EPA/635/R -15/ 034 a P ublic Comment Draft **www.epa.gov/iris**



## T **oxicological Review of Hexahydro**

**- 1,3,5** 

**- trinitro** 

**- 1,3,5** 

**- triazine (RDX)** 

(CASRN 121

- 82

- 4)

**In Support of Summary Information**  **on the Integrated Risk Information System (IRIS)** 

March 2016

NOTICE

This document is a **Public Comment 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 implicati ons.

National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Washington, DCToxicological Review of Hexahydro

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**DISCLAIMER**

This document is a preliminary draft for review purposes only . T his 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. I t 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 o r recommendation for use.

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# CONTENTS

AUTHORS | CONTRIBUTORS | REVIEWERS

..................................................................................... ix

PREFACE

................................................................................................ .......................................... xi

PREAMBLE TO IRIS TOX ICOLOGICAL REVIEWS

............................................................................... xiv

EXECUTIVE SUMMARY

................................................................................................................ ES -1 LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION

.................................. LS -1

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- 38

1.2.4. Liver Effects

........................................................................................................ 1- 49

1.2.5. Carcinogenicity

................................................................................................... 1- 60

1.2.6. Other Noncancer Effects

.................................................................................... 1- 69

1.3. INTEGRATION AND EVALUATION

.................................................................................. 1- 69

1.3.1. Effects Other Than Cancer

.................................................................................. 1- 69

1.3.2. Carcinogenicity

................................................................................................... 1- 72

1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes . 1- 73

2. DOSE

- RESPONSE ANALYSIS

.................................................................................................... 2-1

2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THA N CANCER

......................................... 2-1

2.1.1. Identification of Studies for Dose

- Response Analysis of Selected Effects

........... 2-1

2.1.2. Methods of Analysis

............................................................................................. 2-5

2.1.3. Derivation of Candidate Values

.......................................................................... 2- 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 LD 01 s

....................................................................... 2- 18
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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

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# TABLES

Table ES

- 1. Organ/system

- specific RfDs and overall RfD for RDX

.............................................. ES -2 Table ES

- 2. Summary of reference dose (RfD) derivation

......................................................... ES -3 Table LS

- 1. Inclusion

- exclusion criteria for health effect studies

.............................................. LS -5 Table LS

- 2. Studies determined not to be informative because of significant issues with design, conduct, or reporting

.............................................................................................. LS -7 Table LS

- 3. Considerations and relevant experimental information for evaluation of experimental animal studies

................................................................................... LS -8 Table LS

- 4. Summary of experimental animal database

.......................................................... LS

- 10 Table LS

- 5. Experimental animal studies considered less informative because of certain study design, conduct, or reporting limitations

.............................................................. LS

- 14 Table 1

- 1. Chemical identity and physicochemical properties of RDX

........................................ 1-1 Table 1

- 2. Ev idence 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- 24 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 ( Lev ine 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- 32 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- 33 Table 1

- 9. Evidence pertaining to male reproductive effects in animals

.................................. 1- 41 Table 1

- 10. Evidence pertaining to reproductive and developmental effects in animals

......... 1- 45 Table 1

- 11. Evidence pertaining to liver effects in humans

....................................................... 1- 52 Table 1

- 12. Evidence pertaining to liver effects in animals

....................................................... 1- 53 Table 1

- 13. Liver tumors observed in chronic animal bioassays

............................................... 1- 63 Table 1

- 14. Lung tumors obs erved in chronic animal bioassays

................................................ 1- 66 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 T able 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 2- 20 Table 2

- 7. Model predictions and OSFs for hepatocellular and alveolar/ bronchiolar adenomas or carcinomas in female B6C3F 1 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

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# FIGURES

Figure LS

- 1. Summary of literature search and screening process for RDX.

.............................. LS -4 Figure 1

- 1. Exposure response array of nervous system effects following oral exposure

........ 1- 16 Figure 1

- 2. Exposure

- response array of kidney and other urogenital system effects.

.............. 1- 35 Figure 1

- 3. Exposure response array of male reproductive effects following oral exposure.

... 1- 44 Figure 1

- 5. Exposure response array of liver effects following oral exposure.

.......................... 1- 59 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

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# ABBREVIATIONS

AAP Army ammunition plant ACGIH American Confere nce of Governmental Industrial Hygienists AChE acetylcholinesterase ADAF age- dependent adjustment factor AIC Akaike’s information criterion ALP alkaline phosphatase ALT alanine aminotransferase AST aspartate aminotransferase
atm atmosphere ATSDR Agency for Toxic Substances and Disease Registry AUC area under the curve BDNF brain

- derived neurotrophic factor BHC beta

- hexachlorocyclohexane BMC benchmark concentration BMCL benchmark concentration lower confidence limit BMD benchmark dose BMDL benc hmark dose lower confidence limit BMDS Benchmark Dose Software BMDU benchmark dose upper bound BMR benchmark response BUN blood urea nitrogen BW body weight CASRN Chemical Abstracts Service Registry Number CCL Contaminant Candidate List CI confi dence interval CICAD Concise International Chemical Assessment Document CNS central nervous system CSF cerebrospinal fluid CYP450 cytochrome P450 DAF dosimetric adjustment factor DDT dichlorodiphenyltrichloroethane d.f. degrees of freedom DMSO dimethylsulfoxide DNA deoxyribonucleic acid DNX 1- nitro -3,5- dinitroso

1,3,5- triazacyclohexane DTIC Defense Technical Information Center EEG electroencephalogram EHC Environmental Health Criteria EPA Environmental Protection Agency ER extra risk FDA Food and Drug Administration FOB functional observational battery FUDS Formerly Used Defense Sites GABA gamma
- amino butyric acid GD gestational day GI gastrointestinal GLP good laboratory practices HED human equivalent dose HERO Health and Environmental Research Online HGPRT hypoxanthine

- guanine phosphoribosyltransferase HMX octahydro -1,3,5,7- tetranitro

1,3,5,7- tetrazocine IARC International Agency for Research on Cancer i.p. intraperitoneal IPCS International Programme on Chemical Safety IRIS Integrate d Risk Information System IUR inhalation unit risk i.v. intravenous LDH lac tate dehydrogenase LOAEL lowest

- observed

- adverse

- effect level LOD limit of detection miRNA microRNA MNX hexahydro -1- nitroso -3,5- dinitro

1,3,5- triazine MOA mode of action MRL Min imal 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 N OAEL no
- observed

- adverse

- effect level NOEL no
- observed

- effect level NPL National Priorities List NRC Nuclear Regulatory Commission NSCEP Natio n al Service Center for Environmental Publications NTP National Toxicology Program NZW New Zealand White OR odds ratio
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ORD Office of Research and Development OSF oral slope factor OSHA Occupational Safety and Health Administration PBPK physiologically based pharmacokinetic PCB polychlorinated biphenyl PCE polychromatic erythrocyte PEL Permissible Exposure Li mit PND postnatal day POD point of departure PWG Pathology Working Group RBC red blood cell RDX Royal Demolition eXplosive (hexahydro

- 1,3,5

- trinitro

1,3,5- triazine) REL Recommended Exposure Limit RfC inhalation reference concentration RfD oral reference dose SDMS spontaneous death or moribund sacrifice SDWA Safe Drinking Water Act SGOT glutamic oxaloacetic transaminase, also known as AST SGPT glutamic pyruvic transaminase, also known as ALT SLE systemic lupus erythematosus SS scheduled sacrifice TLV Threshold Limit Value TNT trinitrotoluene TNX hexahydro -1,3,5- trinitroso

1,3,5- triazine TSCATS Toxic Substances Control Act Test Submissions TWA time

- weighted average U.S. United States of America UCM Unregulated Contaminant Monitoring UF uncertainty factor UF A animal

- to

- human uncertainty factor UF D database deficiencies uncertainty factor UF H human variation uncertainty factor UF L LOAEL

- to

- NOAEL uncertain factor UF S subchronic

- to

- chronic uncertainty factor WBC white blood cell WHO World Health Organization

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# AUTHORS | CONTRIBUTORS | REVIEWERS

**Assessment Team** Louis D’Amico, Ph.D. ( Assessment Manager ) U.S. EPA /ORD/NCEA Todd Blessinger , Ph.D. Washington, DC Ravi Subramaniam, Ph.D.

Christopher Brinkerhoff, Ph.D. ORISE Postdoctoral Fellow at U.S. EPA/ORD/NCEA Currently at U.S. EPA/Office of Chemical Safety and Pollution Prevention Washington, DC

**Contributors** Rob DeWoskin, Ph.D. U.S. EPA /ORD/NCEA Karen Hogan, MS Washington, DC Jordan Trecki, Ph.D.

Belinda Hawkins , Ph.D. U.S. EPA/ORD/NCEA Scott Wesselkamper , Ph.D. Cincinnati, OH

Tammy Stoker, Ph.D. U.S. EPA/ORD/National Health and Environmental Charles Wood, DVM, PhD, DACVP Research Laboratory (NHEERL) Research Triangle Park, NC

Anne Loccisano, Ph.D. ORISE Postdoctoral Fellow at U.S. EPA/ORD/NCEA Washington, DC

**Production Team** Maureen Johnson U.S. EPA/ORD/NCEA Vicki Soto Washington, DC

**Contractor Support** Heather Carlson

- Lynch, S.M

. , DABT SRC
, Inc
., North Syracuse, NY Julie Melia, Ph.D. , DABT Megan Riccardi , M .S.

Pam Ross , M.S. ICF International , Fairfax, VA Robin Blain , Ph.D.

**Executive Direction** Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director) U.S. EPA /ORD/NCEA John Vandenberg, Ph.D, (National Program Director, HHRA) Washington, DC Lynn Flowers, Ph.D., DABT (Associate Director for Health) Vincent Cogliano, Ph.D. (IRIS Program Director ) Samantha Jones, Ph.D. (IRIS Associate Director for Science) Susan Rieth, MPH ( Quantitative Modeling Branch Chief)

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**Internal Review Team** General Toxicity /Immunotoxicity /Cancer Workgroup U.S. EPA/ORD/NCEA Epidemiology Workgroup Neurotoxicity Workgroup Pharmacokinetics Workgroup Reproductive and Developmental Toxicity Workgroup Scoping and Problem Formulation Workgroup Statistics Workgroup Toxicity Pathways Workgroup

**Reviewers**

This assessment was provided for review to scientist s 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 Program s Office of Land and Emergency Management Office of Land and Emergency Management/Federal Facilities Forum Office of Water Region 2, New York, NY Region 8, Denver, CO



### 1 2 3 4 5 6 7 8 9 0 1 2

3 1 1 1

1 This assessment was provided for review to other federal agencies and the Executive Office of the President. Comments were submitted by:

Department of Defense Department of Energy Department of Health and Human Services/Agency for Toxic Substances and Disease Registry Dep a rtment 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 M a nagement and Budget

This document is a draft for review purposes only and does not constitute Agency policy.

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# PREFACE

This Toxicological Review critically reviews the publicly available studies on hexahydro

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- triazine (RDX , Royal Demolition eXplosive , or cyclonite ) in order to identify its adverse health effects and to characterize exposure

- response relationships . I t was prepared under the auspices of the U.S. Environmental Prote ction Agency ( EPA ) Integrated Risk Information System (IRIS) Program . T his 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 carcinogenici ty 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
- r esponse arrays prior to the development of the IRIS assessment. All public comments

p rovided were taken int o consideration in developing the draft assessment. The complete set of public comments are available on the docket at http://www.regulations.gov (Docket ID No. EPA

HQ

- ORD

- 2013

- 0430). 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 b iological system.
Th is assessment was conducted in accordance with EPA guidance, which is summarized in the Preamble to IRIS Toxicological Reviews an d 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 ( h ttp ://www.epa.gov/iris ) . Appendices containing information on assessments by other health agencies, details of the literature search strategy , toxi cokinetic information,
summaries of supplementary t o xicity information, and dose- response modeling are provided as Supplemental Information to this assessment (see Appendices A to D). For additional information about this assessment or for general question s regarding IRIS, please contact EPA’s IRIS Hotline at 202

- 5 6 6

- 1676 (phone), or hotline.iris@epa.gov

.

**Uses and Environmental Occurrence** 

R D X is a military munitions explosive with limited civilian uses ( Gadagbui et al., 2012 ) . I n the United States, RDX is produced at Army ammunition plants (AAP s ) and is not produced

comme r cially . R D X p roduction peaked in the 1960 s; 180 million pounds per year were produced
from 1 969 to 1971 . Y early total production dropped to 16 million pounds in 1984 ( ATSDR, 2012 )

.
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According to the U.S. EPA Chemical Data Access Tool ( http://java.epa.gov/oppt_chemical_search/ ), 

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. RDX c an be released into environmental media (air, water, soil) as a result of waste generated during manufacture, packing

, or d isposal of the pure product, or use and disposal of RDX

containing munitions ( A T SDR, 2012 ; Gadagbui et al., 2012 ; ATSDR, 1999
, 1993
, 1992 ) . R DX is mobile in soil; leaching into groundwater has been reported in samples from military facilities ( Best et al., 199 9a ; 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 su rface
runoff ( U.S. EPA, 2012c )

. An extensive discussion of RDX properties and fate and transport is
available in U.S. EPA (2012c)

. De tectable levels of RDX have been observed in plants irrigated or grown wit h RDX

- contaminated water ( Best et al., 1999b ; Simini and Checkai, 1996 ; Harvey et al.,
1991 ) . R DX 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

- us e d military facilities where RDX is or was produced, stored, or used

. O r al, inhalation, and dermal routes of exposure may be relevant.
A s of 2015, R D X was detected in surface water, groundwater, sediment, or soil at 34 current U.S. EPA National Priorities List (NPL) sites. T h e NPL serves as a list of sites with known or threatened releases of hazardous substances, pollutants, or contaminants through o u t the United
States and its territories

. T h e NPL aids the Agency in identifying the most serious sites that may warrant cleanup. Th e m ajority of the NPL sites where RDX was listed are associated with military
facilities . Based on Department of Defense r e cords, Gadagbui et al. (2012) reported that RDX contamination is present on 76 active military sites, 9 c losed 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 wa s 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) R ule by EPA’s Office of Water from 2007 to 2011 . C ontaminants included in the UCM

program ar e suspected of be ing present in drinking water, but do not have existing health

- based
standards set under the SDWA. R D X has also been included on the Office of Water’s Drinking Water Contaminant Candidate List (CCL) since the initial listing was published in 1998 . Th e p resence 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**

To x icity information on RDX ha s been e valuated by the Agency for Toxic Substanc es and Disease Registry (ATSDR), National Institute for Occupational Safety and Health (NIOSH), Occupational Safety and Health Ad ministration (OSHA) , and Australian National Industrial
Chemicals Notification a n d Assessment Scheme (NICNAS). T he results of the se assessments (as of

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2015 ) a re presented in Appendix A of the Supplemental Information . I t is important to recognize 

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4 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.

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# PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS

Note: The Preamble to IRIS assessments is being revised based on comments received from external peer reviewers and the public, and based on IRIS Program experience with the implementation of systematic review methods. Subsequent drafts of the RDX assessment will include the revised Preamble.



## 1. Scope of the IRIS Program 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 Soon after the EPA was established in 1970 , it was at the forefront of developing risk assessment as a science and applying it in decisions to protect human healt h an d the environment . The C lean Air Act, for example, mandates that the EPA provide “an ample margin of safety to protect public health”; the Safe Drinking Water Act, that “no adverse effects on the health of persons may reasonably be anticipated to occu r, a llowing an adequate margin of safety.” Accordingly, the EPA uses information on the adverse effects of chemicals and on exposure levels below which these effects are not anticipated
to occur. IRIS assessments critically review the publicly available st ud i es to identify adverse health effects from exposure to chemicals and to characterize exposure

- resp onse relationships . I n te r ms set forth by the National Research Council ( NRC , 1983 ) , IRIS assessments cover the hazard identification and dose

- response assessment steps of risk

as s essment, not the exposure assessment or risk characterization steps that are conducted by the EPA’s program and regional offices and by other federal, state, and local health agencies that evaluate risk in specific populations and exposure scenarios. I RIS

a ssessments are distinct from and do not address political, economic, and technical considerations that influen ce th e design and selection of risk management alternatives. An IRIS assessment may cover a single chemical, a group of structurally or toxicologically related chemicals, or a 

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62

63 64 65 66 67 68 69 70 71 72 73 74 complex mixture . These agents may be found in air, water, soil, or sediment. E xceptions are chemicals currently used exclusively as pesticides, ionizing and non

- ionizing radiation, and criteria air pollutants listed under Section 108 of the Clean Air Act (carbon monoxide, lead, nitrogen oxides, ozone, particulate matter, and sulfur oxides). Periodically, the IRIS Program asks other EP A programs and regions, other federal agencies, state health agencies, and the general public to nominate chemicals and mixtures for future assessment or
reassessment . A gent s may be considered for

reas sessment as significant new studies are p u blished . S ele ction is based on program and regional office priorities and on availability of adequate information to evaluate the potential for adverse effects. Other agents may also be assessed in response to an ur ge nt public health need.



## 2. Process for developing and peer

reviewing IRIS assessments
The process for developing IRIS assessments (revised in May 2009 and

e nhanced in July 201 3 ) involves critical analysis of the pertinent studies, opportunities for publi c in put, and multiple levels of scientific review . The EP A revises draft assessments after each review, and external drafts and comments become part of the public record ( U.S. EP A, 2009 ). Before beginning an assessment, the IRIS Program discusses the scope with other EPA programs and regions to ensure that the

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a 

### 1 p 

### 2 in 

### 3
st 

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c 

### 5

**St** 

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**St** 

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**St** 

### 24 25 26 27 28 29 30 31 32

**St** 

### 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ssessment will meet their needs. T hen a ubli c meeting on problem formulation vites discussion of the key issues and the udies and analytical approaches that might ontribute to their resolution.

**ep 4. Public review and comment,** 

**fo l**  **lowed by external peer review** . T he EPA releases the draft assessment for public review and comment . A publ ic meeting provides an opportunity to discuss the assessment prior to peer review . Then the EPA releases a draft for
ext er nal peer review. The peer r eview meeting is open to the public and includes time for oral public comments . The peer

r eviewers ass es s whether the evidence has been assembled and evaluated according to guidelines and whether the conclusions are justified by the evidence

. T he peer review draft, written public com ments, and peer review report

**ep 1**  **. D e**  **velopment of a draft**

**Tox i**  **cological Review** . T he dr aft assessment considers all pert inent p ublicly available studies and applies consistent criteria to evaluate study quality, identify health effects, identify mechanistic events and pathways, integrate the evidence of causation for each effect, and derive toxicity values. A
p u blic meetin g pr i or to the integration of evidence and derivation of toxicity values promotes public discussion of the literature search, evidence, and key issues.

**ep 2. Internal review by scientists in** 

**EPA p**  **rograms and regions** . T he dr aft assessment is revised to a ddr e ss the comments from within the EPA.

**ep 3. Interagency science consultation** 

**wit h o**  **ther federal agencies and the Executive Offices of the President** 

**.** T he d raft assessment is revised to address the interagency comments . T he s cience consultation draft, inter agency comments, and the EPA’s response to major comments become part of the public record. 

### 48 49

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60 61 62 63 64 65 66 67 68 69

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74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

90
91

92 become part of the public record. **Step 5**  **. R evision of draft Toxicological**  **Re view and development of draft IRIS**  **summary** . The draft assessment is revised to reflect the peer review comments, public comments, and newly published studies that are critical to the conclusions of the assessment . T he d i sposition of peer review comments and public comments becomes part of the public record. **Step 6. Final EPA review and interagency** 

**sc ie**  **nce discussion with other federal agencies and the Executive Offices of**  **the President** The dr aft assessment and summary a re re vised to address the EPA and interagency comments. T he sc i ence discussion draft, written interagency comments, and EPA’s response to major comments become part of the public record. **Step 7.**  **Co mpl**  **etion and posting** . T he Toxicological Review and IRIS summary are posted on the IRIS website ( http://www.epa.gov/iris/ ).

The remainder of this Preamble addresses step 1, the development of a draft Toxicological Review . I RIS ass essments follow standard practices of evi denc e

ev aluation and peer review, many of which are discussed in EPA guidelines ( U.S. EPA, 2005a
, b
, 2000b
, 1998
, 1996
, 1991
, 1986a
, b ) and other methods ( U.S. EPA, 2012a
, b
, 2011
, 2006a
, b
, 2002
, 1994 ) . Transparent application of scientific judgment is of paramount importance . T o pr o vide a harmonized approach across IRIS assessments, this Preamble summarizes concepts from these guidelines and emphasizes principles of general applicability.



## 3. Identifying and selecting pertinent studies

**3.1. Identifying studies** 

This document is a draft for review purposes only and does not constitute Agency policy.

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Before beginning an assessment, the EPA 

### 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 conducts a comprehensive search of the primary scientific literature . The literature search follows standard practices and includes the PubMed and ToxNet databases of the National Library of Medicine, Web of Science, and other databases listed in the EPA’s HERO system (Health and Envir onmental Research Online, http://hero.epa.gov/ ) . Searches for information on mechanisms of toxicity are inherently specialized and may include studies on other agents that act through related mechanisms. Each assessmen t specifies the search strategies, keywords, and cut

- off dates of its literature searches. T he EPA posts the results of the literature search on the IRIS website and requests information from the public on additional studies and ongoing research. The EPA also considers studies received through the IRIS Submission Desk and studies (typically unpublished) submitted under the Toxic Substances Control Act or the Federal Insecticide, Fungicide, and Rodenticide Act . Mat er ial submitted as Confidential Business I nf ormation is considered only if it includes health and safety data that can be publicly released. If a study that may be critical to the conclusions of the assessment has not been peer

reviewed, the EPA will have it peer

- rev iewed. The EPA also examines t h e toxicokinetics of the agent to identify other chemicals (for example, major metabolites of the agent) to include in the assessment if adequate information is available, in order to more fully explain the toxicity of the agent and to suggest dose metrics f or subsequent modeling. In assessments of chemical mixtures
, mixture studies are preferred for their ability to reflect interactions among components. The literature search seeks, in decreasing order of preference ( U.S. EPA, 2000b, §2.2 ; 1986b, §2.1 )]:

– Studies of the mixture being assessed.

– S tudies of a sufficiently similar mix ture . In evaluating similarity, the assessment considers the alteration 

### 50 51 52 53

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88 89 of mixtures in the environment through partitioning and transformation.

– S tudies of individual chemical components of the mixture, if there are not adequate studies of sufficiently similar mixtures.



### 3.2. **Sel ecting pertinent epidemiologic**  **studies** Study design is the key consideration for selecting pertinent epidemiologic studies from the results of the literature search

.

– Cohort studies, case

- control studies, and some population

- bas ed surveys (for example, NHANES) provide the strongest epidemiologic evidence, especially if they collect information about individual exposures and effects.

– Ec ological studies (geographic

c orrelation st udies) relate exposures and effects by geographic area . T h e y can provide strong evidence if there are large exposure contrasts between geographic areas, relatively little exposure variation within study areas, and population migration is limited.

– C ase r e ports of high or accidental exposure lack definition of the population at risk and the expected number of cases . T he y can provide information about a rare effect or about the relevance of analogous results in animals. The assessment briefly reviews ecolo g ical studies and case reports but

r eports details only if they suggest effects not identified by other studies.



### 3.3. **Sel ecting pertinent experimental**  **studies** Exposure route is a key design consideration for selecting pertinent

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experimental animal studies or hu man 

### 1 2

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29 30 31 32 33 34 35 36 37

38 39 40 41 42 43 clinical studies.

– S tudies of oral, inhalation, or dermal exposure involve passage through an absorption barrier and are considered mo st pertinent to human environmental exposure.

– I njection or implantation studies are

o ften considered less pertinent but may provide valuable toxicokinetic or mechanistic information . T h ey also may be useful for identifying effects in animals if deposition or absorption is problematic (for example, for particles and fibers). Exposure duration is also a key design considera t ion for selecting pertinent

ex perimental animal studies.

– S tudies of effects from chronic

ex posure are most pertinent to lifetime human exposure.

– S tudies of effects from less

- th an
chronic exposure are pertinent but less preferred for identifying effects fr om lifetime human exposure

. S uch studies may be indicative of effects from less

- tha n
- lifetime human
exposure.
Short- d uration studies involving animals

o r humans may provide toxicokinetic or mechanistic information. For developmental toxicity and reproductive toxicity, irreversible effects may result from a brief exposure during a critical period of development . A c c ordingly, specialized study designs are used for these effects ( U.S. EPA, 2006b
, 1 998
, 1996
, 1991 ).



## 4. Evaluating the quality of individual studies
After the subsets of pertinent epidemiologic and experimental studies have been selected from the literature searches, the assessment evaluates the quality of e ach individual study

. T h is evaluation considers the design, methods, conduct, and d 

### 44 45

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e 

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**4**

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e 

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78 79 80 81 82 83 84 ocumentation of each study, but not whether the results are positive, negative, or

ull . T he objective is to identify the stronger, more informative studies based on a uni form

valuation of quality characteristics across tudies of similar design.



### .1. **Ev aluating the quality of**  **epidemiologic studies** The assessment evaluates design and methodological aspects that can increase or

ecrease the weight given to each pidemiologic stu d y i n the overall evaluation U.S. EPA, 2005a
, 1998
, 1 996
, 1994
, 1991 ):

– Documentation of study design, methods, population characteristics
, and results.

– Defi nition and selection of the study g roup and comparison group.

– A scertainment of exposure to the ch emical or mixture.

– A scertainment of disease or health

e ffect.

– D uration of exposure and follow

- u p and adequacy for assessing the

occurrence o f e ffects.

– Cha racterization of exposure during

c ritical periods.

– S ample size and statistical power to d etect anticipated effects.

– P articipation rates and potential for

se lection bias as a result of the achieved participation rates.

– Me asurement error (can l ead to misclassification of exposure, health outcomes, and other factors) and other types of information bias.

– P otential confounding and other

so urces of bias addressed in the study design or in the analysis of results

. T h e basis for consideration of

c onf ounding is a reasonable

ex pectation that the confounder is related to both exposure and

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outcome and is sufficiently prevalent 

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32 33

34 35

36 37 38 39 40 41 42 to result in bias. For developmental toxicity, reproductive
t oxicity, neurotoxicity, and cancer there is further guidance on the n uan ces of evaluating epidemiologic studies of these effects ( U.S. EPA, 2005a
, 1998
, 1996
, 1991 ).



### 4.2. **Evaluating the quality of experimental studies**  The assessment evaluates design and methodological aspects that can increase or decrease the weight given to each experimental animal study, in

- vi tro study, or human clinical study ( U

. S. EPA, 2005a
, 1998
, 1996
, 1991 ) . Research inv olving human subjects is considered only if conducted according to ethical principles.

– Documentation of study design, animals or study population, methods, basic data, and results.

– N ature of the assay and validity for its i ntended purpose.

– C haracterization o f the nature and extent of impurities and contaminants of the administered chemical or mixture.

– C haracterization of dose and dosing

r egimen (including age at exposure) and their adequacy to elicit adverse effects, including latent effects.

– S ample sizes a n d statistical power to detect dose

- re lated differences or trends.

– A scertainment of survival, vital signs, d isease or effects, and cause of death.

– Con trol of other variables that could i nfluence the occurrence of effects.

The assessment uses statistical tests to

ev aluate whether the observations may be due to chance. T he s tandard for determining statistical significance of a response is a trend test or comparison of outcomes in the exposed groups against those of concurrent controls . I n so me situations, e xamination of historical control data from the same 

### 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 laboratory within a few years of the study may improve the analysis . F or an uncommon effect that is not statistically significant compared with concurrent controls, historical controls may show that the effect is unlikely to be due to chance . F or a response that appears significant against a concurrent control response that is unusual, historical controls may offer a different interpretation ( U.S. EPA, 2005a, §2.2.2.1.3 ). For developmental toxicity, reproductive toxicity, neurotoxicity, and cancer there is further guidance on the nuances of evaluating experimental studies of these effects ( U.S. EPA, 2005a
, 1998
, 1996
, 1991 ) . I n multi

g eneration studies, agents that produce developmental effects at doses that are not toxic to the maternal animal are of special

concern . E f fects that occur at doses associated with mild maternal toxicity are not assumed to result only from maternal toxicit y . Mor eover, maternal effects may be reversible, while effects on the offspring may be permanent ( U.S. EPA, 1998, §3.1.2.4.5.4 ; 1991, §3.1.1.4 )

,.


### 4.3. **Reporting study results**

The assessment uses evidenc e tables to present the design and key results of pertinent studies . Ther e may be separate tables for each site of toxicity or type of study. If a large number of studies observe the same effect, the assessment considers the study quality characteristics in t his section to identify the strongest studies or types of study . The tables present details from these
st udies, and the assessment explains the reasons for not reporting details of other studies or groups of studies that do not add new information

. S up plemental information provides references to all studies considered, including those not summarized in the tables. The assessment discusses strengths and limitations that affect the interpretation of each study. If th e interpretation of a study in the a s sessment differs from that of the study

a uthors, the assessment discusses the basis for the difference.

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### 1 of 

### 2

r 

### 3

a 

### 4



## 5

**5.**

5

e 

### 6 d 

### 7

c 

### 8

a 

### 9 in 

### 10 p 

### 11

m 

### 12 P 

### 13

w 

### 14 15 ju 

### 16

n 

### 17

a 

### 18

c 

### 19 S 

### 20 th 

### 21 C 

### 22 ( I 

### 23 2 

### 24 di 

### 25 si 

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st 

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s 

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a 

### 30 ( 

### 31 1 

### 32

**St** 

### 33 34 35 36 37 38 39 40 41 42 As a check on the selection and evaluation pertinent studies, the EPA asks peer eviewers to identify studies that were not dequately considered.



## . E valuating the overall evidence of each effect

**1. Concepts of causal inference** 

For each health effect, the assessment valuates the evidence as a whole to etermine whether it is reasonable to infer a ausal association between exposure to the gent and the oc cur rence of the effect . T his ference is based on information from ertinent human studies, animal studies, and echanistic studies of adequate quality

. o sitive, negative, and null results are given

e ight according to study quality.

Causal inference i nvol ves scientific dgment, and the considerations are uanced and complex . S ever al health gencies have developed frameworks for ausal inference, among them the U.S

. u rgeon General ( C DC, 2004 ; HEW, 1964 )

,

e International Agency for Research on

ancer ( IARC, 2006 ) , the Institute of Medicine OM, 2008 )

, and the ( U.S. EPA (2010), §1.6 ; 005a), §2.5 )

. A lthough developed for fferent p ur poses, the frameworks are milar in nature and provide an established ructure and language for causal inference

. ach considers aspects of an association that

u ggest causation, discussed by Hill (1965) nd elaborated by Rothman and Greenland 1998) , and ( U .S. EPA (2005a), §2.2.1.7 ; 994), Appendix C ). **rength of association:** The finding of a

large relative risk with narrow confidence intervals strongly suggests that an association is not due to chance, bias, or other factors

. M o dest relative risks, however, ma y reflect a small range of exposures, an agent of low potency, an increase in an effect that is common, exposure misclassification, or other sources of bias. **Consistency of association:** A n inference of 

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81 82 83 84 85 86 87

88 89 causation is strengthened if elevated risks are obs erved in independent studies of different populations and exposure scenarios . R eproducibility of findings constitutes one of the strongest arguments for causation. D iscordant results sometimes reflect differences in study design, exposure, or confounding factors. **Specificity of association:** A s originally intended, this refers to one cause associated with one effect . C u rrent understanding that many agents cause multiple effects and many effects have multiple causes make this a less informative aspect of c au sation, unless the effect is rare or unlikely to have multiple causes. **Temporal relationship:** A c ausal interpretation requires that exposure precede development of the effect. **Biologic gradient (exposure**

**- r**  **esponse relationship):**  E x posure
- response relation ships strongly suggest causation

. A mo notonic increase is not the only p attern consistent with causation . T he presence of an exposure

- r e sponse gradient also weighs against bias and confounding as the source of an association. **Biologic plausibility:** A n i n ference of causation is strengthened by data demonstrating plausible biologic mechanisms, if available . P l a usibility may reflect subjective prior beliefs if there is insufficient understanding of the biologic process involved.

**Coherence:** A n inference of c ausation is strengthened by supportive results from animal experiments, toxicokinetic studies, and short

- te rm tests . C oherence may also be found in other lines of evidence, such as changing disease patterns in the population. **“Natural experiments”:** A c h an ge in exposure that brings about a change in

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disease frequency provides strong 

### 1 2 3 4 5 6 7

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25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

43 44 45 46 evidence, as it tests the hypothesis of causation . A n example would be an intervention to reduce exposure in the workplace or environment that is followed by a reduction of an adverse effect. **Analogy:** In formation on structural analogues or on chemicals that induce similar mechanistic events can provide insight into causation.

These considerations are consistent with guidelines for systematic reviews that evaluate the quality and w ei ght of evidence

. C o nfidence is increased if the magnitude of effe ct is large, if there is evidence of an exposure
- r esponse relationship, or if an

as sociation was observed and the plausible biases would tend to decrease the magnitude of the reported ef f e ct . C onfidence is decreased for study limitations, inconsistency of results, indirectness of evidence, imprecision, or reporting bias ( G uyatt et al., 2008b ; G uyatt et al., 2008a ).



### 5.2. **Evaluating evidence in humans**

For eac h effect, the assessment evaluates the evidence from the epidemiologic studies as a whole

. T h e objective is to determine whether a credible association has been observed and, if so, whether that association is consistent with causation . I n d o ing this, th e assessment explores alternative explanations (such as chance, bias, and confounding) and draws a conclusion about whether these alternatives can satisfactorily explain any observed association. To make clear how much the epidemiologic evidence contribut e s to the overall weight of the evidence, the assessment may select a standard descriptor to characterize the epidemiologic evidence of association between exposure to the agent and occurrence of a health effect. Sufficient epidemiologic evidence of an

as s ociation consistent with causation: T he evidence establishes a causal association for which alternative explanations such as chance, bias, and 

### 47 48 49

S 

### 50 51 52 53 54 55

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### 56 57 58 59 60

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### 61 62 63 64 65 66 67 68 69

**5**

70
t 

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### 86 87 88 89 confounding can be ruled out with reasonable confidence. uggestive epidemiologic evidence of an

as sociation consi s tent with causation: The evidence suggests a causal association but chance, bias, or confounding cannot be ruled out as explaining the association. nadequate epidemiologic evidence to infer a causal association: T h e available studies do not permit a con cl usion regarding the presence or absence of an association. pidemiologic evidence consistent with no causal association: S e veral adequate studies covering the full range of human exposures and considering susceptible populations, and for which alternative explanations such as bias and confounding can be ruled out, are mutually consistent in not finding an association.



### .3. **Eva lu**  **ating evidence in animals**
For each effect, the assessment evaluates he evidence from the animal experiments as whole to determine the extent to which they ndicate a potential for effects in humans

. o nsistent results across various species and

r ains increase confidence that similar

e sults would occur in humans . S ev er al oncepts discussed by Hi ll (1965) are ertinent to the weight of experimental esults: consistency of response, dose

esponse relationships, strength of response, iologic plausibility, and coherenc e ( U.S. EPA, 005a, §2.2.1.7 ; 1 994, Appendix C ). In weighing evidence from multiple xperiments, U .S . EPA (2005a), §2.5 istinguishes: onflicting evidence (t hat is, mixed positive and negative results in the same sex and strain using a similar study protocol) from

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Differi ng results (that is, positive results and 

### 1 2 3

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25 26 27 28 29

30 31 32

33 34 35 36

37 38 39 40 41

42 43 negative results are in different sexes or strains or use different study protocols). Negative or null results do not invalidate
p ositive results in a different experimental system . T he EPA regards all as valid

o b servations and looks to explain differing

r esults using mechanistic information (for example, physiologic or metabolic differences across test systems) or methodological differences (for example, relative sensitivity of the tests, differences in dose leve ls, insufficient sample size, or timing of dosing or data collection). It is well established that there are critical periods for some developmental and reproductive effects ( U

. S. EPA, 2006b
, 2005a
, b
, 1998
, 1996
, 1991 ) . A ccordingly, the assessment determines whether critical periods have been adequately investigated

. S imilarly, the assessment determines

w hether the database is adequate to evaluate other critical sites and effects. In evaluating evidence of genetic toxicity:

– Dem onstration of gene mutations,

c hromosome aberrations, or aneuploidy in humans or experimental mammals ( in v i vo ) provides the strongest evidence.

– Th is is followed by positive results in l ower organisms or in cultured cells ( in v itro ) or for other genetic events.

