Robinette, 1988). Functional GABAA receptors have been identified in the anterior pituitary (Zemkova et al., 2008; Mayerhofer et al., 2001), 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. (1983) 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 et al. (1983) did not evaluate serum endocrine measures or pituitary weights, and pituitary adenomas that could account for higher 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-1,3,5-trinitro-1,3,5-triazine 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 prolactin levels were not observed. A MOA hypothesis based on pituitary-mediated alterations in endocrine signaling also does not explain the other urogenital lesions observed by Levine et al. (1983). 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 (Reyes-García et al., 2007; Tian et al., 2004), and GABAA receptor agonists have decreased cytotoxic immune responses and hypersensitivity reactions (Tian et al., 1999; Bergeret et al., 1998). In a mouse autoimmune model of multiple sclerosis, Bhat et al. (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 (Johnson, 2015b). If an autoimmune mechanism was contributing to the effects observed with RDX exposure, it is unclear why inflammation would be limited to the prostate. RDX has also tested negative in the only battery of immunotoxicity tests to which it was subjected (Crouse et al., 2006). If the urogenital effects are mediated through localized interaction with GABAA receptors, another possibility would be that that effects would result from direct interactions with GABAA receptors located on the prostate. GABAA receptors have been identified on the prostate (Napoleone et al., 1990), 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 (Johnson, 2015b). No evidence was found to support this potential pathway leading to prostate inflammation. In summary, there are no studies available that inform mechanistically how RDX might lead to kidney and other urogenital effects. There is evidence that RDX binds to GABAA receptors in neuronal tissues (Williams et al., 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 urogenital effects. Among the mechanistic information presented above, MOAs that require direct action on the prostate are considered less likely because the available information suggests that the prostatitis is a secondary effect. However, the ways that GABAA receptors work in non-neuronal tissues and organs is still not well understood, and the MOA by which RDX induces kidney and other urogenital effects is unknown. Integration of Kidney and Urogenital System Effects 35 36 37 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-38 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 accidental exposure (ingestion and inhalation) to unknown amounts of RDX. No RDX-related changes in kidney parameters were found in a small cross-sectional study of RDX-exposed workers (Hathaway and Buck, 1977). A dose-related increase in the incidence of suppurative prostatitis in male rats (Levine et al., 1983) provides the strongest evidence of RDX-associated kidney and other urogenital system effects. As discussed above, the incidence of suppurative prostatitis is considered to be an indicator for the broader array of kidney and other urogenital effects seen in this study. Levine et al. (1983) identified other histopathological effects (papillary necrosis, pyelitis, luminal distension, and cystitis) in the kidney and bladder, but at the highest dose only. A second 2-year study in SpragueDawley rats found no histopathological changes in the kidney or urogenital system (Hart, 1976), but exposure levels used in this study were low compared to Levine et al. (1983). Other measures of kidney effects, specifically 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 suppurative prostatitis and the lack of support for an alternative (i.e., non-RDXrelated) basis for this effect, kidney and urogenital effects are a potential human hazard of RDX exposure. 1.2.3. Reproductive and Developmental Effects 17 18 19 20 21 22 23 24 25 26 No human studies were identified that evaluate the potential of RDX to cause reproductive or developmental effects. Information relevant to an examination of the association between RDX exposure and reproductive and developmental effects comes from a 2-generation reproductive toxicity study in rats and developmental studies in rats and rabbits involving oral administration of RDX during gestation. In addition, oral subchronic and chronic studies in experimental animals provide information useful for examining the association between RDX exposure and effects specifically on the male reproductive system. A summary of the reproductive and developmental effects associated with RDX exposure is presented in Tables 1-9 and 1-10 and Figures 1-3 and 1-4. Studies are ordered in the evidence tables and exposure-response arrays by duration of exposure and then by species. Reproductive Effects 27 28 29 30 31 32 33 34 35 Evidence of male reproductive toxicity is provided by the finding of testicular degeneration in male mice. An increased incidence of testicular degeneration (10−11%) was observed in male B6C3F1 mice exposed to ≥35 mg/kg-day RDX for 2 years in the diet compared to concurrent (0%) and historical (1.5%) controls (Lish et al., 1984). Reductions in absolute testicular weight were observed, but the magnitude of this effect was small (≤6% compared to controls) and not doserelated. An increased incidence of germ cell degeneration was observed in rats exposed to 40 mg/kg-day (40%) compared with controls at 12 months (0%); by 24 months, almost all male rats (including controls) had testicular masses (interstitial cell tumors), and no instances of germ cell degeneration were identified in control or RDX-treated groups (Levine et al., 1983). No doseThis document is a draft for review purposes only and does not constitute Agency policy. 1-39 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 related histopathological changes in the testes were identified in other studies in rats (Crouse et al., 2006; Levine et al., 1990; Levine et al., 1981a, b; Hart, 1976) or dogs (Hart, 1974). Changes in testicular weight were inconsistent across studies, with an equivalent number of studies identifying decreases (Crouse et al., 2006; Lish et al., 1984; Cholakis et al., 1980) or increases (Levine et al., 1990; Levine et al., 1981a, b; Cholakis et al., 1980; Hart, 1976, 1974) in testicular weight; in most cases, the changes in testicular weight were small (≤10% change compared to control) and not dose-related. Reproductive function was assessed in two separate studies reported by Cholakis et al. (1980). No specific effects on reproductive function were observed in F0 and F1 CD rats exposed to ≤16 mg/kg-day RDX in the Cholakis et al. (1980) two-generation study. The highest dose tested, 50 mg/kg-day, was associated with reductions in fertility (specifically a decreased number of pregnancies) in the F0 generation, although these changes were not statistically significant. The finding of lower fertility rates only at the 50 mg/kg-day dose, a dose associated with reduced body weight and feed consumption and increased mortality (9% in male rats and 27% in female rats), suggests that effects on reproductive function were likely due to the general toxicity of RDX rather than a direct effect of RDX on reproduction. In the dominant lethal mutation study, which used the F0 males from the two-generation reproductive toxicity study, no effects on fertility were observed in male rats exposed to ≤16 mg/kg-day RDX. Pregnancy rates were lower in untreated females mated to males exposed to 50 mg/kg-day RDX for 15 weeks prior to mating; the authors attributed this effect to a treatment-related decrease in the well-being of the males in this high-dose group (Cholakis et al., 1980). Developmental Effects 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 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 et al., 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 (Cholakis et al., 1980), although no effect on live fetuses was observed in a developmental toxicity study in Sprague-Dawley rats at the same dose (Angerhofer et al., 1986); 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) (Cholakis et al., 1980). Both of these 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 (U.S. EPA, 1991a), 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-40 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 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 (Cholakis et al., 1980), 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 (Angerhofer et al., 1986). 14 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 (Cholakis et al., 1980). Dose-related reductions in fetal body weight were not observed in NZW rabbits at doses up to 20 mg/kg-day (Cholakis et al., 1980). 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 et al., 1986; Cholakis et al., 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 (Cholakis et al., 1980). Although increased incidences of enlarged frontal fontanel and unossified sternebrae were observed in fetuses of all groups of NZW rabbits administered RDX (Cholakis et al., 1980), 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 treatmentrelated by the study authors (Cholakis et al., 1980). This interpretation is supported by the following additional considerations. Neither individual litter data nor historical control data from the performing laboratory were available to assist in the interpretation of these findings. 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 (MTA, 1992) 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 unossified sternebrae in Cholakis et al. (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 et al. (1980) 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 et al. (1980) was similar to the pattern of decreases in fetal body 14The 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, 1991b) recommend that fetal data be analyzed on a per litter (rather than per fetus) basis. In a reanalysis of the Angerhofer et al. (1986) data by EPA on a per litter basis, fetal body weight and length showed statistically significant decreasing trends. This document is a draft for review purposes only and does not constitute Agency policy. 1-41 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 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 1-9. Evidence pertaining to male reproductive effects in animals Reference and study design 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 µm 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 Results Doses 0 1.5 35 175/100 2/62 6/59 3/27a Testicular degeneration (incidence) 0/63 2/60 Absolute testes weight; wk 105 (percent change compared to control) 0% −6% 0% −2% −6% Relative testes weight; wk 105 (percent change compared to control) 0% Hart (1976) Rats, Sprague-Dawley, 100/sex/dose Purity and particle size not specified 0, 1.0, 3.1, or 10 mg/kg-d Diet 2 yrs 7.0 Doses −4% 0 2% 1.0 −2% 3.1 −2% 10 Absolute testes (with epididymis) weight; wk 104 0% −2% 2% 5% Relative testes (with epididymis) weight; wk 104 0% −1% 7% 9% Testes were examined microscopically in control and 10 mg/kg-d groups; no degeneration or other treatment-related effects were observed. Levine et al. (1983) 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Doses 0 0.3 1.5 8.0 40 Testes, germ cell degeneration; 12 mob (incidence) SS SDMS 0/10 0/10 0/10 0/10 4/10* − − 1/3 − 4/19 Testes, germ cell degeneration; 24 mo (incidence) SS 0/38 0/36 0/25 0/29 0/4 SDMS 0/16 0/19 0/27 0/26 0/27 Testes weights were not measured at termination due to testicular masses in nearly all males. This document is a draft for review purposes only and does not constitute Agency policy. 1-42 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Results Cholakis et al. (1980) Doses 0 10 14 20 28 40 Mice, B6C3F1, 10−12/sex/group Absolute testes weight (percent change compared to control) 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0% − − − −4% −4% Experiment 1: 0, 10, 14, 20, 28, or Relative testes weight (percent change compared to control) 40 mg/kg-d Diet 0% − − − 2% −1% 13 wks Experiment 2: 0, 40, 60, or 80 mg/kg-d for Doses 0 80 160 320 2 wks followed by 0, 320, 160, or Absolute testes weight (percent change compared to control) 80 mg/kg-d (TWA doses of 0, 79.6, 147.8, or 256.7 mg/kg-d for males and 0, 82.4, 0% 4% −4% −8% 136.3, or 276.4 mg/kg-d for females)c Relative testes weight (percent change compared to control) Diet 13 wks 0% 1% −4% −9% Testes were examined microscopically in control and 320 mg/kg-d groups; no effects were observed. Cholakis et al. (1980) Doses 0 10 14 20 28 Rats, F344, 10/sex/dose Absolute testes weight (percent change compared to control) 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0% − − − −2% 0, 10, 14, 20, 28, or 40 mg/kg-d Relative testes weight (percent change compared to control) Diet 13 wks 0% − − − 2% 40 0% 9% Testes were examined microscopically in control and 40 mg/kg-d groups; no effects were observed. Cholakis et al. (1980) Rats, CD, two-generation study; F0: 22/sex/group; F1: 26/sex/group; F2: 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size F0 and F1 parental animals: 0, 5, 16, or 50 mg/kg-d Diet F0 exposure: 13 wks pre-mating, and during mating, gestation, and lactation of F1; F1 exposure: 13 wks after weaning, and during mating, gestation, and lactation of F2; F2 exposure: until weaning In F2 weanling offspring of 0, 5, and 16 mg/kg-d groups. No highdose F2 animals available. Doses 0 5 16 50 Absolute testes weight (percent change compared to control) 0% 3% −31% − Testes of F2 weanlings were examined microscopically in all groups; no effects were observed. This document is a draft for review purposes only and does not constitute Agency policy. 1-43 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design 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 Levine et al. (1990); Levine et al. (1981a); Levine et al. (1981b)d Rats, F344, 10/sex/group; 30/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 0, 10, 30, 100, 300, or 600 mg/kg-d Diet 13 wks e Hart (1974) Dogs, Beagle, 3/sex/dose 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Results Doses 0 4 8 10 12 15 Absolute testes weight (percent change compared to control) 0% −3% −5% −4% −4% −8% Relative testes weight (percent change compared to control) Doses 0% 4% 5% 0% −6% −10%* 0 10 30 100 300 600 1/9 1/10 Testes, germ cell degeneration (incidence) 0/10 0/10 0/10 0/10 Absolute testes weight (percent change compared to control) 0% 1% 1% −2% − − Relative testes weight (percent change compared to control) 0% Doses 4% 0 5% 19%* 0.1 − − 1 10 Absolute testes (with epididymis) weight (percent change compared to control) 0% − − 51% Testes were not examined microscopically. *Statistically significant (p < 0.05) based on analysis by study authors. a Although the study authors did not observe a statistically significant increase in the incidence of testicular degeneration, they determined that the incidences at the 35 and 175/100 mg/kg-day dose groups were “notable” when compared to concurrent (0%) and historical (1.5%) incidences. b Testicular atrophy was observed at 12 months along with a statistically reduced mean testes weight (compared with controls). By 24 months, almost all male rats (including controls) had testicular masses (interstitial cell tumors); testes weights were not recorded, and an increased incidence of testicular degeneration was not observed. c Doses were calculated by the study authors. d Levine 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. e Because testes weight was reported for only three treated animals in this study, organ data from this study were considered less informative than other studies; therefore, testes weights from Hart (1974) were not presented in the exposure-response array for male reproductive effects. 