EPA/635/R-15/034b

Public Comment Draft

www.epa.gov/iris 

Toxicological 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

March 2016

NOTICE

National Center for Environmental Assessment

Office of Research and Development

U.S. Environmental Protection Agency

Washington, DC 

This document is a Public Comment Draft. This information is distributed solely for the purpose 

of pre-dissemination peer review under applicable information quality guidelines. It has not been 

formally disseminated by EPA. It does not represent and should not be construed to represent any 

Agency determination or policy. It is being circulated for review of its technical accuracy and 

science policy implications.
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

DISCLAIMER

This document is a preliminary draft for review purposes only. This information is

distributed solely for the purpose of pre-dissemination peer review under applicable information 

quality guidelines. It has not been formally disseminated by EPA. It does not represent and should 

not be construed to represent any Agency determination or policy. Mention of trade names or 

commercial products does not constitute endorsement of recommendation for use.

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

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CONTENTS

APPENDIX A. ASSESSMENTS BY OTHER NATIONAL AND INTERNATIONAL HEALTH AGENCIES .A1

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

AND EVALUATION...............................................................................................B-1

B.1. DEFENSE TECHNICAL INFORMATION CENTER (DTIC) LITERATURE SEARCH 

AND SCREEN..............................................................................................B-1

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

ANALYSIS.............................................................................................................C-1

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

C.1.1. Absorption....................................................................................C-1

C.1.2. Distribution ..................................................................................C-6

C.1.3. Metabolism..................................................................................C-7

C.1.4. Excretion ......................................................................................C-9

C.1.5. Physiologically Based Pharmacokinetic (PBPK) Models.............C-11

C.2. HUMAN STUDIES.....................................................................................C-31

C.3. OTHER PERTINENT TOXICITY INFORMATION..........................................C-37

C.3.1. Mortality in Animals...................................................................C-37

C.3.2. Other Noncancer Effects............................................................C-42

C.3.3. Genotoxicity...............................................................................C-62

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 and Model Selection............................. D-3

D.1.2. Modeling Results......................................................................... D-4

D.1.3. Mortality: Dose-Response Analysis and BMD Modeling 

Documentation ......................................................................... D-36

D.2. BENCHMARK DOSE MODELING SUMMARY FOR CANCER ENDPOINTS . D-46

D.2.1. Evaluation of Model Fit and Model Selection for Mouse Tumor 

Data........................................................................................... D-46

D.2.2. Modeling Results for Mouse Tumor Data................................. D-47

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

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D.2.3. Dose-response Analysis and BMD Modeling Documentation for 

Other Tumor Data Sets ............................................................. D-61

REFERENCES FOR APPENDICES .....................................................................................................R-1

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

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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 C-1. Distribution of RDX or radiolabel from administered RDXa ........................................C-7

Table C-2. Principal urinary metabolites of RDX in miniature swine 24 hours after dosing 

with RDX...........................................................................................................................................C-9

Table C-3. Elimination t1/2 values for RDX or radiolabeled RDX...................................................C-10

Table C-4. Parameter values used in the Sweeney et al. (2012a) and Sweeney et al. (2012b) 

PBPK models for RDX in rats, humans, and mice .........................................................C-12

Table C-5. Parameters values used in the EPA application of the rat, human, and mouse 

models ............................................................................................................................................C-15

Table C-6. Doses, dosing formulations, and absorption rate constants in animal and human 

studies ............................................................................................................................................C-20

Table C-7. Sensitivity coefficients for rat and human RDX PBPK models .................................C-26

Table C-8. Summary of case reports of exposure to RDX.................................................................C-31

Table C-9. Occupational epidemiologic studies of RDX: summary of methodologic featuresC35

Table C-10. Evidence pertaining to mortality in animalsa...............................................................C-38

Table C-11. Evidence pertaining to other noncancer effects (hematological) in humans .C-46

Table C-12. Evidence pertaining to other noncancer effects in animalsa..................................C-48

Table C-13. Summary of in vitro studies of the genotoxicity of RDX ..........................................C-63

Table C-14. Summary of in vivo studies of the genotoxicity of RDX ...........................................C-67

Table C-15. Summary of in vitro and in vivo studies of the genotoxicity of RDX 

metabolites...................................................................................................................................C-68

Table D-1. Noncancer endpoints selected for dose-response modeling for RDX....................D-1

Table D-2. Convulsion or mortality endpoints from Crouse et al. (2006) selected for doseresponse modeling for RDX.....................................................................................................D-2

Table D-3. Model predictions for convulsions in female F344 rats exposed to RDX by gavage 

for 90 days (Crouse et al., 2006); BMR = 10% ER..........................................................D-4

Table D-4. Model predictions for convulsions in female F344 rats exposed to RDX by gavage 

for 90 days (Crouse et al., 2006); BMR= 5% ER..............................................................D-7

Table D-5. Model predictions for convulsions in female F344 rats exposed to RDX by gavage 

for 90 days (Crouse et al., 2006); BMR= 1% ER..............................................................D-9

Table D-6. Model predictions for convulsions in male F344 rats exposed to RDX by gavage 

for 90 days (Crouse et al., 2006); BMR = 10% ER....................................................... D-11

Table D-7. Model predictions for convulsions in male F344 rats exposed to RDX by gavage 

for 90 days (Crouse et al., 2006); BMR = 5% ER.......................................................... D-13

Table D-8. Model predictions for convulsions in male F344 rats exposed to RDX by gavage 

for 90 days (Crouse et al., 2006); BMR = 1% ER.......................................................... D-15

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 = 10% ER.................................. D-17

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 = 5% ER .......................... D-19

Table D-11. Model predictions for convulsions in male and female F344 rats exposed to 

RDX by gavage for 90 days (Crouse et al., 2006); BMR = 1% ER .......................... D-21

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Table D-12. Model predictions for convulsions in female F344 rats exposed to RDX by 

gavage on gestational days (GDs) 6−19 (Cholakis et al., 1980); BMR = 10% ER.D23

Table D-13. Model predictions for convulsions in female F344 rats exposed to RDX by 

gavage on GDs 6−19 (Cholakis et al., 1980); BMR = 5% ER.................................... D-25

Table D-14. Model predictions for convulsions in female F344 rats exposed to RDX by 

gavage on GDs 6−19 (Cholakis et al., 1980); BMR = 1% ER.................................... D-27

Table D-15. Model predictions for combined incidence of convulsion and mortality in male 

and female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 

2006); BMR = 1% ER............................................................................................................... D-29

Table D-16. Model predictions for testicular degeneration in male B6C3F1 mice exposed to 

RDX by diet for 24 months (Lish et al., 1984); BMR = 10% ER.............................. D-31

Table D-17. Model predictions for prostate suppurative inflammation in male F344 rats 

exposed to RDX by diet for 24 months (Levine et al., 1983b); BMR = 10% ER ....D34

Table D-18. Mortality data selected for dose-response modeling for RDX ............................. D-36

Table D-19. Model predictions for combined mortality in male and female F344 rats 

exposed to RDX by diet for 13 weeks (Levine et al., 1981); BMR = 1% ER ...... D-39

Table D-20. Summary of BMD modeling results for model predictions for mortality (number 

found dead) in rats exposed to RDX in the diet for 13 weeks (von Oettingen et al., 

1949).............................................................................................................................................. D-41

Table D-21. Model predictions for combined mortality (number found dead) in male and 

female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 2006); 

BMR= 1% ER............................................................................................................................... D-42

Table D-22. Model predictions for mortality in female Sprague-Dawley rats exposed to RDX 

by gavage on gestation days 6–15 (Angerhofer, 1986); BMR = 1% ER.............. D-44

Table D-23. Cancer endpoints selected for dose-response modeling for RDX....................... D-46

Table D-24. Model predictions for combined alveolar/bronchiolar adenoma and carcinoma 

in female B6C3F1 mice exposed to RDX by diet for 24 months (Lish et al., 1984); 

BMR = 10% ER........................................................................................................................... D-47

Table D-25. Model predictions for combined alveolar/bronchiolar adenoma and carcinoma 

in female B6C3F1 mice exposed to RDX by diet for 24 months (Lish et al., 1984); 

BMR = 5% ER.............................................................................................................................. D-50

Table D-26. Model predictions for combined hepatocellular adenoma and carcinoma in 

female B6C3F1 mice exposed to RDX by diet for 24 months (Parker et al., 2006); 

BMR = 10% ER........................................................................................................................... D-52

Table D-27. Model predictions for B6C3F1 female mouse combined hepatocellular adenoma 

and carcinoma in mice exposed to RDX by diet for 24 months (Parker et al., 

2006); BMR = 5% ER............................................................................................................... D-54

Table D-28. Model predictions for combined hepatocellular adenoma and carcinoma in 

female B6C3F1 mice exposed to RDX by diet for 24 months, using incidence 

frequencies from Parker et al. (2006) and sample sizes from Lish et al. (1984); 

BMR = 10% ER........................................................................................................................... D-58

Table D-29. Liver carcinoma data from Levine et al. (1983b)...................................................... D-61

Table D-30. Model predictions and oral slope factor for alveolar/bronchiolar carcinomas in 

male B6C3F1 mice exposed to RDX by diet for 2 years (Lish et al., 1984)........ D-62

Table D-31. Summary of BMD modeling results for model predictions for 

alveolar/bronchiolar carcinoma in male B6C3F1 mice exposed to RDX by diet 

for 2 years (Lish et al., 1984); BMR = 10% ER.............................................................. D-62

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Table D-32. Model predictions and oral slope factor for hepatocellular carcinomas in male 

F344 rats administered RDX in the diet for 2 years (Levine et al., 1983b)...... D-65

Table D-33. Model predictions for combined hepatocellular adenoma and carcinoma in 

F344 rats exposed to RDX by diet for 24 months (Levine et al., 1983b); BMR = 

5% ER ............................................................................................................................................ D-65

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FIGURES 

Figure C-1. PBPK model structure for RDX in rats and humans. ..................................................C-11

Figure C-2. EPA rat PBPK model predictions fitted to observed RDX blood concentrations in 

male and female Sprague-Dawley rats following i.v. 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-18

Figure C-4. EPA rat model predictions fitted to observed RDX blood concentrations 

following oral exposure to RDX in dry capsules. ..........................................................C-18

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-19

Figure C-6. EPA rat model predictions fitted to observed RDX brain tissue concentrations 

following oral exposure to RDX. ..........................................................................................C-19

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-21

Figure C-8. Comparison of EPA rat model predictions with data from Schneider et al. (1978)

for the subchronic gavage study..........................................................................................C-22

Figure C-9. Comparison of EPA rat model predictions with data from Schneider et al. (1978)

for the subchronic drinking water study. ........................................................................C-22

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

Figure C-11. EPA human model predictions fitted to observed RDX blood concentrations 

resulting from accidental exposure to adults assumed to be 70 kg (Özhan et al., 

2003)...............................................................................................................................................C-24

Figure C-12. Comparison of EPA mouse PBPK model predictions with data from oral 

exposure to RDX dissolved in water. .................................................................................C-27

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-5

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 (Crouse 

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 fitted curve for selected model, for 

convulsions in male F344 rats exposed to RDX by gavage for 90 days (Crouse et 

al., 2006)....................................................................................................................................... D-11

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-13

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-15

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-17

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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 90 days 

(Crouse et al., 2006)................................................................................................................. D-19

Figure D-9. Plot of incidence rate by dose, with the fitted curve of the multistage 3° model, 

for convulsions in male and female F344 rats exposed to RDX by gavage for 90 

days (Crouse et al., 2006). BMR = 1% ER; dose shown in mg/kg-day............... D-21

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 GDs 6−19 

(Cholakis et al., 1980).............................................................................................................. D-23

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 GDs 6−19 

(Cholakis et al., 1980).............................................................................................................. D-25

Figure D-12. 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 GDs 6−19 

(Cholakis et al., 1980).............................................................................................................. D-27

Figure D-13. Plot of incidence rate by dose with fitted curve for Multistage 3° model for 

Model predictions for combined incidence of convulsion and mortality in male 

and female F344 rats exposed to RDX by gavage for 90 days (Crouse et al., 

2006).............................................................................................................................................. D-29

Figure D-14. Plot of incidence rate by dose, with fitted curve for selected model, for 

testicular degeneration in male B6C3F1 mice exposed to RDX by diet for 24 

months (Lish et al., 1984)...................................................................................................... D-32

Figure D-15. 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., 1983b)........................................................................................ D-35

Figure D-16. Plot of incidence rate by dose, with the fitted curve of the multistage 2° model, 

for combined mortality in male and female F344 rats exposed to RDX by diet for 

13 weeks (Levine et al., 1981); BMR = 1% ER.............................................................. D-39

Figure D-17. Plot of incidence rate by dose with fitted curve for Dichotomous-Hill model for 

Model predictions for mortality (number found dead) in rats exposed to RDX in 

the diet for 13 weeks (von Oettingen et al., 1949); dose shown in mg/kg-day....D41

Figure D-18. Plot of incidence rate by dose, with the fitted curve of the multistage 2° model, 

for mortality in male and female F344 rats exposed to RDX by gavage for 90 

days (Crouse et al., 2006). BMR = 1% ER; dose shown in mg/kg-day............... D-43

Figure D-19. Plot of incidence rate by dose, with the fitted curve of the multistage 3° model, 

for mortality in female Sprague-Dawley rats exposed to RDX by gavage on 

gestation days 6–15 (Angerhofer, 1986); BMR = 1% ER. ........................................ D-45

Figure D-20. Plot of incidence rate by dose, with the fitted curve for the selected model, for 

combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F1 mice 

exposed to RDX by diet for 24 months (Lish et al., 1984)........................................ D-48

Figure D-21. Plot of incidence rate by dose, with fitted curve for selected model, for 

combined alveolar/bronchiolar adenoma and carcinoma in female B6C3F1 mice 

exposed to RDX by diet for 24 months (Lish et al., 1984)........................................ D-50

Figure D-22. Plot of incidence rate by dose, with fitted curve for selected model, for 

combined hepatocellular adenoma and carcinoma in female B6C3F1 mice 

exposed to RDX by diet for 24 months (Parker et al., 2006). ................................. D-52

Figure D-23. Plot of incidence rate by dose, with fitted curve for selected model, for B6C3F1

female mouse combined hepatocellular adenoma and carcinoma in mice 

exposed to RDX by diet for 24 months (Parker et al., 2006). ................................. D-54

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Figure D-24. Plot of incidence rate by dose with fitted curve for Multistage-Cancer 1° model 

for model predictions for combined hepatocellular adenoma and carcinoma in 

female B6C3F1 mice exposed to RDX by diet for 24 months, using incidence 

frequencies from Parker et al. (2006) and sample sizes from (Parker et al., 

2006).............................................................................................................................................. D-58

Figure D-25. Plot of incidence rate by dose with fitted curve for Multistage-Cancer 1° model 

for Model predictions for alveolar/bronchiolar carcinoma in male B6C3F1 mice 

exposed to RDX by diet for 24 months (Lish et al., 1984)........................................ D-62

Figure D-26. 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., 1983b). ................................................................ D-66

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ABBREVIATIONS

AAP Army ammunition plant

ACGIH American Conference of Governmental 

Industrial Hygienists 

AChE acetylcholinesterase

ADAF age-dependent adjustment factor

AIC Akaike’s information criterion 

ALP alkaline phosphatase

ALT alanine aminotransferase 

AST aspartate aminotransferase 

atm atmosphere

ATSDR Agency for Toxic Substances and 

Disease Registry 

AUC area under the curve

BDNF brain-derived neurotrophic factor

BHC beta-hexachlorocyclohexane

BMC benchmark concentration

BMCL benchmark concentration lower 

confidence limit

BMD benchmark dose 

BMDL benchmark dose lower confidence limit 

BMDS Benchmark Dose Software 

BMDU benchmark dose upper bound 

BMR benchmark response 

BUN blood urea nitrogen 

BW body weight 

CASRN Chemical Abstracts Service Registry 

Number 

CCL Contaminant Candidate List

CI confidence interval

CICAD Concise International Chemical 

Assessment Document

CNS central nervous system 

CSF cerebrospinal fluid

CYP450 cytochrome P450 

DAF dosimetric adjustment factor 

DDT dichlorodiphenyltrichloroethane

d.f. degrees of freedom

DMSO dimethylsulfoxide 

DNA deoxyribonucleic acid 

DNX 1-nitro-3,5-dinitroso1,3,5-triazacyclohexane 

DTIC Defense Technical Information Center

EEG electroencephalogram

EHC Environmental Health Criteria

EPA Environmental Protection Agency 

ER extra risk

FDA Food and Drug Administration 

FOB functional observational battery

FUDS Formerly Used Defense Sites

GABA gamma-amino butyric acid

GD gestational day

GI gastrointestinal

GLP good laboratory practices

HED human equivalent dose 

HERO Health and Environmental Research 

Online

HGPRT hypoxanthine-guanine 

phosphoribosyltransferase

HMX octahydro-1,3,5,7-tetranitro1,3,5,7-tetrazocine

IARC International Agency for Research on 

Cancer

i.p. intraperitoneal

IPCS International Programme on Chemical 

Safety

IRIS Integrated Risk Information System 

IUR inhalation unit risk

i.v. intravenous

LDH lactate dehydrogenase

LOAEL lowest-observed-adverse-effect level 

LOD limit of detection

miRNA microRNA

MNX hexahydro-1-nitroso-3,5-dinitro1,3,5-triazine 

MOA mode of action

MRL Minimal Risk Level

NAPDH nicotinamide adenine dinucleotide 

phosphate 

NAS National Academy of Science

NCE normochromatic erythrocyte

NCEA National Center for Environmental 

Assessment 

NCI National Cancer Institute

NCTR National Center for Toxicological 

Research

NHANES National Health and Nutrition 

Examination Survey

NICNAS National Industrial Chemicals 

Notification and Assessment Scheme

NIEHS National Institute of Environmental 

Health Sciences

NIOSH National Institute for Occupational 

Safety and Health

NOAEL no-observed-adverse-effect level 

NOEL no-observed-effect level 

NPL National Priorities List

NRC Nuclear Regulatory Commission

NSCEP National Service Center for 

Environmental Publications

NTP National Toxicology Program 

NZW New Zealand White

OR odds ratio

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ORD Office of Research and Development 

OSF oral slope factor

OSHA Occupational Safety and Health 

Administration

PBPK physiologically based pharmacokinetic 

PCB polychlorinated biphenyl

PCE polychromatic erythrocyte

PEL Permissible Exposure Limit

PND postnatal day

POD point of departure 

PWG Pathology Working Group

RBC red blood cell

RDX Royal Demolition eXplosive 

(hexahydro-1,3,5-trinitro1,3,5-triazine)

REL Recommended Exposure Limit

RfC inhalation reference concentration 

RfD oral reference dose 

SDMS spontaneous death or moribund 

sacrifice

SDWA Safe Drinking Water Act

SGOT glutamic oxaloacetic transaminase, also 

known as AST 

SGPT glutamic pyruvic transaminase, also 

known as ALT 

SLE systemic lupus erythematosus

SS scheduled sacrifice

TLV Threshold Limit Value

TNT trinitrotoluene

TNX hexahydro-1,3,5-trinitroso1,3,5-triazine 

TSCATS Toxic Substances Control Act Test 

Submissions

TWA time-weighted average 

U.S. United States of America

UCM Unregulated Contaminant Monitoring

UF uncertainty factor 

UFA animal-to-human uncertainty factor 

UFD database deficiencies uncertainty factor 

UFH human variation uncertainty factor 

UFL LOAEL-to-NOAEL uncertain factor

UFS subchronic-to-chronic uncertainty 

factor 

WBC white blood cell

WHO World Health Organization

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APPENDIX A. ASSESSMENTS BY OTHER NATIONAL 

AND INTERNATIONAL HEALTH AGENCIES

1 Table A-1. Assessments by other national and international health agencies 

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 [physiologically based 

pharmacokinetic or PBPK modeling] and 10 for human variability)

Intermediate oral MRL—0.1 mg/kg-d 

Basis: 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 variability)

Chronic oral MRL—0.1 mg/kg-d 

Basis: tremors and convulsions in rats (Levine et al., 1983b); 

application of a composite UF of 30 (3 for extrapolation from animals 

to humans with dosimetric adjustments [PBPK modeling] and 10 for 

human variability)

National Institute for Occupational 

Safety and Health (NIOSH, 2012) 

Recommended Exposure Limit (REL)—1.5 mg/m3 TWA for up to a 10-hr 

workday during a 40-hr workweek; short-term (15-min) limit—

3 mg/m3

Basis: agreed with Occupational Safety and Health Administration

(OSHA)-proposed Permissible Exposure Limit (PEL) in 1988 PEL 

Hearings

Skin designation indicates potential for dermal absorption

Basis: agreed with OSHA proposal for skin notation in 1988 PEL 

Hearings

Occupational Safety and Health 

Administration (OSHA, 2012a, b) 

PEL—1.5 mg/m3 time-weighted average (TWA) for an 8-hr workday in a 

40-hr workweek

Basis: adopted from the American Conference of Governmental 

Industrial Hygienists (ACGIH) Threshold Limit Value (TLV) established 

in 1969

Skin designation indicates that cutaneous exposure may contribute to 

overall exposure and measures should be taken to prevent skin 

absorption 

Basis: adopted from ACGIH

Hazardous Substances Information 

System (Safe Work Australia, 2014) 

Exposure standard—1.5 mg/m3 TWA for an 8-hr workday

Basis: adopted from the ACGIH TLV established in 1991

Skin absorption notice indicates that absorption through the skin may be 

a significant source of exposure

Basis: adopted from ACGIH

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APPENDIX B. ADDITIONAL DETAILS OF LITERATURE 

SEARCH STRATEGY | STUDY SELECTION AND 

EVALUATION

1 The literature search for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was conducted in 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

five online scientific databases through January, 2015. The detailed search strategy used to search 

four of these databases—PubMed, Toxline, Toxcenter, and Toxic Substances Control Act Test 

Submissions (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 searches of two recent reviews (Table B-3). Forward searching 

was used to identify articles that cited the selected studies (i.e., the two reviews identified in 

Table B-3), and backward searching was used to identify articles that the selected studies cited. 