– N egative results carry less weight, p artly because they cannot exclude the possibility of effects in other tissues ( IARC, 2006 ). For germ

- cell mutagenicity, t he EPA has defined categories of evidence, ranging from positive results of human germ

- ce ll mutagenicity to negative results for all effects of concern ( U.S . EPA, 1986a, §2.3 ).



### 5.4. **Evaluating mechanistic data**

Mechanistic data can be useful in answering several questions. – Th e biologic plausibility of a causal 

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### 79 80 81 82 83 84 85 86 87 88 89 interpretation of human studies.

– Th e generalizability of animal studies to humans.

– Th e susceptibility of particular p opulations or lifestages.

The focus o f the analysis is to describe, if ossible, mechanistic pathways that lead to a ealth effect . Th e se pathways encompass:

– T oxicokinetic processes o f absorption, distribution, metabolism, and elimination that lead to the formation of an active agent and its p r esence at the site of initial biologic interaction.

– T oxicodynamic processes th at lead to a health effect at this or another site (also known as a mode of action )

. F or each effect, the assessment discusses he available information on its modes of ction an d associated key events ( key events eing empirically observable, necessary recursor steps or biologic markers of such eps; mode of action b eing a series of key vents involving interaction with cells, perational and anatomic changes, and esulting i n disease). P e rtinent information may also come from studies of metabolites or

f compounds that are structurally similar or hat act through similar mechanisms

. n formation on mode of action is not required

or a conclusion that the agent is causally elat ed to an effect ( U .S. EPA, 2005a, §2.5 ). The assessment addresses several uestions about each hypothesized mode of ction ( U.S. EPA, 2005a, §2.4.3.4 ). ) **Is the hypothesized mode of action**

**s ufficiently supported in test animals?**  Strong support for a key event being necessary to a mode of action can come from experimental challenge to the hypothesized mode of action, in which studies that suppress a key event observe suppression of the effect . S up p ort for a mode of action is mean ing fully strengthened by consistent results in different experimental models, much

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more so than by replicate experiments in 

### 1 2 3 4

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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 the same model. T he assessment may consider various aspects of causation in addressing this question. 2) **I s the hypothesized mode of**  **action relevant to humans?**  The assessment reviews the key events to identify critical similarities and differences between the test animals and humans . S i t e concordance is not assumed between animals and humans, though it may hold for certain effects or m ode s of action

. I n formation suggesting quantitative d ifferences in doses where effects would occur in animals or humans is considered in the dose

- res ponse analysis . C urrent levels of human exposure are not used to rule out human relevance, as IRIS

assess ments may be used in evaluating new or unforeseen circumstances that may entail higher exposures. 3) **W hich populations or lifestages can be**  **pa rticularly susceptible to the**  **hypothesized mode of action?** Th e assessment reviews the key events to identify populati on s and lifestages that might be susceptible to their occurrence

. Q uantitative differences may result in

s eparate toxicity values for susceptible populations or lifestages.

The assessment discusses the likelihood that an agent operates through multiple

mo d es of action

. A n uneven level of support for different modes of action can reflect disproportionate resources spent investigating them ( U.S. EPA, 2005a, §2.4.3.3 ) . I t should be no ted that in clinical reviews, the credibility of a series of studies is reduced if evidence is limited to studies funded by one interested sector ( Guyatt et al., 2008a ). For cancer, the assessment evaluates evidenc e o f a mutagenic mode of action to g uide extrapolation to lower doses and consideration of susceptible lifestages . K ey d ata include the ability of the agent or a metabolite to react with or bind to DNA, positive results in multiple test systems, or si 

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### 84 85 86 87 88 89 90 91 92 mila r properties and structure

- activity elationships to mutagenic carcinogens ( U.S. PA, 2005a ,§2.3.5 ).



### .5. **Characterizing the overall weight**

**o f the evidence**  After evaluating the human, animal , and mechanistic evidence pertinent to an effect, he assessment answers the question: Does he agent cause the adverse effect? ( NRC, 009
, 1983 ). I n doing this, the assessment evelops a narrative that integrates the vidence pertinent to causation. T o p rovide larity and consistency, the narrative ncludes a st andard hazard descriptor . F or xample, the following standard descriptors ombine epidemiologic, experimental, and mechanistic evidence of carcinogenicity ( U.S. PA, 2005a, §2.5 ). arcinogenic to humans: Th ere is convincing epidemiologic evidence of a causal association (that is, there is reasonable confidence that the association cannot be fully explained by chance, bias, or confounding); or there is strong human evidence of cancer o r its precursors, extensive animal evidence, identification of key precursor events in animals, and strong evidence that they are anticipated to occur in humans. ikely to be carcinogenic to humans: T h e evidence demonstrates a potential hazard to humans bu t does not meet the criteria for carcinogenic . Ther e may be a plausible association in humans, multiple positive results in animals, or a combination of human, animal, or other experimental evidence. uggestive evidence of carcinogenic potential: T h e ev idence raises concern for effects in humans but is not sufficient for a stronger conclusion . T h is descriptor covers a range of evidence, from a positive result in the only available study to a single positive result in an extensive database that includes n e gative results in other species.

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I 

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22 i 

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39 40 41 42 43 44 45 46 47 nadequate information to assess carcinogenic potential: No ot her descriptors apply

. C o nflicting evidence can be classified as in adequate information i f all positive results are opposed by negative studies of equal quali ty in the same sex and strain

. D iffering results

, however, can be classified as suggestive evidence o r as likely to be carcinogenic

. Not likely to be carcinogenic to humans: There is robust evidence for concluding that there is no basis for concern. T h e r e may be no effects in both sexes of at least two appropriate animal species; positive animal results and strong, consistent evidence that each mode of action in animals does not operate in humans; or convincing evidence that effects are not likely by a pa r t icular exposure route or below a defined dose. Multiple descriptors may be used if there s evidence that carcinogenic effects differ by dose range or exposure route ( U.S. EPA, 2005a, §2.5 ). Another example of standard descriptors comes from the EPA’s Integrated Science Assessments, which evaluate causation for the effects of the criteria pollutants in ambient air ( U .S . EPA, 2010 ). Causal relationship: Sufficient evidence to conclude that there is a causal relationship . O bse rvational studies cannot be explained by plausible alternatives, or they are supported by other lines of e vi dence, for example, animal studies or mechanistic information. Likely to be a causal relationship: S ufficient evidence that a causal relationship is likely, but important uncertainties remain . For example, observational

s tudies show an association but c oexposures are difficult to address or other lines of evidence are limited or inconsistent; or multiple animal studies from different laboratories demonstrate effects and there are limited or no human 

### 48 49

50 51 52 53

54 55 56 57

58 59 60 61 62 63

64 65 66 67

68 69 70 71 72 73 74 75 76

77 78 79 80 81

82 83 84 85

86 87 88 data. Suggestive of a causal relationship: A t least

on e high

- q uality epidemiologic study shows an association but other studies are inconsistent. Inadequate to infer a causal relationship: The studies do not permit a conclusion regarding the presence or absence of an association. Not likely to be a causal rel ati o nship: Several adequate studies, covering the full range of human exposure and considering susceptible populations, are mutually consistent in not showing an effect at any level of exposure. The EPA is investigating and may on a trial basis use these or other standard descriptors to characterize the overall weight of the evidence for effects other than cancer.



## 6. Selecting studies for derivation of toxicity values

For each effect where there is credible evidence of an association with the agent, the

asses sment derives toxicity values if there

ar e suitable epidemiologic or experimental data . T he decision to derive toxicity values

m ay be linked to the hazard descriptor. Dose

- response analysis requires q uantitative measures of dose and response

. T h en, other f actors being equal:

– E pidemiologic studies are preferred

o ver animal studies, if quantitative measures of exposure are available and effects can be attributed to the agent.

– A mong experimental animal models,
t hose that respond most like humans

are preferred, if the comparability of

r esponse can be determined.

– S tudies by a route of human

en vironmental exposure are preferred, although a validated

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toxicokinetic model can be used to 

### 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 extrapolate across exposure routes.

– S tudies of longer exposure duration and follow

- up are preferred, to minimize uncertainty about whether effects are representative of lifetime
exposure.

– S tudies with multiple exposure levels

ar e preferred for their ability to provide information about the shape of the exposure

- r e sponse curve

.

– S tudies with adequate power to d etect effects at lower exposure levels are preferred, to minimize the extent of extrapolation to levels found in the environment.

Studies with non

- m on otonic exposure

response relationships are not necessarily excluded from th e analysis . A diminished effect at higher exposure levels may be satisfactorily explained by factors such as competing toxicity, saturation of absorption or metabolism, exposure misclassification, or selection bias. If a large number of studies are sui t a ble for dose

- r esponse analysis, the assessment

co nsiders the study characteristics in this section to focus on the most informative data

. T he assessment explains the reasons for not

a nalyzing other groups of studies . A s a check on the selection of stud ie s for dose

- response analysis, the EPA asks peer reviewers to identify studies that were not adequately considered.



## 7. Deriving toxicity values

**7.1. General framework for dose** 

**** **response analysis**

The EPA uses a two

- step approach that distinguishes analysis of the o b s erved dose

response data from inferences about lower doses ( U.S. EPA, 2005a, §3 ). Within the observed range, the preferred approach is to use modeling to incorporate a wide range of data into th e analysis. T he modeling yields a point of departure ( a n exposure level near the lower end of the 

### 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 observed range, without significant extrapolation to lower doses) (Sections 7.2

- 7.3). Extrapolation to lower doses considers

w hat is known about the modes of action for each effect (Sections 7.4

- 7.5 ) . I f response estimates at lower doses are not required, an alternative is to derive reference values

, which are calculated by applying factors to the point of departure in order to account for sources of uncert ai nty and variability (Section 7.6). For a group of agents that induce an effect through a common mode of action, the dose

response analysis may derive a relative potency factor fo r each agent . A full dose

response analysis is conducted for one well

studi ed i ndex chemical in the group, then the potencies of other members are expressed in relative terms based on relative toxic effects, relative absorption or metabolic rates, quantitative structure

- a ct ivity relationships, or receptor binding characteristics ( U.S. EPA, 2005a, §3.2.6 ; 2000b, §4.4 ). Increasingly, the EPA is basing toxicity values on combined analyses of multiple data sets or multiple responses . The EP A also considers multiple dose

- re sponse approaches if t h ey can be supported by robust data.



### 7.2. **Mod eling dose to sites of biologic**  **effects** The preferred approach for analysis of dose is toxicokinetic modeling because of its ability to incorporate a wide range of data

. T he preferred dose metric would refer to th e active agent at the site of its biologic effect or to a close, reliable surrogate measure . T he active agent may be the administered chemical or a metabolite . C onf idence in the use of a toxicokinetic model depends on the robustness of its validation pro cess and on the results of sensitivity analyses ( U.S. EPA, 2006a ; 2005a, §3.1 ; 1994, §4.3 ). B ecause toxicokinetic modeling can require many parameters and more data than are typically available, the EPA has developed

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standard approaches that can be applied to 

### 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 typical data sets . T hese standard approaches also facilitate comparison across exposure patterns and species.

– I ntermittent study exposures are standardized to a daily average over the duration of exposure . F or chronic effects, daily exposures are averaged over the lifespan. E x posures during a critical period, however, are not averaged over a longer duration ( U.S. EPA, 2005a, §3.1.1 ; 1991, §3.2 ).

– Doses are standardized to equivalent human terms to facilitate comparison of results from different species.

– O ral doses are scaled allometrically

u sing mg/kg 3/4

- day a s the equivalent dose metric across species

. A llometric scaling pertains to

e quivalence across species, not across lifestages, and is not used to scale doses from adult humans or mature animals to infants or children ( U .S. EPA, 2011 ; 2 005a, §3.1.3 ).

– Inhalation exposures are scaled using dosimetry models that apply species

specific physiologic and anatomic factors and consider whether the effect occurs at the site of f ir st contact or after systemic circulation ( U.S. EPA, 2012a ; 1994, §3 ).

It can be informative to convert doses across exposure routes

. I f this is done, the assessment describes the underlying data, algorithms, and assumptions ( U.S. EPA, 2005a, §3.1.4 ). In the absence of study

- specific dat a on, for example, intake rates or body weight, the EPA has developed recommended values for use in dose

- re sponse analysis ( U.S. EPA, 1988 ).



### 7.3. **Modeling response in the range of observation**  Toxicodynamic (“biologically based”) modeling can incorporate data on biologic processes leading to an effect. S uch m odels require sufficient data to ascertain a mode of 

### 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

81 82 83 84 85 86 87 88 89 90

91 92 93 action and to quantitatively support model parameters associated with its key events

. B ecause different models may provide equivalent fits to the observed data but diverge substantially at lower doses, critical biologic parameters should be measured from laboratory studies, not by model fitting

. C onfidence in the use of a toxicodynamic model depends on the robustness of its validation process and on the results of sensitivity analyses . Peer review of the scientific basis and performance of a model is essential ( U. S. EPA, 2005a, §3.2.2 ). Because toxicodynamic modeling can require many parameters and more knowledge and data than are typically available, the EPA has developed a standard set of empirical (“curve

- fit ting”) models ( h ttp://www.epa.gov/ncea/bmds/ ) that can be applied to typical data sets, including those that are nonlinear

. T h e EPA has also developed guidance on modeling dose

response data, assessing model fit, selecting suitable mode ls , and reporting modeling results ( U.S. EPA, 2012b ) . A dditional judgment or alternative analyses are used if the procedure fails to yield reliable results, for example, if the fit is poor, modeling may be restricted to the lower doses, especially if there is competing toxicity at higher doses ( U.S. EPA, 2005a, §3.2.3 ). Modeling is used to derive a point of departure ( U.S . EPA, 2012b ; 2005a, §3.2.4 )

. (See Section 7.6 for alternatives if a point of departure cannot be derived by modeling.):

– If linear extrapo lation is used, selection of a response level corresponding to the point of departure is not highly influential, so standard values near the low end of the observable range are generally used (for example, 10% extra risk for cancer bioassay data, 1% for
ep i demiologic data, lower for rare

c ancers).

– Fo r nonlinear approaches, both
st atistical and biologic considerations are taken into account.

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### 1 2 3 4 5 6 7 8 9 10 11 12 13

14 b 

### 15

s 

### 16

**7**

17 d 

### 18 b 

### 19

e 

### 20

c 

### 21

a 

### 22

1 

### 23 24 25 26 27 28

29 30 31 32

33 34 35 36 37 38 39

40 41 42 – For dichotomous data, a response level of 10% extra risk is generally used for minimally adverse effects, 5% or lower for more severe effects.

– F or continuous data, a response level i s ideally based on an established definition of biologic significance

. I n the absence of such definition, one control standard deviation from the control mean is often used for minimally adve r s e effects, one

- half standard deviation for more severe effects. The point of departure is the 95% lower ound on the dose associated with the elected response level.



### .4. **Ex trapolating to lower doses and**  **response levels** The purpose of extrapolating to lower oses is to estimate responses at exposures elow the observed data. L ow
- do se xtrapolation, ty pically used for cancer data, onsiders what is known about modes of ction ( U.S. EPA, 2005a, §3.3.1 and §3.3.2 ). ) If a biologically based model has been d eveloped and validated for the agent, extrapolation may use the fitted model below the observed range if significant model uncertainty can be ruled out with reasonable co nfi dence. 2) L inear extrapolation is used if the dose

r esponse curve is expected to have a linear component below the point of departure . T hi s includes:

– A gents or their metabolites that are DN A

- r eactive and have direct

mu tagenic activity.

– Agents or their met a bolites for which human exposures or body burdens are near doses associated with key events leading to an effect. Linear extrapolation is also used when data are insufficient to establish mode of action and when scientifically plausible. The result of li n ear extrapolation is 

### 43 44 45 46 47

48 49 50 51 52 53 54 55 56 57 58 59 60

61 62 63 64 65 66 67

68 69 70 71 72 73 74 75 76 77 78 79 80

81 82 83 84 85 described by an oral slope factor or an inhalation unit risk, which is the slope of the dose

- response curve at lower doses or concentrations, respectively. 3) N onlinear models are used for extrapolation if there are sufficient data
to as certain the mode of action and to

c onclude that it is not linear at lower doses, and the agent does not demonstrate mutagenic or other activity consistent with linearity at lower doses

. N o nlinear approaches generally should

n ot be used in cases where mode of a ction has not ascertained

. I f nonlinear extrapolation is appropriate but no model is developed, an alternative is to calculate reference values. 4) B oth linear and nonlinear approaches

m ay be used if there a multiple modes of action . F or example, model i ng to a low response level can be useful for estimating the response at doses where a high

- d ose mode of action would be less i mportant. If linear extrapolation is used, the assessment develops a candidate slope factor or unit risk for each suitable data s et . T h e se results are arrayed, using common dose metrics, to show the distribution of relative potency across various effects and experimental systems. T h e assessment then derives or selects an overall slope factor and an overall unit risk for the agent, con sidering the various dose

- r e sponse analyses, the study preferences discussed in Section 6, and the possibility of basing a more robust result on multiple data sets.



### 7.5. **Co nsidering susceptible**  **populations and lifestages** The assessment analyzes the availabl e information on populations and lifestages that may be particularly susceptible to each effect . A t iered approach is used ( U.S. EPA, 2005a, §3.5 ).

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1) If an epidemiologic or experimental study 

### 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 reports quantitative results for a susceptible population or lifestage, these data are analyzed to derive separate toxicity values for susceptible individuals. 2) If data on risk

- related parameters allow comparison of the general population and susceptible i ndividuals, these data are used to adjust the general

- pop ulation toxicity values for application to susceptible individuals. 3) I n the absence of chemical

- sp ecific data, the EPA has developed age -dep endent adjustment factors f o r early

- life exposure to potenti al c arcinogens that have a mutagenic mode of action . T he re is evidence of early

- l i fe susceptibility to various carcinogenic agents, but most epidemiologic studies and cancer bioassays do not include early

- li fe
exposure . To ad dress the potential for early- life susceptibility, the EPA

r ecommends ( U

. S. EPA, 2005b, §5 ):

– 10

- fold adjustment for exposures before age 2 years.

– 3- f old adjustment for exposures between ages 2 and 16 years.



### 7.6. **Ref erence values and uncertainty**  **factors**

An oral reference dose or an inhalation reference concentration is an estimate of an exposure (including in susceptible subgroups) that is likely to be without an appreciab le ri sk of adverse health effects over a lifetime ( U

. S. EPA, 2002, §4.2 )

. R eference values are typically calculated for effects other than cancer and for suspected carcinogens if a well characterized mode of action indicates that a necessary key event does not occur below a specific dose

. R efer ence values provide no information ab out risks at higher exposure levels. The assessment characterizes effects that form the basis for reference values as adverse, considered to be adverse, or a precursor to an adverse effect. F or d 

### 46

a 

### 47

a 

### 48 ( 

### 49 50 in 

### 51

e 

### 52

a 

### 53

ca 

### 54 fa 

### 55 d 

### 56

n 

### 57

o 

### 58 T 

### 59

c 

### 60

v 

### 61

**H** 

### 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

**A** 

### 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94

oses only a evelopmental toxicity, reproductive toxicity, nd neurotoxicity there is guidance on dverse effects and their biologic markers U.S. EPA, 1998
, 1 996
, 1991 ). To account for uncertainty and variability the derivation of a lifetime human xposure where adverse effects are not nticipated to occur, reference values are

lculated by applying a series of uncertainty
ctors to the poi n t of departure . I f a point of eparture cannot be derived by modeling, a

o
- observed

- ad verse
- effect level or a lowest

bserved

- ad v erse
- effect level is used instead

. he assessment discusses scientific

o nsiderations involving several areas of ariabilit y or uncertainty.

**uman variation**

**.** T h e assessment accounts for variation in susceptibility across the human population and the possibility that the available data may not be representative of individuals who are most susceptible to the effect . A f a ctor o f 10 is generally used to account for this variation . T h is factor is reduced only if the point of departure is derived or adjusted specifically for susceptible individuals (not for a general population that includes both susceptible and non

susceptible individuals) ( U.S. EPA, 2002, §4.4.5 ; 1998, §4.2 ; 1996, §4 ; 1994, §4.3.9.1 ; 1991, §3.4 )

. **nimal**

**- to** 

**- human extrapolation.**  I f animal results are used to make inferences about humans, the assessment adjusts for

cross
- sp ecies differences . T h ese may arise from d iff erences in toxicokinetics or toxicodynamics . A c c ordingly, if the point of departure is standardized to equivalent human terms or is based on toxicokinetic or dosimetry modeling, a factor of 10 1/2 (rounded to 3) is applied to account for the remaining u nce rtainty involving toxicokinetic and toxicodynamic differences . I f a b iologically based model adjusts fully for toxicokinetic and toxicodynamic differences across species, this factor is not used . I n mo st other cases, a factor of 10 is applied ( U.S . EPA, 2011 ;

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### 1 2

**A** 

### 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

**S** 

### 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

**I** 

### 35 36 37 38 39 40 41 42 43 44 45 46 47 48 2002, §4.4.5 ; 1998, §4.2 ; 1996, §4 ; 1994, §4.3.9.1 ; 1991, §3.4 ).

**dverse**

**- effect level to no** 

**- observed** 

**** **adverse**

**- effect level** 

**.** I f a point o f departure is based on a lowest

- o bs erved

adverse- effect level, the assessment must i nfer a dose where such effects are not expected . T h is can be a matter of great uncertainty, especially if there is no evidence available at lower doses. A factor of 10 i s a pplied to account for the uncertainty in making this inference . A factor other than 10 may be used, depending on the magnitude and nature of the response and the shape of the dose

response curve ( U.S . EPA, 2002, §4.4.5 ; 1998, §4.2 ; 1996, §4 ; 1994, §4.3.9.1 ; 1991, §3.4 ).

**ubchronic**

**- to** 

**- chronic exposure**  . I f a point of departure is based on sub ch r onic studies, the assessment considers whether lifetime exposure could have effects at lower levels of exposure. A factor of 10 is applied to account for the uncertainty in using subchronic studies to make inferences about lifetime exposure . T h i s fact or may also be applied for developmental or reproductive effects if exposure covered less than the full critical period

. A factor other than 10 may be used, depending on the duration of the studies and the nature of the
response ( U. S. EPA, 2002, §4.4.5 ; 1998, §4.2 ; 1994, §4.3.9.1 ). **ncomplete database.** I f an incomplete database raises concern that further studies might identify a more sensitive effect, organ system , o r lifestage, the assessment may apply a database uncertainty factor ( U.S . EPA, 2002, §4.4.5 ; 1998, §4.2 ; 1996, §4 ; 1994, §4.3.9.1 ; 1991, §3.4 )

. T he size of the factor depends on the nature of the database deficiency . For example, the EPA typically follows the suggestion that a factor of 10 be applied if both a prenatal toxicity study and a two

- gen eration reproduction study are missing and a factor of 10 1/2 if either is missing ( U.S. 

### 49 50

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

81 82 83 84 85 86 87 88 89 90 91

92 93 EPA, 2002, §4.4.5 ). In this way, the assessment derives candidate values for each suitable data set and effect that is credibly associated with the agent . T he se results are arrayed, using common dose metrics, to show where effects occur across a range of exposures ( U.S. EPA, 1994, §4.3.9 ). The assessment derives or selects an organ - o r system

- s pecific reference value for each organ or system affected by the agent

. T he assessment explains the r ationale for each organ/system

- sp ecific reference value (based on, for example, the highest quality studies, the most sensitive outcome, or a clustering of values)

. B y providing these organ/system

- s p ecific reference values, IRIS assessments facilitate s ub sequent cumulative risk assessments that consider the combined effect of multiple agents acting at a common site or through common mechanisms ( NRC, 2009 ). The assessment then selects an overall reference dose and an overall reference concentration for the agent to represent lifetime human exposure levels where e ffects are not anticipated to occur. T his is generally the most sensitive organ/system

- sp ecific reference value, though consideration of study quality and confidence in each value may lead to a different selection.



### 7.7. **Con fidence and uncertainty in the**  **reference values**

The assessment selects a standard descriptor to characterize the level of confidence in each reference value, based on the likelihood that the value would change with further testing . Co n fidence in reference values is based on quality of the st u d ies used and completeness of the database, with more weight given to the latter. The l evel of confidence is increased for reference values based on human data supported by animal data ( U.S. EPA, 1994, § 4.3.9.2 ). **High confidence:** The reference value is not likely to c ha nge with further testing,

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except for mechanistic studies that might 

### 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 affect the interpretation of prior test results.

**Medium confidence:** T his is a matter of judgment, between high and low confidence.

**Low confidence:** T h e reference value is especially vulne rab le to change with further testing.

These criteria are consistent with guidelines for systematic reviews that evaluate the quality of evidence. T h e se also focus on whether further research would be likely to change confidence in the estimate of effect ( G uyatt et al., 2008b ). All assessments discuss the significant uncertainties encountered in the analysis

. T he EPA provides guidance on

c haracterization of uncertainty ( U.S. EPA, 2005a, §3.6 ) . For example, the discussion distinguishes model uncertainty (lack of knowledge about the most appropriate experimental or analytic model) and parameter uncertainty (lack of knowledge about the parameters of a model)

. A sse ssments also discuss human variation ( interpersonal differences in biologic susceptibility or in exposures that modify the effects of the agent).

Aug ust 2013

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# E XECUTIVE SUMMARY

Summary of Occurrence and Health Effects

Hexahydro- 1,3,5

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- 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 . E xposure to RDX is likely limited to individuals in or around military f acilities where RDX is or was produced, used , or stored . O ral exposure may occur from drinking contaminated groundwater or ingesti ng crops irrigated with contaminated
water . I nhalation or dermal exposures are more likely in occupational settings. Epidem iological 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 . A nimal studies of ingested RDX demonstrate toxicity, including nervous system effects, kidney and other urogenital effects, and male reproductive effects. Results from animal studies provide suggest ive evidence of carcinogenic potential for RDX based on evidence of positive trends in liver and lung tumor incidence i n experimental animals . T here are no data on the carcinogenicity of RDX in humans. 

### 1

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17 **Effects Other Than Cancer Observed Following Oral Exposure**

N ervous system effects are a human hazard of RDX exposure. S everal human case reports and animal studies provide consistent ev i dence of an association between RDX exposure and effects on the nervous system, including seizures or convulsions
, t r emors, hyperirritability, hyper

reactivity, and behavioral changes. Mec h a nistic data support the hypothesis that RDX -induced hyperactivity and seizures likely result from inhibition of GABAergic signaling in the limbic system.
K i dney and other urogenital effects ar e a potential human hazard of RDX exposure based on
observati on s i n 2

- y ear 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. A n increased incidence of suppurative prostatitis was identified, and is considered a marker for RDX

related urogenital effects . Th er e is no established mode of action ( MOA ) for RDX

- related effects on
the u rogenital system.
Th ere is suggestive evidence of male reproductive effects associated with RDX exposure
b as ed on t h e find ing of testicular degeneration in male mice exposed to RDX in the diet for 2 years, in the only mouse s t udy conducted of that duration . T here is no known MOA for male reproductive effects of RDX exposure. Ev i dence for effects on other organs/systems, inc luding the liver a nd develop m ent a l effects

, was more limited than for the endpoints summarized above

.

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**Oral Reference Dose (RfD) for Effects Other Than Cancer** Organ

- specific RfDs were derived for hazards associated with RDX exposure (see 

### 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 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** 

**. O**  **rgan/system**

**- specific RfDs and overall RfD for RDX** 

Effect Ba sis RfD (mg/kg

- d) Study e xposure description Confidence

Nervous system Convulsions 3 × 10 − 3 Subchronic Medium
Kidney/urogenital Suppurative prostatitis 8 × 10 − 3 Chronic Low
Male reproductive Testicular degeneration 8 × 10 − 2 Chronic Low

O verall RfD Nervous system effects 3 × 10 − 3 Subchronic Medium

The overall RfD (s e e Table ES -2 ) is derived to be protective of all types of hazards associated with RDX exposure. Th e e ffect of RDX on the nervous system was chosen as the basis for
the overall Rf D b ecause nervous system effects were observed most consistently across studies, species, and exposure durations, and because they rep r esent the most sensitive human hazard of RDX exposure. I nc i dence of seizures or convulsions as reported in a subchron ic g avage study ( Crouse et al., 2006 ) was selected for derivation of the overall RfD as th is endpoint was measured in

a study that was well

- conducted, utilized a test material of higher p urity than other studies, and ha d five closely

- sp a ced dose groups that allowed characterization of the dose

- response curve

. B enchmark dose (BMD) modeling was utilized to derive t h e point of departure (POD) for RfD derivation (expressed as the BMDL 01 ) . A 1% response level was chosen because of the severity of the endpoint . F u rther, the doses associated with nervous system effects in the Crouse et al. (2006) study also caused increased mortality in the animals. Experimental animal studies provide some
evidence of an association between RDX

- ind u ced convulsions and mortality. In three studies in rats ( Crouse et al., 2006 ; Levine et al., 1983b ; Cholakis et al., 1980 ) , investigators noted that early deaths

we re frequently preceded by neurotoxic signs such as tremors and convulsions; however, the
recorded data from Crouse et al. (2006) do not show as clear a correspondence between convulsions (and other neurotoxic signs) and mort ality (Section 1.2.1).
A physiologically

- based pharmacokinetic (PBPK) model was used to extrapolate the
BMDL 01 derived from a rat study to a human equivalent dose (HED) based on RDX arterial blood
concentration
, wh ic h was then used for RfD derivation. The overall RfD was calculated by dividing the BMDL 01 -H ED for nervous system effects by a composite uncertainty factor (U F ) of 1 00 to account for extrapolation from animals to humans (3), interindividual differences in human susceptibility (10), and uncertai nt y in the database (3)

.

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**Table ES -2** 

**. Summary of**  **r eference**  **d ose**  **(RfD) derivation**  

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Nervous system effects (convulsions) 90

- d F344 rat study Crouse et al. (2006) BMDL 01

- HED : 0.28 mg/kg

- d 100 3 × 10 − 3 mg/kg

- d

a A benchmark response (BMR) of 1% was used to derive the BMD and BMDL given the severity of the endpoint

.

T he resulting POD was converted to a BMDL 01

- HED using a PBPK model based on modeled arterial blood concentration . T he c o ncentration 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 durati on

n or malized to 24 hours.

**Effects Other Than Cancer Observed Following Inhalation Exposure**  No studies were identified that provided useful information on the effects observed following inhalation exposure to RDX . Of th e av ailable human epidemiological st udies of RDX, none provided data that could be used for dose

- re s ponse analysis of inhalation exp o sures . The single experimental animal study involving inh al a tion exposure is not public ly available, and was excluded from consideration due to significant st ud y limitations, including small numbers of animals tested, lack of controls, and incomplete reporting of exposure levels. T he r efore

, the available health effects literature does not support the identification of hazards following inhalation exposure to R DX

.

**Inhalation Ref**  **er ence Concentra**  **tion (RfC ) for**  **Effects Other Than Cancer**

An RfC for RDX could not be derived based on the available health effects data. While inhalation absorption of RDX particulates is a plausible route of exposure, there are no toxi cok in etic studies of RDX inhalation absorption to support an inhalation model. Therefore, a PBPK model for inhaled RDX was not developed to support route

- to

- r out e extrapolation of an RfC from the RfD.

**Evidence for Human Carcinogenicity**

Under EPA’s cancer g u idelines ( U.S. EPA, 2005a ) , there is suggestive evidence of carcinogenic potential for RDX. RDX induced benign and malignant tumors in the liver and lung s of mice ( Parker et al., 2006 ; Lish et al., 1984 ) or rats ( Levine et al., 1983b ) following long

- term administration in the diet. The potential for carcinogenicity a pplies to all routes of human exposure.

**Quantitative Estimate**  **of Carcinogenic Risk**  **f rom Oral Exposure** 

A quantitative estimate of carcinogenic risk from oral exposure to RDX was based on the

i ncreased in c idence of hepatocellular adenomas or carcinomas and alveolar/bronchiolar adenomas
or carcinomas in female B6C3F 1 mice observed in the carcinogenicity bioassay in mice ( Lish et al.,
1984 ) . T his 2- year dietary study included four dose groups and a control group
, adequate numbers
of a nimals 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. ES -3 DRAFT

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12 months), and detailed reporting of methods and results (including individual animal data). T he 

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- day) was reduced t o 100 mg/kg

- day at week 11 due to high mortality. Considering these data along with the uncertainty associated with the suggest i ve nature of the weight of the evidence f or RDX carcinogenicity
, quantitative analys is of the mouse tumor data may be useful for providing a sense of the magnitude of p otential carcinogenic risk.
An or al slope factor ( OSF) that considered the combination of female mouse liver and lung

tumors w as derived f rom BMD and BMDL estimates that correspond to a 10% extra ri sk (ER) of
either tumor . Th e B MDL 10 so derived was extrapolated to the HED using BW 3/4 scaling, and a n OSF was derived by linear extrapolation from the BMDL 10

- HED . T he OSF is 0.0 4 per mg/kg -d ay , based on the liver and lung tumor response in female mice ( L i sh et al., 1984 ).

**Quantitative Estimate**  **of Carcinogenic Risk**  **f rom Inhalation Exposure**  An inhalation unit risk (IUR) value was not calculated because inhalation carcinogenicity
data for RDX are not available. While inhalation abs o rption 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 extrapolatio n of an IUR from the OSF. Thus, a quantitative cancer assessment was not
conducted.

**Susceptible Populations and Lifestages f**  **o r Cancer and Noncancer Outcomes**  Little information is available on populations that may be especially vulnerable to the toxic
eff ects of RDX . L i f estage, and in particular childhood
, susceptibility has not been observed in human or animal studies of RDX toxicity

. I n rats, t ransfer of RDX from the dam to the fetus during gestation an d 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

. D ata to suggest that males may be more susceptible than females to noncancer toxicity associated with R DX ex posure are limited. S pecifically, u rogenital effects have
been noted at lower doses in males than in females

. Dat a on the incidence of convulsions and mortality provide some indication that pregnant animals may be a susceptible population, although
the evidence is unclear. S o m e 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 . S i m ilarly, indivi duals with epilepsy or other seizure syndromes that have their basis in genetic mutation to GABA A receptors may represent another group that may be susceptible to RDX exposure ; h o w ever, there is no information to indicate how genetic polymorphisms may affect s usceptibility to RDX.

**Key Iss**  **ues Addressed in Assessment**

S

election of a 1% benchmark response ( BMR ) for convulsions. In most instances, the spectrum of effects associated with chemical exposure will range in se verity , with relatively less

This document is a draft for review purposes only and does not constitute Agency policy. ES -4 DRAFT

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severe effects generally occurring at doses lower than those associated with more severe or “frank” 

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38 toxicity . C onvulsions in rats were selected as the basis for derivation of the RDX RfD ; less severe nervous system effects were generally not observed at l ower doses

. U. S. EPA (2012b) recommends considering the statistical and biological characteristics of the dataset when selecting a BMR

, including t he severity of the effect

. Fo r convulsions, a BMR leve l of 1% ER was selected for modeling, balancing th e q u antitative limitations of the available animal bioassays and the severity
of th is ef fect

. Modeling convulsion incidence from Crouse et al. (2006) using this BMR resulted in a moderate extrapolation of th e 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 w i t h the influence of the method of oral dosing on the magnitude of dose required
to induc e nervous system effects. Findings from animal studies suggest that gavage administration

g enerally induced convulsions in experimental animals at lower doses than di d d ietary
administration
, p o ssibly due to the bolus dose re ceived from gavage administration resulting in a comparatively faster absorption and higher peak blood concentrations of RDX ( s ee Section 1. 2 .1)

. The difference in neurotoxic response associated with gavage versus dietary administration is i n
pa rt r eflected in the 14- fo ld difference in the candidate POD HED values derived from the Crouse et al. (2006) (gavage administration) and Levine et al. (1983b) (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 a cr oss studies in test materials and experimental designs (e.g., test article purity and particle size, number and spacing of dose groups, e x posure duration, frequency of clinical observations, and thoroughness of t h e reporting of observations) that could also have contributed to differences in response. As d ie tary 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.
S uppurative prostatitis as a marker for kidney and other urogenital effects. The candidate RfD for kidney and other urogenital effects is based on a d o se
- related increase in the
incidence of suppurative prostatitis f r om a 2

- year feeding study in male F344 rats ( Levine et al.,
1983b )

. This study is the only 2

- year study in rats that examined the prostate. Some reports have hypothesized that the observed suppurative prostatitis i s a secondary effect from a bacterial
infection unrelate d to R DX toxicity ( ATSDR, 2012 ; Sweeney et al., 2012a ; Crouse et al., 2006 )

. W hile an opportunistic bacterial infection may have been the proximal cause of the suppu rative prostatitis, the infection was considered se co ndary to urogenital effects associated with RDX

exposure . Hi s topathological findings for the bladder are not definitive because the design of the principal study called for histopathological examination o f the bladder only if gross abnormalities
were observed. Alt h ough the pathogenesis of kidney and urogenital effects is unclear, suppurative prostatitis was considered to be a marker for the broader array of kidney and other urogenital
effects observed b y Levine et al. (1983b)

.

