## DRAFT DELIBERATIVE: FOR INTERAGENCY REVIEW ONLY. DO NOT DISTRIBUTE OUTSIDE YOUR AGENC Y. **EPA/635 /R** 

**- 14/303**  **Interagency Review Draft www.epa.gov/iris** 



## Tox **icological 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)**
**Supplemental Information**

September 2014

**NOTICE**

This document is an **Intera gency Review**  **D raft.**  This information is distributed solely for the purpose of pre

- dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. It is being circulated for review of its technical accuracy and science policy implications.

National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Washington, DCSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. ii DRAFT―DO NOT CITE OR QUOTE **DISCLAIMER**
This document is a preliminary draft for review purposes only. This information is distributed solely for the purpose of pre

- dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. Mention of trade names or commercial products does not constitute endorsement of recommendation for use.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. iii DRAFT―DO NOT CITE OR QUOTE 

# CONTENTS

APPENDIX A. ASSESSMENTS BY OTHER NATIONAL AND INTERNATIONAL HEALTH AGENCIES

............ A-1

APPENDIX B. ADDITIONAL DETAILS OF LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION

............................................................................................................ B-1
APPENDIX C. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE

- RESPONSE ANALYSIS

.......................................................................................................................... C-1
C.1. CHEMICAL PROPERTIES

.......................................................................................... C-1
C.2. TOXICOKINETICS

..................................................................................................... C-2
C.2.1. A bsorption

................................................................................................ . C-2
C.2.2. Distribution

............................................................................................... C-6
C.2.3. Metabolism

............................................................................................... C-8
C.2.4. Excretion

................................................................................................ . C- 10
C.2.5. Phy siologically

- Based Pharmacokinetic (PBPK) Models

......................... C- 12
C.3. HUMAN STUDIES

.................................................................................................. C- 30
C.4. OTHER PERTINENT TOXICITY INFORMATION

....................................................... C- 38

APPENDIX D. DOSE

- RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES FOR EFFECTS OTHER THAN CANCER AND THE DERIVATION OF CANCER RISK ESTIMATES

...................................................................................................................... D-1

D.1. BENCHMARK DOSE MODELING SUMMARY FOR NONCANCER ENDPOINTS

......... D-1

D.1.1. Evaluation of Model Fit

............................................................................ D-2
D.1.2. Model Selection

....................................................................................... D-2
D.1.3. Modeling Results

...................................................................................... D-3
D.2. BENCHMARK DOSE MODELING SUMMARY FOR CANCER ENDPOINTS

.............. D- 39
D.2.1. Evaluation of Model Fit and Model Selection

........................................ D- 39
D.2.2. Modeling Results

.................................................................................... D- 40
REFERENCES FOR APPENDICES

.................................................................................................................. R-1Supplemental Information― Hexahydro

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# TABLES

Table A

- 1. Assessments by Other National and International Health Agencies

....................................... A-1 Table B

- 1. Summary of detailed search strategies for RDX (Pubmed, Toxline, Toxcenter, TSCATS)

........ B-3 Table B

- 2. Summary of detailed search strategies for RDX (DTIC)

......................................................... B- 10 Table B

- 3. Processes used to augment the search of core databases for RDX

....................................... B- 11 Table B

- 4. Summary disposition of DTIC database citations

.................................................................. B- 13 Table C

- 1. Chemical identity and physicochemical properties of RDX

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

- 2. Distribution of RDX or radiolabel from administered RDX a

..................................................... C-8 Table C

- 3. Principal urinary metabolites of RDX in miniature swine 24 hours after dosing with RDX

................................................................................................ ................................ . C- 10 Table C

- 4. Elimination t 1/2 values for RDX or radiolabeled RDX

.............................................................. C- 11 Table C

- 5. Parameter values used in the Sweeney et al. (2012a) and Sweeney et al. (2012b) PBPK models for RDX in rats, humans, and mice

........................................................... C- 14 Table C

- 6. Parameters values used in the EPA application of the rat, human, and mouse models

....... C- 17 Table C

- 7. Doses, dosing formulations, and absorption rate constants in animal and human studies

............................................................................................................................ C- 19 Table C

- 8. Sensitivity coefficients for rat and human RDX PBPK models

............................................... C- 27 Table C

- 9. Summary of case reports of exposure to RDX

....................................................................... C- 31 Table C

- 10. Epidemiologic studies of RDX: summary of methodologic features

.................................... C- 35 Table C

- 11. Summary of in vitro studies of the genotoxicity of RDX

...................................................... C- 41 Table C

- 12. Summary of in vivo studies of the genotoxicity of RDX

....................................................... C- 44 Table C

- 13. Sum mary of in vitro and in vivo studies of the genotoxicity of RDX metabolites

............... C- 45 Table D

- 1. Noncancer endpoints selected for dose

- response modeling for RDX

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

- 2. Model predictions for convulsions in female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 10% extra risk

...................................................... D-3 Table D

- 3. Model predictions for convulsions in female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR= 5% extra risk

......................................................... D-6 Table D

- 4. Model predictions for convulsions in fem ale F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR= 1% extra risk

......................................................... D-9 Table D

- 5. Model predictions for convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 10% extra risk

......................................................... D- 11 Table D

- 6. M odel predictions for convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 5% extra risk

........................................................... D- 14 Table D

- 7. Model predictions for convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 1% extra risk

........................................................... D- 17 Table D

- 8. Model predictions for convulsions in male and female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 10% extra risk

.................................. D- 19 Table D

- 9. Model predictions for convulsions in male and female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 5% extra risk

.................................... D- 22 Table D

- 10. Model predictions for convulsions in male and female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 1% extra risk

.................................... D- 25 Table D

- 11. Model predictions for convulsions in female F344 rats exposed to RDX by gavage on gestation days 6−19 (Cholakis, 1980); BMR = 10% extra risk

....................................... D- 27 Table D

- 12. Model predictions for convulsions in female F344 rats exposed to RDX by gavage on g estation days 6−19 (Cholakis, 1980); BMR = 5% extra risk

......................................... D- 29Supplemental Information― Hexahydro

- 1,3,5

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

v DRAFT―DO NOT CITE OR QUOTE Table D

- 13. Model predictions for convulsions in female F344 rats exposed to RDX by gavage on gestation days 6−19 (Cholakis, 1980); BMR = 1% extra risk

......................................... D- 31 Table D

- 14. Model predictions for tes ticular degeneration in male B6C3F 1 mice exposed to RDX by diet for 24 months (Lish et al., 1984); BMR = 10% extra risk

................................... D- 33 Tabl e D

- 15. Model predictions for prostate suppurative inflammation in male F344 rats exposed to RDX by diet for 24 months (Levine et al., 1983); BMR = 10% extra risk

................... D- 36 Table D

- 16. Cancer endpoints selected for dose

- response modeling for RDX

...................................... D- 39 Table D- 17. Model predictions for combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F 1 mice exposed to RDX by diet for 24 months (Lish et al., 1984); BMR = 10% extra risk

.................................................................................................... D- 40 Table D

- 18. Model predictions for combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F 1 mice exposed to RDX by diet for 24 months (Lish et al., 1984); BMR = 5% extra risk

...................................................................................................... D- 43 Table D

- 19. Model predictions for combined hepatocellular adenoma and carcinoma in female B6C3F 1 mice exposed to RDX by diet for 24 months (Parker et al., 2006); BMR = 10% extra risk

................................................................................................................ D- 45 Table D

- 20. Model predictions for B6C3F 1 female mouse combined hepatocellular adenoma and carcinoma in mice exposed to RDX by diet for 24 months (Parker et al., 2006); BMR = 5% extra risk

...................................................................................................... D- 47 Table D -2 1. Model predictions and oral slope factor for hepatocellular carcinomas in male F344 rats administered RDX in the diet for 2 years (Levine et al., 1983)

.............................. D- 52 Table D

- 22. Model predictions for combined hepatocellular aden oma and carcinoma in F344 rats exposed to RDX by diet for 24 months (Levine et al., 1983); BMR = 5% extra risk

................................................................................................ ................................ . D- 53



# FIGURES

Figure C

- 1. PBPK model structure for RDX in rats and humans.

............................................................. C- 12 Figure C

- 2. EPA rat PBPK model predictions fitted to observed RDX blood concentrations in male and female SD rats following intravenous exposure.

.................................................... C- 17 Figure C

- 3. EPA rat PBPK model predictions fitted to observed RDX blood concentrations following oral exposure to RDX dissolved in water.

...................................................... C- 20 Figure C

- 4. EPA rat model predictions fitted to observed RDX blood concentrations following oral exposure to RDX in dry capsules..

.......................................................................... C- 20 Figure C

- 5. Effect of varying oral absorption parameters on EPA rat model predictions fitted to observed RDX blood concentrations following oral exposure to coarse grain RDX.

................................................................................................ ................................ C- 21 Figure C

- 6. EPA rat model predictions fitted to observed RDX brain tissue concentrations following oral e xposure to RDX..

................................................................................... C- 21 Figure C

- 7. EPA rat model predictions fitted to observed RDX blood concentrations following oral exposure to fine grain RDX in a saline slurry

.......................................................... C- 22 Figure C

- 8. Comparison of EPA rat model predictions with data from Schneider et al. (1978) for the subchronic gavage study

. ........................................................................................ C- 24 Figure C

- 9. Comparison of EPA rat model predictions with data from Schneider et al. (1978) for the subchronic drinking water study

............................................................................. C- 24Supplemental Information― Hexahydro

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- 10. EPA human model predictions fitted to observed RDX blood concentrations resulting from an accidental ingestion of RDX by a 14.5

- kg boy (Woody et al., 1986)..

............................................................................................................................ C- 26 Figure C

- 11. EPA human model predictions fitted to observed RDX blood concentrations resulting from accidental exposure to adults assumed to be 70 kg (Oz han et al., 2003)

. ............................................................................................................................. C- 26 Figure C

- 12. Comparison of EPA mouse PBPK model pre dictions with data from oral exposure to RDX dissolved in water.

................................................................................................ . C- 28 Figure D

- 1. Plot of incidence rate by dose, with fitted curve for selected model, for convulsions in female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006)

. ......... D-4 Figure D

- 2. Plot of incidence rate by dose, with fitted curve for selected model, for convulsions in female F344 rats exposed to RDX by gavage for 90 days (C rouse et al., 2006).

......... D-7 Figure D

- 3. Plot of incidence rate by dose, with fitted curve for selected model, for convulsions in female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006)

.......... D-9 Figure D

- 4. Plot of incidence rate by dose, with fi tted curve for selected model, for convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006).

.......... D- 12 Figure D

- 5. Plot of incidence rate by dose, with fitted curve for selected model, for convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006).

.......... D- 15 Figure D

- 6. Plot of incidence rate by dose, with fitted curve for selected model, for convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006).

.......... D- 17 Figure D

- 7. Plot of incidence rate by dose, with fitted curve for selected model, for convulsions in male and female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006).

...................................................................................................................... D- 20 Figure D

- 8. Plot of incidence rate by dose, with the fitted curve of the selected model, for convulsions in male and female F344 rats exposed to RDX by gavage for 9 0 days (Crouse et al., 2006).

..................................................................................................... D- 23 Figure D

- 9. Plot of incidence rate by dose, with the fitted curve of the selected model, for convulsions in male and female F344 rats exposed to RDX by gavage fo r 90 days (Crouse et al., 2006)

...................................................................................................... D- 25 Figure D

- 10. Plot of incidence rate by dose, with the fitted curve of the selected model, for convulsions in female F344 rats exposed to RDX by gavage on gesta tion days 6

– 19 (Cholakis, 1980)

........................................................................................................ D- 27 Figure D

- 11. Plot of incidence rate by dose, with the fitted curve of the selected model, for convulsions in female F344 rats exposed to RDX by gavage on gestati on days 6

– 19 (Cholakis, 1980).

. ...................................................................................................... D- 29 Figure D

- 12. Plot of incidence rate by dose, with the fit ted curve of the selected model, for convulsions in female F344 rats exposed to RDX by gavage on gestation days 6

– 19 (Cholakis, 1980).

....................................................................................................... D- 31 Figure D

- 13. Plot of incidence rate by dose, with fitted curve for selected model, for testicular degeneration in male B6C3F 1 mice exposed to RDX by diet for 24 months (Lish et al., 1984)

....................................................................................................................... D- 34 Figure D

- 14. Plot of incidence rate by dose, with fitted curve for selected model, for prostate suppurative inflammation in male F344 rats exposed to RDX by diet for 24 months (Levine et al., 1983)

......................................................................................... D- 37 Figure D

- 15. Plot of incidence rate by dose, with the fitted curve for the selected model, for combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F 1 mice exposed to RDX by diet for 24 months (Lish et al., 1984); BMR = 10% e xtra r isk; dose shown in mg/kg

- day.

............................................................................................ D- 41Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. vii DRAFT―DO NOT CITE OR QUOTE Figure D

- 16. Plot of incidence rate by dose, with fitted curve for selected model, for combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F 1 mice exposed to RDX by diet for 2 4 months (Lish et al., 1984)

............................................................... D- 43 Figure D

- 17. Plot of incidence rate by dose, with fitted curve for selected model, for combined hepatocellular adenoma and carcinoma in female B6C3F 1 mice exposed to RDX by diet for 24 months (Parker et al., 2006).

.................................................................. D- 45 Figu re D

- 18. Plot of incidence rate by dose, with fitted curve for selected model, for B6C3F 1 female mouse combined hepatocellular adenoma and carcinoma in mice exposed to RDX by diet for 24 months (Parker et al., 2006)

........................................ D- 47 Figure D

- 19 . Plot of incidence rate by dose, with fitted curve for selected model, for combined hepatocellular adenoma and carcinoma in F344 rats exposed to RDX by diet for 24 months (Levine et al., 1983)

.................................................................................... D- 53Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

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This document is a draft for review purposes only and does not constitute Agency policy. viii DRAFT―DO NOT CITE OR QUOTE 

# ABBREVIATIONS

ACGIH Amer ican Conference of Governmental Industrial Hygienists AIC Akaike’s information criterion ALT alanine aminotr ansferase AST aspartate aminotransferase ATSDR Agency for Toxic Substances and Disease Registry AUC area under the curve BMD benchmark dose BMDL benchmark dose lower confidence limit BMDS Benchmark Dose Software BMDU benchmark dose upper bound BMR benchmark response BUN blood urea nitrogen CAS Chemical Abstracts Service CASRN Chemical Abstracts Service Registry Number CICADS Concise International Chemical Assessment Documents CNS central nervous system CSF cerebrospinal fluid CYP450 cy tochrome P450 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 electroencephalography EHC Environmental Health Criteria EPA Environmental Protection Agency ER extra risk FDA Food and Drug Administration FM Fort Meade GI gastrointestinal HERO Health and Environmental Research Online HGPRT hypoxanthine

- guanine phosphoribosyltransferase HMX cyclotetramethylene

- tetranitramine i.p. intraperitoneal i.v. intravenous IARC International Agency for Research on Cancer IH industrial hygiene IPCS International Programme on Chemical Safety IRIS Integrated Risk Information System LAAP Louisiana Army Ammunition Plant LC 50 median leth al concentration LD 50 median lethal dose MNX hexahydro

‐ 1

‐ nitroso

‐ 3,5

‐ dinitro

‐ 1,3,5- triazine MRL Minimal Risk Level NADPH nicotinamide adenine dinucleotide phosphate NAP National Academies Press NAS National Academy of Science NCE normochromatic erythro cytes NCEA National Center for Environmental Assessment NCI National Cancer Institute NCTR National Center for Toxicological Research ND not detected NIEHS National Institute of Environmental Health Sciences NIOSHTIC National Institute for Occupational Safety and Health Technical Information Center NR n ot r eported NSCEP National Service Center for Environmental Publications NTP National Toxicology Program ORD Office of Research and Development PBPK physiologically based pharmacokinetic PCE polychr omatic erythrocytes PEL permissible exposure limit PND postnatal day POD point of departure REL recommended exposure limit ROD record of decision SD standard deviation STEL short

- term exposure limit TLV threshold limit value TNT trinitrotoluene TNX 1,3,5- trinitroso -1,3,5- triazacyclohexane TSCATS Toxic Substances Control Act Test Submissions TWA time

- weighted average UF uncertainty factor WHO World Health OrganizationSupplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. A-1 DRAFT―DO NOT CITE OR QUOTE 

# APPENDIX A. ASSESSMENTS BY OTHER NATIONAL AND INTERNATIONAL HEALTH AGENCIES

**Table A**

**- 1.**  **Assessments by Other National and International Health Agencies** 1

Organization Toxicity value Agency for Toxic Substances and Disease Registry ( ATSDR, 2012 ) Acute oral minimal risk level (MRL)

— 0.2 mg/kg

- d Basis: tremors and convulsions in rats ( Crouse et al., 2006 ) ; application of a composite uncertainty factor (UF) of 30 (3 for extrapolation from animals to humans with dosimetric adjustments [PBPK modeling] and 10 for human variability) Intermediate oral MRL

— 0.1 mg/kg -d B as is: convulsions in rats ( Crouse et al., 2006 ) ; application of a composite UF of 30 (3 for extrapolation from animals to humans with dosimetric adjustments [PBPK modeling] and 10 for human variabi lity) Chronic oral MRL

— 0.1 m g/kg -d Ba sis: tremors and convulsions in rats ( Levine et al., 1983 ) ; application of a composite UF of 30 (3 for extrapolation from animals to humans with dosimetric adjustments [PBPK modeling] and 10 for human variability)Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. A-2 DRAFT―DO NOT CITE OR QUOTE Organization Toxicity value

American Conference of Governmental Industrial Hygienists ( ACGIH, 2011 , 2001 ) Threshold Limit Value (TLV)

— 0.5 mg/m 3 , time weighted average (TWA) for an 8

- hr workday in a 40

- hr workweek Basis: Intended to minimize the potential for adverse hepatic, prostate, and hematop oietic effects reported in long

- term oral studies in experimental animals. ACGIH documentation does no t describe how dose

- response data from oral studies was extrapolated to inhalation exposures, or whether other factors were applied to account for extrapolation of animal data to humans. Skin notation indicates potential for systemic exposure and/or toxici ty via dermal absorption. Basis: A report of five cases of RDX

- exposed munition workers with convulsions and/or loss of c onsciousness in a plant where mechanical ventilation was absent, material handling was poorly controlled, and rules regarding wearing of respirators and hand

- washing were often ignored {Kaplan, 1965, 630095}. A uthors identified inh alation, ingestion, and possibly skin absorption as exposure routes ; dermal exposure was not specifically documented

. {ACGIH, 2001, 630056@@author

- year} acknow ledged that data related to dermal exposure and toxic effects we re very limited, but that “a conservative approach to minimize potential toxic effects warrants inclusion of a skin notation for cyclonite.” Class A4 (Not classifiable as a human carcinogen) Basis: S tatistically significantly increased incidence of hepatocellular adenomas and carcinomas in B6C3F 1 mice {Lish, 1984, 630027@@author

- year} were determined to be of little biological significance.
Australian National Industrial Chemicals Notification and Assessment Scheme (NICNAS Health Substances Information System Database http://www.nicnas.gov.au/industry/ai cs/search.asp , accessed May 2, 2012) Exposure Standard

— 1.5 mg/m 3 TWA for an 8

- hr workday Basis: adopted fro m the ACGIH TLV established in 1969 Skin absorption notice indicates that absorption through the skin may be a significant source of exposure Basis: adopted from ACGIH

Nati onal Institute of Occupational Safety and Health (NIOSH Pocket Guide online http://www.cdc.gov/niosh/npg/defaul t.html , accessed May 2, 2012) Recommended Exposure Limit (REL)

— 1.5 mg/m 3 TWA for up to a 10

- hr workday during a 40

- hr workweek Basis: adopted fro m the ACGIH TLV established in 1969 Skin designation indicates potential for dermal absorption Basis: adopted from ACGIH Occupational Safety and Health (OSHA PEL for Maritime and Construction Industries; 29 CFR 1915.1000 Table Z

Shipyards and 29 CFR 1926.55 Appendix A) Permissible Exposure Limit (PEL)

— 1.5 mg/m 3 TWA for an 8

- hr workday in a 40

- hr workweek Basis: adopted fro m the ACGIH TLV established in 1969 Skin designation indicates that cutan eous exposure may contribute to overall exposure and measures should be taken to prevent skin absorption. Basis: adopted from ACGIHSupplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. B-1 DRAFT―DO NOT CITE OR QUOTE 1

2
3
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13
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15
16
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19
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22 23

24 25 

# APPENDIX B. ADDITIONAL DETAILS OF LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION

The literature search for RDX was conducted in five online scientific databases; the most recent update was conducted in January, 2014. The detailed search strategy used to search four of these databases

— Pubmed, Toxline, Toxcenter, and TSCATS

— is provided in Table B

- 1. The search strategy used to search the Defense Technical Information Center (DTIC) database is described in Table B

- 2. The computerized database searches were augmented by review of online regulatory sources as well as “forward” and “backward” Web of Science search es of two recent reviews (Table B- 3).

**Defense Technical Information Center (DTIC) Literature Search and Screen** Eight hundred sixty

- seven RDX

- related citations were identified in the DTIC database; 510 were the full

- text documents with unlimited distribution, 307 were classified as “dis tribution limited to U.S. Government agencies only,” and 50 were classified as “distribution limited to Department of Defense only.” Of the 867 citations, eight citations with unlimited distribution and 10 citations with limited distribution were selected for further review. The eight citations with unlimited distribution (that were not duplicated in other databases) were uploaded to the Health and Environmental Research Online (HERO) website 1 ( http://hero.epa.gov ). The 10 limited

di stribution citations were evaluated for pertinence to the health effects of RDX (i.e., with a focus on whether they provided additional primary health effects data) to determine whether EPA should seek authorization for public distribution an d upload to HERO. A review of the abstract or full

- text of the documents associated with the citation resulted in the following determinations:

• 4 of the 10 citations were excluded from further consideration because the reports were not specific to RDX, or addressed environmental properties (e.g., leaching);

• 3 of the citations were excluded because they did not provide additional primary health effects data. The citations either described a study plan for, or reported data from, experiments that

1 HERO is a database of scientific studies and other references used to develop EPA’s risk assessments aimed at understanding the health and environmental effects of pollutants and chemicals. It is developed and managed in EPA’s Office of Research and Development (ORD) by the National Center for Environmental Assessment (NCEA). The database includes more than 300,000 scientific articles from the peer

- reviewed literature. New studies are added continuously to HERO.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. B-2 DRAFT―DO NOT CITE OR QUOTE were subsequently published ( Williams et al., 2011 ; Hathaway and Buck, 1977 ) and had 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 already been identified by the literature search strategy;

• 1 citation was identified as actually having unlimited distribution (duplicate record in DTIC database), and was added to the HERO database ( Lish et al., 1984 );

• 1 citation provided animal inhalation data and was considered pertinent, but was not brought forward for further review because flaws in the design of study were such that results would not be considered credible. These study design issues included lack of a control group, small numbers of animals, incomplete information on dosage or exposure levels, and inadequate reporting;

• 1 citation did not have an abstract o r full text available outside of the Department of Defense. Based on the title, this report appeared to deal specifically with the manufacture and chemical/explosive properties of RDX. Given the available information, it was determined that it was unlikely the report would provide primary health effects data that warranted further review.

The rationales for exclusion of the other 849 references that were not selected for further consideration are summarized in Table B

- 4.

**Disposition of Studies Kept for Further Review in July 2013 Preliminary Materials**  In EPA’s 2013 Preliminary Materials for the Integrated Risk Information System (IRIS) Toxicological Review of Hexahydro

- 1,3,5

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- 1,3,5

- triazine (RDX) , 15 studies were identified as “kept for possible further review,” including papers with no abstract, inadequate information in the abstract, or available only in a foreign language. EPA sought input from the public on the utility of these studies in the development of the Toxicological Review. No publi c comments on the usefulness of these 15 papers to assessing the health effects of RDX were received at the December 2013 bimonthly meeting discussion of the RDX preliminary materials. Upon further review, none of the 15 references were determined to be pertinent to an assessment of the health effects of RDX following chronic exposure or a source of information significantly different from other studies identified through the literature search. Of the 15 citations reviewed:

• 5 of the 15 references describe d case reports of animal poisoning.

• 5 of the 15 articles were in a foreign language and either due to their age (published before 1960) or title were determined not to provide additional information to substantively inform the toxicity of RDX.

• 2 of the 1 5 references were not published in the peer

- reviewed literature and did not appear to significantly add to other literature sources.

• 1 of the 15 references, based on limited information, appeared to be a site

- specific investigation of poisonings that did n ot substantively address the chronic toxicity of RDX.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. B-3 DRAFT―DO NOT CITE OR QUOTE • 1 of the 15 references, based on limited information, appeared to be a review/annotated 1 2 3

4

5
6
7
8
9
10
11
12
13

14 15 bibliography of other literature and would not significantly add to the literature already i dentified through the literature search process.

• 1 o f the 15 references was a meeting abstract.
**Additional References Not Specific to RDX** During assessment development, 93 additional references were cited as sources of information used to help explain or clarify an issue raised in assessing the health effects of RDX, but were not specific to RDX and were not identified through the chemical

- specific literature search strategies described in Figure LS -1 . Other references cited in the Toxicological Review that are not speci fic to RDX include EPA guidelines and related documents . These references were tracked in HERO as an additional search strategy (“references added during assessment development”). For transparency, those articles are then automatically included as Second ary Sources of Health Effects Information in the overall literature search strategy.