B.1. DEFENSE TECHNICAL INFORMATION CENTER (DTIC) LITERATURE 

SEARCH AND SCREEN 

Among the RDX-related citations that were identified in the DTIC database, 826 (722 after 

duplicate removal within DTIC) were classified with the distribution "Approved for Public Release", 

239 (217 after duplicate removal) were classified as “distribution limited to U.S. Government 

agencies and their Contractors,” and 199 (181 after duplicate removal) were classified as 

“distribution limited to U.S. Government agencies only.” A preliminary screen of the 1,120 unique 

citations was performed; 85 citations with unlimited distribution and 10 citations with limited 

distribution were selected for further review as potential sources of health effects data or 

supporting information. The remaining 1,025 unlimited and limited-distribution DTIC references 

not selected for further consideration were not studies of RDX or did not contain information 

pertinent to the assessment of the health effects of RDX (e.g., documents were related to 

environmental properties such as leaching, explosive properties, fuel and propellant properties, 

weapons systems, treatment of wastewater containing explosives, and disposal technologies).

The 85 unique citations with unlimited distribution were uploaded to the Health and 

Environmental Research Online (HERO) website1 (http://hero.epa.gov). The 10 citations with 

1HERO is a database of scientific studies and other references used to develop EPA’s assessments aimed at 

understanding the health and environmental effects of pollutants and chemicals. It is developed and 

managed in EPA’s Office of Research and Development (ORD) by the National Center for Environmental 

Assessment (NCEA). The database includes more than 1.6 million scientific references, including articles 

from the peer-reviewed literature. New studies are added continuously to HERO. 

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

A-1 DRAFT―DO NOT CITE OR QUOTE

 
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 limited distribution were subject to a more in-depth screen to determine whether the references 

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3 

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5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

provided additional primary health effects data and whether the U.S. Environmental Protection 

Agency (EPA) should seek authorization for public distribution and upload to HERO. A review of 

the abstract or full-text of the documents associated with the limited-distribution citations resulted 

in the following determinations:

• one citation was excluded because it did not provide additional primary health effects data. 

The citation reported data from a study that was subsequently published (Hathaway and 

Buck, 1977) and had already been identified by the literature search strategy. 

• one citation (dated 1944) provided human and animal inhalation data and was considered 

pertinent, but was not brought forward for further review because flaws in the design of 

both the human and animal studies were such that results would not be considered 

credible. Experimental animal study design issues included lack of a control group, small 

numbers of animals, incomplete information on dosage or exposure levels, and inadequate 

reporting. The human study described a case series and lacked a referent group and 

measures of RDX exposure. 

• eight citations were regulatory documents, reviews, or risk assessments that did not 

specifically identify RDX and did not appear to contain primary health effects data. 

Based on these determinations, none of the 10 limited distribution citations that were 

subject to further review were selected for further consideration or added to HERO.

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

A-2 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

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

2 Toxcenter, TSCATS)

Database Terms Hits

PubMed

Date: 4/2012

((((121-82-4) OR (Cyclonite[tw] OR Cyclotrimethylenetrinitramine[tw] OR 

"cyclotrimethylene trinitramine"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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-Triaza1,3,5-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-Trinitrohexahydros-triazine"[tw] OR "1,3,5-Trinitroperhydro-1,3,5-triazine"[tw] OR "Esaidro1,3,5-trinitro-1,3,5-triazina"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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-trinitro1,3,5-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-Triaza1,3,5-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-Trinitrohexahydros-triazine"[tw] OR "1,3,5-Trinitroperhydro-1,3,5-triazine"[tw] OR "Esaidro1,3,5-trinitro-1,3,5-triazina"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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])) 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])) 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])))) 

NOT (((((121-82-4) OR (Cyclonite[tw] OR Cyclotrimethylenetrinitramine[tw] OR 

"cyclotrimethylene trinitramine"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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-Triaza1,3,5-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-Trinitrohexahydros-triazine"[tw] OR "1,3,5-Trinitroperhydro-1,3,5-triazine"[tw] OR "Esaidro1,3,5-trinitro-1,3,5-triazina"[tw] OR "Hexahydro-1,3,5-trinitro337

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

B-3 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Database Terms Hits

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-trinitro1,3,5-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-Triaza1,3,5-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-Trinitrohexahydros-triazine"[tw] OR "1,3,5-Trinitroperhydro-1,3,5-triazine"[tw] OR "Esaidro1,3,5-trinitro-1,3,5-triazina"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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])) 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])) 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

(Cyclonite[tw] OR RDX[tw] OR Cyclotrimethylenetrinitramine[tw] OR 

"cyclotrimethylene trinitramine"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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-Triaza1,3,5-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-Trinitrohexahydros-triazine"[tw] OR "1,3,5-Trinitroperhydro-1,3,5-triazine"[tw] OR "Esaidro1,3,5-trinitro-1,3,5-triazina"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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

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

B-4 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Database Terms Hits

PubMed

Date limit: 

11/2012−

1/2014

(Cyclonite[tw] OR RDX[tw] OR Cyclotrimethylenetrinitramine[tw] OR 

"cyclotrimethylene trinitramine"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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-Triaza1,3,5-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-Trinitrohexahydros-triazine"[tw] OR "1,3,5-Trinitroperhydro-1,3,5-triazine"[tw] OR "Esaidro1,3,5-trinitro-1,3,5-triazina"[tw] OR "Hexahydro-1,3,5-trinitro1,3,5-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 (("2012/11/01"[Date - MeSH] : 

"3000"[Date - MeSH]) OR ("2012/11/01"[Date - Entrez] : "3000"[Date -

Entrez]) OR ("2012/11/01"[Date - Create] : "3000"[Date - Create]))

138

PubMed

Date limit: 

11/2013−

1/2015

("cyclonite"[nm] OR 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-trinitros-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-Trinitro1,3,5-triazacyclohexane"[tw] OR "1,3,5-Trinitrohexahydro-1,3,5-triazine"[tw] 

OR "1,3,5-Trinitrohexahydro-s-triazine"[tw] OR "1,3,5-Trinitroperhydro1,3,5-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-trinitro1,3,5-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 (2013/11/01 : 3000[crdat] OR 2013/11/01 : 3000[edat])

76

Toxline

Date: 4/2012

Notes: Searched CASRN or synonyms; removed invertebrates, aquatic 

organisms, amphibians, earthworms.

507

Toxline

Date limit: 

2011−2/2013

@OR+("Cyclonite"+"RDX"+"Cyclotrimethylenetrinitramine"+"cyclotrimethylen

e trinitramine"+"Hexahydro-1,3,5-trinitro-1,3,5-triazine"+"Hexahydro1,3,5-trinitro-s-triazine"+"Hexogen"+"1,3,5-trinitro1,3,5triazine"+"1,3,5-Triaza-1,3,5-trinitrocyclohexane"+"1,3,5-Trinitro1,3,5-triazacyclohexane"+"1,3,5-Trinitrohexahydro-1,3,5-triazine"+

"1,3,5-Trinitrohexahydro-s-triazine"+@term+@rn+121-82-4)+

@AND+@range+yr+2011+2013+@NOT+@org+pubmed+pubdart+crisp+tscats

5

@OR+("1,3,5-Trinitroperhydro-1,3,5-triazine"+"Esaidro-1,3,5-trinitro1,3,5-triazina"+"Hexahydro-1,3,5-trinitro-1,3,5-triazin"+"Perhydro1,3,5-trinitro-1,3,5-triazine"+"Cyclotrimethylenenitramine"+

"Trimethylenetrinitramine"+"Trimethylene+trinitramine"+

"Trimethyleentrinitramine"+"Trinitrocyclotrimethylene+triamine"+

"Trinitrotrimethylenetriamine"+"CX+84A"+"Cyklonit"+"Geksogen"+

0

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

B-5 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Database Terms Hits

"Heksogen"+"Hexogeen"+"Hexolite"+"KHP+281")+@AND+@range+yr+2011+

2013+@NOT+@org+pubmed+pubdart+crisp+tscats

Toxline

Date limit: 

2012−1/2014

@OR+("Cyclonite"+"RDX"+"Cyclotrimethylenetrinitramine"+"cyclotrimethylen

e trinitramine"+"Hexahydro-1,3,5-trinitro-1,3,5-triazine"+"Hexahydro1,3,5-trinitro-s-triazine"+"Hexogen"+"1,3,5-trinitro-1,3,5-triazine"+

"1,3,5-Triaza-1,3,5-trinitrocyclohexane"+"1,3,5-Trinitro1,3,5-triazacyclohexane"+"1,3,5-Trinitrohexahydro-1,3,5-triazine"+

"1,3,5-Trinitrohexahydro-s-triazine"+@term+@rn+121-82-4)+ @AND+

@range+yr+2012+2014+@NOT+@org+pubmed+pubdart+crisp+tscats

10

@OR+("1,3,5-Trinitroperhydro-1,3,5-triazine"+"Esaidro-1,3,5-trinitro1,3,5-triazina"+"Hexahydro-1,3,5-trinitro-1,3,5-triazin"+"Perhydro1,3,5-trinitro-1,3,5-triazine"+"Cyclotrimethylenenitramine"+

"Trimethylenetrinitramine"+"Trimethylene+trinitramine"+

"Trimethyleentrinitramine"+"Trinitrocyclotrimethylene+triamine"+

"Trinitrotrimethylenetriamine"+"CX+84A"+"Cyklonit"+"Geksogen"+

"Heksogen"+"Hexogeen"+"Hexolite"+"KHP+281")+@AND+@range+yr+2012+

2014+@NOT+@org+pubmed+pubdart+crisp+tscats

0

Toxline

Date limit: 

2013-1/2015

@SYN0+@OR+(RDX+"cyclotrimethylene+trinitramine"+"1,3,5-trinitro1,3,5-triazine"+"1,3,5-Triaza-1,3,5-trinitrocyclohexane"+"1,3,5-Trinitro1,3,5-triazacyclohexane"+"1,3,5-Trinitrohexahydro-1,3,5-triazine"+

"1,3,5-Trinitrohexahydro-s-triazine"+"1,3,5-Trinitroperhydro-1,3,5-triazine"+

"CX+84A")+@AND+@range+yr+2012+2015+@NOT+@org+pubmed+pubdart+"

nih+reporter"+tscats+crisp

19

@SYN0+@OR+("Cyclonite"+"Cyclotrimethylenenitramine"+"Cyclotrimethylene

trinitramine"+"Hexahydro-1,3,5-trinitro-1,3,5-triazine"+"Hexahydro1,3,5-trinitro-s-triazine"+"Hexogen"+"Hexolite"+"KHP+281"+"PBX+(af)+108"+

"PBXW+108(E)"+"Pbx(AF)+108"+"Perhydro-1,3,5-trinitro-1,3,5-triazine")+

@AND+@range+yr+2012+2015+@NOT+@org+pubmed+pubdart+

"nih+reporter"+tscats+crisp

9

@SYN0+@OR+("Research+Development+Explosive"+"Royal+Demolition+eXpl

osive+"Trimethylenetrinitramine"+"Trinitrocyclotrimethylene+triamine"+"Trini

trotrimethylenetriamine"+"sym-Trimethylene+trinitramine"+@term+

@rn+121-82-4+@term+@rn+204655-61-8+@term+@rn+50579-23-

2+@term+@rn+53800-53-6+@term+@rn+57608-45-4+@term+@rn+82030-

42-0)+ @AND+@range+yr+2012+2015+@NOT+@org+pubmed+pubdart+

"nih+reporter"+tscats+crisp

0

TSCATS

Date: 2/2013

@term+@rn+121-82-4+@AND+@org+tscats 4

TSCATS 2

Date: 1/2015

121-82-4 from EPA receipt date 01/01/2000 0

TSCATS 

8e/FYI

Date: 1/2015

("121-82-4" OR "1,3,5-Triazine, hexahydro-1,3,5-trinitro-") tsca (8e OR FYI) 0

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

B-6 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Database Terms Hits

Toxcenter

Date: 4/2012

((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-trinitro1,3,5-triazine" OR "1,3,5-Triaza-1,3,5-trinitrocyclohexane" OR "1,3,5-Trinitro1,3,5-triazacyclohexane" OR "1,3,5-Trinitrohexahydro-1,3,5-triazine" OR 

"1,3,5-Trinitrohexahydro-s-triazine" OR "1,3,5-Trinitroperhydro-1,3,5-triazine" 

OR "Esaidro-1,3,5-trinitro-1,3,5-triazina" OR "Hexahydro-1,3,5-trinitro1,3,5-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 

((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 

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 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 titles 

screened 

(20 selected for 

full records and 

added to HERO)

Toxcenter 

Date limit:

1/2012−

2/2013

(((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-trinitro1,3,5-triazine" OR "1,3,5-Triaza-1,3,5-trinitrocyclohexane" OR "1,3,5-Trinitro1,3,5-triazacyclohexane" OR "1,3,5-Trinitrohexahydro-1,3,5-triazine" OR 

"1,3,5-Trinitrohexahydro-s-triazine" OR "1,3,5-Trinitroperhydro-1,3,5-triazine" 

OR "Esaidro-1,3,5-trinitro-1,3,5-triazina" OR "Hexahydro-1,3,5-trinitro1,3,5-triazin" OR "Perhydro-1,3,5-trinitro-1,3,5-triazine" OR 

26 titles screened 

(6 selected for 

full records and 

added to HERO)

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

B-7 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Database Terms Hits

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>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 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 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 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 removed.

Toxcenter 

Date limit:

11/2012−

1/2014

(((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-trinitro1,3,5-triazine" OR "1,3,5-Triaza-1,3,5-trinitrocyclohexane" OR "1,3,5-Trinitro1,3,5-triazacyclohexane" OR "1,3,5-Trinitrohexahydro-1,3,5-triazine" OR 

"1,3,5-Trinitrohexahydro-s-triazine" OR "1,3,5-Trinitroperhydro-1,3,5-triazine" 

OR "Esaidro-1,3,5-trinitro-1,3,5-triazina" OR "Hexahydro-1,3,5-trinitro1,3,5-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 adverse OR poisoning)/st,ct,it OR inhal? OR pulmon? OR nasal? OR 

20 titles screened

(0 selected for 

full records; none 

added to HERO)

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

B-8 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Database Terms Hits

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 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 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 removed.

Toxcenter 

Date limit: 

11/2013−

1/2015

(((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-trinitro1,3,5-triazine" OR "1,3,5-Triaza-1,3,5-trinitrocyclohexane" OR "1,3,5-Trinitro1,3,5-triazacyclohexane" OR "1,3,5-Trinitrohexahydro-1,3,5-triazine" OR 

"1,3,5-Trinitrohexahydro-s-triazine" OR "1,3,5-Trinitroperhydro-1,3,5-triazine" 

OR "Esaidro-1,3,5-trinitro-1,3,5-triazina" OR "Hexahydro-1,3,5-trinitro1,3,5-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 >2013 OR ed>20131101)) 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 

80 titles screened

(3 selected for 

full records and 

added to HERO)

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

B-9 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Database Terms Hits

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 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))

Note: Duplicates were removed.

1 

2

3 

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

Database Terms Hits

DTIC Online 

Access 

Controlled

Date 

searched: 

1/2015

Synonyms in all fields search box

("121-82-4" OR "RDX" OR "Cyclotrimethylenetrinitramine" OR "Cyclonite" OR 

"cyclotrimethylene trinitramine" OR "Hexogen" OR "Hexahydro-1,3,5-trinitro1,3,5-triazine" OR "Hexahydro-1,3,5-trinitro-s-triazine" OR "Trimethylene 

trinitramine" OR "Trimethylenetrinitramine" OR "Hexolite" OR 

"Trinitrotrimethylenetriamine")

Keywords in citation box 

("toxicity" OR "toxicology" OR "poisoning" OR "cancer" OR "carcinogens" OR 

"carcinogen" OR "neoplasms" OR "neoplasm" OR "oncogenesis" OR 

"teratogenic compounds" OR "lethality" OR "death" OR "body weight" OR 

"immunology" OR "genotoxicity" OR "mutagenicity" OR "mutagens" OR 

"mutations" OR "oral" OR "gavage" OR "inhalation" OR "dermal" OR 

"metabolism" OR "pharmacokinetics" OR "pharmacokinetic" OR "PBPK" OR 

"pharmacology" OR "organs" OR "skin" OR "tissues" OR "body fluids" OR 

"toxic agents" OR "rats" OR "mice" OR "mouse" OR "rat") 

Limited to 

Content type: Documents

Distribution: Approved for Public Release 826 (85 selected 

and added to 

HERO)

Distribution: U.S. Gov't and Contractors 239 (0 selected and 

added to HERO)

Distribution: U.S. Gov't Only 199 (0 selected and 

added to HERO)

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

B-10 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

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

Selected key reference(s) or sources Date

Additional references 

identified

“Forward” and “backward” Web of Science searchesa

Sweeney et al. (2012a). Assessing the non-cancer risk for RDX (hexahydro1,3,5-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 citations added

Sweeney et al. (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 (backwards search)

0 search results

3/2013 0 citations added

Sweeney et al. (2012a). Assessing the non-cancer risk for RDX (hexahydro1,3,5-trinitro-1,3,5-triazine) using physiologically based pharmacokinetic 

(PBPK) modeling. Regul Toxicol Pharmacol 62(1):107−114.

(review of 35 references cited in this paper)

3/2013 0 citations added

Sweeney et al. (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

(review of 69 references cited in this paper)

3/2013 3 citations added

Online regulatory sources

Combination of Chemical Abstracts Registry Number (CASRN) and 

synonyms searched on the following websites:

Agency for Toxic Substances and Disease Registry (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)

California Environmental Protection Agency (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) 

EPA—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 National Service Center for Environmental Publications (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/indexeng.php)

4/11/2012

1/27/2014

1/22/2015

15 citations added

1 citation added

0 citations added

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

B-11 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Selected key reference(s) or sources Date

Additional references 

identified

Health Canada Second Priority List Assessments

(http://www.hc-sc.gc.ca/ewh-semt/pubs/contaminants/psl2-lsp2/indexeng.php) 

International Agency for Research on Cancer (IARC) 

(http://monographs.iarc.fr/htdig/search.html) 

International Programme on Chemical Safety (IPCS) INCHEM 

(http://www.inchem.org/) 

National Academy of Science (NAS) via the National Academies Press

(http://www.nap.edu/) 

National Cancer Institute (NCI) 

(http://www.cancer.gov) 

National Center for Toxicological Research (NCTR) 

(http://www.fda.gov/AboutFDA/CentersOffices/OC/OfficeofScientificand

MedicalPrograms/NCTR/default.htm) 

National Institute of Environmental Health Sciences (NIEHS)

(http://www.niehs.nih.gov/)

National Institute for Occupational Safety and Health (NIOSH) NIOSHTIC 2 

(http://www2a.cdc.gov/nioshtic-2/) 

National Toxicology Program (NTP)—RoC, status, results, and management 

reports 

(http://ntpsearch.niehs.nih.gov/query.html) 

World Health Organization (WHO) assessments—Concise International 

Chemical Assessment Documents (CICADs), Environmental Health Criteria 

(EHC) 

(http://www.who.int/ipcs/assessment/en/)

1 

2 

3 

4 

a

Sweeney et al. (2012a) and Sweeney et al. (2012b) were selected for forward and backward searching in the Web 

of Science as the two more recent reviews of the health effects of RDX toxicity in the published literature.

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

B-12 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

APPENDIX C. INFORMATION IN SUPPORT OF 

HAZARD IDENTIFICATION AND DOSE-RESPONSE 

ANALYSIS

1 C.1. TOXICOKINETICS

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

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28

Hexahydro-1,3,5-trinitro-1,3,5-triazine (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 transferred 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.1.1. Absorption

Absorption of RDX following oral exposure has been demonstrated in humans and 

laboratory animals (rats, mice, swine, and voles) through measurement of radiolabeled 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 to 90% 

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

administered. Studies 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 absorption of RDX is 

provided by in vitro studies showing that RDX can be absorbed through excised skin of humans and 

animals. 

Oral Absorption

Quantitative information on blood levels following accidental exposure to RDX is limited to 

two studies of accidental oral exposures (Küçükardali et al., 2003; Woody et al., 1986) and one 

study of mixed dermal and inhalation exposure (Özhan et al., 2003). A number of qualitative case 

studies of accidental exposures with similar toxic effects provide additional support that RDX is 

absorbed into the body (Hett and Fichtner, 2002; Harrell-Bruder and Hutchins, 1995; Goldberg et 

al., 1992; Ketel and Hughes, 1972; Hollander and Colbach, 1969; Stone et al., 1969). The oral 

absorption 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 

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

B-1 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 al., 1986). RDX was measured in serum, urine, cerebrospinal fluid, and feces. Based on a kinetic 

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

analysis of the serum RDX concentrations, the dose was estimated to be 85 mg/kg and the firstorder absorption rate constants were estimated to be 0.34−2.20 hour−1 (Woody et al., 1986)2. 