This document is a draft for review purposes only and does not constitute Agency policy. ES -5 DRAFT

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# LITE RATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION

**L iterature Search and Screening Strategy**  A literature search and screening strategy was applied to identify literature related to 

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31 characterizing the health effects of hexadydro- 1,3,5

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- 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 i n four online scientific databases

— Pub Med, Toxline, Toxcenter, and T o xic Substances Control Act Test Submissions ( TSCATS )

. The initial search was performed in April 2012 , an d literature search updates were conducted in February
2013 , J anuary 2014 , an d January 2015 . Searches of TSCATS were perfor med in February 2013 and January 2 015 o nly. The detailed search approach for these databases, including the query strings
, and th e n umber s of citations identified per database are provided in Appendix B, Table B

- 1. The Department of Defense has conducted se v eral unpublished toxicological studies on RDX ; to ensure that all such studies were located, t h e Defense Technical Information Center (DTIC) database, a central online repository of defense

- r elat ed scientific and technical information within the Department of Defense, w as 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

. S earch es of the five online databases identified 1,143 c itations (after electronically eliminating duplicates). The computerized database searches

w ere supplemented by review i ng on line regulatory sources, performing “forward” and “backward”
searches of W eb of Science ( see Appendix B, Table B

- 3), and adding additional references that were
identified d uring th e development of the T oxicological R eview ( including submissions from the Department of Defense ); 3 4 citations were obtained using these additional sea rch strategies. In total, 1,177 c it ations were identified using online scientific databases and additional search strategies. The U.S. Environmental Protection Agency ( E PA ) requested public submissions of
additional information in 2 0 10 (75 FR 76982; De cember 10, 2010 )

. EPA also issued a request to the public for additional information in a Federal Register Notice in 2013 ( 7 8 FR 48674; August 9,
2013 ) , and established a docket for public comment (EPA

- HQ

- O RD

- 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.

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The citations identified using the search strategy described above were screened using the 

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18 title, abstract, and in limited instances, full text for pertinence to examining the health effects of
chronic RDX exposure. The process for screening the literatur e is described below and is shown graphically in Figure LS

- 1. 1 The objective of this manual screen was to identify sources of primary human health effects data and sources of primary data that inform t h e assessment of RDX health effects (i.e., the bottom three bo x es 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 applie d in identifying sources of mechanistic and toxicokinetic data. The number of such studies for RDX is not large, and therefore, all s tudies that provided data on adsorption, distribution, metabolism, or elimination, physiologically- b a sed pharmacokinetic ( PBPK) models, or relevant RDX mode of action (MOA)
were considered. Studies that met one or more of the exclusion criteria i n Table LS -1 were binned as “Excluded/Not on Topic” and were not further considered in this assessment. A final group of
studies c o nsisted of reviews and other sources of RDX i nformation (e.g., exposure , ecosystem effects ) that did not meet the inclusion criteria in Table LS

- 1 . These studies were binned into a category called “ S e condary Literature and Sources of Other RDX Informatio n,” 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 :

• 25 r eferences (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. 2

• 25 references were identified as sources of supplementary health effects data, including experimental animal studie s 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.

1 S tudies 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, s tudies 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) d id 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.

2 On the HERO project page, 27 records are a ssociated with “Sources of Health Effects Data,” rather than the 25 identified in Figure LS

- 1. Two of the records in HERO are not unique studies or reports. Rather, these two records provide links to the multi

- volume laboratory reports for the 2

- year stu dies in rats by Levine et al. (1983b) and mice by Lish et al. (1984)

This document is a draft for review purposes only and does not constitute Agency policy. LS -2 DRAFT

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### 1 • 47 references were identified as sources of mechanistic and toxicokinetic data; these included 1 9 studies describing PBPK models and other toxicokinetic information, 11 studies providing genotoxicity information, and 17 studies pertaining to other mechanisti c information. Information from these studies was not extracted into evidence tables; however, these studies supplemented the assess ment of RDX health effects ( e.g., evaluation of MOA and extrapolation of experimental animal findings to humans ).

• 2 80 refer ences 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 refe rences were kept as additional resources for development of the Toxicological Review.

• 80 1 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 consideratio n (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 potenti ally relevant health effects data.

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### 1 2 3 4 5 6

7 The n umbers on this figure match the HERO project page as of 1/12/2016; subsequent changes may not be reflected. A limited number of references were assigned more than one tag; therefore, the sum of the references i n bo xes 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** 

**.**
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**Table LS -1**  **. Inclusion**

**- exclusion**  **criteria for health effect studies**  

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Inclusion criteria Exclusion criteria
Population • Humans

• Standard mammalian animal models,

including rat, mouse, rabbit, guinea pig, monkey, dog • Ecological species a

• Nonmammalian species a

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 on e 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 tre atment 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 (s tudies that do not involve

testing of RDX )

• Other studies not informa tive 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. LS -5 DRAFT

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Inclusion criteria Exclusion criteria
was considered small (e.g., Chinese paper of case reports of RDX poisonings)

-- duplicate studies not previously identified 

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20 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 effe cts 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: ( h ttp://hero.epa.gov/index.cfm/project/page/project_id/2216 ).

**S election of Critical Studies for**  **Presentation in Evidence Tables** 

Selection of Critical Studies In order to systematically summarize the important information from the primary health
effects st udies in the RDX d atabase, 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 “ S ources of H ealth E ffects D ata” (Figure LS -1 , Table LS -1) and were considered for extraction into evidence tables for hazard identification in Chapter 1.
A s tud y w as not presented in the evidence tables if flaws in its design, conduct, or reporting

we r e so great that the results would not be considered credible ( e.g., studies where concurrent control information is lacking) . S u c h study design flaws are discussed in a number of EPA’s guidelines (see h ttp ://www.epa.gov/iris/backgrd.html ) or summarized in the Pr eamble . F or 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. T h e specific studies and basis for considering the studies t o be uninformative are summarized in Table LS -2.

This document is a draft for review purposes only and does not constitute Agency policy. LS -6 DRAFT

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**Table LS -2** 

**. Studies determined not to be informative because of significant**  

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12 **issues with design, conduct, or reporting**

Reference 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 re ported.

ATSDR (1996) ; Disease prevalence study in residential population Study of a population residing in two neighborhoods where RDX had been detected in well water . T he study was conducted 7 yrs after residents were provided the opportunity to connect to a municipal water supply . O nly one target

- area household reported using private well water for bathing and cooking at the time of the health study . T he 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 . I ssues 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), a ll human and experime ntal animal studies of RDX involving repeated exposure were considered in assessing the evidence for health effects associated with chronic exposure to RDX

. S t udies that contain pertinent information for the toxicological review and augment hazard
id e nti fication conclusions, such as genotoxicity and other mechanistic studies, studies describing
the to xicokinetics of RDX, hum an case reports , and experimental animal studies involving exposures of acute/ sho r t- term duration or routes of exposure other than or al and inhalation, were
not included in evidence tables. Nevert h eless, 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. LS -7 DRAFT

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**S tudy Evaluation** 
For this assessment, primary sources of health effects data consisted of three human 

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15 studies 3 and 21 reports 4 presenting results of e xperimental animal studies. These studies were evaluated using the study quality considerations outlined in the Preamble, considering aspects of design, conduct, or reporting that could affect the inte rpretation 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 iden tify 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. Mu ch of the key information for conducting this evaluation can be determined based on study methods and how the study results were reported. Im p ortantly, the evaluation at this stage does not consider the direction or magnitude of any reported effects.

**Table LS -3** 

**. C**  **on siderations**  **and relevant experimental information for evaluation of experimental animal studies** 

Methodological feature Considerations (relevant information extracted into evidence tables)

Test animal Suitability of the species, strain, sex, and source of the test animals Experimental design Suitability of animal age/lifestage at exposure and endpoint testing; periodicity and duration of exposure (e.g., hr s /d, d/wk); timing of endpoint evaluations; and sample size and experimental unit (e.g., animals
, dams
, litters) Exposure Cha ra cterization 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); verif ication 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 intere st (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)

3 Two 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 b ecause 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 i dentified in the literature search (see Figure LS -1).

4 The 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. (1983b) 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. LS -8 DRAFT

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Information on study features related to this evaluation is reported in evidence tables and 

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35 was considered in the synthesis of evidence. Discussion of study strengths and limitations (that ultimately supported preferences for the studies and data relied upon) were included in the text
where relevant. If EPA’s i nterpreta tion of a study differed from that of the study authors, the
assessment discusses the basis for the difference.
T h e general findings of this evaluation are presented in the remainder of this section . S tudy evaluation considerations that are outcome specifi c are discussed in the relevant health effect
sections in Section 1. 2

.

Human Studies

T he body of literature on RDX includes three studies of populations occupationally exposed to RDX (one case

- c o ntrol and two cross

- sectional studies) ( West and Stafford, 1997 ; Hathaway and Buck, 1977 ) . To varying degrees, these epidemiol ogy 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 p recise definition of the study population

, while others d i d 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

- co n trol study), which limits their sta tistical 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 informati on 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 em ploy ment or other metrics that c ould be used to ascertain duration of exposure. In the case

- c o ntrol 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

- e x posure to other chemicals that may elicit the observed effects. The methodological limi t a tions 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 c ase reports that describe effects following acute exposure to RDX. Case reports can suggest

o rgan systems and health outcomes that might be related to RDX exposure but are often anecdotal, and typically describe unusual or extreme exposure situations; t h us, 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 Append i x C, Section C. 2.

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Experimental Animal Studies The oral toxicity database for RDX includes three chronic studies in rats and mice, eight 

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19 subchronic studies in rats, mice, dogs, and monkeys, two short er
- term studies in dogs and rats
, one
two

- generation reproductive toxicity study in the rat, four developmental toxicity studies in rats
and rabbits , and a single

- exposure study of audiogenic seizures in rats (Table LS

- 4)

.

**Table L**  **S -4** 

**. S**  **ummary of experimental animal d atabase** 

Study c ategor y Study duration, s pecies / strain , and oral administration method

Chronic 2

- Y r study in B6C3F 1 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., 1983b )
Subchronic 13

- Wk study in B6C3F 1 mice , experiment 1 (diet) ( Cholakis et al., 1980 ) 13

- Wk study in B6C3F 1 mice, experiment 2 (diet) ( Cholakis et al., 1980 ) 13

- Wk study in F344 rat s (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 st udy 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

- G eneration reproductive toxicit y study in CD rats (diet) ( Cholakis et al., 1980 ) Developmental Developmental study ( gestational days [ GD s] 6

− 19) in F344 rats (gavage) ( Cholakis et al., 1980 ) Developmental study (GD s 6

− 15) in Sprague

- Dawley rats , range

- finding (gavage) ( Angerhofer et al., 1986 ) Developmental study (GD s 6

− 15 ) in Sprague

- Dawley rats (gavage) ( Angerhofer et al., 1986 ) Developmental study (GD s 7

− 29) in New Zealand W hite (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 stu d i es ( Levine et al., 1990 ; von Oettingen et al., 1949 ) , these toxicity studies are available only as unpublished contract laboratory reports. Pe er reviews of

t hree unpublished studies identified as most informative to the assessment of the health effects of
RD X

— th e 2

- ye ar bioassays by Levine et al. (1983b) and Lish et al. (1984) and 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, 2012d ) 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 em p loyed

. 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 i mmunotoxicity ( U.S. EPA, 2012d ).
O nly one unpublished inhalation study of RDX (dated 1944 ) was identifi ed . A s discussed in Appendix B and Table LS -2
, t his inhalation study was considered uninformative and was excluded

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from consideration in the development of the Toxicological Review because of study design issues 

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34 (including lack of a control group, incompl ete information on exposure levels, and inadequate reporting) . Therefore, evaluation of the experimental animal database for RDX is limited to studies of oral toxicity . An evaluation of the oral toxicity literature, organized by general methodological
fe ature s , is provided in the remainder of this section.

Test animal

Th e RDX database consists of health effect studies conducted in multiple strains of rats (F344, Sprague

- Daw ley , CD ), mice (B6C3F 1 ), dog s (beagle), and monkey s . T he species and strains
of an imals used are consistent with those typically used in laboratory studies. All of these species

o r strains were considered relevant to assessing the potential human health effects of RDX. S ev er al studies in the RDX database provided inadequate informat i o n on test animals . T he strain of monkey ( R h e sus or C ynomolgus) used in the study by Martin and Hart (1974) was not clear ly specified . I n 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

. O ther studies of RDX were identified that used nonstandard species

, including deer mice ( P eromys c us maniculatus ), western fence lizards ( Sceloporus occidentalis ), prairie voles ( Microtus ochrogaster ), a nd northern bobwhite quail ( Colinus virginianus ) . T hese studies provide information
relevant to RDX t oxicokinetics and mechanis m of action on the ner vous system , but not health
effects data. T h e r efore, these studies are not included in evidence tables, but are discussed where
relevant in the assessment.
Experimental setup

Gen eral aspects of study design and experimental setup were evaluated for all studies that
included health effect s d a ta to determine if they were appropriate for evaluation of specific endpoints . K ey features of the experimental setup, including the periodicity and duration of

exposure , t iming of exposure (e.g., gestational days fo r developmental studies)

, experimental group sample sizes
, a n d interim sacrifices are summarized in the evidence tables . N ote that sample size
was not a basis for exclud i ng a study from con sideration
, as studies with a small number of animals

can st ill inform the consistency of effects observed for a specific endpoint

. N evertheless, the informativeness of studies with small sample sizes, e.g., three a n imals/sex/group in the case of Hart (1974) and Martin and Hart (1974) , was reduced. E lements of the experimental setup that could influence interpretation of study findings are discussed in the relevant hazard identification
sections of the assessment.

Exposure

P roperties of the test material were also considered in determin ing whether the exposures were sufficiently specific to the compound of interest . T w o properties of the RDX test materials that varied across experimental animal studies and that were t ak en into consideration in evaluating
the evidence for RDX hazard s ar e t he particle size and purity of the test material . T he purity of RDX

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used in health effects studies varied from 84 to 99.99% . The major contaminants were octahydro



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- tetranit ro- 1,3,5,7

- tetrazocine (HMX) and water, which are the primary contaminants of RDX produced during manufacturing . T he majority of studies used RDX with ~10% impurities; only Crouse et al. (2006) used 99.99% pure RDX as a test material in their study . T he toxicity of HMX

was assessed by the Integrated Risk Information System ( IRIS ) P rogram 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 c hronic studies were available to evaluate the carcinogenici ty of HMX . T he presence of the impurities introduces some uncertainty in attribution of toxicity to RDX . H o w ever, consistenc y in t he doses at which some toxic effects were seen across studies suggest s 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

− 1 5 mg/kg

- day;
studies w ith less pure RDX reported similar symptoms at doses ≥ 20 mg/kg

- day . I t should be noted
that the test material s employed in these studies (i.e., with ~10% impurities) are co n sistent with the purity of RDX that would be released into the environment. Differences in milling procedures used to generate the test material result ed in the use of RDX of varying particle sizes across studies . S o m e studies utilized a test material with a relatively fine particle size (majority of particles <66 μm in size)

, w h ile others used a test material with comparatively coarse particle size (~20 0 μm particle size). D ifferences in particle size across studies could result in different rates of absorption of RDX into the blood stream, which could
account for differences in res p o nse observed across studies, including neurotoxicity . I nformation

on te st m aterial purity and particle size
, as provided by study authors
, is reported in the evidence tables, and was considered in evaluating the toxicity of RDX. T h e l ack of characterization of the test material in the studies by Hart (1974)

, H a rt (1976) , and Martin and Hart (1974) was considered a deficiency.
Endpoint evaluation procedures Some methodological considerations used to evaluate studies of RDX toxicity are outcome specific

— i n particular
, effects on the nervous system and development . O utcome

- specific methodological considerations are discussed in the relevant health effect sections in Section 1. 2

. F or example, many of the studies that noted neurotoxicity in the form of seizures or convulsions

w ere not designed to assess that specific endpoin t a nd reported the number of animals with
seizures a s part of clinical observations th at , in general
, were recorded only once daily. This frequency of observations could have missed neurobehavioral events. Wh i le these studies can provide qualitative evide n ce of neurotoxicity, they may have underestimated the true incidence of seizures or convulsions because they were not designed to systematically evaluate neurotoxic

outcomes.

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Outcomes and data reporting In evaluating studies, consideration was given to whether data were reported for all 

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36 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

wa s limited to control and high

- dose groups, a study reporting feature that limited the ability to
identif y dose

- related trends . I n limited cases, EPA performed additional statistical analysis to identify trends or refine analyses consistent with EPA guidance (e.g., analyzing deve lopmental data sets on a per litter basis rather than by individual fetus)

. S tud y results have been extracted and presented in evidence tables.
Notable features of the RDX database

T hree 2- year toxicity bioassays of RDX are available as unpublished labora tory studies ( Lish et al., 1984 ; Levine et al., 1983b ; Hart, 1976 ) . T he bioassays by Levine et al. (1983b) in the rat and by Lish et al. (1984) in the mouse were conducted in accordance with Food and Drug Administration (FDA) Good Labora tory Practices (GLPs) in place at the time of the studies . B oth studies included interim sacrifices (at 6 and 12 mo n ths) . C omplete histopathological examinations were performed on all animals in the control and high

- d o se groups; however, only a subset of
tissues was examined in the mid

- d o se groups, limiting the ability to identify dose

- related trends for tissues with incomplete histopathology

. A dditionally, i n the mouse bioassay by Lish et al. (1984)

, the initial hig h 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

- d o se animals on study for the full 2 years of dosing (see Table LS -5 )

. A n earlier unpublished 2- year study in rats by Hart (1976) used a dose range that was
low er than the Levine et al. (1983b) 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 a bility to interpret the histopathology data. I n a ddition, a heating system malfunction on day s 7 5

- 7 6 of the study resulted in the death of 59 rats from the control and treatment groups, thereby reducing the number of animals i n t he 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 t w o of the eight short

- term and subchronic toxicity studies of RDX are available as unpubl ished laboratory studies; p u blished studies include von Oettingen et al. (1949) and Levine et al. (1981a) , a laboratory report of a 13

- week study of RD X 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

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information on the toxicity of RDX, including an array of endpoints for neurotoxicity and 

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9 10 i mmunotoxicity ( U.S. EPA, 2012d )

. The assessment of neurotoxicity in the study could have been improved with more histological evaluation as well as additional behavioral assessment. Some of the more important limitations in study design, conduct, and reporting of experimental animal toxicity studies of RDX are summarized in Table LS -5

. Limitations of these
studies a s well as the study evaluation consideration described in this section were taken into

c onsideration in evaluating and synthesizing the evidence for each of the health effects in
Section 1

. 2.

**Table LS**

**- 5** 

**. Experimental animal studies considered less inform**  **ative because of certain study design**  **, con duct,**  **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 . M ortality 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 . D ead animals were subsequently eliminated from the analysis. Interpretation of the h istopathology findings w as 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 d ose range was too low to produce effects in mice . H istopathological 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 wk s

. F or the next 11 wk s, 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 Analysi s 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 po orly characterized; purity was not reported.
von Oettingen et al. (1949) 12

- wk rat study The s train of rat was not reported. Only gross observations were made at autopsy.
von Oettingen et al . (1949) 6

- wk dog study The breed of dog was not reported. Only gross observations were made at autopsy.

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# 1. HAZARD IDENTIFICATION

**1.1. Overview of Chemical Properties and Toxicokinetics** 

**1.1.1. Chemical Properties**  Hexahydro- 1,3,5

- trinitro

- 1,3,5

- triazine ( RDX ) is a member of the nitramine class of organic 

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10 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 slow ly vo latiliz es from
water or moist soil ( ATSDR, 2012 ) . The normalized soil organic carbon/water partition coefficient (K OC ) values for RDX indicate a potential for RDX to be mobile in soil ( Spanggord et al., 1980a ) . Th e
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 ; Spangg ord 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 H exahydro

- 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 C 3 H 6 N 6 O 6 NLM (2011)
Molecular weight 222.12 Lide (2005)
Density (g/cm 3 at 20°C) 1.82 Lide (2005)
Boiling point (°C) 276−280 Bingham et al. (2001)

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Characteristic or property Value Reference
Melting point (°C) 205.5 Lide (2005)
Heat of formation (kJ/g)

− 0.277 Ryon et al. (1984)
Log K OW 0.87−0.90 Hansch et al. (1995)

K OC 42−167 Spanggord et al. (1980b)
Henry’s law constant (atm

- m 3 /mole at 25°C) 2.0 × 10 − 11 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) 59.7 Yalkowsky and He (2003)



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25 **1.1.2. Toxicokinetics**  RDX is absorbed following exposure by oral and inhalation routes (see Appendix C

, Sect i on C .1.1 ). Studies in experimental animals indicate that oral absorption rates can range from approximately 50 to 9 0% ( Krishnan et al., 2009 ; Guo et al., 1985 ; Schneider et al., 1978
, 1977 ) , with the rate and extent of absorption dependent on the physica l form of RDX (i.e., the increased surface area associated with finely powdered RDX allows for increased absorption) a nd the dosing preparation or matrix ( Bann on 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 dis tributed, including to the brain ( i.e.
, RDX can cross the blood:brain barrier), heart, kidney, liver, and fat ( M u sick 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) ( M u sick et al., 2010 ; Major et al.,
2007 ; Fellows et al., 2006 ; Bhushan et al., 2003 ; Schn eider et al., 1978
, 1977 )

. RDX and metabolites are elimi nated primarily via urinary excretion and exhalation of CO2 ( S weeney et al., 2012a ; Musick et al., 2010 ; Kr ishnan et al., 2009 ; Major et al., 2007 ; Schneider et al.,
1977 ) . E stimated e limination half

- lives (t 1/2 ) indicate that RDX is more rapidly metabolized in mice than in rats and humans; e stimated t 1/2 values were 1.2 hours for mice, 5

− 10 hours for rats, and
15

− 2 9 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

.

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**1.1.3. Description of Toxicokinetic Models** 

A physiologicall y based pharmacokinetic ( PBPK ) model to simulate the pharmacokinetics of 

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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

- o r der uptake from the gastrointestinal ( GI ) tract into the liver, with 100% of the dose absorbed. RDX is assumed to be cle ared by first

- o r der 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 subsequent ly modified by the U.S. Environmental Protection Agency ( EPA ) for use in dose

- response modeling in this assessment ( s e e Appendix C, Section C.1.5 ).



## 1.2. **PRESENTATION AND SYNTHESIS OF EVIDENCE**  **BY ORGAN/SYSTEM**
In experimental animal studies, RDX test material administered in toxicology studies included formulations that ranged in purity (from 84 to 99.99%) and in particle size (from <66 to ~200 μm particle size). D i ffer ences in test material purity and particle size were taken into
consideration w h ile evaluating RDX toxicity findings ; this is discussed in the l iterature s earch
section an d incorporated in the synthesis of evidence.
Mo rtality has been reported in the animal toxicology studies conducted for RDX. D u e to the serious nature associated with a frank effect such as mortality, EPA specifically evaluated the database with respect to mortality ( s e e Appendix C, Section C.3.1). In brief, mortality was observed following exposure to a range of doses in chroni c- d u ration studies, in studies up to 6 months in duration, and during gestation. In further analyzing the available evidence, mo r tality occurred at lower doses in rats compared with mice and following gavage administration compared with dietary administra t i on. Additionally, mortality occur red to a greater extent with administration of
RDX in the form of relatively finer particle sizes, p o tentially due to the reduced ability of larger particles of RDX to enter the bloodstream. S om e i nvestigators attributed the mortality to RDX

related cancer or noncancer effects (e.g., ki d n ey or nervous system effects); others identified no cause for the animal deaths. Typically

, ev i dence related to various hazards is presented and synthesized in distinct organ - o r 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 f all under o ther specific hazards. M ortality evidence is considered in discussions of the
evidence for organ/ sy st em
- specific hazard s where applicable.

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**1.2.1. Nervous System**  **E ffects** 
In humans, n ervous system effects following RDX exposure have been observed in multiple 

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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 st udies 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 s tudies are ordered in the evidence table and exposure

- response array by duration of exposure and then species.

Observational Studies in Humans

I n a cross
- sectional study by Ma and Li (1993) , neurobehavioral effects were evaluated in Chinese workers occupationally exposed to RDX. M emory retention and block design scores 5 were significantly lower among exposed workers (mean concentration s of RDX in two exposed groups: 0.407 and 0.672 mg/m 3 ) 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 lett e r c ancellation test) (Table 1 -2). T his study did not consider potential confounders such as alcohol consumption or co

- ex p osure to trinitrotoluene ( T NT ) , an d there was limited infor mation characterizing exposure to RDX

. Case reports suggest an a ssociation between RDX exposure (via ingestion, inhalation, and possibly dermal exposure) and neurological effects (see Appendix C, S e ction C. 2) . S evere neurological disturbances include tonic

- cl o nic seizures ( formerly known as grand mal seizures) in factory workers ( T e stud 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, 1 972 ; 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

Ner vous 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 exposu re to RDX (see Table 1 -3

5 The memory quotient index measured short

- term hearing memory, visual memory, combined hearing and visual memory, and learning ability. The block design index measured visual perception and design replication, and the ability to analyze spatial relationships.

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and Figure 1

- 1). Although study authors interchangeably used the terms seizures and convulsions, seizures, which result from abnormal electrical activity in the brain, can outwardly manifest in a variety of ways. While seizures can be detected in the form of convulsions, they can also manifest as facial twitches, tremors

, or increas ed 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 (B6C3F 1 ), convulsions were observed beginning at doses of 35

− 40 mg/kg

- day, but not at lower doses ( Lish et al., 1984 ; Levine et al., 1983b ; Hart, 1976 ). 6 Subchronic dietary exposure to RDX was also associated with convulsions in the rat, although doses reported t o 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 ; vo n Oettingen et al., 1949 ) . In three other rat studies involving exposure durations of 30

− 90 days, no evidence of seizures, convulsio ns, 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 ( Lev ine 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 t hat 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 weels ( von Oettingen et al., 1949 ) , but not 10 mg/kg

- day for
the 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 

### 1
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4 seizur e occurrence was more frequent in Long Evans rats exposed to a single dose of 50 or
60 m g/kg RDX by gavage when challenged with an audiogenic stimulus 8 and 16 hours after

t reatment . Ho w ever, no audiogenic seizures were observed at the earlier 2 - and 4

- ho ur post

6 The 2

- year dietary studies in F344 rats by Levine et al. (1983b) and B6C3F 1 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 per formed 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, 2012d ). The 2

- year dietary study in Sprague

- Dawley rats by Hart (1976) is available as an unpublished technical report.

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dosing test periods even though RDX plasma concentrations were elevated throughout the testing 

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31 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

. T h e 90

- day study by
Crouse et al. (2006) 7 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)

. Tr e m ors were reported following administration of ≥12 mg/kg

- day, persisting throughout the
90

- d ay study . C o nvulsions were observed at ≥8 mg/kg

- day in male and female rats; information on duration and onset was not reported ( Crouse et al., 2006 )

. I n general, gavage dosing induced convulsions a t lower doses than did dietary
administration . F o r 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 dos es of
15

− 20 mg/kg

- day, convuls ion s were observed in approximately 60

− 70% of the animals. In contrast, in a 2

- ye a r dietary study by Levine et al. (1983b) , convulsions were reported only at a
dose of 40 mg/kg

- day; no convulsions were observed at lower doses (≤8 mg/k g- day). The difference in response between gavage and dietary administration may be du e to the bolus dosing resulting from gavage a d ministration 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. I n a 2

- y ear study in rats, Levine et al. (1983b) noted that
tremors and /or convulsions were often seen in high

- dose animals prior to their death

. I n a rat developmental to x icity 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 a r oun d the mouth and nose) in other dams that
died d uring the s tudy

. Convulsions preceding death were also observed in pregnant Sprague

Dawley rats exposed to RDX during gestation ( Angerhofer et al., 1986 ).

7 The 13

- week gavage study in F344 rats by Crouse et al. (2006) was available only as a laboratory report. An

ex ternal peer review was conducted by Versar, Inc. in July 2012 to evaluate the accuracy of experimental procedures, results, and interpretation and discussio n of the findings presented. The U.S. EPA (2012d) 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

.

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The 90

- day Crouse et al. (2006) study provides the most detailed information on the 

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35 relationship between convulsions and mortality (see Appendix C, Table C

- 10 for additional information on evidence of mortality associated with RDX exposure)

. C onvulsions (3/20) and pre

term deaths (2/20) 8 were observed in male and female rats exposed to 8 mg/kg

- day RDX; the
incidence s o f both convulsions and pre

- term deaths were higher in dose groups with greater

exposures

. I nvestigators stated that nearly all observed pre

- term deaths in rats exposed to t he three higher doses (10, 12, and 15 mg/kg

- d a y RDX ) for 90 days were preceded by neurotoxic signs
such as rearing behavior, t r emors and convulsions ; however
, pre
- term death did not occur in all
animals that convulsed. Some uncertainty exists in that conv ulsions were not t y p i cally observed during a functional observational battery (F OB ) t es t conducted after exposure, likely due to the
time needed t o complete exposures prior to beginning behavioral testing. Of the 100 RDX

- treated
rats in the C r ouse 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 p r ovided as a memorandum by study investigators ( Johnson, 2015a ) , 26
of the se 34 rats (7 6 %) 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 e xhibited conv ulsions prior to pre

- te rm death , convulsions were documented anywhere
from t he same day that t he 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. T h us, while an increase in mortality was observed in the Crouse et al. (2006) study at th e 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 m a y have occurred at times when animals were not
observed and are therefore undercounted i n t h e individual animal data ; however, Johnson (2015a)
noted that it is unlikely that seizure observations were missed
, since seizures generally occurred

soon a fter dosing.
A few studies reported mortality that was not specifically or directly associated with neurological effects ( s ee 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 hav e observed convulsive activity prior to death. In c ase reports of c onvulsions and other nervous system effects in workers exposed to RDX during

m anufacture an d in individuals exposed acutely as a result of accidental or intentional ingestion,
there were n o r e ports 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

- r e activity to approach , fighting, and irritability at doses similar to those that

8 At the 8 mg/kg

- day dos e level, the three rats that convulsed survived to the end of the study; no convulsions were observed in the two rats that died before study termination.

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induced tremors, convulsions, and seizures ( 20

− 100 mg/kg

- day) ( Levine et al., 1990 ; Angerhofer et 

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38 al., 1986 ; Levine et al., 1983b ; Levine et al., 1981a
, b ; von Oettingen et al., 1949 ) . H yperactivity and nervousness were also reported in m ale 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 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 [ 1 2 .5

− 50 mg/kg ] were noted) , but doses were relatively low (≤10 mg/kg

- d a y) ( MacPhail et al., 1985 ) , and no significan t 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 incre ased barbering in female F344 rats receiving 15 mg/kg

- day may have been caused by the oral dosing procedure ( g avage) alone.
C hanges in absolute and relative brain weight were mixed across studies. E levated absolute brain weights were reported in subchronic assays in B6C3F 1 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 . R elative brain weights in some studies showed correspondingly greater increases compared to absolute brain weight ( Crouse et al., 2006 ; Levine et al., 1983b ; 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 B6C3F 1 mice (3−4% relative to control) was reported at doses ≥35 mg/kg

- d a y ( Lish et al., 1984 ) , where as an increase in absolute brain weight (2% relative to control) was observed in F344 rats at a dos e of 40 mg/kg

- day ( Levine et al., 1983b )

. Less weight is placed on evidence of organ weight changes from chronic (2

- year) studies because normal physiological changes assoc iated with aging and intercurrent disease may contribute to
inter

- an imal variability that could confound organ weight interpretation ( Sellers et al., 2007 ).
I n som e studies, seizures appeared soon after dosing, suggesting that seizure induction was more strongly correlated with dose level than with duration of exposure. C o nsistent with this observation are the findings of W i lliams et al. (2011) , who demonstrated that RDX is rapidly
absorbed and c rosses the blood : brain barrier following oral administration in rats, and that distribution of RDX (8 μg/g wet weight ) t o the brain correlated with seizure onset.
While a dose

- response relationship was observed consistently within studies, a dose that
ind uc ed convulsions in a nimals in one study did not necessarily induce convulsions at the same dose in another study

. T h is lack of consistency may be attributed , at least in part, to d ifferences in the purity or particle size of the test material across stud ie s . A ssuming 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 expecte d to produce less neurotoxicity in test animals . T h e 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. (1983b) that used a smaller (<66 μm) particle size . T his could contribute to why the Cholakis et al.

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(1980) subchronic dietary study in the mouse (doses up to 320 mg/kg

- day RDX) and rat (doses up 

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6

7

8 9 10 to 40 mg/kg

- day) failed to report seizures or convulsions . F inally, differences in study design may
have co ntributed to differences in reported neurological responses in subchronic and chronic
d uration studies . In p articular, the protocols for observation for clinical signs (e.g., observations performed once daily in the morning i n Levine et al. (1983b) ) may not have been sufficiently frequent to accurate ly measure the incidence of seizures or other nervous system effects.

**Table 1 -2** 

**. Evidence pertaining to nervous system effects in humans** 

Reference and s tudy d esign Results
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 concentration s in air (basis for dividing workers into two exposure groups was not provided ). Concentration (mg/m 3 ) (mean
± stan dard deviation ): Group A 0.407 (± 0.332) Group B 0.672 (± 0.556) Effect measures a : Five neurobehavioral function tests and five additional memory subtests. Analysis : Variance (F

- test); unadjusted linear regression, multiple regression, and correlation ana lysis. 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 (milliseconds) 493 (199) 539 (183) 578 (280)

Choice reaction time (milliseconds) 763 (180) 775 (161) 770 (193)

Block design* (elapsed time) 18.0 (5.4) 16.0 (4.3) 13.5(6.7)

Letter cancellation (quality per unit time) 1,487 (343) 1,449 (331) 1,484 (443)

*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).

a Symptom data were not included in evidence table because of incomplete reporting.

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**Table 1 -3** 

**. Evidence pertaining to nervous system effects in animals** 

Reference and s tudy d esign Results

Convulsions and neurobehavioral effects
Lish et al. (1984) Mice, B6C3F 1 , 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 One male in the 35 mg/kg

- d dose group and one female in the 175/100 mg/kg

- d group convulsed near the end of the study.

Hart (1976) Rats, Sprague

- Dawley, 100/sex/group Purity and particle size not specified 0, 1.0, 3.1, or 10 mg /kg -d Diet 2 yrs No ne rvous system effects, as evidenced by clinical signs or changes in appearance or behavior, were reported.

Levine et al. (1983b) 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 Tremors, convulsions, and hyper

- responsiveness to stimuli were noted in males and females at 40 mg/kg

- d; no incidence data were reported.

Cholakis et al. (1980) Mice, B6C3F 1 , 10−12/sex/group 88.6% pure, with 9% HMX and 2.2% water 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 Hyperactivity and/or nervousness observed in 50% of the high

- dose males; no signs observed in females b ; no incidence data were reported.

Cholakis et al. (1980) Rats, F344, 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 μm particle size 0, 10, 14, 20, 28, or 40 mg/kg -d Diet 13 wks No nervous system effects, as evidenced by clinical signs or changes in appearance or behavior, were reported.

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Reference and s tudy d esign 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 No nervous system effects were reported.

Crouse et al. (2006) Rats, F344, 10/sex/group 99.99% pure 0, 4, 8, 10, 12, or 15 mg/kg -d Gavage 13 wks Doses 0 4 8 b 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)
, 1981b) ) c Rats, F344, 10/sex/group; 30/s ex 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; p article size not specified 0, 15, 25, or 50 mg/kg -d Diet 13 wks Hyperirritability and convulsions were observed in the 25 and 50 mg/kg

- d groups b ; no incidence data were reported.

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Reference and s tudy d esign Results
Hart (1974) Dogs, Beagle, 3/sex/group
Pre

- mix with ground dog chow containing 20 mg RDX/g

- chow, 60 g dog food ; p urity and particle size not specified 0, 0.1, 1, or 10 mg/kg -d Diet 13 wks No nervous system effects, as evidenced by clinical signs or changes in appearance or behavior, were reported.

Martin and Hart (1974) Monkeys, Cynomolgus or Rhesus e
, 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 b

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

v on Oettingen et al. (1949) Dogs, breed not specified, 5 females/group (control);
7 females/group (exposed) 90−97% pure, with 3

− 10% HMX; p article size not specified 0 or 50 mg/kg -d Diet 6 d/wk for 6 wks Treated dogs exhibited convulsions, excitability, ataxia, and hyperactive reflexes b ; 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.