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-44 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Figure 1-3. Exposure response array of male reproductive effects following oral exposure. This document is a draft for review purposes only and does not constitute Agency policy. 1-45 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 Table 1-10. Evidence pertaining to reproductive and developmental effects in animals Reference and study design Results Offspring survival Cholakis et al. (1980) Rats, CD, two-generation study; F0: 22/sex/group; F1: 26 sex/group; F2: 10 sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size F0 and F1 parental animals: 0, 5, 16, or 50 mg/kg-d Diet F0 exposure: 13 wks pre-mating, and during mating, gestation, and lactation of F1; F1 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) F1 8/207 6/296 4/259 16/92* F2 6/288 6/290 2/250 24/46* Offspring survival at birth (percent of fetuses) F1 96% 98% 98% 83%* F2 98% 98% 99% 48%* Survival at weaning (percent of liveborn pups) F1 87% 96% 90% 8% F2 79% 86% 79% 0% F0 maternal deaths occurred at 50 mg/kg-d. Only six F1 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) Doses 0 0.2 Rabbits, NZW, 11−12/group Early resorptions (mean percent per dam) 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 6% 5% 0, 0.2, 2.0, or 20 mg/kg-d Late resorptions (mean percent per dam) Gavage GDs 7−29 8% 5% 2 20 4% 1% 3% 3% 77% 94% 2.0 20 4.8% 15.3% 0.3% 1.6% 0 2 94.9% 81.4% Viable fetuses (mean percent per dam) 85% 82% Cholakis et al. (1980) Doses 0 0.2 Rats, F344, 24−25 females/group Early resorptions (mean percent per dam) 88.6% pure, with 9% HMX and 2.2% water as contaminants. 6.0% 2.5% 0, 0.2, 2.0, or 20 mg/kg-d Late resorptions (mean percent per dam) Gavage GDs 6−19 0.5% 0.5% Complete litter resorptions (number of litters) 0 0 Viable fetuses (mean percent per dam) 93.2% 97.6% 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. 1-46 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design 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 Results Doses 0 2 6 20 5.9% 6.4% Resorptions (percent of total implantations) 4.8% 6.1% 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% 100% 100% Live fetuses (mean percent per litter) 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) Doses 0 0.2 2.0 Rabbits, NZW, 11−12/group Fetal body weight (percent change compared to control) 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0% −6.7% −2.3% 0, 0.2, 2.0, or 20 mg/kg-d Gavage GDs 7−29 20 −9.3% Cholakis et al. (1980) Doses 0 0.2 2.0 20 Rats, F344, 24−25 females/group Fetal body weight (percent change compared to control) 88.6% pure, with 9% HMX and 2.2% water as contaminants. 0% 2% 3% −7% 0, 0.2, 2.0, or 20 mg/kg-d Significant maternal mortality (7/24 dams) occurred at 20 mg/kg-d. Gavage GDs 6−19 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) 0% −1% −1% −5%a Significant maternal mortality (16/51) occurred at 20 mg/kg-d. This document is a draft for review purposes only and does not constitute Agency policy. 1-47 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Results Morphological development Cholakis et al. (1980) Rabbits, NZW, 11−12/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm 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 0/88 0/99 0/94 3/110 Litters 0/11 0/11 0/11 2/12 Misshapen eye bulges (incidence) Fetuses 0/88 0/99 0/94 3/110 Litters 0/11 0/11 0/11 1/12 Cleft palate (incidence) Fetuses 0/39 1/46 2/44 2/52 Litters 0/11 1/11 1/11 1/12 Enlarged front fontanel (incidence) Fetuses 0/49 5/53 2/50 8/58 Litters 0/11 2/11 2/11 2/12 Unossified sternebrae (incidence) 1 2 3 4 Fetuses 4/49 12/53 8/50 12/58 Litters 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 0, 2, 6, or 20 mg/kg-d Gavage GDs 6−15 No treatment-related increase in the incidence of anomalies was observed. Doses 0 2 6 20 Total malformations (percent of fetuses with malformations) 1% 1% 0% 2% Significant maternal mortality (16/51) occurred at 20 mg/kg-d. *Statistically significant (p < 0.05) based on analysis by study authors. a 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-48 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 100 signficantly changed Dose (mg/kg-day) not signifcantly changed 10 Offpsring growth ↓ Offspring survival Angerhofer et al., 1986 (rat) Cholakis et al., 1980 (rat) Cholakis et al., 1980 (rabbit) Angerhofer et al., 1986 (rat) (1) Cholakis et al., 1980 (rat) Cholakis et al., 1980 (rabbit) Angerhofer et al., 1986 (rat) Cholakis et al., 1980 (rat) Cholakis et al., 1980 (rabbit) 0.1 Cholakis et al., 1980 (rat, F₁ and F₂) 1 Morphological development 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. Figure 1-4. Exposure response array of reproductive and developmental effects following oral exposure. This document is a draft for review purposes only and does not constitute Agency policy. 1-49 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Integration of Reproductive and Developmental Effects 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Testicular effects were reported in male B6C3F1 mice chronically exposed to RDX in the diet for 24 months (Lish et al., 1984). No other studies of equivalent duration were performed in mice to determine the consistency of this effect. Germ cell degeneration was observed in F344 rats at 12 months, but not at 24 months, in a 2-year study (Lish et al., 1984); therefore, the biological significance of the 12-month findings is uncertain. Other testicular effects were inconsistent across rat studies. Based on the evidence of testicular degeneration in male mice reported by Lish et al. (1984), there is suggestive evidence of male reproductive effects associated with RDX exposure. Developmental studies in rats (Angerhofer et al., 1986; Cholakis et al., 1980) 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 et al., 1980). As noted in EPA’s Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991a), where adverse developmental effects are produced only at doses that cause minimal maternal toxicity, developmental effects should not be discounted as being secondary to maternal toxicity; however, at doses causing excessive toxicity, as is the case with RDX, information on developmental effects may be difficult to interpret and of limited value. Therefore, at this time, no conclusions are drawn regarding developmental effects as a human hazard of RDX exposure. 1.2.4. Liver Effects 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 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 (Küçükardali et al., 2003; Woody et al., 1986; Knepshield and Stone, 1972; Hollander and Colbach, 1969; Stone et al., 1969; Merrill, 1968) (see Appendix C, Section C.2). Liver biopsies did not reveal any abnormal observations (Stone et al., 1969). In other case reports, no significant changes in serum levels of liver enzymes were observed (Testud et al., 1996a; Ketel and Hughes, 1972). 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: This document is a draft for review purposes only and does not constitute Agency policy. 1-50 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 undetectable (<0.01 mg/m3) to 1.6 mg/m3) (Hathaway and Buck, 1977), serum chemistry analysis (including the serum liver enzymes AST, ALT, and alkaline phosphatase [ALP]) revealed no statistically significant differences between exposed and unexposed workers (Table 1-11). In experimental animals, some, but not all, subchronic studies reported increased liver weight associated with RDX exposure (Table 1-12 and Figure 1-5). Dose-related increases in relative liver weight 15 (11−25% in high-dose groups) were observed in male and female B6C3F1 mice given RDX in the diet for 90 days (Cholakis et al., 1980) and in female F344 rats in two separate 90-day dietary studies of RDX (Levine et al., 1990; Levine et al., 1981a, b; Cholakis et al., 1980); however, relative liver weights were not increased in female F344 rats in another 90-day gavage study (Crouse et al., 2006). Male F344 rats exhibited an increase in relative liver weight only in one of these subchronic studies (Levine et al., 1990; Levine et al., 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) (Martin and Hart, 1974) and in male, but not female, beagle dogs (53% relative to control in male dogs at 10 mg/kg-day) (Hart, 1974). 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 (Lish et al., 1984) was reduced when mice with liver adenomas or carcinomas were removed from the analysis. In a 2-year rat study (Levine et al., 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 weight 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 et al., 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 et al., 2006; Levine et al., 1990; Lish et al., 1984; Levine et al., 1983; Levine et al., 1981a, b; Hart, 1976, 1974; Martin and Hart, 1974), including 2-year oral studies in mice at doses up to 100 mg/kg-day (Lish et al., 1984) and in rats at doses up to 40 mg/kg-day (Levine et al., 1983). The few findings of liver lesions were reported in studies with more limited histopathological analyses, and were not confirmed in the 15Based on an evaluation of the relationship between organ weight and body/brain weight to determine which endpoint (organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio) is likely to more accurately detect target organ toxicity, Bailey et al. (2004) 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 (Table 1-12). This document is a draft for review purposes only and does not constitute Agency policy. 1-51 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 studies with more complete histopathologic examination and longer exposure durations (Lish et al., 1984; Levine et al., 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 (Cholakis et al., 1980). There was an increase in the incidence of mild liver microgranulomas in female mice only (Cholakis et al., 1980) and karyomegaly of hepatocytes in male mice only exposed to 320 mg/kg-day RDX in the diet for 90 days (Cholakis et al., 1980). Because both the rat and mouse studies by Cholakis et al. (1980) used relatively small group sizes (n = 10/sex/group) and provided histopathologic findings for the control and high-dose groups only, less weight 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 B6C3F1 mice exposed to RDX in the diet for 2 years, although increases were not dose-related (Lish et al., 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 et al., 1990; Levine et al., 1981a, b). In a chronic toxicity study by the same investigators (Levine et al., 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 B6C3F1 mice exposed to RDX in the diet for 2 years (Lish et al., 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 (Levine et al., 1983), in rats exposed to RDX by gavage for 90 days at doses up to 15 mg/kg-day (Crouse et al., 2006), and in monkeys exposed to RDX in the diet for 90 days (Martin and Hart, 1974) were relatively small (within 38% of control mean) and were not dose related. This document is a draft for review purposes only and does not constitute Agency policy. 1-52 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Table 1-11. Evidence pertaining to liver effects in humans Reference and study design Hathaway and Buck (1977) (United States) Cross-sectional study, 2,022 workers, 1,491 participated (74% response rate). Analysis group: limited to whites; 69 exposed to RDX alone and 24 exposed to RDX and HMX; 338 not exposed to RDX, HMX, or TNT. Exposure measures: Exposure determination based on job title and industrial hygiene evaluation. Exposed subjects assigned to two groups: 0.01 mg/m3 (n = 45) LDH 173 191 174 ALP 82 78 80 ALA (SGOT) 22 25 21 AST (SGPT) 21 26 18 Bilirubin 0.5 0.4 0.4 Test ‡ 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. Liver function tests in men (prevalence of abnormally elevated values) Test (abnormal range) RDX exposed‡ 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) (>35) 20/237 4/22 2/45 ALT (SGPT) (>35) 15/237 2/22 0/45 Bilirubin (>1.0) 5/237 1/22 1/45 ‡ 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. 2 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. 1-53 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Table 1-12. Evidence pertaining to liver effects in animals Reference and study design Results Liver weight 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 µm 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 Hart (1976) Rats, Sprague-Dawley, 100/sex/group Purity and particle size not specified 0, 1.0, 3.1, or 10 mg/kg-d Diet 2 yrs Doses 0 1.5 7.0 35 175/100 Absolute liver weight at 104 wks (percent change compared to control) M 0% 28%* 11% 12% 35%* F 0% 7% 7% 15% 18%* Relative liver weight at 104 wks (percent change compared to control) M 0% 32%* 12% 14% 46%* F 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. 0 1.0 3.1 10 Absolute liver weight (percent change compared to control) M 0% −6% −6% −6% F 0% 7% −11% 1% Relative liver weight (percent change compared to control) M 0% −5% −2% −3% F 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Doses 0 0.3 1.5 8.0 40 Absolute liver weight at 105 wks (percent change compared to control) M 0% 3% −7% 1% −8% F 0% 1% −4% 3% 0% Relative liver weight at 105 wks (percent change compared to control) M 0% 1% 0% 2% 11% F 0% 1% −2% 6% 18%* Cholakis et al. (1980) Mice, B6C3F1, 10−12/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size Doses 0 Experiment 1: 0, 10, 14, 20, 28, or 40 mg/kg-d Diet 13 wks Relative liver weight (percent change compared to control) 10 14 20 28 40 Absolute liver weight (percent change compared to control) M 0% − − − −6% −5% F 0% − − − −4% −1% M 0% − − − −4% −4% F 0% − − − −6% 1% This document is a draft for review purposes only and does not constitute Agency policy. 