Sweeney et al. (2012a) estimated the absorption rate constant for this same subject to be 

0.060 hour−1. The large range in the calculated absorption rate constants resulted from uncertainty 

in the dose and time to peak serum RDX levels, and the models that were used to simulate the RDX 

toxicokinetics. Özhan et al. (2003) summarized plasma RDX levels in five military personnel who 

were accidentally exposed to toxic levels of RDX. Although Özhan et al. (2003) reported that 

personnel were exposed through dermal and inhaled routes, secondary oral exposure may have 

occurred. Based on physiologically based pharmacokinetic (PBPK) model fits to the plasma RDX 

concentration data, Sweeney et al. (2012a) estimated a first-order absorption rate constant of 

0.033 hour−1. Küçükardali et al. (2003) summarized 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 and 

repeated 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 [14C]-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 

includes 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 

absorbed from the GI tract. Similar results were observed following repeated daily oral exposure of 

Sprague-Dawley rats to [14C]-RDX by gavage (in DMSO) or drinking water for 1 week. Based on 

recovery of carbon-14 in urine and expired air and the 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 

intravenous (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 based on the reported blood RDX concentrations, 

terminal elimination rate constants (estimated for this review by fitting the serum RDX data with a 

first-order exponential function, see Table C-5 in Section C.1.4, Excretion) and the blood area under 

the curve (AUC) values (calculated for this review using the trapezoid rule extrapolated to infinite 

2Woody 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. 

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

B-2 DRAFT―DO NOT CITE OR QUOTE

 
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 time). Calculated dose-adjusted AUC values were 9.6 and 8.4 hours-kg/L for the i.v. studies and 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

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19

20

21

22

23

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25

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31

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38

4.7 and 6.0 hours-kg/L for the oral dosing studies. These AUC values correspond 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 

possible that oral bioavailability is actually higher than 83−87%. Guo et al. (1985) reported data on 

blood tritium kinetics in mice that received i.v. (0.055 mg RDX or ~2.5 mg/kg body weight) or oral 

(50 mg/kg) doses of [3H]-RDX. Based on the reported blood tritium concentrations (% of dose/g) 

and terminal t1/2 values for concentrations of tritium in blood (1.1 days for i.v. and 2.2 days for 

oral), the corresponding AUCs of the blood concentration versus time curves were calculated 

(calculated for this review using the trapezoid rule extrapolated to infinite time) to be 30 and 

16 hours-% dose/g for i.v. and oral dosing, respectively. This corresponds to an oral bioavailability 

of RDX-derived tritium concentration of approximately 50% (i.e., 16/30). 

In Yucatan miniature swine administered a single dose of [14C]-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 

oral absorption of a single 100 mg/kg gavage dose of coarse granular [14C]-RDX as a slurry in 

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

in Sprague-Dawley rats. Plasma carbon-14 levels were measured for 24 hours after dosing. For 

both [14C]-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 the 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 powdered preparation than for the coarse 

granular preparation consistent with a greater surface area available for absorption with finely 

powdered RDX. However, blood levels were only measured 24 hours after dosing, and the lower 

24-hour carbon-14 plasma concentration for the coarse 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)

investigated the oral bioavailability of RDX in contaminated soils relative to pure RDX by 

comparison of the AUC for the RDX blood concentration versus 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 

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1 Plant (AAP) or Fort Meade. Blood concentrations for rats dosed with Louisiana AAP soil 

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(1.24 mg/kg) and neat RDX (1.24 mg/kg) peaked at approximately 6 hours. The AUC and 6-hour 

RDX blood concentration were both approximately 25% lower for Louisiana AAP soil than for neat 

RDX (p ≤ 0.003 for AUC), suggesting that oral bioavailability of RDX from Louisiana AAP soil was 

25% lower than neat RDX. For Fort Meade 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 Fort 

Meade soil was approximately 15% lower than for neat RDX, although the AUC for Fort Meade 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. 

Fellows et al. (2006) showed that plants (alfalfa shoots and corn leaves) incorporated 

[14C]-RDX grown on [14C]-RDX-amended soils. [14C]-RDX and plant metabolites of [14C]-RDX were 

absorbed by voles following oral administration (Fellows et al., 2006). In adult male prairie voles 

(Microtus 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 CO2) and 3−5% was retained in the carcass. 

Feces, urine, and CO2 contained 74−79, 13−14, and 8−12% of the total carbon-14 in excreta, 

respectively. Based on carbon-14 elimination in urine and CO2 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 is 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 concentrations were proportional to the administered dose (Bannon, 2006). However, the 

potential 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., 2009a; Crouse et al., 2008; 

Bannon, 2006; Guo et al., 1985; MacPhail et al., 1985; Schneider et al., 1977). Oral absorption of 

RDX was rapid in LACA mice following stomach perfusion with [3H]-RDX (50mg/kg in methyl 

cellulose) (Guo et al., 1985). The tritium radiolabel was detected in blood 15 minutes following 

dosing 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 mg/kg) of RDX 

in gelatin capsules, peak blood RDX concentrations were observed between 2.5 and 6 hours

(Bannon et al., 2009a; Krishnan et al., 2009; Crouse et al., 2008). Peak blood concentrations 

occurred at 24 hours after Sprague-Dawley rats were administered a single oral dose (100 mg/kg) 

of coarse granular RDX in saline (Schneider et al., 1977). Similarly, peak RDX blood concentrations 

in swine 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., 2009a), compared to 24 hours after a 

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1 single dose (100 mg/kg) of RDX administered as a finely powdered in saline (Bannon et al., 2009a; 

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Schneider et al., 1977). Peak plasma concentrations in Yucatan miniature swine administered a 

single dose of [14C]-RDX (45 mg/kg as a suspension in carboxymethylcellulose) were reached 

within 6−12 hours after dosing (Musick et al., 2010). Krishnan et al. (2009) and Sweeney et al. 

(2012a) estimated absorption rates in rats dosed with higher doses of coarse granular RDX to be 

approximately 100 times slower than absorption rates in rats dosed with lower doses of finely 

powdered neat RDX preparations or neat RDX dissolved in aqueous vehicles. For example,

Krishnan et al. (2009) estimated the absorption rate constant to be 0.75 hour−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 or 

1.24 mg/kg) (Crouse et al., 2008), compared to 0.0075 hour−1 for rats dosed with coarse granular 

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., 1996a; Testud et al., 1996b; Ketel and Hughes, 1972; Hollander and 

Colbach, 1969; Kaplan et al., 1965; Barsotti and Crotti, 1949). These reports suggest that RDX may 

be absorbed by the respiratory system. However, in several cases, the study authors were unable 

to clearly identify the primary route of exposure. In some 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 skin

(Reddy et al., 2008; Reifenrath et al., 2008). Reddy et al. (2008) reported that 5.7% of the applied 

RDX dose (in acetone) was absorbed into excised human skin in 24 hours. Dermal absorption of 

[14C]-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 

carbon content, and soil aging on the in vitro percutaneous penetration of [14C]-labeled RDX in 

excised pig skin. Mean 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-dinitrotoluene) 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).

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1 C.1.2. Distribution

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Information on the distribution of absorbed RDX in humans is limited to a few cases of 

accidental exposures to RDX that provide data on the kinetics of RDX in blood and cerebrospinal 

fluid (Küçükardali et al., 2003; Özhan et al., 2003; Woody et al., 1986). Concentrations of RDX in 

serum and cerebrospinal 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 

tissue 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). In 

these studies, RDX or radiolabeled RDX ([14C] or [3H) was administered by the oral, intraperitoneal 

(i.p.), i.v., or 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 of radiolabel will not necessarily reflect the distribution of RDX (Schneider et al., 1977). 

To compare tissue distributions in studies in which animals received different doses by different 

routes of administration, distribution data are expressed as ratios of tissue RDX or radiolabel to 

that of either whole blood or plasma, whichever was reported. RDX in blood distributes into red 

blood cells (RBCs) and plasma to achieve concentration ratios that are close to unity. The 

plasma:whole blood carbon-14 ratio in swine that received a single oral dose of [14C]-RDX 

(45 mg/kg) was approximately 1.3 (Musick et al., 2010), and whole rat blood incubated in vitro

with RDX had a plasma:RBC RDX ratio of approximately 1.0 (Krishnan et al., 2009). As a result of 

the similarity between plasma and whole blood concentrations, tissue distribution 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 distribution of RDX to be 

approximately 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. 

However, Bannon et al. (2006) observed tissue:blood concentrations for RDX of approximately 4 in 

brain hippocampus and 3.5 in brain cortex of swine that received a single oral dose of 10 mg/kg 

[14C]-RDX, although this is the only study that reported distribution for brain regions. Reported 

tissue:blood (or plasma) concentration ratios of RDX 24 hours following a single dose (oral or i.p.) 

were 1−9 for kidney, 1−7 for liver, and 1−3 for heart (Table C-1) (Bannon, 2006; Schneider et al., 

1977). With repeated oral dosing (e.g., 30−90 days), tissue:blood ratios of RDX for these tissues are 

consistently greater than unity (Schneider et al., 1978). There is no consistent evidence that RDX 

accumulates 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). 

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1 Table C-1. Distribution of RDX or radiolabel from administered RDXa

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Animal Route Dose (mg/kg) Time (hrs) Brain Heart Kidney Liver Fat Source

Swine Oral 45b 24 0.6c 0.7 2.4 7.3 0.4 Musick et al. (2010)

Swine Oral 10d 3 3.5−4.0d 2 ≤1 <1 NAg Bannon et al. (2006)

Swine Oral 100d 24 1.5c 1.1 1.2−1.9 0.9 1.8 Schneider et al. (1977)

Rat Oral 100d 24 3.4c 2.9 6.6 0.7 NA Schneider et al. (1977)

Rat i.p. 50d 2 3.4c 2.6 8.8 5.7 NA Schneider et al. (1977)

Rat i.p. 500d ≤6.5 2.5c 2.1 4.8 3.3 NA Schneider et al. (1977)

Mouse Oral 50e 24 1c 0.8 1 1.4 0.8 Guo et al. (1985)

Mouse i.v. 2.5e 24 0.6f 0.8 0.7 1.6 0.4 Guo et al. (1985)

a

Values are tissue:blood or tissue:plasma ratios following a single dose of either RDX, [14C]-RDX, or [3H]-RDX. b

Carbon-14. c

Tissue:plasma. d

RDX. e

Tritium. f

Tissue: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 

postnatal 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 

gestational day (GD) 6 to PND 10. RDX was also detected in maternal milk (concentrations ranged 

from 3 to 5.7 μg/mL on PND 1 and from 0.7 to 3.1 μg/mL on PND 10).

C.1.3. 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 CO2. Predominant metabolic 

pathways and major organs involved in RDX metabolism have not been identified, although results 

of in vitro studies suggest a role for CYP450.

RDX undergoes metabolism through processes that generate CO2. In Sprague-Dawley rats 

administered a single 50 mg/kg gavage dose of [14C]-RDX, 43% was recovered as exhaled [14CO2] 

after 4 days (Schneider et al., 1977). Similarly, approximately 30−50% of the radioactivity was 

recovered as exhaled [14CO2] in rats administered [14C]-RDX in saturated drinking water or daily 

gavage for up to 3 months (Schneider et al., 1978). Metabolism of RDX to CO2 was also observed in 

prairie voles following dietary exposure (average RDX dose per animal of 2.3 mg/kg-day) to 

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[ 1 14C]-RDX incorporated plant materials for 5−7 days, with approximately 9% of the administered 

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[14C]-RDX dose eliminated as exhaled [14CO2] (Fellows et al., 2006). 

Terminal metabolites of RDX have been identified in the urine of rats and swine, with very 

little urinary excretion of parent compound, indicating extensive metabolism of RDX. Following 

oral administration of a single 50 mg/kg gavage dose of [14C]-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 unmetabolized RDX contributed 3−5% of total 

urinary radioactivity in rats exposed to [14C]-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 [14C]-RDX, with approximately 1−3.5% of the urinary radioactivity as parent 

RDX (Major et al., 2007). Urinary metabolites were not characterized in these studies (Schneider et 

al., 1978, 1977). However, Schneider et al. (1978) cited unpublished findings in their laboratory 

that, 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 less well understood. N-Nitroso RDX metabolites have been identified as derived through 

anaerobic metabolism (ATSDR, 2012; Pan et al., 2007b). Based on characterization of RDX 

metabolites 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 

metabolites identified in miniature swine 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-2). Bhushan et al. (2003)

suggested 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, only trace amounts of the 

nitrosamine RDX metabolites, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and 1-nitro3,5-dinitroso-1,3,5-triazacyclohexane (DNX), were found in urine (Musick et al., 2010; Major et al., 

2007). 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 study 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 concentrations 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., 2007b). MNX and DNX were also measured in various organs of female B6C3F1 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. The authors concluded that RDX can be metabolized into its N-nitroso compounds 

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1 in mice, but did not identify a mechanism for the formation of the metabolites. Comparing RDX 

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with MNX and TNX, RDX was the most potent compound at causing overt signs of toxicity (seizures 

and mortality) as determined through identification of the median lethal dose using the EPA upand-down procedure in deer mice of varying ages (Smith et al., 2009; Rispin et al., 2002). 

Table C-2. 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-Nitro-2,4-diazabutanal

Urine peak 2 M2 4-Nitro-2,4-diaza-butanamide

Sources: Major et al. (2007); Musick et al. (2010). 

Although the metabolic pathways and major tissues involved in RDX metabolism have not 

been identified, there is some evidence for the involvement of the liver and CYP450 enzymes. 

Comparison of hepatic radioactivity to liver concentrations of RDX after a single gavage dose to rats 

suggested the presence of RDX metabolites and a possible role for hepatic metabolism of RDX

(Schneider et al., 1977). In vitro data indicated that CYP450 may be involved in the metabolism of 

RDX (Bhushan et al., 2003). Incubation of RDX with nicotinamide adenine dinucleotide phosphate

(NADPH) and rabbit liver CYP450 2B4 under anaerobic conditions produced nitrite, 4-nitro2,4-diazabutanal, formaldehyde, and ammonium ion (Bhushan et al., 2003). The reaction rate 

under aerobic conditions was approximately one-third of that observed under anaerobic 

conditions. Several CYP450 inhibitors (ellipticine, 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.1.4. Excretion

The primary routes of elimination of absorbed RDX are excretion of RDX and metabolites in 

urine, and exhalation of CO2 liberated from metabolism of RDX (Sweeney et al., 2012a; Musick et al., 

2010; Krishnan et al., 2009; Major et al., 2007; Schneider et al., 1977). Tritium derived from 

administered [3H]-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 

been 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 

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[ 1 14C]-RDX, with RDX accounting for <5% of the urinary carbon-14 (Musick et al., 2010; Schneider et 

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al., 1977). 

Data on kinetics of elimination of absorbed RDX from blood are available from reports of 

accidental exposures of humans to RDX (Table C-3). Woody et al. (1986) estimated the elimination 

t1/2 to be approximately 15 hours in a child who ingested approximately 85 mg of RDX per kg of 

body weight. The t1/2 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 (measurements made between 24 and 96 hours following 

exposure) (Özhan et al., 2003), a first-order elimination t1/2 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 

accidental, acute exposures and, in particular, the data for the child are based on a single set of 

measurements for one individual.

Table C-3. Elimination t1/2 values for RDX or radiolabeled RDX

Animal Route Dose (mg/kg) Timea t1/2 (hrs) Source

Human (child) Oral 85b 24−120 hrs 15.0c Woody et al. (1986)

Human (adult) Oral NA 24−96 hrs 21−29c,d Özhan et al. (2003)

Rat i.v. 5−6 0.5 min−6 hrs 10b Schneider et al. (1977)

Rat i.v. 0.8−1.0 30 min−10 hrs 4.6c,d Krishnan et al. (2009)

Rat Oral 1.53−2.07 1−10 hrs 6.9c,d Krishnan et al. (2009)

Mouse Oral 35, 60, 80 45 min−4 hrs 1.2d Sweeney et al. (2012b)

a

Observation period following exposure on which the t1/2 values were based. b

Reported estimate of dose based on blood kinetics. c

Value for blood RDX. d

Calculated for this review based on reported blood RDX concentrations. 

The kinetics of elimination of absorbed RDX from blood has been evaluated in rats and 

mice. In rats elimination kinetics were biphasic (Krishnan et al., 2009; Guo et al., 1985; Schneider et 

al., 1977). As shown in Table C-3, estimated t1/2 values for the terminal elimination phase in rats 

range from 5 to 10 hours (Krishnan et al., 2009; Schneider et al., 1977). Blood concentration time 

course measurements of RDX can be used to estimate an apparent metabolism and elimination of 

RDX from blood. The RDX blood concentrations reported in Sweeney et al. (2012b) after gavage 

dosing of 35, 60, and 80 mg/kg RDX found a consistent terminal elimination t1/2 of approximately 

1.2 hours. The elimination t1/2 estimated for rats (Krishnan et al., 2009; Schneider et al., 1977) is as 

much as an order of magnitude longer than mice (Sweeney et al., 2012b). 

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1 C.1.5. Physiologically Based Pharmacokinetic (PBPK) Models

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Overview 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., 

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

compartments: blood, brain, fat, liver, and lumped compartments for rapidly perfused tissues and 

slowly perfused tissues (Figure C-1). The model can simulate RDX exposures via the i.v. 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 GI 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, Aegis, Inc., Huntsville, Alabama) was obtained from the authors of Sweeney et 

al. (2012a). 

Exposure to RDX is by the i.v. or oral route, and clearance occurs by metabolism in the liver. See Table C-4 for 

definitions of parameter abbreviations. The GI tract is represented as one compartment in Krishnan et al. (2009)

(on the left) and two compartments in Sweeney et al. (2012a) (on the right).

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

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1 The parameter values used in the Sweeney et al. (2012a) rat model are listed in Table C-4. 

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The physiological model parameter values for cardiac output, tissue volumes, and blood perfusion 

of tissues were obtained from the literature (Timchalk et al., 2002; Brown et al., 1997). RDX 

tissue: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 tissues 

(PS), as well as the metabolic rate constant (KfC), were simultaneously optimized to fit rat blood 

RDX concentrations following i.v. doses of 0.77 or 1.04 mg/kg RDX (Krishnan et al., 2009) 

producing values of 5.57, 0.15, and 2.6 kg0.33/hour for PF, PS, and KfC, respectively. While the 

optimized value for PS is much smaller than that used by Krishnan et al. (2009) (1.0 kg0.33/hour), 

the optimized values for PF and KfC were fairly similar to those used by Krishnan et al. (2009)

(7.55, and 2.2 kg0.33/hour). The rat model with these parameter values also had good agreement 

with blood RDX concentrations after a 5−6 mg/kg i.v. exposure (Schneider et al., 1977). 

Table C-4. Parameter values used in the Sweeney et al. (2012a) and Sweeney 

et al. (2012b) PBPK models for RDX in rats, humans, and mice

Parameter (abbreviation; 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/hr/kg0.74) 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)

Blood (KVV) 0.06 0.079 0.06 Timchalk et al. (2002); Brown et al. (1997)

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)

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Parameter (abbreviation; units) Rat Human Mouse Source

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 (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

First-order metabolic rate constant 

(KfC; kg0.33/hr)

2.6 9.87 (child); 

11.2 (adult)

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

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a

Predicted from n-octanol:water partition coefficient. b

Optimized from rat i.v. data.

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Optimized from human data of Özhan et al. (2003) and Woody et al. (1986). d

Optimized from mouse oral data. 

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Optimized from rat oral data of Bannon et al. (2009a), Crouse et al. (2008), Krishnan et al. (2009), and Schneider 

et al. (1977). 

Note: Parameter values used in the Sweeney et al. (2012a) and Sweeney et al. (2012b) PBPK models for RDX in 

rats, humans, and mice.

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 

one-compartment GI tract. KAS was fit to the RDX blood concentrations in Krishnan et al. (2009),

and the 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 RDX 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 

other oral dosing studies, the two-compartment GI model did not improve the model fit to the data, 

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1 so KT was set equal to zero making the GI submodel equivalent to a one-compartment model. The 

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value of KAS was adjusted separately to fit the oral studies with RDX in capsules (Bannon et al., 

2009a; 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 

mostly 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 

constants were optimized to fit observed RDX blood concentrations from a case study of ingestion 

by a 3-year-old boy (Woody et al., 1986), and a study where five soldiers were intentionally or 

accidentally exposed to RDX powder via inhalation or dermal contact (Özhan et al., 2003). The 

amounts of RDX ingested in both studies were unknown, so Sweeney et al. (2012a) estimated the 

dose amount by optimizing this parameter to fit the data (Table C-4). Sweeney et al. (2012a)

initially simulated each individual soldier’s blood level data separately. The resulting parameter 

values were similar, so data from the five soldiers were combined and the rate constants were reestimated using the combined data. For comparison, the rat metabolic rate constant (KfC) was 

scaled 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 et al., 2008; Lipscomb and Poet, 2008). The scaling from rats yielded a human in 

vivo metabolic rate constant of 12.4 kg-BW0.33/hour, which is similar to the values that Sweeney et 

al. (2012a) derived by fitting the combined Özhan et al. (2003) adult data (11.2 kg-bw0.33/hour) and 

the Woody et al. (1986) child data (9.87 kg-bw0.33/hour).

Mouse RDX toxicokinetics were also modeled by Sweeney et al. (2012b) using the same 

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

and 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 algorithm 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 KfC and KAS were 

optimized to fit measured mouse RDX blood concentrations (Sweeney et al., 2012b). The KfC value 

estimated for the mouse (102 kg0.33/hour) is much higher than those estimated for rats and humans 

(2.6 and 11.2 kg0.33/hour, respectively); however, the KAS value (0.51/hour) fit to mouse data is 

similar to the value (0.83/hour) used in the RDX rat model for gavage in water. The Sweeney et al. 

(2012b) model predictions of blood RDX concentrations were in good agreement with the 

experimental mouse gavage data reported in the same study. 

The above PBPK model was evaluated and subsequently modified by EPA for use in doseresponse modeling in this assessment. This is detailed in the following section.