Cholakis et al. (1980) Rats, F344, 24−25 females/group 88.6% pure, with 9% HMX and 2.2% water as contaminants 0, 0.2, 2.0, or 20 mg/kg -d Gavage GDs 6−19 Doses 0 0.2 2.0 20

Convulsions (incidence)

F 0/24 0/24 1/24 18/25

Angerhofer et al. (1986) (range

- finding study) Rats, Sprague

- Dawley, 6 pregnant females/group Purity 90%; 10% HMX and 0.3% acetic acid occurred as contaminants 0, 10, 20, 40, 80, or 120 mg/kg -d Gavage GDs 6−15 Convulsions preceding death were observed at ≥40 mg/kg

- d;

no incidence data were reported.

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Reference and s tudy d esign Results
Angerhofer et al. (1986) Rats, Sprague

- Dawley, 39−51 mated females/group Purity 90%; 10% HMX and 0.3% acetic acid occurred as contaminants 0, 2, 6, or 20 mg/kg -d Gavage GDs 6−15 Convulsions and hyperactivity b were observed at 20 mg/kg

- d;

no incidence data were reported.

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 25 50 60
Prevalence of audiogenic seizures (%) †

M 0 9 0 29 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, B6C3F 1 , 85/sex/group; interim sacrifices (10/sex/group) at 6 and 12 mo 89.2−98.7% pure, with 3−10% HMX as

c ontaminant; 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 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%*
Levine et al. (1983b) Rats, F344, 75/sex/group; in terim 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 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

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Reference and s tudy d esign Results
Cholakis et al. (1980) Mice, B6C3F 1 , 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 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%
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 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%
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 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%
Crouse et al. (2006) Rats, F344, 10/sex/group 99.99% pure 0, 4, 8, 10, 12, or 15 mg/kg -d Gavage 13 wks Doses 0 4 8 10 12 15
Absolute brain weight (percent change compared to control)

M 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

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Reference and s tudy d esign Results
( Levine et al. (1990) ; Levine et al. (1981a)
, 1 981b) ) c Rats, F344, 10/sex/group; 30/sex for control 84.7 ± 4.7% purity, ~10% HM X, median particle diameter 20 μm, ~90% of particles ≤66 μm 0, 10, 30, 100, 300, or 600 mg/kg -d Diet 13 wks Doses 0 10 30 100 300 600
Absolute brain weight (percent change compared to control)

M 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%

− − 

### 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 * 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

- r e sponse array are those provided in the results section of the technical r e port ( Levine et al., 1981a ) and in the published paper ( Levine et al., 1990 ) . In other se ctions of the technical report, the authors reported that hyperactivity to approach and convulsions were observed in rats receiving ≥30 mg/kg

- da y ( abstract and executive summary), or that mortality was observed in rats receiving 100 mg/kg -d ay a nd that hype ractivity 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

m e thods section) or R hesus (in th e s u mmary).

CNS = central nervous system; GD = gestational day; HMX = octahydro

- 1,3,5,7

- tetranitro

- 1,3,5,7

- tetrazocine ; TWA = t ime

- w eighted average

Note: A dash (“‒”) indicates that the study authors did not measure or report a value for that dose group.

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**Figure 1-1** 

**. Exposure response array o**  **f nervous system effects following oral exposure**

**. 1**  

### 1
2 3 4 1 Because 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 wh ere there was an y

occ urrence of convulsions and/or seizures reported in the study, whether or not the incidence was statistically significantly el evat ed over the control.

This document is a draft for review purposes only and does not constitute Agency policy. 1- 16 DRAFT

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M

echanistic Evidence

S tudies that have explored the mode of action ( MOA ) of RDX on the central nervous system 

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37 ( 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 GABA A receptor. GAB A is a major
inhibitor y neurotransmitter in the brain, and the GABA A receptor has been implicated in susceptibility to seizures ( G alanopoulou, 2008 )

. In research conducted by the U.S. Army Center for Health Promotion and Preventative Medicine, W illiams et al. (2011) and Bannon et al. (2009a) showed a correlation between blood and brain concentrations of RDX in rats that received a single o ral 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 leve l s increased ( Williams et al., 2011 ) . Similarly, oral exposure to RDX (via a gel

capsu le: 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 aff inity for GABA A 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 GABA A - mediated inhibitory postsynaptic currents and initiated seizure

- like neuronal discharges. RDX exposure may reduce the inhibitory effects of GABAergic neurons, resulting in enha n ced 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 tha t amygdaloid kindled rats (rats subjected to patterns of electrical stimulation to promote the development of seizures) e xhibited

pro
- c onvulsant activity at a dose that was approximately half of the dose necessary for RDX to induce spontaneous seizures ( r ats 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

- r esponsiveness to approach , and irritability noted across
several studies of RDX to x icity ( Levine et al., 1990 ; Levine et al., 1983b ; Levine et al., 1981a
, b ; Cholakis et al., 1980 ; von Oettingen et al., 1949 ). RDX binding at the picrotoxin convulsant site of the GABA channel may also inform the relationship between exposure to the chemical and the time when a seizure w as observed . In general ac ross the RDX database , induction of convulsions and seizures appears to be mo re strongly

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correlated with dose than duration of exposure. However, Gerkin et al. (2010) demonstrated that 

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38 young C57/B l6 mice injected intraperitoneally (i.p.) with picrotoxin to induce seizures had a significantly increased frequency of elevated neuronal activity (“Up state”)

, a nd firing rates were significantly increased in neocortical neurons up to 24 h our s after exposure. It is possible that this period of elevated neuronal activity could increase the l ikeli hood 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 b inding at the same site as picrotoxin, t hrough a general increase in brain tissue with elevated neuronal activity, could increase the likelihood of seizures developing over time , o r have other longer

- term effects on
normal brain function

. I t is possible to construct a hypothetical MOA for RDX

- induced neurotoxicity based on the evidence summarized above. Following absorption and transport to the brain:

1) P arent RDX acts as a receptor antagonist (supported by Schneider et al. (1977) and Williams et al. (2011) ), binding noncompetitively to the picrotoxin convuls ant site of the
GABA A receptor (supported by Williams and Bannon (2009) and Williams et al. (2011) ). 2) RDX binding to the GABA A receptor results in decreased conduction of chloride through
the ion channel. 3) R educed chloride conduction results in depolarization of the neuronal membrane, thereby reducing spontaneous inhibitor y p ostsynaptic 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

exposur e to RDX. In addition, RDX treatment of slices inhibited GABA

- induced currents. 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 discharg es in vitro from slices of the basolateral amydgala in rats after adding RDX and noted that t he
effects were not reversible after 40 minutes of washout.

T he steps above provide a biologically plausible sequence of mechanistic events that result in the g en eration of seizure

- like neuronal activity. Reduction of the inhibitory GABAergic signaling is common to many convulsants, as summarized in Kalu eff (2007) . Some organochlorine insecticides, including alpha

- endosulfan, dieldrin, and lindane, also exert neurotoxic effects through
interaction with the GABA A receptor, and can produce a range of hyperex c itability effects (including convulsions) in mammals ( Vale et al., 2003 ; Bloomquist, 1992 ; Suñol e t al., 1989 ) . The interaction
of RDX with the GABA A 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

This document is a draft for review purposes only and does not constitute Agency policy. 1- 18 DRAFT

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( Quinn et al., 2013 ; McFarland et al., 2009 ; Johnson et al., 2007 ; Bruchim et al., 2005 ; Küçükardali et 

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38 al., 2003 ; Woody et al., 1986 ; Lish et al., 1984 ; Berry et al., 1983 ; Levine et al., 1983b ) . A more

recent meta

- analysis of toxicogenomic data across a phylogenetically diverse set of organisms (rat, quai l, f athead minnow, earthworm, and coral) demonstrated that neurotoxic responses are conserved in more highly

- r elated species and that binding to the GABA A 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 abo ve in the development of RDX neurotoxicity.
The GABA A receptor is also a target of many anticonvulsant therapies (e.g., benzodiazepines, propofol, barbiturates) ( M eldrum and Rogawski, 2007 ; Möhler, 2006 ) . Additional support for the
invol vement of GABAergic signaling in the neurotoxicity of RDX comes from human case reports. In multiple case reports, medical intervention included treatment with benzodiazepines (commonly diazepam or lorazepam) to treat seizing patients ( Kasuske 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 GABA A 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 acetyl c holinesterase (AChE) activity ( M cDonough 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, 2 009 ) ; 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 t he 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 B6C3F 1 mice fed 5 mg RDX/kg diet ( estimated dose: 0.75−1.5

This document is a draft for review purposes only and does not constitute Agency policy. 1- 19 DRAFT

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mg/kg

- day; Bannon et al., 2009b ) for 28 days. One miRNA, miR

- 206, was upregulated 26

- fold in 

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36 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

- ex posed brains (miR

- 3 0a 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 s ystem 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

an d downstream targets of miRNA require verification ( Bannon et al., 2009b ) . Thus, the contribution, if any, of aberrant expression of a suite of miRNAs to the MOA for RDX neurotoxicity is
unknown. Recent research has provided greater insight to inform a mechanistic basis of RDX neurotoxicity. While other possible MOA(s) m ay contribute to the overall neurotoxicity of RDX, the demonstrated affinity of RDX for the GABA A receptor, evidence of supportiv e electrophysiological changes w ith direct application of RDX, and toxicokinetic evidence of distribution of RDX to the
brain p rovides a mechanistic basis for the association of seizures with exposure to RDX. The available information supports that RDX

- in d uced hyperactivity and seizures likely result from inhibition of GABAergic signaling in the limbic system.

Integration o f Nervous System Effects

Evidence fo r nervous system effects associated with exposure to RDX comes from studies in
both humans and anim a ls . O ne occupational study reported memory impairment and decrements
in ce rtain 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 exposure and neurological effects

. There was consistent evidence of neurotoxicity associated with exposure to R D X; 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., 1983b ; Levine et al., 1981b ; Cholakis et al., 1980 ; von Oettingen et al., 1949 ) . I n most of these studies, the occurrence of neurological effects was dose

- related . I n those studies that
found no evidence of RDX

- as sociated 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 G A BAergic signaling due to the interaction of RDX with the GABA A receptor. Convulsant receptor binding leading to a decreased seizure threshold, considered with kindling
studies
, s uggests that the effect is specific to CNS toxicity.

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Together, toxicological information in animals and humans

, supported by toxicokinetic and 

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37 mechanistic information , provides a coherent identification of ne rvous system effects as a human
hazard of RDX exposure

.

**1.2.2. Kidney and**  **Other Urogenital**  **S ystem**  **Effects**

T he 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
, of fer some information on the potential for acute exposure to RDX to
affect t he kidney in humans. Organ weight and histopathology findings from experimental animal studies involving subchronic and chronic ex posure to ingested RDX also provide data relevant to an

ex am ination 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 exposure

response array by duration of exposure and then by sp ecies. 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 ur og enital system . R e ported 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 ) , eleva ted blood urea nitrogen (BUN) levels ( Hollander and Colbach, 1969 ; Merrill, 1968 ) , and one case of acute renal f ailure 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 ) . I n 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 octahydro1,3,5,7

- te tranitro

- 1,3,5,7

- tetrazocine ( HMX ) ; RDX exposure range: undetectable [ <0.01 mg/m 3 ] to
1. 6 mg/m 3 ), no statistically significant differences in BUN or total serum protein between nonexposed and RDX

- ex posed 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 assoc iated with kidney and other ur ogenital system effects (Table 1 -5 and Figure 1 -2). Histopathological changes in the urogenital system were as sociated with exposure to RDX in a 2- year bioassay. Specifically, i ncreased incidences of kidney medullary papilla ry necrosis and pyelitis, uremic mineralization, bladder distention and/or cystitis, and suppurative prostatitis were observed in high

- d ose (40 mg/kg

- day) male rats that died spontaneously or were sacrificed in moribund
condition ( L evine et al., 1983b ). These renal effects were considered the principal cause of

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treatment

- related morbidity and mortality in these high

- dose males. Similar kidney lesions were 

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38 not observed in female rats in this study. An increased incidence of tubular nephrosis was
observed in male B6C3F 1 mice exposed to 320 mg/kg

- day RDX in feed for 90 days, but not in female mice in this study ( C holakis et al., 1980 ) . In other chronic and subchronic oral studies in rats and mice, no histopathological changes in the kidney wer e 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 1 0 mg/kg

- day RDX for 90 days by gavage ( Martin and Hart, 1974 ) , b ut 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 ( D MSO ) 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, ra t s, 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.
T he findings of suppurative prostatitis provide the str ongest evidence of urogenital toxicity. A significant, dose

- r elated increase in the total incidence of suppurative prostatitis was reported in male F344 rats exposed to ≥1.5 mg/kg

- da y RDX in the diet for 2 years ( Levine et al., 1983b )

. 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. (1983b) report is the only 2

- year study that reported examination of the prostate in rats. Some reports have hypothesized that the observation of prostate inflammation in Levine et al. (1983b) is second ary 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 dea th or moribund sacrifice (SDMS), it was most likely that the condition was a result of an incidental bacterial infection. However, Levine et al. (1983b) distinguish ed between nonsupp urative 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 b o th 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 c ombined. Additionally, the dose

- related nature of the increased incidence suggests that the primary ca u se (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

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related lesion. The dose

- responsiveness could be explained if the infections were secondary to 

### 1
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38 treatment

- related immunotoxicity, but there is no evidence from Levine et al. (1983b) 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 )

. As noted above, Levine et al. (1983b) documented an array of ki dney 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

, a nd cystitis were observed mainly in the male r ats exposed to 40 mg/kg

- day RDX for 24 months, although one
rat in the 0.3 m g/kg

- day dose group also exhibited these lesions. Treatment

- related effects on the kidney (necrosis) and bladder (distension/obstruction and hemorrhagic cystitis) were also
iden ti fied in the 12

- month pathology report (see Tables 1 -6 to 1 -8 ). The absence of these
observations in the 6

- m onth interim pathology report suggests that an exposure duration
>6 m onths may be required before RDX

- induced effects on the urogenital system are observed. Suppurative prostatitis was observed with increasing incidence in each dose group in the study at 24 months. Considered as a group, treatment

- r elated kidney and urogenital lesions may have led to a blockage that resulted in urinary stasis. Re duced urinary flow and/or retrograde flow may have contributed to an environment that allowed bacterial infection of the prostate. Thus

, w hile 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 is 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 t hat suppurative prostatitis would occur a fter other kidney or bladder lesions that resulted in the initial blockage and urinary stasis. The incidence of suppurative prostatitis reported in Levine et al. (1983b) 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 t he bladders were only examined following observat io n of a gross abnormality. Bladder distension was reported sporadically among the lower dose groups (0.3, 1.5, or 8.0 mg/kg

- d ay), but the bladder was not routinely examined in these groups ( Levine et al., 1983b ) . 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 and as a marker for the broader array of kidney and uroge ni tal system effects observed by Levine et al. (1983b)

. 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

- r elated 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 c h ronic (2

- year) studies ( Lish et al., 1984 ; Hart, 1976 ) because normal physiological changes

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associated with aging and intercurrent disease may contribute to in ter

- animal variability that could 

### 1
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3
4
5
6
7
8
9
10
11
12

13

14 15 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, r at s fed doses ≥450 mg/kg

- day HMX for 13 weeks (see the Integrated Risk Information System [ IR IS ] 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 m g/kg

- d ay in the studies of RDX toxicity, the contribution of HMX to the observed kidney toxicity in studies of RDX is expected to be negligible. F urther, differences in the pattern of toxicity (i.e., kidney effects observed only in RDX

- ex p osed 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 s tudy d esign Results 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 (<LOD) or ≥0.01 mg/m 3 (mean for employees with exposures ≥LOD: 0.28 mg/m 3 ). Effect measures : Renal function tests (blood) Analysis : Type s of statistical tests were not reported (assumed to be t

- tests for comparison of means and χ 2 tests for comparison of proportions). Renal function tests: mean (standard deviation not reported)

Test Referent (n = 237) RDX exposed males * Undetected ( <LOD) (n = 22) >0.01 mg/m 3 (n = 45)
BUN 15.5 15.6 16.4 Total protein 7.2 7.2 7.3 RDX exposed females* Referent (n = 101) Undetected (<LOD) (n = 1) >0.01 mg/m 3 (n = 25)
BUN Total protein 13.2 7.3 8 7.6 12.6 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

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**Table 1 -5** 

**. Evidence pertaining to kidney and other urogenital**  **system effects**  

### 1 2 **in animals**

Reference and s tudy d esign Results
Histopathological lesions
Lish et al. (1984) Mice, B6C3F 1 , 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 . H owever, at 12 and 24 mo of treatment, this lesion was observed as frequently in controls as male s treated with RDX. There was no increase in incidence of this lesion in females at any time point.

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. (1983b) 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

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Reference and s tudy d esign Results
Urinary bladder, luminal distention; 24 mo (incidence)
(SS) 0/38 0/36 0/25 0/29 1/4*
(SDMS) 0/16 2/19 1/27 3/22 24/28*
(Sum) 0/54 2/55 1/52 3/51 25/32*
Urinary bladder, cystitis hemorrhagic/suppurative; 24 mo (incidence)
(SS) 0/38 0/36 0/25 1/29 0/4
(SDMS) 0/16 2/19 1/27 0/22 18/27*
(Sum) 0/54 2/55 1/52 1/51 18/31*
Prostate, suppurative inflammation (prostatitis); 24 mo (incidence)

SS 0/38 1/36 2/25* 4/29* 0/4

SDMS 2/16 3/19 7/27* 8/26 19/27*
(Sum) 2/54 4/55 9/52* 12/55* 19/31*
Cholakis et al. (1980) Mice, B6C3F 1 , 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 Doses 0 80 160 320
Tubular nephrosis (incidence)

M 0/10

− − 4/9*

F 0/11

− − 1/11

Cholakis et al. (1980) Rats, F344, 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 μm particle size 0, 10, 14, 20, 28, or 40 mg/kg

- d Diet 13 wks Histopathological examination of kidney did not reveal any significant differences compared to controls; incidence data were reported only for control and 40 mg/kg

- d groups.

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Reference and s tudy d esign 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 l actation of F1; F1 exposure: 13 wks after weaning, and during mating, gestation, and lactation of F2; F2 exposure: until weaning Data were reported only for F2 generation controls and 5 and 16 mg/kg

- d groups.

Doses 0 5 16 50
Cortical cysts (incidence)

M 4/10 4/10 8/10

−

F 3/10 4/10 8/10

−

Crouse et al. (2006) Rats, F344, 10/sex/group 99.99% pure 0, 4, 8, 10, 12, or 15 mg/kg

- d Gavage 13 wks Doses 0 4 8 10 12 15
Prostate, mild subacute inflammation (incidence)

M 0/10

− − − − 1/8
Histopathological examination of kidney did not reveal any significant differences compared to controls; incidence data were reported only for control and 15 mg/kg

- d groups.
( Levine et al. (1990) ; Levine et al. (1981a)
, 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 availa b le for the 300 or 600 mg/kg

- d dose groups b ecause all rats in these groups died before the 13

- wk necropsy.

Hart (1974) Dogs, Beagle, 3/sex/g roup Pre

- mix with ground dog chow containing 20 mg RDX/g

- chow, 60 g dog food ; p urity and particle size not specified 0, 0.1, 1, or 10 mg/kg

- d Diet 13 wks Histopathological examination of kidney did not reveal any significant differences compared to control s; incidences were reported only for control and 10 mg/kg

- d groups.

Martin and Hart (1974) Monkeys, Cynomolgus or Rhesus c
, 3/sex/group Purity of test material not specified 0, 0.1, 1, or 10 mg/kg

- d Gavage 13 wks Doses 0 0.1 1 10
Medulla; mineralization, minimal to mild (incidence)

M + F 0/6 1/6 0/6 4/6

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Reference and s tudy d esign Results
Kidney weight d
Lish et al. (1984) Mice, B6C3F 1 , 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, o r 175/100 mg/kg

- d (high dose reduced to 100 mg/kg

- d in wk 11 due to excessive mortality) Diet 2 yrs Doses 0 1.5 7.0 35 175/100
Absolute kidney weight at 104 wks (percent change compared to control)

M 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%*
Hart (1976) Rats, Sprague

- Dawley, 100/sex/group Purity and particle size not specified 0, 1.0, 3.1, or 10 mg/kg

- d Diet 2 yrs Doses 0 1.0 3.1 10
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%
Levine et al. (1983b) 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 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%*
Cholakis et al. (1980) Mice, B6C3F 1 , 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 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%

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Reference and s tudy d esign Results
Experiment 2: 0, 40, 60, or 80 mg/kg

- d for 2 wks followed by 0, 320, 160, or 80 mg/kg

- d (TWA doses of 0, 79.6, 147.8, or 256.7 mg/kg

- d for males and 0, 82.4, 136.3, or 276.4 mg/kg

- d for females) a Diet 13 wks 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%
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 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%
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

a s 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
Absolute kidney weight (percent change compared to control)

M 0% 6%

− 12%

−

F 0%

− 4%

− 21%*

−

Crouse et al. (2006) Rats, F344, 10/sex/group 99.99% pure 0, 4, 8, 10 , 12, or 15 mg/kg

- d Gavage 13 wks Doses 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%*

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- 29 DRAFT

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Reference and s tudy d esign Results
( Levine et al. (1990) ; Levine et al. (1981a)
, 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 0 10 30 100 300 600
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%

− − Hart (1974) e Dogs, Beagle, 3/sex/group
Pre

- mix with ground dog chow containing 20 mg RDX/g

- chow, 60 g dog food ; p urity and particle size not specified 0, 0.1, 1, or 10 mg/kg -d Diet 13 wks Doses 0 0.1 1 10
Absolute kidney weight (percent change compared to control)

M 0%

− − 38%

F 0%

− − − 18%

Martin and Hart (1974) e 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 0 0.1 1 10
Absolute kidney weight (percent change compared to control)

M + F 0%

− 2%

− 3% 4%



### 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 *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 C ynomolgus (in the

m e thods section) or R hesus (in the s ummary).

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 K idney weight data from the Hart (1974) and Martin and Ha rt (1974) s tud ies were considered less informative than other studies

. Hart (1974) reported orga n weight data for high

- dose dogs (3/sex/group) only, and the kidney weight s 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). T herefore, kidney weight data from these two studies we re not presented in the exposure

- r espo nse 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

-- s pontaneous death or moribund sacrifice ; SS

-- scheduled sacrifice

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**Table 1 -6**  **. Six**

**- , 12** 

**- , and 24** 

**- month incidence of kidney endpoints in male F344**  

### 1 2 **rats reported fo r statistical evaluation in**  **Levine et al. (1983b)**

Doses (mg/kg

- d) 0 0.3 1.5 8.0 40
Medullary papillary necrosis (incidence)

6 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − − − 0/5

Sum 0/10 0/10 0/10 0/10 0/15

12 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − 0/3

− 15/19*

Sum 0/10 0/10 0/13 0/10 15/29*

24 mo

SS 0/38 0/36 0/25 0/29 0/4

SDMS 0/17 1/19 0/27 0/26 18/27*

Sum 0/55 1/55 0/52 0/55 18/31*
Pyelitis (incidence)

6 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − − − 0/5

Sum 0/10 0/10 0/10 0/10 0/15

12 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − 0/3

− 1/19

Sum 0/10 0/10 0/13 0/10 1/29

24 mo

SS 0/38 0/36 0/25 0/29 0/4

SDMS 0/17 1/19 0/27 1/26 5/27*

Sum 0/55 1/55 0/52 1/55 5/31*
Pyelonephritis (incidence)

6 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − − − 0/5

Sum 0/10 0/10 0/10 0/10 0/15

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Doses (mg/kg

- d) 0 0.3 1.5 8.0 40

12 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − 0/3

− 1/19

Sum 0/10 0/10 0/13 0/10 1/29

24 mo

SS 0/38 0/36 0/25 1/29 0/4

SDMS 0/17 0/19 2/27 1/26 1/27

Sum 0/55 0/55 2/52 2/55 1/31



### 1 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

-- s pontaneous death or moribund sacrifice ; SS

-- scheduled sacrifice

Source: Levine et al. (1983b)

.

**Table 1 -7**  **. Six**

**- , 12** 

**- , and 24** 

**- month incidence of urinary bladder endpoints in**  **male F344 rats reported for statistical e**  **valuation in Levine et al. (1983b)** 

Doses (mg/kg

- d) 0 0.3 1.5 8.0 40

Luminal distention (incidence)

6 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − − − 0/5

Sum 0/10 0/10 0/10 0/10 0/15

12 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − 0/3

− 18/19*

Sum 0/10 0/10 0/13 0/10 18/29

24 mo

SS 0/38 0/36 0/25 0/29 1/4*

SDMS 0/16 2/19 1/27 3/22 24/28*

Sum 0/54 2/55 1/52 3/51 25/32*
Cystitis, hemorrhagic/suppurative (incidence)

6 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − − − 0/5

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Doses (mg/kg

- d) 0 0.3 1.5 8.0 40

Sum 0/10 0/10 0/10 0/10 0/15

12 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − 0/3

− 17/19*

Sum 0/10 0/10 0/13 0/10 17/29

24 mo

SS 0/38 0/36 0/25 1/29 0/4

SDMS 0/16 2/19 1/27 0/22 18/27*

Sum 0/54 2/55 1/52 1/51 18/31* 

### 1

* 

### 2 3
N 

### 4
S 

### 5 6
S 

### 7

8 9 Statistically significant ( p < 0.05) based on analysis by study authors.

ote: A dash (“‒”) indicates that the study authors did not measure or report a value for that dose group. DMS

-- s pontaneous death or moribund sacrifice ; SS

-- sched uled sacrifice

ource: Levine et al. (1983b)

.

**Table 1 -8**  **. Six**

**- , 12** 

**- , and 24** 

**- month incidence of prostate endpoints in male**  **F344 rats reported for statistical evaluation in Levine et al. (1983b)** 

Doses (mg/kg

- d) 0 0.3 1.5 8.0 40
Spermatic granuloma (incidence)

6 mo

SS 0/10 2/10 2/10 1/10 6/10*

SDMS

− − − − 2/5

Sum 0/10 2/10 2/10 1/10 8/15*

12 mo

SS 0/10 0/10 1/10 1/10 0/10

SDMS

− − 0/3

− 0/19

Sum 0/10 0/10 1/13 1/10 0/29

24 mo

SS 0/38 0/36 0/25 0/29 0/4

SDMS 0/16 0/19 0/27 0/26 0/27

Sum 0/54 0/55 0/52 0/55 0/31
Suppurative inflammation (incidence)

6 mo

SS 0/10 0/10 0/10 0/10 0/10

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Doses (mg/kg

- d) 0 0.3 1.5 8.0 40

SDMS

− − − − 0/5

Sum 0/10 0/10 0/10 0/10 0/15

12 mo

SS 0/10 0/10 0/10 0/10 0/10

SDMS

− − 0/3

− 0/19

Sum 0/10 0/10 0/13 0/10 0/29

24 mo

SS 0/38 1/36 2/25* 4/29* 0/4

SDMS 2/16 3/19 7/27* 8/26 19/27*

Sum 2/54 4/55 9/52* 12/55* 19/31* 

### 1 2 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

-- s pontaneous death or moribund sacrifice ; SS

-- scheduled sacrifice

Source: L evine et al. (1983b)

.

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**F igure**  **1-2**

**. Exposure** 

**- response array of kidney a**  **nd other**  **urogenital system**  **effects**

**.** 

### 1 0.1 1 10 100 1000Lish et al., 1984 (mouse, male)Lish et al., 1984 (mouse, female)Hart, 1976 (rat)Levine et al., 1983b (rat)Cholakis et al., 1980 (mouse)Cholakis et al., 1980 (mouse)Cholakis et al., 1980 (rat)Cholakis et al., 1980 (F0 rat, male)Cholakis et al., 1980 (F0 rat, female)Crouse et al., 2006 (rat, male)Crouse et al., 2006 (rat, female)Levine et al., 1990, 1981a,b (rat)Lish et al., 1984 (mouse)Hart, 1976 (rat)uremic mineralization; Levine et al., 1983b (rat)necrosis, pyelitis; Levine et al., 1983b (rat) (1)luminal distention; Levine et al., 1983b (rat)cystitis; Levine et al., 1983b (rat) (2)suppurative prostitis: Levine et al., 1983b (rat)tubular nephrosis, Cholakis et al., 1980 (mouse)Cholakis et al., 1980 (rat)cortical cysts, Cholakis et al., 1980 (rat)prostate inflammation, Crouse et al., 2006 (rat)Levine et al., 1990, 1981a,b (rat)Hart, 1974 (dog)mineralization, Martin & Hart, 1974 (monkey)

Chronic Subchronic Chronic Subchronic
Absolute kidney weight Histopathological lesionsDose (mg/kg-day) significantly changed not significantly changed

Note: Filled circle indicates that response was statistically significantly different from the control. (1) Statistical significance determined from incidence at time of scheduled sacrfice. (2) Statistical significance determin ed from incidence at spontaneous death.

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M

echanistic Evidence

N o MOA information is available for RDX

- induced kidney and other urogenital effects. 

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37 H owever , mechanistic information underlying the neurotoxicity observed with RDX exposure, and the specific affinity of RDX to the GABA A receptor

- convulsant site ( W illiams et al., 2011 ; Williams and Bannon, 2009 ) , suggests a biolog ically plausible role for the GABA A receptor in RDX

- related effects on the urogenital system

, and provides some potential MOA hypotheses for the effects reported in Levine et al. (1983b)

. On e hypothesis is that urogenital effects of RDX are caused by interactions with GABA A receptors mediating inputs to the urogenital system. GABA an d GABA receptors have been identified in a number of peripheral tissues ( Er dö et al., 1991 ; Ong and Kerr, 1990 ; Erdo, 1985 )

. Brar et al. (2014) demonstrated that pretreatment with picrotoxin reduced the renoprotect ive effects of sodium valproate (which acts on both GABA A and GABA B receptors) in a rat model of
ischemia

- in duced acute kidney injury, suggesting that GABA A receptors may be important in renal
function. GABA is believed to play a role in the regulation of u rination and bladder capacity ( r eviewed in Fowler et al. (2008) and Yoshimura and de Groat (1997) ) . In rats, injection of a GABA A receptor ag onist inhibits the urination reflex ( Igawa et al., 1993 ; Kontani et al., 1987 ) . GABA A 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 GABA A receptor antagonist is not known. Evidence of GABAergic signaling regulating
blad d er function, and the hypothesized disruption of that regulation by RDX via interaction with
GABA A receptors, may plausibly account for the kidney and other urogenital lesions, including suppurative prostatitis, observed by Levine et al. (1983b) ; however, no evidence to support this hypothesized MOA is available. Other potential mechanisms by which RDX, through GABA A binding, may lead to kidney and urogenital effects ar e less apparent. Alterations in hormonal signaling or circulating levels of estrogen or prolactin may lead to prostatitis . P r ostate inflammation has been associated with endocrine disruptors in the environment ( C owin et al., 2010 ) , and inc reased prolactin has been shown to cause lateral lobe prostatitis ( Stoker et al., 1999b ; Stoker et al., 1999a ; Tangbanluekal and Robinette, 1993 ; Robinette, 1988 ) . T ypically, the inflammation seen is chronic and does not reverse
over time ( Robinette, 1988 ) . F unctional GABA A receptors ha ve 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 t he 2

- year study by Levine et al. (1983b) could have been produced by disruption of pituitary prolactin or an other hormonal signal via interference with normal regulatory GABA

- related horm onal control . However, no direct evidence for this hypothesized MOA i s available. Levine et al. (1983b) did not evaluate serum endocrine measures or pituitary weights, and pituitary adenomas tha t could account for higher

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prolactin levels were not observed. A MOA hypothesis based on pituitary

- mediated alterations in 

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37 endocrine signaling also does not explain the other urogenital lesions observed by Levine et al. (1983b)

. Another hypothesis is that the prostate effects could be mediated through an autoimmune inflammatory response

. G ABA A receptor transcripts have been identified in immune cells of mouse models ( R eyes
- García et al., 2007 ; Tian et al., 2004 ) , and GABA A receptor agonists have decreased cytotoxic immune responses and hypersensitivity reaction s ( Tian et al., 1999 ; Bergeret et al., 1998 )

. I n 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 bind s to the same site) was able to reduce this e ffect . H o wever, picrotoxin on its own did not significantly alter cytokine production, suggesting th at the effects are limited to reversal of agonist -induced GABAergic activity ( Johnson, 2015b ) . I f an autoimmune mechanism was contributing to the effects observed with RDX exposure, it is unclear why inflammation would be limited to the prostate . R DX 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 GABA A receptors, another possibility w ould be that that effects would result from direct interactions with G ABA A receptors located on the prostate. G AB A A receptors have been identified on the prostate ( N apoleone 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 bi nding to GABA A receptor

- convulsant sites on the prostate would result in a reduction of the inhibitory effects of the GABA receptor

, l eading to increased
inflammation ( Johnson, 2015b ) . No evidence was found to support this potential pathway leading to prostate inflammation. In summary, t here are no studies available that inform mechanistically how RDX might lead to kidney and o ther urogenital effects. T here is evidence that RDX binds to GABA A receptors in
neuronal tissues ( W illiams et al., 2011 ; Williams and Bannon, 2009 ) , and i t is biologically plausible
that binding to the GABA receptor could occur in other tissues as well
, contributing to the observed kidney and urogenital effects. Am ong the mechanistic information presented above, MOAs that require direct action on the prostate are considered less likely because the available i nformation
suggests th at the prostatitis is a secondary effect . However, the ways that GABA A receptors work in

non
- n euronal tissues and organs is still not well understood, and the MOA by which RDX induces kidney and other ur ogenital effects is unknown.

Integration of Kidney and Urogenital System Effects Evidence for kidney effects resulting from RDX exposure consists of human case reports and findings of histopathological changes in rodents . In humans, e vidence for kidney effects (including decreased urine output, blood in urine, and proteinuria ) is limited to individuals with acute

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accidental exposure (ingestion and inhalation) to unknown amounts of RDX . N o RDX

- related 

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35 changes in kidney parameters were found in a small cross

- sectional study of RDX

- exposed workers ( H athaway and Buck, 1977 )

. A dose- related increase in the incidence of suppurative prostatitis in male rats ( Levine et al.,
1983b ) 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 u rogenital effects seen in this study

. Levine et al. (1983b) identified other histopathological effects (papillary necrosis, pyelitis, luminal distension, and cystitis) i n the kidney and bladder
, but at the high est dose only. A second 2

- year study in Sprague

Dawley 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. (1983b)

. Other measures of kidney effects , specifically kidney weights and serum chemistry parameters, did not provide
consistent evidence of dose

- r elated changes associated with RDX exposure. I n light of the dose

related incr e ase in suppurative prostatitis and the lack of support for an alternative (i.e., non

- RDX

related) basis for this effect, k idney and urogenital effects are a potential human hazard of RDX

exposure

.

**1.2.3. Reproductive and Developmental**  **Effects** No human studies were identified that evaluate the potential of RDX to cause reproductive or developmental effects. Information relevant to an examination of the association between RDX exposure and reproductive and developmental effects comes from a 2

- ge n eration reproductive toxicity study in rats and d evelopmental 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 exami n ing the association between RDX exposure and effects specifically o n 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 a re ordered in the evidence tables and exposure

- response arrays by duration of exposure
and then by species.