**Table B-**  **1. Summary of detailed search strategies for RDX (Pubmed, Toxline,**  **Toxcenter, TSCATS)**

Database Set # Terms Hits

PubMed Date: 4/2012 1A 1 ((((121

- 82

- 4) OR (Cyclonite[tw] OR Cyclotrimethylenetrinitramine[tw] OR "cyclotrimethylene trinitramine"[tw] OR "Hexahydro

- 1,3,5

- trinitro

1,3,5

- triazine"[tw] OR "Hexahydro

- 1,3,5

- trinitro -s- triazine"[tw] OR Hexogen[tw] OR "1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Triaza

1,3,5

- trinitrocyclohexane"[tw] OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane"[tw] OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Trinitrohexahydro -s- triazine"[tw] OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"[tw] OR "Esaidro

- 1,3,5

- tr initro

- 1,3,5

- triazina"[tw] OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin"[tw] OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR Cyclotrimethylenenitramine[tw] OR Trimethylenetrinitramine[tw] OR "Trimethylene trinitramine"[tw] OR Trimethyleentrinitramine[tw] OR "Trinitrocyclotrimethylene triamine"[tw] OR Trinitrotrimethylenetriamine[tw] OR "CX 84A"[tw] OR Cyklonit[tw] OR Geksogen[tw] OR Heksogen[tw] OR Hexogeen[tw] OR Hexolite[tw] OR "KHP 281"[tw] OR "PBX (af) 108"[tw] OR "PBXW 108(E)"[tw] OR "Pbx(AF) 108"[tw]) OR (rdx[tw])) NOT medline[sb]) OR (((121

- 82

- 4) OR (Cyclonite[tw] OR Cyclotrimethylenetrinitramine[tw] OR "cyclotrimethylene trinitramine"[tw] OR "Hexahydro

- 1,3,5

- trinitro

1,3,5

- triazine"[tw] OR "Hexahydro

- 1,3,5

- trinitro -s- triazine"[tw] OR Hexogen[tw] OR "1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Triaza

1,3,5

- trinitrocyclohexane"[tw] OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane"[tw] OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Trinitrohexahydro -s- triazine"[tw] OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"[tw] OR "Esaidro

- 1,3,5

- trinitro

- 1,3,5

- triazina"[tw] OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin"[tw] OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR Cyclotrimethylenenitramine[tw] OR Trimethylenetrinitramine[tw] OR 33 7Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B-4 DRAFT―DO NOT CITE OR QUOTE Database Set # Terms Hits "Trimethylene trinitramine"[t w] OR Trimethyleentrinitramine[tw] OR "Trinitrocyclotrimethylene triamine"[tw] OR Trinitrotrimethylenetriamine[tw] OR "CX 84A"[tw] OR Cyklonit[tw] OR Geksogen[tw] OR Heksogen[tw] OR Hexogeen[tw] OR Hexolite[tw] OR "KHP 281"[tw] OR "PBX (af) 108"[tw] OR "PBXW 108(E)"[tw] OR "Pbx(AF) 108"[tw]) OR (rdx[tw])) AND (to[sh] OR po[sh] OR ae[sh] OR pk[sh] OR (me[sh] AND (humans[mh] OR animals[mh])) OR ci[sh] OR bl[sh] OR cf[sh] OR ur[sh] OR ((pharmacokinetics[mh] OR metabolism[mh]) AND (humans[mh] OR mammals[mh])) O R "dose

response relationship, drug"[mh] OR risk[mh] OR "toxicity tests"[mh] OR noxae[mh] OR cancer[sb] OR "endocrine system"[mh] OR "endocrine disruptors"[mh] OR "Hormones, Hormone Substitutes, and Hormone Antagonists"[mh] OR triazines/ai OR ("Inhalation Exposure"[Mesh] OR "Maternal Exposure"[Mesh] OR "Maximum Allowable Concentration"[Mesh] OR "Occupational Exposure"[Mesh] OR "Paternal Exposure"[Mesh] OR "Environmental Exposure"[Mesh:noexp])))) NOT (((((121

- 82

- 4) OR (Cyclonite[tw] OR Cyclotrimethylenetrini tramine[tw] OR "cyclotrimethylene trinitramine"[tw] OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR "Hexahydro

- 1,3,5

- trinitro -s- triazine"[tw] OR Hexogen[tw] OR "1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Triaza

- 1,3,5

- trinitrocyclohexane"[tw] OR "1,3, 5- Trinitro

- 1,3,5

- triazacyclohexane"[tw] OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Trinitrohexahydro -s- triazine"[tw] OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"[tw] OR "Esaidro

- 1,3,5

- trinitro

- 1,3,5

- triazina"[tw] OR "Hexahydro

- 1,3,5

- trinitro -1 ,3,5

- triazin"[tw] OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR Cyclotrimethylenenitramine[tw] OR Trimethylenetrinitramine[tw] OR "Trimethylene trinitramine"[tw] OR Trimethyleentrinitramine[tw] OR "Trinitrocyclotrimethylene triamine"[tw] OR Trinitrotr imethylenetriamine[tw] OR "CX 84A"[tw] OR Cyklonit[tw] OR Geksogen[tw] OR Heksogen[tw] OR Hexogeen[tw] OR Hexolite[tw] OR "KHP 281"[tw] OR "PBX (af) 108"[tw] OR "PBXW 108(E)"[tw] OR "Pbx(AF) 108"[tw]) OR (rdx[tw])) NOT medline[sb]) OR (((121

- 82

- 4) OR (Cycl onite[tw] OR Cyclotrimethylenetrinitramine[tw] OR "cyclotrimethylene trinitramine"[tw] OR "Hexahydro

- 1,3,5

- trinitro

1,3,5

- triazine"[tw] OR "Hexahydro

- 1,3,5

- trinitro -s- triazine"[tw] OR Hexogen[tw] OR "1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Triaza

1,3, 5- trinitrocyclohexane"[tw] OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane"[tw] OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Trinitrohexahydro -s- triazine"[tw] OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"[tw] OR "Esaidro

- 1,3,5

- trinitro

- 1,3,5

- triazina" [tw] OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin"[tw] OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR Cyclotrimethylenenitramine[tw] OR Trimethylenetrinitramine[tw] OR "Trimethylene trinitramine"[tw] OR Trimethyleentrinitramine[tw] OR "Trinitrocyclotrime thylene triamine"[tw] OR Trinitrotrimethylenetriamine[tw] OR "CX 84A"[tw] OR Cyklonit[tw] OR Geksogen[tw] OR Heksogen[tw] OR Hexogeen[tw] OR Hexolite[tw] OR "KHP 281"[tw] OR "PBX (af) 108"[tw] OR "PBXW 108(E)"[tw] ORSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B-5 DRAFT―DO NOT CITE OR QUOTE Database Set # Terms Hits "Pbx(AF) 108"[tw]) OR (rdx[tw])) AND (t o[sh] OR po[sh] OR ae[sh] OR pk[sh] OR (me[sh] AND (humans[mh] OR animals[mh])) OR ci[sh] OR bl[sh] OR cf[sh] OR ur[sh] OR ((pharmacokinetics[mh] OR metabolism[mh]) AND (humans[mh] OR mammals[mh])) OR "dose

response relationship, drug"[mh] OR risk[mh] OR " toxicity tests"[mh] OR noxae[mh] OR cancer[sb] OR "endocrine system"[mh] OR "endocrine disruptors"[mh] OR "Hormones, Hormone Substitutes, and Hormone Antagonists"[mh] OR triazines/ai OR ("Inhalation Exposure"[Mesh] OR "Maternal Exposure"[Mesh] OR "Maximum Allowable Concentration"[Mesh] OR "Occupational Exposure"[Mesh] OR "Paternal Exposure"[Mesh] OR "Environmental Exposure"[Mesh:noexp])))) AND (invertebrates OR aquatic organisms OR fish OR fishes OR amphibians OR earthworm*))
PubMed Date limit: 1/2012

− 2/2013 1A 2 (Cyclonite[tw] OR RDX[tw] OR Cyclotrimethylenetrinitramine[tw] OR "cyclotrimethylene trinitramine"[tw] OR "Hexahydro

- 1,3,5

- trinitro

1,3,5

- triazine"[tw] OR "Hexahydro

- 1,3,5

- trinitro -s- triazine"[tw] OR Hexogen[tw] OR "1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Triaza

1,3,5

- trinitrocyclohexane"[tw] OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane"[tw] OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Trinitrohexahydro -s- triazine"[tw] OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"[tw] OR "Esaidro

- 1,3,5

- trinitro

- 1,3,5

- triazina"[tw] OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin"[tw] OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR Cyclotrimethylenenitramine[tw] OR Trimethylenetrinitramine[tw] OR "Trimethylene trinitramine"[tw] OR Trimethyleentrinitramine[tw] OR "Trinitrocyclotrimethylene triamine"[tw] OR Trinitrotrimethylenetriamine[tw] OR "CX 84A"[tw] OR Cyklonit[tw] OR Geksogen[tw] OR Heksogen[tw] OR Hexogeen[tw] OR Hexolite[tw] OR "KHP 281"[tw] OR "PBX (af) 108"[tw] OR "PBXW 108(E)"[tw] OR "Pbx(AF) 108"[tw]) AND (("2012/01/01"[Date - MeSH] : "3000"[Date - MeSH]) OR ("2012/01/01"[Date - Entrez] : "3000"[Date - Entrez]) OR ("2012/01/01"[Date

- Create] : "3000"[Date

- Create])) 112

PubMed Date limit: 11/2012

− 1/2014 1A3 (Cycl onite[tw] OR RDX[tw] OR Cyclotrimethylenetrinitramine[tw] OR "cyclotrimethylene trinitramine"[tw] OR "Hexahydro

- 1,3,5

- trinitro

1,3,5

- triazine"[tw] OR "Hexahydro

- 1,3,5

- trinitro -s- triazine"[tw] OR Hexogen[tw] OR "1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Triaza

1,3,5

- trinitrocyclohexane"[tw] OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane"[tw] OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine"[tw] OR "1,3,5

- Trinitrohexahydro -s- triazine"[tw] OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"[tw] OR "Esaidro

- 1,3,5

- trinitro

- 1,3, 5- triazina"[tw] OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin"[tw] OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"[tw] OR Cyclotrimethylenenitramine[tw] OR Trimethylenetrinitramine[tw] OR "Trimethylene trinitramine"[tw] OR Trimethyleentrinitramine[tw] OR "Trinitr ocyclotrimethylene triamine"[tw] OR Trinitrotrimethylenetriamine[tw] OR "CX 84A"[tw] OR Cyklonit[tw] OR Geksogen[tw] OR Heksogen[tw] OR Hexogeen[tw] OR Hexolite[tw] OR "KHP 281"[tw] OR "PBX (af) 108"[tw] OR "PBXW 108(E)"[tw] OR "Pbx(AF) 108"[tw]) AND (("20 12/ 1 1/01"[Date

- MeSH] : "3000"[Date

- 138Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B-6 DRAFT―DO NOT CITE OR QUOTE Database Set # Terms Hits MeSH]) OR ("2012/ 1 1/01"[Date

- Entrez] : "3000"[Date

- Entrez]) OR ("2012/ 1 1/01"[Date

- Create] : "3000"[Date

- Create]))
Toxline Date: 4/2012 1B 1 Notes: Searched CASRN or synonyms ;

- removed invertebrates, aquatic organisms, amphibians, earthworms 507

Toxline Date limit: 2011

− 2/2013 1B 2 @OR+("Cyclonite"+"RDX"+"Cyclotrimethylenetrinitramine"+"cyclotrim ethylene trinitramine"+"Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"+"Hexahydro

- 1,3,5

- trinitro -s- triazine"+"Hexogen"+"1,3,5

- trinitro

- 1,3,5

- triazine"+"1,3,5

- Triaza

- 1,3,5

- trinitrocyclohexane"+"1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane"+"1,3,5

- Trinitrohexahydro

- 1,3,5

- triazin e"+"1,3,5

- Trinitrohexahydro -s- triazine"+@term+@rn+121

- 82

- 4)+@AND+@range+yr+2011+2013+@NOT+@org+pubmed+pubdart+cr isp+tscats 5

1B 3 @OR+("1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"+"Esaidro

- 1,3,5

- trinitro

1,3,5

- triazina"+"Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin"+"Perhydro

1,3,5

- trinitro

- 1,3,5

- triazine"+"Cyclotrimethylenenitramine"+"Trimethylenetrinitramine"+" Trimethylene+ trinitramine"+"Trim ethyleentrinitram ine"+"Trinitrocyclo trimethylene+triamine"+"Tr initrotrimethylenetriamine"+"CX+ 84A"+"Cy klonit"+"Geksogen"+"Heksogen"+"Hexogeen"+"Hexolite"+"KHP+ 281") +@AND+@range+yr+2011+2013+@NOT+@org+pubmed+pubdart+cris p+tscats 0

Toxline Date limit: 2012

− 1/2014 1B 4 @OR+("Cyclonite"+"RDX"+"Cyclotrimethylenetrinitramine"+"cyclotrim ethylene trinitramine"+"Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine"+"Hexahydro

- 1,3,5

- trinitro -s- triazine"+"Hexogen"+"1,3,5

- trinitro

- 1,3,5

- triazine"+"1,3,5

- Triaza

- 1,3,5

- trinitrocycloh exane"+"1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane"+"1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine"+"1,3,5

- Trinitrohexahydro -s- triazine"+@term+@rn+121

- 82

- 4)+@AND+@range+yr+201 2 +201 4 +@NOT+@org+pubmed+pubdart+cr isp+tscats 10

1B 5 @OR+("1,3,5

- Trinitroperhydro

- 1,3,5

- triazine"+"Esaidro

- 1,3,5

- trinitro

1,3,5

- triazina"+"Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin"+"Perhydro

1,3,5

- trinitro

- 1,3,5

- triazine"+"Cyclotrimethylenenitramine"+"Trimethylenetrinitramine"+" Trimethylene+ trinitramine"+"Trim ethyleentrinitra mine"+"Trinitrocyclo trimethylene+triamine"+"Tr initrotrimethylenetriamine"+"CX+ 84A"+"Cy klonit"+"Geksogen"+"Heksogen"+"Hexogeen"+"Hexolite "+"KHP+281") +@AND+@range+yr+2012+201 4 +@NOT+@org+pubmed+pubdart+cris p+tscats 0

TSCATS Date: 2/2013 1C1 @term+@rn+121

- 82

- 4+@AND+@org+tscats 4

TSCATS Date limit: 2012

− 1/2014 1C 2 @OR+(@term+@rn+121

- 82

- 4)+@AND+@range+yr+2012+2014+@AND+@org+tscats 0Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B-7 DRAFT―DO NOT CITE OR QUOTE Database Set # Terms Hits

Toxcenter Date: 4/2012 1D 1 ((121

- 82

- 4 OR Cyclonite OR RDX OR Cyclotrimethylenetrinitramine OR "cyclotrimethylene trinitramine" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR "Hexahydro

- 1,3,5

- trinitro -s- triazine" OR Hexogen OR "1,3,5

- trinitro

- 1,3,5

- triazine" OR "1,3,5

- Triaza

- 1,3,5

- trinitrocyclohexane" OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane" OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine" OR "1,3,5

- Trinitrohexahydro -striazine" OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine" OR "Esaidro

- 1,3,5

- trinitro

- 1,3,5

- triazina" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin" OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR Cyclotrimethylenenitramine OR Trimethylenetrinitramine OR "Trimethylene trinitramine" OR Trimethyleentrinitramine OR "Trinitrocyclotrimethylene triamine" OR Trinitrotrimethylenetriamine OR "CX 84A" OR Cyklonit OR Geksogen OR Heksogen OR Hexogeen OR Hexol ite OR "KHP 281" OR "PBX (af) 108" OR "PBXW 108(E)" OR "Pbx(AF) 108") NOT (patent/dt OR tscats/fs))AND ((chronic OR immunotox? OR neurotox? OR toxicokin? OR biomarker? OR neurolog? OR pharmacokin? OR subchronic OR pbpk OR epidemiology/st,ct,it) OR acute OR subacute OR ld50# OR lc50# OR (toxicity OR adverse OR poisoning)/st,ct,it OR inhal? OR pulmon? OR nasal? OR lung? OR respir? OR occupation? OR workplace? OR worker? OR oral OR orally OR ingest? OR gavage? OR diet OR diets OR dietary OR drinking(w)water OR (maximum and concentration? and (allowable OR permissible)) OR (abort? OR abnormalit? OR embryo? OR cleft? OR fetus? OR foetus? OR fetal? OR foetal? OR fertil? OR malform? OR ovum OR ova OR ovary OR placenta? OR pregnan? OR prenatal OR perinatal? OR postnatal? OR reproduc? OR steril? OR teratogen? OR sperm OR spermac? OR spermag? OR spermati? OR spermas? OR spermatob? OR spermatoc? OR spermatog? OR spermatoi? OR spermatol? OR spermator? OR spermatox? OR spermatoz? OR spermatu? OR spermi? OR spermo? OR

neon at? OR newborn OR development OR developmental? OR zygote? OR child OR children OR adolescen? OR infant OR wean? OR offspring OR age(w)factor? OR dermal? OR dermis OR skin OR epiderm? OR cutaneous? OR carcinog? OR cocarcinog? OR cancer? OR precancer? OR neoplas? OR tumor? OR tumour? OR oncogen? OR lymphoma? OR carcinom? OR genetox? OR genotox? OR mutagen? OR genetic(w)toxic? OR nephrotox? OR hepatotox? OR endocrin? OR estrogen? OR androgen? OR hormon? OR rat OR rats OR mouse OR mice OR muridae OR dog OR dogs OR rabbit? OR hamster? OR pig OR pigs OR swine OR porcine OR goat OR goats OR sheep OR monkey? OR macaque? OR marmoset? OR primate? OR mammal? OR ferret? OR gerbil? OR rodent? OR lagomorpha OR baboon? OR bovine OR canine OR cat OR cats OR feline OR pigeon? OR occupation? OR worker? OR epidem?) AND ((biosis/fs AND py>1999) OR caplus/fs))

Notes: Duplicates were removed; Biosis subfile results were date limited to avoid extensive overlap with Toxline 337 (20 selected and added to HERO)

Toxcenter

Date limit: 1D 2 (( (121

- 82

- 4 OR Cyclonite OR RDX OR Cyclotrimethylenetrinitramine OR "cyclotrimethylene trinitramine" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR "Hexahydro

- 1,3,5

- trinitro -s- triazine" OR Hexogen OR "1,3,5

- trinitro

- 1,3,5

- triazine" OR "1,3,5

- Triaza

- 1,3,5

- 26 (6 selected and added to HERO)Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B-8 DRAFT―DO NOT CITE OR QUOTE Database Set # Terms Hits 1/1/2012

− 2/2013 trinitrocyclohexane" OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane" OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine" OR "1,3,5

- Trinitrohexahydro -striazine" OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine" OR "Esaid ro

- 1,3,5

- trinitro

- 1,3,5

- triazina" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin" OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR Cyclotrimethylenenitramine OR Trimethylenetrinitramine OR "Trimethylene trinitramine" OR Trimethyleentrinitramine OR "Trinitrocyclo trimethylene triamine" OR Trinitrotrimethylenetriamine OR "CX 84A" OR Cyklonit OR Geksogen OR Heksogen OR Hexogeen OR Hexolite OR "KHP 281" OR "PBX (af) 108" OR "PBXW 108(E)" OR "Pbx(AF) 108") NOT (patent/dt OR tscats/fs) ) AND (py>2012 OR ed>20120101) ) AND (chronic OR immunotox? OR neurotox? OR toxicokin? OR biomarker? OR neurolog? OR pharmacokin? OR subchronic OR pbpk OR epidemiology/st,ct, it) OR acute OR subacute OR ld50# OR lc50# OR (toxicity OR adverse OR poisoning)/st,ct,it OR inhal? OR pulmon? OR nasal? OR lung? OR respir? OR occupation? OR workplace? OR worker? OR oral OR orally OR ingest? OR gavage? OR diet OR diets OR dietary OR drinking(w)water OR (maximum and concentration? and (allowable OR permissible)) OR (abort? OR abnormalit? OR embryo? OR c left? OR fetus? OR foetus? OR fetal? OR foetal? OR fertil? OR malform? OR ovum OR ova OR ovary OR placenta? OR pregnan? OR prenatal OR perinatal? OR postnatal? OR reproduc? OR steril? OR teratogen? OR sperm OR spermac? OR spermag? OR spermati? OR spermas? OR spermatob? OR spermatoc? OR spermatog? OR spermatoi? OR spermatol? OR spermator? OR spermatox? OR spermatoz? OR spermatu? OR spermi? OR spermo? OR neonat? OR newborn OR development OR developmental? OR zygote? OR child OR children OR adolescen? OR infan t OR wean? OR offspring OR age(w)factor? OR dermal? OR dermis OR skin OR epiderm? OR cutaneous? OR carcinog? OR cocarcinog? OR cancer? OR precancer? OR neoplas? OR tumor? OR tumour? OR oncogen? OR lymphoma? OR carcinom? OR genetox? OR genotox? OR mutagen? OR genetic(w)toxic? OR nephrotox? OR hepatotox? OR endocrin? OR estrogen? OR androgen? OR hormon? OR rat OR rats OR mouse OR mice OR muridae OR dog OR dogs OR rabbit? OR hamster? OR pig OR pigs OR swine OR porcine OR goat OR goats OR sheep OR monkey? OR macaque? OR marmoset? OR primate? OR mammal? OR ferret? OR gerbil? OR rodent? OR lagomorpha OR baboon? OR bovine OR canine OR cat OR cats OR feline OR pigeon? OR occupation? OR worker? OR epidem?) AND (biosis/fs OR caplus/fs))

Notes: Duplicates were removedSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B-9 DRAFT―DO NOT CITE OR QUOTE Database Set # Terms Hits

Toxcenter

Date limit: 11/1/2012

− 1/2014 1D 3 (( (121

- 82

- 4 OR Cyclonite OR RDX OR Cyclotrimethylenetrinitramine OR "cyclotrimethylene trinitramine" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR "Hexahydro

- 1,3,5

- trinitro -s- triazine" OR Hexogen OR "1,3,5

- trinitro

- 1,3,5

- triazine" OR "1,3,5

- Triaza

- 1,3,5

- trinitrocyclohexane" OR "1,3,5

- Trinitro

- 1,3,5

- triazacyclohexane" OR "1,3,5

- Trinitrohexahydro

- 1,3,5

- triazine" OR "1,3,5

- Trinitrohexahydro -striazine" OR "1,3,5

- Trinitroperhydro

- 1,3,5

- triazine" OR "Esaidro

- 1,3,5

- trinitro

- 1,3,5

- triazina" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazin" OR "Perhydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR Cyclotrimethylenenitramine OR Trimethylenetrinitramine OR "Trimethylene trinitramine" OR Trimethyleentrinitramine OR "Trinitrocyclotrimethylene triamine" OR Trinitrotrimethylenetriamine OR "CX 84A" OR Cyklonit OR Geksogen OR Heksogen OR Hexogeen OR Hexolite OR "KHP 281" OR "PBX (af) 108" OR "PBXW 108(E)" OR "Pbx(AF) 108") NOT (patent /dt OR tscats/fs) ) AND (py>2012 OR ed>20121101)) AND (chronic OR immunotox? OR neurotox? OR toxicokin? OR biomarker? OR neurolog? OR pharmacokin? OR subchronic OR pbpk OR epidemiology/st,ct, it) OR acute OR subacute OR ld50# OR lc50# OR (toxicity OR advers e OR poisoning)/st,ct,it OR inhal? OR pulmon? OR nasal? OR lung? OR respir? OR occupation? OR workplace? OR worker? OR oral OR orally OR ingest? OR gavage? OR diet OR diets OR dietary OR drinking(w)water OR (maximum and concentration? and (allowable OR per missible)) OR (abort? OR abnormalit? OR embryo? OR cleft? OR fetus? OR foetus? OR fetal? OR foetal? OR fertil? OR malform? OR ovum OR ova OR ovary OR placenta? OR pregnan? OR prenatal OR perinatal? OR postnatal? OR reproduc? OR steril? OR teratogen? OR spe rm OR spermac? OR spermag? OR spermati? OR spermas? OR spermatob? OR spermatoc? OR spermatog? OR spermatoi? OR spermatol? OR spermator? OR spermatox? OR spermatoz? OR spermatu? OR spermi? OR spermo? OR neonat? OR newborn OR development OR developmental? OR zygote? OR child OR children OR adolescen? OR infant OR wean? OR offspring OR age(w)factor? OR dermal? OR dermis OR skin OR epiderm? OR cutaneous? OR carcinog? OR cocarcinog? OR cancer? OR precancer? OR neoplas? OR tumor? OR tumour? OR oncogen? OR lymphom a? OR carcinom? OR genetox? OR genotox? OR mutagen? OR genetic(w)toxic? OR nephrotox? OR hepatotox? OR endocrin? OR estrogen? OR androgen? OR hormon? OR rat OR rats OR mouse OR mice OR muridae OR dog OR dogs OR rabbit? OR hamster? OR pig OR pigs OR swine OR porcine OR goat OR goats OR sheep OR monkey? OR macaque? OR marmoset? OR primate? OR mammal? OR ferret? OR gerbil? OR rodent? OR lagomorpha OR baboon? OR bovine OR canine OR cat OR cats OR feline OR pigeon? OR occupation? OR worker? OR epidem?) AND (bios is/fs OR caplus/fs))

Notes: Duplicates were removed 20 (0 selected)

1Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B- 10 DRAFT―DO NOT CITE OR QUOTE **Table B-2.**  **Summary of detailed search strategies for RDX (DTIC)**  1

Database Set # Terms Hits
DTIC Online Access Controlled

Date: 2/11/2013 2A1 key:((toxicity OR phramacokinetics OR toxicology OR pharmacology OR poisoning OR toxic hazards OR radiation hazards OR radiation effects OR toxic diseases OR toxic agents OR lethal agents OR antidotes OR death OR "signs and symptoms" OR cancer OR carinogens OR physiology OR biochemistry OR body weight OR anatomy OR body fluids OR metabolism OR immunology OR mutagens OR teratogenic compounds OR mutations OR antimetabolites) NOT (aquatic animals OR Invertebrates OR venomous animals OR wildlife OR biodegradation)) and ("121

- 82

- 4" OR Cyclonite OR RDX OR Cyclotrimethylenetrinitramine OR "cyclotrimethylene trinitramine" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR "Hexahydro

- 1,3,5

- trinitro -s- triazine" OR Hexogen OR Cyclotrimethylenenitramine OR Trimethylenetrinitramine OR "Trimethylene trinitramine " OR Trimethyleentrinitramine OR "Trinitrocyclotrimethylene triamine" OR Trinitrotrimethylenetriamine OR Hexolite ) and distco:(A) | Report Date: All dates 504 (8 selected and added to HERO)

2A2 distco:(govt) and key:((toxicity OR phramacokinetics OR tox icology OR pharmacology OR poisoning OR toxic hazards OR radiation hazards OR radiation effects OR toxic diseases OR toxic agents OR lethal agents OR antidotes OR death OR "signs and symptoms" OR cancer OR carinogens OR physiology OR biochemistry OR body weight OR anatomy OR body fluids OR metabolism OR immunology OR mutagens OR teratogenic compounds OR mutations OR antimetabolites) NOT (aquatic animals OR Invertebrates OR venomous animals OR wildlife OR biodegradation)) and ("121

- 82

- 4" OR Cyclonite OR RDX OR Cyclotrimethylenetrinitramine OR "cyclotrimethylene trinitramine" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR "Hexahydro

- 1,3,5

- trinitro -s- triazine" OR Hexogen OR Cyclotrimethylenenitramine OR Trimethylenetrinitramine OR "Trimethylene trinitramine" O R Trimethyleentrinitramine OR "Trinitrocyclotrimethylene triamine" OR Trinitrotrimethylenetriamine OR Hexolite ) | Report Date: All dates 304 (7 selected for further consideration)

2A3 distco:(dod) and key:((toxicity OR phramacokinetics OR toxicology OR pharmacology OR poisoning OR toxic hazards OR radiation hazards OR radiation effects OR toxic diseases OR toxic agents OR lethal agents OR antidotes OR death OR "signs and symptoms" OR cancer OR carinogens OR physiology OR biochemistry OR body weight OR an atomy OR body fluids OR metabolism OR immunology OR mutagens OR teratogenic compounds OR mutations OR antimetabolites) NOT (aquatic animals OR Invertebrates OR venomous animals OR wildlife OR biodegradation)) and ("121

- 82

- 4" OR Cyclonite OR RDX OR Cyclotri methylenetrinitramine OR "cyclotrimethylene trinitramine" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR "Hexahydro

- 1,3,5

- trinitro -s- triazine" OR Hexogen OR Cyclotrimethylenenitramine OR Trimethylenetrinitramine OR "Trimethylene trinitramine" OR Trimethyleentrinitramine OR "Trinitrocyclotrimethylene triamine" OR Trinitrotrimethylenetriamine OR Hexolite ) | Report Date: All dates 50 (3 selected for further consideration)Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B- 11 DRAFT―DO NOT CITE OR QUOTE Database Set # Terms Hits
DTIC R&E Gateway Search

Date limit: 11/1/2012

− 1/23/2014 2B1 ((toxicity OR phramacokinetics OR toxicology OR pharmacology OR poisoning OR toxic hazards OR radiation hazards OR radiation effects OR toxic diseases OR toxic agents OR lethal agents OR antidotes OR death OR "signs and symptoms" OR cancer OR carinogens OR physiology OR biochemistry OR body weight OR anatomy OR body fluids OR metabolism OR immunology OR mutagens OR teratogenic compounds OR mutations OR antimetabolites) NOT (aquatic animals OR Invertebrates OR venomous animals OR wildlife OR biodegradation)) and ("121

- 82

- 4" OR Cyclonite OR RDX OR Cyclotrimethylenetrinitramine OR "cyclotrimethylene trinitramine" OR "Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine" OR "Hexahydro

- 1,3,5

- trinitro -s- triazine" OR Hexogen OR Cyclotrimethylenenitramine OR Trimethylenetrinitramine OR "Trime thylene trinitramine" OR Trimethyleentrinitramine OR "Trinitrocyclotrimethylene triamine" OR Trinitrotrimethylenetriamine OR Hexolite )

Notes: Date limited to items created after October 31,2012. Searched all distribution limits individually: 6 results fo und in "approved for public release", 3 results found in "Gov't and Gov't contractors only" 9 (0 selected)

**Table B-**  **3. Processes used to augment the search of core databases for RDX**  1

Selected Key Reference(s) or Sources Date Additional References Identified 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) using physiologically based pharmacokinetic (PBPK) modeling. Regul Toxicol Pharmacol 62 (1) :107

− 114. (forward search) 1 search result 3/2013 0 citati ons added

Sweeney, LM; Okolica, MR; Gut, CP, Jr; Gargas, ML. ( 2012b ) . Cancer mode of action, weight of evidence, and proposed cancer reference value for hexahyd ro

- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX). Regul Toxicol Pharmacol 64 (2):205

− 224 (backwards search) 0 search results 3/2013 0 citations added

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) using physiologically based pharmacokinetic (PBPK) modeling. Regul Toxicol Pharmacol 62 (1) :107

− 114. 35 cited papers 3/2013 0 citations added

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 Toxicol Pharmacol 64 (2):205

− 224 69 cited papers 3/2013 3 citations addedSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B- 12 DRAFT―DO NOT CITE OR QUOTE Selected Key Reference(s) or Sources Date Additional References Identified
Combination of CASRN and synonyms searched on the following websites: ATSDR http://www.atsdr.cdc.gov/substances/index.asp (Note: the reference list for the ATSDR toxicological profile for RDX was compared to the search results and relevant references were added) CalEPA (Office of Environmental Health Hazard Assessment) ( http://www.oehha.ca.gov/risk.html ) eChemPortal ( http://www.echemportal.org/echemportal/participant/page.action? pageID=9 ) EPA Acute Exposure Guideline Levels ( http://www.epa.gov/oppt/aegl/pubs/chemlist.htm ) EP A

− IRISTrack/New Assessments and Reviews ( http://cfpub.epa.gov/ncea/iristrac/ ) to find dates ( http://www.epa.gov/ncea/iris/index.html ) to find data EPA NSCEP ( http://www.epa.gov/ncepihom/ ) EPA Science Inventory ( http://cfpub.epa.gov/si/ ) Federal Docket www.regulations.gov Health Canada First Priority List Assessments ( http://www.hc

- sc.gc.ca/ewh

- semt/pubs/contaminants/psl1

- lsp1/index

- eng.php ) Health Canada Second Priority List Assessments ( http://www.hc

- sc.gc.ca/ewh

- semt/pubs/contaminants/psl2

- lsp2/index

- eng.php ) IARC ( http://monographs.iarc.fr/htdig/search.html ) IPCS INCHEM ( http://www.in chem.org/ ) NAS via NAP ( http://www.nap.edu/ ) NCI ( http://www.cancer.gov ) NCTR ( http://www.fda.gov/AboutFDA/CentersOffices/OC/OfficeofScientific andMedicalPrograms/NCTR/default.htm ) National Institute for Environmental Heal th Sciences (NIEHS) http://www.niehs.nih.gov/ NIOSHTIC 2 ( http://www2a.cdc.gov/nioshtic

- 2/ ) NTP

− RoC, status, results, and management reports ( http://ntpsearch.niehs.nih.gov/query.html ) WHO assessments

− CICADS, EHC ( http://www.who.int/ipcs/assessment/en/ ) 4/11/2012

1/27/2014 15 citations added

1 citat ion added

1Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. B- 13 DRAFT―DO NOT CITE OR QUOTE **Table B-**  **4. Summary disposition of DTIC database citations**  1

Criteria Percent of c itations

Exclusion

— Not chemical

- specific ~50%
Exclusion

— Bioremediation or biodegradation 5%
Exclusion

— Chemical/physical properties of explosive properties <5%
Exclusion

— Physical or chemical treatment <5%
Exclusion

— Miscellaneous, including:

• Superfund RODs for which the abstract did not specify whether RDX was a contaminant of concern

• Meeting minutes and conference proceedings for which only general categories of topics were included in the DTIC record

• DTIC records containing only a title containing inadequate information with which to classify the citation ~35%

Exclusion Total 98% (847 total)

Additional Resource

— Regulatory documents <5%
Additional Resource

— Reviews <5%
Additional Resource

— Ecosystem effects <5%
Additional Resource

— Risk assessments <5%
Additional Resource

— Exposure levels <5%
Additional Resource

— Measurement methods <5%
Additional Resource

— Mixture only <5%
Additional Resource

— Toxicokinetics <5%
Possible Further Review

— No abstract <5%
Possible Further Review

— inadequate reporting in abstract <5%
Inclusion Total ~2% (20 total)

TOTAL NUMBER OF DTICS CITATIONS (including 10 limited distribution for further review) 867

2Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-1 DRAFT―DO NOT CITE OR QUOTE 1 

# APPENDIX C. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE

- RE S PONSE ANALYSIS

**C.1. CHEMICAL PROPERTIES**  2

**Table C**  **-1. C**  **hemical identity and physicochemical properties of RDX** 

Characteristic or property Value Reference

Chemical s tructure NLM, 2011

CASRN 121

- 82

- 4 Color/form White, crystalline solid Bingham et al. (2001)
Molecular formula C 3 H 6 N 6 O 6 ACGIH (2001) Molecular weight 222.12 Lide (2005) Density (g/cm 3 at 20°C) 1.82 Lide (2005) Melting point (°C) 205.5 Lide (2005)
Heat of fo rmation (kJ/g)

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

K OC 42−167 Spanggord et al. (1980) Boiling point (°C) 276−280 Bingham et al. (2001) Henry’s law constant (atm

- m 3 /mole at 25°C) 2.0 × 10 − 1 U.S. EPA (2003)

Solubility in water (mg/L at 25°C) 59.7 Yalkowsky and He (2003)

Vapor pressure (mm Hg at 20°C) 4.10 × 10 − 9 Spanggord et al. (1980)Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-2 DRAFT―DO NOT CITE OR QUOTE 

## C.2. **TOXICOKINETICS** 1

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34 RDX is absorbed following exposure by inhalation and oral routes

. The rate and extent of absorption are dependent upon the dosing preparation. RDX is systemically distributed, can be transfered from mother to fetus and can transfer in maternal milk. Metabolism of RDX is extensive and includes denitration, ring cleavage, and generation of CO2 possibly through cytochrome P450 (CYP450)

. RDX metabolites are eliminated primarily via urinary excretion and exhalation of CO2.