1-54 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design 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)a Diet 13 wks Cholakis et al. (1980) Rats, F344, 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0, 10, 14, 20, 28, or 40 mg/kg-d Diet 13 wks Results Doses 0 80 160 320 Absolute liver weight (percent change compared to control) M 0% 2% 12% 26%* F 0% 4% 9% 29%* Relative liver weight (percent change compared to control) M 0% 0% 9% 25%* F 0% 4% 4% 22%* Doses 0 10 14 20 28 40 Absolute liver weight (percent change compared to control) M 0% − − − −2% −5% F 0% − − − 6% 4% Relative liver weight (percent change compared to control) M 0% − − − 2% 3% F 0% − − − 10% 11% Cholakis et al. (1980) Rats, CD, two-generation study; F0: 22/sex/group; F1: 26/sex/group; F2: 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size F0 and F1 parental animals: 0, 5, 16, or 50 mg/kg-d Diet F0 exposure: 13 wks pre-mating, and during mating, gestation, and lactation of F1; F1 exposure: 13 wks after weaning, and during mating, gestation, and lactation of F2; F2 exposure: until weaning Doses 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 5 16 50 Absolute liver weight (percent change compared to control) M 0% 7% −16% − F 0% 0% −14% − 0 4 8 10 12 15 Absolute liver weight (percent change compared to control) M 0% −6% −9% 0% 7% 5% F 0% 1% 7% 18%* 15% 28%* Relative liver weight (percent change compared to control) M 0% 0% −1% 2% 5% 2% F 0% 1% −2% 2% −3% 2% This document is a draft for review purposes only and does not constitute Agency policy. 1-55 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Results Levine et al. (1990); Levine et al. (1981a); Levine et al. (1981b)b Rats, F344, 3−4 wks old; 10/sex/group; 30/sex/group for controls 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 0, 10, 30, 100, 300, or 600 mg/kg-d Diet 13 wks Doses Hart (1974)c 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 Martin and Hart (1974)c Monkeys, Cynomolgus or Rhesus d, 3/sex/group Purity of test material not specified 0, 0.1, 1, or 10 mg/kg-d Gavage 13 wks Doses 0 10 30 100 300 600 Absolute liver weight (percent change compared to control) M 0% 5% −1% −2% − − F 0% 2% 4% 16%* − − Relative liver weight (percent change compared to control) M 0% 9% 6% 20% − − F 0% 3% 5% 19%* − − 0 0.1 1 10 Absolute liver weight (percent change compared to control) M 0% − − 53% F 0% − − 3% 0 0.1 1 10 Absolute liver weight (percent change compared to control) M+F 0% 2% 6% 16% Histopathological lesions 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 µm 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-56 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Doses Cholakis et al. (1980) Mice, B6C3F1, 10−12/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm 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)a Diet 13 wks Doses Cholakis et al. (1980) Rats, F344, 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 µm particle size 0, 10, 14, 20, 28, or 40 mg/kg-d Diet 13 wks Doses 0 0.3 1.5 8.0 40 Microgranulomas (incidence) M 0/38 0/36 0/25 0/29 0/4 F 10/43 19/45 12/42 17/41 4/28 0 80 160 320 Liver microgranulomas; mild (incidence) M 2/10 − − 1/9 F 2/11 − − 7/11* Increased karyomegaly of hepatocytes (incidence) M 0/10 − − 5/9* F − − − − 0 10 14 20 28 40 Liver granulomas; mild (incidence) M 0/10 − − − − 1/10 F − − − − − − Liver portal inflammation (incidence) M 2/10 − − − − 3/10 F 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 et al. (1981b)b Rats, F344, 10/sex/group; 30/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-57 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-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 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, 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 µm 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 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Doses 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 0 1.5 7.0 35 175/100 Serum cholesterol at 105 wks (percent change compared to control) M 0% 11% −11% 5% 39% F 0% 5% 15% 25% 38% Serum triglycerides at 105 wks (percent change compared to control) M 0% 21% −20% 10% −25% F 0% 34% 28% 41% 28% 0 0.3 1.5 8.0 40 Serum cholesterol at 104 wks (percent change compared to control) M 0% 15% 38% 19% −6% F 0% 6% 3% −7% −9% Serum triglycerides at 104 wks (percent change compared to control) M 0% 14% −15% −12% −52% F 0% 18% 5% −42% −51%* Doses 0 4 8 10 12 15 Serum cholesterol (percent change compared to control) M 0% −3% −10%* −16%* −18%* −11%* F 0% −1% −8% −4% −4% −1% Serum triglycerides (percent change compared to control) M 0% 1% 1% −7% −2% This document is a draft for review purposes only and does not constitute Agency policy. 1-58 −19% DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design Results F 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0% −16% −21% 7% −37% 18% 0 10 30 100 300 600 Levine et al. (1990); Levine et al. (1981a); Levine et al. (1981b)b Rats, F344, 10/sex/group; 30/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 µm, ~90% of particles ≤66 µm 0, 10, 30, 100, 300, or 600 mg/kg-d Diet 13 wks Doses 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. Serum triglyceride levels (percent change compared to control) M 0% −14% −34% −62%* − − F 0% −12% −29% −50%* − − Doses 0 0.1 1 10 Serum cholesterol (percent change compared to control) M 0% −17% −2% −7% F 0% 7% 7% 7% *Statistically significant (p < 0.05) based on analysis by study authors. a Doses were calculated by the study authors. b Levine 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. c Liver 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). d The 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-59 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Figure 1-5. Exposure response array of liver effects following oral exposure. This document is a draft for review purposes only and does not constitute Agency policy. 1-60 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Integration of Liver Effects 1 2 3 4 5 6 7 8 9 10 11 There is limited evidence from human studies and from studies in experimental animals that RDX may affect the liver. The observation of short-term 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, no conclusions are drawn regarding liver effects as a human hazard of RDX exposure. 1.2.5. Carcinogenicity 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 The relationship between exposure to RDX and cancer in human populations has not been investigated. The carcinogenicity of RDX has been examined in one oral chronic/carcinogenicity bioassay in mice (Lish et al., 1984) and two bioassays in rats (Levine et al., 1983; Hart, 1976). The 2-year studies by Lish et al. (1984) and Levine et al. (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). The earlier Hart (1976) study is largely limited by the lack of characterization of the test material and the pathology 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 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 29 30 31 32 33 34 An increased incidence of liver tumors was observed in one chronic mouse study (Lish et al., 1984) and one of two chronic rat studies (Levine et al., 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, B6C3F1 mice as compared to concurrent controls in a 2-year dietary study (Lish et al., This document is a draft for review purposes only and does not constitute Agency policy. 1-61 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 1984). In female B6C3F1 mice, Lish et al. (1984) observed that the liver tumor incidence in the concurrent female control mice was relatively low (1/65), and significantly lower than the 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%) (Haseman et al., 1985). 16 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 Pathology Working Group (PWG) with substantial participation by NTP pathologists reviewed the slides of female mouse liver lesions from the Lish et al. (1984) study (Parker et al., 2006; Parker, 2001). 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 et al., 1999). There remained a statistically significant positive trend in the combined incidence of hepatocellular adenomas or carcinomas, consistent with the original findings of Lish et al. (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 et al. (2006) were considered the more reliable measure of liver tumor response in female mice from the Lish et al. (1984) bioassay. In male mice from the Lish et al. (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 the range for the same mouse strain reported by NTP (8−32%) (Haseman et al., 1985). Similarly, the combined incidences of liver adenoma or carcinoma in control (32%) and treated groups 16Comparison of control incidences of hepatocellular adenomas or carcinomas between Lish et al. (1984) and Haseman et al. (1985) 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. (1985) did not include the lab contracted to perform the Lish et al. (1984) study, and it is not clear if the diet used in the Lish et al. (1984) 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. (1984) study. EPA Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005b) 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-62 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 (27−48%) were within the range for the same mouse strain reported by NTP (14−58%) (Haseman et al., 1985). 17 The PWG did not re-analyze liver tumor slides from male mice. A statistically significant positive trend with dose was observed in the incidence of hepatocellular carcinomas in male, but not female, F344 rats exposed to RDX in the diet for 2 years (Levine et al., 1983). In the Levine et al. (1983) 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 liver tumors are rare tumors in the rat 18, some perspective is obtained by considering historical control data. In a paper published concurrently with the Levine et al. (1983) study, NTP reported an incidence of liver carcinomas in untreated control male F344 rats of 0.7% (12/1,719; range: 0−2%) (Haseman et al., 1985). In Levine et al. (1983), 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., 1985) are comparable with Levine et al. (1983) 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 et al. (1983) study, the mortality in the highest dose group is 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. Nonmalignant liver tumors (neoplastic nodules) in F344 male rats in NTP historical controls were reported more frequently than carcinomas, with an average incidence of 3.5% (61/1,719; range: 0−12%) (Haseman et al., 1985); Levine et al. (1983) reported an incidence of neoplastic nodules of 7.3% in their control male rats, consistent with the NTP historical control data, and a decline in incidence with increasing RDX exposure. The combined incidence of liver neoplastic nodules or carcinomas did not show a significant trend with dose. In a second 2-year dietary study in the rat study using a different strain (Sprague-Dawley), the combined incidence of hepatocellular adenomas or carcinomas was not increased with dose in rats of either sex at doses up to 10 mg/kg-day (Hart, 1976). However, interpretation of results 17Ibid. 18NTP historical control data for hepatocellular carcinomas F344 rats as reported in Haseman et al. (1985): 12/1,719 (0.7%) in males; 3/1,766 (0.17%) in females. Historical control data for Charles River SpragueDawley rats as reported in Chandra et al. (1992): 6/1,340 (0.45%) in males; 1/1,329 (0.08%) in females. This document is a draft for review purposes only and does not constitute Agency policy. 1-63 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 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 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 µm 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 Resultsa Doses 0 1.5 7.0 35 175/100b Hepatocellular adenomas (incidence)c M 8/63 6/60 1/62* 7/59 7/27 F 1/65 1/62 6/64 6/64 3/31 (12.7) (1.5) (10.0) (1.6) (1.6) (9.4) (11.9) (9.4) (25.9) (9.7) Hepatocellular carcinomas (incidence)c M 13/63 20/60 16/62 18/59 6/27 F 0/65 4/62 3/64 6/64 3/31d (20.6) (0.0) (33.3) (6.5) (25.8) (4.7) (30.5) (9.4) (22.2) (9.7) Hepatocellular adenoma or carcinoma combined (incidence)c M 20/63 26/60 17/62 25/59 13/27 F 1/65 5/62 9/64* 12/64* 6/31*d (31.7) (1.5) (43.3) (8.1) (27.4) (14.1) (42.4) (18.8) (48.1) (19.4) PWG reanalysis of liver lesion slides from female mice (Parker et al., 2006; Parker, 2001).e Doses 0 7.0 35 175/100b 2/63 8/64 2/31 2/64 2/31 1.5 Hepatocellular adenomas (incidence)c F 1/67 (1.5) 3/62 (4.8) (3.2) (12.5) (6.5) Hepatocellular carcinomas (incidence) c F 0/67 (0.0) 1/62 (1.6) 3/63 (4.8) (3.1) (6.5) Hepatocellular adenoma or carcinoma combined (incidence)c F 1/67 (1.5) 4/62 (6.5) 5/63 (7.9) 10/64 (15.6) This document is a draft for review purposes only and does not constitute Agency policy. 1-64 4/31d (12.9) DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference and study design 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 Resultsa Doses 0 1.0 3.1 10 − 3/77 − 1/81 Neoplastic nodules (incidence) c M 0/82 − F 1/72 − Hepatocellular carcinomas (incidence) c M 1/82 − − 1/77 F 1/72 − − 1/81f Neoplastic nodules or hepatocellular carcinomas combined (incidence)c 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs M 1/82 − − 4/77 F 2/72 − − 2/81 Doses 0 0.3 1.5 8.0 40 Neoplastic nodules (incidence)c M 4/55 3/55 0/52 2/55 1/31 F 3/53 1/55 1/54 0/55 4/48 (7.3) (5.6) (5.5) (1.8) (0.0) (1.9) (3.6) (0.0) (3.2) (8.3) Hepatocellular carcinomas (incidence) c M 1/55 0/55 0/52 2/55 2/31d F 0/53 1/55 0/54 0/55 0/48 (1.8) (0.0) (0.0) (1.8) (0.0) (0.0) (3.6) (0.0) (6.5) (0.0) Neoplastic nodules or hepatocellular carcinomas combined (incidence)c 1 2 3 4 5 6 7 8 9 10 11 12 13 M 5/55 3/55 0/52 4/55 3/31 F 3/53 2/55 1/54 0/55 4/48 (9.1) (5.6) (5.5) (3.6) (0.0) (1.9) (7.3) (0.0) (9.7) (8.3) *Statistically significant difference compared to the control group (p < 0.05), identified by the authors. a Selected percent incidences are provided in parentheses below the incidences to help illustrate patterns in the responses. b The lower dose of 100 mg/kg-day was started in week 11, resulting in a duration-weighted average dose of 107 mg/kg-day. c The incidences reflect the animals surviving to month 12. d Statistically significant trend (p < 0.05) was identified using a one-sided Cochran-Armitage trend tests performed by EPA. e The 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-65 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 discrepancy (email to Louis D’Amico, U.S. EPA, from Mark Johnson, U.S. Army Public Health Command, February 13, 2015). f Hart (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. Lung Tumors 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Lung tumors were observed in female and male B6C3F1 mice exposed to RDX in the diet for 2 years (Lish et al., 1984) (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 (McConnell et al., 1986). 