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

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al. (2012b) Models 

EPA evaluated and performed a quality control check of the PBPK models for RDX in rats, 

humans, and mice published by Sweeney and colleagues (Sweeney et al., 2012a; Sweeney et al., 

2012b). The conclusions from these analyses are summarized below and then discussed in more 

detail: 

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 two-compartment GI submodel and for other oral exposures (e.g., gelatin 

capsule), the model used a one-compartment GI submodel. The model was revised to have 

a one-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 

model calibration as well as for independent comparison against model predictions. 

4) In addition to the sensitivity analysis conducted by Sweeney et al. (2012b) on the mouse 

model, a sensitivity analysis in the rat and human models was performed.

5) The Sweeney et al. (2012b) mouse model used the same physiological parameters scaled to 

body 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 

rate constants (as discussed below) and the partition coefficients for the fat and slowly perfused 

tissues (PF and PS) as shown in Table C-5. 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:

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

The fits to RDX blood time course data after i.v. 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. 

Table C-5. Parameters values used in the EPA application of the rat, human, 

and mouse models 

Parameter (abbreviation; 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/hr/kg0.74) 15 14 15 Timchalk et al. (2002); Brown et al. (1997)

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Parameter (abbreviation; units) Rat Human Mouse Source

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. (1997)

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)

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 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; kg0.33/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-6 1.75 0.6 Fit to rat, human, and mouse oral data

Absorption from lung to blood 

(Klung; /hr)

0.75 Fit to human data

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Predicted from n-octanol:water partition coefficient. b

Optimized from rat i.v. data. c

Optimized from human data of Özhan et al. (2003) and Woody et al. (1986). d

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

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A) data from Krishnan et al. (2009) (0.4 kg rats) and B) data from Schneider et al. (1977)

(simulation of 0.25 kg rats and 5.5 mg/kg dose for 0.2−0.25 kg rats and 5−6 mg/kg dose).

Figure C-2. EPA rat PBPK model predictions fitted to observed RDX blood 

concentrations in male and female Sprague-Dawley rats following i.v. 

exposure. 

Absorption of RDX from the GI Tract

As discussed above in the oral absorption section under toxicokinetics (Section C.1.1), 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 doses of 100 mg/kg of course, granular RDX 

and 50 mg/kg finely powdered RDX, and observing that 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 one-compartment GI tract submodel 

for all simulations. To account for the differences in absorption due to the physical form of RDX, 

separate rate constants for RDX oral absorption were optimized to fit measured blood 

concentrations of RDX according to the type of dosing formulation; the model fits obtained with the 

EPA’s revised parameters for rats are shown in Figures C-3 to C-5. The oral dosing formulations 

were grouped into four categories: RDX dissolved in water, RDX in capsules, fine-grain RDX, 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 optimized to the data in the Crouse et al. (2008) and Bannon et al. (2009a) studies 

(Figure C-4). The absorption rate constant for fine-grain RDX was optimized to the data described 

below (Additional RDX Time-Course Data) in the MacPhail et al. (1985) and Schneider et al. (1977)

studies (Figure C-7). The Schneider et al. (1977) study was used to estimate the absorption rate 

constants for coarse-grain 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 timeThis document is a draft for review purposes only and does not constitute Agency policy.

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

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to RDX brain time course data after oral exposure to RDX in capsules (Figure C-6A) are similar to 

the fits of the Sweeney et al. (2012a) rat model. The absorption rate constants for each dosing 

formulation are listed in Table C-6. 

Male and female Sprague-Dawley rats (0.4 kg) were dosed by gavage (Krishnan et al., 2009). 

Figure C-3. EPA rat PBPK model predictions fitted to observed RDX blood 

concentrations following oral exposure to RDX dissolved in water. 

The ingested RDX doses were: A) 0.2 and 1.24 mg/kg RDX in male Sprague-Dawley rats (0.4 kg, 

data from Crouse et al. (2008)) and B) 3 and 18 mg/kg RDX in male and female Sprague-Dawley 

rats (0.35 kg, data from Bannon et al. (2009a)) for KAS = 0.35/hour.

Figure C-4. EPA rat model predictions fitted to observed RDX blood 

concentrations following oral exposure to RDX in dry capsules. 

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Symbols denote observed RDX blood concentrations measured in male Sprague-Dawley rats 

(0.225 kg) resulting from oral doses of 100 mg/kg RDX (Schneider et al., 1977). The KAS fit to 

these data assuming 100% bioavailability resulted in the same estimate (KAS = 0.00497/hour) 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/hour fit to data from Schneider et al. (1977) and MacPhail et al. 

(1985); Figure C-7), the estimate of oral bioavailability fit to the RDX blood concentrations was 

35%. A bioavailability of 40% and KAS = 0.019/hour is also shown for comparison.

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. 

A) 3 and 18 mg/kg RDX in dry capsules (0.35 kg male and female rat data from Bannon et al. 

(2009a); best fit KAS = 0.35/hour. 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/hour.

Figure C-6. EPA rat model predictions fitted to observed RDX brain tissue 

concentrations following oral exposure to RDX. 

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1 Table C-6. Doses, dosing formulations, and absorption rate constants in 

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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 capsulesa Crouse et al. (2008) 0.2, 1.24 mg/kg, single dose 0.35

Bannon et al. (2009a) 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

a

Capsules were filled with dry RDX from stock solution of acetone, and acetone was evaporated off. b

RDX particle size was ≤66 μm in diameter suspended in a 2% solution of carboxymethylcellulose.

An alternative to varying the 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 an 

analysis of model sensitivity to oral bioavailability was conducted as discussed further in the 

section, Sensitivity Analysis of the Rat PBPK Model. 

Additional RDX Time-Course Data

The EPA revised models were simultaneously fitted against additional RDX time-course 

data (not used in the original Sweeney et al. (2012a) model calibration). These data came from

(1) two studies in which animals received oral doses of fine-grain RDX (MacPhail et al., 1985; 

Schneider et al., 1977) (Figure C-7) and (2) RDX brain time-course data from a study in which 

animals received oral doses of fine-grain RDX (MacPhail et al., 1985) (Figure C-6B). Overall, the 

calibrated EPA rat model predictions are within a factor of 1.5 of the measured values from 

different data sets, and are therefore likely to provide a more robust estimated parameter.

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Oral doses of 50 mg/kg RDX were administered to: A) male Sprague-Dawley rats (0.225 kg) 

(Schneider et al., 1977) and B) male and female Sprague-Dawley rats (0.25 kg) data (MacPhail et 

al., 1985). Best fit KAS = 0.019/hour.

Figure C-7. EPA rat model predictions fitted to observed RDX blood 

concentrations following oral exposure to fine-grain RDX in a saline slurry. 

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 

gavage 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 μg/mL) ad libitum for which 

the study authors estimated a daily dose between 5 and 8 mg RDX/kg body weight. It is striking 

that the observed RDX blood concentrations in the gavage study (Figure C-8, symbols) were 

virtually the same, or only slightly elevated, as compared to the blood concentrations reported in 

the drinking water exposures, with an approximately threefold lower daily administered 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. 

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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 Sprague-Dawley rats (0.225 kg). The RDX in saline 

slurry was assumed to be coarse-grained with an oral absorption rate constant 

KAS = 0.00497/hour.

Figure C-8. Comparison of EPA rat model predictions with data from 

Schneider et al. (1978) for the subchronic gavage study. 

Model fits and mean observed RDX blood concentrations resulting from a daily estimated dose of 

6.5 mg RDX/kg-day for 90 days to male and female Sprague-Dawley 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 waking state. The oral absorption rate constant 

for RDX dissolved in water was used (KAS = 1.75/hour).

Figure C-9. Comparison of EPA rat model predictions with data from 

Schneider et al. (1978) for the subchronic drinking water study. 

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1 EPA’s modified PBPK model was set up to simulate drinking water exposures with a 

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noncontinuous sipping pattern based on Spiteri (1982), which assumed 80% of the consumption to 

occur episodically at night when the rats were awake3. The model predicts blood concentrations to 

increase in proportion to the total dose; for the gavage study, the model predictions yielded an RDX 

blood concentration approximately threefold higher than the reported mean blood concentrations 

(Figure 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 study. 

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, higher doses 

this elimination route could be significant. Schneider et al. (1978) found similar RDX 

concentrations in the feces of rats in the gavage and drinking water studies (3.1 ± 2.0 and 

2.7 ± 1.3 μg RDX per g 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 two 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 observed RDX blood concentrations. The reasonably good fits 

of the model to the drinking water data set demonstrated achievement of regular periodic levels, 

and 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 versus 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 the use of the model in estimating and extrapolating blood levels following 

chronic exposure at or below this exposure range (5−8 mg/kg-day), particularly in drinking water. 

Simulating Exposures in Humans

The Sweeney et al. (2012a) model for humans was modified in the same ways as the rats, by 

changing the partition coefficients for the fat and slowly perfused tissues (PF and PS) as shown in 

Table C-5 and fitting the rate constants for oral absorption and metabolism to RDX blood 

concentration data. In the studies of humans with measured RDX blood concentrations by Woody 

et al. (1986) and Özhan et al. (2003), the RDX doses were unknown and the doses were therefore 

also optimized to fit the data. The model predictions for the Woody et al. (1986) data using the best 

fit values of dose = 58.9 mg/kg, KAS = 1.75/hour, and KfC = 9.87 kg0.33/hour are shown in 

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

12-hour awake period consisted of eight 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 

four cycles with regular sipping periods of 30 minutes followed by 2.5 hours of no ingestion.

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1 Figure C-10. The model predictions for the Özhan et al. (2003) data using the best fit values of an 

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oral dose = 3.5 mg/kg, KAS = 1.75/hour, and KfC = 9.87 kg0.33/hour are shown in Figure C-11.

The best fit values were KAS = 1.75/hour, dose = 58.9 mg/kg, and KfC = 9.87 kg0.33/hour.

Figure 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). 

For an assumed oral exposure, the best fit values were KAS = 1.75/hour, dose = 3.5 mg/kg, and 

KfC = 9.87 kg0.33/hour. 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/hour.

Figure C-11. EPA human model predictions fitted to observed RDX blood 

concentrations resulting from accidental exposure to adults assumed to be 

70 kg (Özhan et al., 2003). 

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1 EPA’s calibration of the model differed in another important respect from that carried out 

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by Sweeney et al. (2012a). As previously mentioned, Sweeney et al. (2012a) simulated the soldiers’ 

exposure from the Özhan et al. (2003) study as an oral exposure, although the study report states 

that the exposure was via inhalation and dermal routes. An inhalation 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 was therefore not modeled. This decision is supported by a 

study that used excised human skin and reported that 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 

inhalation exposure and compared with the data from Özhan et al. (2003). There are insufficient 

data on blood:air partitioning to modify the Sweeney et al. (2012a) model with a lung 

compartment; 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 exposed 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 

assumed that 100% of the inhaled dose was absorbed and that 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 Özhan et al. (2003) as shown in 

Figure C-11.

Sensitivity Analysis of the Rat and Human PBPK Models 

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-term (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 AUC. Parameters with sensitivity coefficients >0.1 in absolute value 

(i.e., considered sensitive) are presented in Table C-7. For the blood AUC dose-metric, the only 

sensitive RDX-specific parameter is the 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 absorbed 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 transport 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 body weight and by liver volume, the 

blood AUC is also sensitive to these parameters. The sensitivity analysis by Sweeney et al. (2012b)

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1 for the AUC of RDX in the liver found the model was sensitive to the liver:blood partition coefficient 

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(PL) in addition to the same parameters (KfC, KVL, and BW) found for the blood AUC.

Table C-7. Sensitivity coefficients for rat and human RDX PBPK models

Parameter Rat sensitivity coefficient Human sensitivity coefficient

Fractional liver volume (KVL) −1 −1

Body weight (BW) 0.3 0.3

Metabolic 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, KQL; fat, KQF; slowly perfused tissues, KQS; brain, KQB)

• fractional tissue volume of fat (KVF), brain (KVB), and blood volume (KVV)

• blood partition coefficients to all tissues (liver, PL; fat, PF; rapidly perfused, PR; slowly perfused, PS; brain, PB)

• absorption rates from GI (KAS, KT, KAD)

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 

(Section C.1.1), 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 Figure 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 was acknowledged as an uncertainty. 

Simulating Exposures in Mice

Physiological parameters specific to mice were obtained from the literature (Brown et al., 

1997) and are shown in Table C-5. The partition coefficients calculated for mice by Sweeney et al. 

(2012b) were used, and include the liver, brain, and richly perfused tissues. The partition 

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

were not used because they were estimated via optimization of fits to rat i.v. 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 perfused tissues (0.9) was calculated for 

mouse tissues using the same methodology as Krishnan et al. (2009). The rate constants for oral 

absorption 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. 

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Model fits and mean and standard deviation of observed RDX blood concentrations in female 

B6C3F1 mice (0.0205 kg) for doses of 35, 60, and 80 mg/kg with KAS = 0.6/hour and 

KfC = 77 kg0.33/hour. Experimental data from Sweeney et al. (2012b). 

Figure C-12. Comparison of EPA mouse PBPK model predictions with data 

from oral exposure to RDX dissolved in water. 

The only information on RDX metabolism in the mouse comes from a study by Pan et al. 

(2013). Pan et al. (2013) measured nitrosamine RDX metabolites of RDX (MNX, DNX, and TNX, the 

latter representing a minor metabolic pathway) in mice at the end of a 28-day exposure to RDX in 

feed (ad libitum). These measurements were a single time point without controlling the time 

between the last RDX ingestion and measurement, and were therefore judged not to be sufficient 

for use in parameterizing a PBPK model of the nitrosamine metabolites. 

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1 Rat to Human Extrapolations

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The rat and human PBPK models as described above were applied to derive human 

equivalent doses (HEDs) for candidate points of departure (PODs) for endpoints selected from rat 

bioassays. The rat and human PBPK models were used to estimate two dose metrics—the AUC and 

the peak concentration (Cmax) for RDX concentration in arterial blood. The relationships between 

administered dose and both internal metrics (AUC and Cmax) were evaluated with the rat PBPK 

model over the range of 1 μg/kg-day to 100 mg/kg-day and with the human PBPK model over the 

range of 0.05 μg/kg-day to 200 mg/kg-day, ranges that encompass the PODs. The times to reach 

steady state for the dose metrics were shorter than the duration of the toxicity studies, so the 

steady state values were considered representative of the study and were used. To calculate 

steady-state values for daily exposure, the simulations were run until the daily average had a <1% 

change between consecutive days. For both the rat and human PBPK models, both dose metrics 

correlated linearly with the administered dose. For rats dosed via gavage, the slope of 

administered dose versus AUC was 6.800 mg/L-day / mg/kg-day and that for Cmax was 

0.4718 mg/L / mg/kg-day. For a continuous dose, the slope of dose versus AUC was the same 

(6.800 mg/L-day / mg/kg-day) and for Cmax was 0.3951 mg/L / mg/kg-day. For humans, assuming 

a drinking water dose sipping pattern, the slope of administered dose versus AUC was 

13.95 mg/L-day / mg/kg-day and that for Cmax was 0.7316 mg/L / mg/kg-day. Given this linearity 

in internal metrics and assuming that equal internal metrics in rats and humans are associated with 

the same degree of response, the HEDs could then be directly determined by multiplying the lower 

bound on the benchmark dose (BMDL) in rats by the ratio of these slopes. For a gavage dose in rats 

converted to a human drinking water dose, the ratio for AUC was 6.800 / 13.95 = 0.487 and Cmax

was 0.4718 / 0.7316 = 0.645. For a continuous dose in rats converted to a human drinking water 

dose, the ratio for AUC was 6.800 / 13.95 = 0.487 and for Cmax was 0.3951 / 0.7316 = 0.540. These 

ratios were applied in Table 2-2 to calculate the PODHED from the rat benchmark dose lower 

confidence limits (BMDLs) and no-observed-adverse-effect levels (NOAELs) for each endpoint. 

Mouse to Human Extrapolations

The mouse and human PBPK models as described above were applied to derive HEDs for 

candidate PODs for endpoints selected from mouse bioassays. The mouse and human PBPK models 

were used to estimate two dose metrics—the area under the curve (AUC) and peak concentration 

(Cmax) for RDX concentration in arterial blood. The relationships between administered dose and 

both internal metrics (AUC and Cmax) were evaluated with the mouse PBPK model over the range 

10 μg/kg-day to 100 mg/kg-day and with the human PBPK model over the range 0.05 μg/kg-day to 

200 mg/kg-day, ranges that encompass the PODs. The times to reach steady state for the dose 

metrics were shorter than the duration of the toxicity studies, so the steady state values were 

considered representative of the study and were used. To calculate steady state values for daily 

exposure, the simulations were run until the daily average had a <1% change between consecutive 

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1 days. For both the mouse and human PBPK models, both dose metrics correlated linearly with the 

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administered dose. For mouse dosed via gavage, the slope of administered dose versus AUC was 

0.0656 mg/L-day / mg/kg-day and that for Cmax was 0.0273 mg/L / mg/kg-day. For a continuous 

dose, the slope of dose versus AUC was the same 0.0656 mg/L-day / mg/kg-day and for Cmax was 

0.0081 mg/L / mg/kg-day. For humans, assuming a drinking water dose sipping pattern, the slope 

of administered dose versus AUC was 13.95 mg/L-day / mg/kg-day and that for Cmax was 

0.7316 mg/L / mg/kg-day. Given this linearity in internal metrics and assuming that equal internal 

metrics in mice and humans are associated with the same degree of response, the HEDs could then 

be directly determined by multiplying the BMDL in mice by the ratio of these slopes. For a gavage 

dose in mice converted to a human drinking water dose, the ratio for AUC was 0.0656 / 13.95 = 

0.0047 and Cmax was 0.0273 / 0.7316 = 0.373, respectively. For a continuous dose in mice 

converted to a human drinking water dose, the ratio for AUC was 0.0656 / 13.95 = 0.0047 and for 

Cmax was 0.0081 / 0.7316 = 0.011. These ratios were applied in Table 2-2 to calculate the PODHED

from the mouse BMDLs and NOAELs for each endpoint. 

Summary of Confidence in PBPK Models for RDX

Overall, good fits to the rat, mouse, and human time-course data for RDX internal 

concentrations 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. 

The metabolic rate constant used in the human model was fit to limited data from 

accidentally exposed humans; however, the value of the metabolic rate constant has additional 

support from in vitro experimental data. The rat metabolic rate constant, fit to multiple 

experimental data sets, was scaled to humans using the ratio of human to rat rate constants 

measured with in vitro 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 predicting 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 types 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 in vitro measurements of RDX metabolism by mouse cells and 

quantification of potential routes of RDX elimination in mice. Overall, these uncertainties result in 

lower confidence in the mouse model than in the rat and human models. 

1) RDX 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 

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1 metabolites (a minor metabolic pathway) in mice, but the data are too sparse to help better 

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parameterize a PBPK model. Consequently, the PBPK models used in this assessment do 

not incorporate the kinetics of RDX metabolites. 

2) The 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 that a lower bioavailability is likely.

3) The human model is based on single accidental exposures, and the exposure concentrations 

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 to indicate that the role 

of metabolism in RDX toxicity may be different across species. This uncertainty decreases 

the confidence in the mouse PBPK model. 

5) The 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) The analytical detection limit in the mouse pharmacokinetic study is too high to enable 

detection at the lower doses. The lowest dose at which RDX was detected was 35 mg/kg; 

this concentration was 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 animal (others were non-detects or excluded as outliers). This 

decreases the confidence in the calibration of the mouse PBPK model.

7) The 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 that 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 CYP450 enzymes. Alternatively, mice may 

have other unknown metabolic pathways responsible for metabolizing RDX. Identifying the 

specific CYP450 enzymes and 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 the confidence in using the mouse PBPK model for predicting mouse 

blood RDX concentrations. 

Model Code for RDX PBPK Model Used in the Assessment

The PBPK acslX model code is made available electronically through the Health and 

Environmental Research Online (HERO) database. All model files may be downloaded in a zipped 

workspace from HERO (U.S. EPA, 2014). 

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1 C.2. HUMAN STUDIES

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Table C-8 presents a summary of case reports of humans acutely exposed to RDX. Table C-9 

provides a chronological summary of the methodologic features of the available epidemiology 

studies of RDX.

Table C-8. Summary of case reports of exposure to RDX

Reference, 

number of cases, 

exposure 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 (epileptic) 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 convulsions 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 

intoxication may have 

been masked by viral 

illness with nonspecific 

symptoms (e.g., 

headache, weakness, 

upset 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 

moderate amounts of alcohol

Coma, vomiting, hyperirritability, 

muscle twitching, convulsions, 

mental confusion, and amnesia; 

kidney damage (oliguria, gross 

hematuria, proteinuria, elevated 

BUN); liver or muscle damage 

(high 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)

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Reference, 

number of cases, 

exposure setting Exposure Effects observed Comments

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 laboratory 

findings, muscle injury, elevated 

AST; no kidney damage

For the patient who ingested the 

highest dose, anemia and loss of 

recent memory present after 30 d

Troops ingested small 

quantities of RDX to get 

a feeling of inebriation 

similar to that induced 

by ethanol

Hollander and 

Colbach (1969)

5 males (June 

1968−January 1969)

Wartime, Vietnam

Inhalation (all five cases) and 

ingestion of unknown 

quantity of C-4 (two 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; 

symptoms cleared by the next day 

except for amnesia (in case 2), 

oliguria (lasted for 4 d), and gross 

hematuria (decreased by 

9th hospital day)

Knepshield and 

Stone (1972)

6 males

Wartime, Vietnam

Ingestion of C-4, range 

25−180 g, average 77 g

Generalized seizures, coma, 

lethargy, severe neuromuscular 

irritability with twitching and 

hyperactive reflexes, myalgia, 

headache, nausea, vomiting, 

oliguria, gross hematuria, lowgrade fever; abnormal laboratory 

findings (neutrophilic 

leukocytosis, azotemia, elevated 

AST)

Includes data on 

two 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 

CNS signs (confusion, marked 

hyperirritability, involuntary 

twitching of the extremities, 

severe prolonged generalized 

seizures, prolonged postictal 

mental confusion, amnesia); renal 

effects (oliguria and proteinuria,

one case of acute renal failure 

requiring hemodialysis); GI 

toxicity (nausea, vomiting)

C-4 was cut with the 

same knife used to 

stir/prepare food

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Reference, 

number of cases, 

exposure setting Exposure Effects observed Comments

Woody et al. (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; 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 hrs; within 24 hrs, 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 form

Goldberg et al. 