**C.2.1. Absorption**  Absorption of RDX following oral exposure has been demonstrated in humans and labo ratory animals (rats, mice, swine
, and voles) through measurement of radiolabel ed RDX and/or metabolites in excreta (urine and expired air) and tissues (including blood)

. Quantitative estimates of oral absorption (e.g., oral bioavailability or fractional absorption) are not available in humans

. Results of animals studies indicate that oral bioavailability ranges from approximately 50

− 90 % and may vary based on the physical form of RDX and the matrix (e.g., soil, plants) in which it is administered

. Studie s investigating absorption of RDX following inhalation exposure were not identified . Results of an intratracheal administration study in rats provide limited evidence of absorption of RDX from the respiratory tract . The only data evaluating dermal absorp tion of RDX is provided by in vitro studies showing that RDX can be absorbed through excised skin of humans and animals.

Oral Absorption Q uantitative information on blood levels following accidental exposure to RDX is limited to
two studies of accidental oral exposure s ( Küçükardali et al., 2003 ; Woody et al., 1986 ) and one

st udy of mixed dermal and inhalation exposure ( Ozhan et a l., 2003 )

. A number of qualitative case
stu dies of accidental exposures with similar toxic effects provide additional support that RDX is absorbed into the bod y ( Hett and Fichtner, 2002 ; Harrell- Bruder and Hutchins, 1995 ; Goldberg et al., 1 99 2 ; Ketel and Hughes, 1972 ; Hollander and Colbach, 1969 ; Stone et al., 1969 )

. The oral abs orption of RDX in humans was demonstrated in a case report of a 3

- year
- old male child who ingested plasticized RDX material that adhered to his mother’s work boots and clothing ( Woody et al., 19 86 ) . RDX was measured in serum
, urine , cerebrospinal fluid, and feces

. Based on a kinetic analy sis of the serum RDX concentrations, the dose was estimated to be 85 mg/kg and the first

order absorption rate constant s w ere estimate d to be 0.34

− 2.20 h ou r − 1 ( Woody et al., 1986 ) 2

. Sw eeney et al. (2012a) estimated the absorption rate constant for this same subject to be 0. 060 h ou r − 1 . The large range in the calculated absorption rate constant s resulted from uncertainty in the dose and time to peak serum RDX levels, and the models that were used to simulate the RDX toxicokinetics. Ozhan et al. (2003) summarized plasma RDX levels in five military personnel who

2 Woody et al. (1986) reported the absorption rate constants in units of L/hour; however, this appears to have been a typographical error for 1/hour or hour

− 1

.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-3 DRAFT―DO NOT CITE OR QUOTE were accidentally exposed to toxic l evels of RDX. Although Ozhan et al. (2003) reported that 1
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38 personnel were exposed through dermal and inhaled routes, secondary oral exposure may have occurred

. Based on PBPK model fits to the plasma RDX concentration data
, Swee n ey et al. (2012a)

est imate d a first

- order absorption rate constant of 0.033 h ou r − 1

. Küçükardali et al. (2003)

summ arized plasma RDX levels in five military personnel who ingested toxic levels of RDX (doses were not reported)

. RDX was detected in plasma of all patients within 3 hours after ingestion. Quantitative data to directly support estimates of oral bioavailability are available from studies in rats and mice ( Guo et al., 1985 ; Schneider et al., 1978
, 1977 )

. Results of single a nd

re peated oral dose studies in adult Sprague

- Dawley rats indicate that approximately 83

− 87% of the administered dose is absorbed from the gastrointestinal (GI) tract

. Following gavage administration of 50 mg/kg [ 14 C]

- RDX dissolved in dimethylsulfoxide ( DMSO), approximately 90% of the administered carbon

- 14 was recovered 4 days after dosing, with ~3% in feces, 34% in urine, 43% in expired air
, and 10% in the carcass ( Schneider et al., 1977 )

. It is unclear if the carcass incl udes the GI tract, which may include unabsorbed RDX

. Assuming that all of the carbon

- 14 in feces represents unabsorbed RDX (rather than RDX that was absorbed and subsequently secreted into the intestine), results of this study indicate that at least 87% of the administered dose was

a bsorbed from the GI tract

. Similar results were observed following repeated daily oral exposure of Sprague

- Dawley rats to [ 14 C]

- RDX by gavage (in DMSO) or drinking water for 1 week. Based on recovery of carbon

- 14 in urine and expired air and th e carbon -14 retained in carcass, approximately 83% (drinking water) to 85 % (gavage) of the administered dose was absorbed ( Schneider et al., 1978 ). An estimate of oral bioavailability in rats can also be obtained from data on blood RDX concentrations reported in Krishnan et al. (2009) . Male Sprague

- Dawley rats received a single
int ravenous (i.v.) (0.77 or 1.04 mg/kg) or oral (1.53 or 2.07 mg/kg, dissolved in water) dose of RDX. Estimates of bioavailability were obtained b ased on the reported blood RDX conce ntrations , terminal elimination rate constants ( estimated for this review by fitting the serum RDX data with a first

- order exponential function, see Table C -4 in the Elimination section be low) and the blood area under the curve ( AUC ) values (calculated for this review using the trapezoid rule extrapolated to infinite time)

. Calculated dose

- adjusted AUC values were 9.6 and 8.4 hours

- kg/L for the i.v. studies and 4.7 and 6.0 h ou rs- kg/L for the oral dosing studies

. These AUC values cor respond to estimated oral bioavailability ranging from 50 to 70%

. Recovery of administered radiolabel was incomplete (~90% of the administered carbon

- 14 ) in the studies ( Schneider et al., 1978
, 1977 ) ; therefore, it is
po ssible that oral bioava ilability is actually higher than 83

− 87%. Guo et al. (1985) reported data on bl ood tritium kinetics in mice that receive d i.v. ( 0.055 mg RDX or ~ 2.5 mg/kg body weight ) or oral doses (50 mg/kg) of [ 3 H]

- RDX

. Based on the reported blood tritium concentrations (% of dose/g) and terminal t 1/2 values for concentrations of tritium in blood (1.1 days for i.v. and 2.2 days for oral ), the corresponding AUC s of the blood concentration v ersus time curve s were calculated (calculated for this review using the trapezoid rule extrapolated to infinite time) to be 30 andSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-4 DRAFT―DO NOT CITE OR QUOTE 16 h ou rs- % dose/g for i .v. and oral dosing, respectively

. This corresp onds to an oral bioavailability 1
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37 of RDX

- derived tritium concentration of approximately 50% (i.e., 16/30).
In Yucatan miniature swine administered a single dose of [ 14 C]

- RDX (43

− 45 mg/kg as a suspension in carboxymethylcellulose), approximately 0.8

− 6% of the administered carbon

- 14 was eliminated in feces 24 hours after dosing ( Musick et al., 2010 ; Major et al., 2007 )

. Although results of

swine studies suggest that GI absorption of RDX was nearly complete, data cannot be used to determine a quantitative estimate of oral bioavailability because it is unlikely that fecal excretion of unabsorbed RDX was complete 24 hours after dosing ( Snoeck et al., 2004 ). Oral bioavailability of RDX appears to vary depending upon the physical form of RDX and the matrix (e.g., soil, vegetation) in which it is administered. Schneider et al. (1977) compared the

or al absorption of a single 100 mg/kg gavage dose of coarse granular [ 14 C]

- RDX as a slurry in isotonic saline with a single 50 mg/kg gavage dose of a finely powdered [ 14 C]

- RDX solution in saline in Sprague

- Dawley rats. Plasma carbon

- 14 levels were measured for 24 hours after dosing. For both [ 14 C]

- RDX preparations, peak plasma levels of carbon

- 14 were observed 24 hours after oral administration, with a higher 24

- hour plasma concentration for the 50 mg/kg dose (~4.7 μg/mL) compared to t he 100 mg/kg dose (3.12 μg/mL)

. Results of this study indicate that the oral bioavailability of RDX may be greater for the finely powered preparation than for the coarse granular preparation consistent with a greater surface area available for absorption with finely powdered RDX

. H owever, blood levels were only measured 24 hours after dosing, and the lower 24

- hour carbon

- 14 plasma concentration for the co a rse granular preparation could be due to slower absorption of coarse RDX granules compared with fine RDX powder , rather than lower overall bioavailability. Oral bioavailability of RDX is lower when administered as RDX

- contaminated soil or when RDX is in plant materials that were grown on RDX

- contaminated soils. Crouse et al. (2008)
inv estigated the oral bioavailability of RDX in contaminated soils relative to pure RDX by comparison of the AUC for the RDX blood concentration v ersu s time curves

. Adult male Sprague

- Dawley rats were administered oral doses (in gelatin capsules) of pure RDX (99.9% purity; neat) or an equivalent amount of RDX in contaminated soils from the Louisiana Army Ammunition Plant (LAAP) or Fort Meade (FM)

. Blood concentrations for rats dosed with LAAP soil (1.24 mg/kg) and neat RDX (1.24 mg/kg) peaked at approximately 6 hours . The AUC and 6 -h ou r RDX blood concentration were both approximately 25% lower for LAAP soil than for neat RDX ( p ≤ 0.003 for AUC), suggesting that oral bioavailability of RDX from LAAP soil was 25% lower than n eat RDX

. For FM soil (0.2 mg/kg), RDX blood concentrations peaked at 6 hours compared to 4 hours for neat RDX (0.2 mg/kg). The 4

- hour blood concentration for FM soil was approximately 15% lower than for neat RDX, although the AUC for FM soil was only 5% lower than for neat RDX (not statistically significant) . Collectively, these results suggest that RDX in soil is absorbed following oral exposure and that it has a lower bioavailability than neat RDX.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-5 DRAFT―DO NOT CITE OR QUOTE F ellows et al. (2006) showed that plants (alfalfa shoots and corn leaves) incorporated 1
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37 [ 14 C]

- RDX grown on [ 14 C]

- RDX

- amended soils

. [ 14 C]

- RDX and plant metabolites of [ 14 C]

- RDX were absorbed by voles following oral administration ( Fellows et al., 2006 )

. In adult male prairie voles ( Mi crotus ochrogaster ) fed diets containing RDX incorporated in plants for 5 or 7 days (average RDX dose per animal of 2.3 mg/kg

- day), 94.8 and 96 .6 % (respectively) of the administered carbon

- 14 was eliminated in excreta (combined feces, urine, and CO 2 ) and 3

− 5 % was retained in the carcass

. Feces, urine, and CO 2 contained 74

− 79, 13

− 14, and 8

− 12% of the total carbon

- 14 in excreta, respectively

. Based on carbon

- 14 eliminat ion in urine and CO 2 plus that retained by the carcass, the study authors estimated the oral bioavailability of plant

- derived RDX to be >20% . However, if biliary excretion of RDX and/or RDX metabolites i s a major excretory pathway in voles (as is the case with mice), estimates of bioavailability of plant

- derived RDX could be substantially higher. In Yorkshire piglets administered single doses of 5 or 10 mg/kg in gelatin capsules, peak plasma concentration s were proportional to the administered dose ( Bannon, 2006 ) . However, the
po tential for dose

- dependence has not been evaluated over a wide range of doses. RDX appears in blood within 1 hour following oral dosing ; however, the rate of absorption may depend upon the physical form or dose of RDX ( Bannon et al., 2009 ; Crouse et al., 2008 ; Bannon, 2006 ; Guo et al., 1985 ; MacPhail et al., 1985 ; Schneider et al., 1977 )

. Oral absorption of
RD X was rapid in LACA mice following stomach perfusion with [ 3 H]

- RDX (50mg/kg in met hyl cellulose) ( Guo et al., 1985 )

. The tritium radiolabel was detected in blood 15 minutes following do sing and the highest concentrations in blood were observed 30 minutes after dosing. Based on

an analysis of the blood tritium concentration kinetics, the authors estimated an absorption rate constant of 8.7 hour −1

. In Sprague

- Dawley rats administered single doses (0.2

− 18.0 m g/kg) of RDX in gelatin capsules, peak blood RDX concentrations were observed between 2.5 and 6 hours ( Bannon et al., 2009 ; Krishnan et al., 2009 ; Crouse et al., 2008 )

. Peak blood concentrations

oc curred at 24 hours af ter Sprague

- Dawley rats were administered a single oral dose (100 mg/kg) of co a rse granular RDX in saline ( Schneider et al., 1977 )

. Similarly, peak RDX blood concentrations
in sw ine administered single doses (5

− 10 mg/kg) of finally powdered (>98% pure) RDX in gelatin capsules occurred at 3

− 8 hours after dosing ( Bannon et al., 2009 ) , compared to 24 hours after a sing le dose (100 mg/kg) of RDX administered as a finely powdered in saline ( Bannon et al., 2009 ; Schneider et al., 1977 ) . Peak plasma concentrations in Yucatan miniature swine administered a sing le dose of [ 14 C]

- RDX (45 mg/kg as a suspension in carboxymethylcellulose) were re ached within 6

− 12 hours after dosing ( Musick et al., 2010 ). Krishnan et al. (2009) and Sweeney et al. (2 012a) estimated absorption rates in rats dosed with higher doses of co a rse granular RDX to be
app roximately 100 times slower than absorption rates in rats dosed with lower doses of finely powdered neat RDX preparations or neat RDX dissolved in aqueous vehicl es

. For example, Krishnan et al. (2009) estimated the absorption rate constant to be 0.75 h ou r −1 for rats dosed with

neat RDX dissolved in water (1.53 or 2.07 mg/kg) or neat RDX in a gelatin capsule (0.2 orSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-6 DRAFT―DO NOT CITE OR QUOTE 1.24 mg/kg, Crouse et al. (2008) ) , compared to 0.0075 h ou r − 1 for rats dosed with co a rse granular 1
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36 RDX (100 mg/kg, Schneider et al. (1977) )

. Inhalation Absorption Studies evaluating absorption of RDX in humans following inhalation exposure were not identified . Several case reports have documented seizures and other neurological effects in individuals exposed to RDX either in a manufacturing setting or in the course of using RDX as a cooking fuel ( Testud et al., 1996b ; Testud et al., 1996a ; Ketel and Hughes, 1972 ; Hollander and Co lbach, 1969 ; Kaplan et al., 1965 ; Barsotti and Crotti, 1949 ) . These reports suggest that RDX may be a bsorbed by the respiratory system . However, in several cases, the study authors were unable to clearly identify the primary route of exposure. In som e cases, incidental oral exposure was suggested . Studies in laboratory animals have not investigated the absorption of RDX following inhalation exposure.

Dermal Absorption
In vitro studies have demonstrated the dermal absorption of RDX in human and pig s kin ( Reddy et al., 2008 ; Reifenrath et al., 2008 ). Reddy et al. (2008) reported that 5.7% of the applied
RD X dose (in acetone) was absorbed into excised human skin in 24 hours

. Dermal absorption of [ 14 C]

- RDX from both a low

- carbon (1.9%) and a high

- carbon (9.5%) soil was also assessed in this system

. Approximately 2 .6 % of the RDX applied in the low

- carbon soil and 1.4 % applied in the high

- carbon soil was absorbed after 24 hours

. Thus, the dermal absorption of RDX from soils was reduced when compared with absorption from acetone, and absorption was lower in the high

- carbon soil than in the low

- carbon soil. Reifenrath et al. (2008) investigated the influence of skin surface moisture conditions, soil

ca rbon content
, and soil aging on the in vitro percutaneous penetration of [ 14 C]

- labeled RDX in excised pig skin

. Mea n skin absorption of RDX was higher for low

- carbon soil (1.2%), regardless of soil age and skin surface moisture

. Absorption and evaporation were < 1% for RDX regardless of soil type and age. While dermal absorption of certain munitions (e.g., 2,6

- dinitro toluene) is greatly enhanced by hydration of the skin surface, hydration had minimal effect on RDX mostly due to the lack of RDX volatility (e.g., < 0.5% evaporation).

**C.2.2. Distribution** 
Information on the distribution of absorbed RDX in humans is limited to a fe w cases of accidental exposures to RDX that provide data on the kinetics of RDX in blood and cerebr o spinal fluid ( Küçükardali et al., 2003 ; Ozhan et al., 2003 ; Woody et al., 1986 )

. Concentrations of RDX in

ser um and cerebr o spinal fluid were similar (11 and 9 mg/L, respectively) in a child 24 hours after ingesting an estimated dose of 85 mg/kg RDX ( Woody et al., 1986 )

. More extensive information on
ti ss ue distribution is available for animals, including mice, rats, and swine ( Musick et al., 2010 ; Bannon, 2006 ; Reddy et al., 1989 ; Guo et al., 1985 ; MacPhail et al., 1985 ; Schneider et al., 1977 )

. InSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-7 DRAFT―DO NOT CITE OR QUOTE these studies, RDX or radiolabeled RDX ([ 14 C] or [ 3 H) was administered by the oral, intraperitoneal 1
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29 (i.p.), i.v., o r intratracheal route and the distribution of the RDX or radiolabel was measured. Since metabolism of RDX can result in loss of carbon

- 14 or tritium from the parent compound, the distribution o f radiolabel will not necessarily reflect the distribution of RDX ( Sc hneider et al., 1977 )

. T o c ompare tissue distributions in studies in which animals received different doses by different routes of administration, distribution data are expressed as rati os of tissue RDX or radiolabel to that of either whole blood or plasma, whichever was reported. RDX in blood distributes into red blood cells and plasma to achieve concentration ratios that are close to unity. The plasma:whole blood carbon

- 14 ratio in sw ine that received a single oral dose of [ 14 C]

- RDX (4 5 mg/kg) was approximately 1 .3 ( Musick et al., 2010 ), and whole rat blood incubated in vitro with RDX had pl asma:red blood cell RDX ratios of approximately 1.0 ( Krishnan et al., 2009 ) . As a result of the
simi larity between plasma and whole blood concentrations, tissue distri bution is approximately equivalent when expressed as ratios of blood or plasma. Studies conducted in rats, mice
, and swine indicate that absorbed RDX distributes to many different tissues. Schneider et al. (1977) estimated the volume of dist ribution of RDX to be
app roximately 2.18 L/kg in rats, based on plasma RDX kinetics in rats that received a single i.p. dose of RDX (5

− 6 mg/kg)

. Consistent with this estimate are observations of tissue:blood (or plasma) concentration ratios that exceed 1 in various tissues, including brain (showing that RDX can cross the blood:brain barrier) , heart, kidney
, and liver ( Musick et al., 2010 ; Bannon et al., 2006 ; MacPhail et al., 1985 ; Schneider et al., 1977 )

. Distribution within the brain may not be uniform

. Ho wever, Bannon et al. (2006) observed tis sue:blood concentrations for RDX of approximately 4 in br ain hippocampus and 3.5 in brain cortex of swine that received a single oral dose of 10 mg/kg [ 14 C]

- RDX , although this is the only study that reported distribution for brain regions

. Reported
tissue :blood (or plasma) concentration ratios of RDX 24 hour s following a single dose (oral or i.p. ) were 1

− 9 for kidney, 1

− 7 for liver, and 1

− 3 for heart (Table C -2; Bannon (2006) ; ( Schneider et al., 19 77 ) . With repeated oral dosing (e.g.
, 30

− 90 days), tissue:blood ratios of RDX for these tissues are

co nsistently greater than unity ( Schneider et al., 1978 )

. There is no con sistent evidence that RDX

acc umulates in fat, although estimates of the fat:blood partition coefficient range from 6 to 8 and exceed that of other tissues ( Sweeney et al., 2012a ; Krishnan et al., 2009 ).Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-8 DRAFT―DO NOT CITE OR QUOTE **Table C-2.**  **Distribution of RDX or radiolabel from administered RDX a**  1

Animal Route Dose (mg/kg) Time (hrs) Brain Heart Kidney Liver Fat S ource Swine O ral 45 b 24 0.6 c 0.7 2.4 7.3 0.4 Musick et al. (2010)
Swine O ral 10 d 3 3.5

− 4.0 d 2 ≤1 <1 NA g Bannon et al. (2006)
Swine Oral 100 d 24 1. 5 c 1. 1 1. 2

− 1.9 0.9 1.8 Schneider et al. (1977)
Rat O ral 100 d 24 3.4 c 2.9 6.6 0.7 NA Schneider et al. (1977)
Rat i.p. 50 d 2 3.4 c 2.6 8.8 5.7 NA Schneider et al. (1977)
Rat i.p. 500 d ≤6.5 2.5 c 2.1 4.8 3.3 NA Schneider et al. (1977)
Mouse O ral 50 e 24 1 c 0.8 1 1.4 0.8 Guo et al. (1985)
Mouse i.v. 2.5 e 24 0.6 f 0.8 0.7 1.6 0.4 Guo et al. (1985) 2 3 4 5 6 7 8 9 10 11
12
13
14
15
16

17
18
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20
21
22
23
24
25
26
27
28
29
30 a Values are tissue:blood or tissue:plasma ratios following a single dose of either RDX, [ 14 C]

- RDX, or [ 3 H]

- RDX.
b Carbon

- 14

. c T issue:plasma

. d RDX

. e Tritium

. f T issue:blood

. g Not available

.

In rats, RDX can cross the placental:blood barrier resulting in exposure to the fetus, and can also be transported into maternal milk. Hess

- Ruth et al. (2007) detected RDX in the brain tissue of
po stnatal day ( PND ) 1 rat pups (concentrations ranged from 0.64 to 7.6 μg/g brain tissue, with no differences between males and females) after maternal exposure to 6 mg/kg RDX via gavage from gestation day 6 to PND 10 . RDX was also detected in maternal milk (concentra tions ranged from 3 to 5.7 μg/mL on PND 1 and from 0.7 to 3.1 μg/mL on PND 10).

**C.2.3. Metabolism** 
The metabolism of RDX is not well characterized. No studies investigating the metabolism of RDX in humans were identified. Studies in animals indicate that RDX undergoes extensive metabolism, including denitration, ring cleavage
, and generation of CO 2 . Predominant metabolic pathways and major organs involved in RDX metabolism have not been identified, although results of in vitro studies suggest a role for CYP45 0. RDX undergoes metabolism through processes that generate CO 2 . In Sprague

- Dawley rats administered a single 50 mg/kg gavage dose of [ 14 C]

- RDX, 43% was recovered as exhaled [ 14 CO 2 ] after four days ( Schneider et al., 1977 )

. Similarly, approximately 30−50 % of the radioactivity was

rec overed as exhaled [ 14 CO 2 ] in rats administered [ 14 C]

- RDX in saturated drinking water or daily
gavage for up to three months ( Schneider et al., 1978 )

. Metabolism of RDX to CO 2 was also ob served in prairie voles following dieta ry exposure (average RDX dose per animal of 2.3 mg/kg

- day) to [ 14 C]

- RDX incorporated plant materials for 5

− 7 days, with approximately 9% of the administered [ 14 C]

- RDX dose eliminated as exhaled [ 14 CO 2 ] ( Fellows et al., 2006 )

.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C-9 DRAFT―DO NOT CITE OR QUOTE Terminal metabolites of RDX have been identified in the urine of rats and swine, with very 1
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38 little urinary excretion of parent compound, indicating extensive metabolism of RDX

. Following oral administration of a single 50 mg/kg gavage dose of [ 14 C]

- RDX, 3.6% of the urinary radioactivity was identified as unmetabolized RDX ( Schneider et al., 1977 ) . Total urinary radiolabel accounted for about one third of the administered label and u nmetabolized RDX contributed 3−5% of total urinary radioactivit y in rats exposed to [ 14 C]

- RDX

- saturated drinking water for 1 or 13 weeks ( Schneider et al., 1978 )

. Similar results were observed in Yucatan swine administered a single 45 mg/kg oral dose of [ 14 C]

- RDX, with approximately 1

− 3.5% of the urinary radioactivit y as parent RDX ( Major et al., 2007 ) . Urinary metabolites were not characterized in these studies ( Schneider et al., 19 78 , 1977 )

. However, Schneider et al. (1978) cited unpublished findings in their laboratory tha t , in addition to carbon dioxide, other one

- carbon intermediates were produced including bicarbonate and formic acid. In the environment, the predominant breakdown products of RDX are methylenedinitramie and 4

- nitro -2- diazbutanal ( Sweeney et al., 2012b ; Paquet et al., 2011 )

. RDX metabolism in animals is le ss wel l understood

. N- nitroso RDX metabolites have been identified as derived through anaerobic metabolism ( ATSDR, 2012 ; Pan et al., 2007b ) . Based on characterization of RDX
met abolites in urine and plasma of miniature swine, metabolism of RDX appears to involve loss of nitro groups and ring cleavage ( Musick et al., 2010 ; Major et al., 2007 )

. The two principal urinary

met abolites identified in miniature swin e following a single oral dose of 43 or 45 mg/kg were 4- nitro

- 2,4

- diazabutanal and 4

- nitro

- 2,4

- diaza

- butanamide ( see Table C -3)

. Bhushan et al. (2003)

su ggested that the formation of the 4

- nitro

- 2,4

- diazabutanal metabolite occurred via denitration followed by hydroxylation and spontaneous hydrolytic decomposition resulting in ring cleavage and aldehyde formation . In the miniature swine gavage studies, o nly trace amounts of the nitrosamine RDX metabolites hexahydro -1- nitroso

- 3,5

- dinitro

- 1,3,5

- triazine (MNX) and 1

- nitro

3,5

- dinitroso

- 1,3,5 -triazacycloh exane (DNX) were found in urine ( Musick et al., 2010 ; M ajor et al., 20 07 ) . In plasma, most of the radioactivity existed as RDX, with trace levels of MNX, DNX, and 1, 3,5

- trinitroso

- 1,3,5

- triazacyclohexane (TNX). The s tudy authors suggested that the trace levels of MNX, DNX, and TNX in plasma may have been formed within the GI tract via sequential nitrogen reduction by intestinal bacteria ( Major et al., 2007 )

. The low levels of these compounds in urine and plasma were attributed to the nearly complete absorption of RDX from the GI tract, leaving little parent compound available for bacterial metabolism within the GI tract

. In a study of female deer mice ( Peromyscus maniculatus ) fed diets containing RDX at concent r ations of 12 and 120 mg/kg for 9 days
, MNX and DNX were identified in the stomach but TNX was not detected ( Pan et al., 20 07b ) . MNX and DNX were also measured in various organs of female B6C3F 1 mice provided
RDX in feed at doses of 0.38

− 522 mg/kg; TNX was also detected in some organ compartments, but not in the liver

. T he authors concluded that RDX can be m etabolized into its N

- nitroso compounds in mice, but did not identify a mechanism for the formation of the metabolites. Comparing RDX with MNX and TNX, RDX was the most potent compound at causing overt signs of toxicity (seizuresSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 10 DRAFT―DO NOT CITE OR QUOTE and mortality) as determi ned through identification of the median lethal dose using the EPA up

1
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3 4

6 7 8
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31

5 and

- down procedure in deer mice of varying ages ( Sm ith et al., 2009 ; Rispin et al., 2002 ).