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 et al. (1984) noted an increase in the combined incidences of primary pulmonary neoplasms in males of all dose groups and in females in the 7.0, 35, and 175/100 mg/kg-day dose groups, 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, 1976) or in F344 rats of either sex exposed chronically to RDX at doses up to 40 mg/kg-day (Levine et al., 1983). Alveolar/bronchiolar carcinomas are rare tumors in both species of rats, male or female (Chandra et al., 1992; Haseman et al., 1985). This document is a draft for review purposes only and does not constitute Agency policy. 1-66 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Table 1-14. Lung tumors observed in chronic animal bioassays Reference and study design 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 µm 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 Resultsa Doses 0 1.5 7.0 35 175/100b Alveolar/bronchiolar adenomas (incidence)c M 6/63 5/60 5/62 7/59 1/27 F 4/65 2/62 5/64 9/64 3/31 (9.5) (6.2) (8.3) (8.1) (3.2) (11.9) (7.8) (3.7) (14.1) (9.7) Alveolar/bronchiolar carcinomas (incidence)c M 3/63 6/60 3/62 7/59 5/27d F 3/65 1/62 3/64 3/64 4/31d (4.8) (4.6) (10.0) (4.8) (1.6) (11.9) (4.7) (18.5) (4.7) (12.9) Alveolar/bronchiolar adenoma or carcinoma combined (incidence)c 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 M 9/63 11/60 8/62 14/59 6/27 F 7/65 3/62 8/64 12/64 7/31d 0 1.0 (14.3) (10.8) Doses (18.3) (12.9) (4.8) (23.7) (12.5) (22.2) (18.8) 3.1 (22.6) 10 Alveolar/bronchiolar adenoma (incidence) M 2/83 − − 1/77 F 0/73 − − 0/82 No alveolar/bronciolar carcinomas reported by study authors. 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 µm 0, 0.3, 1.5, 8.0, or 40 mg/kg-d Diet 2 yrs Doses 0 0.3 1.5 8.0 40 Alveolar/bronchiolar adenomas (incidence)c M 1/55 0/15 1/17 0/16 1/31 F 3/53 0/7 0/8 1/10 0/48 Alveolar/bronchiolar carcinomas (incidence)c M − − − − − F 0/53 0/7 1/8 0/10 0/48 Alveolar/bronchiolar adenoma or carcinoma combined (incidence)c 2 3 4 5 6 7 M − − − − − F 3/53 0/7 1/8 1/10 0/48 a Selected percent incidences are provided in parentheses below the incidences to help illustrate patterns in the responses. b The lower dose of 100 mg/kg-day was started in week 11, resulting in a duration-weighted average dose of 107 mg/kg-day. c The incidences reflect the animals surviving to month 12. This document is a draft for review purposes only and does not constitute Agency policy. 1-67 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 d Statistically significant trend (p < 0.05) was identified using a one-sided Cochran-Armitage trend test performed by EPA. Note: A dash (“‒”) indicates that the study authors did not measure or report a value for that dose group. Mechanistic Evidence 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 37 38 There are few mechanistic data to inform a MOA determination for either liver or lung tumors induced by exposure to RDX. The available in vitro and in vivo genotoxicity assay results are largely negative for parent RDX or its oxidative metabolites (see Appendix C, Section C.3.2), supporting the hypothesis that parent RDX or its oxidative metabolites do not interact directly with deoxyribonucleic acid (DNA). In contrast, there are some positive genotoxicity results for the N-nitroso metabolites of RDX, specifically hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso1,3,5-triazine (TNX). MNX and TNX have been identified from minipigs; minipigs were chosen as the animal model for investigation of RDX metabolism because the GI tract of pigs more closely resembles that of humans (Musick et al., 2010; Major et al., 2007). MNX has tested positive in some in vitro assays, including unscheduled DNA synthesis in primary rat hepatocytes and the mouse lymphoma forward mutation assay (Snodgrass, 1984), although MNX tested negative in the only in vivo test performed, a mouse dominant lethal mutation test (Snodgrass, 1984). 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 (Pan et al., 2007b; Snodgrass, 1984). When S. typhimurium strains TA97a and TA102, strains sensitive to frame shift and oxidative DNA damage, were used in conjunction with elevated concentrations of the metabolizing system (S9), MNX and TNX were mutagenic. 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 et al., 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, 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 (Major et al., 2007). 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 B6C3F1 mice fed 5 mg RDX/kg in the diet for 28 days (Zhang and Pan, 2009b) (dose of 0.75−1.5 mg/kg-day estimated by Bannon et al. (2009b)), 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) (Sweeney et al., 2012b). miRNAs have been associated with several cancers (Wiemer, 2007; Zhang et al., 2007), but the utility of miRNAs as predictive of carcinogenesis has not been demonstrated (Bannon et al., 2009b). Further, it is unknown whether or not aberrant expression of a specific miRNA (or suite of This document is a draft for review purposes only and does not constitute Agency policy. 1-68 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 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 (Bannon et al., 2009a); however, the relevance of this finding to the carcinogenicity of RDX is unclear. • 7 8 9 10 11 12 13 14 15 16 17 18 • 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 et al. (2012b) to conclude that this MOA is not plausible for RDX or its 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 et al. (2012b) to conclude that this MOA is not sufficiently plausible. Cell proliferation. The key events in this hypothesized MOA are GI-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 et al. (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, there are other results that do not support a metabolite-based proliferative response as the MOA for carcinogenesis. • 19 20 • 21 • 22 • 23 24 25 26 27 28 29 Sweeney et al. (2012b) hypothesized a set of MOAs for the liver tumors: The absence of significant liver histopathology in mice after subchronic or chronic exposure to RDX at doses that induced liver tumors (Lish et al., 1984; Cholakis et al., 1980) suggests that cellular toxicity is not a precursor to these tumors. 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. No studies were available that directly measured RDX-induced cell proliferation rates. 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 et al., 2007b) and minipig (Musick et al., 2010; Major et al., 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-69 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 toxicokinetic information on RDX metabolites, neither a cell proliferative MOA nor a mutagenic N- 5 1.2.6. Other Noncancer Effects 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 events leading to the observed liver and lung tumor response in RDX-exposed rodents and lack of nitroso metabolite MOA is supported. Thus, the MOA leading to the increased incidence of liver and lungs tumors is not known. There are isolated reports of RDX inducing systemic effects in several organs/systems, including the eyes and the musculoskeletal, cardiovascular, immune, and GI systems. However, there is less evidence for these effects compared to organ systems described earlier in Section 1.2. Generally, evidence for toxicological effects in these organ systems was limited to human case reports, lacked reproduction or were not observed in other studies of similar duration in the same species, or lacked consistent, dose-related patterns of increasing or decreasing effect. A longer discussion of the evidence for each of the other noncancer effects noted above is provided in Appendix C.3.2. At this time, no conclusions are drawn regarding the other noncancer effects as human hazards of RDX exposure. 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 features, 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 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 hazards 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 et al., 2006; Angerhofer et al., 1986; Levine et al., 1983; Levine et al., 1981a, b; Cholakis et al., 1980; von Oettingen et al., 1949). Human studies provide supporting evidence for RDX as a neurotoxicant and that the nervous system effects observed in experimental animals are plausible in, and relevant to, humans. A cross-sectional study described memory impairment and visual-spatial decrements in RDX- exposed workers (Ma and Li, 1993), although confidence in these findings is relatively low because of issues with design and reporting. Several case reports provide additional evidence of associations between exposure to RDX (via ingestion, inhalation, and possibly dermal exposure) and seizures and convulsions (Kasuske et al., 2009; Küçükardali et al., 2003; Testud et al., 1996a; This document is a draft for review purposes only and does not constitute Agency policy. 1-70 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 Testud et al., 1996b; Woody et al., 1986 and others, see Appendix C.2). Other nervous system effects identified in human case reports include dizziness, headache, confusion, and hyperirritability. Additional support for an association between RDX exposure and nervous system effects comes from consistent evidence of neurotoxicity across taxa, including several species of wildlife (Quinn et al., 2013; Garcia-Reyero et al., 2011; McFarland et al., 2009; Gogal et al., 2003). The association between RDX and neurological effects is biologically plausible, with studies demonstrating a correlation between blood and brain concentrations of RDX and the time of seizure onset (Williams et al., 2011; Bannon et al., 2009a). 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 et al., 2011). Induction of convulsions and seizures appears to be more strongly correlated with dose than with duration of exposure. However, there is some mechanistic information to suggest that repeated exposure to a chemical binding to the receptor convulsant site of GABAA may promote a state of increased neuronal activity that could increase the likelihood of subsequent neurological effects (Gerkin et al., 2010). Some uncertainty remains as to whether the available mechanistic information adequately addresses potential neurotoxicity resulting from longer-duration exposure to RDX. It is unclear if nervous system effects progressed in severity (e.g., from behavioral change to seizures and convulsions) 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. 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. 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 kidney and other urogenital toxicity is more limited than evidence for neurotoxicity. Histopathological changes in the urogenital system (suppurative prostatitis, medullary papillary necrosis, suppurative pyelitis, uremic mineralization, and luminal distention and cystitis of the urinary bladder) were reported in male rats exposed to RDX in the diet for 2 years (Levine et al., 1983). Similar histopathological changes of the urogenital system were not observed in mice, and no other rat studies of similar duration that examined the prostate were available. As discussed earlier, among the lesions identified in the rat, the incidence of suppurative This document is a draft for review purposes only and does not constitute Agency policy. 1-71 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 prostatitis is considered a surrogate marker for RDX-related urogenital effects as it is plausibly associated with effects resulting from other kidney or bladder lesions. The plausibility of a MOA that shares a common molecular initiating event (binding to the GABAA receptor convulsant-site) with the neurotoxic effects of RDX provides some support for an association between RDX exposure and kidney and other urogenital effects. Kidney and other urogenital system effects are a potential human hazard of RDX exposure and were carried forward for consideration for dose-response analysis. Prostatitis, considered a surrogate marker for the kidney and urogenital effects, was selected as a sensitive endpoint representative of the urogenital system effects. There is some evidence for male reproductive toxicity that comes from the finding of testicular degeneration in male B6C3F1 mice chronically exposed to RDX in the diet (Lish et al., 1984) in the only mouse study conducted of that duration (24 months). The effect was noted by the study authors at both the penultimate and highest dose tested in the study. However, studies in different rat strains did not consistently observe testicular effects. Although the available data are limited, given the dose-related findings of mouse testicular degeneration, there is suggestive evidence of male reproductive effects associated with RDX exposure; these effects were carried forward for consideration for dose-response analysis. Testicular degeneration, the only endpoint observed, was selected as the endpoint representative of male reproductive 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 (Angerhofer et al., 1986; Cholakis et al., 1980). Evidence for potential hepatic effects comes from observations of increases (generally dose-related) in liver weight in some subchronic oral animal studies (Lish et al., 1984; Levine et al., 1983; Levine et al., 1981a, b; Cholakis et al., 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, no conclusions are drawn regarding developmental and liver toxicity as human hazards of RDX exposure; these effects were not considered further for dose-response analysis and derivation of reference values. As discussed in Section 1.2, mortality is not addressed in this assessment as a hazard by itself, but rather in the context of nervous and urogenital system hazards. Histopathological changes in the urogenital system observed in male rats exposed to 40 mg/kg-day in the diet for 2 years were considered the principal cause of treatment-related morbidity and mortality (Levine et al., 1983). However, the incidence of suppurative prostatitis, considered a surrogate marker of the urogenital effects, was increased at doses of ≥1.5 mg/kg-day. Therefore, the mortality characterized as secondary to renal effects in Levine et al. (1983) is a less sensitive endpoint (by This document is a draft for review purposes only and does not constitute Agency policy. 1-72 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 more than 10-fold) than the effect that is the selected as the basis dose-response analysis (i.e., suppurative prostatitis). 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 et al., 2006; Angerhofer et al., 1986; Levine et al., 1983; Levine et al., 1981a; Cholakis et al., 1980; von Oettingen et al., 1949). In a 90-day study that recorded nervous system effects and survival more thoroughly than earlier studies, Crouse et al. (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 et al. (2006) 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; however, 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, in general, 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 (2002), 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 or kidney/urogenital systems), 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). In addition, the relationship between convulsions and mortality is not clear and raises concerns for the potential underreporting of convulsions (see Section 1.2.1). 