Reproductive Effects

E vidence of male reproductive toxicity is provided by the finding of testicular degeneration
in male mice . A n increased incid ence of testicular degeneration (10−11%) was observed in male
B6C3F 1 mice exposed to ≥ 35 mg/kg

- day RDX for 2 years in the diet compared to concurrent (0%) and historical (1.5%) controls ( L ish et al., 1984 ) . R eductions in absolute testicular weight were observed, but the magnitude of th is effect was small (≤6% compared to controls) and not dose

related

. An increased incidence of g erm cell degeneration was observed in rats exposed to
40 m g/kg

- day (40%) compared with controls at 12 m onths (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

- t reated groups ( Le vine et al., 1983b ) . N o dose

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related histopathological changes in the testes were identified in other studies in rats ( Crouse et al., 

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37 2006 ; Levine et al., 1990 ; Levine et al., 1981a
, b ; Hart, 1976 ) or dogs ( Hart, 1974 ) . C hanges 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 no t dose- r elated

. 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 t hese changes were not statistically significant. The finding of lower fertility rates only at the 50 mg/kg

- d ay dose, a dose associated with reduced body weight and feed consumption and increased mortality (9% in male r ats 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. I n the dominant lethal mutation study , which used the
F0 males from the two

- g eneration reproductive toxicit y study , no effects on fertility were observed
in male r ats exposed to ≤ 16 mg/kg -day RDX . Pregnancy rates were lower in untreated females mated to males exposed to 50 mg/kg

- d ay RDX for 15 weeks prior to mating ; the authors attributed
this effect to a trea tment -related decrease in the well

- being of the males in this high

- dose group ( Cholakis et al., 1980 )

.
Developmental Effects Animal studies have reported decreases in offspring survival following administration of RDX. Pup survival rates in the F0 and F1 generations (including both st i llborn pups and postnatal deaths through the age of weaning) were statistically significantly decreased in RDX

- ex posed CD rats compared to controls in the only available two

- g eneration reproductive toxicity study of RDX ( C holakis et al., 1980 ) . This observation was noted only at the hi ghest dose tested (50 mg/kg

- day) that also produced toxicity in adults (mortality [ 18 %], reduced body weights [8

− 14 %], and reduced food consumption [ 1 0

− 17% ] ). Decreased fetal viability was observed at the highest dose tested, 20 mg/kg

- d ay, 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 resor ptions were similarly limited to the highest dose tested (20 mg/kg

- d ay) ( Cholakis et al., 1980 ) . Both of these studies started treatment with RDX on gestation al day (GD) 6, which may contribute to the incidence of resorptions observed in the control and treated groups. As noted in EPA’ s G uidelines for Developmental Toxicity Risk Assessment ( U.S. EPA,
1991 ) , 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 evide nce of

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maternal toxicity, embryotoxicity
, or decreased fetal viability in a teratology study of pregnant New 

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34 Zealand White (NZW) rabbits administered RDX by gavage from GD 7 to 29 at doses up to 20 mg/kg

- day ( C holakis et al., 1980 ) , suggesting that rabbits may be less sensitive to RDX to xicity than

rats. Statistically significant, dose

- related reductions in fetal body weight and length were reported in Sprague

- D awley rats administered RDX by gavage from GD 6 to 15 ( Angerhofer et al.,
1986 ). 9 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

- d ay, 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 morphologica l development have been reported in rats exposed to a dose as high as 20 m g/kg

- day RDX, a dose that resulted in 29

− 31% maternal mortality ( A ngerhofer 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 rabb its 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 treatment

related by the study authors ( Cholakis et al., 1980 ) . This interpretation is supported by the
followi ng 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 observati ons in NZW rabbits for 224 prenatal developmental toxicology studies conducted in 8 contract research laboratories during the period
of 1 988

− 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 patt ern of decreases in fetal body

9 The statistical analyses presented by the study authors were performed on a per fetus basis; EPA’s Gu idelines for Developmental Toxicity Risk Assessment ( U.S. EPA, 1991 ) 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.

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weight in the same study, suggesting a possible link between these particular sternebral and 

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7 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 s tudy d esign Results
Lish et al. (1984) Mice, B6C3F 1 , 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 re duced to 100 mg/kg

- d in wk 11 due to excessive mortality) Diet 2 yrs Doses 0 1.5 7.0 35 175/100
Testicular degeneration (incidence)
0/63 2/60 2/62 6/59 3/27 a
Absolute testes weight; wk 105 (percent change compared to control)

0%

− 6% 0%

− 2%

− 6%
Relative testes weight; wk 105 (percent change compared to control)

0%

− 4% 2%

− 2%

− 2%
Hart (1976) Rats, Sprague

- Dawley, 100/sex/dose Purity and particle size not specified 0, 1.0, 3.1, or 10 mg/kg -d Diet 2 yrs Doses 0 1.0 3.1 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. (1983b) Rats, F344, 75/sex/g roup; 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 mo b (incidence)

SS 0/10 0/10 0/10 0/10 4/10*

SDMS

− − 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.

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Reference and s tudy d esign Results
Cholakis et al. (19 80) Mice, B6C3F 1 , 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 Doses 0 10 14 20 28 40
Absolute testes weight (percent change compared to control)

0%

− − − − 4%

− 4%
Relative testes weight (percent change compared to control)

0%

− − − 2%

− 1%

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) c Diet 13 wks Doses 0 80 160 320
Absolute testes weight (percent change compared to control)

0% 4%

− 4%

− 8%
Relative testes weight (percent change compared to control)

0% 1%

− 4%

− 9%
Testes were examined microscopically in control and 320 mg/kg

- d groups; no effects were observed.
Cholakis et al. (1980) Rats, F344, 10/sex/dose 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 μm particle size 0, 10, 14, 20, 28, or 40 mg/kg

- d Diet 13 wks Doses 0 10 14 20 28 40
Absolute testes weight (percent change compared to control)

0%

− − − − 2% 0%
Relative testes weight (percent change compared to control)

0%

− − − 2% 9% Testes were examined microscopically in control and 40 mg/kg

- d groups; no effects were observed.
Cholakis et al. (1980) Rats, CD, two

- generation study; F0: 22/sex/group; F1: 26/sex/group; F2: 10/sex/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 μm p article 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: unti l weaning In F2 weanling offspring of 0, 5, and 16 mg/kg

- d groups. No high

dose F2 animals available.

Doses 0 5 16 50
Absolute testes weight (percent change compared to control)

0% 3%

− 31%

−

Testes of F2 weanlings were exa mined microscopically in all groups; no effects were observed.

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Reference and s tudy d esign Results
Crouse et al. (2006) Rats, F344, 10/sex/group 99.99% pure 0, 4, 8, 10, 12, or 15 mg/kg -d Gavage 13 wks Doses 0 4 8 10 12 15
Absolute testes weight (percent change compared to control)

0%

− 3%

− 5%

− 4%

− 4%

− 8%
Relative testes weight (percent change compared to control)

0% 4% 5% 0%

− 6%

− 10%*
Levine et al. (1981a) ; Levine et al. (1990) ; Levine et al. (1981b) d Rats, F344, 10/sex/group; 30/sex for control 84.7 ± 4.7% purity, ~10% HMX, median particle diameter 20 μm, ~90% of partic les ≤66 μm 0, 10, 30, 100, 300, or 600 mg/kg -d Diet 13 wks Doses 0 10 30 100 300 600
Testes, germ cell degeneration (incidence)
0/10 0/10 0/10 0/10 1/9 1/10
Absolute testes weight (percent change compared to control)

0% 1% 1%

− 2%

− − Relative testes weight (percent change compared to control)

0% 4% 5% 19%*

− − Hart (1974) e Dogs, Beagle, 3/sex/dose
Pre

- mix with ground dog chow containing 20 mg RDX/g

- chow, 60 g dog food ; p urity and particle size not specified 0, 0.1, 1, or 10 mg/kg -d Diet 13 wks Doses 0 0.1 1 10
Absolute testes (with epididymis) weight (percent change compared to control)

0%

− − 51%
Testes were not examined microscopically.



### 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 *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

- d ay dose groups were “notable” when compared to concurrent (0%) and historical (1.5%) incidences.

b Testicular atrophy was observed at 12 month s along with a statistically reduced mean testes weight (compared

with controls). By 24 mo nth s, 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 t he 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 weight s fr om 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

-- s pontaneous death or moribund sacrifice; SS

-- scheduled sacrifice

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**F igure**  **1-3 . Exposure response array of male reproductive effects following oral exposure.**  

### 1

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**Table 1**

**- 10**  **. Evidence pertaining to reproductive and developmental effects in** 

### 1 2 **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) 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 20
Early resorptions (mean percent per dam)

6% 5% 4% 1%
Late resorptions (mean percent per dam)

8% 5% 3% 3%

Viable fetuses (mean percent per dam)

85% 82% 77% 94%
Cholakis et al. (1980) Rats, F344, 24−25 females/group 88.6% pure, with 9% HMX and 2.2% water as contaminants. 0, 0.2, 2.0, or 20 mg/kg -d Gavage GDs 6−19 Doses 0 0.2 2.0 20
Early resorptions (mean percent per dam)

6.0% 2.5% 4.8% 15.3%
Late resorptions (mean percent per dam)

0.5% 0.5% 0.3% 1.6%
Complete litter resorptions (number of litters)

0 0 0 2

Viable fetuses (mean percent per dam)

93.2% 97.6% 94.9% 81.4% Significant maternal mortality (7/24 dams) occurred at 20 mg/kg

- d.

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Reference and study design Results
Angerhofer et al. (1986) R ats, Sprague

- Dawley, 39−51 mated females/group (25−29 pregnant dams/group) Purity 90%; 10% HMX and 0.3% acetic acid occurred as contaminants 0, 2, 6, or 20 mg/kg

- d Gavage GDs 6−15 Doses 0 2 6 20
Resorptions (percent of total implantations)

4.8% 6.1% 5.9% 6.4%
Early resorptions (percent of total implantations)

4.8% 6.1% 5.9% 6.2%
Late resorptions (percent of total implantations)

0% 0% 0% 0.27%

Live fetuses (mean percent per litter)

100% 100% 100% 100%
Significant maternal mortality (16/51) occurred at 20 mg/kg

- d. Percent resorptions and live fetuses based on number of surviving females at time of necropsy.
Offspring growth
Cholakis et al. (1980) Rabbits, NZW , 11−12/group 88.6% pure, with 9% HMX and 2.2% water as contaminants; ~200 μm particle size 0, 0.2, 2.0, or 20 mg/kg

- d Gavage GDs 7−29 Doses 0 0.2 2.0 20
Fetal body weight (percent change compared to control)

0%

− 6.7%

− 2.3%

− 9.3%

Cholakis et al. (1980) Rats, F344, 24−25 females/group 88.6% pure, with 9% HMX and 2.2% water as contaminants. 0, 0.2, 2.0, or 2 0 mg/kg

- d Gavage GDs 6−19 Doses 0 0.2 2.0 20
Fetal body weight (percent change compared to control)

0% 2% 3%

− 7% Significant maternal mortality (7/24 dams) occurred at 20 mg/kg

- d.

Angerhofer et al. (1986) Rats, Sprague

- Dawley, 39−51 mated females/group (25−29 pregnant dams/group) Purity 9 0%; 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.

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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 )

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.



### 1 2 3 4 *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.

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**Figure 1** 

**- 4** 

**. Exposure response array of reproductive and developmental effects following oral exposure.**  0.1

1 10 100Cholakis et al., 1980 (rat, F1 and F2)Cholakis et al., 1980 (rabbit)Cholakis et al., 1980 (rat)Angerhofer et al., 1986 (rat)Cholakis et al., 1980 (rabbit)Cholakis et al., 1980 (rat)Angerhofer et al., 1986 (rat) (1)Cholakis et al., 1980 (rabbit)Cholakis et al., 1980 (rat)Angerhofer et al., 1986 (rat)

↓ Offspring survival Offpsring growth Morphological developmentDose (mg/kg-day) signficantly changed
not signifcantly changed

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.

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Integration of Reproductive and Developmental Effects

Te sticular effects were reported in male B6C3F 1 mice chronically exposed to RDX in the diet 

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35 for 24 months ( L ish 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 m onths, 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 . O ther testicular effects were inconsistent across
rat studies . Bas ed 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

- r elated developmental effects were observed in
rabbits ( Cholakis et al., 1980 ) . As noted in EPA’s Guidelines for Developmental Toxicity Risk
Assessment ( U.S. EPA, 1991 ) , where adverse developmental effects are produced only at doses that cause minimal maternal toxicity, developmental effects should not be discounted as being secondary to maternal toxicity; however, at doses causing excessive toxicity, as is the case with RDX, information on developmental effects may be difficult to interpret and of limited value. Therefore, at this time, no conclusions are drawn regarding de v elopmental effects as a human
hazard of RDX exposure.

**1.2.4. Liver Effects**  One occupational epidemiology study examined t he association between RDX exposure and changes in s erum liver enzymes . Case reports involving accidental exposure to RDX provide
informati on on the potential for acute exposure to RDX to a ffe ct the liver in humans. In addition,

o r gan weight
, histopathology , and serum chemistry findings from experimental animal studies involving subchronic and chronic exposure to ingested RDX provide data re l evant to an examination of the association between RDX exposure and liver e ffects. A summary of the liver effects associated with RDX exposure is presented in Tables 1 -1 1 and 1 -12 and Figure 1

- 5. Experimental animal studies are ordered in the evidence t ab le and exposure

- response array by duration of exposure and then by sp ecies. Reports in humans provide inconsistent e vidence of liver toxicity associated with acute exposure to RDX . E levated 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 ( Kü çükardali 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)

. L iver biopsies did not reveal any abnormal observations ( Stone et al., 1969 ) . In other case reports, no significant changes in serum levels of liver en zymes were observed ( Testud et al., 1996a ; Ketel and Hughes, 1972 ) . I n 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:

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undetectable (<0.01 mg/m 3 ) to 1.6 mg/m 3 ) ( Hathaway and Buck, 1977 ) , serum chemistry an alysis 

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30
31
32 (including the serum liver enzymes AST, ALT, and alkaline phosphatase [ ALP ] ) revealed no statistically significant differences between exposed and unexposed workers (Table 1

- 1 1 ). In experimental animals, some, but not all, subchronic studies reported increased liver weight a ssociated with RDX exposure (Table 1 -12 and Figure 1 -5) . D ose
- related increases in relative liver weight 10 (11

− 25% in high

- dose groups) were observed in male and female B6C3F 1 mice giv en RDX in the diet for 90 days ( Cho lakis 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 the se subchronic studies ( Levine et al., 1990 ; Levine et al., 1981a
, b )

. I n subchronic studies in other species , absolute liver weights were increased in male and female monkeys (6

− 1 6% relative to control at 1 and 10 mg/kg

- day) ( Martin and Hart, 197 4 ) and in male, but not
female

, beagle dogs (53% relative to control in male dogs at 10 mg/kg

- day) ( Hart, 1974 )

. C hronic RDX exposures in B6C3F 1 mice and F344 or Sprague

- Dawley rats showed a less consistent pattern of liver weight increases

. I nterpretation of liver weight increases in the 2- year

mouse s tud y 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 ( L ish et al., 1984 )
was reduced when mice with liver adenomas or c arcinomas were removed from the analysis. I n a 2- y ear rat study ( Levine et al., 1983b ) , relative liver weights were increased in high

- dose (40 mg/kg

- day) males and females (by 11 and 18% compared to controls, respectively)

, likely
reflect i ng 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, l ess weight is placed on evidence of organ weight changes from chroni c (2

- year) studies because normal physiological changes associated with aging and intercurrent disease contribute s 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 ( C rouse et al., 2006 ; Levine et al., 1990 ; Lish et al., 1984 ; Levine et al., 1983b ; 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., 1983b ) . The few findings of liver lesions were

10 Based on an evaluation of the relationship between organ weight and body/brain weight to determine which endpoint (organ weight, organ

- to

- body weight ratio, or organ

- to

- brain weight ratio) is likely to more accurately detec t target organ toxicity, Bailey et al. (2004) concluded th at 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).

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reported in studies with more limited histopathological analyses, and were not confirmed in the 

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37 studies with more complete histopa thologic examination and longer exposure durations ( Lish et al
., 1984 ; Levine et al., 1983b ) . For example, the incidence of liver portal inflammation was in creased
in female rats , but not male rats , exposed to 40 mg/kg

- day in the diet for 90 days ( Cholakis et al.,
1980 ) . T here 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 die t for 90 days ( Cholakis et al., 198 0 )

. 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- y ear bioassays. It should be noted that exposure to HMX, the primary contaminant in several of the RDX studies, w as associated with histopathological changes
in the livers of male rats fed doses ≥ 4 50 mg/kg

- day for 13 weeks. However, s imilar findings we re not observed in the RDX studies, where the doses of RDX employed in the studies would h ave result ed in HMX exposure s of ≤60 mg/kg -day . The contribution of HMX exposure to the overall
liver f indings 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

. S tatistically significant changes in these parameters in some subchronic and chronic toxicity studies in r ats and mice were relatively small (generally <50% of the control mean), were not dose

- r elated 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 mi ce reported dose

- r elated 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 . Sp ecifically, serum triglyceride levels were elevated (up to 41%) in female B6C3F 1 mice exposed to RDX in the diet for 2 years, although increases were not dose

- r elated ( Lish et al.,
1984 ) ; male mice in the same study did not show a similar increase in triglycerides . I n 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 ) . I n a chronic toxicity study by the same investigators ( Levine et al., 1983b ) , serum triglyceride levels were generally decreased in male and female rats (52 and 51%, r espectively, at the highest dose of
40 m g/kg

- day); however, triglyceride levels across the four dose groups in this study did not show
a dose

- r elated response.
Serum cholesterol levels showed a dose

- related increase (38% at the high dose of
100 m g/kg

- day) in female B6C3F 1 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. C hanges in serum cholesterol in male and female F344 rats expos ed to RDX in the diet for 2 years at doses up to 40 mg/kg -d ay ( Levine et al., 1983b ) , in rats exposed to RDX by gavage for 90 days at doses up to

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15 mg/kg -day ( Crouse et al., 2006 ) , and in mo nkeys exposed to RDX in the diet for 90 days ( Martin 

### 1
2

3

4 and Hart, 1974 ) were relatively small (within 38% of control mean) and were not dose related.

**Table 1 -11**  **. Evidence pertaining to liver effects in humans** 

Reference and study design Results
Hathaway and Bu ck (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 : Ex posure determination based on job title and industrial hygiene evaluation. Exposed subjects assigned to two groups: < LOD or ≥0.01 mg/m 3 (mean for employees with exposures ≥LOD: 0.28 mg/m 3 ). Effect measures : Liver function tests. Analysis : Types of statistical tests were not reported (assumed to be t

- tests for comparison of means and χ 2 tests for comparison of proportions). Mean l aboratory v alues of l iver e nzymes in men ( mean ; standard deviation not reported)

Test Referent (n = 237) RDX exposed ‡

Undetected (<LOD) (n = 22) >0.01 mg/m 3 (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

‡ 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 abnorma lly elevated values )

Test (abnormal

range) Referent RDX exposed ‡

Undetected (<LOD) >0.01 mg/m 3
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.

LDH = lactate dehydrogenase; SGOT = glutamic oxaloacetic transaminase; SGPT = glutamic pyruvic transaminase

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**Table 1 -12**  **. Evidence pertaining to liver effects in animals** 

### 1

Reference and study design Results

Liver weight
Lish et al. (1984) Mice, B6C3F 1 , 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 (hi gh dose reduced to 100 mg/kg

- d in wk 11 due to excessive mortality) Diet 2 yrs Doses 0 1.5 7.0 35 175/100
Absolute liver weight at 104 wks (percent change compared to control)

M 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

. Hart (1976) Rats, Sprague

- Dawley, 100/sex/g roup Purity and particle size not specified 0, 1.0, 3.1, or 10 mg/kg -d Diet 2 yrs Doses 0 1.0 3.1 10
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. (1983b) 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, B6C3F 1 , 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 Doses 0 10 14 20 28 40
Absolute liver weight (percent change compared to control)

M 0%

− − − − 6%

− 5%

F 0%

− − − − 4%

− 1%
Relative liver weight (percent change compared to control)

M 0%

− − − − 4%

− 4%

F 0%

− − − − 6% 1%

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Reference and study design Results

Experiment 2: 0, 40, 60, or 80 mg/kg

- d for 2 wks followed by 0, 320, 160, or 80 mg/kg

- d (TWA doses of 0, 79.6, 147.8, or 256.7 mg/kg

- d for males and 0, 82.4, 136.3, or 276.4 mg/kg

- d for females) a Diet 13 wks 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%*
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 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 0 5 16 50
Absolute liver weight (percent change compared to control)

M 0% 7%

− 16%

−

F 0% 0%

− 14%

−

Crouse et al. (2006) Rats, F344, 10/sex/group 99.99% pure 0, 4, 8, 10, 12, or 15 mg/kg

- d Gavage 13 wks Doses 0 4 8 10 12 15
Absolute liver weight (percent change compared to control)

M 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%

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Reference and study design Results
( Levine et al. (1990) ; Levine et al. (1981a)
, 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 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%*

− − Hart (1974) c Dogs, Beagl e, 3/sex/group
Pre

- mix with ground dog chow containing 20 mg RDX/g

- chow, 60 g dog food ; p urity and particle size not specified 0, 0.1, 1, or 10 mg/kg

- d Diet 13 wks Doses 0 0.1 1 10
Absolute liver weight (percent change compared to control)

M 0%

− − 53%

F 0%

− − 3%

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 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, B6C3F 1 , 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 re duced to 100 mg/kg

- d in wk 11 due to excessive mortality) Diet 2 yrs Histopathological lesions in liver other than adenomas and carcinomas were not significantly different compared to controls, as reported by study authors.

Hart (1976) Rats, Sprague

- Dawley, 100/sex/group Purity and particle size not specified 0, 1.0, 3.1, or 10 mg/kg

- d Diet 2 yrs Histopathological examination performed only for controls and 10 mg/kg

- d rats; no significant differences compared to controls were reported by study authors.

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Reference and study design Results
Levine et al. (1983b) 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 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

Cholakis et al. (1980) Mice, B6C3F 1 , 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 o f 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 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

− − − −

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 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)
, 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 findi ngs available for the 300 or 600 mg/kg

- d dose groups because all rats in these groups died before the 13

- wk necropsy.

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Reference and study design Results
Hart (1974) Dogs, Beagle, 3/sex/group
Pre

- mix with ground dog chow containing 20 mg RDX/g

- chow, 60 g dog food ; p urity 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 Rhesus d

, 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 cyto plasm 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 i n the study report, the toxicological significance of this finding could not be determined.

Serum chemistry
Lish et al. (1984) Mice, B6C3F 1 , 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 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%

Levine et al. (1983b) 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

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%*
Crouse et al. (2006) Rats, F344, 10/sex/group 99.99% pure 0, 4, 8, 10, 12, or 15 mg/kg

- d Gavage 13 wks Doses 0 4 8 10 12 15

Serum cholesterol (percent change compared to control)

M 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%

− 19%

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Reference and study design Results

F 0%

− 16%

− 21% 7%

− 37% 18%
( Levine et al. (1990) ; Levine et al. (1981a)
, 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 0 10 30 100 300 600
Serum triglyceride levels (percent change compared to control)

M 0%

− 14%

− 34%

− 62%*

− −

F 0%

− 12%

− 29%

− 50%*

− −

Martin and Hart (1974) 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 Serum biochemistry analysis revealed scattered deviations, but study authors indicated they appear to have no toxicological significance.

Doses 0 0.1 1 10

Serum cholesterol (percent change compared to control)

M 0%

− 17%

− 2%

− 7%

F 0% 7% 7% 7% 

### 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 *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) s tudies 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 1 41 g). Therefore, liver weight data from these two studies we re not presented in the
exposure

- r esponse array for liver effects ( Figure 1 -5).

d The species of monkey used in this study was inconsistently reported in the study as either C ynomolgus (in the

m e thods section) or R hesus (in the s ummary).

Note: A dash (“‒”) indicates that the study authors did not measure or report a value for that dose group.

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**Figure 1-5** 

**. Exposure response array of liver**  **effects following oral exposure.** 

### 1

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Integration of Liver Effects

Th ere is limited evidence from human studies and from studies in experimental animals 

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34 that RDX may affect the liver. The observation of short

- term elevat ions of serum liver enzymes in several human case reports of individuals who ingested unknown amounts of RDX suggest s 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 experiment al 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

. C hanges in serum chemistry were not consistent across studies and the magnitude of change
relative to concurrent controls was not indicat ive of liver damage . N onneoplastic histopathologic
lesions of the liver were also not consistently associated with RDX exposure. At this time, no conclus ions are drawn regarding liver effects as a human hazard of RDX exposure.

**1.2.5. Carcinogenicity**  The relationship between exposure to RDX and cancer in human populations has not been investigated. The carcinogenicity of RDX has been examined in one oral chronic/carcinogenicity bioassay in mice ( L ish et al., 1984 ) and two bioassays in rats ( Levine et al., 1983b ; Hart, 1976 ) . The
2- year studies by Lish et al. (1984) and Levine et al. (1983b) included comprehensive histopathological examination of major organs, multiple dose groups and a control, and

> 50 an imals/ dose group ( plus additional interim sacrifice groups ). In both studies, the maximum tolerated dose was reached or exceeded in high -d o se animals (based on decreased terminal body weight in high

- d ose 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). Th e 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
≥5 0 rats/sex/group at termination, and it seems unlikely that the mortality associated with the temperature spike w ould 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 T umors
An i ncreased 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., 1983b ) . Incidences of hepatocellular tumors are presented in Table 1 -13 and discussed in further detail below.
T he incidence of hepatocellular carcinomas and the combined incidence of hepatocellular
adenomas or carcinomas sh o wed a statistically significant positive trend with RDX dose in female, but not male, B 6C3F 1 mice as compared to concurrent controls in a 2- year dietary study ( Lish et al.,

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1984 ). In female B6C3F 1 mice
, Lish et al. (1984) observed that the liver tumor incidence in the 

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28 concurrent female control mice was relatively low (1/65), and significantly lower than the incidence from historical controls (historical incidence data n ot provided by study authors). T he study authors also compared liver tumor incidence in RDX

- e xposed female mice to mean historical
control incidence for female mice o f the same strain from National Toxicology Program (NTP) studies conducted during the sam e time period (147/1

, 7 81 or 8%; range: 0−20%) ( Haseman et al.,
1985 ). 11 T he 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 hi st orical control data originati ng 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 t he 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 adenoma s were changed to non

- neoplastic lesions based on more recent diagnostic criteria used by the PWG ( Harada et al., 1999 )

. T here remained a stati stically 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 histop athological 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.
I n male mice from the Lish et al. (1984) study , t he 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 t reated groups than in the control; however, there were no statistically significant trends in either case. Th e incidences of
liver carcinoma i n control (21%) and treated groups o f male mice (22

− 33%) were generally within the range for the same mo use strain reported by NTP (8

− 32%) ( Haseman et al., 1985 ). Similarly,
the combined incidence s of liver adenoma or carcinoma in control (32%) and treated groups

11 Comparison 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. Speci fically, 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 includ ed 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, 2005a ) also note that, unless the tumor is rare, the standard for determining statistical significance of tumor incidence is a comparison of dosed animals with the concurrent controls.

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(27

− 48%) were within the range for the same mouse strain reported by NTP (14

− 58%) ( Haseman 

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33 et al., 1985 ). 12 T he PWG did n ot 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 ( L evine et al., 1983b )

. In the Levine et al. (1983b) study
, t here 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
inference s m ade from such a sparse response are uncertain

. Because liver tumors are rare tumors
in the rat 13 , s ome perspective is obtained by considering historical control data . In a paper published concurrently with the Levine et al. (1983b) 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. (1983b) , t he incidence of liver carcinomas in control male rats (1/55 or 1.8%) was at the up per end of th is NTP range , and the incidence in RDX

- treated male F344 rats in the highest two dose groups (3.6 and 6.4%) exceeded t he NTP historical control range. Using
incidence data from NTP h istorical controls, the trend for carcinoma in the RDX

- treated F344 rats was statistically significant (p- v alue = 0.003 ; one
- sided exact Cochrane

- Armitage trend test )

. It
should b e noted that a lthough t he NTP historical controls ( Haseman et al., 1985 ) are comparable
with Levine et al. (1983b) 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 f rom male F344 rats, both recent and of the time period of the Levine study, indicate
similar low in c idences of liver carcinomas ( 0.36%, ( NTP, 2009 ) ; 0.31%, ( Maita et al., 1987 ))

. I n the Levine et al. (1983b) 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

. I t was not possible to estimate mortality

- adjusted incidences because no time

to

- d eath 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. (1983b) reported a n incidence of neoplastic nodules of 7.3% in their control male rats, consistent with the NTP historical control data, a nd 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 co mbined incidence of hepatocellular adenomas or carcinomas was not increased with dose in rats of either sex at doses up to 10 mg/kg

- d ay ( Hart, 1976 ). However, interpretation of results

12 I bid

. 13 NTP 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 Sprague

D awley rats as reported in Chandra et al. (1992) : 6/1,340 (0.45%) in males; 1/1,329 ( 0.08% ) in females

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from this study is limited by the comparatively lower doses employed in the study, and the 

### 1
2

3 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 Results a
Lish et al. (1984) Mice, B6C3F 1 , 85/sex/group; interim sacrifices (10/sex/group) at 6 and 12 mo 89.2−98.7% pure, with 3−10% HMX as
conta minant; 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 0 1.5 7.0 35 175/100 b
Hepatocellular adenomas (incidence) c

M 8/63 (12.7) 6/60 (10.0) 1/62* (1.6) 7/59 (11.9) 7/27 (25.9)

F 1/65 (1.5 ) 1/62 (1.6 ) 6/64 ( 9.4 ) 6/64 ( 9.4 ) 3/31 (9 .7) Hepatocellular carcinomas (incidence) c

M 13/63 ( 20.6 ) 20/60 ( 33.3 ) 16/62 ( 25.8 ) 18/59 ( 30.5 ) 6/27 ( 22.2 )

F 0/65 ( 0.0 ) 4/62 ( 6.5 ) 3/64 (4 .7) 6/64 ( 9.4 ) 3/31 d (9 .7) Hepatocellular adenoma or carcinoma combined (incidence) c

M 2 0 /63 (31 .7) 26/60 (43.3 ) 17/62 (27.4 ) 25/59 (42.4 ) 13/27 (48.1 )

F 1/65 (1.5 ) 5/62 (8.1 ) 9/64* (14.1 ) 12/64* (18.8 ) 6/31* d (19.4 )

P WG reanalysis of liver lesion slides from female mice ( Parker et al., 2006 ; Parker, 2001 )

. e

Doses 0 1.5 7.0 35 175 /100 b
Hepatocellular adenomas (incidence) c

F 1/67 (1.5 ) 3/62 (4.8 ) 2/63 (3.2 ) 8/64 (12.5 ) 2/3 1 (6.5 ) Hepatocellular carcinomas (incidence) c

F 0/67 (0.0 ) 1/62 (1.6 ) 3/63 (4.8 ) 2/64 (3.1 ) 2/3 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 ) 4/3 1 d (12.9 )

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Reference and study design Results a
Hart (1976) Rats, Sprague

- Dawley, 100/sex/group Purity and particle size not specified 0, 1.0, 3.1, or 10 mg /kg -d Diet 2 yrs Doses 0 1.0 3.1 10
Neoplastic nodules (incidence) c

M 0/82

− − 3/77

F 1/72

− − 1/81
Hepatocellular carcinomas (incidence) c

M 1/82

− − 1/77

F 1/72

− − 1/81 f
Neoplastic nodules or hepatocellular carcinomas combined (incidence) c

M 1/82

− − 4/77

F 2/72

− − 2/81
Levine et al. (1983b) 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 p articles <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
Neoplastic nodules (incidence) c

M 4/55 (7.3) 3/55 (5.5) 0/52 (0.0) 2/55 (3.6) 1/31 (3.2)

F 3/53 (5.6) 1/55 (1.8) 1/54 (1.9) 0/55 (0.0) 4/48 (8.3) Hepatocellular carcinomas (incidence) c

M 1/55 (1.8) 0/55 (0.0) 0/52 (0.0) 2/55 (3.6) 2/31 d (6.5)

F 0/53 (0.0) 1/55 (1.8) 0/54 (0.0) 0/55 (0.0) 0/48 (0.0) Neoplastic nodules or hepatocellular carcinomas combined (incidence) c

M 5/55 (9.1) 3/55 (5.5) 0/52 (0.0) 4/55 (7.3) 3/31 (9.7)

F 3/53 (5.6) 2/55 (3.6) 1/54 (1.9) 0/55 (0.0) 4/48 (8.3) 

### 1 2 3 4 5 6 7 8 9 10 11 12 13 *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 -d ay was started in week 11, resulting in a duration

- weighted average dose of

107 mg /kg -d ay

.
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 -d ay dose group

(n = 63) as re ported 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

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discrepancy (email to Louis D’Amico, U.S. EPA, from Mark Johnson, U.S. Army Public Health Command, February 

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28 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 Lung tumors were observed in female and male B6C3F 1 mice exp osed to RDX in the diet for 2 years ( L ish et al., 1984 ) (see Table 1-14 ). I ncidence of alveolar/bronchiolar carcinomas and the
combined incidence of alveolar/bronchiolar adenomas or carcinomas showed a statistically significant pos i tive trend (one

- sided p
- value s of 0.016 and 0.009, respectively, for the Cochran

Armitage trend test) i n female mice. I ncidence of alveolar/bronchiolar carcinomas in male mice showed a statistically s ignificant positive trend ( p
- value = 0.015 ; one -sided Cochran

- Armitage trend test) . H owever, the combined incidence of adenomas and carcinomas was not elevated in male
mice

. I n such a ca se, NTP policy recommends analyzing the tumors both separately and in
combination ( McConnell et al ., 1986 ) . This recommendation a rose out of concern that combining benign and malignant neoplasms can result in a false n egative 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

- d ose 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

- d ay 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 . T h e incidence of alveolar/bronchiolar adenomas or carcinomas was not
increased i n either sex of Sprague

- Dawley rats exposed chronically to RDX at doses up to
10 m g/kg -day ( Hart, 1976 ) or in F344 rats of either sex exposed chronically to RDX at doses up to
40 mg/k g-day ( Levine et al., 1983b )

. A lveolar/bronchiolar carcinomas are rare tumors in both species of rats, male or female ( Chandra et al., 1992 ; Haseman et al., 1985 ).

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**Table 1 -14**  **. Lung tumors observed in chronic animal bioassays** 

### 1

2 3 4 5 6 7 Reference and study design Results a
Lish et al. (1984) Mice, B6C3F 1 , 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 0 1.5 7.0 35 175/100 b
Alveolar/bronchiolar adenomas (incidence) c

M 6/63 (9.5) 5/60 (8.3) 5/62 (8.1) 7/59 (11.9) 1/27 (3.7)

F 4/65 (6.2) 2/62 (3.2) 5/64 (7.8) 9/64 (14.1) 3/31 (9.7) Alveolar/bronchiolar carcinomas (incidence) c

M 3/63 (4.8) 6/60 (10.0) 3/62 (4.8) 7/59 (11.9) 5/27 d (18.5)

F 3/65 (4.6) 1/62 (1.6) 3/64 (4.7) 3/64 (4.7) 4/31 d (12.9) Alveolar/bronchiolar adenoma or carcinoma combined (incidence) c

M 9/63 (14.3) 11/60 (18.3) 8/62 (12.9) 14/59 (23.7) 6/27 (22.2)

F 7/65 (10.8) 3/62 (4.8) 8/64 (12.5) 12/64 (18.8) 7/31 d (22.6) Hart (1976) Rats, Sprague

- Dawley, 100/sex/group Purity and particle size not specified 0, 1.0, 3.1, or 10 mg /kg -d Diet 2 yrs Doses 0 1.0 3.1 10 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. (1983b) 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

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.

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d Statistically significant trend ( p < 0.05) was identified using a one

- sided Cochran

- Armitage trend test performed by 

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38 EPA.

Note: A dash (“‒”) indicates that the study authors did not measure or report a value for that dose group.

M

echanistic Evidence

There are few mechanistic data to inform a MOA determination for either liver or lung tumors induced by exposure to RDX.
T he 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), support i ng 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

- n itroso metabolites of RDX, specifically hexahydro -1

- nitroso

- 3,5 -dinitro- 1,3,5

- triazine (MNX) and hexahydro

- 1,3,5

- trinitroso

1,3,5

- t riazine (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

resem b les 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 mut at ion 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

- n itroso 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

- ni troso derivatives ( Pan et al., 2007b ) . Exposure to potentially mutagenic N

- nitroso metabolites of RDX generated in the GI tract of mice may occur i n the liver (and subsequently in the systemic circulation) via enterohepatic circulation. However, in pigs, the N

- n itroso metabolites of RDX have been identified only in trace amounts in urine compared to the major metabolites,
4- n itro

- 2,4

- diazabutanal an d 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 B6C3F 1 mice fed 5 mg RDX/kg in the diet for 28 days ( Z hang 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

- t reated mice (e.g., m iR- 92a was downregulated in liver tissue samples when it is typically upregulated in hepatocellular carcinomas) ( Sweeney et al.,
2012b ) . miRN As 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

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miRNAs) plays a role in the MOA of RDX carcinogenicity. Microarray analysis of gene expression in 

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- 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 ( B annon et al., 2009a ) ; however, the relevance of thi s finding to the carcinogenicity of RDX is
unclear. Sweeney et al. (2012b) hypothesized a set of MOAs for the liver tumors:

• Genotoxicity mediated by either: (1 ) RDX ; (2 ) tissue

- generated oxidative metabolites ; or (3) N- nitroso metabolites generated anaerobically in the GI tract

. The key events in this hypothesized MOA are: production of DNA damage, gene mutation, formation of neoplastic lesions, and p romotion/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- n itroso metabolites, absorption, distribution to the liver, cytoxocity (optional), and enhanced cell proliferation, leading to preneoplastic foci that progress to hepatocellular adenomas and carcinomas. S weeney 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. T hey 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 f or carcinogenesis.