(1992)

1 male

Nonwartime

Ingestion after chewing a 

piece (unknown size) of 

“Semtex” plastic explosive 

4 hrs before first seizure

Frontal headache and two tonicclonic 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 × 109

/dL (87% 

neutrophils); hemoglobin, platelet 

count, coagulation screen, serum 

and CSF biochemistry all within 

normal limits; CSF and blood 

bacteriologically unremarkable

Shortly following admission, 

headache and rash disappeared; 

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. 

(1996a)

1 male

Manufacturing

Inhalation and possible 

dermal exposure during the 

RDX manufacturing process

Malaise with dizziness, headache, 

and nausea progressing to 

unconsciousness and generalized 

seizures without involuntary 

urination or biting of the tongue; 

blood chemistries were in the 

normal range and blood alcohol 

content was null 

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Reference, 

number of cases, 

exposure setting Exposure Effects observed Comments

Testud et al. 

(1996b)

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 seizures

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 seizures, 

hyperactive deep tendon reflexes, 

sinusal tachycardia; elevated 

serum levels of AST and ALT; 

kidney damage; plasma RDX 

levels 3 hrs 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 abuse 

Seizures, headache, nausea, and 

vomiting; hypokalemia and 

elevated creatine kinase, lactate 

dehydrogenase, and phosphate 

noted in all but two patients; 

metabolic acidosis only occurred 

in two patients directly following 

seizures

Patient histories may 

have been affected 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 WBC count and 

elevated creatine phosphokinase; 

proteinuria and gross hematuria 

observed

1 

2 

3 

ALT = alanine aminotransferase; AST = aspartate transaminase; BUN = blood urea nitrogen; CNS = central nervous 

system; CSF = cerebrospinal fluid; EEG = electroencephalogram; WBC = white blood cell

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1 Table C-9. Occupational epidemiologic studies of RDX: summary of methodologic features

Reference, 

setting and 

design

Participants, 

selection, 

follow-up

Consideration 

of likely 

selection bias

Exposure measure and 

range

Outcome 

measure

Consideration 

of likely 

confounding

Analysis and 

results details Sample size 

Ma and Li 

(1993)

(China)a

Industrial 

workers

(translated 

article) 

Details of industrial 

process and subject 

selection framework 

not reported; 

referents chosen 

from same plant; 

age, employment 

duration, and 

education similar 

across groups; 

participation rate 

not reported

Sparse reporting 

of details on 

subject 

recruitment and 

participation 

Details of exposure 

monitoring not reported. 

Two groups of exposed 

subjects: 

Group A, intensity, 

0.407 (0.332) mg/m3

[mean (standard 

deviation)], daily 

cumulative, 

2.66 (1.89) mg/m3

. 

Group B, intensity, 

0.672 (0.556) mg/m3

; daily 

cumulative, 

2.53 (8.40) mg/m3

. 

Neurobehavioral 

battery 

administered by 

trained personnel:

memory 

retention, simple 

reaction time, 

choice reaction 

time, letter 

cancellation, and 

block design 

No adjustment 

for other risk 

factors (e.g., 

alcohol 

consumption); 

no consideration 

or attempt to 

distinguish TNT

Comparisons of 

mean scaled score 

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

2,022 workers 

(1,017 exposed to 

open explosives 

(TNT, RDX, HMX), 

1,005 referents) at 

five U.S. Army 

ammunition plants 

(Iowa, Illinois, 

Tennessee); 

participation rate: 

76% exposed, 71% 

referents 

Potential healthy 

worker effect 

Atmospheric samples of all 

operations with potential 

exposure to open 

explosives taken in 1975. 

Range: not detected to 

1.57 mg/m3

. Seventy 

exposed workers with RDX 

at >0.01 mg/m3 [the LOD]; 

mean: 0.28 mg/m3

[standard deviation not 

presented]. Job title used 

to initially identify exposed 

or unexposed status and 

reassigned to one of two 

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 males, 

26 females), 

24 RDX/HMX 

exposed (all 

males), 

338 referents 

(237 males, 

101 females)

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Reference, 

setting and 

design

Participants, 

selection, 

follow-up

Consideration 

of likely 

selection bias

Exposure measure and 

range

Outcome 

measure

Consideration 

of likely 

confounding

Analysis and 

results details Sample size 

exposed groups 

(nondetected, 

>0.01 mg/m3

) based on 

subject’s industrial hygiene 

monitoring results.

West and 

Stafford 

(1997)

(United 

Kingdom)

Ammunition 

workers

(casecontrol 

study)

378 of 404 subjects 

(excluded 3 deaths 

and 23 subjects with 

unknown addresses) 

previously studied in 

1991, 32 cases with 

abnormal 

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 

adverse health 

outcome were 

not included in 

the 1991

prevalence study

Semiquantitative 

assessment; source of 

industrial hygiene data not 

reported. Interviews with 

current and past 

employees and job title

analysis were conducted to 

identify potential exposure 

to 100 agents, including 

RDX. Exposure surrogate 

was >50 hrs in duration 

and intensity was low 

(1−10 ppm), moderate 

(10−100 ppm), or high 

(100−1,000 ppm). RDX 

exposure prevalence 

(males) was 83%.

Abnormal 

hematology value 

in 1991 survey 

indicating possible 

myelodysplasia:

neutropenia 

(2.0 x 109/L), low 

platelet count 

(<150 x 109/L), or 

macrocytosis 

(mean 

corpuscular 

volume = 99 fL or 

>6% macrocytes) 

Cases and 

controls were 

not matched and 

statistical 

analyses were 

not adjusted for 

other risk factor 

or occupational 

exposures; no 

consideration 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)

1 

2 

3 

4 

5 

a

Ma and Li (1993) describe symptoms reported by subjects during a physical examination, but these are not included in the evidence table because responses 

for individual symptoms were not identified. 

CI = confidence interval; HMX = octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; LOD = limit of detection; TNT = trinitrotoluene

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1 C.3. OTHER PERTINENT TOXICITY INFORMATION

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

C.3.1. Mortality in Animals

Evaluations of the evidence for specific health effects associated with RDX exposure are 

provided in Sections 1.2.1 to 1.2.6. In addition to these specific organ/system health effects, 

reduced survival associated with RDX exposure has been observed in experimental animals across 

multiple studies of varying exposure duration and study design (Table C-10). Evidence pertaining 

to mortality in experimental animals exposed to RDX is summarized in Table C-10; studies are 

ordered in the evidence table by duration of exposure and then species.

Following chronic dietary exposure, an increased rate of mortality in F344 rats, and in 

particular male rats, at 40 mg/kg-day was largely attributed to RDX-related effects on the kidneys 

(Levine et al., 1983b)4; see further discussion in Section 1.2.2. In a comparable chronic study, mice 

were less sensitive than rats with respect to mortality following RDX exposure. After the high dose 

was reduced from 175 to 100 mg/kg-day at week 11 in a 2-year dietary study in B6C3F1 mice 

because of high mortality, the mortality curve at 100 mg/kg-day in surviving mice was not 

significantly different from the control group for the duration of the 2-year study (Lish et al., 1984). 

The investigators did not identify the probable cause of death at 175 mg/kg-day.

Increased rates of mortality were also observed in experimental animals that ingested RDX 

for durations up to 6 months (Lish et al., 1984; Levine et al., 1983b; Levine et al., 1981a; Cholakis et 

al., 1980; von Oettingen et al., 1949). The most detailed data on RDX-related mortality come from a 

90-day gavage study in F344 rats by Crouse et al. (2006). Across groups of rats exposed to 

8−15 mg/kg-day RDX, pre-term deaths occurred in male rats as early as day 26−78 and in female 

rats as early as day 8−16 (Johnson, 2015; Crouse et al., 2006). Treatment-related mortality was 

also observed in the dams of rats exposed gestationally by gavage at doses ranging from 20 to 

120 mg/kg-day (Angerhofer et al., 1986; Cholakis et al., 1980). Deaths were additionally reported 

in one of 40 pregnant dams in both 2 and 6 mg/kg-day groups in the rat developmental toxicity 

study by Angerhofer et al. (1986)

In general, the evidence suggests that mortality occurs at lower doses in rats than in mice

(e.g., comparison of rates from the 2-year dietary studies in mice by Lish et al. (1984) and in rats by 

Levine et al. (1983b)), and at lower doses following gavage administration than dietary 

administration (e.g., comparison of rates from the 13-week rat studies using gavage (Crouse et al., 

2006) and dietary (Levine et al., 1981a) administration). An RDX formulation with a larger particle 

size (e.g., ~200 μm) (Cholakis et al., 1980), which could potentially reduce the ability of RDX to 

4 Deaths in high-dose (40 mg/kg-day) male rats were reported as early as week 4 (estimated from Figure 4 in 

Levine et al. (1983a)); the cause of death in rats that died prior to 6 months on study was generally not 

determined (Levine et al., 1983c). Survival rates in both male and female rats at doses ≤8 mg/kg-day RDX 

were similar to the control. 

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1 enter the bloodstream, appears to produce less mortality than formulations with finer particle sizes

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

(e.g., median particle diameter of 20 μm) (Levine et al., 1981a). There is evidence that mortality 

may be associated with nervous system effects; several investigators reported that unscheduled 

deaths were frequently preceded by convulsions or seizures (Crouse et al., 2006; Levine et al., 

1983b; Cholakis et al., 1980). In a number of studies, treatment-related mortality was observed at 

doses as low as doses associated with nervous system effects (Crouse et al., 2006; Angerhofer et al., 

1986; Levine et al., 1983b; Levine et al., 1981a; Cholakis et al., 1980; von Oettingen et al., 1949). 

The evidence for an association between nervous system effects and mortality is discussed in more 

detail in Section 1.2.1, Nervous System Effects. 

In humans, there were no reports of mortality in case reports involving workers exposed to 

RDX during manufacture or in individuals exposed acutely as a result of accidental or intentional 

ingestion (see Appendix C, Section C.2). 

Table C-10. Evidence pertaining to mortality in animalsa

Reference and study design Results

Lish et al. (1984)

Mice, B6C3F1, 85/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles <66 μm

0, 1.5, 7.0, 35, or 175/100 mg/kg-d (high 

dose reduced to 100 mg/kg-d in wk 11 due 

to excessive mortality)

Diet

2 yrs (mortality incidence also provided for 

mice through week 11 when the high dose 

was dropped because of high mortality at 

that dose, and from the report of the 6-

month interim sacrifice)

Doses 0 1.5 7.0 35 175/100

Mortality at 11 wks (incidence)

M 1/85 0/85 0/85 0/85 30/85

F 0/85 0/85 0/85 0/85 36/85

Mortality at 6 mo (incidence)

M 1/85 2/85 3/85 3/85 34/85

F 0/85 1/85 0/85 0/85 36/85

Mortality at 2 yrs (incidence)

M 20/65 23/65 25/65 29/65 41/65

F 16/65 21/65 14/65 21/65 42/65

After the high dose was reduced to 100 mg/kg-d, survival was 

similar to controls.

Levine et al. (1983b)

Rats, F344, 75/sex/group; interim sacrifices 

(10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant: 83−89% of particles <66 μm

0, 0.3, 1.5, 8.0, or 40 mg/kg-d 

Diet

2 yrs (mortality incidence also provided for 

mice through week 13, and from the report 

of the 6-month scheduled sacrifice)

Doses 0 0.3 1.5 8.0 40

Mortality at 13 wks (incidence)

M 0/75 0/75 0/75 0/75 0/75

F 0/75 0/75 0/75 0/75 0/75

Mortality in 6-mo interim sacrifice animals (incidence)#

 #

includes spontaneous death and moribund sacrifice

M 0/75 0/75 0/75 0/75 5/75

F 0/75 0/75 0/75 0/75 0/75

Mortality at 2 yrs (incidence)

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Reference and study design Results

M 17/55 19/55 30/55* 26/55 51/55*

F 12/55 10/55 13/55 14/55 27/55*

Cholakis et al. (1980)

Mice, B6C3F1, 10−12/sex/group

88.6% pure, with 9% HMX and 2.2% water 

as contaminants; ~200 μm particle size

0, 40, 60, or 80 mg/kg-d for 2 wks followed 

by 0, 320, 160, or 80 mg/kg-d (TWA doses 

of 0, 79.6, 147.8, or 256.7 mg/kg-d for 

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

for females)b

Diet

13 wks

Doses 0 79.6 147.8 256.7

Mortality (incidence)

M 0/10 0/10 0/10 4/10*

F 0/11 0/12 0/10 2/12

Cholakis et al. (1980)

Rats, F344, 10/sex/group

88.6% pure, with 9% HMX and 2.2% water 

as contaminants; ~200 μm particle size

0, 10, 14, 20, 28, or 40 mg/kg-d

Diet

13 wks

Doses 0 10 14 20 28 40

Mortality (incidence)

M 0/10 0/10 0/10 0/10 0/10 0/10

F 1/10

(accidental 

death)

0/10 0/10 0/10 0/10 0/10

Cholakis et al. (1980)

Rats, CD, two-generation study; 

F0: 22/sex/group; F1: 26/sex/group; 

F2: 10/sex/group 

88.6% pure, with 9% HMX and 2.2% water 

as contaminants; ~200 μm particle size

F0 and F1 parental animals: 0, 5, 16, or 

50 mg/kg-d

Diet

F0 exposure: 13 wks pre-mating, and during 

mating, gestation, and lactation of F1; 

F1 exposure: 13 wks after weaning, and 

during mating, gestation, and lactation of 

F2; F2 exposure: until weaning

Doses 0 5 16 50

Mortality in F0 adults (incidence)c

M (F0) 0/22 0/22 0/22 2/22

F (F0) 0/22 0/22 0/22 6/22

M + F 

(F0)

0/44 0/44 0/44 8/44*

Crouse et al. (2006)

Rats, F344, 10/sex/group

99.99% pure

0, 4, 8, 10, 12, or 15 mg/kg-d

Gavage

13 wks

Doses 0 4 8 10 12 15

Mortality (incidence)

M 0/10 0/10 1/10 3/10 2/10 3/10

F 0/10 0/10 1/10 2/10 5/10 4/10

(Levine et al. (1990); Levine et al. (1981a), 

1981b))

d

Rats, F344, 10/sex/group; 30/sex for 

control

Doses 0 10 30 100 300 600

Mortality (incidence)e

M 0/30 0/10 0/10 8/10 10/10 10/10

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Reference and study design Results

84.7 ± 4.7% purity, ~10% HMX, median 

particle diameter 20 μm, ~90% of particles 

≤66 μm

0, 10, 30, 100, 300, or 600 mg/kg-d

Diet

13 wks

F 0/30 1/10 0/10 5/10 10/10 10/10

von Oettingen et al. (1949)

Rats, sex/strain not specified, 20/group

90−97% pure, with 3−10% HMX; particle 

size not specified

0, 15, 25, or 50 mg/kg-d

Diet

13 wks

Doses 0 15 25 50

Mortality (incidence)

0/20 1/20

(probably 

not related 

to RDX)

8/20 8/20

Hart (1974)

Dogs, Beagle, 3/sex/group

Pre-mix with ground dog chow containing 

20 mg RDX/g-chow, 60 g dog food; purity 

and particle size not specified

0, 0.1, 1, or 10 mg/kg-d

Diet

13 wks

Doses 0 0.1 1 10

Mortality (incidence)

M 0/3 0/3 1/3

(not related to 

RDX)

0/3

F 0/3 0/3 0/3 0/3

Martin and Hart (1974)

Monkeys, Cynomolgus or Rhesusf, 

3/sex/group

Purity and particle size not specified

0, 0.1, 1, or 10 mg/kg-d

Gavage

13 wks

Doses 0 0.1 1 10

Mortality (incidence)

M 0/3 0/3 0/3 0/3

F 0/3 0/3 0/3 1/3

(animal exhibited 

neurological 

effects; euthanized)

von Oettingen et al. (1949)

Dogs, breed not specified, 5 females/group 

(control); 7 females/group (exposed)

90−97% pure, with 3−10% HMX; particle 

not specified

0 or 50 mg/kg-d

Diet

6 d/wk for 6 wks

Doses 0 50

Mortality (incidence)

F 0/5 1/7

MacPhail et al. (1985)

Rats, Sprague-Dawley derived CD, 

8−10 males or females/group 

Purity 84 ± 4.7%; ≤66 μm particle size

0, 1, 3, or 10 mg/kg-d

Gavage

30 d

No mortality was reported (incidence data were not provided).

Cholakis et al. (1980) Doses 0 0.2 2.0 20

Mortality (incidence)

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Reference and study design Results

Rabbits, New Zealand White (NZW), 

11−12 pregnant females/group

88.6% pure, with 9% HMX and 2.2% water 

as contaminants; ~200 μm particle size

0, 0.2, 2.0, or 20 mg/kg-d 

Diet

GDs 7−29

F 0/11 0/11 0/11 0/12

Cholakis et al. (1980)

Rats, F344, 24−25 females/group

88.6% pure, with 9% HMX and 2.2% water 

as contaminants

0, 0.2, 2.0, or 20 mg/kg-d 

Gavage

GDs 6−19

Doses 0 0.2 2.0 20

Mortality (incidence)

F 0/24 0/24 0/24 5/24

(1 rat accidentally 

killed; removed from 

analysis)

Angerhofer et al. (1986) (range-finding 

study)

Rats, Sprague-Dawley, 6 pregnant 

females/group

Purity 90%; 10% HMX and 0.3% acetic acid 

occurred as contaminants

0, 10, 20, 40, 80, or 120 mg/kg-d 

Gavage

GDs 6−15

Doses 0 10 20 40 80 120

Mortality (incidence)

F 0/6 0/6 0/6 6/6 6/6 6/6

Angerhofer et al. (1986)

Rats, Sprague-Dawley, 39−51 mated 

females/group

Purity 90%; 10% HMX and 0.3% acetic acid 

occurred as contaminants

0, 2, 6, or 20 mg/kg-d 

Gavage

GDs 6−15

Doses 0 2 6 20

Mortality (incidence)

F 0/39 1/40 1/40 16/51

Not reported whether 

deaths at 2 and 6 mg/kg-d 

related or likely related to 

RDX exposure

1 

2 

3 

4 

5 

6 c 7 

8 

9 

10

11 f 12

13

14

15

*Statistically significant (p < 0.05) based on analysis by the study authors. a

The 2-year rat study by Hart (1976) was not included in this evidence table because a malfunctioning heating 

system incident resulted in the premature deaths of 59 animals on study days 75−76 across groups, thereby 

confounding mortality findings.

b

Doses were calculated by the study authors.

Data for male and female rats were combined for statistical analysis.

d

Levine et al. (1981a) is a laboratory report of a 13-week study of RDX in F344 rats; two subsequently published 

papers (Levine et al., 1990; Levine et al., 1981b) present subsets of the data provided in the full laboratory report. e

Animals receiving 300 mg/kg-day died by week 3 of the study; animals receiving 600 mg/kg-day died by week 1 of 

the study. 

The species of monkey used in this study was inconsistently reported in the study as either Cynomolgus (in the 

methods section) or Rhesus (in the summary).

TWA = time-weighted average

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1 C.3.2. Other Noncancer Effects

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

There are isolated reports of RDX inducing systemic effects in several organs/systems, 

including the eyes and the musculoskeletal, cardiovascular, immune, and GI systems. However, 

there is less evidence for these effects compared to organ systems described in Section 1.2. Overall, 

at the present time, the evidence does not support identifying these other systemic effects as 

human hazards of RDX exposure. Summaries of the evidence for other systemic effects in humans 

and animals are shown in Tables C-11 and C-12, respectively. Experimental animal studies are 

ordered in the evidence table by type of effect, and then by duration of exposure and by species.

Ocular Effects

There are no reports of ocular effects in human case reports or epidemiological studies. In 

experimental animals, evidence of ocular effects comes from cataract findings in one 2-year 

bioassay. Specifically, the incidence of cataracts was 73% in female F344 rats exposed to 

40 mg/kg-day RDX in the diet for 2 years, compared with 32% in the control group (Levine et al., 

1983b). After 76 weeks of exposure, the incidence of cataracts in female rats at 40 mg/kg-day 

(23%) was also elevated compared to controls (6%). The incidence of cataracts was not increased 

in RDX-exposed male rats in the same study (Levine et al., 1983b), and other studies have not 

observed ocular effects associated with RDX exposure. Only two rats (dose groups not reported) 

were observed to have mild cataracts in a 90-day study of male and female F344 rats exposed to 

RDX at doses up to 15 mg/kg-day by gavage; however, the authors noted that these observations 

are common in F344 rats at 4 months of age and should not be attributed to treatment (Crouse et 

al., 2006). Furthermore, cataracts were not observed in male or female F344 rats exposed to 

40 mg/kg-day RDX by diet for 90 days (Cholakis et al., 1980) or in male or female B6C3F1 mice 

exposed to RDX in the diet for 2 years at doses up to approximately 100 mg/kg-day (Lish et al., 

1984). A statistically significant increase in the incidence of cataracts in male mice was initially 

noted by Lish et al. (1984), but was not confirmed when mice used for orbital bleedings were 

excluded from the analysis, suggesting that the effect was not treatment related. No ocular effects 

were observed in monkeys exposed by gavage for 90 days at doses up to 10 mg/kg-day (Martin and 

Hart, 1974). 