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 35 36 37 As presented in Section 1.2.5, dietary administration of RDX induced dose-related increases in the incidence of hepatocellular adenomas or carcinomas in male and female B6C3F1 mice (Parker et al., 2006; Lish et al., 1984). In the same study, RDX also induced dose-related increases in the This document is a draft for review purposes only and does not constitute Agency policy. 1-73 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 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 yielded a statistically significant trend in the incidence of hepatocellular carcinomas 19 in males, but not in females (Levine et al., 1983). A 2-year dietary study in Sprague-Dawley rats was negative in both sexes Hart (1976), 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 (1976), versus hepatocellular carcinomas in male rats at 8 and 40 mg/kg-day in the Levine et al. (1983) study). The human studies are not informative. This evidence leads to consideration of two hazard descriptors under the EPA’s cancer guidelines (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 et al. (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 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. As noted in the EPA’s cancer guidelines (U.S. EPA, 2005a), 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 B6C3F1 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 19Hepatocellular 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. (1984) 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-74 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 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 the 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 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 37 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. Lifestage, and in particular childhood, susceptibility has not been observed in human or animal studies of RDX toxicity. Transfer of RDX from dam to the fetus during gestation has been reported, and the presence of RDX in the milk of dams administered 6 mg/kg-day by gavage has been documented (Hess-Ruth et al., 2007); however, 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 et al., 1980). Thus, the existing toxicity literature does not provide evidence of early lifestage susceptibility to RDX. 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). Urogenital effects have been observed in males at lower doses than in females (Levine et al., 1983; Levine et al., 1981a, b; Cholakis et al., 1980), suggesting a possible sex-based difference in susceptibility to RDX 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 et al. (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 (Angerhofer et al., 1986), mortality and convulsions were reported in pregnant Sprague-Dawley rats at a dose of ≥40 mg/kg-day, but not at ≤20 mg/kgday, 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 (Angerhofer et al., 1986). In comparison, increased mortality and convulsions were reported at ≥8 mg/kg-day in a 90-day gavage study in F344 rats (Crouse et al., 2006). The instances of one convulsion and two deaths in This document is a draft for review purposes only and does not constitute Agency policy. 1-75 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pregnant rats in the Cholakis et al. (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. This document is a draft for review purposes only and does not constitute Agency policy. 1-76 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 2. DOSE-RESPONSE ANALYSIS 2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER 1 2 3 4 5 6 7 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 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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-1,3,5-trinitro-1,3,5-triazine (RDX) exposure, and kidney and other urogenital system effects are a potential human hazard of RDX exposure. There is suggestive evidence of male reproductive 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. Rather, the mortality evidence was evaluated in the context of that system-specific hazard (see Sections 2.1.6 and 2.1.7 for further discussion). The effects selected to best represent each of the hazards (see discussion in Section 1.3.1) are noted 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-1 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 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 the health effects are summarized below. Nervous System Effects 12 13 14 15 16 17 18 19 Convulsions, a severe adverse effect, were selected for dose-response analysis as a consistent and sensitive endpoint of nervous system effects (see Section 1.3.1 for discussion). This endpoint was reported in seven studies (Crouse et al., 2006; Lish et al., 1984; Levine et al., 1983; Levine et al., 1981a; Cholakis et al., 1980; Martin and Hart, 1974; von Oettingen et al., 1949). 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. Table 2-1. Information considered for evaluation of studies that examined convulsions Study design and size Study reference Crouse et al. (2006) Design Toxicity study Cholakis et al. Developmental study (1980) Measurement of endpoint Relevance of exposure paradigm Number of animals Route Duration Number of Levels Purity Incidence data dose a (mg/kg-d) (%) reported groups 10 rats/sex/ group Gavage 13-wk 5 4−15 99.99 Yes 24−25 female rats/group Gavage 14-d 3 0.2−20 89 Yes Martin and Hart (1974) Toxicity study 3 monkeys/ sex/group Gavage 13-wk 3 0.1−10 Not specified Yes Levine et al. (1983) Toxicity and carcinogenicity bioassay 75 rats/sex/ group Diet 2-yr 4 0.3−40 89−99 No Lish et al. (1984) Toxicity and carcinogenicity bioassay 85 mice/sex/ group Diet 2-yr 4 1.5−175 89−99 No Levine et al. (1981a) Toxicity study 10 rats/sex/ group Diet 13-wk 5 10−600 85 No This document is a draft for review purposes only and does not constitute Agency policy. 1-2 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Study design and size Study reference Design von Oettingen Toxicity study et al. (1949) a 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 Number of animals Route Duration 20 rats/group Measurement of endpoint Relevance of exposure paradigm Diet 13-wk Number of Levels Purity Incidence data dose (%) reported groupsa (mg/kg-d) 3 15−50 90−97 No Excluding the control group. Incidence of convulsions was reported in three studies of RDX—all involving gavage administration: Crouse et al. (2006), Cholakis et al. (1980) (developmental toxicity study), and Martin and Hart (1974). Qualitative findings of nervous system effects were reported in other chronic and subchronic studies—all involving dietary administration: Lish et al. (1984), Levine et al. (1983), Levine et al. (1981a), and von Oettingen et al. (1949). Incidence data on neurotoxic effects of RDX were not collected in any of the dietary studies. For example, Levine et al. (1983) 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 et al. (2006) reported a dose-related increase in convulsions and tremors in both male and female F344 rats following a 90-day oral (gavage) exposure to RDX. This study used a test material of high purity and six dose groups (including the control) that provided good resolution of the doseresponse curve. Cholakis et al. (1980) 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 (1974) 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 et al., 1984; Levine et al., 1983; Levine et al., 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-3 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 gavage studies (Crouse et al., 2006; Cholakis et al., 1980; Martin and Hart, 1974). 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 reference values. In the 2-year study by Levine et al. (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 et al. (1984) and 50 mg/kg-day in the 3-month rat study by von Oettingen et al. (1949)), and, with the exception of Lish et al. (1984), used shorter exposure durations. The unusual dosing regimen in the Cholakis et al. (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 et al. (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 et al. (2006) and Cholakis et al. (1980), and one dietary study, Levine et al. (1983), were selected for dose-response analysis. Kidney and Other Urogenital System Effects 17 18 19 20 21 22 23 24 Suppurative prostatitis was selected for dose-response analysis. It is considered to be a surrogate marker for the broader range of urogenital effects observed in F344 male rats in a 2-year study by Levine et al. (1983). The Levine et al. (1983) study: (1) included a histopathological examination of the kidney and other urogenital system tissues at 6-, 12-, and 24-month time points; (2) 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 (3) reported individual animal data. This study, the only one to identify suppurative prostatisis, was selected for dose-response analysis. Male Reproductive Toxicity 25 26 27 28 29 30 31 Testicular degeneration was selected for dose-response analysis. Lish et al. (1984) observed a dose-related increase in the incidence of testicular degeneration in mice following chronic administration of RDX in the diet. This 2-year study: (1) included histopathological examination of male reproductive organs; (2) included four dose groups and a control group, and adequate numbers of animals per dose group (85/sex/group, with interim sacrifice groups of 10/sex/group at 6 and 12 months); and (3) reported individual animal data. This study, the only one to identify testicular degeneration, was selected for dose-response analysis. 2.1.2. Methods of Analysis 32 33 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-4 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 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 animalto-human extrapolation through the use of physiologically based pharmacokinetic (PBPK) models or the application of a dosimetric adjustment factor, depending on the data available. 11 12 Figure 2-1. Conceptual approach to dose-response modeling for oral exposure. 13 This document is a draft for review purposes only and does not constitute Agency policy. 1-5 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Nervous System Effects 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Incidence data for convulsions from Crouse et al. (2006) and Cholakis et al. (1980) were amenable to BMD modeling. For Crouse et al. (2006), statistical analysis conducted by EPA indicated no significant difference in convulsion rates of male and female rats (Mantel-Haenszel test for independence; see Table 2-2); thus, combined incidence data from male and female rats were used for modeling convulsion data from this study. A BMR of 1% extra risk (ER) for convulsions was used to address the severity of this endpoint; the BMD and BMDL estimates for 5 and 10% ER for the selected model are provided in Appendix D (see Section D.1.2, Tables D-3 to D-6) for comparative purposes. In general, for noncancer effects, severe endpoints are not typically used as the basis of a noncancer risk value because of relatively high uncertainty in extrapolating to a level of exposure likely to be without appreciable risk. The use of a 1% ER BMR for convulsions in Crouse et al. (2006) resulted in extrapolation below the range of the experimental doses. However, the BMD of 3.02 mg/kg-day was not far below the dose range of 4−15 mg/kg-day used in the study; thus, this extrapolation was considered moderate. In addition to uncertainty from extrapolation, model uncertainty from the use of the 1% ER BMR can be a concern. However, the BMDLs from Crouse et al. (2006) ranged from 0.54 to 2.90, a 5.4-fold difference, which is also not considered large, so the use of a 1% ER BMR did not result in substantial model uncertainty. Because incidence data for convulsions were not provided by Levine et al. (1983), 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. Kidney/Urogenital System Effects 21 22 23 24 25 Incidence data on suppurative prostatitis as reported by Levine et al. (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. Male Reproductive Effects 26 27 28 29 30 31 Incidence data on testicular degeneration as reported by Lish et al. (1984) 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 date on the incidence of testicular degeneration. More detailed BMD modeling information is available in Appendix D. This document is a draft for review purposes only and does not constitute Agency policy. 1-6 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Table 2-2. Summary of derivation of PODs following oral exposure to RDX Endpoint and reference (exposure duration/route) PODHED (mg/kg-d) Species/sex Modela BMR AdminBMD BMDL istered RDX RDX (mg/kg-d) (mg/kg-d) doseb AUCc Cmaxd Nervous system Incidence of convulsions Crouse et al. (2006) (90-d/gavage) Male and Multistage 2° female F344 rat, combinede Incidence of Female F344 convulsions rat Cholakis et al. (1980) (GDs 6−19/gavage) Incidence of convulsions Levine et al. (1983) (2-yr/diet) Male and female F344 rat 1% ER 3.02 0.57 0.14 0.28 0.37 1% ER 0.18 0.12 0.03 0.06 0.08 LOAEL = 40 mg/kg-d; NOAEL = 8 mg/kg-df 1.9 3.9 4.3 Quantallinear Kidney/urogenital system 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 LogProbit 10% ER 56.0 16.3 2.4 0.08 0.18 Male reproductive system Incidence of testicular degeneration Lish et al. (1984) (2-yr/diet) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Male B6C3F1 mouse a For modeling details, see Appendix D. POD was converted to an HED using a standard DAF based on BW3/4. c POD 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. d POD 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. e Exact Mantel-Haenszel test for independence between convulsion incidence and sex, stratified by dose, yielded p-value >0.05. f 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. b AUC = area under the curve; BW = body weight; DAF = dosimetric adjustment factor; ER = extra risk; HED = human equivalent dose This document is a draft for review purposes only and does not constitute Agency policy. 1-7 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Human Extrapolation 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 EPA guidance (U.S. EPA, 2011) 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 ¾ 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 et al., 2012a; Sweeney et al., 2012b; Krishnan et al., 2009) based on RDXspecific data. EPA evaluated and further developed these models for extrapolating doses from animals to humans (see Appendix C, 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 (McLanahan et al., 2012). 