**Table C-3.**  **Principal urinary metabolites of RDX in miniature swine 24 hours**  **after dosing with RDX**

Sample origin Metabolite name Metabolite structure Urine peak 1 M1 4

- N itro

- 2,4

- diazabutanal

Urine peak 2 M2 4

- N itro

- 2,4

- diaza

- butanamide

Source s: Major et al . (2007) ; Musick et al. (2010)

.
Al though the metabolic pathways and major tissues involved in RDX metabolism have not been identified , the re is some evidence for the involvement of the liver and CYP450 enzymes. Comparison of hepatic radioactivity to liver concentrations of RDX after a sin gle gavage dose to rats suggest ed the presence of RDX metabolites and a possible role for hepatic metabolism of RDX ( Schneider et al., 1977 )

. In vitro data indicate d that CYP450 may be involved in the metabolism of
RDX ( Bhushan et al., 2003 )

. Incu b ation of RDX with NADPH and rabbit liver CYP450 2B4 under

ana erobic condi tions produced nitrite, 4

- nitro

- 2,4

- diazabutanal, formaldehyde, and ammonium ion ( Bhushan et al., 2003 ) . The reaction rate under aerobic conditions was approximately one

- third of th at observed under anaerobic conditions . Several CYP450 inhibitors (el lipticine, metyrapone, phenylhydrazine, 1

- aminobenzotriazole, and carbon monoxide) decreased the formation of RDX metabolites (55−82% inhibition), providing support for the role of CYP450 in RDX metabolism.

**C.2.4. Excretion**  The primary routes of elimination of ab sorbed RDX are excretion of RDX and metabolites in urine, and exhalation of CO 2 liberated from metabolism of RDX ( Sweeney et al., 2012a ; Musick et al., 20 10 ; Krishnan et al., 2009 ; Major et al., 2007 ; Schneider et al., 1977 )

. Tritium derived from ad minist ered [ 3 H]

- RDX has been detected in mouse gall bladder contents, suggesting biliary secretion in this species ( Guo et al., 1985 ) ; however , biliary secretion of RDX or metabolites has not be en confirmed in other animal species

. Studies conducted in the rat and swine suggest that metabolism is the dominant mechanism of elimination of absorbed RDX

. In both species, metabolites dominated the carbon

- 14 distribution in urine of animals that received doses of [ 14 C]

- RDX, with RDX accounting for < 5% of the urinary carbon

- 14 ( Musick et al., 2010 ; Schneider et al., 19 77 ). Data on kinetics of elimination of absorbed RDX from blood are available from reports of accidental exposures of hu mans to RDX ( Table C -4 ). Woody et al. (1986) estimated the eliminationSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 11 DRAFT―DO NOT CITE OR QUOTE t 1/2 to be approximately 15 hours in a child who ingested approximately 85 mg of RDX per kg of 1
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9

10

11 12 13 14 15 16 17 18
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28 body weight

. The t 1/2 estimate was based on measured serum concentrations of RDX made between 24 and 120 hours following ingestion for RDX

. Based on plasma RDX concentration data from five adults exposed to RDX (m easurements made between 24 and 96 hours following
exposure ) ( Ozhan et al., 2003 ), a first

- order elimination t 1/2 of 20

− 30 hours was derived (calculated for this review by fitting the serum RDX data to a first

- order exponential function). It needs to be noted that it is not possible to draw reliable inferences from these values since they are based on accid ental, acute exposures and, in particular, the data for the child is based on a single set of

mea surements for one individual.

**Table C-4.**  **Elimination t 1/2**  **values for RDX or radiolabeled RDX**

Animal Route Dose (mg/kg) Time a t 1/2 (hr s ) S ource Human (child) O ral 85 b 24

− 120 hr s 15.0 c Woody et al. (1986) Human (adult) O ral NA 24

− 96 hr s 21

− 29 c, d Ozhan et al. (2003)

Rat i.v. 5

− 6 0.5 min

− 6 hr s 10 b Schneider et al. (1977)

Rat i.v. 0.8

− 1.0 30 min

− 10 hr s 4.6 c, d Krishnan et al. (2009)

Rat O ral 1.53

− 2.07 1

− 10 hr s 6.9 c, d Krishnan et al. (2009)

Mouse i.v. 2.5 1.5 min

− 24 hr s 26 d,e Guo et al. (1985)

Mouse O ral 50 1.5 min

− 168 hr s 53 d.e Guo et al. (1985)

a Observation period following exposure on which the t 1/2 values were based

. b Reported estimate of dose based on blood kinetics

. c Val u e for blood RDX

. d Calculated for this review based on reported plasma RDX or tritium data

. e Value for blood tritium

.
Th e kinetics of elimination of absorbed RDX from blood has been evaluated in rats and
mice ; in both species
, elimination kinetics were bi

- phasic ( Krishnan et al., 2009 ; Guo et al., 1985 ; Schneider et al., 1977 )

. As shown in Table C -4, e stimated t 1/2 values for the terminal elimination ph ase in rats range from 5 to 10 hours ( Krishnan et al., 2009 ; Schneider et al., 1977 ). Guo et al. (1 985) estimated the terminal elimination t 1/2 for RDX -derived tritium in mice to be approximately 26 hours following a single i.v. dose of [ 3 H]

- RDX (2.5 mg/kg)

. The elimination t 1/2 for tritium in mice following an oral dose of [ 3 H]

- RDX (50 mg/kg) was approximately 53 hours ( Guo et al., 1985 )

. The shorter elimination t 1/2 estimated for rats ( Krishnan et al., 2009 ; Schneider et al., 1977 )
comp ared to mice ( Guo et al., 1985 ) may reflect a real spe cies difference in elimination kinetics of
RDX , or may reflect a difference between the kinetics of elimination of RDX and of tritium derived from [ 3 H]

- RDX , which would include RDX metabolites.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 12 DRAFT―DO NOT CITE OR QUOTE **C.2.5. Physiologically**  **-B ased**  **P harmacokinetic**  **(PBPK) M**  **odels** 1

2
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13
1

15

16 17 18 19 20

4 Over view of Available PBPK Models
A PBPK model to simulate the pharmacokinetics of RDX in rats was first developed by Krishnan et al. (2009) and improved upon to extend the model to humans and mice ( Sweeney et al., 201 2a ; Sweeney et al., 2012b )

. The Sweeney et al. (2012a) model consists of six main

com partments: blood, brain, fat, liver, and lumped compartments for rapidly perfused tissues and slowly perfused tissues ( Figure C-1 ) . The model can simulate RDX exposures via the IV or oral
route

. Distribution of RDX to tissues is assumed to be flow

- limited . Oral absorption is represented in this model as first

- order uptake from the gastrointestinal tract into the liver, with 100% of the dose absorbed

. RDX is assumed to be cleared by first

- order metabolism in the liver

. However, there is no representation of the kinetics of any RDX metabolites. The acslX model code (Advanced Continuous Simulation Language, A egis, Inc., Huntsville, Alabama) was obtained from the authors of Sweeney et al. (2012a)

.

**Figur e**  **C-1. PBPK model structure for RDX in rats and humans** 

**.** Exposure to RDX is by the IV or oral route and clearance occurs by metabolism in the liver. See Table C-5 for definitions of parameter abbreviations. The GI tract is represented as 1- compartment in Krishnan et al. (2009) (on the left) and 2- compartments in Sweeney et al. (2012a) (on the right)

.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 13 DRAFT―DO NOT CITE OR QUOTE The parameter values used in the Sweeney et al. (2012a) rat model are listed in Table C-5

. 1
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32
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35
36
37
38 The physiological model parameter values for cardiac output, tissue volumes
, and blood perfusion of tissues were obtained from the literature ( Timch alk et al., 2002 ; Brown et al., 1997 )

. RDX tis sue:blood partition coefficients for liver (PL), brain (PB), and richly perfused tissues (PR) were estimated with an algorithm that relates the measured n

- octanol: water partition coefficient for RDX to reported compositions of water and lipids in specific rat tissues ( Poulin and Theil, 2000 ; Poulin and Krishnan, 1995 )

. Tissue:blood partition coefficients for fat (PF) and slowly perfused
tis sues (PS), as well as the m etabolic rate constant (KfC) were simultaneously optimized to fit rat blood RDX concentrations following IV doses of 0.77 or 1.04 mg/kg RDX ( Krishnan et al., 2009 )
pr oducing values of 5.57, 0.15, and 2.6 kg 0.33 /h ou r for PF, PS, and KfC
, respectively . While, the optimized value for PS is much smaller than that used by Krishnan et al. (2009) of 1.0
, the
op timized values for PF and KfC were fairly similar to those used by Krishnan et al. (2009) of 7.55, and 2.2 kg 0.33 /h ou r . The rat model with these parameter values also had good agreement with blood RDX concentrations after a 5

− 6 mg/kg IV exposure ( Schneider et al., 1977 ). The GI tract oral absorption rate constant (KAS) was optimized to fit the time

- course concentration data for rat oral dosing studies

. The Krishnan et al. (2009) model used a 1- c ompartment GI tract

. KAS was fit to the RDX blood concentrations in Krishnan et al. (2009) and th e model with this parameter value had good agreement with the blood RDX concentrations after 0.2 and 1.24 mg/kg oral exposures ( Crouse et al., 2008 ) . The value of KAS was adjusted to fit the
RD X blood concentrations in the Schneider et al. (1977) study. Sweeney et al. (2012a) modified the GI tract description by adding a second GI compartment and corresponding oral absorption parameters (KAS, KAD, and KT) to fit the blood concentrations from Krishnan et al. (2009)

. For the

ot her oral dosing studies the 2

- compartment GI model did not improve the model fit to the data, so KT was set equal to zero making the GI submodel equivalent to a 1

- compartment model. The value of KAS was adjusted separately to fit the oral studies with RDX in capsules ( Bannon et al., 2009 ; Crouse et al., 2008 ) and coarse grain RDX in a saline slurry ( Schneider et al., 1977 ). The Sweeney et al. (2012a) model fits to blood and brain RDX concentrations for rats were

mo stly within a factor of 1.5 of the experimentally measured values indicating a tightly calibrated model. Human RDX toxicokinetics were modeled with the same model structure as for rats. Values for the human physiological parameters such as tissue volumes and blood perfusion of tissues were obtained from the literature ( Brown et al., 1997 ) . Human absorption and metabolic clearance rate

con stants were optimized to fit observed RDX blood concentrations from a case study of inges tion by a 3

- year
- old boy ( Woody et al., 1986 ) , and a study where five soldiers were intentionally or

acc identally exposed to RDX powder via inhalation or dermal contact ( Ozhan e t al., 2003 )

. The

amo unts of RDX ingested in both studies were unknown
, so Sweeney et al. (2012a) estimated the do se amount by optimizing this parameter to fit the data ( Table C-5 ). Sweeney et al. (2012a)
in itially simulated each individual soldier’s blood level data separately . The resulting parameterSupplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 14 DRAFT―DO NOT CITE OR QUOTE values were similar, so data from the five soldiers were combined and the rate constants re

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23 24 estimated using the combined data . For comparison, the rat metabolic rate constant (KfC) was scale d to humans ; the rat KfC (from fitting to in vivo data) was multiplied by the ratio of the human to rat metabolic rate constants measured in vitro and by the ratio of human to rat microsomal protein levels ( Cao , 2008 ; Lip scomb and Poet, 2008 )

. The scaling from rats yielded a human in vivo
met abolic rate constant of 12.4 kg

- bw 0.33 /h ou r which is similar to the values Sweeney et al. (2012a) de rived by fitting the combined Ozhan et al. (2003) adult data (11.2 kg

- bw 0.33 /h ou r) and the Woody et al. ( 1986) child data (9.87 kg

- bw 0.33 /h ou r). Mouse RDX toxicokinetics were also modeled by Sweeney et al. (2012b) using the same

mo del structure as for rats . Values for the mouse physiological parameters such as tissue volumes

a nd blood perfusion of tissues were assumed to be the same as the body weight normalized parameter values in the rat model. RDX tissue:blood partition coefficients for liver (PL), brain (PB), and richly perfused tissues (PR) were estimated with an algorith m that relates the measured

n- octanol: water partition coefficient for RDX to reported compositions of water and lipids in specific mouse tissues ( Poulin and Theil, 2000 ; Poulin and Krishnan, 1995 ) . The rate constant for

met abolism (KfC), and the oral absorption rate constant (KAS), were optimized to fit measured mouse RDX blood concentrations ( Sweeney et al., 2012b ) . The KfC value estimated for the mouse (1 02 kg 0.33 /h ou r) is much higher than thos e estimated for rats and humans (2.6 and 11.2 kg0 .33 /h ou r, respectively); however, the KAS value (0.51/h ou r) fit to mouse data is similar to the value (0.83/h ou r) used in the RDX rat model for gavage in water. The Sweeney et al. (2012b)

mo del predictions of blood RDX concentrations were in good agreement with the experimental mouse gavage data reported in the same study.

**Table C-5.**  **Parameter values used in th e**  **Swe eney**  **et al. (2012**  **a )**  **and Sweeney**  **et al. (**  **2012 b)**  **PBPK models for RDX in rats, hum ans**  **, and mice** 

Parameter ( a bbreviation; units) Rat Human Mouse Source Body weight (BW; kg) 0.3 70 0.0206 Default values; study

- specific values used if available Cardiac output (KQC, L/h/kg 0.74 ) 15 14 15 Timchalk et al. (2002) ; Brown et al. (1997) Tissue volumes (fraction of BW)

Liver (KVL) 0.04 0.026 0.04 Timchalk et al. (2002) ; Brown et al. (1997) Brain (KVB) 0.012 0.02 0.012 Timchalk et al. (2002) ; Brown et al. (1997) Fat (KVF) 0.07 0.21 0.07 Timchalk et al. (2002) ; Brown et al. (1997) Richly perfused tissues (KVR) 0.04 0.052 0.04 Timchalk et al. (2002) ; Brown et al. (1997)Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 15 DRAFT―DO NOT CITE OR QUOTE Parameter ( a bbreviation; units) Rat Human Mouse Source Blood (KVV) 0.06 0.079 0.06 Timchalk et al. (2002) ; Brown et al. (19 97) Slowly perfused tissues (KVS) 0.688 0.523 0.688 0.91

- (KVL+KVB+KVF+KVR+KVV) Blood flows (fraction of cardiac output) Liver (KQL) 0.25 0.175 0.25 Timchalk et al. (2002) ; Brown et al. (1997) Brain (KQB) 0.03 0.114 0.03 Timchalk et al. (2002) ; Brown et al. (1997) Fat (KQF) 0.09 0.085 0.09 Timchalk et al. (2002) ; Brown et al. (1997) Slowly perfused tissues (KQS) 0.2 0.2449 0.2 Timchalk et al. (2002) ; Brown et al. (1997) Richly perfused tissues (KQR) 0.43 0.3811 0.43 1

- (KQL+KQB+KQF+KQS) Tissue:blood partition coefficients Liver (PL) 1.2 1.3 1.3 Krishnan et al. (2009) a Brain (PB) 1.4 1.6 1.6 Krishnan et al. (2009) a Richly perfused tissues (PR) 1.4 1.6 1.6 Krishnan et al. (2009) a Fat:blood PC (PF) 5.57 5.57 5.57 Sweeney et al. (2012a) b Slowly perfused tissues (PS) 0.15 0.15 0.15 Sweeney et al. (2012a) b

Metabolism

F irst

- order metabolic rate constant (KfC; kg 0.33 /hr) 2.6 9.87 (child); 11.2 (adults) 102 Sweeney et al. (2012a) b,c ; Sweeney et al. (2012b) d
GI absorption Dosing via gavage

Absorption from compartment 1 (KAS, /hr) 0.83 0.033 0.51 Sweeney et al. (2012a) c,d,e

Transfer from compartment 1 to compartment 2 (KT, /hr) 1.37 0 0 Sweeney et al. (2012a) c,d

Absorption from compartment 2 (KAD, /hr) 0.0258 0 0 Sweeney et al. (2012a) c,d

Dosing via capsule (KAS, /hr) 0.12 NA NA Sweeney et al. (2012a) e “coarse” RDX formulation (KAS, /hr) 0.005 NA NA Sweeney et al. (2012a) e 1 2 3 4 5 6 7 a P redicted from n

- octanol:water PC

. b O ptimized from rat IV data

. c O ptimized from human data of Ozhan et al. (2003) and Woody et al. (1986)

. d O ptimized from mouse oral data

. e O ptimized from rat oral data of Bannon et al. (2009)
, Crouse et al. (2008)
, Krishnan et al. (2009) , and Schneider et

al. (1977)

.Supplemental Information― Hexahydro

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The above PBPK model was evaluated and subsequently modified by EPA for use in dose

response modeling in this assessment

. This is detailed in the following section.

PBPK Model Evaluation and Further Development of the Sweeney et al. (2012a) and Sweeney et al

. (2012b) Models EPA evaluated and performed a quality control check of the PBPK models for RDX in rats, humans
, and mice published by Swe eney and colleagues ( Sweeney et al., 2012a ; Sweeney et al., 20 12b ) . The conclusions from these analyses are summarized below and then discussed in more de tail:

1) The model code and the parameter values matched the published reports.
2) The absorption of RDX from the GI tract did not use a consistent structure; for gavage doses the model used a 2

- compartment GI submodel and for other oral exposures (e.g., gelatin caps ule) the model used a 1

- compartment GI submodel . The model was revised to have a 1- compartment GI submodel to simulate all oral exposures with a consistent set of absorption parameters for each dosage formulation of administered RDX.
3) Additional oral rat data were identified from single dose studies ( MacPhail et al., 1985 ; Schneider et al., 1977 ) and subchronic studies ( Schneider et al., 1978 ) and were used for mod el calibration as well as independent comparison against model predictions.
4) In addition to the sensitivity analysis conducted by Sweeney et al. (2012b) on the mouse

mo del, a s ensitivity analysis in the rat and human models was performed.
5) The Sweeney et al. (2012b) mouse model used the same physiological parameters scaled to bo dy weight as the rat model. This mouse model was revised to use mouse specific physiological parameters.

The Sweeney et al. (2012a) model for rats was modified by changing the oral absorption

ra te constants (as discussed below) and the partition coefficients for the fat and slowly perfused tissues (PF a nd PS) as shown in Table C-6 . The partition coefficients for the fat and slowly perfused tissues were set to the values calculated by Krishnan et al. (2009) relating the measured n

- octanol:

wa ter partition coefficient for RDX to reported compositions of water and lipids in those tissues

. The fits to RDX blood time course data after iv exposure ( Figure C-2 ) are slightly worse than the Sweeney et al. (2012a) rat model because the Sweeney et al. (2012a) rat model optimized the fat: blood and slowly perfused tissue partition coefficients to fit the data.Supplemental Information― Hexahydro

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8 9 **F igure**  **C-2. EPA rat PBPK model predictions fitted**  **to observed RDX blood concentrations in male and female SD rats**  **following intravenous exposure.** A) data from Krishnan et al. (2009) (0.4 kg rats) and B) data from Schneider et al. (19 77) (simulation of 0.25 kg rats and 5.5 mg/kg dose f or 0.2

− 0.25 kg rats and 5

− 6 m g/kg dose).

**Table C-6.**  **Parameters values used in the EPA application of the rat, human ,**  **and mouse models**

Parameter ( a bbreviation; units) Rat Human Mouse Source Body weight (BW; kg) 0.3 70 0.0206 Default values shown; study

- specific values used if available Cardiac output (KQC; L/h/kg 0.74 ) 15 14 15 Timchalk et al. (2002) ; Brown et al. (1997)

Tissue volumes (fraction of BW) Liver (KVL) 0.04 0.026 0.055 Timchalk et al. (2002) ; Brown et al. (1997) Brain (KVB) 0.012 0.02 0.017 Timchalk et al. (2002) ; Brown et al. (1997) Fat (KVF) 0.07 0.21 0.07 Timchalk et al. (2002) ; Brown et al. (1 997) Richly perfused tissues (KVR) 0.04 0.052 0.071 Timchalk et al. (2002) ; Brown et al. (1997) Blood (KVV) 0.06 0.079 0.049 Timchalk et al. (2002) ; Brown et al. (1997) Slowly perfused tissues (KVS) 0.688 0.523 0.648 0.91

- (KVL+KVB+KVF+KVR+KVV) Blood flows (fraction of cardiac output)Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 18 DRAFT―DO NOT CITE OR QUOTE Parameter ( a bbreviation; units) Rat Human Mouse Source Liver (KQL) 0.25 0.175 0.25 Timchalk et al. (2002) ; Brown et al. (1997) Brain (KQB) 0.03 0.114 0.03 Timchalk et al. (2002) ; Brown et al. (1997) Fat (KQF) 0.09 0.085 0.09 Timchalk et al. (2002) ; Brown et al. (1 997) Slowly perfused tissues (KQS) 0.2 0.2449 0.2 Timchalk et al. (2002) ; Brown et al. (1997) Richly perfused tissues (KQR) 0.43 0.3811 0.43 1

- (KQL+KQB+KQF+KQS) Tissue:blood partition coefficients and metabolism Liver (PL) 1.2 1.3 1.3 Krishnan et al. (2009) a Brain (PB) 1.4 1.6 1.6 Krishnan et al. (2009) a Richly perfused tissues (PR) 1.4 1.6 1.6 Krishnan et al. (2009) a Fat:blood PC (PF) 7.55 7.55 7.55 Krishnan et al. (2009) a Slowly perfused tissues (PS) 1.0 1.0 0.9 Krishnan et al. (2009) a
First

- order metabolic rate constant (KfC; kg 0.33 /hr) 2.6 9.87(small boy); 11.2(soldiers) 77 Sweeney et al. (2012a) b,c ; Sweeney et al. (2012b) d

Absorption Absorption from GI to liver (KAS; /hr) Table C

- 7 1.75 0.6 F it to rat, human , and mouse oral data

Absorption from lung to blood (Klung; /hr) 0.75 F it to human data

1 2 3 4 5 6

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16 a P redicted from n

- octanol:water PC

. b O ptimized from rat IV data

. c O ptimized from human data of Ozhan et al. (2003) and Woody et al. (1986)

. d O ptimized from mouse oral data, and differs from that obtained by Sweeney et al. (2012b)

.

Absorption of RDX from the GI T ract As discussed above in the oral absorption section under toxicokinetics ( Section C .2.1) the
rate of oral absorption depends on the physical form of RDX

. This was demonstrated by comparing the Schneider et al. (1977) studies which used gavage do s es of 100 mg/kg of coarse granular RDX and 50 mg/kg finely powdered RDX and observing the 50 mg/kg finely powdered RDX had a higher peak plasma level. These results are likely explained by the smaller surface area to mass ratio of the coarse grain RDX leading to slower dissolution and absorption. To follow the rule of model parsimony (i.e., use no more parameters than needed for the best fit to all of the data), oral absorption was modeled with a 1

- compartment GI tract sub -model for all simulations

. To account for the differences in absorption due to the physical form of RDX
,Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 19 DRAFT―DO NOT CITE OR QUOTE separate rate const ants for RDX oral absorption were optimized to fit measured blood 1
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22 23 concentrations of RDX according to the type of dosing formulation and the model fits obtained with
t he EPA’s revised parameters for rats are shown in Fi gur e C-3 to C -5

. The oral dosing formulations were grouped into four categories; RDX dissolved in water, RDX in capsules, fine grain and coarse grain RDX. The absorption rate constant for RDX dissolved in water was optimized to the data in the Krishnan et al. (2009) study ( Figure C-3 )

. The absorption rate constant for RDX in capsules was
op timized to the data in the Crouse et al. (2008) and Bannon et al. (2009) studies ( Figure C-4 )

. The abs orption rate constant for fine grain RDX was optimized to the data descri bed below ( A dditional RDX time

- course data ) in the MacPhail et al. (1985) and Schneider et al. (1977) studies ( Figure C-7 )

. Th e Schneider et al. (1977) study was used to estimate the absorption rate constants for coarse
gr ain RDX ( Figure C-5 ; as represented by the fit to the data obtained by the solid curve at 100% bioavailability)

. Overall, the fits of the EPA revised model to the blood time

- course data of these studies are similar to the fits of the Sweeney et al. (2012a) rat model

. The fits to RDX brain time

co urse data after oral exposure to RDX in capsules ( Figure C-6 A) are similar to the fits of the Sweeney et al. (2012a) rat model . The absorption rate constants for each dosing formulation are list ed in Table C -7

. A n alternative to varying the absorption rate constant (KAS) for each RDX formulation would be to vary the oral bioavailability, in effect modifying the administered exposure
concentration

. Therefore, the sensitivity of the model fit to variations in oral bioavailability was examined in Figure C- 5 and a n analysis of model sensitivity to oral bioavailability was conducted as discussed further in the s ection , S ensitivity analysis of the rat PBPK model

.

**Table C-7.**  **Doses, dosing formulations, and absorption rate constants in animal and human studies** 

Formulation Study Dose Estimated KA (/hr)

RDX dissolved in water Krishnan et al. (2009) 1.53, 2.07 mg/kg single gavage 1.75

Schneider et al. (1978) ~5

− 8 mg/kg

- d drinking water 90 d Dry RDX in capsules a Crouse et al. (2008) 0.2, 1.24 mg/kg sing le dose 0.35 Bannon et al. (2009) 3, 18 mg/kg single dose Fine grain RDX in saline slurry Schneider et al. (1977) 50 mg/kg single gavage 0.19

MacPhail et al. (1985) b 50 mg/kg single gavage Coarse grain RDX in saline slurry Schneider et al. (1977) 100 mg/kg single gavage 0.00497

24Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 20 DRAFT―DO NOT CITE OR QUOTE a Capsules were filled with dry RDX from stock solution of acetone and acetone was evaporated off. 1 2 b RDX particle size was ≤ 66 μm in diameter suspended in a 2% solution of carboxymethylcellulose.

3

4 5 6

7 **Figur e**  **C-3. EPA rat PBPK model predictions fitted to observed RDX**  **blood concentrations following oral exposure to RDX dissolved in water.**  Male and female SD rats (0.4 kg) were dosed by gavage ( Krishnan et al., 2009 ).

8

9 10 11 12 13

14 **F igure**  **C-4. EPA rat model predictions fitted to observed**  **RDX blood concentrations following oral exposure to RDX in dry capsules.**  The ingested RDX doses were A) 0.2 and 1.24 mg/kg RDX in male SD rats (0.4 kg, data from Crouse et al. (2008) ) and B) 3 and 18 m g/kg RDX in male and female SD rats (0.3 5 kg, data from Bannon et al. (2009) ) for KAS = 0.35/h ou r.Supplemental Information― Hexahydro

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2 3 4 5 6 7 8 9 10 11 12

13

14

15 16 17 18 19 **F igure**  **C -5.**  **Effect of varying oral absorption parameters on EPA rat model**  **predictions fitted to observed RDX blood concentrations following oral exposure to coarse grain RDX. Symbols denote observed RDX blood**  **concentrations measured in male SD rats (0.225 kg) resulting from oral doses**  **of 1 00**  **mg/kg RDX (**  **Schneider et al., 1977 )** 

**.** The absorption rate constant (KAS) fit to these data assuming 100% bioavailability resulted in the same estimate (KAS = 0.00497/h ou r) as obtained by Sweeney et al. (2012a) . Alternatively, for KAS fixed at the value fit to fine grain RDX in a saline slurry (KAS = 0.019/h ou r fit to data from Schneider et al. (1977) and MacPhail et al. (1985)
, Figure C-7 ) the estimate of oral bi oavailability fit to the RDX blood concentrations was 35%. A bioavailability of 40% and KAS = 0.019/h ou r is also shown for comparison.

**F igure**  **C -6.**  **EPA rat model predictions fitted to observed RDX brain tissue concentrations following oral exposure to RDX.**  A) 3 and 18 mg/kg RDX in dry capsules (0.35 kg male and female rats data from Bannon et al. (2009) ); best fit KAS = 0.3 5/h ou r

. B) 50 mg/kg fine grain RDX in a saline slurry (0.25 kg male and female rats data from MacPhail et al. (1985) ); best fit KAS = 0.019/h ou r.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 22 DRAFT―DO NOT CITE OR QUOTE Additional RDX T ime -C ourse D ata 1
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9 The EP A revised models were simultaneously fitted against additional RDX time course data (not used in the original Sweeney et al. (2012a) model calibration) from two studies in which
anim als received oral dos es of fine grain RDX ( MacPhail et al., 1985 ; Schneider et al., 1977 ) in Figure C-7 and RDX brain time course data from a study in which animals received oral dose s of fine grain RDX ( Figure C-6 B)

. Overall the calibrated EPA rat model predictions are within a factor of 1.5 of the measured values from different data sets, and therefore likely provides a more robust estimated paramete r.