### • T he absence of significant liver histopathology in mice after subchronic or chronic exposure to RDX at doses that induced liver tumors ( L ish 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 patter n 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 ( s ee 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 ( P an et al., 2007b ) and minipig ( Musick et al., 2010 ; Major e t 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

- d erived oxidative metabolites of RDX ha s been proposed, but is not supported by the available data. In light of limited information on precursor

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events leading to the observed liver and lung tumor response in RDX

- exposed rodents and lack of 

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nitroso metabolite MOA is supported. Thus, the MOA leading to the increased incidence of liver and lungs tumors is not known.

**1.2.6. Other Noncancer**  **Effects**

There a re isolated reports of RDX inducing systemic effects in several organs/systems, including the eyes and the musculoskeletal, cardiovascular, immune, and GI sy stems

. However, there is less evidence for these effects compared to organ systems described earlier in Section 1. 2

. Generally, evide nce 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

- r elated patterns of increasing or decreasi ng effect. A longer
discussion o f 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 A ND EVALUATION** 

**1.3.1. Effects**  **Other Than Cancer**

T h e majority of evidence for the health effects of RDX comes from oral toxicity studies in
animal s

. T he three e pidemiology studies that document possible inhalation exposure are limited by various study design feat ures, including inability to distinguish exposure to TNT ( associated with liver and hematological system toxicity )

, i nability to adequately characterize exposure levels, small sample sizes, and inadequate reporting. T h e single animal inhalation study identified in the
literature search h ad deficiencies (e.g., lack of a control and incomplete exposure information) that precluded its inclusion in this assessment ( s ee l i terature s earch section ). The strongest evidence for hazard s f ollowing exposure to RDX is for ner vous system effects.
T o xicity studies in multiple animal species involving chronic, subchronic
, and gestational exposures provide consistent evidence of nervous system effects following oral exposure . E ffects included
dose- r elated increases in seizures and convulsions, as well as observations of tremors, hyperirritability, hyper

- r eactivity, and other behavioral changes ( Crouse et al., 2006 ; Angerhofer et al., 1986 ; Levine et al., 1983b ; 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

, a nd relevant to
, humans.
A cross

- se ctional study describe d memory impairment and visual

- spatial decrements in RDX

exposed workers ( M a and Li, 1993 ) , although confidence in these findings is relatively low because of issues with design and reporting. S everal case reports provide additional evidence of associations between exposure to RDX ( via ingestion, inhalation, and possibly dermal exposure)

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and seizures and convulsions ( Kasuske et al., 2009 ; Küçükardali et al., 2003 ; Testud et al., 1996a ; 

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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 dizz iness, 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 ( Q uinn 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 GABA A channel suggests that the resulting disinhibition could lead
to the onset of seizures ( Williams et al., 2011 )

. I nduction of convulsions and seizures appears to be more strongly correlated with dose than with duration of exposure

. However, th ere is some mechanistic information to suggest that repeated exposure to a chemical binding to the receptor convulsant site of GABA A may promote a state of increased neuronal activity that could increase the likelihood of subsequent neurological
effects ( G erkin et al., 2010 )

. I t 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 det a iled dose

- response information

, a nd 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 RD X a nd in multiple experimental animal studie s 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. Th is 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
convuls i ons. Nervous system effects are a human hazard of RDX exposure and are carried forward
for consideration for dose

- r esponse analysis. Convulsions, considered a severe adverse effect, were selected as a consistent and sensitive endpoint representative of nervous system effects. Evidence for kidney and ot her urogenital toxicity i s more limited than evidence for neurotoxicity

. H istopathological changes in the urogenital system ( suppurative prostatitis, medullary papillary necrosis, suppurative pyelitis, uremic mineralization, and luminal distention and cystitis of the urinary bladder) w ere reported in male rats exposed to RDX in the diet for
2 y ears ( Levine et al., 1983b )

. S imilar histopathological changes of the urogenital system were not observed in mice, and no other rat studies of similar duration tha t examined the prostate were
available

. As discussed earlier, a mong the lesions identified in the rat, the incidence of suppurative

pro st atitis is considered a marker for RDX -related urogenital effects as it is plausibly associated

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with a progression of effects resulting from other kidney or bladder lesions . T he plausibility of a 

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38 MOA that shares a common molecular initiating event (binding to the GABA A recept or 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 marker for the kidney and urogenital effects, was selected as a sensitive endpoint representative of the urogenital system effects.
There is some e vidence for male rep roductive toxicity that comes from the finding of testicular degeneration in male B6C3F 1 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 high est dose tested in the study . H owever, studies in different rat strains did not consistently observe t esticular effects . Although the available data are limited, gi ven the dose

- related findings of mouse testicular degeneration

, there is suggestive evidence of male reproductive effects associated with RDX exposure ; t hese effects were carried
forward for consideration for dose

- r esponse 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 . I n a n imal studies
, developmental effects , including offspring survival, growth, and morphological development, w ere observed only at doses associated with maternal mortality ( A ngerhofer et al., 1986 ; Cholakis et al., 1980 ) . Evidence for potential h epat ic 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., 1983b ; 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 i nterpretation 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

- r esponse analysis and derivation of reference values

. As discussed in Section 1.2, mortality is not addressed in this ass essment as a hazard by
itself

, but rather in the context of nervous and urogenital system hazards. Histopathological changes in the urogenital system observed i n male rats exposed to 40 mg/kg

- day in the diet for 2

years w ere considered the principal cause of treatment

- related morbidity and mortality ( Levine et al., 1983b )

. However, the incidence of suppurative prostatitis , considered a sensitive 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. (1983b) is a less sensitive endpoint (by
more than 10

- fold) than th e effect that is the selected as the basis dose

- response analysis ( i.e., suppurative prostatitis).

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In a number of the animal studies reporting nervous system effects, unscheduled deaths 

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37 occurred at RDX doses as low as those that induced nervous system eff ects ( C rouse et al., 2006 ; Angerhofer et al., 1986 ; Levine et al., 1983b ; 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. Convulsion s 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 wi th symptoms of neurotoxicity e xposed 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 popu lat ions exposed chronically to RDX. 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 exp o sure 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 a nd, 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, t h e relationship between convulsions and mortality is
not clear and raises concerns for the potential underreporting of convulsions (see Section 1.2.1).
An a nalysis
, described in Section 2 .1. 7 , addresses the possibility that the analyses of convulsions brought forward for dose

- r esponse analysis resulted in an underestimate of the toxicity for RDX.

**1.3.2. Carcinogenicity**  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 B6C3F 1 mice mice ( P arker et al., 2006 ; Lish et al., 1984 )

. In the same study, RDX also induced dose -relate d increases in the incidence of alveolar/bronchiolar adenomas or carcinomas in both sexes. Some of these trends in liver and lung were statistically significant. In Fischer 344 rats, dietary administration of RDX yielded a statistically significant trend in the incidence of hepatocellular carcinomas in males,
but not in females ( Levine et al., 1983b )

. A 2

- year dietary study in Sprague

- Dawley rats was

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negative in both sexes Hart (1976) , although doses in this study were somewhat lower (no 

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37 carcinomas at doses up to 10 mg/kg

- d in Hart (1976) , versus hepatocellular carcinomas at 8 and
40 mg/kg

- d in the Levine et al. (1983b) 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 desc riptor 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 . O ne 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

- r elated increases in tumors in two species (mouse and rat), in both sexes, and at two sites (liver and lung). Alternatively, the descriptor suggestive evidence of carcinogenic potential is appropriate when the evidence raises “a concern for potential carcinogenic effects in humans” b u t is not sufficient for a stronger conclusion. The hepatocellular carcinoma result in male F344 rats is based on a small number of tumors (two each at 8 and 40 mg/kg

- d ay, versus one in controls), and RDX
did not increase the incidence of carcinomas at an y 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 f or RDX. Although the evidence includes dose

- related tumor increases in two species, two sexes, and two sites, the evidence of carcinogenicity o u tside the
B6C3F 1 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 , t he evidence for RDX is strong. There are well

- c onducted studies that tested large numbers of animals at multiple dose levels, making the cancer response suitable for dose

- r esponse 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
infor ma tion provides sufficient basis to apply the cancer descriptor developed from oral studies to other exposure routes.

**1.3.3. S**  **usceptible Populations and Lifestages for Cancer and Noncancer Outcomes**  Susceptib il ity refers to factors such as lifestage, genetics, sex, and health status that may predispose a group of individuals to greater response to an exposure. Th is greater response c ould be achieved either through differences in exposure to the chemical underlying RD X toxicokinetics

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or differences in RDX toxicodynamic s between susceptible and other populations. L ittle 

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38 information is available on populations that may be especially vulnerable to the toxic effects of RDX

. L ifestage, and in particular childhood

, susceptibility has not been observed in human o r animal studies of RDX toxicity . T r ansfer of RDX from dam to the fetus du ring gestation has been reported
, and the presence of RDX in the milk of dams administered 6 mg/kg

- day by gavage has
been documented ( H ess
- Ruth et al., 2007 ) ; however, reproductive and developmental toxicity
studies generally did not identify effects in offspring at do ses 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.
L imited 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 br ain weight changes )

. U r ogenital effects have been observed in males at lower doses than in females ( Levine et al., 1983b ; Levine et al., 1981a
, b ; Cholakis et al., 1980 ) , suggesting a possible sex

- based difference in susceptibility to RDX toxicity

. D ata on the incidence of convulsions and mortality from gavage studies of RDX in the rat provide some indication that pregnant animals may be a s usceptible 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
- f inding 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/kg

day, although the relatively small g roup 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

- d ay, but one death (in dose groups of 40 rats) was reported at both 2 and 6 mg/kg

- d ay ( 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 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 pregnants animals may be more susceptible to the effects of RDX; however, direct comparison between the available gavage studies in pregnants and

nonpr e gnant 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.
T here is limited evidence that CYP450 or similar enzymes are involved in the metabolism of
RDX ( B hushan 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. I n dividuals with epilepsy or other seizure syndromes
, and in particular those that have their basis in genetic mutation to GABA A receptors , may represent another group that may be susceptible to RDX exposure. Ho wever, there is currently

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no information to support predictions of how genetic polymorphisms or the presence of seizure 

### 1
2 syndromes may affect susceptibility to RDX exposure

.

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# 2. DOSE

- RESPONSE ANALYSIS

**2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER** 

The oral reference dose ( RfD

, expressed in units of mg/kg

- day) is defined as an estimate 

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30 (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. I t can be derived from a no
- observed

- adverse

- effect level (NOAEL), lowest

- observed

- adverse

- effect level (LOAEL), or the 95% lower bound on the benchmark dose (BMDL), with uncertainty factors (UFs) generally applied to reflect limitations of
the data used.

**2.1.1. Identification of Studies**  **for Dose**

**- Response Analysis**  **of Selected Effects**

As discussed in Section 1. 3 .1, based on findings from oral studies in experimental animals,
nervous system e ffects are a human hazard of hexahydro- 1,3,5

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exposure , a nd kidney and o the r u rogenital 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 conduc t ed for RDX, it w as not considered a hazard by itself or as the basis for the derivation of a reference
value. R ather, the mortality evidence was evaluated in the context of that system

- specific hazard (see Section s 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

- r esponse 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 s t udies of acute exposure durations. S tudies 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

- r esponse relationship. Additionally, with respect to measur ement 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 o f worker populations exposed

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to RDX examined the relationship between certain health endpoints and inhalation exposure; 

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18 19 however, no epidemiological studies of ingested RDX are available. Multiple case reports support
the identification of hazards associ ated 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-re sponse 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.
E xperimental animal studies considered for each health effect were evaluated using general study quality con siderations discussed in Section 6 of the Preamble and in the literature search
section , an d the attributes described above. T he rationales for selecting the strongest studies that reported t he health effects are summarized below.

Nervous S y

stem E ffects

C onvulsions 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 ( C rouse et al., 2006 ; Lish et al., 1984 ; Levine et al., 1983b ; Levine et al., 1981a ; Cho lakis 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., convulsion s ) evaluated
for dose

- r esponse analysis.

**Table 2**

**- 1.**  **Information consider**  **ed for evaluation of studies that examined**  **convulsions**

Study reference Study design and size Relevance of exposur e paradigm Measurement of endpoint

Design Number of animals Route Duration Number of dose
groups 1 Levels (mg/kg

- d) Purity (%) Incidence data reported

Crouse et al. (2006) Toxicity study 10 rats /sex/
group Gavage 13

- wk 5 4

− 15 99.99 Yes

Cholakis et al. (1980) Developmental study 24−25 female
rat s/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 specifie d Yes

Levine et al. (1983b) Toxi city 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 8 5 mice /sex/
group Diet 2

- yr 4 1.5

− 175 89

− 99 No

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Study reference Study design and size Relevance of exposur e paradigm Measurement of endpoint

Design Number of animals Route Duration Number of dose
groups 1 Levels (mg/kg

- d) Purity (%) Incidence data reported

Levine et al. (1981a) Toxicity study 10 rats /sex/
group di et 13

- wk 5 10

− 600 85 No

von Oettingen et al. (1949) Toxicity study 20 rats /group die t 13

- wk 3 15

− 50 90−97 No

1 Excluding the control group. 

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28 Incidence of convulsions was reported in t h ree studies of RDX

— all involving gavage
administration : C rouse et al. (2006)

, Cholakis et al. (1980) (developmental toxicity st udy), 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)

, Le vine et al. (1983b)

, 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. (1983b) 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 conside red. 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 s i x dose groups (including the control) that provided good resolution of the dose

response 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 (G Ds ) 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 charac terized 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

- r esponse analysis because of small group sizes (n = 3/sex) and uncertainty in

measure s o f 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 ( L ish et al., 1984 ; Levine et al., 1983b ; Levine et al., 1981a ; von Oettingen et al., 1949 ) that

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did not provide incidence data , it is important to note that the r eported neurotoxic effects in the 

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33 dietary studies were observ ed at dose levels higher than the doses at which effects were observed in the gavage studies ( C rouse 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

- ye ar study by Levine et al. (1983b)

, a LOAEL for nervous system effects (convulsions, tremors

, and hyper

- irritability) of 40 mg/kg

- day and a NOAEL of 8 mg/kg

- d ay were identified. Other studies identified higher effect levels (i.e., 100 mg/kg

- day in
the 2

- y ear mo use 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. (194 9) 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 d ose level at which convulsions occurred; therefore, a reliable NOAEL and LOAEL from this study could not be ident i fied. Therefore, two gavage studies, Crouse et al. (2006) and Cholakis et al. (1980) , and one dietary study
, Levine et al. (1983b) , were selected for dose

- response analysis.

Kidne y and O ther U rogenital System E ffects Suppurative prostatitis was selected for dose

- response analysis. It is considered to be a sensitive marker for the broader range of urogenital effects observed in F344 male rats in a 2

- y ear study by Levine et al. (1983b)

. The Levine et al. (1983b) study : (1 ) included a histopathological examination of the kidney and other urogenital s ystem 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 ( 7 5/sex/group, with interim sacrifice groups of 10/sex/group at 6 and 12 months) ; and (3 ) r eported individ ual animal d ata. T his study, the only one to identify suppurative prostatisis,
was selected for dose

- r esponse analysis.

Male R eproductive T oxicity

T esticular 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

- y ear study : (1) included histopathological examination of male reproductive organs ; (2 ) inclu ded four dose groups and a control group, and adequate numbers of animals per dose group ( 8 5 /sex/group, with interim sacrifice groups of 10/sex/group at 6 and 12 months) ; a nd (3) reported individual animal data. This study, the only one to identify testic u lar degeneration , was selected for dose

- response analysis.

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**2.1.2. Methods of Analysis**  No biologically based dose

- response models are available for RDX. I n this situation, the U.S. 

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15 Environmental Protection Agency ( EPA ) evaluates a range of dose

- response models thought to be consistent with underlying biological processes to determine how best to empirically model the
dose- response relationship in the range of the observed data. C onsistent with this approach, EPA
evaluated dose

- response information with the mod els available in EPA’s Benchmark Dose Software (BMDS, versions 2.4 and 2.5). EPA estimated the benchmark dose ( BMD ) and BMDL using a benchmark response (BMR) selected for each effect

. A conceptual model of the analysis approach used for RDX is provided in Figure 2

- 1. In this assessment, points of departure ( PODs ) are identified through BMD modeling (preferred) or identification of a NOAEL, and followed by animal

to

- human extrapolation through the use of physiologically based pharmacokinetic ( PBPK ) models or the application of a dosimetric adjustment factor, depending on the data available.

**F igure 2** 

**- 1.**  **Concep t**  **ual approach to dose** 

**- response modeling**  **for oral exposure**

**.**

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Nervous S ystem E ffects

Incidence data for convulsions from Crouse et al. (2006) and Cholakis et al. (1980) were 

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31 amenable to BMD modeling. For Crouse et al. (2006) , statistical analysis conducted by EPA indicated no significa nt 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) f or convulsions was used to address the severity of this endpoint ; modeling with 5 and 10% ER is provided in Appendix D (see Section D .1.2, Tables D

- 3 to D

- 14 ) for comparative purposes . In general, for noncancer effects, severe endpoints are not typically u sed 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. T he use of a 1% ER BMR for convulsions in Crouse et al. (2006) resulted in extrapolation below the range of the experimental doses

. However, t he BMD of 3.02 mg/kg

- day

was n ot far below the dose range of 4

− 15 mg/kg

- day used in the study ; thus, this extrapolation was
considered moderate. I n addition to uncertainty from extrapolation, model uncertainty from the use of the 1% ER BMR can be a concern. H owever, 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 su bstantial model uncertainty

. Because incidence data for convulsions were not provided by Levine et al. (1983b) , 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. M ore detailed BMD modeling information is available in Appendix D.

Kidney/ U r

ogenital System E ffects

Incidence data on suppurative prostatitis as reported by Levine et al. (1983b) were amenable to BMD modeling . A BMR of 10% ER was applied under the assumption that it represents
a minimal l y biolo gically 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 Incidence data on testicular degeneration as reported by Lish et al. (1984) were amenable to modeling. A BMR of 10% ER was applied under the assumption that it represents a minimally biologically significant lev el 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.

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**Table 2-2**  **. Summary of derivation of PODs following oral exposure to RDX** 

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2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Endpoint and reference (exposure duration/route) Species/sex Model a BMR BMD (mg/kg

- d) BMDL (mg/kg

- d) POD HED (mg/kg

- d)

Admin

istered dose b RDX AUC c RDX C max d

Nervous system

Incidence of convulsions Crouse et al. (2006) (90

- d/gavage) Male and female F344 rat, combined e Multistage 2° 1% ER 3.02 0.57 0.14 0.28 0.37

Incidence of convulsions Cholakis et al. (1980) (GDs 6

− 19/gavage) Female F344
rat Quantal

linear 1% ER 0.18 0.12 0.03 0.06 0.08

Incidence of convulsions Levine et al. (1983b) (2

- yr/diet) Male and female F344
rat LOAEL = 40 mg/kg

- d; NOAEL = 8 mg/kg

- d f 1.9 3.9 4.3

Kidney/urogenital system

Incidence of suppurative prostatitis Levine et al. (1983b) (2

- yr/diet) Male F344 rat LogProbit 10% ER 1.67 0.47 0.11 0.23 0.25

Male reproductive system

Incidence of testicular degeneration Lish et al. (1984) (2

- yr/diet) Male B6C3F 1
mouse LogProbit 10% ER 56.0 16.3 2.4 0.08 0.18

a For modeling details, see Appendix D.

b POD was converted to an HED using a standard DAF based on BW 3/4

.
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, C max ) derived using PBPK models.

e Exact Mantel

- Haenszel test for independence between convulsion incidence and sex, stratified by dose, yielded
p

- v alue >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.

AUC = area under the curve; BW = body weight; DAF = dosimetric adjustment fa ctor; ER = extra risk ; HED = human

equivalent dose

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Human E xtrapolation

EPA guidance ( U.S. EPA, 2011 ) describes a hierarchy of approaches for deriving h uman 

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36 equivalent doses (HEDs) from data in laboratory animals, with the preferred approach being PBPK modeling . O t her approaches can include using chemical

- specific information in the absence of a complete PB PK model . In lieu of either reliable, chemical

- spec ific models or data to inform the derivation of human equivalent oral exposures, a body weight scaling to the 3⁄4 power (i.e., BW 3/4 ) approach is g enerally applied to extrapolate toxicologically equivalent doses of orally administered agents from adult labor at ory animals to adult humans. Candidate PODs for endpoints selected from rat and mouse bioassays were expressed as HEDs. HEDs were derived using both PBPK model ing ( with alternative measures of internal dose),
and a BW 3/4 scaling approach. These approaches are outlined in Figure 2 -1 , and the resulting
POD HED v alues are presented in Table 2 -2.

Extrapolation using PBPK modeling. PBPK models for RDX in rats, humans, and mice have been published ( S weeney et al., 2012a ; Sweeney et al., 2012b ; Krishnan et al., 2009 ) based on RDX

specific data . EPA evaluated and further developed these models for extrapolating doses from animals to humans (see Appendix C, Section C. 1 .5) . I n 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 ) . T herefore, 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.
T h e PBPK models for RDX were used to estimate two dose metrics : the area under the curve ( AUC )
and th e peak concentration (C max ) for RDX concentration in arterial blood

. The AUC represents the average blood RDX concentration for the exposure duration normalized to 24 hours an d the C max represents the maximum RDX concentration for the e xposure duration

. It appears logical to use RDX concentration levels in the brain as the internal dose metric for analyzing convulsion da ta . Nevertheless, the blood concentration of RDX was preferred as the dose metric due to greater confidence in modeli n g this variable. This is because of the substantially greater number of measurements of RDX blood levels used in calibrating model parameters. Additionally, predictions of RDX concentrations in the brain are highly correlated with RDX blood
concentratio n s because the brain compartment does not have absorption, metabolism , or elimination of RDX. G r eater 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 constan t for oral absorption ( KAS) is uncertain , and peak concentrations are more sensitive to variations in this parameter than average values. RDX

- in duction 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 p e ak blood concentration, comes from mechanistic information on RDX binding at the

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picrotoxin convulsant site of the gamma
- amino butyric acid ( GABA ) channel. T here is evidence 

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37 from examination of picrotoxin binding to GABA A that a resulting period of eleva ted 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). Therefore,
the AUC for RDX concentration in arteria l blood was selected as the internal dose metric for analyzing dose

- response data for convulsions. Nevertheless, the POD HED values based on both
blood AUC and peak blood concentration (C max ) are presented in Table 2 -2 for completeness.
The rodent PBPK mod el was applied to the BMDLs generated from BMD modeling to
determine the animal internal dos e , expressed as the AUC of RDX blood concentration, and representing the cross

- s pecies toxicologically equivalent (internal) dose. T he human PBPK model

was t hen 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 m odeling is performed. Because the sequence of the calculation does not influence the results, a pplying 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 t o 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
selecte d 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 ( S weeney 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 at which RDX was detected was 35 mg/kg, an exposure level high enough to manifest some toxicity in the chronic mouse bioassay, and except for measurements from a single animal, all other data points were non

- d etects or excluded as outliers ( 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 is low. Further, there are no data to enable characterizing the fraction of RDX that is metabolize d 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 P4 50 [ CYP 450 ]

enzymes ) . G iven the high sensitivity of the model to the metabolic rate constant, the uncertainty in mouse toxicokinetics significantly decreases confidence in using the mouse PBPK model for predicting mouse blood RDX concentrations. (See Summary of C o nfidence i n PBPK M odels for RDX in Appendix C, Section C. 1 .5 for further discussion of confidence in the mouse model.) Given the low confidence in the mouse PBPK model, the preferred approach for determining candidate oral

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values for endpoints selected from the mou se bioassay is that based on the administered dose of 

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30 RDX extrapolated to humans using allometric BW 3/4 scaling.

Extrapolation using BW 3/4 scaling. HEDs were also calculated using a BW 3/4 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) derive d as follows:

DAF = (BW a 1/4 /BW h 1/4 ),
where
BW a = animal body weight
BW h = human body weight

Using BW a values of 0.25 kg for rats and 0.035 kg for mice and a BW h of 70 kg for humans ( U .S. EPA, 1988 ) , the resulting DAFs for rats and mice are 0.24 and 0.15, respectively. A pplying the DAF to the POD identified for effects in adult rats or mice yields a POD HED as follows (see Table 2 -2 ):

POD HED = l abor atory 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 . D etails of the
PBPK model evaluation used fo r extrapolation from BMDL values can be found in Appendix C,
Section C. 1 .5 . T able 2-2 summarizes the results of the BMD modeling and the POD HED for each data
set discussed above.

**2.1.3. De**  **rivation of Candidate Values**

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 variability were considered when determining the application of UFs to the PODs presented in
Table 2 -2 . A n explanation follows : An intraspecies uncertainty factor

, UF H , of 10 was applied to all PODs to account for potential differences in toxicokinetics and toxicodynamics in the absence of information on the
varia b ility 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

- sp ecific UF for intraspecies uncertainty. An interspecies unce rta inty factor

, UF A , of 3 (10 1/2 = 3.16, rounded to 3) was applied to all
PODs t o 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 B W 3/4 scaling (see rationale in
Section 2 .1.2

— Human Extrapolation) , which addresses predominantly toxicokinetic and some

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toxicodynamic aspects of cross
- species extrapolation; r esidual uncertainty in toxicokinetic and 

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37 toxicodynamic extrapolation remains . In the absence of chemical

- specific data to quantify this uncertainty, EPA’s BW 3/4 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 d oses 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 UF A of 3 was applied to account for this remaining toxicodynamic and any residual toxicokinetic uncertain t y not accounted for by the PBPK
model

. A subchronic to chronic uncertainty factor, UF s , 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 used to
derive a POD were of 2

- y ear duration. Although EPA guidance recommends a default UF S of 10 on the assumption that effects in a subchonic study w ould occur at a pproximately 10

- fold higher concentration than in a corresponding (but absent) ch ronic study ( U.S. EPA, 2002 ) , the RDX database does not support a UF S of 10. As discussed in Section 1.2.1, although Gerkin et al. (2010) introduces the possibility of effects develo ping 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 t h ose induced by subchronic exposure. In addition, chronic dietary doses associated
with convulsions were ≥35 mg/kg

- d ay and were at least fourfold higher than gavage doses that
induced convulsions in 14 - an d 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). Th is may be due to differences between dietary and gavage administration (see Section s 2.1.1 and 2.1. 7 ). Nevertheless,
the se s tudies do not sup port the default expectation of observing effects in chronic studies at approximately 10

- f old lower exposure levels than in subchronic studies. Accordingly, a UF s of 1 was applied to PODs derived from studies of less

- th an
- chronic duration.
A LOAEL to NOA EL uncertainty factor

, UF L , of 1 was applied to all POD values because the
PO D w as a BMDL or a NOAEL. When the POD is a BMDL, the current approach is to address this
factor a s one of the considerations in selecting a BMR for BMD modeling . I n this case, the BMR for modeled endpoints w as selected under the assumption that the BMR represents a minimal, biologically significant change for these effects. A database uncertainty factor, UF D , of 3 was applied to all POD values. The oral toxicity database for RDX includes subchronic and chronic toxicity studies in the rat and mouse, a two

generation reproductive toxicity study in the rat, developmental toxicity studies in the rat and rabbit, a nd subchronic studies (with study design limitations) in the dog and monkey. As discussed below, some uncertainty is associated with characterization of the RDX n eurotoxicity

.

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Analyses presented in Section 2.1.6 note that reference values derived from mo rtality data 

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38 would be similar to the RfD for RDX based on convulsions. EPA prefers to identify reference values based on upstream (less severe) effects that would precede frank effects like convulsions and mortality. Some uncertainty remains in our under standing of RDX

- induced neurotoxicity. In part, this is due to limitations in study design to assess neurotoxicity across the RDX database; the frequency of animal observations in the available studies raises concerns that there may be underreporting of t he 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 neurotoxici ty. 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 a ltered brain activity, ranging from involuntary limb and facial twitches to tonic

- cl onic 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 ther e ar e 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

- in duced 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.

• Ad ditional 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

- i nduced convulsant activity, but could also be u sed to identify and quantify sub

- c onvulsive 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.

• A FOB conducted by Crouse et al. (2006) provides information on neurobehavioral effects associated with RDX exposure, yet the results of that study d id 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 stimuli

induced 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 additiona l measures of RDX neurotoxicity.

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### • Further evaluation of potential developmental neurotoxicity (and specifically seizure 

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25 induction) associated with RDX exposure. Models for examining seizure

- related behaviors during development exist, mainly involving manipulation and analyses in pre

- weanling
rodents. Hess

- R uth 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 endp o ints has not been conducted in studies of RDX toxicity, the available testing, including a two

- g eneration 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
charact erize 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 a s 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 s ome concern that additional studies described above may lead to identification of a more sensitive endpoint or a lower POD. Accordingly, a UF D of 3 was applied to all derived PODs.
Table 2 -3 is a continuation of Table 2 -2 and summarizes the applicatio n of UFs to each
POD HED to derive a candidate value for each data set. T he candidate values presented in T able 2-3 are preliminary to the derivation of the organ/system

- s pecific reference values. These candidate values are considered individually in the s election of a representative oral reference value for a specific hazard and subsequent overall RfD for RDX.

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**Table 2-3** 

**. Effects and corresponding derivation of candidate values**  

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2 3 4 5

6
7 Endpoint and reference POD H ED a POD type UF A UF H UF L UF S UF D Composite UF Candidate value (mg/kg

- d)

N ervous system (rat)

Incidence of c onvulsions Crouse et al. (2006) 0.2 8 BMDL 01 3 10 1 1 3 100 2. 8 × 10 − 3

Incidence of c onvulsions Cholakis et al. (1980) 0. 06 BMDL 01 3 10 1 1 3 100 6.0 × 10 − 4

Incidence of c onvulsions Levine et al. (1983b) 3.9 NOAEL 3 10 1 1 3 10 0 3.9 × 10 − 2

K

idney/urogenital system (rat)
Incidence of p rostate suppurative inflammation Levine et al. (1983b) 0.23 BMDL 10 3 10 1 1 3 10 0 2.3 × 10 − 3

M ale reproductive system (m ouse )

Incidence of t esticular degeneration Lish et al. (1984) 2.4 BMDL 10 3 10 1 1 3 10 0 2.4 × 10 − 2

a POD HED values based on data from the rat were derived using PBPK modeling; the POD HED based on data from the

mouse was derived using BW 3/4 adjustment (see S ection 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 POD HED values , with each bar corresponding to one data set described in Tables 2 -2 a nd 2 -3.

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**Figure 2-2** 

**. Candidate values with corresponding POD and composite UF.**  

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**2.1.4. Derivation of Organ/System** 

**- Specific Reference Doses** 

Table 2-4 distills the candidate values from Table 2-3 into a single value for each organ or 

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. O rgan - 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 Basis RfD (mg/kg

- d ) Study e xposure description Confidence

Nervous system Incidence of c onvulsions ( Crouse et al., 2006 ) 3 × 10 − 3 Subchronic Medium

Kidney/urogenital system Incidence of s uppurative prostatitis ( Levine et al., 1983b ) 2 × 10 − 3 Chronic Low

Male reproductive system Incidence of t esticular degeneration ( Lish et al., 1984 ) 2 × 10 − 2 Chronic Low

O verall RfD Nervous system 3 × 10 − 3 Subchronic Medium

Nervous S ystem E ffects The organ/system

- specific RfD for nervous system effects was based on the incidence of convulsions in F344 rats reported in C r ouse 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

- r esponse curve for convulsions, and an endpoint (convulsions) that was replicated across

m ultiple studies. Although the candidate value derived from the developmental t o x icity study in F344 rats by Cholakis et al. (1980) is lower (by approximately five fold), there is greater certainty in
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 resolution of the dose

response curve for RDX

- induced convulsions, whereas Cholakis et al. (1980) used only thre e dose group (plus the control) with order of magnitude dose spacing, resulting in a less well defined
characterization of the dose

- r es ponse curve for this endpoint. Further, Crouse et al. (2006) used a higher purity test m aterial than did Chol akis 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

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this study as the basis for the RfD; however, as discussed in Se ction 1.3.3, the available studies in 

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33 pregnant and nonpregnants 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. (1983b) 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 fr o m dietary administration (recognizing that differences in particle size and purity of the test material may confound direct comparisons between gavage and dietary administration)

. Convul s ion data from Levine et al. (1983b) yielded a POD HED 14- fold higher than the POD HED derived
from Crouse et al. (2006)

. The POD derived from the Levine et al. (1983b) study is considered less
certain than that derived from Crouse et al. (2006)

. Levine et al. (1983b) did not provide
information on the incidence of neurotoxic effects, and BMD analysis was thus not support ed (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. (1983b) 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 thes e r easons, and in light of the fact that data from the Levine et al. (1983b) s tudy yielded a higher POD, Levine et al. (1983b) was not u sed as the basis for the organ/system

- specific RfD for nervous system effects.

Kidney/ U rogenital E ffects A single data set for incidence of suppurative prostatitis in male F344 rats as reported in a
2- y ear dietary study by Levine et al. (1983b) was brought forward for quantitative analysis as a sensitive marker for the broader array of RDX

- associated effects observed in the urogenital system.
T h e RfD for kidney and o ther urogenital effects is based on this dataset

.

Male R eproductive E ffects A single dataset for male reproductive effects , specifically the incidence of testicular degeneration as reported in male B6C3F 1 mice exposed to RDX in diet for 24 months ( Lish et al.,
1984 ), was brought forward for quantitative analysis. T he RfD for male reproductive effects is
based on this dataset.

**2.1.5. Selection of the Overall Reference Dose**  Multiple organ/system

- specific reference doses were derived for effects identified as potential hazards from RDX exposure, including nervous system effects

, k idney and other urogenital effects, and male reproductive effects . E v idence for nervous system effects, and specifically convulsions, was observed in multiple stu dies, in multiple species, and following a range of exposure durations . I n addition, the organ/system

- specific RfD for nervous system effects was the lowest among the organ/system

- s pecific RfDs derived for RDX. E vidence for dose -related
effects on the u rogenital system co mes primarily from a single 2

- year toxicity study in male rats

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( Levine et al., 1983b ) , and evidence for male reproductive effects comes primarily from a single 

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The organ/system

- specific RfD of **3 × 10** 

**− 3**  **mg/kg**

**- day**  for nervous system effects in the rat as reported by C rouse 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 as the lowest organ/system

specific RfD . T h is overall RfD provide s an exposure level below which effects associated with RDX exposure are not expected to occur. The overall RfD is derived to be protective of all types of effects for a given duration of exposure, and is intended to protect the population as a whole, including potentially susceptible subgroups ( U .S. EPA, 2002 ) . D ecisions concerning averaging exposures over time for comparison
with the RfD should conside r the types of toxicological effects and specific lifestages of concern

. F lu ctuations 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 d uration 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 LD**  **01 s**  As previously discussed, mortality was considered in discussions of ot her organ/system

specific toxicity (and in particular, effects on the nervous system and kidney). EPA did not develop a candidate RfC 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 )
identi f ied 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) )

.

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**Table 2**

**- 5. Comparison of dose levels associated with mortality and**  

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17 **convulsions in selected studies**

Stu dy Doses associated with mortality Doses associated with convulsions
Crouse et al. (2006) Rats, F344, 10/sex/group 0, 4, 8, 10, 12, or 15 mg/kg -d 13 wks/gavage ≥8 mg/kg

- d ≥8 mg/kg

- d

von Oettingen et al. (1949) Rats, sex/strain not specified, 20/group 0, 15, 25, or 50 mg/kg -d 13 wks/diet ≥25 mg/kg

- day ≥25 mg/kg

- d

Cholakis et al. (1980) Rats, F344, 24−25 females/group 0, 0.2, 2.0, or 20 mg/kg -d GDs 6−19/gavage 20 mg/kg

- d Primarily 20 mg/kg

- d; 1 convulsion at 2 mg/kg

- d

Angerhofer et al. (1986) Rats, Sprague

- Dawley, 39−51 mated females/group 0, 2, 6, or 20 mg/kg -d GDs 6−15/gava ge Primarily at 20 mg/kg

- d, but one death each at 2 and 6 mg/kg -d 20 mg/kg

- d

A discussion of mortality evidence for RDX is presented in Appendix C, Section C.3.1, and the

r elat ionship 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

- d ay g avage stud y ( Crouse et al., 2006 ) and in development toxicity studies with exposure durations of two weeks ( Angerhofer et al. (1986) ; Cholakis et al. (1980) )

. Given the proxi mity in the dose at which mortality and nervous system effects were observed in several studies, the dose

- re sp onse relationships for mortality were compared across
studies with similar durations to those in Table 2

- 5 by c omparing the LD 01 (the dose expecte d to be lethal to 1% of the animals) or NOAELs derived from each study. In addition, these LD 01 s and NOAELs were compare to the BMD 01 for convulsions used to derive the RfD. 14 Interpretation of mortality data from chronic exposure studies in mice and rats is c omplicated 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 an a ly sis are not presented in Table 2

- 6. 15

14 BMDs were compared, as opposed to BMDLs, because, as stated on p. 20 of the BMD Technical Guidance ( U.S. EPA, 2012b ) , “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...”