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-8 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 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 GABAA that 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 et al. (2006) 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. PODHED values based on both blood AUC and peak blood concentration (Cmax) are presented in Table 2-2 for completeness. 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 2-2). 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. A published PBPK model for the mouse was evaluated (Sweeney et al., 2012b); however, major uncertainties were identified in this model. 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-9 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 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.) Comparison of the PODHED values obtained using the mouse PBPK model and a BW¾ scaling approach show a 30-fold difference (see Table 2-2). The BW3/4 approach takes into consideration the fact that the mouse is smaller than the rat and human and on that basis is expected to be a faster metabolizer. The 30-fold difference in PODHED values based on limited mouse data suggests a difference beyond what would be expected from the mouse as a faster metabolizer, but this difference cannot be explained by the available data. For these reasons and the relatively low confidence in the mouse PBPK model, the preferred approach for determining candidate oral values for the endpoint selected from the mouse bioassay (testicular degeneration) is that based on the administered dose of RDX extrapolated to humans using allometric BW3/4 scaling. Extrapolation using BW3/4 scaling. HEDs were also calculated using a BW3/4 scaling approach consistent with EPA guidance (U.S. EPA, 2011). 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: where DAF = (BWa1/4/BWh1/4), BWa = animal body weight BWh = human body weight Using BWa values of 0.25 kg for rats and 0.035 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) × 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.1. 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. 2.1.3. Derivation of Candidate Values 28 29 Under EPA’s A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA, 2002) (Section 4.4.5), and as described in the Preamble, five possible areas of uncertainty and This document is a draft for review purposes only and does not constitute Agency policy. 1-10 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 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. For the testicular degeneration dataset from the mouse bioassay, mouse to human extrapolation was accomplished using BW3/4 scaling (see rationale in Section 2.1.2—Human Extrapolation), which addresses predominantly toxicokinetic and some toxicodynamic aspects of cross-species extrapolation; residual uncertainty in toxicokinetic and toxicodynamic extrapolation remains. In the absence of chemical-specific data to quantify this uncertainty, EPA’s BW3/4 guidance (U.S. EPA, 2011) 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. This is because (1) in studies of subchronic or gestational exposure used to derive a POD, effects were seen at lower doses in the studies of shorter duration than in chronic studies, and (2) other studies upon which a POD was derived were of 2-year duration. Although EPA guidance recommends a default UFS of 10 on the assumption that effects in a subchonic study would occur at approximately 10-fold higher concentration than in a corresponding (but absent) chronic study (U.S. EPA, 2002), the RDX database does not support a UFS of 10. As discussed in Section 1.2.1, although some uncertainty remains about the possibility of effects developing over longer-term exposures to RDX, in general, seizure induction appears to be more strongly correlated with dose level than with exposure duration. The available bioassays suggest that chronic exposure would not lead to effects at lower doses than those induced by subchronic exposure. In addition, chronic dietary doses associated with convulsions were ≥35 mg/kg-day and were at least fourfold higher than gavage doses that induced convulsions in 14- and 90-day studies (i.e., 2 mg/kg-day in Cholakis et al. (1980) and 8 mg/kg-day in Crouse et al. (2006)) (also see Table 1-3 and Figure 1-1). This may be due to differences between dietary and gavage administration (see Sections 2.1.1 and 2.1.7). Nevertheless, these studies do not support the default expectation of observing effects in chronic studies at This document is a draft for review purposes only and does not constitute Agency policy. 1-11 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 approximately 10-fold lower exposure levels than in subchronic studies. Accordingly, a UFs of 1 was applied to PODs 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 3 was applied to all POD values. The oral toxicity database for RDX includes subchronic and chronic toxicity studies in the rat and mouse, a twogeneration 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. EPA prefers to identify reference values based on upstream (less severe) effects that would precede frank effects like convulsions, and some uncertainty remains in our understanding of RDXinduced 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. Uncertainties in the database for RDX neurotoxicity could be addressed by: • Analysis of “convulsions” using more detailed behavioral scoring methods. In the available studies, “convulsion” can 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 (Racine, 1972), employed • in Burdette et al. (1988) would provide a much more accurate, complete, and possibly 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 et al. (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. This document is a draft for review purposes only and does not constitute Agency policy. 1-12 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 • A FOB conducted by Crouse et al. (2006) provides 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 et al. (2006) would be expected to give a screening-level evaluation of some stimuliinduced behaviors that have the potential to be related (e.g., response to handling, touch, click or open field), additional studies addressing whether RDX exposure alters the susceptibility to seizures elicited by traditional means could be informative. Burdette et al. (1988) examined seizure susceptibility in gavaged male Long Evans rats, but at doses ≥10 mg/kg. Further evaluation of seizure susceptibility at doses lower than 10 mg/kg, and with longer exposure durations, may identify additional measures of RDX neurotoxicity. Further evaluation of potential developmental neurotoxicity (and specifically seizure induction) associated with RDX exposure. Models for examining seizure-related behaviors during development exist, mainly involving manipulation and analyses in pre-weanling rodents. Hess-Ruth et al. (2007) 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. Although examination of specific developmental neurotoxicity endpoints has not been conducted in studies of RDX toxicity, the available testing, including a two-generation reproductive toxicity study in the rat (Cholakis et al., 1980), did not report any evidence of neurobehavioral effects in offspring exposed during gestation or lactation. However, confidence in the observation is reduced as there is a question if the extent of observation in Cholakis et al. (1980) was sufficient to accurately characterize neurobehavioral effects. Additional 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. 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 some concern that additional studies described above may lead to identification of a more sensitive endpoint or a lower POD. Accordingly, a UFD of 3 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-13 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Table 2-3. Effects and corresponding derivation of candidate values Endpoint and reference PODHEDa POD type UFA UFH UFL UFS UFD Composite UF Candidate value (mg/kg-d) Nervous system (rat) Incidence of convulsions Crouse et al. (2006) 0.28 BMDL01 3 10 1 1 3 100 2.8 × 10−3 Incidence of convulsions Cholakis et al. (1980) 0.06 BMDL01 3 10 1 1 3 100 6.0 × 10−4 Incidence of convulsions Levine et al. (1983) 3.9 NOAEL 3 10 1 1 3 100 3.9 × 10−2 0.23 BMDL10 3 10 1 1 3 100 2.3 × 10−3 BMDL10 3 10 1 1 3 100 2.4 × 10−2 Kidney/urogenital system (rat) Incidence of prostate suppurative inflammation Levine et al. (1983) Male reproductive system (mouse) Incidence of testicular degeneration Lish et al. (1984) 2 3 4 5 6 7 a 2.4 PODHED values based on data from the rat were derived using PBPK modeling; the PODHED based on data from the mouse was derived using BW3/4 adjustment (see Section 2.1.2 and discussion of the PBPK models above and in Appendix C, Section C.1.5). Figure 2-2 presents graphically the candidate values, UFs, and PODHED values, with each bar corresponding to one data set described in Tables 2-2 and 2-3. This document is a draft for review purposes only and does not constitute Agency policy. 1-14 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 Figure 2-2. Candidate values with corresponding POD and composite UF. This document is a draft for review purposes only and does not constitute Agency policy. 1-15 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 2.1.4. Derivation of Organ/System-Specific Reference Doses 1 2 3 4 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 subsequent cumulative risk assessments that consider the combined effect of multiple agents acting at a common site. Table 2-4. Organ/system-specific RfDs and overall RfD for RDX Effect RfD (mg/kg-d) Basis Study exposure description Confidence 3 × 10−3 Subchronic Medium Kidney/urogenital system Incidence of suppurative prostatitis (Levine et al., 1983) 2 × 10−3 Chronic Low Male reproductive system Incidence of testicular degeneration (Lish et al., 1984) 2 × 10−2 Chronic Low Overall RfD 3 × 10−3 Subchronic Medium Nervous system Incidence of convulsions (Crouse et al., 2006) Nervous system Nervous System Effects 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 The organ/system-specific RfD for nervous system effects was based on the incidence of convulsions in F344 rats reported in Crouse et al. (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 et al. (1980) is lower (by approximately fivefold), there is greater certainty than the value derived from Crouse et al. (2006). Crouse et al. (2006) was specifically designed to assess the nervous system effects of RDX (including a functional observational battery), whereas Cholakis et al. (1980) 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 et al. (2006) 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. Further, Crouse et al. (2006) used a higher purity test material than did Cholakis et al. (1980) (99.99% versus 88.6%, respectively). Finally, the Crouse et al. (2006) study used a longer exposure duration (90 days) than did the Cholakis et al. (1980) study (14 days), and is more representative of a chronic exposure duration. The lower candidate reference value from the Cholakis et al. (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 This document is a draft for review purposes only and does not constitute Agency policy. 1-16 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 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. (1983) 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 et al. (1983) yielded a PODHED 14-fold higher than the PODHED derived from Crouse et al. (2006). The POD derived from the Levine et al. (1983) study is considered less certain than that derived from Crouse et al. (2006). Levine et al. (1983) 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 et al. (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 the fact that data from the Levine et al. (1983) study yielded a higher POD, Levine et al. (1983) was not used as the basis for the organ/system-specific RfD for nervous system effects. Kidney/Urogenital Effects 19 20 21 22 A single data set for incidence of suppurative prostatitis in male F344 rats as reported in a 2-year dietary study by Levine et al. (1983) was brought forward for quantitative analysis as a surrogate marker for the broader array of RDX-associated effects observed in the urogenital system. The RfD for kidney and other urogenital effects is based on this dataset. Male Reproductive Effects 23 24 25 26 A single dataset for male reproductive effects, specifically the incidence of testicular degeneration as reported in male B6C3F1 mice exposed to RDX in diet for 24 months (Lish et al., 1984), was brought forward for quantitative analysis. The RfD for male reproductive effects is based on this dataset. 2.1.5. Selection of the Overall Reference Dose 27 28 29 30 31 32 33 Multiple organ/system-specific reference doses were derived for effects identified as potential hazards from RDX exposure, including nervous system effects, kidney and other urogenital effects, and male reproductive effects. Evidence for nervous system effects, and specifically convulsions, was observed in multiple studies, in multiple species, and following a range of exposure durations. In addition, the organ/system-specific RfD for nervous system effects of 3 × 10−3 mg/kg-day was smaller than the organ/system-specific RfD for male reproductive system effects (2 × 10−2 mg/kg-day) and similar to the value for kidney/urothelial system effects This document is a draft for review purposes only and does not constitute Agency policy. 1-17 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 (2 × 10−3 mg/kg-day). Evidence for dose-related effects on the urogenital system comes primarily from a single 2-year toxicity study in male rats (Levine et al., 1983), and evidence for male reproductive effects comes primarily from a single 2-year toxicity study in mice (Lish et al., 1984); neither a second chronic study in the rat that evaluated prostate histopathology nor a second mouse study was available to validate and replicate these findings. The organ/system-specific RfD of 3 × 10−3 mg/kg-day for nervous system effects in the rat as reported by Crouse et al. (2006) 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 evidence for nervous system effects as a sensitive human hazard of RDX exposure. The overall RfD is derived to be protective of all types of effects for a given duration of exposure, and is intended to protect the population as a whole, including potentially susceptible subgroups (U.S. EPA, 2002). 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 LD01s 18 19 20 21 22 23 24 25 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 a candidate RfD from 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 et al. (2006); Angerhofer et al. (1986); Cholakis et al. (1980); von Oettingen et al. (1949)). This document is a draft for review purposes only and does not constitute Agency policy. 