10

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27 **Figur e**  **C-7. EPA rat model predictions fitted to observed RDX blood**  **concentrations following oral exposure to fine grain RDX in a saline slurry.** Oral doses of 50 mg/kg RDX were administered to A) male SD rats (0.225 kg) ( Schneider et al., 1977 ) and B) male and female SD rats (0.25 kg) data ( MacPhail et al., 198 5 ) . Best fit KAS = 0.019/h ou r. Following calibration, the EPA model was further tested by comparison with results from two other subchronic oral studies in male and female rats ( Schneider et al., 1978 ) . These were a
gavag e study where 20 mg/kg RDX was administered in saline slurry and a drinking water study where rats were provided with RDX

- saturated drinking water (50

− 70 μ g/mL) ad libitum for which the study authors estimated a daily dose between 5 and 8 mg RDX/kg BW

. It is striking that the observed RDX blood concentrations in the gavage study ( Figure C-8 , symbols) were virtually the same as or only slightly elevated compared to the blood concentrations reported in the drinking water exposures , with a n approximately threefold lower daily admin istered dose in the drinking water study ( Figure C-9 , symbols)

. This is counter to the expectation that higher doses cause higher blood levels and is discussed further below. EPA’s modified PBPK model was set up to simulate drin king water exposures with a non

continuous sipping pattern based on Spiteri (1982) which assumed 80% of the consumption toSupplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 23 DRAFT―DO NOT CITE OR QUOTE occur episodically at night when the rats were awake 3

. The model predicts blood concentrations to 1
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25 increase in proportion to the total dose; for the gavage study the model predictions yielded an RDX bl ood concentration approximately three fold higher than the reported mean blood concentrations ( Fi gure C-8 ) while for the subchronic drinking water study the model fit the data reasonably well ( Figure C-9 ). It is possible that multiple mechanisms such as elimination of unabsorbed RDX or metabolic induction may explain why the observed RDX blood concentrations did not increase in proportion to the higher administered dose in the gavage studies compared to the drinking water. Elimination of unmetabolized RDX may be an insignificant factor in the single

- dose studies used for calibration of the absorption constant for the RDX in saline slurry, but for repeated, hi gher doses this elimination route could be significant

. Schneider et al. (1978) found similar RDX concentrations in th e feces of rats in the gavage and drinking water studies (3.1 ± 2.0 and 2.7 ± 1.3 micrograms RDX per gram dry weight feces, respectively)

. The total recovery of radioactivity in feces was also similar in the gavage study (4.8 ± 0.8%, week 1 only) and drinking water study (4.4 ± 0.6%, measured over the course of the study)

. Thus
, the difference in fecal elimination for the tw o routes does not appear significant. It is also possible that metabolic induction occurred during the repeated dosing of RDX in the gavage study leading to the lower RDX blood concentrations seen. The reasonably good fits to the model to the drinking wa ter data set demonstrated achievement of regular periodic levels, indicate a lack or much lower extent of metabolic induction over time from those repeated doses
, possibly because the dose rate was lower: 5

− 8 mg/kg

- day vs. 20 mg/kg

- day in the gavage study

. Overall the reasonable agreement of the modified EPA RDX rat model with the subchronic drinking water data support use of the model in estimating and extrapolating blood levels following chronic
exposure at or below this exposure range (5

− 8 mg/kg

- day), p articularly in drinking water

.

3 A constant drinking water ingestion rate interspaced between episodes of no ingestion was assumed. Each 12

- hour awake period consisted of 8 cycles that alternated between 1.5

- hour cycles of frequent sipping (continuous ingestion) and zero ingestion for 45 minutes each. Each 12

- hour sleeping period consisted of 4 cycles with regular sipping periods of 30 min utes followed by 2.5 hours of no ingestion.Supplemental Information― Hexahydro

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17 **Figure C-8.**  **Comparison of EPA rat model predictions with data from** Schneider et al. (19 78) **for the subchronic gavage study**

**.** Model fits and mean observed RDX blood concentrations resulting from daily gavage doses of 20 mg/kg RDX for 90 days to male and female SD rats (0.225 kg)

. The RDX in saline slurry was assumed to be coarse grained with oral absorption rate constant KAS = 0.00497/h ou r.

**Figure C-9.**  **Comparison of EPA rat model predictions with data from** Schneider et al. (19 78) **for the subchronic drinking water study**

**.** Model fits and mean obse rved RDX blood concentrations resulting from a daily estimated dose of 6.5 mg RDX/kg

- day for 90 days to male and female SD rats (0.225 kg) . The large peak to trough change in the simulation results from model representation of the daily oral ingestion of drinking water primarily during the waki ng state

. The oral absorption rate constant for RDX dissolved in water was used (KAS = 1.75/h ou r).Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 25 DRAFT―DO NOT CITE OR QUOTE Simulating E xposures in H umans 1
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. In the studies of humans with measured RDX blood concentrations by Woody et al. ( 1986) and Ozhan et al. (2003) the RDX doses were unknown and so the doses were also
op timized t o fit the data

. The model predictions for the Woody et al. (1986) data using the best fit valu es of dose = 58.9 mg/kg, KAS = 1.75/h ou r, and KfC = 9.87 kg 0.33 /h ou r are shown in Figure C- 10

. The model predictions for the Ozhan et al. (2003) data using the best fit values of an oral dose = 3. 5 mg /kg, KAS = 1.75/h ou r, and KfC = 9.87 kg 0.33 /h ou r are shown in Figure C- 11

. EPA’s calibration of the model differed in another important respect from that carried out by Sweeney et al. (2012a)

. As previously mentioned, Sweeney et al. (2012a) simulated the soldier s’

ex posure from the Ozhan et al. (2003) study as an oral exposure, although the study report states th at the exposure was via inhalation and dermal routes. An inhalat ion or dermal exposure could change the amount of RDX reaching the blood compared with an oral exposure due to first pass metabolism in the liver after oral absorption. Dermal absorption was not considered by EPA to be a significant route of RDX exposure and therefore was not modeled

. This decision is supported by a study that used excised human skin and reported only 5.7% of the applied dose was absorbed into the skin by 24 hours post dosing ( Reddy et al., 2008 )

. The model was modified to simulate an
in halation exposure and compared with the data from Ozhan et al. (2003)

. There are insufficient da ta on blood: air partitioning to modify the Sweeney et al. (2012a) model with a lung

co mpartment; therefore, inhalation exposure was modeled in an approximate manner as a direct input to the blood with an optimized absorption rate to represent absorption from air containing RDX into the blood

. The soldiers’ inhalation exposure was simulated as a continuous 8

- hour exposure (i.e., assuming that the soldiers were ex posed occupationally during an 8

- hour workday), and for the same dose of 3.5 mg RDX/kg that was estimated by Sweeney et al. (2012a)

. The model

ass umed 100% of the inhaled dose was absorbed and the absorption rate constant was optimized to fit the measured blood concentrations of RDX . The model predictions were in good agreement with the RDX blood concentrations reported by Ozhan et al. (2003) as shown in Figure C- 11

.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 26 DRAFT―DO NOT CITE OR QUOTE 1

2 3 4 5

7

8 9 10 11 12 13

14
15
16
17
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20

6 **F igure**  **C**

**- 10** 

**. EPA human model predictions fitted to observed RDX blood**  **concentrations resulting from an accidental ingestion of RDX by a 14.5**

**- kg boy**  **(** Woody et al., 1986 **)**

**.** The best fit values were KAS = 1.75/h ou r, dose = 58.9 mg/kg and KfC = 9.87 kg 0.33 /h ou r.

**F igure**  **C**

**- 11** 

**. EPA human model predictions fitted to observed RDX blood**  **concentrations resulting from accidental exposure to adults assumed to be**  **70 kg**  **(** Ozhan et al., 2003 **)**

**.** For an assumed oral exposure the best fit values were KAS = 1 .75/h ou r, dose = 3.5 mg/kg and Kf C = 9.87 kg 0.33 /h ou r . For the same 3.5 mg/kg dose and metabolism rate constant an inhalation exposure found a best fit value for Klung of 0.75/h ou r.
Sensitivity A nalysis of the R at and H uman PBPK M odel s A sensitivity analysis was performed to see how each model parameter affects the model output

. A sensitivity coefficient, defined as the change in a specified dose metric due to a 1% increase in the value of a parameter, was calculated for each parameter in the rat and human models

. This analysis was carried out for both short (24 hours following a single oral dose of 1.5 mg/kg RDX) and longer term (90 days of repeated oral dosing with 1.5 mg/kg RDX) exposures for the dose metric of blood area

- under

- the

- curve (AUC)

. Parameters with sensitivity coeffic ientsSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 27 DRAFT―DO NOT CITE OR QUOTE greater than 0.1 in absolute value (i.e., considered sensitive) are presented in Table C-8

. For the 1
2
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24 25 26 27 28 29 30 31

32
33
34 blood AUC dose metric the only sensitive RD X- s pecific parameter is the metabolic clearance rate (KfC) . This sensitivity is likely because bioavailability was assumed to be 100% and metabolism is the only route of elimination in the model

. These assumptions mean that all administered RDX will be a bsorbed and metabolized, in other words the blood AUC is proportional to the dose and inversely proportional to the metabolic clearance rate constant. For the parameter values in this model the rate of metabolism is relatively slow compared to the transpo rt of RDX between other tissues and the site of metabolism in the liver, so that the blood AUC is not sensitive to parameters that impact transport such as KQC and KQL . Because the metabolic clearance rate constant is scaled to BW and by liver volume, the blood AUC is also sensitive to these parameters. The sensitivity analysis by Sween ey et al. (2012b) for the AUC of RDX in the liver found the model was

sens itive to the liver:blood partition coefficient (PL) in addition to the same parameters (KfC, KVL and BW) found for the blood AUC. The model is also very sensitive to oral bioavailability with a sensitivity coefficient of 0.8 in the case of the rat model

. As discussed above in the oral absorption section of toxicokinetics ( C .2.1), estimates of the bioavailability of RDX range from 50 to 87% or greater and may depend upon the physical form of RDX ( Krishnan et al., 2009 ; Schneider et al., 1978
, 1977 ) . However, as seen in Figu re C- 5, it was not possible to identify the bioavailability and the absorption rate (KAS) as separate parameters by fitting to the available RDX blood concentration time course. Introducing oral bioavailability as an additional unknown parameter and recalibrating the model did not appear to provide an advantage. Therefore, 100% bioavailability was assumed in the model and acknowledged as an uncertainty.

**Table C-8.**  **Sensitivity coefficients for rat and human RDX PBPK models**

Parameter Rat sensitivity coefficient Human sensitivity coefficient

F ractional liver volume (KVL)

− 1

− 1

B ody weight (BW) 0.3 0.3

M etabolic rate constant (KfC)

− 1

− 1

Parameters with sensitivity coefficients < 0.1 in absolute value are considered not sensitive, and are listed below:

cardiac output (KQC); fractional blood flow to all tissues (liver, KQ L ; fat KQF; slowly perfused tissues KQS; brain KQB); fractional tissue volume of fat (KVF)
, br a in (KVB)
, and blood volume (KVV); blood partition coefficie nts t o all tissues (liver PL, fat PF, rapidly perfused PR, slowly perfused PS, brain PB); absorption rates from GI (KAS, KT, KAD)

Simu lating E xposures in M ice Physiological parameters specific to mice were obtained from the literature ( Brown et al., 199 7 ) and are shown in Table C-6

. The partition coefficients calculated for mice by Sweeney et al.Supplemental Information― Hexahydro

- 1,3,5

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- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 28 DRAFT―DO NOT CITE OR QUOTE (2012b) were used, which includes the liver, brain
, and richly perfused tissues

. The partition 1
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17

18

19

20 21 22 coefficients for the fat and slowly perfused tissues from the Sweeney et al. (2012b) mouse model

were n ot used because they were estimated via optimization of fits to rat iv data. Instead the partition coefficient for fat tissues was set equal to the value calculated by Krishnan et al. (2009) for rat fat tissue, 7.55. The partition coefficient for slowly p erfused tissues ( 0.9 ) was calculated for mouse tissues using the same methodology as Krishnan et al. (2009)

. The rate constants for oral abso rption and metabolism were optimized to fit the data from Sweeney et al. (2012b) for mouse blood RDX concentrations

. The model predictions were in good agreement with the RDX blood concentrations reported by Sweeney et al. (2012b) as shown in Figure C- 12

. The only information on RDX metabolism in the mouse c omes from a study by Pan et al. (20 13)

. Pan et al. (2013) measured nitrosamine RDX metabolites of RDX (MNX, DNX, TNX
, the lat ter representing a minor metabolic pathway) in mice at the end of a 28

- day exposure to RDX in feed ( ad lib itum )

. T hese measurements were a single time point without controlling the time between the last RDX ingestion and measurement , and were t herefore judged not to be sufficient for use in parameteriz ing a PBPK model of the nitrosamine metabolites.

**Figur e**  C

**-** 12

**. Comparison of EPA mouse PBPK model predictions with data**  **from oral exposure to RDX dissolved in water** 

**.** Model fits and mean and standard deviation of observed RDX blood concentrations in female B6C3F 1 miceSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

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This document is a draft for review purposes only and does not constitute Agency policy. C- 29 DRAFT―DO NOT CITE OR QUOTE (0.0205 kg) for doses of 35, 60
, and 80 mg/kg with KAS = 0.6/h ou r and KfC = 1 2

3
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24 25 26 27 28

29 30 31

32 33

34 35 36 37 38 77 k g 0.33 /h ou r . Experimental data from Sweeney et al. (2012b)

. Summary of C onfidence in PBPK M odels for RDX Overall, good fits to the rat, mouse, and human time

- course data for RDX internal

c oncentrations were obtained . For the rat and human models, calibration was based generally on fitting to more than one data set obtained from different studies originating in different laboratories or accidental exposure settings. Predictions from the rat model compared well with data from a subchronic drinking water study that was not used in model calibration.
T he metabolic rate constant used in the human model was fit to limited data from accidentally exposed humans ; however
, th e value of the metabolic rate constant has additional support from in vitro experimental data . The rat meta bolic rate constant, fit to mult iple experimental data sets, was scaled to humans using the ratio of human to rat rate constants measured with in vi tro methods . This scaled value of the human metabolic rate constant was very similar to the rate constant estimated by fitting the model to the human data

. The congruence in values increases the confidence in using the EPA

- modified PBPK model for predict ing human blood RDX concentrations. There are several uncertainties in these models, listed below
, the most significant of which pertain to the mouse PBPK model

. The mouse model was based on a single data set, which used high RDX doses to obtain detectable RDX blood concentrations, and the type of additional data that increased the confidence in the rat and human models are not available for mice. The additional data not available for mice are the lack of in vitro measurements of RDX metabolism by mouse cells and lack of quantification of potential routes of RDX elimination in mice. Overall these uncertainties result in lower confidence in the mouse model than i n the rat an human models.

1) R DX is readily metabolized in several species, yet there are no data on the toxicokinetics of RDX metabolites in the rat and human. Some data are available for the n

- nitrosoamine metabolites (a minor metaboli c pathway) in mice
, but the data are too sparse to help better paramet e rize a PBPK model. Consequently, the PBPK models used in this assessment do not inco r porate the kinetics of RDX metabolites.
2) T he available toxicokinetic data are not sufficient to uniquely identify a parameter value for RDX oral bioavailability . Consequently the model assumes 100% bioavailability even though some studies in rats suggest a lower bioavailability is likely.
3) The human model is based on single accidental exposures, and the exposure concentra tions are not known.
4) The only route of clearance of RDX used in the models is that of total metabolism , which appears reasonable for the rat for which only roughly 5% of the RDX was detected unmetabolized in urine and feces. However, no data on the excretion of RDX are available for the mouse

. This inability to properly characterize the fraction of RDX that is metabolized in the mouse is problematic considering some evidence t o indicate that the roleSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

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This document is a draft for review purposes only and does not constitute Agency policy. C- 30 DRAFT―DO NOT CITE OR QUOTE of metabolism in RDX toxicity may be different across s pecies

. This uncertainty decreases 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 the confidence in the mouse PBPK model.
5) T he PBPK model for the mouse is based on a single data set. This single data set is used to fit both the absorption and metabolic rate constants. There are no in vitro data to independently estimate the metabolic rate constant for the mouse. Consequently, the confidence in the mouse model parameter values is low.
6) T he analytical detection limit in the mouse pharmacokinetic study is too high to enable detection at the lower doses. T he lowes t dose at which RDX wa s detected was 35 mg/kg; this concentration wa s high enough to manifest some toxicity in the chronic mouse bioassay . The measured blood concentration at the 35 mg/kg dose is based on the level measured from one single ani mal (others were non

- detects or excluded as outliers)

. This decreases the confidence in the calibration of the mouse PBPK model.
7) T he metabolic rate constant as estimated by the PBPK model for mice was 30

- fold higher than the rat (after accounting for body weight differences)
, suggesting the toxicokinetics of RDX could be significantly different in the mouse than in the rat. Mice may have more efficient or higher expression of the CYP P450 enzymes. Alternatively, mice may have other unknown metabolic path ways responsible for metabolizing RDX. Identifying the specific CYP enzymes, measuring expression levels
, and in vitro metabolic rate constants in mice would allow for in vitro scaling from rats to mice
, which could be used to independently evaluate the mouse metabolic rate constant. Given the high sensitivity of the model to the metabolic rate constant
, this uncertainty in the mouse toxicokinetics significantly decreases confidence in using the mouse PBPK model for predicting mouse blood RDX concentratio ns.

Model C ode for RDX PBPK M odel U sed in the A ssessment The PBPK acslX model code is made available electronically through the HERO database. All mod el files may be downloaded in a zipped workspace from HERO at https://hero.epa.gov/index.cfm/project/page/project_id/2216 ; search for “ RDX PBPK files in acsIX for mat.”



## C.3. **HUMAN STUDIES**
Table C- 9 presents a summary of case reports of humans acutely exposed to RDX

. Table C- 10 provides a summary of the methodologic features of the available epidemiology studies of RDX.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 31 DRAFT―DO NOT CITE OR QUOTE **Table C-9.**  **Summary of case reports of exposure to RDX**  1

Reference,

n umber of c ases,
e xposure setting Exposure Effects observed Comments

Barsotti and Crotti (1949)

17 males among 20 male Italian workers (1939

− 1942)

Manufacturing Inhalation of RDX powder during the drying, cooling, sieving, and packing processes of its manufacture Generalized convulsions of a tonic

- clonic (epilepti c) type followed by postictal coma; loss of consciousness without convulsions; vertigo; vomiting and confusion; transient arterial hypertension

Symptoms occurred either without prodromal symptoms or were preceded by several days of insomnia, restlessness, irritability, or anxiety Tobacco and alcohol use were considered by the study authors to be aggravating factors

Kaplan et al. (1965)

5 males among 26 workers (April

− July 1962)

Manufacturing Inhalation, ingestion, and possible skin absorption as a result of the release of RDX dust into the workroom air during the dumping of dried RDX powder, screening and blending, and cleanup of spilled material without adequate ventilation Sudden convulsion s or loss of consciousness without convulsions; few or no premonitory symptoms (e.g., headache, dizziness, nausea, vomiting); stupor, disorientation, nausea, vomiting, and weakness; no changes in complete blood counts or urinalysis Mild cases of RDX intoxi cation may have been masked by viral illness with nonspecific symptoms (e.g., headache, weakness, upset gastrointestinal [GI] tract)

. No method was available for determining RDX concentrations in air; recovery was complete without sequelae Merrill (1968)

2 males

Wartime, Vietnam Ingestion of unknown quantity of C

- 4 with

m oderate amounts of alcohol Coma, vomiting, hyperirritability, muscle twitching, convulsions, mental confusion, and amnesia; kidney damage (oliguria, gross hematuria, proteinuria, elevated blood urea nitrogen [BUN]); liver or muscle damage (high aspartate t ransaminase [AST]), leukocytosis Confounding factors included ingestion of C

- 4 while intoxicated with ethanol (vodka), which may have caused GI symptoms, and smoking (1−1.5 packs of cigarettes per day) Stone et al. (1969)

4 males (March

− December 1968)

Wartime, Vietnam Ingestion of 180 g (patient 1), or 25 g (patients 2, 3) of C

- 4 (91% RDX) Generalized seizures, lethargy, nausea, vomiting, fever, muscle soreness, headaches, twitching, (semi)comatose, headaches, hematuria, abnormal lab oratory findings, muscle injury, elevated AST; no kidney damage

For the patient who ingested the highest dose, anemia and loss of recent memo ry present after 30 d Troops ingested small quantities of RDX to get a feeling of inebriation similar to that induced by ethanolSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 32 DRAFT―DO NOT CITE OR QUOTE Reference,

n umber of c ases,
e xposure setting Exposure Effects observed Comments

Hollander and Colbach (1969)

5 males (June 1968

− January 1969)

Wartime, Vietnam Inhalation (all 5 cases) and ingestion of unknown quantity of C

- 4 (2 cases) Tonic

- clonic seizures; nausea and vomiting occurred before and after admission; hyperirritability, muscle twitching, convulsions, mental confusion, and amnesia; kidney damage (oliguria, gross hematuria, proteinuria, elevated BUN); liver or muscle damage (high AST), leukocytosis; sympto ms cleared by the next day except for amnesia (in case 2), oliguria (lasted for 4 d), and gross hematuria (decreased by 9 th hospital day) Knepshield and Stone (1972)

6 males

Wartime, Vietnam Ingestion of C

- 4, range 25−180 g, average 77 g Generalized seizure s, coma, lethargy, severe neuromuscular irritability with twitching and hyperactive reflexes, myalgia, headache, nausea, vomiting, oliguria, gross hematuria, low

grade fever; abnormal laboratory findings (neutrophilic leukocytosis, azotemia, elevated AST) Includes data on 2 patients from Merrill (1968)

Ketel and Hughes (1972)

18 males ( December 1968

− December 1969)

Wartime, Vietnam Inhalation while cooking with C- 4 and possible ingestion Central nervous system signs: confusion, marked hyperirritability, involuntary twitching of the extremities, severe prolonged generalized seizures, pro longed postictal mental confusion and amnesia, renal (oliguria and proteinuria;

one case of acute renal failure requiring hemodialysis) and GI toxicity (nausea, vomiting) C

- 4 was cut with the same knife used to stir/prep food

Woody et a l. (1986)

1 male child (August 1984)

Manufacturing Ingestion of plasticized RDX from mother's clothing and/or boots; estimated ingested dose of 1.23 g RDX was normalized to the patient's body weight (84.82 mg/kg) Seizures before and after admission; e lectroencephalogram (EEG) revealed prominent diffuse slowing that was greatest in the occipital regions with no evidence of epileptiform activity; elevated AST on admission and after 24 hr; within 24 hr the child was extubated and intensive care withdrawn; normal mental status and normal neurological examination at discharge Mother worked at an explosive plant in which RDX was manufactured in a plasticized formSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 33 DRAFT―DO NOT CITE OR QUOTE Reference,

n umber of c ases,
e xposure setting Exposure Effects observed Comments Goldberg et al. (1992)

1 male

Nonwartime Ingestion after chewing a piece (unknown size) of “Semtex” plastic explosive 4 hr before first seizure Frontal hea dache and two tonic

clonic seizures; progressively disseminating petechial rash suggestive of meningococcal infection apyrexial; normotensive; no photophobia; no neurological abnormalities; florid petechial rash over the face and trunk; lacerated tongue

. Initial results included: leukocyte count of 10.8 × 10 9 /dl (87% neutrophils); hemoglobin, platelet count, coagulation screen, serum and cerebrospinal fluid (CSF) biochemistry all within normal limits; CSF and blood bacteriologically unremarkable

. Shortly following admission, his headache and rash disappeared

. There were no further seizures
Harrell

- Bruder and Hutchins (1995)

1 male

Nonwartime Ingestion of C

- 4 (chewing on a piece of undetermined size) Tonic

- clonic seizures, postictal state, EEGs were normal; brisk deep tendon reflexes

Testud et al. (1996b)

1 male

Manufacturing Inhalation and possible dermal exposure during the RDX manufacturing process Malaise with dizziness, headache, and nausea progressing to unconsciousness and general ized seizures without involuntary urination or biting of the tongue

. Blood chemistries were in the normal range and blood alcohol content was null.
Testud et al. (1996a)

2 males

Manufacturing Inhalation and possible dermal exposure during the RDX manufacturing process Sudden loss of consciousness and generalized seizures; blood serum level of 2 mg/L RDX measured Smoker and alcohol drinker

Hett and Fichtner (2002)

1 male

Nonwartime Ingestion of a cube (1 cm across) of C

- 4 Nausea and vomiting , tonic

- clonic seizure lasting 2 min, followed by two seizures of about 30 sec each; myoclonic jerks in all limbs; petechial hemorrhages around face and trunk after seizuresSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 34 DRAFT―DO NOT CITE OR QUOTE Reference,

n umber of c ases,
e xposure setting Exposure Effects observed Comments Küçükardali et al. (2003)

5 males

Nonwartime Ingestion (accidental) of 37−250 mg/kg body weight RDX during military training via food contaminated with RDX Abdominal pain, nausea, vomiting, myalgia, headache, generalized weakness, repetitive tonic

- clonic convulsions, lethargic or comatose between se izures, hyperactive deep tendon reflexes, sinusal tachycardia

. Elevated serum levels of AST and ALT

. Kidney damage was observed

. Plasma RDX levels 3 hr after ingestion ranged from 268 to 969 pg/mL. Davies et al. (2007)

17 males

Nonwartime Ingestion of unknown quantity C

- 4 under unclear circumstances, but unrelated to recreational abu se Seizures, headache, nausea and vomiting; hypokalemia and elevated creatine kinase, lactate dehydrogenase, and phosphate were noted in all but two patients

. Metabolic acidosis only occurred in two patients directly following seizures Patient histories may have been affect by the fact that the incident was the focus of a military police investigation

Kasuske et al. (2009)

2 males

Nonwartime Ingestion of C

- 4 after handling explosive ordnance Seizures, postictal state, confusion, drowsiness, headache, nausea and vomiting

. Blood work revealed high white blood cell count and elevated creatine phosphokinase)

. Proteinuria and gross hematuria were observed.
1Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 35 DRAFT―DO NOT CITE OR QUOTE **Table C-**  **10 . E**  **pidemiologic s**  **tudies of**  **RDX : summary of methodologic features**  1

Reference,

s etting and d esign Participants,

s election, f ollow

- up Consideration of l ikely

s election b ias Exposure

m easure and

r ange Outcome

m easure Consideration of l ikely

c onfounding Analysis and

r esults d etails Sample s ize Occupational e xposure s tudies
Ma and Li (1992) (China) a Industrial workers Translated article Details of industrial process and subject selectio n framework not reported

. Referents chosen from same plant; age, employment duration, and education similar across groups. Participation rate, NR. Sparse reporting of details on subject recruitment and participation. Details of exposure monitoring not

r eported

. Two groups of exposed subjects: Group A, intensity, 0.407 (0.332) mg/m 3 [mean (SD)], daily cumulative, 2.66 (1.89) mg/m 3

. Group B, intensity, 0.672 (0.556) mg/m 3 ; daily cumulative, 2.53 (8.40) mg/m 3

. Neurobehavioral battery administered by train ed personnel

— memory retention, simple reaction time, choice reaction time, letter cancellation, block design. No adjustment for other risk factors, e.g., alcohol consumption

. No consideration or attempt to distinguish TNT. Comparisons of mean scaled

sco re on memory retention, letter cancellation, or block design test; mean time on reaction tests; and total behavioral score

. Variance (F test), linear and multiple regression, and correlation analysis. 60 exposed ; Group A (n = 30; 26 males, 4 females) ; Group B (n = 30); 32 referents (27 males, 5 females).

Hathaway and Buck (1977) ; (United States) Ammunition workers 2022 workers (1,017 exposed to open explosives (TNT, RDX, HM X), 1,005 referents) at five U.S. Army ammunition plants (IA, IL, TN)

. Participation rate, 76% Potential h ealthy worker effect. Atmospheric samples of all operations with potential exposure to open explosives taken in 1975

. Range: ND

− 1.57 mg/m 3

. 70 exposed workers with RDX at >0.01 mg/m 3 [the LOD]; mean: 0.28 mg/m 3 [SD not Liver function, renal function, and hematology tests [blood]. Workers with TNT exposure excluded from exposed groups

. Comparison of mean value; prevalence of elevated value on

an individual test. 69 RDX exposed (43 male s, 26 females), 24 RDX/HMX exposed (all males), 338 referents (237 males, 101 females).Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 36 DRAFT―DO NOT CITE OR QUOTE Reference,

s etting and d esign Participants,

s election, f ollow

- up Consideration of l ikely

s election b ias Exposure

m easure and

r ange Outcome

m easure Consideration of l ikely

c onfounding Analysis and

r esults d etails Sample s ize exposed, 71% referents. presented]

. Job title used to initially identify exposed or unexposed status and reassigned to one of two exposed groups (nondetected, >0.01 mg/m 3 ) based on subject’s IH monitoring results.
West and Stafford (1997) ; (United Kingdom) Ammunition workers (Case

- control study) 378 of 404 subjects (excluded 3 deaths and 23 subjects with unknown addresses) previously studied in 1991, 32 cases with

a bnormal hematology test and 322 controls with normal hematology test. Participation rate among eligible subjects, 97% cases, 93% controls. Former employees who were unable to work due to advers e health outcome were not included in the 1991 prevalence study. Semiquantitative assessment; source of IH data not reported

. Interviews with current and past employees and job title analysis to identify potential exposure to 100 agents, including RDX

. Exposure surrogate was >50 hrs duration and intensity (low [1

− 10 ppm], moderate [10

− 100 ppm], and high [100

− 1, 000 ppm])

. RDX exposure Abnormal hematology value in 1991 survey indicating possible meylodysplasia: neutropenia (2.0 x 10 9 /l), low platelet count (<150 x 109/l), or macrocytosis (mean corpuscular volume = 99 fl or >6% macrocytes). Cases and controls are not matched and statistical analyses are not adjusted for other risk factor or occupational exposures

. No considerat ion or attempt to distinguish TNT Unadjusted prevalence odds ratios and 95% CIs

. Analyses limited to males because of low frequency of exposure to females. 32 cases (29 males, 3 females) and 322 controls (282 males, 12 females).Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 37 DRAFT―DO NOT CITE OR QUOTE Reference,

s etting and d esign Participants,

s election, f ollow

- up Consideration of l ikely

s election b ias Exposure

m easure and

r ange Outcome

m easure Consideration of l ikely

c onfounding Analysis and

r esults d etails Sample s ize prevalence (males), 83%. 1 2 3 4 5 6 a Ma and Li (1992) describes symptoms reported by subjects during a physical examination but are not included in the evidence table because resp onses for individual symptoms were not identified.