In summary, the incidence of cataracts was statistically significantly increased in high-dose 

female rats in one chronic oral study; however, this finding was not reproduced in other subchronic 

and chronic studies in rats or mice.

Cardiovascular Effects

Human evidence for cardiovascular effects is limited to case reports that include 

observations of transient arterial hypertension in male workers following inhalation of RDX during 

manufacturing (Barsotti and Crotti, 1949), sinus tachycardia, and in one instance, premature 

ventricular beats in five men following accidental ingestion of RDX at 37−250 mg/kg body weight 

(Küçükardali et al., 2003) (see Appendix C, Section C.2). 

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

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1 Inconsistent observations of cardiovascular effects have been reported in animal studies. 

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An increase in the relative heart-to-body weight ratio was observed at the highest dose tested in 

B6C3F1 mice (male: 13%; female: 17%) and F344 rats (male: 22%; female: 15%) following chronic 

dietary administration of RDX (Lish et al., 1984; Levine et al., 1983b); however, these doses also 

resulted in reductions in body weight in both males and females. Dose-related decreases in 

absolute heart weight in rats were reported in some subchronic (dietary) studies (Levine et al., 

1990; Levine et al., 1981a, b; Cholakis et al., 1980), whereas little change or modest increases in 

absolute heart weight were observed in other subchronic studies in rats or mice (Crouse et al., 

2006; Cholakis et al., 1980). A subchronic study in male dogs reported a 31% increase in absolute 

heart weight at the highest dose tested (10 mg/kg-day) (Hart, 1974). 

Evidence for changes in histopathology associated with RDX exposure is limited to findings 

of an increased incidence of focal myocardial degeneration in female rats (6/10 versus 2/10, 

respectively) and male mice (5/10 versus 0/10, respectively) compared with controls following 

exposure to RDX in the diet for 90 days (Cholakis et al., 1980). With the exception of one male 

mouse, the severity of the lesion was characterized as minimal. In each study, the finding of 

myocardial degeneration was limited to one sex and to the high-dose group only; the high dose in 

the male mouse study caused 40% mortality. Other studies in monkeys (Martin and Hart, 1974) 

and rats (von Oettingen et al., 1949) reported no observable cardiovascular effects. 

In summary, evidence for cardiovascular effects associated with RDX exposure consists of 

two case reports of cardiovascular effects following acute exposure, inconsistent findings of 

changes in heart weight in experimental animals, and one report of minimal histopathological 

changes in a 90-day rat study that was not confirmed in other toxicity studies. 

Musculoskeletal Effects

Evidence of musculoskeletal effects in humans consists of case reports that include 

observations of muscle twitching, myalgia/muscle soreness, and muscle injury as indicated by 

elevated levels of aspartate aminotransferase (AST) or myoglobinuria (Küçükardali et al., 2003; 

Hett and Fichtner, 2002; Hollander and Colbach, 1969; Stone et al., 1969; Merrill, 1968) (see 

Appendix C, Section C.2). Histological evaluations of musculature or skeletal tissue did not reveal 

any alterations in mice (Lish et al., 1984) or rats (Levine et al., 1983b; Hart, 1976) following chronic 

oral exposure to RDX, in mice and rats following subchronic exposure (Cholakis et al., 1980), or in 

dogs following a 90-day dietary exposure (Hart, 1974). 

Immune System Effects

RDX is structurally similar to various drugs known to induce the autoimmune disorder 

systemic lupus erythematosus (SLE). Based on the initial identification of three cases of SLE at 

one U.S. Army munitions plant, further investigation was conducted on a population of 

69 employees at five U.S. Army munitions plants with potential exposure to RDX (Hathaway and 

Buck, 1977); no additional cases of SLE were identified. Increased WBC counts have been reported 

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1 in some case reports of individuals (troops during the Vietnam war) who ingested or inhaled RDX 

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or C-4 (91% RDX) (Knepshield and Stone, 1972; Hollander and Colbach, 1969; Stone et al., 1969; 

Merrill, 1968). 

In animal studies, increased WBC count in female rats following subchronic dietary 

exposure to RDX was the only dose-related immune effect reported (Levine et al., 1990; Levine et 

al., 1981a, b); WBC counts in male rats were unaffected. Conversely, decreased WBC counts were 

reported in male and female rats in a 2-year study (Hart, 1976). Changes in spleen weights were 

observed across studies, but the responses were not consistent and did not appear to be doserelated. For example, in 90-day studies, Cholakis et al. (1980) identified a statistically significant 

decrease in absolute spleen weight in female F344 rats at 40 mg/kg-day, while Crouse et al. (2006)

observed a statistically significant increase in spleen weight at >10 mg/kg-day. Across studies, 

there was no significant or dose-dependent pattern of response to suggest that the WBC changes 

reflect RDX-induced immunotoxicity. No dose-related immune effects from oral exposure to RDX 

were observed in other animal studies, including a 90-day study in F344 rats specifically designed 

to evaluate immunotoxicity (parameters included evaluation of red blood cell [RBC] and WBC 

populations, proportion of cell surface markers, cellularity in proportion to organ weight, B and 

T cells in the spleen, and CD4/CD8 antigens of maturing lymphocytes in the thymus) (Crouse et al., 

2006). Routine clinical and histopathology evaluations of immune-related organs in a twogeneration study in rats (Cholakis et al., 1980) and chronic studies in rats (Levine et al., 1983b) and 

mice (Lish et al., 1984) provide no evidence of immunotoxicity associated with oral (dietary) 

exposure to RDX. 

In summary, evidence for immunotoxicity associated with RDX exposure is limited to 

findings from one study of increased WBC counts in female rats (Levine et al., 1981a, b). Evidence 

that RDX is not immunotoxic comes from several animal studies, including other repeat-dose oral 

studies in mice and rats (Crouse et al., 2006; Lish et al., 1984; Levine et al., 1983b; Cholakis et al., 

1980). 

Gastrointestinal Effects

Clinical signs of nausea and/or vomiting have been frequently identified in case reports of 

accidental or intentional RDX poisonings, and have generally been concurrent with severe 

neurotoxicity (Kasuske et al., 2009; Davies et al., 2007; Küçükardali et al., 2003; Hett and Fichtner, 

2002; Ketel and Hughes, 1972; Knepshield and Stone, 1972; Hollander and Colbach, 1969; Stone et 

al., 1969; Merrill, 1968; Kaplan et al., 1965; Barsotti and Crotti, 1949) (see Appendix C, Section C.2). 

In animal studies, vomiting has also been observed following oral exposure in swine (single-dose 

study) (Musick et al., 2010), dogs (Hart, 1974), and monkeys (Martin and Hart, 1974). One 

subchronic oral (diet) rat study from the early literature reported congestion of the GI tract at 

doses also associated with elevated mortality (von Oettingen et al., 1949); however, none of the 

subsequent subchronic or chronic animal studies reported histological changes of the GI tract 

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1 related to RDX administered via gavage or the diet (Crouse et al., 2006; Lish et al., 1984; Levine et 

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al., 1983b; Hart, 1974; Martin and Hart, 1974). 

In summary, evidence for GI tract effects associated with RDX exposure consists largely of 

reports of nausea and vomiting in humans acutely exposed to RDX and similar reports of vomiting 

in swine, dogs, and monkeys. In general, histopathological changes have not be reported in 

experimental animals exposed to RDX in the diet.

Hematological Effects

Elevated prevalence odds ratios (ORs) for hematological abnormalities (i.e., neutropenia, 

low platelet count, or macrocytosis; see Table C-11 for criteria used to define abnormal) were 

observed in a case-control study of men (24 cases, 199 controls) exposed to RDX in ordnance 

factories (West and Stafford, 1997) (see Table C-11). The prevalence OR for an association between 

RDX exposure and hematological abnormalities was 1.7 (95% confidence interval [CI] 0.7−4.2) for 

men with >50 hours of low-intensity exposure (based on 22 cases), while the prevalence OR was 

1.2 (95% CI 0.3−5.3) for men with >50 hours of high-intensity exposure (based on 2 cases). The 

ORs from this study must be interpreted with caution given the small sample size, wide CIs, and 

lack of identification of co-exposures. No changes in hematological parameters (including 

hemoglobin, hematocrit, and reticulocyte count) were observed in a cross-sectional epidemiologic 

study of 69 workers exposed to RDX by inhalation (RDX exposure range: undetectable 

[<0.01 mg/m3] to 1.6 mg/m3) (Hathaway and Buck, 1977). Humans who ingested or inhaled 

unknown amounts of RDX or C-4 (~91% RDX) for an acute duration displayed temporary 

hematological alterations, including anemia, decreased hematocrit, hematuria, and 

methemoglobinemia (Kasuske et al., 2009; Küçükardali et al., 2003; Knepshield and Stone, 1972; 

Hollander and Colbach, 1969; Stone et al., 1969; Merrill, 1968). In other case reports, normal blood 

counts were observed in accidentally exposed individuals (Testud et al., 1996a; Goldberg et al., 

1992; Woody et al., 1986; Ketel and Hughes, 1972; Kaplan et al., 1965) (see Appendix C, 

Section C.2).

In animals, hematological alterations were observed following oral exposure in chronic and 

subchronic studies in both sexes of rats (F344 or Sprague-Dawley) and B6C3F1 mice (see 

Table C-12). Increases in platelet count were observed in male and female mice and rats in some 

subchronic and chronic studies at doses ranging from 0.3 to 320 mg/kg-day (Lish et al., 1984; 

Levine et al., 1983b; Cholakis et al., 1980); however, changes were generally inconsistent across 

studies and were not generally dose-dependent. Similarly, decreased hemoglobin levels/anemia 

were observed in some chronic and subchronic studies (Levine et al., 1983b; Cholakis et al., 1980; 

von Oettingen et al., 1949), particularly at doses ≥15 mg/kg-day, but trends in hemoglobin levels 

across studies did not show a consistent relationship with dose. Other hematological parameters, 

including WBC counts, reticulocyte counts, and hematocrit, showed conflicting results between 

studies, marginal responses, or inconsistent changes with increasing dose. Other subchronic 

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

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1 studies in rats and dogs (Crouse et al., 2006; Hart, 1974; von Oettingen et al., 1949) did not identify 

2 

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6 

7 

8 

9 

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11

any changes in hematological parameters. 

In summary, evidence for hematological effects associated with RDX exposure in humans 

comes from several case reports that found transient fluctuations in hematological endpoints after 

acute exposures. Hematological findings from the case-control study and the cross-sectional study 

were not consistent. The small number of cases or exposed individuals, respectively, from the casecontrol and cross-sectional study may contribute to the difficulty in interpreting the results across 

studies (Table C-11). In general, animal studies of chronic and subchronic durations showed no 

consistent, dose-related pattern of increase or decrease in hematological parameters. 

Table C-11. Evidence pertaining to other noncancer effects (hematological) in 

humans

Reference and study design Results

Hematological effects

West and Stafford (1997) (United Kingdom)

Case-control study of ordnance factory 

workers, 32 cases with abnormal and 

322 controls with normal hematology test 

drawn from 1991 study of 404 workers at 

ammunitions plant; participation rate 97% of 

cases, 93% of controls. Analysis limited to men 

(29 cases, 282 controls). Analysis specific to 

RDX: 22 low- and 2 high-intensity cases; 

182 low- and 17 high-intensity controls.

Exposure measures: Exposure determination 

based on employee interviews and job title 

analysis; data included frequency (hrs/d, d/yr), 

duration (yrs), and intensity (low [1−10 ppm], 

moderate [10−100 ppm], and high 

[100−1,000 ppm], based on ventilation 

considerations).

Effect measures: Hematology tests; blood 

disorder defined as neutropenia (2.0 × 109

/L), 

low platelet count (<150 × 109

/L), or 

macrocytosis (mean corpuscular volume = 99 fl 

or >6% macrocytes).

Analysis: Unadjusted OR.

Hematological abnormality (neutropenia, low platelet count, or 

macrocytosis) (OR; 95% CI [number of exposed cases])

Low intensity, 50-hr-duration 1.7; 0.7,4.2 [22]

Medium intensity, 50-hr duration 1.6; not reported [not 

reported]

High intensity, 50-hr duration 1.2; 0.3, 5.3 [2]

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Reference and study design Results

Hathaway and Buck (1977) (United States)

Cross-sectional study, 2,022 workers, 

1,491 participated (74% response rate). 

Analysis limited to whites; 69 exposed to RDX 

alone and 24 exposed to RDX and HMX; 338 not 

exposed to RDX, HMX, or TNT.

Exposure measures: Exposure determination 

based on job title and industrial hygiene 

evaluation. Exposed subjects assigned to two 

groups: <LOD or ≥0.01 mg/m3 (mean for 

employees with exposures ≥LOD: 0.28 mg/m3

).

Effect measures: Hematology tests. 

Analysis: Types of statistical tests were not 

reported (assumed to be t-tests for comparison 

of means and χ2 tests for comparison of 

proportions). 

Hematology tests in men (mean; standard deviation not 

reported)

Test

Referent

(n = 237)

RDX exposed*

Undetected 

(<LOD)

(n = 22)

>0.01 mg/m3

(n = 45)

Hemoglobin 15.2 14.7 15.2

Hematocrit 42 45.6 47

Reticulocyte 

count 

0.7 0.9 0.7

*Includes both workers exposed to RDX alone and RDX and 

HMX.

No differences were statistically significant. Similar results in 

women.

Hematology tests in men (prevalence of abnormal values)

Test

(abnormal 

range) Referent

RDX exposed*

Undetected 

(<LOD) >0.01 mg/m3

Hemoglobin 

(<14)

15/237 3/22 4/45

Hematocrit 

(<40)

1/237 1/22 1/45

Reticulocyte 

count (>1.5)

18/237 3/22 2/45

*Includes both workers exposed to RDX alone and RDX and 

HMX.

No differences were statistically significant. Similar results in 

women.

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1 Table C-12. Evidence pertaining to other noncancer effects in animalsa

Reference and study design Results

Ocular effects

Lish et al. (1984)

Mice, B6C3F1, 85/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 1.5, 7.0, 35, or 175/100 mg/kg-d (high 

dose reduced to 100 mg/kg-d in wk 11 

due to excessive mortality)

Diet

2 yrs

Doses 0 1.5 7.0 35 175/100

Cataracts; 103 wks (incidence)b

M 2/47 2/41 0/41 2/37 2/16

F 2/50 1/37 6/52 0/46 1/26

Levine et al. (1983b)

Rats, F344, 75/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 0.3, 1.5, 8.0, or 40 mg/kg-d 

Diet

2 yrs

Doses 0 0.3 1.5 8.0 40

Cataracts; 103 wks (incidence)

M 8/40 6/39 6/31 8/35 2/6

F 14/44 4/48 11/44 8/43 22/30*

Cholakis et al. (1980)

Rats, F344, 10/sex/group

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

0, 10, 14, 20, 28, or 40 mg/kg-d 

Diet

13 wks

No ocular effects were observed (gross examination of eye was 

performed in all animals, and microscopic examination was performed 

in control and 40 mg/kg-d animals).

Crouse et al. (2006)

Rats, F344, 10/sex/group

99.99% pure

0, 4, 8, 10, 12, or 15 mg/kg-d 

Gavage

13 wks

No ocular effects were observed (ophthalmic examinations were 

performed in all animals within 1 wk of sacrifice, and microscopic 

examination of the eye was performed in control and 

15 mg/kg-d animals).

Martin and Hart (1974)

Monkeys, Cynomolgus or Rhesusc, 

3/sex/group

Purity of test material not specified

0, 0.1, 1, or 10 mg/kg-d 

Gavage

13 wks

No ocular effects were observed (ophthalmoscopic examination was 

performed at the end of exposure).

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Reference and study design Results

Cardiovascular effects

Lish et al. (1984)

Mice, B6C3F1, 85/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 1.5, 7.0, 35, or 175/100 mg/kg-d (high 

dose reduced to 100 mg/kg-d in wk 11 

due to excessive mortality)

Diet

2 yrs

Doses 0 1.5 7.0 35 175/100

Absolute heart weight; 104 wks (percent change compared to control)

M 0% 4% 4% 5% 7%

F 0% 1% 5% 2% −5%

Relative heart-to-body weight; 104 wks (percent change compared to 

control)

M 0% 7% 5% 5% 13%*

F 0% 0% 6% 4% 17%*

Body weight was significantly lower at termination in males and 

females exposed to 175/100 mg/kg-d (−5 and −19%, respectively).

Hart (1976)

Rats, Sprague-Dawley, 100/sex/group

Purity and particle size not specified

0, 1.0, 3.1, or 10 mg/kg-d

Diet

2 yrs

Doses 0 1.0 3.1 10

Myocardial fibrosis (percent incidence; number not reported)

M 20% − − 5%

F 5% − − 1%

Endocardial disease (percent incidence; number not reported)

M 1% − − 3%

F 0% − − 0%

Absolute heart weight; 104 wks (percent change compared to control)

M 0% −6% −2% −5%

F 0% 13% 3% 15%

Relative heart-to-body weight; 104 wks (percent change compared to 

control)

M 0% −2% 4% 1%

F 0% 23% 13% 27%

Levine et al. (1983b)

Rats, F344, 75/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 0.3, 1.5, 8.0, or 40 mg/kg-d

Diet

2 yrs

Doses 0 0.3 1.5 8.0 40

Absolute heart weight; 104 wks (percent change compared to control)

M 0% 3% −2% −2% 1%

F 0% −1% 0% −4% −3%

Relative heart-to-body weight; 104 wks (percent change compared to 

control)

M 0% 2% 6% 0% 22%

F 0% −2% 3% −1% 15%

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Reference and study design Results

Cholakis et al. (1980)

Mice, B6C3F1, 10−12/sex/group

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

Experiment 1: 0, 10, 14, 20, 28, or 

40 mg/kg-d

Diet

13 wks

Doses 0 10 14 20 28 40

Absolute heart weight (percent change compared to control)

M 0% − − − 7% 7%

F 0% − − − 0% 0%

Relative heart weight (percent change compared to control)

M 0% − − − 6% 0%

F 0% − − − −4% 0%

Experiment 2: 0, 40, 60, or 80 mg/kg-d 

for 2 wks followed by 0, 320, 160, or 

80 mg/kg-d (TWA doses of 0, 79.6, 

147.8, or 256.7 mg/kg-d for males and 0, 

82.4, 136.3, or 276.4 mg/kg-d for 

females)d

Diet

13 wks

Doses 0 80 160 320

Focal myocardial degeneration (incidence)

M# 0/10 − − 5/10*,‡

F† 0/11 − − 2/11

Absolute heart weight (percent change compared to control)

M 0% 0% 0% 8%

F 0% 0% 0% 8%

Relative heart-to-body weight (percent change compared to control)

M 0% 0% −2% 6%

F 0% 0% −2% 2%

#

Includes one affected and three unaffected animals that died 

prematurely.

†

Includes one unaffected animal that died prematurely. ‡

Minimal severity in four rats, moderate in one.

Cholakis et al. (1980)

Rats, F344, 10/sex/group

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

0, 10, 14, 20, 28, or 40 mg/kg-d

Diet

13 wks

Doses 0 10 14 20 28 40

Focal myocardial degeneration, minimal severity (incidence)

M 3/10 − − − − 1/10

F 2/10 − − − − 6/10

Absolute heart weight (percent change compared to control)

M 0% − − − 0% −8%*

F 0% − − − −6% −11%*

Relative heart-to-body weight (percent change compared to control)

M 0% − − − 3% 0%

F 0% − − − −3% −8%

Relative heart-to-brain weight (percent change compared to control)

M 0% − − − −4% −10%*

F 0% − − − −5% −11%*

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Reference and study design Results

Cholakis et al. (1980)

Rats, CD, two-generation study; F0: 

22/sex/group; F1: 26/sex/group; F2: 

10/sex/group 

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

F0 and F1 parental animals: 0, 5, 16, or 

50 mg/kg-d

Diet

F0 exposure: 13 wks pre-mating, and 

during mating, gestation, and lactation 

of F1; F1 exposure: 13 wks after 

weaning, and during mating, gestation, 

and lactation of F2; F2 exposure: until 

weaning

No cardiac effects were observed (microscopic examination of heart 

was performed in randomly selected F2 animals). 

Doses 0 5 16 50

Absolute heart weight (percent change compared to control)

F2 M 0% 3.2% −6.5% −

F2 F 0% 15% −3.7% −

Crouse et al. (2006)

Rats, F344, 10/sex/group

99.99% pure

0, 4, 8, 10, 12, or 15 mg/kg-d

Gavage

13 wks

Doses 0 4 8 10 12 15

Cardiomyopathy (incidence)

M 2/10 − − − − 3/8

F 0/10 − − − − 1/6

Absolute heart weight (percent change compared to control)

M 0% −2% −7% −1% 1% 11%

F 0% −2% 0% 8% 7% 6%

Relative heart-to-body weight (percent change compared to control)

M 0% 4% 2% 1% −1% 8%

F 0% −2% −7% −6% −9% −16%*

(Levine et al. (1990); Levine et al. 

(1981a), 1981b))

e

Rats, F344, 10/sex/group; 30/sex for 

control

84.7 ± 4.7% purity, ~10% HMX, median 

particle diameter 20 μm, ~90% of 

particles ≤66 μm

0, 10, 30, 100, 300, or 600 mg/kg-d

Diet

13 wks

All animals in the 300 and 600 mg/kg-d groups died prior to study 

termination.

Doses 0 10 30 100 300 600

Chronic focal myocarditis (incidence)

M 8/30 8/10 6/10 1/10 1/10 0/10

F 8/30 3/10 1/10 1/10 1/10 1/9

Absolute heart weight (percent change compared to control)

M 0% −2% −10% −15% − −

F 0% −3% 0% −5% − −

Relative heart-to-body weight (percent change compared to control)

M 0% 2% −4% 3% − −

F 0% −2% 0% −3% − −

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Reference and study design Results

von Oettingen et al. (1949)

Rats (sex/strain not specified); 20/group

Purity and particle size not specified

0, 15, 25, or 50 mg/kg-d

Diet

13 wks

The study authors reported that there were no cardiac effects 

(microscopic examination of the heart was performed in all rats; data 

were not shown).