1-18 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 Table 2-5. Comparison of dose levels associated with mortality and convulsions in selected studies Study Doses associated with mortality Doses associated with convulsions ≥8 mg/kg-d ≥8 mg/kg-d ≥25 mg/kg-day ≥25 mg/kg-d 20 mg/kg-d Primarily 20 mg/kg-d; 1 convulsion at 2 mg/kg-d Primarily at 20 mg/kg-d, but one death each at 2 and 6 mg/kg-d 20 mg/kg-d Crouse et al. (2006) Rats, F344, 10/sex/group 0, 4, 8, 10, 12, or 15 mg/kg-d 13 wks/gavage von Oettingen et al. (1949) Rats, sex/strain not specified, 20/group 0, 15, 25, or 50 mg/kg-d 13 wks/diet Cholakis et al. (1980) Rats, F344, 24−25 females/group 0, 0.2, 2.0, or 20 mg/kg-d GDs 6−19/gavage Angerhofer et al. (1986) Rats, Sprague-Dawley, 39−51 mated females/group 0, 2, 6, or 20 mg/kg-d GDs 6−15/gavage 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 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 et al., 2006) and in developmental toxicity studies with exposure durations of two weeks (Angerhofer et al. (1986); Cholakis et al. (1980)). 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 LD01 (the dose expected to be lethal to 1% of the animals) or NOAELs derived from each study. In addition, these LD01 values and NOAELs were compare to the BMD01 for convulsions used to derive the RfD. 20 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 (e.g., kidney pathology, neoplastic lesions), and was not considered in this analysis. Other studies that were less informative and not considered in this analysis are not presented in Table 2-6. 21 20BMDs were compared, as opposed to BMDLs, because, as stated on p. 20 of the BMD Technical Guidance (U.S. EPA, 2012c), “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...” 21The following less informative studies were not included in the analysis of early mortality: This document is a draft for review purposes only and does not constitute Agency policy. 1-19 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 Table 2-6. Summary of dose-response evaluation for mortality following oral exposure to RDX Reference (exposure duration/route) BMR LD01 (mg/kg-d) LDL01 (mg/kg-d) Species/sex Model Lish et al. (1984) (11-wk data from 2-yr study/diet) Male and female B6C3F1 mouse Not amenable to modeling Levine et al. (1981a) (13-wk/diet) Male and female F344 rat, combined Multistage 4⁰ 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% Crouse et al. (2006) (90-d/gavage) Male and female F344 rat, combined Multistage 2⁰ Cholakis et al. (1980) (GDs 6‒19/gavage) Female F344 rat Not amenable to modeling Female SD rat Multistage 3⁰ a Diet studies NOAEL: 35 mg/kg-d 95% CI for response: 0‒4% 1% ER 7.8 2.2 Gavage studies Angerhofer et al. (1986) (GD 6‒15/gavage) 1% ER 2.1 0.46 NOAEL: 2 mg/kg-day 95% CI for response: 0–12% 1% ER 1.7 0.59 • 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 (1974) and 13-week study in the monkey by Martin and Hart (1974). 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. This document is a draft for review purposes only and does not constitute Agency policy. 1-20 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine Reference (exposure duration/route) Cholakis et al. (1980) (GDs 7‒29/gavage) a Species/sex Modela Female New Zealand white rabbit NA (no mortality at highest dose tested) BMR LD01 (mg/kg-d) LDL01 (mg/kg-d) NOAEL: 20 mg/kg-day 95% CI for response: 0‒22% For modeling details, see Appendix D, Section D.1.3. CI = confidence interval; ER = extra risk; LD01 = dose expected to be lethal to 1% of the animals; LDL01 = lower confidence limit on the LD01. 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 Of the studies in Table 2-6, dose-response analysis was conducted for all studies that showed an increased incidence of unscheduled deaths. LD01 values are provided in Table 2-6, and detailed modeling results are provided in Appendix D, Section D.1.3. Mortality was observed only at the highest dose tested at week 11 in the 2-year mouse study by Lish et al. (1984), in the 13-week rat study by von Oettingen et al. (1949), and in the two-generation reproductive and developmental toxicity studies by Cholakis et al. (1980). In these cases, data were not amenable to LD01 estimation, and a NOAEL (with a confidence interval, CI, on its associated response) was used in this comparative analysis instead. LD01 values for mortality in Table 2-6 range from 1.7 mg/kg-day (10-day gavage exposure in pregnant rats) to 7.8 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 BMD01 for convulsions from Crouse et al. (2006) that was used as basis for the overall RfD for RDX (see Table 2-2). The BMD01 for convulsions of 3.0 mg/kg-day is in the middle of the distribution of calculated LD01s, and the lowest LD01 of 1.7 mg/kg-day is within twofold of the convulsion BMD01 of 3.0 mg/kg-day. The NOAELs from studies where mortality was observed tend to be higher than the LDs. However, NOAELs are not directly comparable to BMD01s 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. (1980) 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 the dose associated with 17% mortality at 20 mg/kg-day [see Table 2-5]). In general, this comparison indicates that reference values derived from mortality data would be similar to the final RfD for RDX based on convulsions, assuming the application of the same extrapolation procedures and uncertainty factors. The proximity of doses associated with mortality and nervous system effects should be taken into consideration when evaluating exposures that exceed the RfD. This document is a draft for review purposes only and does not constitute Agency policy. 1-21 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 2.1.7. Uncertainties in the Derivation of the Reference Dose 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 To derive the RfD, the UF approach (U.S. EPA, 2000a, 1994) 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 subchronic to chronic duration. 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 et al., 2006), Johnson (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 et al. (2006) 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 et al. (2006) 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. The PODHED of 0.24 mg/kg-day for a combined incidence of convulsions and mortality 22 was compared to the PODHED of 0.28 mg/kg-day for convulsions alone, indicating that the addition of mortality incidence did not have a significant impact. Therefore, the RfD based on the incidence of convulsions alone does not appear to underestimate the toxicity associated with RDX. 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 22BMD = 2.56 mg/kg-day; BMDL = 0.49 mg/kg-day (see Appendix D.1.2 for BMD modeling results). The PODHED value was derived using PBPK modeling (see Section 2.1.2 and discussion of the PBPK models in Appendix C, Section C.1.5). This document is a draft for review purposes only and does not constitute Agency policy. 1-22 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 14-fold difference in the candidate PODHED values derived from the Crouse et al. (2006) (gavage administration) and Levine et al. (1983) (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). Although the database is adequate for reference value derivation, uncertainty is associated with the consistency in toxicity results across studies that used RDX test materials that differed in purity, formulation, and particle size. There is evidence that differences in test material formulation and particle size (i.e., the increased surface area associated with finely powdered RDX allows for increased absorption) can affect oral bioavailability of RDX and subsequent toxicity (see discussion in Appendix C, Section C.1.5, Absorption of RDX from the GI Tract). 2.1.8. Confidence Statement 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 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 closelyspaced 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. The database includes three chronic studies in rats and mice; eight subchronic studies in rats, mice, dogs, and monkeys; two short-term studies; and four reproductive/developmental toxicity studies in rats and rabbits (including a two-generation reproductive study). Confidence in the database is reduced largely because of (1) differences in test material used across studies, and (2) uncertainties in the influence of oral dosing methods. 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 dietary administration is more representative of potential human exposures to RDX, the use of toxicity data from a gavage (bolus dosing) study introduces This document is a draft for review purposes only and does not constitute Agency policy. 1-23 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 uncertainty in the RfD. Reflecting high confidence in the principal study and medium confidence in the database, overall confidence in the RfD is medium. 2.1.9. Previous IRIS Assessment 3 4 5 6 7 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 et al. (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 × 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 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 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 (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 (1977) 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-24 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 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 8 An RfC for RDX was not previously derived under the IRIS Program. 2.3. ORAL SLOPE FACTOR FOR CANCER 9 10 11 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 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 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 doseresponse assessment, as the nature of the data generally would not support one; however, when the evidence includes a well-conducted study, quantitative analyses may be useful for some purposes, for example, providing a sense of the magnitude and uncertainty of potential risks, ranking potential hazards, or setting research priorities. In the case of RDX, there are well-conducted studies that tested large numbers of animals at multiple dose levels (Lish et al., 1984; Levine et al., 1983), 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 et al. (1984) were selected for quantitative dose-response analysis. The study by Lish et al. (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 et al. (1984) 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 This document is a draft for review purposes only and does not constitute Agency policy. 1-25 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 incidences from the PWG reanalysis of Lish et al. (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 et al., 1986). Female mouse liver and lung tumor incidences from the Lish et al. (1984) study are summarized in Appendix D, Table D-15. The incidence of hepatocellular carcinomas in male F344 rats from the study by Levine et al. (1983) and the incidence of alveolar/bronchiolar carcinomas in male B6C3F1 mice from the study by Lish et al. (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 et al. (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, at 0, 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 B6C3F1 mice showed a positive trend; however, a positive trend was not observed when the 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 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) 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 (U.S. EPA, 2005a). 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 1.2.5). 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 et al. (1984), the survival curves were determined to be similar across dose groups after the dose was reduced in the high-dose group to 100 mg/kg-day (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. This document is a draft for review purposes only and does not constitute Agency policy. 1-26 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 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 considered not likely to produce substantial error in risk estimates. MS-COMBO analyzes tumor incidence as present if either organ (or both) has a tumor and as absent otherwise. The procedure derives a maximum likelihood estimate of the combined risk at a 95% confidence level based on the parameter values obtained for the individual tumor multistage model fits. EPA’s preferred approach for extrapolating results from animal studies to humans is toxicokinetic modeling. As described in Appendix C, Section C.1.5, PBPK models for RDX in mice and humans published by Sweeney et al. (2012b) were evaluated and further developed by EPA. Consideration was given to whether the available toxicokinetic information supported using an internal dose metric derived by PBPK modeling. The available mechanistic data (Section 1.2.5) 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; EPA’s evaluation of these uncertainties is discussed in Summary of Confidence in PBPK Models for RDX in Appendix C, Section C.1.5. Therefore, the PBPK model developed for the mouse was not used, and consistent with the EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the preferred approach for calculating an HED from the mouse tumors is adjustment of the administered dose by allometric scaling to achieve toxicological equivalence across species. As discussed in Section 2.1.1, the administered dose in animals was converted to an HED on the basis of (body weight)3/4 (U.S. EPA, 1992). This was accomplished by multiplying administered dose by (animal body weight in kg/human body weight in kg)1/4 (U.S. EPA, 1992), where the body weight for the mouse is 0.035 kg and the reference body weight for humans is 70 kg (U.S. EPA, 1988). Details of the BMD modeling can be found in Appendix D, Section D.2. 2.3.3. Derivation of the Oral Slope Factor 36 37 The lifetime cancer OSF for humans is defined as the slope of the line from the BMR (10% ER) at the BMDL to the estimated control response at zero (OSF = 0.1/BMDL10-HED). This slope, a This document is a draft for review purposes only and does not constitute Agency policy. 