ATSDR = A gency for Toxic Substances and Disease Registry, HMX = cyclotetramet hylenetetranitramine, IA = Iowa, IL = Illinois, mg/m 3 = milligram/cubic meter,

ND = n ot detected, SD = standard deviation, TN = Tennessee, TNT = trinitrotolueneSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. C- 38 DRAFT―DO NOT CITE OR QUOTE 

## C.4. **OTHER PERTINENT TOXICITY INFORMATION** 1

2
3
4
5
6
7
8
9
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31
32
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34
35
36
37 **Genotoxicity** R DX

. RDX has tested negative in a variety of in vitro tests for genotoxicity, including mutation assays in multiple strains of Salmonella typhimurium (with or without metabolic activation), recombination in Saccharomyces cerevisiae strain D3, and forward mutations in both V79 Chinese hamster lung cells and mouse lymphoma L5178Y cells. However, in genotoxicity assays designed to be more sensitive, RDX did show some positive results . For example, when the concentration of S9 was doubled, the mutagenicity of RDX was about twice that of background

. RDX also showed positive mutagenic results with metabolic activation in a chemilumine scent assay (Mutatox assay) . In some cases, the interpretation of testing data for RDX was complicated by the tendency of the compound to precipitate out of DMSO solution (the usual vehicle) at concentrations ≥250 μg/mL ( Reddy et al., 2005 )

. As with other studies of RDX, the purity of the test compound

wa s unknown in several (particularly older) studies

. A summary of the results of in vitro genotoxicity studies of RDX is presented in Table C- 11

. RDX has produced negative results in all reverse mutati on assays in S. typhimurium that use standard levels of the metabolic activation system (S9). No evidence of reverse mutation was observed in S. typhimurium (strains TA98, TA100, TA1535, TA1537, and TA1538), either with or without the addition of S9 metab olic activating mixture ( Neuwoehner et al., 2007 ; George et al., 20 01 ; Lachance et al., 1999 ; Tan et al., 1992 ; Minor et al., 1982 ; Cholaki s et al., 1980 ; Whong et al., 19 80 ; Simmon et al., 1977a ) . One exception is a finding of weak mutagenic activity of RDX to S. ty phimurium strain TA97a (mutagenicity index = 1.5

− 2.0) ( Pan et al., 2007a )

. However, this assay

us ed a high percentage of S9 fraction (9% instead of 4%), indicating that extensive metabolic activation is needed to elicit a mutagenic response. RDX did not cause gene recombination in S. cerevisiae strain D3 at concentrations up to 23 μg/mL, with or without metabolic activation ( Simmon et al., 1977a )

. The study authors noted th at the negative findings should be considered in the context of the low concentrations te sted

. Similarly, RDX did not induce forward mutations (HGPRT locus) in V79 Chinese hamster lung cells, with or without metabolic activation, although minimal cytotoxicity was observed at 180 μM ( Lachance et al., 1999 ) . However, RDX produced revertants in two of three trials in the M utatox

ass ay with the bacterium V. fisheri when tested at doses up to 2.5 μg/tube, with and without S9 ( Arfsten et al., 1994 )

. In the pres ence of S9, a dose

- response was observed; in the absence of S9, no do se- response relationship was detected ( Arfsten et al., 1994 )

. RDX did not induce forward

mut ations in mouse lymphoma L5178Y cells with or without metabolic activation ( Reddy et al., 20 05 )

. During an accompanying range

- finding study, precipitates occurred at doses ≥250 μg/mL,
sug gesting that concentrations of RDX in DMS O reported beyond 250 μg/mL may not be accurate. RDX did not induce unscheduled DNA synthesis, with or without metabolic activation, using human diploid fibroblasts (WI

- 38 cells) when tested in DMSO at concentrations up to 4,000 μg/mg;Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 39 DRAFT―DO NOT CITE OR QUOTE however, precipitati on of RDX at high concentrations in cell culture media makes interpretation of 1
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37 these results difficult ( Di lley et al., 1979 ) . Only two in vivo studies are available for the genotoxicity of R DX; these are summarized in Table C- 12

. RDX did not decre ase the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE), nor did it induce micronucleated PCEs in an in vivo mouse bone marrow micronucleus assay in young adult male CD

- 1 mice ( Reddy et al., 20 05 )

. RDX was considered negative for the induction of dominant lethal mutations in male CD rats fe d RDX at nominal doses from 0 to 50 mg/kg

- day for 15 weeks prior to mating with untreated virgin females ( Cholakis et al., 1980 )

. Females sacrificed at midgestation showed no statistically
si gnificant effects on number of corpora lutea, implants, or the number of li ve or dead embryos ( Cholakis et al., 1980 ). Metabolites of RDX

. Several metabolites of RDX, N

- nitroso derivatives of the parent compound (mononitroso, dinitroso, and trinitroso compounds, abbreviated MNX, DNX, and TNX, respectively) ( Musick et al., 2010 ; Major et al., 2007 ) , have been tested directly for genotoxicity ( Pan et al., 200 7a ; George et al., 2001 ; Snodgrass, 1984 ) . Miniature pigs were used to detect these

tr ace metabolites because the swine model of the GI tract more closely resembles that of humans ( Major et al., 2007 ) ; an identification and quantification of RDX metabolites in humans has not been

con ducted

. A summary of the results of in vitro and in vivo genotoxicity studies of metabolites of RDX is p resented in Table C- 13

. Pan et al. (2007a) studied the mutagenicity of two metabolites, MNX and TNX

. These
met abolites were not mutagenic in TA97a at normal levels of S9 but clearly mutagenic at enhanced concentrations of S9 (4% versus 9% S9). The observation that these metabolites were positive in TA97a is likely due to this strain’s higher sensitivity for frameshift mutations that occur at a cluster of cytosine residues in the mutated gene for histidine synthesis in this strain ( Pan et al., 2007a )

. Th ese metabolites were also weakly mutagenic in TA102, again with high levels of S9

. S train TA102 was developed with an A:T base pair at the site of mutation and its sensitivity was increased by the addition of some 30 copies of a plasmid containing the mutant gene that is available for back
mutation

. This strain is sensitive to many oxidative mutagenic compounds ( Levin et al., 1982 )

. Ot her metabolites with potential human relevance identified in the urine of miniature pigs have not been assessed for their genotoxicity ( Major et al., 2007 ). The genotoxicity of MNX was positive in three out of five assays conducted for the U.S. Army ( Snodgrass, 1984 )

. MNX was positive with or without metabolic activation in the mouse lymphoma for ward mutation assay at the thymidine kinase locus, fo r chromosomal aberrations in Chinese hamster ovary cells, and in the primary rat hepatocyte unscheduled DNA synthesis assay. MNX was not considered positive in S. typhimurium (strains TA98, TA100, TA1535, TA1537, and TA1538), either with or without the ad dition of S9 metabolic activating mixture or in an in vivo dominant lethal mutation assay in mice

. However, this study is of limited use due to a significant lack of details including information on dosing, raw data, and statistical reporting.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 40 DRAFT―DO NOT CITE OR QUOTE In summary, RDX is not mutagenic or genotoxic in vitro or in vivo in typical assays used to 1
2
3
4 detect genotoxicity

. In two in vitro studies using more sensitive assays and conditions for detecting mutagenicity, RDX was found to be positive . Several studies showed tha t the N

- nitroso metabolites are genotoxic, but the formation and quantification of these metabolites in humans is not known.Supplemental Information― Hexahydro

- 1,3,5

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This document is a draft for review purposes only and does not constitute Agency policy. C- 41 DRAFT―DO NOT CITE OR QUOTE **Table C-**  **11**

**. Summary of in vitro studies of the genotoxicity of RDX**  1

Endpoint Test system Dose/ concentration a Results b

Comments Reference Without activation With activation
Genotoxicity studies in prokaryotic organisms

Reverse mutation Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 1,000 μg/plate

− − Metabolic activation with S9 Cholakis et al. (1980)

Reverse mutation S. typhimurium TA1535, TA1537, TA1538 TA100, TA98 14 μg/plate

− − Effect of disinfection treatments on mutagenicity tested: RDX was not mutagenic in any strain before or after disinfection treatment with chlorine or ozone Simmon et al. (1977b)

Reverse mutation S. typhimurium TA98, TA100 250 μg/plate

− − Study authors noted that results were consistent with literature George et al. (2001)

Reverse mutation S. typhimurium TA98, TA100 1 mg/plate

− − Metabolic activation with S9 Tan et al. (1992)

Reverse mutation S. typhimurium TA98, TA100 1,090 μg/plate

− − High S9 activation (9%) used Pan et al. (2007a)

Reverse mutation S. typhimurium TA97a 32.7 μg/plate

− ± High S9 activation (9%) used; study authors concluded that RDX “required intensive metabolic activation” to exhibit mutagenicity in this strain Pan et al. (2007a)

Reverse

m utation Vibrio fischeri 0.004 μg/tube ± + Mutatox assay with metabolic activation (S9) Arfsten et al. (1994)

Reverse mutation ( u mu test) Salmonella choleraesius subsp. chol. (prior Salmonella typhimurium ) TA1535/p SK1002 20.6 μg/mL

− − No observed effect concentration; tested at highest concentration where the induction rate was below 1.5 for the first time and the growth factor was below 0.5 Neuwoehner et al. (2007)Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 42 DRAFT―DO NOT CITE OR QUOTE Endpoint Test system Dose/ concentration a Results b

Comments Reference Without activation With activation

Reverse mutation ( NM2009 test) S. choleraesius subsp

. chol

. NM2009, TA1535/pSK1002/pNM12 20.6 μg/mL

− − No observed effect concentration; tested at highest concentration where the induction rate was below 1.5 for the first time and the growth factor was below 0.5 Neuwoehner et al. (2007)

Induction of the sfiA gene (SOS chromotest) Escherichia coli PQ37 20.6 μg/mL

− − No observed effect concentration; tested at highest concentration where the induction rate was below 1.5 for the first time and the growth factor was below 0.5 Neuwoehner et al. (2007)

Reverse mutation S. typhimurium , TA98, TA100 24.8 μg/mL

− − No observed effect concentration; metabolic activation with S9 Neuwoehner et al. (2007)

Reverse mutatio n S. typhimurium TA98, TA100 2.6 μg/mL

− − No observed effect concentration; metabolic activation with S9; minimal cytotoxicity was observed at 180 μM Lachance et al. (1999)

Reverse mutation S. typhimurium TA1535, TA1536, TA1537, TA1538 TA100, TA98 30.8 μg/mL

− − Metabolic activation with S9 Cotruvo et al. (1977)

Genotoxicity studies in nonmammalian eukaryotic organisms
Recombination induction S. cerevisiae D3 23 μg/mL

− − Study authors concluded that this microorganism did not appear to be useful for detecting mutagenicity in several compounds tested Simmon et al. (1977b)

Recombination induction S. cerevisiae D3 30.8 μg/mL

− − Metabolic activation with S9 Cotruvo et al. (1977) Genotoxicity studies in mammalian cells

F orward mutation Chinese hamster lung fibroblasts V79 cells 40 μg/mL

− − Minimal c ytotoxicity observed at 40 μg/mL (limit of solubility) Lachance et al. (199 9)Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 43 DRAFT―DO NOT CITE OR QUOTE Endpoint Test system Dose/ concentration a Results b

Comments Reference Without activation With activation

Mutation L5178Y mouse lymphoma cells 500 μg/mL

− − No or low cytotoxicity seen at these concentrations; however, precipitate was observed >250 μg/mL Reddy et al. (2005)

Unscheduled DNA synthesis; DNA repair WI

- 38 cells , human diploid fibroblasts 4,000 μg/mL

− − Precipitates were observed at concentrations of RDX ≥40 μg/mL Dilley et al. (1979)

1 2 3 a Lowest effective dose for positive results; highest dose tested for negative results.
b + = positive; ± = equivocal or weakly positive; − = negative

.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 44 DRAFT―DO NOT CITE OR QUOTE **Table C-**  **12**

**. Summary of in vivo studies of the genotoxicity of RDX**  1

Endpoint Test system Dose/ concentration Results Comments Reference
In vivo genotoxicity studies in mammalian systems

Micronucleus formation CD

- 1 mouse bone marrow Single dose of 62.5, 125, or 250 mg/kg No significant decrease in PCE:NCE ratios; no induction of micronucleated PCE at any dose 250 mg/kg was maximum tolerated dose determined in dose range

- finding study Reddy et al. (2005)

Dominant lethal mutations Male CD rats dosed and mated with untreated female rats 0, 5, 16, or 50 mg/kg

- d for 15 wk No statistically or biologically significant effects on fertility; determined to be negative for the induction of lethal mutations Males in the high

- dose group experienced lower food consumption and weight gain compared with all other groups Cholakis et al. (1980)

2
3Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. C- 45 DRAFT―DO NOT CITE OR QUOTE **Table C-**  **13**

**. Summary of in vitro and in vivo studies of the genotoxicity of RDX metabolites**  1

Endpoint Test system Dose/ concentration a Results b

Comments Reference Without activatio

n With activatio

n Genotoxicity studies in prokaryotic organisms

Reverse mutation Salmonella typhimurium TA97a, TA102 22 μg/plate

− + Mono and trinitroso metabolites (MNX and TNX); high S9 activation (9%) used Pan et al. (2007a)

Reverse mutation S. typhimurium TA1535, TA1537, TA1538, TA98, TA100 500 μ g/plate + + Positive only for TNX; MNX and DNX were negative George et al. (2001)

Reverse mutation S. typhimurium TA1535, TA1537, TA153 8, TA98, TA100 NR

− − Mononitroso metabolite, MNX; metabolic activation with S9 Snodgrass (1984)

Genotoxicity studies in mammalian cells

— in vitro
Forward mutation Mouse lymphoma thymidine kinase NR + + Mononitroso metabolite, MNX; metabolic activation with S9 Snodgrass (1984)

Chromosomal aberrations Chinese hamster ovary cells NR

− + Mononitroso metabolite, MNX; metabolic activation with S9 Snodgrass (1984)

Unscheduled DNA synthesis; DNA repair Primary rat hepatocytes NR + N D Mononitroso metabolite , MNX; additional metabolic activation not required with S9 Snodgrass (1984)

In vivo genotoxicity studies in mamm alian systems

Dominant lethal mutations Male mice dosed and mated with untreated female mice NR

− N D Mononitroso metabolite , MNX; additional metabolic activation not required with S9 Snodgrass (1984)

2 3 4 5 6 a Lowest effective dose for positive results; highest dose tested for negative results.
b + = positive; ± = equivocal or weakly positive; − = negative

.
ND = not determined , N R = not reportedSupplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. D-1 DRAFT―DO NOT CITE OR QUOTE 1 

# APPENDIX D. DOSE

- RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES FOR EFFECTS OTHER THAN CANCER AND THE DERIVATION OF CANCER RISK ESTIMATES

This appendix provides technical detail on dose

- response evaluation and determination of 2
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11

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13

14 points of departure (POD) for relevant toxicological endpoints. T he endpoints were modeled using the EPA’s Benchmark Dose Software (BMDS, Versions 2.4 ). Sections D.1 (noncancer) and D.2 **Erro r!**  **Reference source not found.** (cancer) describe the common practices used in evaluating the model fit and selecting the appropriate model for determining the POD, as outlined in the Benchmark Dose Technical Guidance Document ( U.S. EPA, 2012 ). In some cases it may be
app ropriate to use alternative methods, based on statistical judgement; exceptions are noted as necessary in the summary of the modeling results.



## D.1. **BENCHMARK DOSE MODELING SUMMARY FOR N ONCANCER**  **E NDPOINTS** 

The noncancer endpoints that were selected for dose

- response modeling are presented in Table D-1. For each endpoint, the doses and response data used for the modeling are presented.

**Table D-1.**  **Noncancer endpoints selected for dose** 

**- response modeling for**  **RDX**

Endpoint and

reference Species/sex Dose Incidence/ t otal (%) or

m ean ± SD (n umber of animals)

Convulsions Crouse et al. (2006) a Female F344 rat 0 mg/kg

- d 4 8 10 12 15 0/10 (0%) 0/10 (0%) 2/10 (20%) 3/10 (30 %) 5/10 (50%) 5/10 (50%)

Male F344 rat 0 mg/kg

- d 4 8 10 12 15 0/10 (0%) 0/10 (0%) 1/10 (10%) 3/10 (30%) 8/10 (80%) 7/10 (70%)Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. D-2 DRAFT―DO NOT CITE OR QUOTE Endpoint and

reference Species/sex Dose Incidence/ t otal (%) or

m ean ± SD (n umber of animals)

Male and female F344 rat, combined 0 mg/kg

- d 4 8 10 12 15 0/20 (0%) 0/20 (0%) 3/20 (15%) 6/20 (30%) 13/20 (65%) 12/20 (60%)

Convulsions Cholakis et al. (1980) Female F344 rat (gestational exposure) 0 mg/kg

- d 0.2

2 20 0/24 (0%) 0/24 (0%) 1/24 (4%) 18/24 (75%) Testicular degeneration Lish et al. (1984) ; Male B6C3F 1 mouse 0 mg/kg

- d 1.5 7 35 107 0/63 (0%) 2/60 (3%) 2/62 (3%) 6/59 (10%) 3/27 (11%)

Prostate suppurative inflammation Levine et al. (1983) Male F344 rat 0 mg/kg

- d 0.3 1.5 8 40 2/54 (4%) 4/55 (7%) 9/52 (17%) 12/55 (22%) 19/31 (61%)
1 2 3 4 5 6

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16 a For convulsions in Crouse et al. (2006) , the incidence rates across doses were determined to be not statistically significantly different between the males and females using an exact Cochran

- Mantel

- Haenszel test ( p ≥ 0.10). The data were combined across sex for this e ndpo int prior to modeling.
b The high dose group was excluded from modeling because a large proportion (approximately half) of the mice in that group died before wk 11, when the dose was reduced from 175 to 100 mg/kg

- d.

**D. 1.1.**  **Evaluation of Model Fit**
For each dichotomous endpoint, BMDS dichotomous models 4 were fitted to the data using the maximum likelihood method. Each model was tested for goodness

- of

- fit using a chi

- square goodness

- of

- fit test (χ 2 p
- value < 0.10 indicates lack of fit). Other factors were al so used to assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the low

- dose region and in the vicinity of the BMR.

**D.1.2. Model Selection**  For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as estimated by the profile likelihood method) and AIC value were used to select a best

- fit model from among the models exhibiting adequate fit. If the BMDL estimates were “sufficiently close,” that is,

4 Unless otherwise specified, all available BMDS dichotomous models besides the alternative and nested dichotomous models were fitted. The following parameter restrictions were applied: For the log

- logistic model, res trict slope ≥ 1; for the gamma and Weibull models, restrict power ≥1.Supplemental Information― Hexahydro

- 1,3,5

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This document is a draft for review purposes only and does not constitute Agency policy. D-3 DRAFT―DO NOT CITE OR QUOTE differed by at most three fold, the model selected was the one that yielde d the lowest AIC value. If 1
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11 12

13 14 15 16 17 18 the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the POD.

**D. 1.3.**  **Modeling Results** Below are tables summarizing the modeling results for the noncancer endpoints modeled.

Nervous System Effects
Tables D

- 2 to D

- 10 present the BMD model results for incidence of convulsions for female, male, and male and female F344 rats combined based on data from Crouse et al. (2006), using BMRs of 10%, 5%, and 1% extra risk. Tables D

- 11 to D

- 13 present the BMD model result s for incidence of convulsions for female F344 rats based on data from Cholakis (1980), using BMRs of 10%, 5%, and 1% extra risk.

**Table D-2** 

**. Model predictions for convulsions in female F344 rats exposed to**  **RDX by gavage for 90 days (Crouse et al.**  **, 2006); BMR = 10% e**  **xtra r**  **isk**

Model a Goodness of fit BMD 10Pct (mg/kg/d) BMDL 10Pct (mg/kg/d) Basis for model selection p - value AIC

Gamma 0.923 55.085 6.46 2.92 The quantal

- linear model was selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.733 56.607 6.76 4.75 LogLogistic 0.929 55.076 6.42 3.04
Probit 0.793 56.086 6.64 4.54 LogProbit 0.952 54.798 6.54 3.39
Weibull 0.892 55.420 6.16 2.62 Multistage 2° 0.954 53.595 5.46 2.47
Quantal

- Linear 0.733 56.131 2.76 1.84 Multistage 5° b 0.885 55.525 5.98 2.47 Multistage 4° 0.885 55.525 5.98 2.47 Multistage 3° 0.885 55.525 5.98 2.49
Dichotomous

- Hill 0.964 56.265 7.10 5.75

a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg -d were 0.00,

− 1. 2 9 , − 0.46,

− 0.12, 0.8 7 , and 0.4 1 , respectively.
b The Multistage 5° model may appear equivalent to the Multistage 4° model, however differences exist in digits

not displayed in the t a b le.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. D-4 DRAFT―DO NOT CITE OR QUOTE 1

2 3 4

5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 2 4 6 8 10 12 14Fraction Affected

dose Quantal Linear Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the

14:08 02/27 2014 BMDL BMD Quantal Linear

**Figur e**  **D-1**

**. Plot of incidence rate by dose, with fitted curve for selected model**  **, for convulsions in female F344 rats exposed to RDX by gavage fo**  **r 90 days (Cr**  **ouse et al.**  **, 2006).** BMR = 10% e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

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This document is a draft for review purposes only and does not constitute Agency policy. D-5 DRAFT―DO NOT CITE OR QUOTE **Quantal Linear Model using Weibull Model**

**.** (Version: 2.16; Date: 2/28/2013) 1
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9 10

11 12

13 14 15 16

17 18 1 2 9

0 The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP( -slope*dose)]

**Benchmark Dose Computation** BMR = 10% Extra ri sk BMD = 2.75544 BMDL at the 95% confidence level = 1.84489

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0.0833333 Slope 0.0382372 0.0404091

Power n/a 1

Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 24.9756 6
Fitted model

− 27.0654 1 4.17949 5 0.5239
Reduced model

− 33.7401 1 17.529 5 0.003598

AIC: = 56.1307

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 10 0

4 0.1418 1.418 0 10

− 1.286

8 0.2635 2.635 2 10

− 0.456

10 0.3178 3.178 3 10

− 0.121

12 0.368 3.68 5 10 0.866

15 0.4365 4.365 5 10 0.405

Chi^2 = 2.79 d.f = 5 P- value = 0.7325Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. D-6 DRAFT―DO NOT CITE OR QUOTE **Table D-3** 

**. Model predictions for convulsions in female F344 rats exposed to**  1 2 **RDX by gavage for 90 days (Crouse et**  **al. , 2006); BMR= 5%**  **e xtra**  **r isk** 

Model a Goodness of fit BMD 5Pct (mg/kg

- d) BMDL 5Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.923 55.085 5.09 1.54 The quantal

- linear model was selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.733 56.607 4.76 2.83 LogLogistic 0.929 55.076 4.99 1.71
Probit 0.793 56.086 4.80 2.69 LogProbit 0.952 54.798 5.33 2.17
Weibull 0.892 55.420 4.55 1.30 Multistage 2° 0.954 53.595 3.81 1.21
Quantal

- Linear 0.733 56.131 1.34 0.898 Multistage 3° 0.885 55.525 4.30 1.21 Multistage 4° b 0.885 55.525 4.30 1.21 Multistage 5° 0.885 55.525 4.30 1.20
Dichotomous

- Hill 0.964 56.265 6.25 2.30 3 4 5 6 7 8 9 a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg -d were 0.00,

− 1. 2 9 , − 0.46,

− 0.12, 0.8 7 , and 0.4 1 , respectively. The BMD 10 and BMDL 10 f or the selected model were 2.76 and 1.84 mg/kg

- d, r espectively.
b The Multistage 4° model may appear equivalent to the Multistage 3° model, however differences exist in digits

not displayed in the table.Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. D-7 DRAFT―DO NOT CITE OR QUOTE 1

2 3 4

5 6
7
8
9
10
11
12
13
14 15

16 17 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 2 4 6 8 10 12 14Fraction Affected

dose Quantal Linear Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B

14:07 02/27 2014 BMDL BMD Quantal Linear

**Figur e**  **D-2**

**. Plot of incidence rate by dose, with fitted curve for selected model**  **, for convulsions in female F344 rats exposed to RDX by gavage fo**  **r 90 days (Crouse**  **et al. , 2006).**  BMR= 5% e xtra r isk; dose shown in mg/kg

- day.

**Quantal Linear Model using Weibull Mode l.**  (Version: 2.16; Date: 2/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP( -slope*dose)]

**Benchmark Dose Computation** BMR = 5% Extra risk BMD = 1.34145 BMDL at the 95% confidence level = 0.89816

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0.0833333 Slope 0.0382372 0.0404091

Power n/a 1

Anal ysis of Deviance Table Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 24.9756 6
Fitted model

− 27.0654 1 4.17949 5 0.5239
Reduced model

− 33.7401 1 17.529 5 0.003598Supplemental Information― Hexahydro

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This document is a draft for review purposes only and does not constitute Agency policy. D-8 DRAFT―DO NOT CITE OR QUOTE 1 2 3 4 AI C: = 56.1307

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 10 0

4 0.1418 1.418 0 10

− 1.286

8 0.2635 2.635 2 10

− 0.456

10 0.3178 3.178 3 10

− 0.121

12 0.368 3.68 5 10 0.866

15 0.4365 4.365 5 10 0.405 5 6 7 Ch i^2 = 2.79 d.f = 5 P- value = 0.7325Supplemental Information― Hexahydro

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- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D-9 DRAFT―DO NOT CITE OR QUOTE **Table D-4** 

**. Model predictions for convulsions in female F344 rats exposed to**  1 2 **R DX by gavage for 90 days (Crouse**  **et a l.**  **, 2006); BMR= 1**  **% e**  **xtra r**  **isk**

Model a Goodness of fit BMD 1Pct (mg/kg/d) BMDL 1Pct (mg/kg/d) Basis for model selection p - value AIC

Gamma 0.923 55.085 3.10 0.355 The quantal

- linear model had a BMD more than 10 times lower than the lowest dose, so this model was excluded from consideration. Of the remaining models, the multistage model was selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.733 56.607 1.60 0.681 LogLogistic 0.929 55.076 2.87 0.468
Probit 0.793 56.086 1.86 0.649 LogProbit 0.952 54.798 3.63 0.919
Weibull 0.892 55.420 2.30 0.259 Multistage 2° 0.954 53.595 1.69 0.236
Quantal

- Linear 0.733 56.131 0.263 0.176 Multistage 3° 0.885 55.525 1.99 0.238 Multistage 4° 0.885 55.525 1.99 0.236 Multistage 5° 0.885 55.525 1.99 0.235
Dichotomous

- Hill 0.964 56.265 4.77 0.778 3 4 5 6 7 a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg

- d were 0.00 ,

− 0.67, 0.14, 0.1 1 , 0. 64, and

− 0.51, respectively. The BMD 10 and BMDL 10 f or the selected model were 5.98 and 2.47, respectively.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 2 4 6 8 10 12 14Fraction Affected

dose Multistage Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:06 02/27 2014 BMD BMDL Multistage

8

9 10 11 **Figur e**  **D-3**

**. Plot of incidence rate by dose, with fitted curve for selected model**  **, for**  **convulsions in female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006).**  BMR= 1 % e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 10 DRAFT―DO NOT CITE OR QUOTE **Multistage Model.** (Version: 3.3; Date: 02/28/2013) 1
2
3
4
5
6
7
8
9 10

11 12

13 14 15 16

17 18 The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)]

**Benchmark Dose Computation** BMR = 1% Extra risk BMD = 1.98616 BMDL at the 95% confidence level = 0.235433

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0 Beta(1) 0 0.0172961 Beta(2) 0.00234798 0.002476 Beta(3) 0.000100566 0 Beta(4) 0 0 Beta(5) 0 0

Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 24.9756 6
Fitted model

− 25.7624 2 1.57351 4 0.8135
Reduced model

− 33.7401 1 17.529 5 0.003598

AIC: = 55.5247

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 10 0

4 0.043 0.43 0 10

− 0.671

8 0.1827 1.827 2 10 0.141

10 0.2849 2.849 3 10 0.106

12 0.4006 4.006 5 10 0.641

15 0.5801 5.801 5 10

− 0.513

Chi^2 = 1.16 d.f = 4 P

- value = 0.8854Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 11 DRAFT―DO NOT CITE OR QUOTE **Table D-5** 

**. Model predictions for convulsions in male F344 rats exposed to**  1 2 **RDX by gavage for 90 days (Crouse et**  **al. , 2006)**  **; BMR = 10% extra risk** 

Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.482 48.534 7.40 5.11 Of the models that provided an adequate fit, the multistage 3° model was selected based on lowest AIC. Logistic 0.335 49.692 7.18 5.03 LogLogistic 0.522 48.248 7.48 5.29
Probit 0.363 49.460 7.17 4.86 LogProbit 0.530 48.224 7.53 5.38
Weibull 0.376 49.496 6.84 4.47 Multistage 2° 0.307 50.335 4.54 2.95
Quantal

- Linear 0.0553 56.530 1.98 1.38 Multistage 5° b Multistage 4° 0.361 49.607 6.85 3.91

Multistage 3° 0.515 47.803 6.17 3.95
Dichotomous

- Hill 0.701 48.408 8.34 6.32 3 4 5 6 7 8 a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg -d were 0.00,

− 0. 5 4 , − 0.82,

− 0.40, 1.6 6 , and

− 0.6 1 , respectively.
b For the Multistage 5° model, the b4 coefficient estimates was 0 (boundary of parameters space). The models in this row reduced to the Multistage 4° model.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 12 DRAFT―DO NOT CITE OR QUOTE 1 2

3 4 5

6 7
8
9
10
11
12
13
14
15 16 0 0.2 0.4 0.6 0.8

1

0 2 4 6 8 10 12 14Fraction Affected

dose Multistage Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:11 02/27 2014 BMD BMDL Multistage

**Figure D-4.**  **Plot of incidence rate by dose, with fitted curve for selected model, for convulsions in male F344 rats exposed to RDX by gavage for 90 days**  **(Crouse et al.**  **, 2006)**

**.** BMR = 10% e xtra r isk; dose shown in mg/kg

- day.