15 The following less informative studies were not included in the analysis of early mortality:

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**Table 2**

**- 6. Summary of dose** 

**- response evaluation for mortality following oral**  

### 1 2 **exposure to RDX**

Reference (exposure duration/route) Species/sex Model a BMR LD 01 (mg/kg

- d) LDL 01 (mg/kg

- d)

Diet studies
Lish et al. (1984) (11

- week data from 2

- yr study/diet) Male and female B6C3F 1 mouse Not amenable to modeling NOAEL: 35 mg/kg

- day 95% CI for response: 0

- 4%

Levine et al. (1981a) (13

- wk/diet) Male and female F344 r at, combined Multistage 40 1% ER 7.8 2.2

von Oettingen et al. (1949) (13

- wk/diet) Rats, sex/ strain not specified Not amenable to modeling NOAEL: 15 mg/kg

- day 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

- day 95% CI for response: 0

– 13%
Levine et al. (1983b) (13

- week data from 2

- yr study/diet) Male and female F344 rat NA (no mortality at highest dose tested) NOAEL: 40 mg/kg

- day 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

- day 95% CI for response: 0

- 25%

Gavage studies
Crouse et al. (2006) (90

- d/gavage) Male and female F344 rat, combined Multistage 20 1% ER 2.1 0.46

Cholakis et al. (1980) (GDs 6‒19/gavage) Female F344 rat Not amenable to modeling NOAEL: 2 mg/kg

- day 95% CI for response: 0

– 12%
Angerhofer et al. (1986) (GD 6‒15/gavage) Female SD rat Multistage 30 1% ER 1.7 0.59

Cholakis et al. (1980) (GDs 7‒29/gavage) Female New Zealand white rabbit NA (no mortality at highest dose tested) NOAEL: 20 mg/kg

- day 95% CI for response: 0

- 22%

• 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- we ek 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

m ortality, but reporting was limited.

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Reference (exposure duration/route) Species/sex Model a BMR LD 01 (mg/kg

- d) LDL 01 (mg/kg

- d)

a For modeling details, see Appendix D, Section D.1.3. ER = extra risk LD 01 = dose expected to be lethal to 1% of the animals LDL 01 = lower confidence limit on the LD 01 

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31 Of the studies in Table 2

- 6, dose

- response analysis was conducted for all studies that showed an increased incidence of unscheduled deaths. LD values are provided in Table 2

- 6 , and detailed modeling results are provided in Appendix D, Section D.1.3. Mort a lity was observed only at the highest dose tested at week 11 in the 2

- ye ar 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 LD 01 estimation, and a NOAEL (with its associated confidence interval, CI) was used in this comparative analysis
instead.
LD 01 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

- d ay (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
BMD for convulsions from Crouse et al. ( 2 006) that was used as basis for the overall RfD for RDX. The BMD for convulsions of 3.0 mg/kg

- day is in the middle of the distribution of calculated LD 01 s, and the lowest LD 01 of 1.7 mg/kg

- day is within twofold of the convulsion BMD 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 BMD 01 s for several reasons. Confidence intervals for the observed responses of 0% characterize some statistical uncertain t y for NOAELs from studies that could not be modeled (note that the upper bound on an observed response of 0% is not directly comparable to a lower bound on a benchmark dose). The confidence intervals suggest that comparable 1% levels for these datasets co uld be somewhat lower than the NOAELs. On the other hand, dose

- s pacing can affect the interpretation of NOAELs, such 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 r eported NOAEL is 10

- fold lower than the dose associated with 17% mortality at 20 mg/kg

- d ay). 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 th e ap plication of the same extrapolation procedures and uncertainty factors. However, the similarity in RDX doses associated with both mortality and nervous system effects should be taken into consideration when using this RfD, and in particular in evaluati ng exposures that exceed the RfD in light of the severity of mortality.

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**2.1.7. Uncertainties in the Derivation of**  **the Reference Dose** 

T o derive the RfD, the UF approach ( U.S. EPA, 2000a
, 1994 ) was applied to a POD HED based 

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32 on nervous system effects in rats expos ed to RDX for a subchronic duration . U Fs were applied to
the POD HED values to account for uncertai nties in extrapolating from an animal bioassay to human exposure, the likely existence of a diverse human p opulation 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 PB PK models are considered in Appendix C, Section C. 1 .5. Nervous system effects have been document ed 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 t he
RfD ( C rouse 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 sig ns of neurotoxic ity . 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 convu l s ions that may not have been observed) c ould result in an overestimation
of the incidence of convu l sio ns. A dose

- response analysis of the combined incidence of seizure s and mortality from Crouse et al. (2006) was conducted to evaluate the impact of these as sumptions
, as the true convulsion incidence would likely fall somewhere between the observed convulsion incidence and the combined incidence of convulsions and mortality. T he POD HED of 0.24 mg/kg

- day
for a combined incidence of co nvulsions and mortality 16 was compared to the POD HED of
0.2 8 mg/kg -day for convulsions alone, indicating that the addition of mortality incidence did not have a significant impact. Therefore, the RfD based o n the incidence of convulsions alone does not appear to underestimate the t oxicity associated with RDX. Some uncertainty is also associated with the influence of the m ethod of oral dosing on the magnitude of dose required to induce nervous system effects. As noted in Section 1. 2 .1, gavage

16 BMD = 2.56 mg/kg

- day; BMDL = 0.49 mg/kg

- day (see Appendix D.1.2 for BMD modeling results). The

POD HED value was derived using PBPK modeling (see Section 2.1.2 and discussion of the PBPK models in Appendix C, Section C.1.5).

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administration generally induced conv ulsions in experimental animals at lower doses than did 

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37 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 POD HED values
derived from the Crouse et al. (2006) ( gavage administration) and Levine et al. (1983b) (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 cou ld also have contributed to differences in response (e.g., test article purity and particle size, number and spacing of dose groups, exposure duration, f requency of clinical observations, and thoroughness of the reporting of observations). Although the da tabase 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 i n test material formulation and particle size ( i.e., the increased surface area associated with finely powdered RDX allows for increased absorption) c an 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. C**  **onfidence Statement** A confidence level of high, medium, or low is assigned to the study used to derive the RfD, the overall database, and the RfD itself, as described in Section 4 .3.9.2 of EPA’s Methods for
Derivat i on of Inhalation Reference Concentrations and Application of Inhalation Dosimetry ( U.S. EPA,
1994 ). The overall confidence in this RfD is medium. C onfidence in the principal study ( Crouse et al., 2006 ) is high . The study was well

- cond ucted, utilized 99.99% pure RDX, and had five closely

spaced dose groups that allowed characterization of dose

- response curves for convulsions in the dose range of interest. One limitation identified by study authors was the limited ability of the FOB

to f ully identify neurobehavioral effects at doses ≥8 mg/kg

- day due to the timing of the dosing procedure and timing of the FOB screening. C o nfidence in the database is medium . The database includes three chronic studies in rats and mice; eight subchronic s tudies in rats, mice, dogs, and monkeys; two short

- t erm studies; and four reproductive/ developmental toxicity studies in rats and rabbits (including a two

- g eneration reproductive study). C onfidence in the database is reduced largely b ecause of (1) differences in test material used across studies, and (2) uncertainties in the influence of oral dosing methods. A s discussed in Section 2.1. 7 and Appendix C, Section C. 1 .5, differences in test material formulation and particle size may affect RDX ab s orption and subsequent toxicity
, w hich in turn c ould 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 neuroto x icity 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 p otential human exposures to R DX
, the use of toxicity data from a gavage (bolus dosing) study introduces

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uncertainty in the RfD. R eflecting high confidence in the principal study and medium confidence in 

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32 the database, overall confidence in the RfD is medium.

**2.1.9. Previous IRIS Assessment**  The previous RfD for RDX, posted to the Integrated Risk Information System ( IRIS ) database
in 1 988 , was based on a 2- year rat feeding study by Levine et al. (1983b) . T he no

- observed

- effect
leve l (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 . A n RfD of 3 × 10

− 3 mg/kg

- day was derived following application of an overall UF of 100 (UF A = 10, UF H = 10).



## 2.2. **INHALATIO N REFERENCE CONCENTRATION FOR EFFECTS OTHER**  **THAN CANCER**

The inhalation reference concentration ( RfC

, expressed in units of mg/m 3 ) is defined as an estimate (with uncertainty spanning perhaps an order of magnitude) of a continuous inhalation exposure to the h uman 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 gene rally 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 c hronic inhalation exposure to
RDX , n or do the s tudies support quantitative dose

- response analysis. O f the available human epidemiological studies of RDX ( W est and Stafford, 1997 ; Ma and Li, 1993 ; Hathaway and Buck,
1977 ) , none provid ed data that could be used for dose

- response analysis. T he studies by Ma and Li (1993) of neurobeh avioral effects in Chinese workers and West and Stafford (1997) of hematological abnormalities in ordnance facto ry workers had numerous methodological limitations that preclude their use for quantitative analysis (see Literature Search Strategy | Study Selection and Evaluation) . T h e study by Hathaway and Buck (1977) found no evidence of adverse
health effects in munition plant workers (based on evaluat ion of liver function, renal function, and hematology) , an d therefore does not identify a POD at which there would be an effect from which to
derive a n RfC . M ultiple 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

- r esponse analysis and subsequent derivation of a chronic reference value b ecause 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 i n the Defense Technical Information Center ( D TIC ) dat abase; the study is not public ly available . H owever, the study would

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not have provided useful data on responses to inhaled RDX, as it was limited by small numbers of 

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T herefore, 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 st udies of RDX inhalation absorption to support an inhalation model. Therefore, a PBPK model for inhaled RDX was not developed to support route

- to

- r oute extrapolation from the
RfD.

**2.2.1. P**  **revious IRIS Assessment**

An RfC for RDX was not previously derived under the IRIS Program.



## 2.3. **ORAL SLOPE FACTOR FOR CANCER**

T he oral slope factor (OSF) is a plausible upper bound on the estimate of risk per mg/kg

- d ay of oral exposure. The OSF can be multiplied by an estimate of lifetime exposure (in mg/kg

- d ay) to estimate the lifetime cancer risk.

**2.3.1. A**  **nalysis of Carcinogenicity Data**

As noted in Section 1. 3 .2, there is “suggestive evidence of carcinogenic potential” for RDX.
T h e Guidelin es for Carcinogen Risk Assessment ( U.S. EPA, 2005a ) state:

When there is suggestive evidence, the Agency generally would not attempt a dose

response assessment, as the nature of the data generally would not support one; however, when the evidence includes a well

- conducted study, quantitative analyses may be useful for some purposes, for example, providing a sense of the magnitude and uncertainty of potential risks, ranking potential hazards, or setting research prioritie s

.
In th e case of RDX

, there are well

- conducted studies that tested large numbers of animals at multiple dose levels ( L ish et al., 1984 ; Levine et al., 1983b ) , making the cancer response suitable for
dose- response analysis. C onsidering the data from these studies , a long 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.
T he incidence s of liver and lung tumors in female mic e from the study by Lish et al. (1984) were selected for quantitative dose

- response analysis . T he study by Lish et al. (1984) : (1 ) included comprehensive histopathological examina tion of major organs ; (2 ) contained four dose groups and
a control ; (3 ) used adequate numbers of animals per dose group ( 8 5/sex/group
, with interim sacrifice groups of 10/sex/group at 6 and 12 months) and a sufficient overall exposure duration (2 y ears) ; and (4) adequately reported methods and results (including individual animal data).
F e male 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) . T he updated liver tumor

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incidences from the PWG reanalysis of Lish et al. (1984) were used for quantitative dose

- response 

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- r esponse 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 l iver and lung tumor
incidence s from the Lish et al. (1984) study are summarized in Appendix D, Table D- 23

. The incidence of hepatocellular carcinomas in male F344 rats from the study by Levine et al. (1983b) and the incidence of alveolar/bronchiolar carcinomas in male B6C3F 1 mice from the study by Lish et al. (1984) were also consider ed 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 ( t wo each at 8 and 40 mg/kg

- day, and one in control s)

. The alveolar/bronchiolar carcinomas in
male B6C3F 1 mice showed a positive trend; however, a positive trend was not observed when the
incidence of adenomas and carcinomas was combined. Modeling re s u lts ar e provided in Appendix D, Section D.2 .3 for comparison.

**2.3.2. Dose** 

**- Response Analysis** 

**— Adjustments and Extrapolation Methods** 

The EPA Guidelines for Carcinogen Risk Assessment ( 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 m ode of action ( MOA ) of the carcinogen and the shape of the cancer
dose- r esponse curve . The linear approach is recommended when there are MOA data to indica te that the dose

- r esponse curve is expected to have a linear component below the POD or when the weight

- of

- ev idence 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)

. T h erefore, 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

- r esponse 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 analy sis was not necessary for this study

. Tumor incidence w as modeled using the multistage

- c ancer models in BMDS (versions 2.4 and 2.5) . A standard BMR of
10% ER w as applied to both tumor sites in the mouse

. G iven 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. Th erefore, an analysis that combines

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the results from the mouse liver a nd lung tumor incidence is preferred. T he MS

- COMBO procedure 

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- cancer models to the case with multiple tumors assuming independence between tumor types

. T here is no known biological relationship between
liver and lung tumors in RDX

- exposed mice , and therefore, as noted by the National Research
Council ( NR C , 1994 ) , t his 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 ab sent otherwise. The procedure derives a maxi mum 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 m odeling . A s described in Appendix C , Section C.1.5 , PBPK models for RDX in mice and humans published by S weeney et al. (2012b) were evaluated and further developed by EPA

. C onsideration was given to whether the available toxicokinetic information supported using an internal dose metric derived by PBPK modeling. T h e available mechanistic data (Section 1. 2.5 ) point to some evidence, although not con c lusive, that RDX

- generated metabolites may be implicated in the observed tumorigenicity in the female mouse

. H owever, 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 P B PK model. In this case, as is to be expected from mass balance principles, the PBPK modeling p rovides no further information; the HED obtained from the model

- estimated amount of total RDX metabolites scaled by B W 3/4 was equal to that calculated using administered dose scaled by BW 3/4

. 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
C o nfidence in PBPK M odels for RDX in Appendix C, Section C. 1 .5. T herefore, the PBPK model developed for the mouse was not used, and co nsistent 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 a llometric scaling to achieve toxicological equivalence across species. As discussed in Section 2.1.1, the administered dose in animals wa s converted to a n HED on the basis of (body weight) 3/4 ( U.S. EPA, 1992 ) . T his was accomplished by multiplying administ ered dose by (animal body weight in kg/human body weight in kg) 1/4 ( U.S. EPA, 1992 ) , where the body

w eight 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** 

The lifetime cancer OSF for humans is defined as the slope of the line from the BMR (10%
ER ) at th e BMD L to the estimated control response at zero ( OSF = 0.1/BMDL 10

- HED ) . T his slope, a 95% upper confidence limit o n the true slope, represents a plausible upper bound on the true slope

or r isk per unit dose . The PODs estimated for each mouse tumor site are summarized in Table 2 -7

.

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U sing linear extrapolation from the BMDL 10

- HED , human equivalent OSFs were derived for each 

### 1
2

3 4 5

6 7 8 9 10 11 12 13 14 15 16 17

18
19
20
21
22

23
24 tumor site individually and both sites combined and are listed in Table 2-7

.

**Table 2**  **-7**

**. Model predictions and**  **OSF s for hepatocellular and alveolar/**  **bronchiolar adenomas or carcinomas in female B6C3F 1**  **mice administered RDX in the**  **diet for 2 year**  **s (**  **Lish et al., 1984 )** 

Tumor type Selected model BMR BMD (mg/kg

- d) BMDL (mg/kg

- d) BMD 10

- HED a (mg/kg

- d) POD = BMDL 10

- HED b (mg/kg

- d) OSF c (mg/kg

- d) −1

Hepatocellular adenomas or carcinomas d Multistage 1° 10% ER 64.2 e 32.6 e 9.56 4.89 0.020

Alveolar/bronchiolar adenomas or carcinomas Multistage 1° 10% ER 52.8 27.7 7.92 4.16 0.024

Liver + lung tumors Multistage 1° (MS

- COMBO) 10% ER 29.0 e 17.7 e 4.35 2.66 0.038

a BMD 10

- HED = BMD 10 × (BW a 1/4 /BW h 1/4 ), where BW a = 0.035 kg, and BW h = 70 kg.

b BMDL 10

- HED = BMDL 10 × (BW a 1/4 /BW h 1/4 ), where BW a = 0.035 kg, and BW h = 70 kg

.
c OSF = BMR /BMDL 10

- HED , where BMR = 0.1 (10% ER ).

d Incidence s 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 w ere 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 a nd BMDLs from the remodeling were 64.8 and 32.8 mg/kg -d ay , respectively, for hepatocellular adenomas and carcinomas and 29.1 and 17.7 mg/kg -d ay , respectively, for liver and lung tumors combined. S ee T able D -23 and the subsequent MS

- COMBO results for details.

An OSF was derived from the BMDL 10

- HED based on a significantly increased trend in the
incidence of hepatocellular and alveolar/bronchiolar adenomas or carcinomas in female B6C3F 1 mice ( i.e., the Liver + Lung BMDL 10

- HED from MS

- COMBO) . T he OSF of **0.0 4**  **(mg/kg**

**- day)** 

**− 1**  is calculated by di v iding the BMR (10% ER ) by the Liver + Lung BMDL 10

- HED and represents an upper
bound on cancer risk p e r unit dose associated with a continuous lifetime exposure:

OSF = 0.1 0 ÷ (Liver + Lung) BMDL 10

- HED = 0.1 0 ÷ 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

The slope of the linear extrapolation from the central estimate of exposure associated with 10% extra cancer risk ( BM D 10

- HED ) from the same data s ets is given by :

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Slope of the linear extrapolation from the central estimate

= 0.1 0 ÷ (Liver + Lung) BMD 10

- HED = 0.1 0 ÷ 4.35 mg/kg -day

= 2.3 × 10 −2 (mg/kg

- day)

− 1

= 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), 

### 1
2
3

4
5
6
7

8 9 because above this level
, the fitted dose

- response model better characterizes what is known about the carcinogenicity of RDX.

**2.3.4. U**  **ncertainties in the Derivation of the Oral Slope Factor**

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 fo r 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 Justification Selection of study The cancer bioassay in the rat ( Levine et al., 1983b ) would provide a lower estimate of the OSF Lish et al. (1984 ) as principal oral study to derive the human cancer risk estimate Lish et al. (1984) was a well

- conducted 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 number s of
tumors. Species/sex Use of data sets from the male mouse or male rat would provide a lower OSF OSF based on tumors in female B6C3F 1
mouse It is assumed that a positive tumor response in animal cancer studies indicates that the agent can have carcinogenic potential in humans in the absence of data indicating that animal tumors are not relevant to humans ( U.S. EPA, 2005a ) . As there are no data to inform whether the response in any given experimental animal species or sex would be most relevant for extrapolating to humans, tumor data from the most sensitive species and sex were selected as the basis for the O SF. Other data sets would provide smaller OSF values, and are not considered any more or less relevant to humans than data from the female mouse (i.e., 0.017 per mg/kg

- day based on hepatocellular carcinomas in male F344 rats,

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Consideration and impact on cancer risk value Decision Justification and 0.018 per mg/kg

- day based on alveolar/bronchiolor carcinomas in male B6C3F 1 mice ; see Appendix D, Section D.2). 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 B6C3F 1 mouse Basing th e 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 li ver and lung tumors, independence between the two tumor sites was assumed. This is not likely to produce substantial error in the risk estimates ( NRC, 1994 )

. Selection of dose metric PBPK models are available for the rat, mouse, and human, and using an appropriate internal metric can ↑ accuracy in human extrapolation Mouse liver and lung
tum ors: administered dose used 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. Althought EPA’s preferred approach for extrapolating results from animal studies to humans is toxicokinetic modeling, the uncertainties associated with use of the mouse PB PK model for RDX were considered higher than use of administered dose.
Cross

- species scaling Alternatives could ↓ or ↑ OSF (e.g., 3.5

- fold ↓ [scaling by body weight] or ↑ 2

- fold [scaling by BW 2/3 ]) BW 3/4 scaling (default approach) There are no data to support alternatives. Because the dose metric was not an AUC, BW 3/4 scaling was used to calculate equivalent cumulative exposures for estimating equivalent human risks. While the true human correspondence is unknown, this overall approach is not expected over

- or underestimate human equivalent risks. BMD model uncertainty Alternative models could ↓ or ↑ OSF Use multistage model to derive a BMD and BMDL for combined tumor incidence No biologically based models for RDX are available, and there is no a priori basis for selecting a model other than the multistage. The multistage model has biological support and is the model most consistently used in EPA
cancer assessments ( Gehlhaus et al., 2011 )

. Low

- dose extrapolation approach ↓ cancer risk would be expected with the application of nonlinear extrapolation Linear extrapolation from the POD Where the available information is insufficient to establish the MOA for tumors at a given site, linear extrapolation is recommended because this extrapolation approach is generally considered to be health

- protective ( U.S. EPA, 2005a ) . Because the MOA for RDX

- induced liver and lung tumors has not been established, linear low

- dose extrapolation was applied
, consistent with EPA guidance. Statistical uncertainty at the POD ↓ OSF by 1.6

- fold if BMD used as the POD rather than the BMDL BMDL (default approach for Lower bound is 95% confidence interval (CI) on administered exposure at 10% ER of liver and lung tumors.

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Consideration and impact on cancer risk value Decision Justification calculating plausible upper bound OSF) Sensitive subpopulations ↑ OSF to an unknown extent Considered qualitatively There is little information on whether some subpopulations may be more or less sensitive to the potential carcinogenicity of RDX (i.e., because of variability in toxicokinetics or toxicodynamics for RDX). The mode of carcinogenic action for liver and lung tumors in experimental animals is unknown, and little information is available on RDX metabolites or variation in metabolic rates that could be used to evaluate human variability in cancer response to RDX. 

### 1

2
3
4
5
6
7

8
9
10
11
12
13
14
15
16
17

18
19
20
21 **2.3.5. Previous IRIS Assessment: Oral**  **Slope Factor** The previous cancer assessment for RDX was posted to the IRIS database in 1990 . T he OSF in the previous cancer assessment was based on the bioassay by L ish et al. (1984) and analysis of
data for hepatocellular adenomas or carcinomas in female mice . A n 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

- s pecies extrapolation. In addition, the previo us assessment dropped the high

dose group because the dose was reduced at week 11 to address high mortality

.



## 2.4. **INHALATION UNIT RISK FOR CANCER**
The carcinogenicity assessment provides information on the carcinogenic hazard potential of the substance in questi on , an d quantitative estimates of risk from oral and inhalation exposure may be derived. Q u antitative risk estimates may be derived from the application of a low

- dose extrapolation procedure

. I f derived, the inhalation unit risk (IUR) is a plausible uppe r bound on the estimate of risk per μg/m 3 air breathed.
An I UR 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 toxico kinetic studies of RDX inhalation absorption to support an inhalation model.
Therefore

, a PBPK model for inhaled RDX was not developed to support route

- to

- route extrapolation of an IUR from the OSF.



## 2.5. **APPLICATION OF AGE**

**- DEPENDENT ADJUSTMENT FACTORS** 

As disc ussed in the Supplemental Guidance for Assessing Susceptibility from Early

- Life Exposure to Carcinogens ( U .S. EPA, 2005b ) , either default or chemical

- specific age

- dependent adjustment factors (ADAFs) are recommended to account for early

- life exposure to carcinogens that act through a mutagenic MOA . B e cause no chemical

- specific data on lifestage susceptibility for

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RDX carcin ogenicity are available, and because the MOA for RDX carcinogenicity is not known (see 

### 1
2

3 Section 1. 2 .5), application of ADAFs is not recommended

.

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# REFERENCES

Angerhofer, R; Davis, G; Balezewski, L. (1986). Teratological assessment of Trinitro - RDX in rats. 

### 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 (75

- 51

- 0573

- 86). Aberdeen Proving Ground, MD: U.S. Army Environmental Hygiene Agency. ATSDR (Agency for Toxic Substances and Disease Registry). (1992). Health assessment for Nebraska Army Ordnance Plant (former), Mead, Saunders County, Nebraska, Region 7. CERCLIS No. NE6211890011. (PB92177815). Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. ATSDR (Agency for Toxic Substances and Disease Registry). (1993). Health Assessment for Milan Army Ammunition Plant, Milan, Carroll and Gibson Counties, Tennessee, Region 4. CERCLIS No. TN0210020582. Atlanta, GA: U.S. Department of Health and Human Services, Pu b lic Health Service. ATSDR (Agency for Toxic Substances and Disease Registry). (1996). Symptom and disease prevalence with biomarkers health study, Cornhuske r Army Ammunition Plant Hall County, Nebraska. (ATSDRHS9672). Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=PB96187760 ATSDR (Agency for Toxic Substances and Disease Registry). (1999). Public health assessment for Iowa Army Ammunition Plant, Middletown, Des Moines County, Iowa. CERCLIS No. IA7213820445. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. ATSDR (Agency for Toxic Substances and Disease Registry). (2012). Toxicological profile for RDX (Update) [ATSDR Tox Profile]. (NTIS/10590213). Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. Bailey, SA; Zid ell, RH; Perry, RW. (2004). Relationships between organ weight and body/brain weight in the rat: What is the best analytical endpoint? Toxicol Pathol 32: 448

- 466. http://dx.doi.org/10.1080/01926230490465874 Bannon, DI. (2006). Toxicology study no. 87

- XE

- 059F

- 06: Biomarkers of RDX exposure in breath of swine, August - September 2006. Aberdeen Proving Ground, MD: U.S. Army Center for Health Promotion and Preventive Medicine. Bannon, DI; Dillman, JF; Hable, MA; Phillips, CS; Perkins, EJ. (2009a). Global gene expression in rat brain and liver after oral exposure to the explosive hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX). Chem Res Toxicol 22: 620

- 6 25. http://dx.doi.org/10.1021/tx800444k Bannon, DI; Dillman, JF; Perkins, EJ; Bao, W; Wolfinger, RD; Chu, T; Phillips, CS. (2006). Acute RDX exposure and gene expression in the rat brain [Abstract]. Oxford, United King dom: Oxford University Press. http://www.toxicology.org/pubs/docs/Tox/2006Tox.pdf Bannon, DI; Johnson, M; Williams, L; Adams, V; Perkins, E; Gust, K; Gong, P. (2009b). RDX and miRNA expression in B6C3F1 mice [Letter]. Environ Health Perspect 117: A98. http://dx.doi.org/10.1289/ehp.0800276 Barsotti, M; Crotti, G. (1949). [Attacchi epileptici come manifestazione di intossicazione professionale da trimetilen

- trinitroamina (T4)]. Med Lav 40: 107

- 112. Bart, JC; Judd, LL; Hoffman, KE; Wilkins, AM; Kusterbeck, AW. (1997). Application of a portable immunosensor to detect the explosives TNT and RDX in groundwater samples. Environ Sci Technol 31: 1505

- 1 511. http://dx.doi.org/10.1021/es960777l

This document is a draft for review purposes only and does not constitute Agency policy. R-1 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Bergeret, M; Khrestchatisky, M; Tremblay, E; Bernard, A; Gregoire, A; Chany, C. (1998). GABA 

### 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 modulates cytotoxicity of immunocompetent cells expressing GABAA receptor subunits. Biomed Pharmacother 52: 214

- 2 19. http: //dx.doi.org/10.1016/S0753

- 3322(98)80019 -X Berry, AP; Arbuckle, JB; Nicol, J. (1983). Cyclonite poisoning in a dog. Vet Rec 113: 449. http://dx.doi.org/10.1136/vr.113.19.449 Best, EP; Sprecher, SL; Larson, SL; Fredrickson, HL; Bader, DF. (1999a). Environmental behavior of explosives in groundwater from the Milan Army Ammunition Plant in aquatic and wetland plant treatments. Removal, mass balances and fate in groundwater of TNT and RDX. Chemosphere 38: 3383

- 3 396. http://dx.doi.org/10.1016/S0045

- 6535(98)00550 -5 Best, EP; Sprecher, SL; Larson, SL; Fredrickson, HL; Bader, DF. (1999b). Environmental behavior of explosives in groundwater from the Milan Army Ammunition Plant in aquatic and wetland plant treatments. Uptake and fate of TNT and RDX in plants. Chemosphere 39: 2057

- 2 072. http://dx.doi.or g/10.1016/S0045

- 6535(99)00117 -4 Bhat, R; Axtell, R; Mitra, A; Miranda, M; Lock, C; Tsien, RW; Steinman, L. (2010). Inhibitory role for GABA in autoimmune infl ammation. PNAS 107: 2580

- 2585. http://dx.doi.org/10.1073/pnas.0915139107 Bhushan, B; Trott, S; Spain, JC ; Halasz, A; Paquet, L; Hawari, J. (2003). Biotransformation of hexahydro- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX) by a rabbit liver cytochrome P450: Insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22. Appl Environ Microbiol 69: 1347

- 1 351. http://dx.doi.org/10.1128/AEM.69.3.1347

- 1351.2003 Bingham, E; Cohrssen, B; Powell, CH

. (2001). Patty's toxicology. In E Bingham; B Cohrssen; CH Powell (Eds.), Patty's toxicology vol 6: Ketones, alcohols, esters, epoxy compounds, organic peroxides (5 ed.). Hoboken, NJ: John Wiley & Sons. http://dx.doi.org/10.1002/0471125474 Bishop, RW; Kennedy, JL; Podolak, GE; Ryea, JL, Jr. (1988). A field evaluation of air sampling methods for TNT and RDX . Am Ind Hyg Assoc J 49: 635

- 638. http://dx.doi.org/10.1080/15298668891380376 Bloomquist, JR. (199 2). Intrinsic lethality of chloride

- channel

- directed insecticides and convulsants in mammals. Toxicol Lett 60: 289

- 298. http://dx.doi.org/10.1016/0378

- 4274(92)90287 -T Boileau, J; Fauquignon, C; Hueber, B; Meyer, H. (2003). Explosives. In Ullmann's Encyclopedia of Industrial Chemistry. Weinheim, Germany: Wiley

- VCH Verlag Gmbh & Co. Kga a. http://dx.doi.org/10.1002/14356007.a10_143 Brar, R; Singh, J; Kaur, T; Arora, S; Singh, A. (2014 ). Role of GABAergic activity of sodium valproate against ischemia -reperfusion- induced acute kidney injury in rats. Naunyn Schmiedebergs Arch Pharmacol 387: 143

- 1 51. http://dx.doi.org/10.1007/s00210

- 013

- 0928 -2 Bruchim, Y; Saragusty, J; Weisman, A; Sternheim, D. (2005). Cyclonite (RDX) intoxication in a police working dog. Vet Rec 157: 354

- 356. http://dx.doi.org/10.1136/vr.157.12.354 Burdette, LJ; Cook, LL; Dyer, RS. (1988). Convulsant properties of cyclo trimethylenetrinitramine (RDX): Spontaneous, audiogenic, and amygdaloid kindled seizure activity. Toxicol Appl Pharmacol 92: 436

- 4 44. http://dx.doi.org/10.1016/0041

- 008X(88)90183 -4 CDC (Centers for Disease Control and Prevention). (2004). The health consequences of smoking: A report of the Surgeon General. Washington, DC: U.S. Department of Health a nd Human Services. http://www.cdc.gov/tobacco/data_statistics/sgr/2004/index.htm Chan dra, M; Riley, MG; Johnson, DE. (1992). Spontaneous neoplasms in aged Sprague

- Dawley rats. Arch Toxicol 66: 496

- 502. http://dx.doi.org/10.1007/BF01970675 Cholakis, JM; Wong, LCK; Van Goethem, DL; Minor, J; Short, R; Sprinz, H; Ellis, HV, III. (1980). Mammalian toxicological evaluation of RDX. (DAMD17

- 78 -C- 8027). Kansas City, MO: Midwest Research Institute. Cowin, PA; Gold, E; Aleksova, J; O'Bryan, MK; Foster, PMD; Scott, HS; Risbridger, GP. (2010). Vinclozolin exposure in utero induces postpubertal prostatitis and reduces sperm

This document is a draft for review purposes only and does not constitute Agency policy. R-2 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

production via a reversible hormone

- regulated mechanism. Endocrinology 151: 783

- 792. 