1-27 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 95% upper confidence limit on the true slope, represents a plausible upper bound on the true slope or risk per unit dose. The PODs estimated for each mouse tumor site are summarized in Table 2-7. Using linear extrapolation from the BMDL10-HED, human equivalent OSFs were derived for each 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 B6C3F1 mice administered RDX in the diet for 2 years (Lish et al., 1984) Selected model BMR Multistage 1° 10% ER 64.2e 32.6e 9.56 4.89 0.020 Alveolar/bronchiolar Multistage 1° adenomas or carcinomas 10% ER 52.8 27.7 7.92 4.16 0.024 Liver + lung tumors 10% ER 29.0e 17.7e 4.35 2.66 0.038 Tumor type Hepatocellular adenomas or carcinomasd 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 POD = BMD BMDL BMD10-HED BMDL10-HEDb OSFc (mg/kg-d) (mg/kg-d) (mg/kg-d) (mg/kg-d) (mg/kg-d)−1 Multistage 1° (MS-COMBO) a a BMD10-HED = BMD10 × (BWa1/4/BWh1/4), where BWa = 0.035 kg, and BWh = 70 kg. BMDL10-HED = BMDL10 × (BWa1/4/BWh1/4), where BWa = 0.035 kg, and BWh = 70 kg. c OSF = BMR/BMDL10-HED, where BMR = 0.1 (10% ER). d Incidences of female mouse liver tumors from Lish et al. (1984) are those reported in the PWG reevaluation (Parker et al., 2006). e Data 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 64.8 and 32.8 mg/kg-day, respectively, for hepatocellular adenomas and carcinomas and 29.1 and 17.7 mg/kg-day, respectively, for liver and lung tumors combined. See Table D-15 and the subsequent MS-COMBO results for details. b An OSF was derived from the BMDL10-HED based on a significantly increased trend in the incidence of hepatocellular and alveolar/bronchiolar adenomas or carcinomas in female B6C3F1 mice (i.e., the Liver + Lung BMDL10-HED from MS-COMBO). The OSF of 0.04 (mg/kg-day)−1 is calculated by dividing the BMR (10% ER) by the Liver + Lung BMDL10-HED and represents an upper bound on cancer risk per unit dose associated with a continuous lifetime exposure: OSF = 0.10 ÷ (Liver + Lung) BMDL10-HED = 0.10 ÷ 2.66 mg/kg-day = 3.8 × 10−2 (mg/kg-day)−1 = 4 × 10−2 (mg/kg-day)−1, rounded to one significant figure 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-1,3,5-trinitro-1,3,5-triazine 1 2 The slope of the linear extrapolation from the central estimate of exposure associated with 10% extra cancer risk (BMD10-HED) from the same data sets is given by: Slope of the linear extrapolation from the central estimate = 0.10 ÷ (Liver + Lung) BMD10-HED = 0.10 ÷ 4.35 mg/kg-day = 2.3 × 10−2 (mg/kg-day)−1 3 4 5 = 2 × 10−2 (mg/kg-day)−1 (rounded to one significant figure) The OSF for RDX should not be used with exposures exceeding the POD (2.66 mg/kg-day), because above this level, the fitted dose-response model better characterizes what is known about the carcinogenicity of RDX. 2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 6 7 8 9 10 11 A number of uncertainties underlie the cancer unit risk for RDX. Table 2-8 summarizes the impact on the assessment of issues such as the use of models and extrapolation approaches particularly those underlying the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the effect of reasonable alternatives, the approach selected, and its justification. Table 2-8. Summary of uncertainty in the derivation of the cancer risk value for RDX Consideration and impact on cancer risk value Decision 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 Species/sex Use of data sets from the male mouse or male rat would provide a lower OSF OSF based on tumors in female B6C3F1 mouse Justification Lish et al. (1984) was a well-conducted study; 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 except highest dose where n = 31). 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. 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 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-1,3,5-trinitro-1,3,5-triazine Consideration and impact on cancer risk value Decision Combined tumor types Human risk would ↓ if OSF was based on analysis using only a single tumor type OSF based on liver and lung tumors in female B6C3F1 mouse Selection of dose metric PBPK models are available for the rat, mouse, and human, and using an appropriate internal metric can ↑ accuracy in human extrapolation Mouse liver and lung tumors: administered dose used Cross-species scaling Alternatives could ↓ or ↑ OSF (e.g., 3.5-fold ↓ [scaling by body weight] or ↑ 2-fold [scaling by BW2/3]) BW3/4 scaling (default approach) BMD model uncertainty Alternative models could ↓ or ↑ OSF Use multistage model to derive a BMD and BMDL for combined tumor incidence Low-dose extrapolation approach ↓ cancer risk would be expected with the application of nonlinear extrapolation Linear extrapolation from the POD Justification 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 0.018 per mg/kg-day based on alveolar/bronchiolor carcinomas in male B6C3F1 mice; see Appendix D, Section D.2). Basing the OSF on one tumor site could potentially underestimate the carcinogenic potential of RDX, so an analysis that included data from the two tumor sites was chosen to calculate the combined risk. Because there is no known biological dependence between the liver and lung tumors, independence between the two tumor sites was assumed. This is not likely to produce substantial error in the risk estimates (NRC, 1994). EPA evaluated a published PBPK model in the mouse (Sweeney et al., 2012b); major 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. 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. No biologically based models for RDX are available, and there is no a priori basis for selecting a model other than the multistage. The multistage model has biological support and is the model most consistently used in EPA cancer assessments (Gehlhaus et al., 2011). 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, 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-1,3,5-trinitro-1,3,5-triazine Consideration and impact on cancer risk value Statistical uncertainty at the POD ↓ OSF by 1.6-fold if BMD used as the POD rather than the BMDL Sensitive subpopulations ↑ OSF to an unknown extent Decision BMDL (default approach for calculating plausible upper bound OSF) Considered qualitatively Justification linear low-dose extrapolation was applied, consistent with EPA guidance. Lower bound is 95% CI on administered exposure at 10% ER of liver and lung tumors. 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. 2.3.5. Previous IRIS Assessment: Oral Slope Factor 1 2 3 4 5 6 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 et al. (1984) and analysis of data for hepatocellular adenomas or carcinomas in female mice. An OSF of 1.1 × 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 highdose group because the dose was reduced at week 11 to address high mortality. 2.4. INHALATION UNIT RISK FOR CANCER 7 8 9 10 11 12 13 14 15 16 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 μg/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 17 18 As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005c), either default or chemical-specific age-dependent 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-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 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 1.2.5), application of ADAFs is not recommended. 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(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 (U.S. Environmental Protection Agency). (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 (U.S. Environmental Protection Agency). (1998). Guidelines for neurotoxicity risk assessment [EPA Report] (pp. 1-89). (EPA/630/R-95/001F). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www.epa.gov/risk/guidelines-neurotoxicity-risk-assessment U.S. EPA (U.S. Environmental Protection Agency). (2000a). Science policy council handbook: Risk characterization (pp. 1-189). (EPA/100/B-00/002). Washington, D.C.: U.S. Environmental Protection Agency, Science Policy Council. http://nepis.epa.gov/Exe/ZyNET.exe/40000006.TXT?ZyActionD=ZyDocument&Client=EPA This document is a draft for review purposes only and does not constitute Agency policy. R-11 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 39 40 41 42 43 44 45 46 47 48 49 &Index=2000+Thru+2005&Docs=&Query=&Time=&EndTime=&SearchMethod=1&TocRest rict=n&Toc=&TocEntry=&QField=&QFieldYear=&QFieldMonth=&QFieldDay=&IntQFieldOp =0&ExtQFieldOp=0&XmlQuery=&File=D%3A%5Czyfiles%5CIndex%20Data%5C00thru05 %5CTxt%5C00000002%5C40000006.txt&User=ANONYMOUS&Password=anonymous&Sor tMethod=h%7C&MaximumDocuments=1&FuzzyDegree=0&ImageQuality=r75g8/r75g8/x150y150g16/i42 5&Display=p%7Cf&DefSeekPage=x&SearchBack=ZyActionL&Back=ZyActionS&BackDesc=R esults%20page&MaximumPages=1&ZyEntry=1&SeekPage=x&ZyPURL U.S. EPA (U.S. Environmental Protection Agency). (2000b). Supplementary guidance for conducting health risk assessment of chemical mixtures (pp. 1-209). (EPA/630/R-00/002). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=20533 U.S. EPA (U.S. Environmental Protection Agency). (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 (U.S. Environmental Protection Agency). (2005a). Guidelines for carcinogen risk assessment [EPA Report] (pp. 1-166). (EPA/630/P-03/001F). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www2.epa.gov/osa/guidelines-carcinogen-risk-assessment U.S. EPA (U.S. Environmental Protection Agency). (2005b). Guidelines for carcinogen risk assessment [EPA Report]. (EPA/630/P-03/001F). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www2.epa.gov/osa/guidelinescarcinogen-risk-assessment U.S. EPA (U.S. Environmental Protection Agency). (2005c). 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 (U.S. Environmental Protection Agency). (2006a). 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=157668 U.S. EPA (U.S. Environmental Protection Agency). (2006b). A framework for assessing health risk of environmental exposures to children (pp. 1-145). (EPA/600/R-05/093F). 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=158363 U.S. EPA (U.S. Environmental Protection Agency). (2009). EPAs integrated risk information system: Assessment development process [EPA Report] (pp. 1-4). Washington, DC: U.S. Environmental Protection Agency, Office of Research and Development. http://epa.gov/iris/process.htm U.S. EPA (U.S. Environmental Protection Agency). (2010). Integrated science assessment for carbon monoxide [EPA Report] (pp. 1-593). (EPA/600/R-09/019F). Research Triangle Park, NC: U.S. Environmental Protection Agency, Office of Research and Development, National Center for Environmental Assessment. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=218686 U.S. EPA (U.S. Environmental Protection Agency). (2011). Recommended use of body weight 3/4 as the default method in derivation of the oral reference dose (pp. 1-50). This document is a draft for review purposes only and does not constitute Agency policy. R-12 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 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 37 38 39 40 41 42 43 44 45 46 47 48 49 (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-derivationoral-reference-dose U.S. EPA (U.S. Environmental Protection Agency). (2012a). Advances in inhalation gas dosimetry for derivation of a reference concentration (RfC) and use in risk assessment (pp. 1-140). (EPA/600/R-12/044). Washington, DC. https://cfpub.epa.gov/ncea/risk/recordisplay.cfm?deid=244650&CFID=50524762&CFTOK EN=17139189 U.S. EPA (U.S. Environmental Protection Agency). (2012b). Benchmark dose technical guidance (pp. 1-99). (EPA/100/R-12/001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. https://www.epa.gov/risk/benchmark-dose-technical-guidance U.S. EPA (U.S. Environmental Protection Agency). (2012c). Benchmark dose technical guidance. (EPA/100/R-12/001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www.epa.gov/raf/publications/benchmarkdose.htm U.S. EPA (U.S. Environmental Protection Agency). (2012d). EPA Federal facilities forum issue paper: Site characterization for munitions constituents [EPA Report] (pp. 1-170). (EPA-505-S-11001). Washington, D.C. http://www2.epa.gov/fedfac/epa-federal-facilities-forum-issuepaper-site-characterization-munitions-constituents U.S. EPA (U.S. Environmental Protection Agency). (2012e). Peer review summary report. External letter peer review of study reports on 1,3,5-trinitro-1,3,5-hexahydrotriazine (RDX). Washington, DC. Vale, C; Fonfría, E; Bujons, J; Messeguer, A; Rodríguez-Farré, E; Suñol, C. (2003). The organochlorine pesticides gamma-hexachlorocyclohexane (lindane), alpha-endosulfan and dieldrin differentially Interact with GABA(A) and and glycine-gated chloride channels in primary cultures cells of cerebellar granule. Neuroscience 117: 397-403. http://dx.doi.org/10.1016/S0306-4522(02)00875-8 von Oettingen, WF; Donahue, DD; Yagoda, H; Monaco, AR; Harris, MR. (1949). Toxicity and potential dangers of cyclotrimethylenetrinitramine (RDX). J Ind Hyg Toxicol 31: 21-31. West, RR; Stafford, DA. (1997). Occupational exposures and haematological abnormalities among ordnance factory workers: A case control study. Leuk Res 21: 675-680. http://dx.doi.org/10.1016/S0145-2126(97)00042-8 Wiemer, EAC. (2007). The role of microRNAs in cancer: No small matter [Review]. Eur J Cancer 43: 1529-1544. http://dx.doi.org/10.1016/j.ejca.2007.04.002 Williams, LR; Aroniadou-Anderjaska, V; Qashu, F; Finne, H; Pidoplichko, V; Bannon, DI; Braga, MFM. (2011). RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptormediated currents in the amygdala: A mechanism for RDX-induced seizures. Environ Health Perspect 119: 357-363. http://dx.doi.org/10.1289/ehp.1002588 Williams, LR; Bannon, DI. (2009). Mechanism of RDX-induced seizures in rats (Toxicology Study No. 87-XE-0BT9-09). (ADA511072. USACHPPM-TSN-87-XE-0BT9-09). Aberdeen Proving Ground, MD: U.S. Army Center for Health Promotion and Preventive Medicine, Health Effects Research Program. http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA511072 Woody, RC; Kearns, GL; Brewster, MA; Turley, CP; Sharp, GB; Lake, RS. (1986). The neurotoxicity of cyclotrimethylenetrinitramine (RDX) in a child: A clinical and pharmacokinetic evaluation. Clin Toxicol 24: 305-319. http://dx.doi.org/10.3109/15563658608992595 Yalkowsky, SH; He, Y. (2003). Entry #172 for cyclonite (RDX). In Handbook of Aqueous Solubility Data (1st ed.). Boca Raton, FL: CRC Press. http://www.worldcat.org/title/handbook-ofaqueous-solubility-data/ This document is a draft for review purposes only and does not constitute Agency policy. R-13 DRAFT—DO NOT CITE OR QUOTE Toxicological Review of Hexahydro-1,3,5-trinitro-1,3,5-triazine 1 2 3 4 5 6 7 8 9 10 11 12 Yoshimura, N; de Groat, WC. (1997). Neural control of the lower urinary tract [Review]. Int J Urol 4: 111-125. http://dx.doi.org/10.1111/j.1442-2042.1997.tb00156.x Zemkova, HW; Bjelobaba, I; Tomic, M; Zemkova, H; Stojilkovic, SS. (2008). Molecular, pharmacological and functional properties of GABA(A) receptors in anterior pituitary cells. J Physiol 586: 3097-3111. http://dx.doi.org/10.1113/jphysiol.2008.153148 Zhang, B; Pan, X. (2009a). RDX and miRNA expression: Zhang and Pan respond. Environ Health Perspect 117: A98-A99. http://dx.doi.org/10.1289/ehp.0800276R Zhang, B; Pan, X. (2009b). RDX induces aberrant expression of microRNAs in mouse brain and liver. Environ Health Perspect 117: 231-240. http://dx.doi.org/10.1289/ehp.11841 Zhang, B; Pan, X; Cobb, GP; Anderson, TA. (2007). MicroRNAs as oncogenes and tumor suppressors [Review]. Dev Biol 302: 1-12. http://dx.doi.org/10.1016/j.ydbio.2006.08.028 This document is a draft for review purposes only and does not constitute Agency policy. R-14 DRAFT—DO NOT CITE OR QUOTE