**Multistage Model.** (Version: 3.3; Date: 02/ 28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)]

**Benchmark Dose Computation** BMR = 10% Extra risk BMD = 6.17392 BMDL at the 95% confidence level = 3.94501

Parameter Estimate s Variable Estimate Default i nitial p arameter v alues
Background 0 0 Beta(1) 0 0 Beta(2) 0 0.00691555 Beta(3) 0.000447707 0 17Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 13 DRAFT―DO NOT CITE OR QUOTE Analysis of Deviance Table 1

2 3 4 5

6 7 Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 20.4721 6
Fitted model

− 22.9013 1 4.85838 5 0.4334
Reduced model

− 37.4599 1 33.9755 5 < 0 .0001

AIC : = 47.8027

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 10 0

4 0.0282 0.282 0 10

− 0.539

8 0.2049 2.049 1 10

− 0.822

10 0.3609 3.609 3 10

− 0.401

12 0.5387 5.387 8 10 1.658

15 0.7793 7.793 7 10

− 0.605

Chi ^2 = 4.24 d.f = 5 P- value = 0.5153Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 14 DRAFT―DO NOT CITE OR QUOTE **Table D-6** 

**. Model predictions for convulsions in male F344 rats exposed to**  1 2 **R DX by gavage for 90**  **days (Crouse et a**  **l. , 2006); BMR = 5%**  **e xtra**  **r isk** 

Model a Goodness of fit BMD 5Pct (mg/kg

- d) BMDL 5Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.482 48.534 6.47 3.96 Of the models that provided an adequate fit, the multistage 3° model was selected based on lowest AIC. Logistic 0.335 49.692 5.74 3.34 LogLogistic 0.522 48.248 6.51 4.14
Probit 0.363 49.460 5.92 3.26 LogProbit 0.530 48.224 6.71 4.40
Weibull 0.376 49.496 5.58 3.16 Multistage 2° 0.307 50.335 3.17 1.63
Quantal

- Linear 0.0553 56.530 0.964 0.670 Multistage 5° b Multistage 4° 0.361 49.607 5.56 1.99

Multistage 3° 0.515 47.803 4.86 2.19
Dichotomous

- Hill 0.701 48.408 7.76 5.27

a 3 4 5 6 7 8 9 Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg -d were 0.000,

− 0.5 4 , − 0. 82,

− 0.40, 1.6 6 , and

− 0.6 1 , respectively. The BMD 10 and BMDL 10 f or the selected model were 6.17 and 3.95 mg/kg

- d, r e spectively.
b For the Multistage 5° model, the beta coefficient estimates were 0 (boundary of parameters space). The models

in this row reduced to the Multistage 4° model.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 15 DRAFT―DO NOT CITE OR QUOTE 0 0.2 0.4 0.6 0.8

1

0 2 4 6 8 10 12 14Fraction Affected

dose Multistage Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:10 02/27 2014 BMD BMDL Multistage

1 2

3 4 5

6 7
8
9
10
11
12
13
14
15 16 **Figure D-5** 

**. Plot of incidence rate by dose, with fitted curve for selected model,**  **for convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse**  **et a l.**  **, 2006).** BMR = 5% e xtra r isk; dose shown in mg/kg

- day.

**Multistage Model.** (Version: 3.3; Date: 02/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)]

**Benchmark Dose Computation**  BMR = 5% Extra risk BMD = 4.85686 BMDL at the 95% confidence level = 2.19244

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0 Beta(1) 0 0 Beta(2) 0 0.00691555 Beta(3) 0.000447707 0 17Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 16 DRAFT―DO NOT CITE OR QUOTE Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 20.4721 6
Fitted model

− 22.9013 1 4.85838 5 0.4334
Reduced model

− 37.4599 1 33.9755 5 <0 .0001 1

2 3 4 5

6 7 AI C: = 47.8027

Good ness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 10 0

4 0.0282 0.282 0 10

− 0.539

8 0.2049 2.049 1 10

− 0.822

10 0.3609 3.609 3 10

− 0.401

12 0.5387 5.387 8 10 1.658

15 0.7793 7.793 7 10

− 0.605

Ch i^2 = 4.24 d.f = 5 P- value = 0.5153Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 17 DRAFT―DO NOT CITE OR QUOTE **Table D-7** 

**. Model predictions for convulsions in male F344 rats exposed to**  **RDX by gavage for 90 days (Crouse et al.**  **, 2006); BMR = 1**  **% e**  **xtra r**  **isk** 1 2

Model a Goodness of fit BMD 1Pct (mg/kg/d) BMDL 1Pct (mg/kg/d) Basis for model selection p - value AIC

Gamma 0.482 48.534 4.96 2.32 The multistage 2 ° model was selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.335 49.692 2.86 0.975 LogLogistic 0.522 48.248 4.79 2.38
Probit 0.363 49.460 3.60 1.01 LogProbit 0.530 48.224 5.41 3.00
Weibull 0.376 49.496 3.52 1.43 Multistage 2° 0.307 50.335 1.40 0.363
Quantal

- Linear 0.0553 56.530 0.189 0.131 Multistage 5° b 0.361 49.607 3.42 0.392 Multistage 4° c 0.361 49.607 3.42 0.392 Multistage 3° 0.515 47.803 2.82 0.457
Dichotomous

- Hill 0.701 48.408 6.64 3.47 3 4 5 6 7 8 9

10

11 12 13 a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg

- d were 0.00 ,

− 0.92,

− 1.26,

− 0.6 5 , 1.76, and 0.11, respectively. The BMD 10 and BMDL 10 for the selected model were 4.54 and 2.95 mg/kg

- d, respectively.
b The Multistage 5° model may appear equivalent to the Multistage 4° model, however differences exist in digits

not displayed in the table.

0 0.2 0.4 0.6 0.8

1

0 2 4 6 8 10 12 14Fraction Affected

dose Multistage Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:09 02/27 2014 BMD BMDL Multistage

**Figure D-6.**  **Plot of incidence rate by dose, with fitted curve for selected model ,**  **for convulsions in male F344 rats exposed to RDX by gavage fo**  **r 90 days (Crouse**  **et al. , 2006).**  BMR = 1 % e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 18 DRAFT―DO NOT CITE OR QUOTE **Multistage Model.** (Version: 3.3; Date: 02/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)]

**Benchmark Dose Computation** BMR = 1% Extra risk BMD = 1.40125 BMDL at the 95% confidence level = 0.363499 1
2
3
4
5
6
7
8
9
10

11 12

13 14

16

17 18 15

19 P arameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0 Beta(1) 0 0 Beta(2) 0.00511858 0.00691555

Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 20.4721 6
Fitted model

− 24.1672 1 7.39017 5 0.1932
Reduced model

− 37.4599 1 33.9755 5 <0 .0001

A IC: = 50.3345

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 10 0

4 0.0786 0.786 0 10

− 0.924

8 0.2793 2.793 1 10

− 1.264

10 0.4006 4.006 3 10

− 0.649

12 0.5215 5.215 8 10 1.763

15 0.6839 6.839 7 10 0.11

Chi^2 = 5.99 d.f = 5 P

- value = 0.3069Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 19 DRAFT―DO NOT CITE OR QUOTE **Table D-8** 

**. Model predictions for convulsions in male and female F344 rats**  1 2 3 **exposed to RDX by gavage for 90 days (Crouse et**  **al. , 2006); BMR = 10%**  **e xtra** 
**r isk** 

Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.484 101.79 6.92 5.09 Of the models that provided an adequate fit, the multistage 3° model was selected based on lowest AIC. Logistic 0.231 104.55 6.86 5.34
LogLogistic 0.512 101.66 6.93 5.15
Probit 0.291 103.61 6.83 5.19 LogProbit 0.557 101.25 7.01 5.31
Weibull 0.369 102.91 6.52 4.62 Multistage 2° 0.364 103.03 4.97 3.75
Quantal

- Linear 0.0369 111.56 2.32 1.77 Multistage 3° b Multistage 4° Multistage 5° 0.502 100.91 6.60 4.59

Dichotomous

- Hill 0.696 101.64 7.73 5.98 4 5 6 7 8 9 a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg -d were 0.00,

− 0. 69,

− 0. 2 5 , − 0.06, 1.62, and

− 1.0 8 , respectively.
b For the Multistage 4° and 5° model s , the b 4 and b5 coefficient estimates were 0 (boundary of parameters space). The models in this row reduced to the Multistage 3° model.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 20 DRAFT―DO NOT CITE OR QUOTE 1 2

3 4 5

6 7
8
9
10
11
12
13
14
15 16

17 0 0.2 0.4 0.6 0.8

0 2 4 6 8 10 12 14Fraction Affected

dose Multistage Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:05 02/27 2014 BMD BMDL Multistage

**Figur e**  **D-7**

**. Plot of incidence rate by dose, with fitted curve for se**  **lected model, for convulsions in male and female F344 rats**  **exposed to RDX by gavage for 90**  **days (Crouse et al.**  **, 2006).** BMR = 10% e xtra r isk; dose shown in mg/kg

- day.

**Multistage Model.** (Version: 3.3; Date: 02/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)]

**Benchmark Dose Computation** BMR = 10% Extra risk BMD = 6.60247 BMDL at the 95% confidence level = 4.59346

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0 Beta(1) 0 0.00163806 Beta(2) 0 0.00485933 Beta(3) 0.000366065 0Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 21 DRAFT―DO NOT CITE OR QUOTE Analysis of Deviance Table 1

3 4 5

7 6 2 Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 47.0806 6
Fitted model

− 49.4567 1 4.75213 5 0.4469
Reduced model

− 71.5289 1 48.8965 5 < 0 .0001

AI C: = 100.913

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 20 0

4 0.0232 0.463 0 20

− 0.689

8 0.1709 3.418 3 20

− 0.248

10 0.3065 6.131 6 20

− 0.063

12 0.4688 9.375 13 20 1.624

15 0.7093 14.186 12 20

− 1.076

Ch i^2 = 4.34 d.f = 5 P- value = 0.5021Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 22 DRAFT―DO NOT CITE OR QUOTE **Table D-9** 

**. Model predictions for convulsions in male and female F344 rats**  1 2 3 **exposed to RDX by gavage for 90 days (Crouse et**  **al. , 2006); BMR = 5%**  **e xtra** 
**r isk** 

Model a Goodness of fit BMD 5Pct (mg/kg

- d) BMDL 5Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.484 101.79 5.78 3.80 Of the models that provided an adequate fit, the multistage 3° model was selected based on lowest AIC. Logistic 0.231 104.55 5.13 3.49
LogLogistic 0.512 101.66 5.74 3.85
Probit 0.291 103.61 5.29 3.43 LogProbit 0.557 101.25 6.01 4.20
Weibull 0.369 102.91 5.11 3.18 Multistage 2° 0.364 103.03 3.47 2.20
Quantal

- Linear 0.0369 111.56 1.13 0.860 Multistage 3° 0.502 100.91 5.19 2.66 Multistage 4° Multistage 5° b 0.502 100.91 5.19 2.65

Dichotomous

- Hill 0.696 101.64 6.98 4.80 4 5 6 7 8 9 10 a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg -d were 0.00,

− 0. 69,

− 0. 2 5 , − 0.06, 1.62, and

− 1.0 8 , respectively. The BMD 10 and BMDL 10 f or the selected model were 6.60 and 4.59 mg/kg

- d, r espectively.
b For the Multistage 5° model, the b 5 coefficient estimate w as 0 (boundary of parameters space). The models in this row reduced to the Multistage 4° model.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 23 DRAFT―DO NOT CITE OR QUOTE 1

2 3 4 5

6
7
8
9
10
11
12
13
14
15 16 0 0.2 0.4 0.6 0.8

0 2 4 6 8 10 12 14Fraction Affected

dose Multistage Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:04 02/27 2014 BMD BMDL Multistage

**Figur e**  **D-8**

**. Plot of incidence rate by dose, with the fitted curve of the selected**  **model, for convulsions in male and female F344 rats exposed to RDX by gavage for 90 days (Crouse**  **et al. , 2006).**  BMR = 5% e xtra r isk; dose shown in mg/kg

- day.

**Multistage Model.** (Version: 3.3; Date: 02/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)]

**Benchmark Dose Computation**  BMR = 5% Extra risk BMD = 5.19399 BMDL at the 95% confidence level = 2.65815

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0 Beta(1) 0 0.00163806 Beta(2) 0 0.00485933 Beta(3) 0.000366065 0 17Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 24 DRAFT―DO NOT CITE OR QUOTE Analysis of Deviance Table 1

2 3 4 5

6 7 8 Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 47.0806 6
Fitted model

− 49.4567 1 4.75213 5 0.4469
Reduced model

− 71.5289 1 48.8965 5 < 0 .0001

AIC : = 100.913

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 20 0

4 0.0232 0.463 0 20

− 0.689

8 0.1709 3.418 3 20

− 0.248

10 0.3065 6.131 6 20

− 0.063

12 0.4688 9.375 13 20 1.624

15 0.7093 14.186 12 20

− 1.076

Chi ^2 = 4.34 d.f = 5 P- value = 0.5021Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 25 DRAFT―DO NOT CITE OR QUOTE 11

12 13 14 15

16 **Ta ble**  **D- 10** 

**. Model predictions for convulsions in male and female F344 rats** 

1 2 3 **exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 1% e**  **xt ra** 
**r isk** 

Model a Goodness of fit BMD 1Pct (mg/kg/d) BMDL 1Pct (mg/kg/d) Basis for model selection p - value AIC

Gamma 0.484 101.79 4.02 2.03 The quantal

- linear model did not fit the data adequately ( p

- value < 0.10), so it was excluded from consideration. Of the remaining models, the multistage 2° model was selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.231 104.55 2.04 0.987
LogLogistic 0.512 101.66 3.79 2.00
Probit 0.291 103.61 2.57 1.03 LogProbit 0.557 101.25 4.50 2.69
Weibull 0.369 102.91 2.94 1.35 Multistage 2° 0.364 103.03 1.53 0.544
Quantal

- Linear 0.0369 111.56 0.222 0.169 Multistage 5° b Multistage 4° 0.502 100.91 3.02 0.549

Multistage 3° 0.502 100.91 3.02 0.569
Dichotomous

- Hill 0.696 101.64 5.62 2.90 4 5 6 7 8 9 10 a Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg

- d were 0.00 ,

− 1.19,

− 0. 93,

− 0. 4 5 , 1.71, and

− 0.1 6 , respectively. The BMD 10 and BMDL 10 f or the selected model were 4.97 and 3.75 mg/kg

- d, r e spectively.
b For the Multistage 5° model, the beta coefficient estimates were 0 (boundary of parameters space). The models

in this row reduced to the Multistage 4° model.

0 0.2 0.4 0.6 0.8

0 2 4 6 8 10 12 14Fraction Affected

dose Multistage Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:03 02/27 2014 BMD BMDL Multistage

**Figur e**  **D-9. Plot of incidence rate by dose, with the fitted curve of the selected**  **model, for convulsions in male and female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006).**  BMR = 1% e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 26 DRAFT―DO NOT CITE OR QUOTE **Multistage Model.** (Versio n: 3.3; Date: 02/28/2013) 1
2
3
4
5
6
7
8
9 The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)]

**Benchmark Dose Computation** BMR = 1% Extra risk BMD = 1.53434 BMDL at the 95% confidence level = 0.544329

Parameter Estimates 10

11 12

13 14

16

17 18 15

19 Variable Estimate Default i nitial p arameter v alues Background 0 0 Beta(1) 0 0.00163806 Beta(2) 0.00426912 0.00485933

Ana lysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 47.0806 6
Fitted model

− 50.5158 1 6.87034 5 0.2305
Reduced model

− 71.5289 1 48.8965 5 < 0 .0001

AIC: = 103.032

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 20 0

4 0.066 1.321 0 20

− 1.189

8 0.2391 4.782 3 20

− 0.934

10 0.3475 6.95 6 20

− 0.446

12 0.4592 9.185 13 20 1.712

15 0.6173 12.346 12 20

− 0.159

Chi^2 = 5.44 d.f = 5 P

- value = 0.3644Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 27 DRAFT―DO NOT CITE OR QUOTE **Table D-**  **11**

**. Model predictions for convulsions in female F344 rats exposed to**  1 2 **R DX by gavage on gestation days 6** 

**− 19 (C**  **holakis, 1980); BMR = 10% e**  **xtra r**  **isk**

Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.989 42.003 3.62 1.56 The quantal

- linear model is selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.526 43.556 8.92 6.14 LogLogistic 0.991 41.996 3.45 1.53
Probit 0.577 43.348 7.64 5.39 LogProbit 1.000 41.963 3.13 1.51
Weibull 0.983 42.026 3.81 1.55 Multistage 3° b 0.960 42.113 4.26 1.54 Multistage 2° 0.960 42.113 4.26 1.54
Quantal

- Linear 0.669 42.077 1.88 1.29 3 4 5 6 7

8

9 10 11 12

13 a Selected model in bold; scaled residuals for selected model for doses 0, 0.2, 2, and 20 mg/k g-d were 0.00,

− 0.52,

− 1.03 , and 0.49 , respectively.
b The Multistage 3° model may appear equivalent to the Multistage 2° model, however differences exist in digits

not displayed in the table.

0 0.2 0.4 0.6 0.8

0 5 10 15 20Fraction Affected

dose Quantal Linear Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the

09:29 06/06 2014 BMDL BMD Quantal Linear

**Figur e**  **D- 10** 

**. Plot of incidence rate by dose, with the fitted curve of the**  **selected model, for convulsions in female F344 rats exposed to RDX by gavage**  **on gestation days 6**

**− 19 (Cholakis, 1980).**  BMR = 10% e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 28 DRAFT―DO NOT CITE OR QUOTE **Quantal Linear Model using Weibull Model** (Version: 2.16; Date: 2/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP( -slope*dose)]

**Benchmark Dose Computation** BMR = 10% Extra risk BMD = 1.87886 BMDL at the 95% confidence level = 1.28909 1
2
3
4
5
6
7
8
9
10

11 12

13 14 15 16

17 18 19 Param eter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0.0384615 Slope 0.056077 0.0588587

Power n/a 1

Analys is of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 18.9808 4
Fitted model

− 20.0384 1 2.11537 3 0.5488
Reduced model

− 47.9793 1 57.9972 3 <0 .0001

AIC: = 42.0769

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 24 0

0.2 0.0112 0.268 0 24

− 0.52

2 0.1061 2.546 1 24

− 1.025

20 0.6742 16.856 18 25 0.488

Chi^2 = 1.56 d.f = 3 P- value = 0.6686Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 29 DRAFT―DO NOT CITE OR QUOTE **Table D-**  **12**

**. Model predictions for convulsions in female F344 rats exposed to**  1 2 **RDX by ga vage on gestation days 6** 

**− 19 (Chol**  **akis, 1980); BMR = 5% e**  **xtra r**  **isk**

Model a Goodness of fit BMD 5Pct (mg/kg

- d) BMDL 5Pct (mg/kg

- d) Basis for model select ion p - value AIC

Gamma 0.989 42.003 2.31 0.759 The quantal

- linear model is selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.526 43.556 6.53 3.90 LogLogistic 0.991 41.996 2.27 0.823
Probit 0.577 43.348 5.41 3.34 LogProbit 1.000 41.963 2.18 0.902
Weibull 0.983 42.026 2.36 0.756 Multistage 2° 0.960 42.113 2.51 0.747
Quantal

- Linear 0.669 42.077 0.915 0.628 Multistage 3° b 0.960 42.113 2.51 0.747 3 4 5 6 7 8 9

10

11 12 13 14 a Selected model in bold; scaled residuals for selected model for doses 0, 0 .2, 2, and 20 mg/kg

- d were 0.00 , − 0.52,

− 1.0 3 , and 0.4 9 , re s pectively. The BMD 10 and BMDL 10 for the selected mod el were 1.88 and 1.29 mg/kg

- d, respectively.
b The Multistage 3 ° model may appear equivalent to the Multistage 2 ° model, however differences exist in digits

not displayed in the table.

0 0.2 0.4 0.6 0.8

0 5 10 15 20Fraction Affected

dose Quantal Linear Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B

15:24 05/23 2014 BMDL BMD Quantal Linear

**Figure D-**  **11**

**. Plot of incidence rate by dose, with the fitted curve of the**  **selected model, for convulsions in female F344 rats exposed to RDX by gavage on gestation days 6** 

**− 19**  **(Cholakis, 1980).** BMR = 5% e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 30 DRAFT―DO NOT CITE OR QUOTE 1 2
3
4
5
6
7
8
9
10 11

12 13

14 15 16 17

18 19 20 **Qu antal Linear Model using Weibull Model**  (Version: 2.16; Date: 2/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP( -slope*dose)]

**Benchmark Dose Computation**  BMR = 5% Extra risk BMD = 0.914694 BMDL at the 95% confidence level = 0.627577

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0.0384615 Slope 0.056077 0.0588587

Power n/a 1

Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 18.9808 4
Fitted model

− 20.0384 1 2.11537 3 0.5488
Reduced model

− 47.9793 1 57.9972 3 < 0 .0001

AIC: = 42.0769

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 24 0

0.2 0.0112 0.268 0 24

− 0.52

2 0.1061 2.546 1 24

− 1.025

20 0.6742 16.856 18 25 0.488

Chi^2 = 1.56 d.f = 3 P- value = 0.6686Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 31 DRAFT―DO NOT CITE OR QUOTE **Table D-**  **13**

**. Model predictions for convulsions in female F344 rats**  **exposed to** 1 2 **R DX by gavage on gestation days 6** 

**− 19 (C**  **holakis, 1980); BMR = 1**  **% e**  **xtra r**  **isk**

Model a Goodness of fit BMD 1Pct (mg/kg

- d) BMDL 1Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.989 42.003 0.866 0.149 The quantal

- linear model is selected based on lowest AIC. Logistic 0.526 43.556 2.46 1.05 LogLogistic 0.991 41.996 0.902 0.201
Probit 0.577 43.348 1.96 0.871 LogProbit 1.000 41.963 1.11 0.335
Weibull 0.983 42.026 0.798 0.148 Multistage 3° b 0.960 42.113 0.638 0.146 Multistage 2° c 0.960 42.113 0.638 0.146
Quantal

- Linear 0.669 42.077 0.179 0.123 3 4 5 6 7 8 9 a Selected model in bold; scaled residuals for selected model for doses 0, 0 .2, 2, and 20 mg/kg

- d were 0.00 , − 0.52,

− 1.0 3 , and 0.4 9 , respectively. The BMD 10 and BMDL 10 for the selected model were 1.88 and 1.29 mg/kg

- d, respectively.
b The Multistage 3° model may appear equivalent to the Multistage 2° model, however differences exist in digits

not displayed in the table.

10 11

12 13 14 15

16 0 0.2 0.4 0.6 0.8

0 5 10 15 20Fraction Affected

dose Quantal Linear Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B

14:19 07/28 2014 BMDLBMD Quantal Linear

**Figur e**  **D- 12** 

**. Plot of incidence rate by dose**  **, with the fitted curve of the selected model,**  **for convulsions in female F344 rats expo**  **sed to RDX by gavage on gestation days 6** 

**− 19 (Cholakis, 1980).**  BMR = 1% e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 32 DRAFT―DO NOT CITE OR QUOTE **Quantal Linear Model using Weibull Model** (Version: 2.16; Date: 2/28/2013) 1
2
3
4
5
6
7
8
9
10

11 12

13 14 15 16

17 18 1 2 9

0 The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP( -slope*dose)]

**Benchmark Dose Computation** BMR = 1% Extra risk BMD = 0.179224 BMDL at the 95% confidence level = 0.122966

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0 0.0384615 Slope 0.056077 0.0588587

Power n/a 1

Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 18.9808 4
Fitted model

− 20.0384 1 2.11537 3 0.5488
Reduced model

− 47.9793 1 57.9972 3 < 0 .0001

AI C: = 42.0769

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0 0 0 24 0

0.2 0.0112 0.268 0 24

− 0.52

2 0.1061 2.546 1 24

− 1.025

20 0.6742 16.856 18 25 0.488

Chi^2 = 1.56 d.f = 3 P

- value = 0.6686Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 33 DRAFT―DO NOT CITE OR QUOTE Male Reproductive Effects 1
2
3

4 5 6

7 8 9 10 11 12 13 14 15 Tab le D

- 14 presents the BMD model results for incidence of testicular degeneration for male B6C3F 1 mice based on data from Lish et al. (1984), using a BMR of 10% extra risk.

**Table D-**  **14**

**. Model predictions for testicular degeneration in male B6C3F**  **1 mice exposed to RDX by diet for 24 months (Lish**  **et al. , 1984); BMR = 10%** 

**e xtra**  **r isk** 

Mode l a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p - value AIC

Gamma b Weibull Quantal

- Linear 0.357 101.10 66.5 35.4 The log

- probit model was selected based on lowest BMDL (BMDLs differed by more than threefold). Logistic 0.159 103.40 97.1 66.1 LogLogistic 0.377 100.91 63.6 32.3
Probit 0.178 103.12 93.1 61.4 LogProbit 0.876 97.564 56.0 16.3 Multistage 2° c Multistage 3° Multistage 4° 0.357 101.10 66.5 35.4

a Selected model in bold; scaled residuals for selected model for doses 0, 1.5, 7, 35, and 107 mg/kg

- d were 0.00,

0.32,

− 0. 6 1 , 0.43, and

− 0.17, respectively.
b For the Gamma and Weibull models, the power parameter estimates were 1 (boundary of parameter space). The

model s i n this row a re equivalent to the Quantal

- Linear model.

c The Multistage 3° and 4° model had b3 and b4 coefficient estimates of 0 (boundary of parameters space). The

models in this row reduced to the Multistage 2° model. T he models in this row may appear equivalent to the Gamma model, ho w ever differences exist in digits not displayed in the table.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 34 DRAFT―DO NOT CITE OR QUOTE 1 2

3 4 5 6

7 8
9
10
11
12
13
14
15
16
17
18 19 0 0.05

0.1 0.15

0.2 0.25

0.3

0 20 40 60 80 100Fraction Affected

dose LogProbit Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B

13:42 02/14 2014 BMDL BMD LogProbit

**Figur e**  **D- 13** 

**. Plot of incidence rate by dose, with fitted curve for selected**  **model, for testicular degeneration in male B6C3F 1**  **mice exposed to RDX by diet for 24 months (Lish**  **et al. , 1984).**  BMR = 10% e xtra r isk; dose shown in mg/kg

- day.

**Probit Model.** (Version: 3.3; Date: 2/28/2013) The form of the probability function is: P[response] = Background + (1

- Background) * CumNorm(Inte rcept+Slope*Log(Dose)),where CumNorm(.) is the cumulative normal distribution function Slope parameter is not restricted

**Benchmark Dose Computation** BMR = 10% Extra risk BMD = 55.9784 BMDL at the 95% confidence level = 16.2787

Parameter Estimates Variable Estimate Default i nitial p arameter v alues background 0 0 intercept

− 2.0054E+00

− 1.9976E+00 slope 0.179828 0.172286 20Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 35 DRAFT―DO NOT CITE OR QUOTE Analysis of Deviance Table 1

2 3 4 5

6 7 Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 46.4212 5
Fitted model

− 46.7817 2 0.721088 3 0.8682
Reduced model

− 52.1663 1 11.4902 4 0.02157

AI C: = 97.5635

Goodness of Fit Table Scaled Dose Est. p rob. Expected Observed Size residuals

0 0 0 0 63 0

1.5 0.0267 1.599 2 60 0.321

7 0.0489 3.033 2 62

− 0.608

35 0.086 5.072 6 59 0.431

107 0.122 3.294 3 27

− 0.173

Chi^2 = 0.69 d.f = 3 P- value = 0.8759Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 36 DRAFT―DO NOT CITE OR QUOTE Kidney Effects 1
2
3
4

5 6 7

8 9 10 11 12 13 14 15 16 Table D

- 1 5 presents the BMD model results for incidence of suppurative inflammation of the prostate in male F344 rats based on data from Levine et al. (1983), using a BMR of 10% extra risk.