Hart (1974)

Dogs, Beagle, 3/sex/group

Pre-mix with ground dog chow 

containing 20 mg RDX/g-chow, 60 g dog 

food; purity and particle size not 

specified

0, 0.1, 1, or 10 mg/kg-d

Diet

13 wks

Doses 0 0.1 1 10

Focal hyalinization of the heart (incidence)

M 0/3 − − 0/3

F 0/3 − − 1/3

Absolute heart weight (percent change compared to control)

M 0% − − 31%

F 0% − − 5.7%

Martin and Hart (1974)

Monkeys, Cynomolgus or Rhesusc,

3/sex/group

Purity of test material not specified

0, 0.1, 1, or 10 mg/kg-d

Gavage

13 wks

Doses 0 0.1 1 10

Myocarditis (percent change compared to control)

M 1/3 − − 1/3

F 0/3 − − 0/3

Absolute heart weight (percent change compared to control)

M 0% 7% −1% 5%

F 0% 10% 12% −12%

Immune effects

Lish et al. (1984)

Mice, B6C3F1, 85/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 1.5, 7.0, 35, or 175/100 mg/kg-d (high 

dose reduced to 100 mg/kg-d in wk 11 

due to excessive mortality)

Diet

2 yrs

No immune effects were observed with routine hematology, clinical 

chemistry, or histopathology evaluations.

Doses 0 1.5 7.0 35 175/100

WBC count; 105 wks (percent change compared to control)

M 0% −13% −8% −16% −30%

F 0% 12% 39%* 28% 0%

Absolute spleen weight; 105 wks (percent change compared to 

control)

M 0% 24% 31% −10% −28%

F 0% 4% 15% −17% 16%

Relative spleen weight; 105 wks (percent change compared to control)

M 0% 26% 32% −11% −21%

F 0% 4% 15% −17% 44%

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

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Reference and study design Results

Hart (1976)

Rats, Sprague-Dawley, 100/sex/group

Purity and particle size not specified

0, 1.0, 3.1, or 10 mg/kg-d

Diet

2 yrs

Doses 0 1.0 3.1 10

WBC count; 104 wks (percent change compared to control)

M 0% −13% −22%* −34%*

F 0% 5% −32%* −12%

Absolute spleen weight; 104 wks (percent change compared to 

control)

M 0% −11% −16% −4%

F 0% 58% 8% 37%

Relative spleen weight; 104 wks (percent change compared to control)

M 0% −11% −14% 1%

F 0% 77% 19% 55%

Levine et al. (1983b)

Rats, F344, 75/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 0.3, 1.5, 8.0, or 40 mg/kg-d

Diet

2 yrs

No immune effects were observed with routine hematology, clinical 

chemistry, or histopathology evaluations.

Doses 0 0.3 1.5 8.0 40

WBC count; 105 wks (percent change compared to control)

M 0% −11% 103% f 184%f 15%

F 0% 7% 12% 354%f 251%f

Absolute spleen weight; 105 wks (percent change compared to 

control)

M 0% 5% −10% −32% −49%

F 0% −28% −44% −35% 17%

Relative spleen weight; 105 wks (percent change compared to control)

M 0% 9% 4% −29% −38%

F 0% −34% −45% −36% 9%

Cholakis et al. (1980)

Mice, B6C3F1, 10−12/sex/group

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

Experiment 1: 0, 10, 14, 20, 28, or 

40 mg/kg-d

Diet

13 wks

Doses 0 10 14 20 28 40

Absolute spleen weight (percent change compared to control)

M 0% − − − 18% 13%

F 0% − − − −2% −8%

Relative spleen weight (percent change compared to control)

M 0% − − − 24% 14%

F 0% − − − −3% −5%

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

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Reference and study design Results

Experiment 2: 0, 40, 60, 80 mg/kg-d for 

2 wks followed by 0, 320, 160, or 

80 mg/kg-d (TWA doses of 0, 79.6, 

147.8, or 256.7 mg/kg-d for males and 0, 

82.4, 136.3, or 276.4 mg/kg-d for 

females)d

Diet

13 wks

Doses 0 80 160 320

WBC count (percent change compared to control)

M 0% −27% −12% 30%

F 0% −17% 3% −3%

Absolute spleen weight (percent change compared to control)

M 0% 17% 0% −17%

F 0% −22% 0% 0%

Relative spleen weight (percent change compared to control)

M 0% 25% 5% 0%

F 0% −12% 0% −3%

Cholakis et al. (1980)

Rats, F344, 10/sex/group

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

0, 10, 14, 20, 28, or 40 mg/kg-d

Diet

13 wks

Doses 0 10 14 20 28 40

WBC count (percent change compared to control)

M 0% − − − −12% 7%

F 0% − − − 17% 30%

Absolute spleen weight (percent change compared to control)

M 0% − − − 2% −4%

F 0% − − − −10% −12%*

Relative spleen weight (percent change compared to control)

M 0% − − − 5% 5%

F 0% − − − −8% −8%

Cholakis et al. (1980)

Rats, CD, two-generation study; F0: 

22/sex/group; F1: 26/sex/group; 

F2: 10/sex/group 

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

F0 and F1 parental animals: 0, 5, 16, or 

50 mg/kg-d

Diet

F0 exposure: 13 wks pre-mating, and 

during mating, gestation, and lactation 

of F1; F1 exposure: 13 wks after 

weaning, and during mating, gestation, 

and lactation of F2; F2 exposure: until 

weaning

No immune effects were observed upon routine histopathology 

evaluation.

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

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Reference and study design Results

Crouse et al. (2006)

Rats, F344, 10/sex/group

99.99% pure

0, 4, 8, 10, 12, or 15 mg/kg-d

Gavage

13 wks

No effects were observed on thymus or spleen histology, RBC or WBC 

populations, or lymphocyte populations.

Doses 0 4 8 10 12 15

WBC count (percent change compared to control)

M 0% −5% −12% −7% 1% −3%

F 0% 22% 45% 12% 52% 29%

Absolute spleen weight (percent change compared to control)

M 0% −3% −6% 3% 1% 5%

F 0% 1% 8% 23%* 17%* 24%*

Relative spleen weight (percent change compared to control)

M 0% 3% 4% 7% −1% 2%

F 0% 1% 0% 6% −1% −2%

Absolute thymus weight (percent change compared to control)

M 0% −1% 3% −10% −12% −25%

F 0% −7% 12% 19% 32% 19%

Relative thymus weight (percent change compared to control)

M 0% −1% 3% −10% −12% −25%

F 0% −7% 4% 4% 12% −6%

(Levine et al. (1990); Levine et al. 

(1981a), 1981b))

e

Rats, F344, 10/sex/group; 30/sex for 

control

84.7 ± 4.7% purity, ~10% HMX, median 

particle diameter 20 μm, ~90% of 

particles ≤66 μm

0, 10, 30, 100, 300, or 600 mg/kg-d

Diet

13 wks

Data were not reported for rats in the 300 or 600 mg/kg-d dose groups 

because all of the rats died before the 13-wk necropsy.

Doses 0 10 30 100 300 600

WBC count (percent change compared to control)

M 0% 4% 7% 15% − −

F 0% 23%* 24%* 62%* − −

Absolute spleen weight (percent change compared to control)

M 0% −11% −16% −34% − −

F 0% 2% 12% 0% − −

Relative spleen weight (percent change compared to control)

M 0% −9% −12% −21% − −

F 0% 2% 12% 3% − −

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

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Reference and study design Results

von Oettingen et al. (1949)

Rats, sex/strain not specified, 20/group

90−97% pure, with 3−10% HMX; particle 

size not specified

0, 15, 25, or 50 mg/kg-d

Diet

13 wks

Doses 0 15 25 50

WBC count (percent change compared to control)

M 0% −30% 7% −6%

Hart (1974)

Dogs, Beagle, 3/sex/group

Pre-mix with ground dog chow 

containing 20 mg RDX/g-chow, 60 g dog 

food; purity and particle size not 

specified

0, 0.1, 1, or 10 mg/kg-d

Diet

13 wks

Doses 0 0.1 1 10

WBC count (percent change compared to control)

M 0% 5% 2% −19%

F 0% −2% 24% 6%

Absolute spleen weight (percent change compared to control)

M 0% − − 123%

F 0% − − −11%

Martin and Hart (1974)

Monkeys, Cynomolgus or Rhesusc, 

3/sex/group

Purity of test material not specified

0, 0.1, 1, or 10 mg/kg-d

Gavage

13 wks

Doses 0 0.1 1 10

WBC count (percent change compared to control)

M 0% −32% 0% −3%

F 0% −38% −1% −41%

Gastrointestinal effects

Lish et al. (1984)

Mice, B6C3F1, 85/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 1.5, 7.0, 35, or 175/100 mg/kg-d (high 

dose reduced to 100 mg/kg-d in wk 11 

due to excessive mortality)

Diet

2 yrs

No GI tract effects were observed as clinical signs or on gross pathology 

or histopathology examination.

Levine et al. (1983b)

Rats, F344, 75/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 0.3, 1.5, 8.0, or 40 mg/kg-d

Diet

2 yrs

No GI tract effects were observed as clinical signs or on gross pathology 

or histopathology examination.

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

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Reference and study design Results

Crouse et al. (2006)

Rats, F344, 10/sex/group

99.99% pure

0, 4, 8, 10, 12, or 15 mg/kg-d

Gavage

13 wks

No GI tract effects were observed on gross pathology or histopathology 

examination. Increased salivation and blood stains around the mouth 

were noted (affected doses and incidences were not reported); it is not 

clear whether these effects occurred in animals also experiencing 

convulsions.

von Oettingen et al. (1949)

Rats (sex/strain not specified); 20/group

90−97% pure, with 3−10% HMX; particle 

size not specified

0, 15, 25, or 50 mg/kg-d

Diet

13 wks

Congestion of the GI tract was observed in 50 and 100 mg/kg-d rats 

that also exhibited mortality (40%) and severe neurotoxicity. 

Martin and Hart (1974)

Monkeys, Cynomolgus or Rhesusc, 

3/sex/group

Purity of test material not specified

0, 0.1, 1, or 10 mg/kg-d

Gavage

13 wks

Vomiting was observed more frequently in the 1 and 10 mg/kg-d 

groups compared to the control or 0.1 mg/kg-d groups, although some 

episodes occurred during the intubation procedure.

Hart (1974)

Dogs, Beagle, 3/sex/group

Pre-mix with ground dog chow 

containing 20 mg RDX/g-chow, 60 g dog 

food; purity and particle size not 

specified

0, 0.1, 1, or 10 mg/kg-d

Diet

13 wks

Some nausea and vomiting were reported (incidences and affected 

dose groups were not reported).

Hematological effects

Lish et al. (1984)

Mice, B6C3F1, 85/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 1.5, 7.0, 35, or 175/100 mg/kg-d (high 

dose reduced to 100 mg/kg-d in wk 11 

due to excessive mortality)

Diet

2 yrs

Doses 0 1.5 7.0 35 175/100

RBC count; 105 wks (percent change compared to control)

M 0% −4% 3% −3% 14%

F 0% 4% −7% 5% 3%

Hemoglobin; 105 wks (percent change compared to control)

M 0% −6% 3% −5% 9%

F 0% 2% −7% 3% 1%

Hematocrit; 105 wks (percent change compared to control)

M 0% −4% 3% −4% 9%

F 0% 3% −6% 3% 1%

Platelets; 105 wks (percent change compared to control)

M 0% 33% 9% 21% 27%

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

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Reference and study design Results

F 0% −14% −7% 1% 5%

Hart (1976)

Rats, Sprague-Dawley, 100/sex/group

Purity and particle size not specified

0, 1.0, 3.1, or 10 mg/kg-d

Diet

2 yrs

Doses 0 1.0 3.1 10

RBC count; 104 wks (percent change compared to control)

M 0% 3% 7% −2%

F 0% −14% 7% 2%

Reticulocyte count; 104 wks (percent change compared to control)

M 0% 250% 500%* 850%*

F 0% 180%* −40% 20%

Hemoglobin; 104 wks (percent change compared to control)

M 0% 3% 4% 0%

F 0% −1% 1% −2%

Levine et al. (1983b)

Rats, F344, 75/sex/group; interim 

sacrifices (10/sex/group) at 6 and 12 mo

89.2−98.7% pure, with 3−10% HMX as 

contaminant; 83−89% of particles 

<66 μm

0, 0.3, 1.5, 8.0, or 40 mg/kg-d

Diet

2 yrs

Doses 0 0.3 1.5 8.0 40

Hemoglobin levels; 105 wks (percent change compared to control)

M 0% 6% 6% 3% −13%

F 0% −5% 1% −9% −14%

RBC count; 105 wks (percent change compared to control)

M 0% 5% 2% −1% −9%

F 0% −2% 2% −9% −13%

Platelet count; 105 wks (percent change compared to control)

M 0% 6% −4% −10% −7%

F 0% 14% −4% 5% 22%

Hematocrit; 105 wks (percent change compared to control)

M 0% 5% 5% 2% −7%

F 0% −5% 0% −8% −12%

Cholakis et al. (1980)

Mice, B6C3F1, 10−12/sex/group 

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

0, 80, 60, or 40 mg/kg-d for 2 wks 

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

(TWA doses of 0, 79.6, 147.8, or 

256.7 mg/kg-d for males and 0, 82.4, 

136.3, or 276.4 mg/kg-d for females)d

Diet

13 wks

Doses 0 80 160 320

RBC count (percent change compared to control)

M 0% −5% −12%* −2%

F 0% −10% −1% 1%

Reticulocytes (percent change compared to control)

M 0% −36% −13% 15%

F 0% 21% 25% −19%

Hematocrit (percent change compared to control)

M 0% −1% −6% 0%

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

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Reference and study design Results

F 0% −8% 2% 1%

Hemoglobin (percent change compared to control)

M 0% −2% −7%* −3%

F 0% −5% 4% 1%

Platelets (percent change compared to control)

M 0% 33% 28% 22%

F 0% 3% 9% 39%

Cholakis et al. (1980)

Rats, F344, 10/sex/group

88.6% pure, with 9% HMX and 2.2% 

water as contaminants; ~200 μm 

particle size

0, 10, 14, 20, 28, or 40 mg/kg-d

Diet

13 wks

Doses 0 10 14 20 28 40

RBC count (percent change compared to control)

M 0% − − − 3% −1%

F 0% − − − −1% −7%

Hemoglobin (percent change compared to control)

M 0% − − − 2% −1%

F 0% − − − −1% −1%

Platelet (percent change compared to control)

M 0% − − − 11% 16%*

F 0% − − − −23% −13%

Reticulocytes (percent change compared to control)

M 0% − − − 26% 76%*

F 0% − − − −2% 17%

Hematocrit (percent change compared to control)

M 0% − − − 3% 0%

F 0% − − − 0% −2%

Crouse et al. (2006)

Rats, F344, 10/sex/group

99.99% pure

0, 4, 8, 10, 12, or 15 mg/kg-d

Gavage

13 wks

Doses 0 4 8 10 12 15

RBC count (percent change compared to control)

M 0% 1% −7% −2% −4% −5%

F 0% 3% 3% −1% 2% −2%

Hemoglobin (percent change compared to control)

M 0% −1% −5% 0% −1% −6%

F 0% 2% 4% −1 4% −4%

Platelet count (percent change compared to control)

M 0% 21% 11% 13% −8% 34%

F 0% 6% 40% 47% 34% −36%

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

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Reference and study design Results

Hematocrit (percent change compared to control)

M 0% 2% −5% 0% −1% −4%

F 0% 3% 4% 0% 4% −2%

(Levine et al. (1990); Levine et al. 

(1981a), 1981b))

e

Rats, F344, 10/sex/group; 30/sex for 

control

84.7 ± 4.7% purity, ~10% HMX, median 

particle diameter 20 μm, ~90% of 

particles ≤66 μm

0, 10, 30, 100, 300, or 600 mg/kg-d

Diet

13 wks

Data were not reported for rats in the 300 or 600 mg/kg-d dose groups 

because all of the rats died before the 13-wk necropsy.

Doses 0 10 30 100 300 600

Hematocrit (percent change compared to control)

M 0% −2% −1% −5% − −

F 0% 0% −4% −7% − −

Hemoglobin (percent change compared to control)

M 0% −3% −1% −6% − −

F 0% 0% −4% −8%* − −

RBC count (percent change compared to control)

M 0% −2% −2% −5% − −

F 0% −1% −4% −5% − −

Reticulocytes (percent change compared to control)

M 0% −4% 10% 28% − −

F 0% 9% 73% 71% − −

von Oettingen et al. (1949)

Rats, sex/strain not specified, 20/group

90−97% pure, with 3−10% HMX; particle 

size not specified

0, 15, 25, or 50 mg/kg-d

Diet

13 wks

Doses 0 15 25 50

RBC count (percent change compared to control)

M + F 0% −23% −12% −14%

Hemoglobin (percent change compared to control)

M + F 0% −25% −7% −11%

Hart (1974)

Dogs, Beagle, 3/sex/group

Pre-mix with ground dog chow 

containing 20 mg RDX/g-chow, 60 g dog 

food; purity and particle size not 

specified

0, 0.1, 1, or 10 mg/kg-d

Diet

13 wks

Doses 0 0.1 1 10

RBC count (percent change compared to control)

M 0% −3% 3% 2%

F 0% 13% 7% 11%

Reticulocyte count (percent change compared to control)

M 0% −66% 0% −50%

F 0% −17% −50% 0%

Hematocrit (percent change compared to control)

M 0% −4% 2% 0%

F 0% 6% 1% 7%

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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Reference and study design Results

Hemoglobin (percent change compared to control)

M 0% 5% −2% 0%

F 0% 8% −2% 8%

Martin and Hart (1974)

Monkeys, Cynomolgus or Rhesusc, 

3/sex/group

Purity of test material not specified

0, 0.1, 1, or 10 mg/kg-d 

Gavage

13 wks

Histopathological examination revealed increased numbers of 

degenerate or necrotic megakaryocytes in all bone marrow sections.

Doses 0 0.1 1 10

RBC count (percent change compared to control)

M 0% −3% 2% −3%

F 0% 0% −1% 2%

Reticulocyte count (percent change compared to control)

M 0% −33% −50% −50%

F 0% −18% −36% 45%

Hematocrit (percent change compared to control)

M 0% −7% −4% −1%

F 0% 10% 7% 3%

Hemoglobin (percent change compared to control)

M 0% −10% −8% −6%

F 0% 6% 6% 3%

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

*Statistically significantly different compared to the control, as determined by study authors (p < 0.05). a

No musculoskeletal evidence is presented in this table as no animal study reported effects on the musculoskeletal 

system and all human effects were in case reports (see summary in Appendix C, Section C.2). 

b

Incidence counts exclude individuals from which blood was obtained via the orbital sinus. c

The species of monkey used in this study was inconsistently reported in the study as either Cynomolgus (in the 

Methods section) or Rhesus (in the Summary). d

Doses were calculated by the study authors.

e

Levine et al. (1981a) is a laboratory report of a 13-week study of RDX in F344 rats; two subsequently published 

papers (Levine et al., 1990; Levine et al., 1981b) present subsets of the data provided in the full laboratory report. f

Standard deviations accompanying the mean response in a given dose group were high, suggesting uncertainty in

the accuracy of the reported percent change compared to control.

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

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

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1 C.3.3. Genotoxicity

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

RDX 

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 chemiluminescent 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 was unknown in 

several (particularly older) studies. A summary of the results of in vitro genotoxicity studies of RDX 

is presented in Table C-13. 

RDX has produced negative results in all reverse mutation 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 metabolic activating mixture (Neuwoehner et al., 2007; George et al., 

2001; Lachance et al., 1999; Tan et al., 1992; Minor et al., 1982; Cholakis et al., 1980; Whong et al., 

1980; Simmon et al., 1977b). One exception is a finding of weak mutagenic activity of RDX to 

S. typhimurium strain TA97a (mutagenicity index = 1.5−2.0) (Pan et al., 2007a). However, this assay 

used 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., 1977b). The study authors noted 

that the negative findings should be considered in the context of the low concentrations tested. 

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 Mutatox 

assay with the bacterium Vibrio fisheri when tested at doses up to 2.5 μg/tube, with and without S9

(Arfsten et al., 1994). In the presence of S9, a dose-response was observed; in the absence of S9, no 

dose-response relationship was detected (Arfsten et al., 1994). RDX did not induce forward 

mutations in mouse lymphoma L5178Y cells with or without metabolic activation (Reddy et al., 

2005). During an accompanying range-finding study, precipitates occurred at doses ≥250 μg/mL, 

suggesting that concentrations of RDX in DMSO reported beyond 250 μg/mL may not be accurate.

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1 Table C-13. Summary of in vitro studies of the genotoxicity of RDX

Endpoint Test system

Dose/

concentrationa

Resultsb

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. 

(1977a)

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 

mutation

Vibrio fischeri 0.004 μg/tube ± + Mutatox assay with metabolic activation (S9) Arfsten et al. 

(1994)

Reverse 

mutation (umu

test)

Salmonella choleraesius subsp. 

chol. (prior S. typhimurium) 

TA1535/pSK1002 

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)

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Endpoint Test system

Dose/

concentrationa

Resultsb

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 

mutation

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

Saccharomyces 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. 