### 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 http://dx.doi.org/10.1210/en.2009

- 0982 Craig, EA; Yan, Z; Zhao, QJ. (2014). The relationship between chemical

- induced kidney weight increases and kidney histopathology in rats. J Appl Toxicol 35: 729

- 736. http://dx.doi.org/10.1002/jat.3036 Crouse, LCB; Michie, MW; Major, M; Johnson, MS; Lee, RB; Paulus, HI. (2006). Subchronic oral toxicity of RDX in rats. (Toxic ology Study No. 85

- XC

- 5131

- 03). Aberdeen Proving Ground, MD: U.S. Army Center for Health Promotion and Preventive Medicine. Crouse, LCB; Michie, MW; Major, MA; Leach, GJ; Reddy, G. (2008). Oral bioavailability of cyclotrimethylenetrinitramine (RDX) from contaminated site soils in rats. Int J Toxicol 27: 317

- 3 22. http://dx.doi.org/10.1080/10915810802366885 Davies, JOJ; Roberts, DM; Hittarage, A; Buckley, NA. (2007). Oral C

- 4 plastic explosive in humans - A case series. Clin Toxicol 45: 454

- 457. http://dx.doi.org/10.1080/15563650601118044 Deng, Y; Ai, J; Guan, X; Wang, Z; Yan, B; Zhang, D; Liu, C; Wilbanks, MS; Escalon, BL; Meyers, SA; Yang, MQ; Perkins, EJ. (2014). MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity. BMC Genomics 15 Suppl 11: S1. http://dx.doi.org/10.1186/1471

- 2164

- 15

- S11

- S1 Erdo, SL. (1985). Peripheral GABAergic mechanisms. Trends Pharmacol Sci 6: 205

- 208. http://dx.doi.org/10.1016/0165

- 6147(85)90096 -3 Erdö, SL; Dobó, E; Párducz, A; Wolf f, JR. (1991). Releasable GABA in tubular epithelium of rat kidney. Experientia 47: 227

- 229. http://dx.doi.org/10.1007/BF01958142 Fellows, RJ; Driver, CR; Cataldo, DA; Harvey, SD. (2006). Bioavailability of hexahydro

- 1,3,5

- trinitro

1,3,5

- triazine (RDX) to the prairie vole (Microtus ochrogaster). Environ Toxicol Chem 25: 1881

- 1 886. http://dx.doi.org/10.1897/05

- 446R1.1 Fowler, CJ; Griffiths, D; de Groat, WC. (2008). The neural control of micturition [Review]. Na t Rev Neurosci 9: 453

- 466. http://dx.doi.org/10.1038/nrn2401 Funk, SB; Roberts, DJ; Crawford, DL; Crawford, RL. (1993). Initial

- phase optimization for bioremediation of munition compound

- contaminated soils. Appl Environ Microbiol 59: 2171

- 2 177. Gadagbui, B; Patterson, J; Rak, A; Kutzman, RS; Reddy, G; Johnson, MS. (2012). Development of a relative source contribution factor for drinking water criteria: The case of hexahydro

- 1,3,5

- trinitro

- 1 ,3,5

- triazine (RDX). Hum Ecol Risk Assess 18: 338

- 354. http://dx.doi.org/10.1080/10807039.2012.650588 Galanopoulou, AS. (2008). GABA(A) receptors in normal development and s eizures: friends or foes? Current Neuropharmacology 6: 1

- 20. http://dx.doi.org/10.2174/157015908783769653 Garcia

- Reyero, N; Habib, T; Pirooznia, M; Gust, KA; Gong, P; Warner, C; Wilbanks, M; Perkins, E. (2011). Conserved toxic responses across divergent phylogenetic lineages: A meta

- analysis of the neurotoxic effects of RDX among multiple species usin g toxicogenomics. Ecotoxicology 20: 580

- 5 94. http://dx.doi.org/10.1007/s10646

- 011

- 0623 -3 Gehlhaus, MW, III; Gift, JS; Hogan, KA; Kopylev, L; Schlosser, PM; Kadry, A,

- R. (2011). Approaches to cancer assessment in EPA's Integrated Risk Information System [Review]. Toxicol Appl Pharmacol 254: 170

- 1 80. http://dx.doi.org/10.1016/j.taap.2010.10.019 Gerkin, RC; Clem, RL; Shruti, S; Kass, RE; Barth, AL. (2010). Cortical up state activity is enhanced af

ter seizures: A quantitative analysis. J Clin Neurophysiol 27: 425

- 432. http://dx.doi.org/10.1097/WNP.0b013e3181fdf8bd Gilbert, ME. (1994). The phenomenology of limbic kindling [Review]. Toxicol Ind Health 10: 343

- 358. Godejohann, M; Preiss, A; Levsen, K; Wollin, K,

- M; Mügge, C. (1998). Determination of polar organic pollutants in aqueous samples of former ammunition sites in lower Saxony by means of

This document is a draft for review purposes only and does not constitute Agency policy. R-3 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

HPLC/photodiode array detection (HPLC/PDA) and proton nuclear magnetic resonance 

### 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 spectroscopy (1H

- NMR). Acta Hydrochim Hydrobiol 26: 330

- 337. http://dx.doi.org/10.1002/(SICI)1521

- 401X(199811)26:6<330::AID

- AHEH330>3.0.CO;2 -T Gogal, OM, Jr; Johnson, MS; Larsen, CT; Prater, MR; Duncan, RB; Ward, DL; Lee, RB; Salice, CJ; Jortner, B; Holladay, SD. (2003). Dietary oral exposure to l,3,5

- trinitro

- 1,3,5

- triazine in the

n orthern bobwhite (Colinus virginianus). Environ Toxicol Chem 22: 381

- 387. http://dx.doi.org/10.1002/etc.5620220220 Goldberg, DJ; Green, ST; Nathwani, D; McMenamin, J; Hamlet, N; Kennedy, DH. (1992). RDX intoxication causing seizures and a widespread petechial rash mimicking meningococcaemia. J R Soc Med 85: 181. Goodman, LS; Gilman, AG; Hardman, JG; Limbird, LE. (1996). Goodman & Gilman's the phamacological basis of therapeutics. In JG Hardman; LE Limbird (Eds.), Goodman & Gilman's the Phamacological Basis of T herapeutics (9th ed. ed.). New York: McGraw -Hill. Guo, L; Xu, H; Chen, Y; Chang, Y. (1985). Distribution and metabolism of tritium

- labeled hexogen in white m ice. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 3: 335

- 339. Guyatt, GH; Oxman, AD; Kunz, R; Vist, GE; Falck

- Ytter, Y; Schünemann, HJ. (2008a). What is "qu ality of evidence" and why is it important to clinicians? [Review]. BMJ 336: 995

- 998. http://dx.doi.org/10.1136/bmj.39490.551019.BE Guyatt, GH; Oxman, AD; Vist, GE; Kunz, R; Falck

- Ytter, Y; Alonso

- Coello, P; Schünemann, HJ. (2008b). GRADE: An emerging consensus on rating quality of evidence and strength of recommendations. BMJ 336: 924

- 9 26. http://dx.doi.org/10.1136/bmj.39489.470347.AD Hansch, C; Leo, A; Hoekman, D. (1995). Exploring QSAR: Hydroph obic, electronic, and steric constants. In Exploring QSAR: Hydrophobic, Electronic, and Steric Constants. Washington, DC: American Chemical Society. Harada, T; Enomoto, A; Boorman, GA; Maronpot, RR. (1999). Liver and gallbladder. In R Maronpot; G Boorman; B Gaul (Eds.), Pathology of the Mouse (pp. 119

- 183). Vienna, IL: Cache River Press. http:/ /www.cacheriverpress.com/books/pathmouse.htm Harrell- Bruder, B; Hutchins, KL. (1995). Seizures caused by ingestion of composition C

- 4. Ann Emerg Med 26: 746

- 74 8. http://dx.doi.org/10.1016/S0196

- 0644(95)70049 -8 Hart, ER. (1974). Subacute toxicity of RDX and TNT in dogs. Final report. (A717530). Kensington, MD: Litton Bionetics, Inc. https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=ADA035717 Hart, ER. (1976). Two

- year chronic toxicity study in rats. (N00014

- 73 -C- 0162). Kensington, MD: Litton Bionetics, Inc. https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=ADA040161 Harvey, SD; Fellows, RJ; Cataldo, DA; Bean, RM. (1991). Fate of the explosive hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX) in soil and bioaccumulation in bush bean hydroponic plants

. Environ Toxicol Chem 10: 845

- 8 55. http://dx.doi.org/10.1002/etc.5620100701 Haseman, JK; Huff, JE; Rao, GN; Arnold, JE; Boorman, GA; McConnell, EE. (1985). Neoplasms observed in untreated and corn oil gavage control groups of F344/N rats and (C57BL/6N X C3H/HeN)F1 (B6C3F1) mice. J Natl Cancer Inst 75: 975

- 9 84. http://dx.doi.org/10.1093/jnci/75.5.975 Haskell Laboratories. (1942). Initial submission: Toxicity of RDX (cyclotrimethylenetrinitramine) with cove r letter dated 101592 [TSCA Submission]. (88920009850). Wilmington, DE: DuPont Chemical Company. https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=OTS0571501 Hathaway, JA; Buck, CR. (1977). Absence of health hazards associated with RDX manufacture and use. J Occup Med 19: 269

- 272.

This document is a draft for review purposes only and does not constitute Agency policy. R-4 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Hess

- Ruth, A; Crous e, L; Roszell, L. (2007). RDX pilot development neurotoxicity test in rats. 

### 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 (Toxicology Study No. 85

- XC

- 064Y

- 07). Aberdeen Proving Ground: U.S. Army Center for Health Promotion and Preventive Medicine. Hett, DA; Fichtner, K. (2002). A plastic explosive by mouth. J R Soc Med 95: 251

- 252. http://dx.doi.org/10.1258/jrsm.95.5.251 HEW (U.S. Department of Health, Education and Welfare). (1964). Smoking and health: Report of the advisory committee to the surgeon general of the public health service. Washington, DC: U .S . Department of Health, Education, and Welfare. http://profiles.nlm.nih.gov/ps/retrieve/ResourceMetadata/NNBBMQ Hill, AB. (1965). The environment and disease: Association or causation? Proc R Soc Med 58: 295

- 300. Hollander, AI; Colbach, EM

. (1969). Composition C

- 4 induced seizures: A report of five cases. Mil Med 134: 1529

- 1530. IARC (International Agency for Research on Cancer). (2006). IARC monographs on the evaluation of carcinogenic risks to humans: Preamble. Lyon, France: World Health Organization. http://monographs.iarc.fr/ENG/Preamble/ Igawa, Y; Mattiasson, A; Andersson, K,

- E. (1993). Effects of GABA

- receptor stimulation and blockade on micturition in normal rats and rats with bladder outflow obstruction. J Urol 150: 537

- 5 42. IOM (Institute of Medicine). (2008). Improving the presumptive disability decision

- making process for veterans. In JM Samet; CC Bodurow (Eds.). Washington, DC: National Academies Press. http://dx.doi.org/10.17226/11908 Jefferys, JGR; Jiruska, P; de Curtis, M; Avoli, M. (2012). Limbic network synchronization and temporal lobe epilepsy. In JL Noebel; HH Jasper (Eds.), Jasper's Basic Mechanisms of the Epilepsies (4th ed ed.). New York, NY: Oxford University Press. http://www.ncbi.nlm.nih.gov/books/NBK98158/ Johnson, MS. (2015a). Memorandum for National Center for Environmental Assessment (8101R/Dr. Louis D'Amico), U.S. EPA - Office of Research and Development, 1200 Pennsylvania Ave., NW, Washington, DC 20460. Subject: Additional data from the oral subchronic toxicity of RDX in rats, 2006. (Toxicology study no. 85

- XC

- 5 131

- 03), U.S. Army Center for Health Promotion and Pr ev entative Medicine, Aberdeen Proving Ground, Maryland; Incidence of seizure relative to mortality events. (MCHB

- IP

- T ). Aberdeen Proving Ground, MD: Department of the Army, US Army Institute of Public Health. Johnson, MS. (2015b). Memorandum for Office of Research and Development (NCEA/Dr. Louis DAmico), National Center for Environmental Assessment, U.S. Environmental Protection Agency, Two Potomac Yard, 2733 South Crystal Drive, Arlington, VA 22202. Subject: Lack of evidence for GABAa receptor

- m ediated MOA of RDX

- induced prostatitis. Aberdeen Proving Ground, MD: Department of the Army, U.S. Army Institute of Public Health. Johnson, MS; Quinn, MJ; Bazar, MA; Gust, KA; Escalon, BL; Perkins, EJ. (2007). Subacute toxicity of oral 2,6

- dinitrotoluene and 1,3,5

- trinitro

- 1,3,5

- triazine (RDX) exposure to the northern bobwhi t e (Colinus virginianus). Environ Toxicol Chem 26: 1481

- 1487. http://dx.doi.org/10.1897/06

- 525.1 Kalueff, AV. (2007). Mapping convulsants' binding to the GABA

- A receptor chloride ionophore: A proposed model for channel binding sites. Neurochem Int 50: 61

- 68. http://dx.doi.org/10.1016/j.neuint.2006.07.004 Kaplan, AS; Berghout, CF; Peczenik, A. (1965). Human intoxication from RDX. Arch Environ Health 10: 877

- 883. Kasuske, L; Schofer, JM; Hasegawa, K. (2009). Two marines with generalized seizure activity. J Emerg Nurs 35: 542

- 543. http://dx.doi.org/10.10 16/j.jen.2008.05.001

This document is a draft for review purposes only and does not constitute Agency policy. R-5 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Ketel, WB; Hughes, JR. (1972). Toxic encephalopathy with seizures secondary to ingestion of 

### 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 composition C

- 4. A clinical and electroencepha lographic study. Neurology 22: 871

- 876. Knepshield, JH; Stone, WJ. (1972). Toxic effects following ingestion of C

- 4 plastic explosive. In W Keup (Ed.), Drug Abuse: Current Concepts and Research (pp. 296

- 300). Springfield, IL: Charles C. Thomas. Kontani, H; Kawabata, Y; Koshiura, R. (1987). In vivo effects of gamma

- aminobutyric acid on the urinary bladder contraction accompanying micturition. Jpn J Pharmacol 45: 45

- 53. http://dx.doi.org/10.1254/jjp.45.45 Krishnan, K; Crouse, LCB; Bazar, MA; Major, MA; Reddy, G. (2009). Physiologically based pharmacokinetic modeling of cyclotrimethylenetrinitramine in male rats. J Appl Toxicol 29: 629

- 6 37. http://dx.doi.org/10.1002/jat.1455 Küçükardali, Y; Acar, HV; Özkan, S; Nalbant, S; Yazgan, Y; Atasoyu, EM; Keskin, O; Naz, A; Akyatan, N; Gökbe n, M; Danaci, M. (2003). Accidental oral poisoning caused by RDX (cyclonite): A report of 5 cases. J Intensive Care Med 18: 42

- 46. http://dx.doi.org/10.1177/0885066602239123 Levine, BS; Furedi, EM; Gordon, DE; Barkley, JJ; Lish, PM. (1990). Toxic interactions of the munitions compounds TNT and RDX in F344 rats. Fundam Appl Toxicol 15: 373

- 380. http://dx.doi.org/10.1016/0272

- 0590(90)90062 -O Levine, BS; Furedi, EM; Gordon, DE; Burns, JM; Lish, PM. (1981a). Thirteen week oral (diet) toxicity study of trinitrotoluene (TNT), hexahydro

- 1, 3, 5 -trinitro- 1, 3, 5

- triazine (RDX) and TNT/ R DX mixtures in the Fischer 344 rat. Final report. (ADA108447. DAMD17

- 79 -C- 9120. DAMD17

- 7 9 -C- 9161). Chicago, IL: IIT Research Institute. http://oai.dtic.mil /oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA108447 Levine, BS; Furedi, EM; Gordon, DE; Burns, JM; Lish, PM. (1981b). Thirteen week toxicity study of hexahydro- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX) in Fischer 344 rats. Toxicol Lett 8: 241

- 245. http://dx.doi.org/10.1016/0378

- 4274(81)90108 -9 Levine, BS; Furedi, EM; Rac, VS; Gordon, DE; Lish, PM. (1983a). Determination of the chronic mammalian toxicological effects of RDX (twenty

- f our month chronic toxicity/carcinogenicity study of hexahydro

- 1 ,3,5 -trinitro- 1,3,5

- triazine (RDX) in the Fischer 344 rat): Final report

-- P hase V. Volume 1. (ADA160774. IITRI

- L6121

- 6. DAMD17

- 79 -C- 9 161). Chicago, IL: IIT Research Institute. http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA16077 4 Levine, BS; Lish, PM; Furedi, EM; Rac, VS; Sagartz, JM. (1983b). Determination of the chronic mammalian toxicological effects of RDX (twenty

- four month chro nic toxicity/carcinogenicity study of hexahydro

- 1 ,3,5 -trinitro- 1,3,5

- triazine (RDX) in the Fischer 344 rat): Final report

-- p hase V. Chicago, IL: IIT Research Institute. Levine, BS; Lish, PM; Furedi, EM; Rac, VS; Sagartz, JM. (1983c). Determination of the chronic mammalian toxicological effects of RDX (twenty

- f our month chronic toxicity/carcinogenicity study of hexahydro

- 1 ,3,5 -trinitro- 1,3,5

- triazine (RDX) in the Fischer 344 rat): Final report

-- p hase V. Volume 3. Chicago, IL: IIT Research Institute. Lide, DR. (2005). Physical constants of organic compounds. In DR Lide (Ed.), CRC Handbook of Chemistry and Physics: A Ready

- Reference Book of Chemical and Physical D ata (86 ed.). Boca Raton, FL: CRC Press. Lish, PM; Levine, BS; Furedi, EM; Sagartz, JM; Rac, VS. (1984). Determination of the chronic mammalian toxicological effects of RDX: Twenty

- four month chronic toxicity/carcinogenicity study of hexahydro

- 1 ,3,5 -trinitro- 1,3,5

- triazine (RDX) in the B6C3F1 hybrid mouse (Volumes1

- 3 ). (ADA181766. DAMD17

- 79 -C- 9161). Fort Detrick, Frederick, MD: U.S. Army Medical Research and Development Command. http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA181766

This document is a draft for review purposes only and does not constitute Agency policy. R-6 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Ma, B; Li, H. (1993). Neurobehavioral effects of hexogen on exposed workers. Gongye Weisheng yu 

### 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 Zhiyebin 19: 20

- 23. MacPhail, RC; Walker, QD; Cook, L. (1985). Neurotoxicology of cyclotrimethylenetrinitramine (RDX). Final report. (ADA168266). Research Triangle Park, NC: U.S. Environmental Protection Agency, Health Effects Research Laboratory, Neurotoxicology Division. https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=ADA168266 Maita, K; Hirano, M; Harada, T; Mitsumori, K; Yoshida, A; Takahashi, K; Nakashima , N; Kitazawa, T; Enomoto, A; Inui, K; Shirasu, Y. (1987). Spontaneous tumors in F344/DuCrj rats from 12 control groups of chronic and oncogenicity studies. J Toxicol Sci 12: 111

- 126. Major, MA; Reddy, G; Berge, MA; Patzer, SS; Li, AC; Gohdes, M. (2007). Metabolite profiling of [14C]hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX) in Yucatan miniature pigs. J Toxicol Environ Health A 70: 1191

- 1 202. http://dx.doi.org/10.1080/15287390701252717 Martin, D; Hart, E. (1974). Subacute toxicity of RDX and TNT in monkeys (pp. 1

- 216). (ADA044650). Kensington, MD: Litton Bionetics, Inc. https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=ADA044650 Mayerhofer, A; Höhne

- Zell, B; Gamel

- Didelon, K; Jung, H; Redecker, P; Grube, D; Urbanski, HF; Gasnier, B; Fritschy, J,

- M; Gratzl, M. (2001). Gamma

- aminobutyric acid (GABA): A para and/or autocrine hormone in the pituitary. FASEB J 15: 1089

- 1091. http://dx.doi.org/10.1096/fj.00

- 0546fje McConnell, EE; Solleveld, HA; Swenberg, JA; Boorman, GA. (1986). Guidelines for c ombining neoplasms for evaluation of rodent carcinogenesis studies. J Natl Cancer Inst 76: 283

- 289. http://dx.doi.org/10.1093/jnci/76.2.283 McCormick, NG; Cornell, JH; Kaplan, AM. (1981). Biodegradation of hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine. Appl Environ Microbiol 42: 817

- 823. McDonough, JH, Jr; Shih, T, -M. (1997). Neuropharmacological mechanisms of nerve agent

- induced seizure and neuropathology [Review]. Neurosci Biobehav Rev 21: 559

- 579. h ttp://dx.doi.org/10.1016/S0149

- 7634(96)00050 -4 McFarland, CA; Quinn, MJ, Jr; Bazar, MA; Talent, LG; Johnson, MS. (2009). Toxic effects of oral hexahydro- 1,3,5

- trinitro

- 1,3,5

- triazine in the Western fence lizard (Sceloporus occidentalis). Environ Toxicol Chem 28: 1043

- 1 050. http://dx.doi.org/10.1897/08

- 419.1 McLanahan, ED; El

- Masri, HA; Sweeney, LM; Kopylev, LY; Clewell, HJ; Wambaugh, JF; Schlosser, PM. (2012). Physiologically based pharmacokinetic model use in risk assessment

-- Why being published is not enough. Toxic o l Sci 126: 5

- 15. http://dx.doi.org/10.1093/toxsci/kfr295 McNamara, BP; Averill, HP; Owens, EJ; Callahan, JF; Fairchild, DG; Ciuchta, HP; Rengstorff, RH; Biskup, RK. (1974). The toxicology of cyclotrimethylenetrinitramine (RDX) and cyclotetramethylenetetranitramine (HMX) solutions in dimethylsulfoxide (DMSO), cyclohexanone, and acetone (pp. 112). (AD780010. C BRNIAC

- CB

- 148341. EB

- TR

- 73040. CPIAC

- 1 975

- 0806). Aberdeen Proving Ground, MD: Edgewood Arsenal, Dept. of the Army. http://www.dtic.mil/dtic/tr/fulltext/u2/780010.pdf Meldrum, BS; Rogawski, MA. (2007). Molecular targets for antiepileptic drug development [Review]. Neurotherapeutics 4: 18

- 61. http://dx.doi.org/10.1016/j.nurt.2006.11.010 Merrill, SL. (1968). Ingestion of an explosive material, composition C

- 4: A report of two cases. USARV Med Bull 40: 5

- 11. Möhler, H. (2006). GABA(A) receptor diversity and pharmacology [Review]. Cell Tissue Res 326: 505

- 516. http://dx.doi.org/10.1007/s00441

- 006

- 0284 -3 MTA (Midwest Teratology Association). (1992). Historical control project 1988

- 1992: Skeletal findings on Sprague

- Dawley CD rats and New Zealand white rabbits. Natural delivery and postnatal data: Sprague

- D awley CD rats. Ann Arbor, MI: Parke

- Davis Pharmaceutical Research.

This document is a draft for review purposes only and does not constitute Agency policy. R-7 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Musick, TJ; Berge, MA; Patzer, SS; Tilch, KR. (2010). Absorption, distribution, metabolism, and 

### 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 excretion of 14C

- RDX following oral administration to minipigs (DAAD05

- 02 -P- 2319). (ADA526472). Madison, WI: Covance Laboratories Inc. http://www.dtic.mil/cgi

bin/GetTRDoc?AD=ADA526472 Napoleone, P; Bronzetti, E; Cavallotti, C; Ament a, F. (1990). Predominant epithelial localization of type A gamma

- aminobutyric acid receptor sites within rat seminal vesicles and prostate glands. Pharmacology 41: 49

- 5 6. http://dx.doi.org/10.1159/000138698 NLM (National Institutes of Health, National Library of Medicine). (2011). ChemIDPlus [Database]. Retrieved from http://chem.sis.nlm.nih.gov/chemidplus/chemidheavy.jsp NRC (National Research Council). (1983). Risk assessment in the federal government: Managing the process. In Risk Assessment in the Federal Government: Managing the Process. Washington, DC: National Academy Press. http://dx.doi .org/10.1080/00139157.1983.9931232 NRC (National Research Council). (1994). Science and judgment in risk assessment (pp. 1

- 652). Washington, DC: National Academy Press. http://www.nap.edu/openbook.php?isbn=030904894X NRC (National Research Council). (2009). Science and decisions: Advancing risk assessment. Washington, DC: National Academy Press. http://www.nap.edu/catalog/12209.html NRC (National Research Council). (2011). Review of the Environmental Protection Agency's draft IRIS assessme nt of formaldehyde (pp. 194). Washington, DC: National Academies Press. http://www.nap.edu/catalog/13142.html NTP (National Toxicology Program). (2009). NTP historical controls report : All routes and vehicles : Rats [NTP]. http://ntp.niehs.nih.gov/ntp/historical_controls/ntp2000_2009/2009

- 05

- 14

- hist -ratsallroutes.pdf Ong, J; Kerr, DI. (1990). GABA

- receptors in peripheral tissues [Review]. Life Sci 46: 1489

- 1501. http://dx.doi.org/10.1016/0024

- 3205(90)90421 -M Özhan, G; Topuz, S; Alpertunga, B. (20 03). Determination of cyclonite (RDX) in human plasma by high

- performance liquid chromatography. Farmaco 58: 445

- 448. http://dx.doi.org/10.1016/S0014

- 827X(03)00069 -7 Pan, X; San Francisco, MJ; Lee, C; Ochoa, KM; Xu, X; Liu, J; Zhang, B; Cox, SB; Cobb, GP. (2007a). Examination of the mutagenicity of RDX and its N

- n itroso metabolites using the Salmonella reverse mutation assay. Mutat Res Genet Toxicol Environ Mutagen 629: 64

- 6 9. http://dx.doi.org/10.1016/j.mrgentox.2007.01.006 Pan, X; Zhang, B; Smith, JN; San Francisco, M; Anderson, TA; Cobb , GP. (2007b). N

- Nitroso compounds produced in deer mouse (Peromyscus maniculatus) GI tracts following hexahydro- 1 ,3,5

- trinitro

- 1,3,5

- triazine (RDX) exposure. Chemosphere 67: 1164

- 1170. http://dx.doi.org/10.1016/j.chemosphere.2006.10.077 Parker, G. (2001). Attachment 1: Pathology Working Group. Chairperson's report. Reevaluation: Twenty

- four month chronic toxicity/carcinogenicity study of hexahydro

- 1,3,5

- trinitro

1,3.5

- t riazine (RDX) in the B6C3F1 hybrid mouse. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Parker, GA; Reddy, G; Major, MA. (2006). Reevaluation of a twenty

- four

- month chronic toxicity/carcinogenicity study of hexahydro

- 1,3,5 -trinitro- 1,3,5

- triazine (RDX) in t he B6C3F1 hybrid mouse. Int J Toxicol 25: 373

- 3 78. http://dx.doi.org/10.1080/10915810600846245 Quinn, MJ; Hanna, TL; Shiflett, AA; McFarland, CA; Cook, ME; Johnson, MS; Gust, KA; Perkins, EJ. (2013). Interspecific effects of 4A

- DNT (4

- amino

- 2,6

- dinitrotoluene) and RDX (1,3,5

- trinitro

- 1 ,3,5

- triazine) in Japanese quail, Northern bobwhite, and Zebra finch . Ecotoxicology 22: 231

- 2 39. http://dx.doi.org/10.1007/s10646

- 012

- 1019 -8

This document is a draft for review purposes only and does not constitute Agency policy. R-8 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Racine, RJ. (1972). Modif ication of seizure activity by electrical stimulation. II. Motor seizure. 

### 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 Electroencephalography and Clinical Neurophysiology 32: 281

- 294. http://dx.doi.org/10.1016/0013

- 4694(72)90177 -0 Reddy, G; Allen, NA; Major, MA. (2008). Absorption of 14C

- cyclotrimethylenetrinitramine (RDX) from soils through excised human skin. Toxicol Mech Meth 18: 575

- 579. http://dx.doi.org/10.1080/15376510701703466 Reifenrath, WG; Kammen, HO; Reddy, G; Major, MA; Leach, GJ. (2008). Interaction of hydration, aging, and carbon content of soil on the evaporation and skin bioavailability of munition contaminants. J Toxicol Environ Health A 71: 486

- 4 94. http://dx.doi.o rg/10.1080/15287390801906956 Reyes

- García, MG; Hernández

- Hernández, F; Hernández

- Téllez, B; García

- Tamayo, F. (2007). GABA(A) receptor subunits RNA expression in mice peritoneal macrophages modulate their IL

- 6 /IL

- 12 production. J Neuroimmunol 188: 64

- 68. http://dx.doi.org/10.1016/j.jneuroim.2007.05.013 Robinette, CL. (1988). Sex

- hormone

- induced inflammation and fibromuscular proliferation in the rat lateral prostate. Prostate 12: 271

- 286. http:/ /dx.doi.org/10.1002/pros.2990120310 Rothman, KJ; Greenland, S. (1998). Modern epidemiology. In Modern Epidemiology (2nd ed.). Philadelphia, PA: Lippincott, Will iams, & Wilkins. Ryon, MG; Pal, BC; Talmage, SS; Ross, RH. (1984). Database assessment of the health and environmental effects of munition production waste pr oducts (pp. 1

- 224). (ORNL6018). Oak Ridge, TN: Oak Ridge National Lab. http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA145417 Schneider, NR; Bradley, SL; Andersen, ME. (1977). Toxicology of cyclotrimethylenetrinitramine: Distribution and metabolism in the rat and the miniature swine. T oxicol Appl Pharmacol 39: 531

- 5 41. http://dx.doi.org/10.1016/0041

- 008X(77)90144 -2 Schneider, NR; Bradley, SL; Andersen, ME. (1978). The distribution and metabolism of cyclotrimethylenetrinitramine (RDX) in the rat after subchronic administration. Toxicol Appl Pharmacol 46: 163

- 1 71. http://dx.doi.org/10.1016/0041

- 008X(78)90147 -3 Sellers, RS; Morton, D; Michael, B; Roome, N; Johnson, JK; Yano, BL; Perry, R; Schafer, K. (2007). Society of toxicologic pathology position paper: Organ weight recommendations for toxicology studies [Review]. Toxicol Pathol 35: 751

- 7 55. http://dx.doi.org/10.1080/01926230701595300 Sikka, HC; Bannerjee, S; Pack, EJ; Appleton, HT. (1980). Environmental fate of RDX and TNT (pp. 1

- 142). (TR 81

- 538). Fort Detrick, MD: U.S. Army Medical Research and Materiel Command. Simini, M; Checkai, RT. (1996). Uptake of RDX and TNT in crop plants irrigated with contaminated water [Abstract]. Phytopathology 86: S86. Snodgrass, HL, Jr. (1984). Preliminary assessment of relative toxicity and mutagenicity potential of 1- nitroso

- 3,5

- dinitro

- 1,3,5

- triazacylcohexane (mononitroso

- RDX) (Final Repo rt ed.). (ADA149351. USAEHA

- 7 5

- 51

- 0345

- 85). Aberdeen Proving Ground, MD: U.S. Army Environmental Hygiene Agency. http://oai.dtic.mil/oai/oai?verb=getRecord& metadataPrefix=html&identifier=ADA149351 Spanggord, RJ; Mill, T; Chou, T,

- W; Mabey, WR; Smith, JH; Lee, S. (1980a). Environmental fate studies on certain muni tion wastewater constituents. Final report, phase II Laboratory studies. Menlo Park, CA: SRI International. http://www.dtic.mil/cgi

bin/GetTRDoc?AD=ADA099256&Location=U2&doc=GetTRDoc.pdf Spanggord, RJ; Mill, T; Chou, T,

- W; Mabey, WR; Smith, JH; Lee, S. (1980b). Environmental fate studies on certain munition wastewater constitutents. Final report, phase i - Literature review. (LSU

- 7 934). Menlo Park, CA: SRI International. http://www.dtic.mil/cgi

bin/GetTRDoc?AD=ADA082372&Location=U2&doc=GetTRDoc.pdf

This document is a draft for review purposes only and does not constitute Agency policy. R-9 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Steuckart, C; Berger

- Preiss, E; Levsen, K. (1994). Determination of explosives and their 

### 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 biodegradation products in contaminated soil and water from former ammunition plants by automated multiple development high

- p erformance thin

- layer chromatography. Anal Chem 66: 2570

- 2 577. http://dx.doi.org/10.1021/ac00087a023 Stoker, TE; Robinette, CL; Britt, BH; Laws, SC; Cooper, RL. (1999a). Prepubertal exposure to compounds that increase prolactin secretion in the male rat: Effects on the adult pros tate. Biol Reprod 61: 1636

- 1 643. http://dx.doi.org/10.1095/biolreprod61.6.1636 Stoker, TE; Robinette, CL; Cooper, RL. (1999b). Perinatal exposure to estrogenic compounds and the subsequent effects on the prostate of the adult rat: Evaluation of inflammation in the ventral and lateral lobes. Reprod Toxicol 13: 463

- 4 72. http://dx.doi.org/10.1016/S0890

- 6238(99)00049 -0 Stone, E; Coote, JH; Allard, J; Lovick, TA. (2011). GABAergic control of micturition within the periaqueductal grey matter of the male rat. J Physiol 589: 2065

- 2078. http://dx.doi.org/10.1113/jphysiol.2010.202614 Stone, WJ; Paletta, TL; Heiman, EM; Bruce, JI; Knepshield, JH. (1969). Toxic effects following ingestion of C

- 4 plastic explosive. Arch Intern Med 124: 726

- 730. http://dx.doi.org/10.1001/archinte.1969.00300220078015 Suñol, C; Tusell, JM; Gelpí, E; Rodrí guez
- Farré, E. (1989). GABAergic modulation of lindane (gamma

hexachlorocyclohexane)

- induced seizures. Toxicol Appl Pharmacol 100: 1

- 8. http://dx.doi.org/10.1016/0041

- 008X(89)90086 -0 Sweeney, LM; Gut, CP, Jr; Gargas, ML; Reddy, G; Williams, LR; Johnson, MS. (2012a). Assessing the

non
- cancer risk for RDX (hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine) u sing physiologically based pharmacokinetic (PBPK) modeling [Review]. Regul Toxicol Pharmacol 62: 107

- 1 14. http://dx.doi.org/10.1016/j.yrtph.2011.12.007 Sweeney, LM; Okolica, MR; Gut, CP, Jr; Gargas, ML. (2012b). Cancer mode of action, weight of evidence, and proposed cancer reference value for hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX). Regul Toxi c ol Pharmacol 64: 205

- 224. http://dx.doi.org/10.1016/j.yrtph.2012.07.005 Tangbanluekal, L; Robinett e, CL. (1993). Prolactin mediates estradiol

- induced inflammation in the lateral prostate of Wistar rats. Endocrinology 132: 2407

- 2416. http://dx.doi.org/10.1210/endo.132.6.8504745 Testud, F; Glanclaude, J,

- M; Descotes, J. (1996a). Acute hexogen poisoning after occupational exposure. J Toxicol Clin Toxicol 34: 109

- 111. http://dx.doi.org/10.3109/15563659609020244 Testud, F; Glanclaude, J; Imperatori, J; Le Meur, B; Descotes, J. (1996b). [Acute hex ogen poisoning after occupational exposure, report of 2 cases]. Arch Mal Prof Med Trav Secur Soc 57: 342

- 346. Tian, J; Chau, C; Hales, TG; Kaufman, DL. (1999 ). GABA(A) receptors mediate inhibition of T cell responses. J Neuroimmunol 96: 21

- 28. http://dx.doi.org/10.1016/S0165

- 5728(98)00264 -1 Tian, J; Lu, Y; Zhang, H; Chau, CH; Dang, HN; Kaufman, DL. (2004). Gamma

- aminobutyric acid inhibits T cell autoimmunity and the development of inflammatory responses in a mouse type 1 diabetes model. J Immunol 173: 5298

- 5 304. http://dx.doi.org/10.4049/jimmunol.173.8.5298 U.S. EPA (U.S. Environmental Protection Ag ency). (1986a). Guidelines for mutagenicity risk assessment. (EPA/630/R

- 98/003). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www.epa.gov/iris/backgrd.html U.S. EPA (U.S. Environmental Protection Agency). (1986b). Guidelines for the health risk assessment of chemical mixtures. (EPA/630/R

- 98/002). Washington, DC: U.S.

This document is a draft for review purposes only and does not constitute Agency policy. R- 10 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Enviro nmental Protection Agency, Risk Assessment Forum. 

### 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 http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=22567 U.S. EPA (U.S. Environmental Protection Agency). (1988). Recommendations for and documentation of biological values for use in risk assessment. (EPA/600/6

- 87/008). Cincinnati, OH: U.S. Environmental Protection Agency, Of fi ce of Research and Development, Office of Health and Environmental Assessment. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=34855 U.S. EPA (U.S. Environmental Protection Agency). (1991). Guidelines for developmental toxicity risk assessment. (EPA/600/FR

- 91/001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment F or um. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=23162 U.S. EPA (U.S. Enviro nmental Protection Agency). (1992). A cross

- species scaling factor for carcinogen risk assessment based on equivalence of mg/kg3/4/day [EPA Report] (pp. 24152

- 2 4161). Washington, DC. U.S. EPA (U.S. Environmental Protection Agency). (1994). Methods for derivation of inhalation reference concentrations (RfCs) and application of inhalation dosimetry [EPA Report]. (EPA/600/8

- 9 0/066F). Washington, DC: U.S. Environment al Protection Agency, Office of Research and Development, Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office. http://cfpub.epa.gov/ncea/cfm/r ecordisplay.cfm?deid=71993 U.S. EPA (U.S. Environmental Protection Agency). (1996). Guidelines for reproductive toxicity risk assessment. (EPA/630/R

- 96/009). Wa shington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www.epa.gov/raf/publications/guidelines -reproductivetox

- risk

- assessment.h tm U.S. EPA (U.S. Environmental Protection Agency). (1998). Guidelines for neurotoxicity risk assessment [EPA Report]. (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. (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 &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. (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 Pr otection Agency). (2002). A review of the reference dose and reference concentration processes. (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]. (EPA/630/P

- 03/001F). Washington, DC: U.S. Environmental

This document is a draft for review purposes only and does not constitute Agency policy. R- 11 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

Protection Agency, Risk Assessment Forum. http:/ /www2.epa.gov/osa/guidelines



### 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 carcinogen

- risk

- assessment U.S. EPA (U.S. Environmental Protection Agency). (2005b). Supplemental guidance for assessing susceptibility from early

- life exposure to carcinogens. (EPA/630/R

- 03/003F). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www.epa.gov/raf/publications/pdfs/childrens_supplement_final.pdf 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]. (EPA/600/R

- 0 5/043F). Washington, DC: U.S. Environmental Pro t ection 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. (EPA/600/R

- 05/093F). Washington, DC: U.S. Envir o nmental 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]. Washington, DC. http://epa.gov/iris/process.htm U.S. EPA (U.S. Environmental Protection Agency). (2010). Integrated science assessment for carbon monoxide [EPA Report]. (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. (EPA/100/R11/0001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum, Office of
t h e Science Advisor. http://www.epa.gov/raf/publications/interspecies

- extrapolation.htm 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. (EPA/600/R

12/044). Washington, DC. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=244650 U.S. EPA (U.S. Environmental Protection Agency). (2012b). 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). (2012c). EPA Federal facilities forum issue paper: Site characterization for munitions constituents [EPA Repor t] (pp. 1

- 170). (EPA

- 505 -S- 11

- 001). Washington, D.C. http://www2.epa.gov/fedfac/epa

- federal

- facilities

- forum

- issue

paper
- site -c haracterization

- munitions

- constituents U.S. EPA (U.S. Environmental Protection Agency). (2012d). 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

- g ated chloride channels in primary cultures cells of cerebellar granule. Neuroscience 117: 397

- 4 03. 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.

This document is a draft for review purposes only and does not constitute Agency policy. R- 12 DRAFT

— DO NOT CITE OR QUOTEToxicological Review of Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

West, RR; Stafford, DA. ( 1997). Occupational exposures and haematological abnormalities among 

### 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 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) receptor

mediated currents in the amygdala: A mechanism for RDX

- i nduced seizures. Environ Health Perspect 119: 357

- 3 63. 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 neu rotoxicity of cyclotrimethylenetrinitramine (RDX) in a child: A clinical and pharmacokinetic evaluation. Clin Toxicol 24: 305

- 3 19. 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

- of

aqueous
- solubility

- data/ Yoshimura, N; de Groat, WC. (1997). Neural contro l 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

- 3 111. 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- 13 DRAFT

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