**Table D-**  **15**

**. Model predictions for prostate suppurative inflammation in male**  **F344 rats exposed to RDX by diet for 24 months (Levine et al.**  **, 1983); BMR**  **= 10%**  **e xtra**  **r isk** 

Model a Goodness of fit BMD 10Pct (mg/kg/d) BMDL 10Pct (mg/kg/d) Basis for model selection p - value AIC

Gamma b Multistage 2° Quantal

- Linear Multistage 3° Multistage 4° 0.288 200.37 4.61 3.24 The log

- probit model is selected based on lowest BMDL (BMDLs differ by more than threefold).

Logistic 0.102 203.50 10.8 8.58 LogLogistic 0.328 200.05 3.33 2.09
Probit 0.116 203.10 9.91 7.96 LogProbit 0.204 202.03 1.67 0.469
Weibull g 0.288 200.37 4.61 3.24

a Selected model in bold; scaled residuals for selected model for doses 0, 0.3, 1.5, 8, and 40 mg/kg

- d were

− 0.289,

0.172, 0.846,

− 1.2 98, and 0.819, respectively.
b The Gamma model had a power parameter estimate was 1 (boundary of parameter space). The multistage 2, 3,

and 4 models had b2, b3, and b4 coefficients of 0 (b oundary of parameter space). Th e mo dels in this row are equivalent to the Quantal

- Li n ear model.

c The Weibull model may appear equivalent to the quantal

- linear model, however differences exist in digits not

displayed in the table.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 37 DRAFT―DO NOT CITE OR QUOTE 1 2

3 4 5 6

7 8
9
10
11
12
13
14
15
16
17
18 19

20 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 5 10 15 20 25 30 35 40Fraction Affected

dose LogProbit Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

13:39 02/14 2014 BMDL BMD LogProbit

**Figur e**  **D- 14** 

**. Plot of incidence rate by dose, with fitted curve for selected**  **model, for prostate suppurative inflammation in male F344 rats exposed to RDX by diet for 24 months (Levine**  **et al. , 1983)** 

**.** BMR = 10% e xtra r isk; dose shown in mg/kg/da y.

**Probit Model.** (Version: 3.3; Date: 2/28/2013) The form of the probability function is: P[response] = Background + (1

- Background) * CumNorm(Intercept+Slope*Log(Dose)),where CumNorm(.) is the cumulative normal distribution function Slope parameter is not restricted

**Benchmark Dose Computation** BMR = 10% Extra risk BMD = 1.67454 BMDL at the 95% confidence level = 0.468688

Parameter Estimates Variable Estimate Default i nitial p arameter v alues background 0.0452202 0.037037 intercept

− 1.4970E+00

− 1.3564E+00 slope 0.417872 0.36341Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 38 DRAFT―DO NOT CITE OR QUOTE Analysis of Deviance Table 1

2 3 4 5

6 7 8 Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 96.3905 5
Fitted model

− 98.0147 3 3.24837 2 0.1971
Reduced model

− 118.737 1 44.6933 4 < 0 .0001

AIC: = 202.029

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0.0452 2.442 2 54

− 0.289

0.3 0.0669 3.682 4 55 0.172

1.5 0.1332 6.927 9 52 0.846

8 0.2982 16.402 12 55

− 1.298

40 0.5396 16.726 19 31 0.819

Chi^2 = 3.18 d.f = 2 P- value = 0.2035Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 39 DRAFT―DO NOT CITE OR QUOTE 

## D.2. **BENCHMARK DOSE MODELING SUMMARY FOR C**  **ANCER E**  **NDPOINTS** 1

2
3

4

5 6 7

8
9
10
11
12
13
14
15
16
17
18
19
20 The cancer endpoints that were selected for dose

- r esponse modeling are presented in Table
D- 16. For each endpoint, the doses and tumor incidence data used for the modeling are presented.

**Table D-**  **16**

**. Cancer endpoints selected for dose** 

**- response modeling for**  **RDX**

Endpoint and reference Species/sex Dose (mg/kg

- d) Incidence/total
Hepatocellular adenomas or carcinomas Parker et al. (2006) Female B6C3F 1 mice 0 1.5 7 35 107 1/67 (1%) 4/62 (6%) 5/63 (8%) 10 /64 (16%) 4/31 (13%) Alveloar/bronchiolar adenomas or carcinomas Lish et al. (1984) Female B6C3F 1 mice 0 1.5 7 35 107 7/65 (11%) 3/62 (5%) 8/64 (13%) 12/64 (19%) 7/31 (23%) Hepatocellular adenomas or carcinomas Levine et al. (1983) Male F344 rats 0 0.3 1.5 8 40 1/55 (2%) 0/55 (0%) 0/5 2 (0%) 2/55 (4%) 2/3 1 a ( 6 %)

a The denominators listed in the table represent the number of animals that were alive one year after dosing

began.

**D. 2.1.**  **Evaluation of Model Fit and Model Selection** For each endpoint, BMDS multistage

- cancer models 5 were fitted to the data using the maximum likelihood method. Each model was tested for goodness

- of

- fit using a chi

- square goodness

- of

- fit test (χ 2 p
- value < 0.05 6 indicates lack of fit). Other factors were used to assess

m odel fit, such as scaled residuals, visual fit, and adequacy of fit in the low

- dose region and in the vicinity of the BMR. For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as estimated by the profile likelihood method) and AIC value were used to select a best

- fit model from among the models exhibiting adequate fit. If the BMDL estimates were “sufficiently close,” that is, differed by more than threefold, the model selected was the one that yielded the lowest AIC value. If the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the POD. After selecting models for the two endpoints, the results were combined using MS

- COMBO in BMDS. This procedure analyzes the incidence of a tumor (adenoma or carcinoma) defined as

5 The coefficients of the multistage

- cancer models were restricted to be nonnegative (beta’s ≥ 0).
6 A significance level of 0.05 is used for selecting cancer models because the model family (multistage) is selected a priori ( Benchmark Dose Technical Guidance Document , U.S. EPA, 2012).Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 40 DRAFT―DO NOT CITE OR QUOTE present if either the hepatocellular or alveolar/bronchiolar tumor (or both) was present, and not 1
2

3
4
5
6
7
8
9
10

11 12 13

14 15 16 17 18 19 present otherwise. The two endpoints were assumed to be independent.

**D. 2.2.**  **Modeling Results** Details of the BMD modeling for mouse tumor data sets are pro vided in Tables D

- 17 to D

- 20 below. In addition, this appendix presents a quantitative dose

- response analysis using rat liver tumor data and detailed BMD modeling results (see Table D

- 22). The analysis of rat liver tumor data and resulting candidate OSF is presented for comparison with other OSF estimates provided in Section 2.3.3 of the Toxicological Review.

**Mouse Tumor Data –**  **BMD Modeling Documentation**

**Table D-**  **17**

**. Model predictions for combined alveolar/bronchiolar adenoma**  **and carcinoma in female B6C3F 1**  **mice exposed to RDX by diet for 24 months (Lish**  **et al. , 1984); BMR = 10%**  **e xtra**  **r isk** 

Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p - value AIC
Multistage 1° b Multistage 2° Multistage 3° Multistage 4° 0.417 218.68 52.8 27.7 All the models reduced to the multistage 1° model, so it was selected.

a Selected model in bold. Scaled residuals for the selected model for doses 0, 1.5 , 7, 35, and 107 mg/kg

- d were

0.40,

− 1. 27, 0.50, 0.73, and

− 0 .52, respectively.
b For the multistage 2°, 3°, and 4° models, the b2, b3 and b4 coefficient estimates were 0 (boundary of parameter

space). The models in this ro w r ed uced to the multistage 1° model.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 41 DRAFT―DO NOT CITE OR QUOTE 1

2 3

4 5 6 7

8

9

10
11
12
13
14
15
16
17
18
19
20
21 22 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0 20 40 60 80 100Fraction Affected

dose Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for t

16:09 02/14 2014 BMD BMDL Multistage Cancer Linear extrapolation

**Figur e**  **D- 15** 

**. Plot of incidence rate by dose, with the fitted curve for the**  **selected model, for combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F**  **1 mice exposed to RDX by diet for 24 months (Lish**  **et al. ,**  **1984)**

**.** BMR = 10% e xtra r isk; dose shown in mg/kg

- day.

**Multistage Cancer Model. (Version: 1.10; Date: 02/28/2013)**
The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)] The parameter betas are restricted to be positive

**Benchmark Dose Computation** BMR = 10% Extra risk BMD = 52.8078 BMDL at the 95% confidence level = 27.748 BMDU at the 95% confidence level = 194.806 Take n together, (27.748, 194.806) is a 90% two

- sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.00360387

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0.093168 0.0998927 Beta(1) 0.00199517 0.00155773Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 42 DRAFT―DO NOT CITE OR QUOTE 1 2

3 4 5 6

7 8 9 Ana lysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 105.777 5
Fitted model

− 107.341 2 3.12764 3 0.3724
Reduced model

− 110.164 1 8.77367 4 0.06701

AI C: = 218.682

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0.0932 6.056 7 65 0.403

1.5 0.0959 5.944 3 62

− 1.27

7 0.1057 6.768 8 64 0.501

35 0.1543 9.877 12 64 0.734

107 0.2675 8.292 7 31

− 0.524

Chi^2 = 2.84 d.f = 3 P- value = 0.4168Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 43 DRAFT―DO NOT CITE OR QUOTE **Table D-**  **18**

**. Model predictions for combined alveolar/bronchiolar adenoma**  1 2 3 **and carcinoma in female B6C3F 1**  **mice exposed to RDX by diet for 24 months (Lish**  **et al. , 1984); BMR = 5%**  **e xtra**  **r isk** 

Model a Goodness of fit BMD 5Pct (mg/kg

- d) BMDL 5Pct (mg/kg

- d) Basis for model selection p - value AIC
Multistage 1° b Multistage 2° Multistage 3° Multistage 4° 0.417 218.68 25.7 13.5 All the models reduced to the multistage 1° model, so it was selected.

4 5 6 7 8 a Selected model in bold. Scaled residuals for the selected model for doses 0, 1.5 , 7, 35, and 107 mg/kg

- d were 0.40,

− 0.40,

− 1. 2 7 , 0.50, 0.73, and

− 0.52 , respectively. The BMD 10 and BMDL 10 for the selected model were 52.8 and 27.7 mg/kg

- d, r e spectively.

9 10

11 12 13 14

15 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0 20 40 60 80 100Fraction Affected

dose Multistage Cancer Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for th

16:11 02/14 2014 BMD BMDL Multistage Cancer Linear extrapolation

**Figur e**  **D- 16** 

**. Plot of incidence rate by dose, with fitted curve for selected**  **model, for combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F**  **1 mice exposed to RDX by diet for 24 months (Lish**  **et al. , 1984).**  BMR = 5% e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 44 DRAFT―DO NOT CITE OR QUOTE **Multistage Cancer Model.** (Version: 1.10; Date: 02/28/2013) 1
2
3
4
5
6
7
8
9
10
11
12
13 14

15 16

17 18 19 20

21 22 23 The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)] The parameter betas are restricted to be positive

**Benchmark Dose Computa tion**  BMR = 5% Extra risk BMD = 25.7088 BMDL at the 95% confidence level = 13.5087 BMDU at the 95% confidence level = 94.8384 Taken together, (13.5087, 94.8384) is a 90% two

- sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.00370131

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0.093168 0.0998927 Beta(1) 0.00199517 0.00155773

Ana lysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 105.777 5
Fitted model

− 107.341 2 3.12764 3 0.3724
Reduced model

− 110.164 1 8.77367 4 0.06701

AI C: = 218.682

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0.0932 6.056 7 65 0.403

1.5 0.0959 5.944 3 62

− 1.27

7 0.1057 6.768 8 64 0.501

35 0.1543 9.877 12 64 0.734

107 0.2675 8.292 7 31

− 0.524

Ch i^2 = 2.84 d.f = 3 P- value = 0.4168Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 45 DRAFT―DO NOT CITE OR QUOTE **Table D-**  **19**

**. Model predictions for combined hepatocellular adenoma and**  1 2 3 **carcinoma in female B6C3F 1**  **mice exposed to RDX by diet for 24 months (Parker**  **et al. , 2006); BMR = 10%**  **e xtra**  **r isk** 

Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p - value AIC
Multistage 1° b Multistage 2° Multistage 3° Multistage 4° 0.160 164.06 64.2 32.6 All the models reduced to the multistage 1° model, so it was selected.

4 5 6 7 8 9 a Selected model in bold. Scaled residuals for the selected model for doses 0, 1.5 , 7, 35, and 107 mg/kg

- d were

− 1.37 , 0.35, 0.5 4 , 1. 3 4 , and

− 1.05 , respectively.
b For the multistage 2°, 3°, and 4° models, the b2, b3 and b4 coefficient estimates were 0 (boundary of parameter

space). The models in this row reduced to the multistage 1° model.

0 0.05

0.1 0.15

0.2 0.25

0.3

0 20 40 60 80 100Fraction Affected

dose Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for t

16:21 02/14 2014 BMD BMDL Multistage Cancer Linear extrapolation

10

11 12 13 14

15 **Figur e**  **D- 17** 

**. Plot of incidence rate by dose, with**  **fitted curve for selected model, for combined hepatocellular adenoma and carcinoma in female B6C3F**  **1 mice exposed to RDX by diet for 24 months (Parker**  **et al. , 2006).**  BMR = 10%

e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 46 DRAFT―DO NOT CITE OR QUOTE **Multistage Cancer Model.** (Version: 1.10; Date: 02/28/2013) 1
2
3
4
5
6
7
8
9
10
11
12
13 14

15 16

17 18 19 20

21 22 23 The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)] The parameter betas are restricted to be positive

**Benchmark Dose Computation** BMR = 10% Extra risk BMD = 64.203 BMDL at the 95% confidence level = 32.6282 BMDU at the 95% confidence level = 281.385 Taken together, (32.6282, 281.385) is a 90% two

- sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.00306483

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0.0520755 0.0658334 Beta(1) 0.00164105 0.000876864

Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 77.1516 5
Fitted model

− 80.0315 2 5.75967 3 0.1239
Reduced model

− 82.5216 1 10.74 4 0.02965

AIC: = 164.063

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0.0521 3.489 1 67

− 1.369

1.5 0.0544 3.373 4 62 0.351

7 0.0629 3.963 5 63 0.538

35 0.105 6.719 10 64 1.338

107 0.2047 6.347 4 31

− 1.045

Chi^2 = 5.17 d.f = 3 P- value = 0.16Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 47 DRAFT―DO NOT CITE OR QUOTE **Table D-**  **20**

**. Model predictions for B6C3F**  **1 female mouse combined**  1 2 3 **hepatocellular adenoma and carcinoma in mice exposed to RDX by diet for 24**  **m onths (Parker**  **et al. , 2006); BMR = 5%**  **e xtra**  **r isk** 

Model a Goodness of fit BMD 5Pct (mg/ kg

- d) BMDL 5Pct (mg/kg

- d) Basis for model selection p - value AIC
Multistage 1° b Multistage 2° Multistage 3° Multistage 4° 0.160 164.06 31.3 15.9 All the models reduced to the multistage 1° model, so it was selected.

4 5 6 7 8 9 10

11

12 13 14 15

16 a Selected model in bold; scaled residuals for selected model for doses 0, 1.5, 7, 35, and 107 mg/kg

- d were

− 1.37, 0.35, 0.54, 1.34,

− 1.05, r espectively. The BMD 10 and BMDL 10 for the selected model were 64.2 and 32.6 mg/kg

- d, respectively.
b For the multistage 2°, 3°, and 4° models, the b2, b3 and b4 coefficient estimates were 0 (boundary of parameter space). The models in this row reduced to the multistage 1° model.

0 0.05

0.1 0.15

0.2 0.25

0.3

0 20 40 60 80 100Fraction Affected

dose Multistage Cancer Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for th

16:21 02/14 2014 BMD BMDL Multistage Cancer Linear extrapolation

**Figur e**  **D**

**- 18** 

**. Plot of incidence rate by dose, with fitted curve for selected**  **model, for B6C3F 1**  **female mouse combined hepatocellular adenoma and carcinoma in mice exposed to RDX by diet for 24 months (Parker**  **et al. , 2006).**  BMR = 5% e xtra r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 48 DRAFT―DO NOT CITE OR QUOTE **Multistage Cancer Model.** (Version: 1.10; Date: 02/28/2013) The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)] The parameter betas are restricted to be positi ve

**Benchmark Dose Computation** BMR = 5% Extra risk BMD = 31.2563 BMDL at the 95% confidence level = 15.8846 BMDU at the 95% confidence level = 136.989 Taken together, (15.8846, 136.989) is a 90% two

- sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.0031477 1
2
3
4
5
6
7
8
9
10
11
12
13 14

15 16

17 18 19 20

21 22 23 Pa rameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0.0520755 0.0658334 Beta(1) 0.00164105 0.000876864

Ana lysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees freedom of p - value

Full model

− 77.1516 5
Fitted model

− 80.0315 2 5.75967 3 0.1239
Reduced model

− 82.5216 1 10.74 4 0.02965

AI C: = 164.063

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0.0521 3.489 1 67

− 1.369

1.5 0.0544 3.373 4 62 0.351

7 0.0629 3.963 5 63 0.538

35 0.105 6.719 10 64 1.338

107 0.2047 6.347 4 31

− 1.045

Ch i^2 = 5.17 d.f = 3 P- value = 0.16Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 49 DRAFT―DO NOT CITE OR QUOTE **Combined results for**  **presence of hepatocellular**  **or alveolar/bronchiolar adenoma**  **or** 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 **carcinoma in B**  **6C3F 1**  **female mice exposed to RDX by diet for 24 months**  **; BMR = 10 %**  **e xtra** 
**r isk** 
BMD = 29.0 mg/kg

- day; BMDL = 17.7 mg/kg

- day

MSCOMBO results
BMR of 10% Extra Risk

**** Start of combined BMD and BMDL Calculations. 

Combined Log

- Likelihood

- 187.3723596892213
Combined Log

- likelihood Constant 166.01737626058841

Benchmark Dose Computation

Specified effect = 0.1
Risk Type = Extra risk

Confidence level = 0.95

BMD = 28.9753

BMDL = 17.6574
Multistage Cancer Slope Factor = 0.00566334Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 50 DRAFT―DO NOT CITE OR QUOTE ** Combined results** **for presence of**  **hepatocellular or alveolar/bronchiolar**  **adenoma or**  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 **carcinoma in B**  **6C3F 1**  **female mice exposed to RDX by diet for 24 months; BMR = 5**  **% e**  **xtra**
**r isk** 
BMD = 29.0 mg/kg

- day; BMDL = 17.7 mg/kg

- day

MSCOMBO results
BMR of 5 % Extra Risk

**** Start of combined BMD and BMDL Calculations. 

Combined Log

- Likelihood

- 187.3723596892213
Combined Log

- likelihood Constant 166.01737626058841

Benchmark Dose Computation

Specified effect = 0.05
Risk Type = Extra risk

Confidence level = 0.95

BMD = 14.1062

BMDL = 8.59627
Multistage Cancer Slope Factor = 0.00581647Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 51 DRAFT―DO NOT CITE OR QUOTE ** Rat** **Tumor Data --**  **Dose**

**- response**  **A nalysis**  **and BMD Modeling Documentation**  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 The incidence of liver carcinomas in male F344 rats from the study by Levine et al. (1983)

wa s consider ed for quantitative dose

- response analysis (see Table D

- 16) for comparison with othe r OSF estimates. The high

- dose male group in Levine et al. (1983) had a markedly lower survival

cu rve than the other dose groups, indicating a substantial number of early deaths in the high

- dose
group. In this case, a time

- to

- tumor analysis is preferred. Although tumor incidence was listed for each animal in this study, the pathology report used a different animal numbering system than the experimental report where the times of death were listed, and the relationship between the two systems was not documented. Therefore, the times of death and the tumor incidence of the animals could not be matched, and a time

- to

- tumor analysis was not possible. Tumor incidence was modeled using the multistage

- cancer models in BMDS (version 2.5). Because the maximum liver tumor response in the male rat was 6.4%, a BMR of 5% was used to model male rat liver tumor data in order to obtain a BMD and BMDL in the range of the experimental data, as recommended in Section 3.2 of Guidelines for Carcinogen Risk Assessment ( U.S. EPA , 2005a ) . To account for the difference in the survival curves across the groups for rats, the

nu mber of animals alive at 12 months was used as the denominator in the analysis. These are the denominators listed in Table D- 16

. To estimate the human equivalent dose at the BMDL, HEDs based on both administered dose scaled by BW 3/4 and PBPK modeling were considered

. C onfidence in the revised rat PBPK model is relatively high (see Appendix C, Section C.2.5); however, the choice of an internal dose is

no t straightforward. First, evidence regarding the involvement of metabolites has been discussed in the literature only in the context of the mouse, and the rate of metabolism (allometrically adjusted) appears to be qualitatively slower for the rat. Second, metabolism in the model represents the total of all pathways, whereas it is only the minor N

- nitroso metabolic route, and not the oxidative route, that has been proposed as a factor in RDX

- induced mouse carcinogenicity. Third, while blood concentration of RDX as an internal dose would be more proximally relevant to the tissue than administered dose, there are no data to indicate that the parent RDX is directly related to its carcinogenicity. Therefore, given the uncertainties, HEDs based on both adminis tered dose scaled by BW 3/4 and AUC of RDX arterial blood concentration (calculated using the PBPK model) are presented. Extrapolation based on the internal dose of the parent compound is accomplished by assuming toxicological equivalence when dose is expressed in terms of the AUC of the RDX blood concentration. The POD estimate s for rat liver carcinomas are provided in Table D-21 , and detailed BMD modeling results are provided in Table D

- 22

. Results based on two dose

- metrics are presented: administe red dose of RDX scaled by BW 3/4 (when dose is expressed in terms of mg/kg

- day, this entails scaling by BW −1/4 ) and AUC of RDX arterial blood concentration (using PBPK modeling). Linear extrapoloation from the POD derived from these two dose

- metrics result ed in candidate OSFs of 0.017 and 0.009 (mg/kg

- day) −1 , respectively. It is important to note that EPA consideredSupplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 52 DRAFT―DO NOT CITE OR QUOTE that the association between RDX exposure and rat liver tumors is not strong, reflecting the 1
2
3
4

5 6 7

8 9 10 11 12

13
14
15
16 relatively low magnitude of the r at liver carcino ma response and reduced confidence that the high

dose group accurately reflects lifetime cancer incidence because, in part, of low survival (see discussion in Section 1.1.5)

.

**Ta ble D**  **-21 . Model predictions and oral slope factor for hepatocellular**  **carcinomas in male F344 rats administered RDX in the diet for 2 years (Levine**  **et al., 1983)**

Tumor type Selected model BMR BMD , mg/kg

- d BMDL , mg/kg

- d POD = BMDL 05

- HED ,

mg/kg

- d Candidate OSF a (mg/kg

- d) -1
Hepatocellular carcinomas Multistage 1° 5% ER 28.5 11.8 2. 88 b , 5. 75 c 0. 017 b , 0. 009 c

a Slope factor = BMR/BMDL 05

- HED , where BMR = 0.05 (5% extra risk).
b Based on allometric scaling of administered RDX dose; BMDL 05

- HED = BMDL 05 × (BW a 1/4 /BW h 1/4 ), BW a = 0.25 kg, and

BW h = 70 kg

. c Based on toxicological equivalence of PBPK model derived AUC of RDX blood concentration

.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 53 DRAFT―DO NOT CITE OR QUOTE **Table D-22.**  **Model predictions for**  **combined hepatocellular adenoma and** 1 2 3 **carcinoma in F344 r**  **ats exposed to RDX by diet for 24 months**  **(Levine et al., 1983)**  **; BMR = 5**  **% e**  **xtra r**  **isk**

Model a Goodness of fit BMD 5 Pct (mg/kg

- d) BMDL 5 Pct (mg/kg

- d) Basis for model selection p - value AIC
Multistage 1° b Multistage 2° Multistage 3° Multistage 4° 0.493 49.095 28.5 11.8 All the models reduced to the multistage 1° model, so it was selected.

4 5 6 7 8 9 a Selected model in bold. Scaled residuals for the selected model for doses 0, 0.3 , 1.5 , 8, and 40 mg/kg

- d were 0.89 ,

− 0.67 , − 0. 74 , 0.74 , and

− 0.26 , respectively.
b For the multistage 2°, 3°, and 4° models, the b2, b3 and b4 coefficient estimates were 0 (boundary of parameter

space). The models in this row reduced to the multistage 1° model.

0 0.05

0.1 0.15

0.2 0.25

0 5 10 15 20 25 30 35 40Fraction Affected

dose Multistage Cancer Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for th

17:39 07/29 2014 BMD BMDL Multistage Cancer Linear extrapolation

10

11 12 13 14

15 **Figur e**  **D- 19** 

**. Plot of incidence rate by dose, with fitted curve for selected**  **model , for**  **combined hepatocellular adenoma and carcinoma in F344 rats**  **exposed to RDX by diet for 24 months (Levine**  **et al., 1983)** 

**.** BMR = 5% e xtra
r isk; dose shown in mg/kg

- day.Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. D- 54 DRAFT―DO NOT CITE OR QUOTE **Multistage Model.** (Version: 3.4; Date: 05/02/2014) 1
2
3
4
5
6
7
8
9
10
11
12
13 14

15 16

17 18 19 20

21 22 23 The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- beta1*dose^1

- beta2*dose^2...)] The parameter betas are restricted to be positive

**Benchmark Dose Computation** BMR = 5% Extra risk BMD = 28.4525 BMDL at the 95% confidence level = 11.8487 BMDU at the 95% confidence level = 235.886 Taken together, (11.8487, 235.886) is a 90% two

- sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.00421987

Parameter Estimates Variable Estimate Default i nitial p arameter v alues Background 0.00766363 0.00949438 Beta(1) 0.00180277 0.00149364

Analysis of Deviance Table

Model Log(likelihood) Number of p aram eter s Deviance Test degrees of freedom p - value

Full model

− 21.0055 5
Fitted model

− 22.5473 2 3.08372 3 0.3789
Reduced model

− 24.4692 1 6.92747 4 0.1398

AIC: = 49.0947

Goodness of Fit Table

Dose Est. p rob. Expected Observed Size Scaled residuals

0 0.0077 0.421 1 55 0.894

0.3 0.0082 0.451 0 55

− 0.674

1.5 0.0103 0.538 0 52

− 0.737

8 0.0219 1.203 2 55 0.735

40 0.0767 2.378 2 31

− 0.255

Chi^2 = 2.4 d.f = 3 P

- value = 0.493Supplemental Information― Hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine

This document is a draft for review purposes only and does not constitute Agency policy. R- 1 DRAFT―DO NOT CITE OR QUOTE 

# REFERENCES FOR APPENDICES 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 ACGIH (American Conference of Governmental Industrial Hygienists). (1991). Threshold limit

va lues and biological exposure indices for 1990

- 1991 [TLV/BEI]. Cincinnati, OH. ACGIH (American Conference of Governmental Industrial Hygienists). (2001). Cyclonite. In Doc umentation of the Threshold Limit Values and Biological Exposure Indices (2nd ed.). Cincinnati, OH. http://www.acgih.org/home.htm ACGIH (American Conference of Governmental Industrial Hygienists). (2011). 2011 TLVs and BEIs: Bas ed on the do cumentation of the threshold limit values for chemical substances and physical agents and biological exposure indices. Cincinnati, OH. http://www.acgih.org/store/ProductDetail.cfm?id=2147 Arfsten, D; Davenport, R; Schaeffer, D. (1994). Reversion of bioluminescent bacteria (Mutatox TM) to t heir luminescent state upon exposure to organic compound s, munitions, and metal salts. Biomed Environ Sci 7: 144

- 149. ATSDR (Agency for Toxic Substances and Disease Registry). (2012). Toxicological Profile for RDX (U pdate) [ATSDR Tox Profile]. (NTIS/10590213). Research Triangle Park, NC.; Agency for Toxic Substances and Disease Registry, Atlanta, GA. <br />: U.S. Department of Health and Human Services, Public Health Service. Bannon, DI. (2006). Toxicology study no. 87

- XE

- 059F

- 06 biomarkers of RDX exposure in breath of swin e 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. (2009). Global gene expression in rat br ain and liver after oral exposure to the explosive hexahydro

- 1,3,5

- trinitro

- 1,3,5

- triazine (RDX). Chem Res Toxicol 22: 620

- 625. http://dx.doi.org/10.1021/tx800444k Bannon, DI; Dillman, JF; Perkins, EJ; Bao, W; Wolfinger, RD; Chu, T; Phillips, CS. (2006). Acute RDX exp osure and gene expression in the rat brain [Abstract]. Toxicologist 90: 392

- 393. Barsotti, M; Crotti, G. (1949). [Attacchi epileptici come manifestazione di intossicazione pr ofessionale da trimetilen

- trinitroamina (T4)]. Med Lav 40: 107

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- triazine

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