(1977a)

Recombination 

induction

S. cerevisiae D3 30.8 μg/mL − − Metabolic activation with S9 Cotruvo et al. 

(1977)

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Endpoint Test system

Dose/

concentrationa

Resultsb

Comments Reference

Without 

activation

With 

activation

Genotoxicity studies in mammalian cells

Forward 

mutation

Chinese hamster lung 

fibroblasts V79 cells

40 μg/mL − − Minimal cytotoxicity observed at 40 μg/mL 

(limit of solubility) 

Lachance et al. 

(1999)

Mutation L5178Y mouse lymphoma cells 500 μg/mL − − No or low cytotoxicity seen at these 

concentrations; however, precipitate was 

observed at >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. 

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1 RDX did not induce unscheduled DNA synthesis, with or without metabolic activation, using 

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

human diploid fibroblasts (WI-38 cells) when tested in DMSO at concentrations up to 4,000 μg/mg; 

however, precipitation of RDX at high concentrations in cell culture media makes interpretation of 

these results difficult (Dilley et al., 1979). Only two in vivo studies are available for the genotoxicity 

of RDX; these are summarized in Table C-14. RDX did not decrease the ratio of polychromatic 

erythrocytes (PCEs) to normochromatic erythrocytes (NCEs), 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., 2005). RDX was considered negative for the induction of dominant lethal mutations in male 

CD rats fed 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 significant effects on number of corpora lutea, implants, or live 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., 2007a; George et al., 

2001; Snodgrass, 1984). Miniature pigs were used to detect these trace 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 conducted. A 

summary of the results of in vitro and in vivo genotoxicity studies of metabolites of RDX is 

presented in Table C-15. 

Pan et al. (2007a) studied the mutagenicity of two metabolites, MNX and TNX. These 

metabolites were not mutagenic in S. typhimurium strain TA97a at normal levels of S9, but were 

clearly mutagenic at enhanced concentrations of S9 (4% versus 9% S9). The observation that these 

metabolites were positive in S. typhimurium strain 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). These metabolites were also weakly 

mutagenic in S. typhimurium strain TA102, again with high levels of S9. Strain 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). 

Other metabolites with potential human relevance identified in the urine of miniature pigs have not 

been assessed for their genotoxicity (Major et al., 2007). 

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1 Table C-14. Summary of in vivo studies of the genotoxicity of RDX

2 

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)

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1 Table C-15. Summary of in vitro and in vivo studies of the genotoxicity of RDX metabolites

2 

3 

4 

Endpoint Test system

Dose/

concentrationa

Resultsb

Comments Reference

Without 

activation

With 

activation

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 μg/plate + + Positive only for TNX; MNX and DNX were 

negative

George et al. 

(2001)

Reverse 

mutation

S. typhimurium TA1535, 

TA1537, TA1538, 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 + ND Mononitroso metabolite, MNX; 

additional metabolic activation not 

required with S9

Snodgrass (1984)

In vivo genotoxicity studies in mammalian systems

Dominant 

lethal 

mutations

Male mice dosed and mated 

with untreated female mice

NR − ND Mononitroso metabolite, MNX; 

additional metabolic activation not 

required with S9

Snodgrass (1984)

a

Lowest effective dose for positive results; highest dose tested for negative results; NR = not reported. b

+ = positive; ± = equivocal or weakly positive; − = negative; ND = not determined.

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1 The genotoxicity of MNX was positive in three out of five assays conducted for the U.S. Army 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

(Snodgrass, 1984). MNX was positive with or without metabolic activation in the mouse lymphoma 

forward mutation assay at the thymidine kinase locus, for 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 addition 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. 

In summary, RDX is not mutagenic or genotoxic in vitro or in vivo in typical assays used to 

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 that the N-nitroso metabolites 

are genotoxic, but the formation and quantification of these metabolites in humans is not known. 

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APPENDIX D. DOSE-RESPONSE MODELING FOR 

THE DERIVATION OF REFERENCE VALUES FOR 

EFFECTS OTHER THAN CANCER AND THE 

DERIVATION OF CANCER RISK ESTIMATES

1 This appendix provides technical detail on dose-response evaluation and determination of 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

points of departure (POD) for relevant toxicological endpoints. The endpoints were modeled using 

the U.S. Environmental Protection Agency (EPA) Benchmark Dose Software (BMDS, Versions 2.4). 

Sections D.1 (noncancer) and D.2 (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, 2012b). In some cases, it may be 

appropriate 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 NONCANCER

ENDPOINTS

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

(mg/kg-d) Incidence/total (%)

Convulsions

Crouse et al. (2006)a 

Female F344 rat 0 

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

4 

8 

10

12

15

0/10 (0%)

0/10 (0%)

1/10 (10%)

3/10 (30%)

8/10 (80%)

7/10 (70%)

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Endpoint and 

reference Species/sex

Dose

(mg/kg-d) Incidence/total (%)

Male and female 

F344 rat, combined

0 0/20 (0%)

4 0/20 (0%)

8 3/20 (15%)

10 6/20 (30%)

12 13/20 (65%)

15 12/20 (60%)

Convulsions

Cholakis et al. (1980)

Female F344 rat

(gestational 

exposure)

0 0/24 (0%)

0.2 0/24 (0%)

2 1/24 (4%)

20 18/24 (75%)

Testicular 

degeneration

Lish et al. (1984)

Male B6C3F1 mouse 0 0/63 (0%)

1.5 2/60 (3%)

7 2/62 (3%)

35 6/59 (10%)

107 3/27 (11%)

Prostate suppurative 

inflammation

Levine et al. (1983b)

Male F344 rat 0 2/54 (4%)

0.3 4/55 (7%)

1.5 9/52 (17%)

8 12/55 (22%)

40 19/31 (61%)

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

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 endpoint prior to modeling. 

In addition to the endpoints presented in Table D-1, the combined incidence of seizure and 

mortality was modeled for Crouse et al. (2006) to determine the effect of possible underestimation

of seizures, as discussed in Section 2.1.6. Table D-2 presents the data on this combined incidence. 

Table D-2. Convulsion or mortality endpoints from Crouse et al. (2006)

selected for dose-response modeling for RDX

Endpoint and 

reference Species/sex

Dose 

(mg/kg-d) Incidence/total (%)

Convulsion or 

mortality

Johnson (2015)

Female F344 rat 0 

4 

8 

10

12

15

0/10 (0%)

0/10 (0%)

3/10 (30%)

5/10 (50%)

9/10 (90%)

8/10 (80%)

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Endpoint and 

reference Species/sex

Dose

(mg/kg-d) Incidence/total (%)

Male F344 rat 0

4 

8 

10

12

15

0/10 (0%)

0/10 (0%)

2/10 (20%)

4/10 (40%)

8/10 (80%)

7/10 (70%)

Male and female 

F344 rat, combined

0

4 

8 

10

12

15

0/20 (0%)

0/20 (0%)

3/20 (15%)

6/20 (30%)

13/20 (65%)

12/20 (60%)

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

a

Incidence was defined for each animal as the presence of convulsion or mortality. The incidence rates across 

doses for this endpoint 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 

endpoint prior to modeling. 

D.1.1. Evaluation of Model Fit and Model Selection

For each dichotomous endpoint, BMDS dichotomous models5 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 (χ2 p-value < 0.10 indicates lack of fit). Other factors were also 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 benchmark response (BMR). 

From among the models exhibiting adequate fit, the best-fit model was selected for 

estimation of the BMD. This model selection was conducted in two stage, first from among only the 

multistage models to determine a representative multistage model, and second from among the 

representative multistage model and the non-multistage models. In each stage, the BMDL estimates 

(95% lower confidence limit on the BMD, as estimated by the profile likelihood method) and AIC 

values of the models considered in that stage were used to make the selection, as follows. If the 

BMDL estimates were “sufficiently close,” that is, differed by threefold or less, the model selected 

was the one that yielded the lowest AIC value. If the BMDL estimates were not sufficiently close, the 

model with the lowest BMDL was selected. The model selected in the second stage was considered 

the best-fit model. 

The BMDL estimate (95% lower confidence limit on the benchmark dose [BMD], as 

estimated by the profile likelihood method) and Akaike’s information criterion (AIC) value were 

5Unless 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, restrict slope ≥1; for the Gamma and Weibull models, restrict power ≥1. 

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1 used to select a best-fit model from among the models exhibiting adequate fit. If the BMDL 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

estimates were “sufficiently close” (i.e., differed by at most 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.

D.1.2. Modeling Results

The tables that follow summarize the modeling results for the noncancer endpoints 

modeled. 

Nervous System Effects

Tables D-3 to D-11 (and Figures D-1 to D-9) present the BMD modeling 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 (ER). Tables D-12 to D-14 (and Figures 

D-10 to D-12) present the BMD modeling results for incidence of convulsions for female F344 rats 

based on data from Cholakis et al. (1980), using BMRs of 10, 5, and 1% ER. Table D-15 (and Figure 

D-13) presents the BMD modeling results for combined incidence of convulsions and mortality for 

male and female rats combined based on data from Crouse et al. (2006). 

Table D-3. Model predictions for convulsions in female F344 rats exposed to 

RDX by gavage for 90 days (Crouse et al., 2006); BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

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-day were 0.00, 

−1.29, −0.46, −0.12, 0.87, and 0.41, respectively.

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b 1 The Multistage 5° model may appear equivalent to the Multistage 4° model; however, differences exist in digits 

2 

 3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

not displayed in the table.

BMR = 10% ER; dose shown in mg/kg-day.

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). 

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% ER

BMD = 2.75544

BMDL at the 95% confidence level = 1.84489

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0.0833333

Slope 0.0382372 0.0404091

Power Not applicable 1

Analysis of Deviance Table

Model Log (likelihood)

Number of

parameters Deviance

Test degrees of 

freedom (d.f.) p-value

Full model −24.9756 6

 0

 0.1

 0.2

 0.3

 0.4

 0.5

 0.6

 0.7

 0.8

 0 2 4 6 8 10 12 14

Fraction 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

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Fitted model −27.0654 1 4.17949 5 0.5239

Reduced model −33.7401 1 17.529 5 0.003598

 1

2

 3 

4 

 5 

a 6 

 7 

8 

 9 

AIC: = 56.1307

Goodness-of-Fit Table

Dose Est. prob.a 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

Estimated probability

Chi^2 = 2.79 d.f. = 5 p-value = 0.7325

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1 Table D-4. Model predictions for convulsions in female F344 rats exposed to 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

RDX by gavage for 90 days (Crouse et al., 2006); BMR= 5% ER

Modela

Goodness of fit BMD5Pct

(mg/kg-d)

BMDL5Pct

p-value AIC (mg/kg-d) Basis for model selection

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

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0.00, 

−1.29, −0.46, −0.12, 0.87, and 0.41, respectively. The BMD10 and BMDL10 for the selected model were 2.76 and 

1.84 mg/kg-day, respectively. b

The Multistage 4° model may appear equivalent to the Multistage 3° model; however, differences exist in digits 

not displayed in the table.

BMR = 5% ER; dose shown in mg/kg-day.

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 

(Crouse et al., 2006). 

 0

 0.1

 0.2

 0.3

 0.4

 0.5

 0.6

 0.7

 0.8

 0 2 4 6 8 10 12 14

Fraction 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

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

C-7 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Quantal Linear Model using Weibull Model (Version: 2.16; Date: 2/28/2013) 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

The form of the probability function is: P[response] = background + 

(1-background)*[1-EXP(-slope*dose)]

Benchmark Dose Computation

BMR = 5% ER

BMD = 1.34145

BMDL at the 95% confidence level = 0.89816

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0.0833333

Slope 0.0382372 0.0404091

Power Not applicable 1

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.7325

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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Table D-5. Model predictions for convulsions in female F344 rats exposed to 

2

3 

4 

5 

6 

7 

8 

9 

10

11

RDX by gavage for 90 days (Crouse et al., 2006); BMR= 1% ER

Modela

Goodness of fit BMD1Pct

(mg/kg-d)

BMDL1Pct

p-value AIC (mg/kg-d) Basis for model selection

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 5° 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

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0.00, 

−0.67, 0.14, 0.11, 0.64, and −0.51, respectively. The BMD10 and BMDL10 for the selected model were 5.98 and 

2.47 mg/kg-day, respectively. 

BMR= 1% ER; dose shown in mg/kg-day.

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). 

 0

 0.1

 0.2

 0.3

 0.4

 0.5

 0.6

 0.7

 0.8

 0 2 4 6 8 10 12 14

Fraction 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

BMDL BMD

 

Multistage

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

C-9 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

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% ER

BMD = 1.98616

BMDL at the 95% confidence level = 0.235433

1 

2 

3 

4 

5 

6 

7 

8 

 9 

10

11

12

13

14

15

16

17

18

Parameter Estimates

Variable Estimate Default initial parameter values

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 parameters Deviance Test d.f. 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. prob. 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 = 0.8854

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

C-10 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Table D-6. Model predictions for convulsions in male F344 rats exposed to 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

RDX by gavage for 90 days (Crouse et al., 2006); BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

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

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0.00, 

−0.54, −0.82, −0.40, 1.66, and −0.61, 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.

BMR = 10% ER; dose shown in mg/kg-day.

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). 

 0

 0.2

 0.4

 0.6

 0.8

 1

 0 2 4 6 8 10 12 14

Fraction 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

BMDL BMD

 

Multistage

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

C-11 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 

2 

3 

4

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

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% ER

BMD = 6.17392

BMDL at the 95% confidence level = 3.94501

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0

Beta(1) 0 0

Beta(2) 0 0.00691555

Beta(3) 0.000447707 0

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.5153

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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

Table D-7. Model predictions for convulsions in male F344 rats exposed to 

RDX by gavage for 90 days (Crouse et al., 2006); BMR = 5% ER

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

Modela

Goodness of fit BMD5Pct

(mg/kg-d)

BMDL5Pct

p-value AIC (mg/kg-d) Basis for model selection

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

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 

0.000, −0.54, −0.82, −0.40, 1.66, and −0.61, respectively. The BMD10 and BMDL10 for the selected model were 

6.17 and 3.95 mg/kg-day, respectively. 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.

BMR = 5% ER; dose shown in mg/kg-day.

 0

 0.2

 0.4

 0.6

 0.8

 1

 0 2 4 6 8 10 12 14

Fraction 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

BMDL BMD

 

Multistage

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). 

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

C-13 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 

2 

3 

4

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

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% ER

BMD = 4.85686

BMDL at the 95% confidence level = 2.19244 

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0

Beta(1) 0 0

Beta(2) 0 0.00691555

Beta(3) 0.000447707 0

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.5153

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

C-14 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Table D-8. Model predictions for convulsions in male F344 rats exposed to 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

RDX by gavage for 90 days (Crouse et al., 2006); BMR = 1% ER

Modela

Goodness of fit BMD1Pct

(mg/kg-d)

BMDL1Pct

p-value AIC (mg/kg-d) Basis for model selection

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

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0.00, 

−0.92, −1.26, −0.65, 1.76, and 0.11, respectively. The BMD10 and BMDL10 for the selected model were 4.54 and 

2.95 mg/kg-day, respectively. b

The Multistage 5° model may appear equivalent to the Multistage 4° model; however, differences exist in digits 

not displayed in the table.

BMR = 1% ER; dose shown in mg/kg-day.

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). 

 0

 0.2

 0.4

 0.6

 0.8

 1

 0 2 4 6 8 10 12 14

Fraction 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

BMDL BMD

 

Multistage

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

C-15 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Multistage Model (Version: 3.3; Date: 02/28/2013) 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

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% ER

BMD = 1.40125

BMDL at the 95% confidence level = 0.363499

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0

Beta(1) 0 0

Beta(2) 0.00511858 0.00691555

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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

AIC: = 50.3345

Goodness-of-Fit Table

Dose Est. prob. 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 = 0.3069

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

C-16 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Table D-9. Model predictions for convulsions in male and female F344 rats 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

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

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0.00, 

−0.69, −0.25, −0.06, 1.62, and −1.08, respectively. b

For the Multistage 4° and 5° models, the b4 and b5 coefficient estimates were 0 (boundary of parameters space). 

The models in this row reduced to the Multistage 3° model.

BMR = 10% ER; dose shown in mg/kg-day.

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). 

 0

 0.2

 0.4

 0.6

 0.8

 0 2 4 6 8 10 12 14

Fraction 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

BMDL BMD

 

Multistage

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

C-17 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 

2

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

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% ER

BMD = 6.60247

BMDL at the 95% confidence level = 4.59346

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0

Beta(1) 0 0.00163806

Beta(2) 0 0.00485933

Beta(3) 0.000366065 0

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.5021

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

C-18 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Table D-10. Model predictions for convulsions in male and female F344 rats 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 5% ER

Modela

Goodness of fit BMD5Pct

(mg/kg-d)

BMDL5Pct

p-value AIC (mg/kg-d) Basis for model selection

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

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0.00, 

−0.69, −0.25, −0.06, 1.62, and −1.08, respectively. The BMD10 and BMDL10 for the selected model were 6.60 and 

4.59 mg/kg-day, respectively. b

For the Multistage 5° model, the b5 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Multistage 4° model.

BMR = 5% ER; dose shown in mg/kg-day.

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 90 days (Crouse et al., 2006). 

 0

 0.2

 0.4

 0.6

 0.8

 0 2 4 6 8 10 12 14

Fraction 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

BMDL BMD

 

Multistage

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

C-19 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

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% ER

BMD = 5.19399 

BMDL at the 95% confidence level = 2.65815

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0

Beta(1) 0 0.00163806

Beta(2) 0 0.00485933

Beta(3) 0.000366065 0

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.5021

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

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1 Table D-11. Model predictions for convulsions in male and female F344 rats 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

exposed to RDX by gavage for 90 days (Crouse et al., 2006); BMR = 1% ER

Modela

Goodness of fit BMD1Pct

(mg/kg-d)

BMDL1Pct

p-value AIC (mg/kg-d) Basis for model selection

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 

multistage models, the 

Multistage 3° model was selected 

based on lowest AIC. From 

among the multistage 3° and 

non-multistage models, the 

multistage 3° 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

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0.00, 

−0.69, −0.25, −0.06, 1.62, and −1.08, respectively. The BMD10 and BMDL10 for the selected model were 6.60 and 

4.59 mg/kg-day, respectively. 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.

Figure D-9. Plot of incidence rate by dose, with the fitted curve of the 

multistage 3° model, for convulsions in male and female F344 rats exposed to 

RDX by gavage for 90 days (Crouse et al., 2006). BMR = 1% ER; dose shown in 

mg/kg-day.

 0

 0.2

 0.4

 0.6

 0.8

 0 2 4 6 8 10 12 14

Fraction 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

BMDL BMD

 

Multistage

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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Multistage Model. (Version: 3.3; Date: 02/28/2013)

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

2

2

2

1

2

3

This document is a draft for review purposes only and doe

C-22

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 = 3.01676

BMDL at the 95% confidence level = 0.569284

Parameter Estimates

Variable Estimate Default Initial 

Parameter Values

Background 0 0

Beta(1) 0 0.00163806

Beta(2) 0 0.00485933

Beta(3) 0.000366065 0

Analysis of Deviance Table

Model Log(likelihood

)

# Param's Deviance Test d.f. 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 <.0001

AIC: = 100.913

Goodness of Fit Table

Dose Est. Prob. Expected Observed Size Scaled Resid

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 = 0.5021

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1 Table D-12. Model predictions for convulsions in female F344 rats exposed to 

2 

3 

4 a 5 

6 b 7 

8 

9 

10

11

12

13

14

RDX by gavage on gestational days (GDs) 6−19 (Cholakis et al., 1980); 

BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

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

Selected model in bold; scaled residuals for selected model for doses 0, 0.2, 2, and 20 mg/kg-day were 0.00, 

−0.52, −1.03, and 0.49, respectively.

The Multistage 3° model may appear equivalent to the Multistage 2° model; however, differences exist in digits 

not displayed in the table.

BMR = 10% ER; dose shown in mg/kg-day.

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 GDs 6−19 (Cholakis et al., 1980). 

 0

 0.2

 0.4

 0.6

 0.8

 0 5 10 15 20

Fraction 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

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1 Quantal Linear Model using Weibull Model (Version: 2.16; Date: 2/28/2013) 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

The form of the probability function is: P[response] = background + 

(1-background)*[1-EXP(-slope*dose)]

Benchmark Dose Computation

BMR = 10% ER

BMD = 1.87886

BMDL at the 95% confidence level = 1.28909

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0.0384615

Slope 0.056077 0.0588587

Power Not applicable 1

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.6686

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

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Table D-13. Model predictions for convulsions in female F344 rats exposed to 

RDX by gavage on GDs 6−19 (Cholakis et al., 1980); BMR = 5% ER

1 

2 

9 

10

11

12

13

3 

4 

5 

6 

7 

8 

Modela

Goodness of fit BMD5Pct

(mg/kg-d)

BMDL5Pct

p-value AIC (mg/kg-d) Basis for model selection

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

a

Selected model in bold; scaled residuals for selected model for doses 0, 0.2, 2, and 20 mg/kg-day were 0.00, 

−0.52, −1.03, and 0.49, respectively. The BMD10 and BMDL10 for the selected model were 1.88 and 

1.29 mg/kg-day, respectively. b

The Multistage 3° model may appear equivalent to the Multistage 2° model; however, differences exist in digits 

not displayed in the table.

Quantal Linear Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B

 0.6

 0.8

Affected

Quantal Linear

BMR = 5% ER; dose shown in mg/kg-day.

 0

 0.2

 0.4

 0 5 10 15 20

Fraction 

dose

15:24 05/23 2014

BMDL BMD

 

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 GDs 6−19 (Cholakis et al., 1980). 

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

C-25 DRAFT―DO NOT CITE OR QUOTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

 1 

2

3 

4

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

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 = 5% ER

BMD = 0.914694

BMDL at the 95% confidence level = 0.627577

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0.0384615

Slope 0.056077 0.0588587

Power Not applicable 1

Analysis of Deviance Table

Model Log likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.6686

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

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1 Table D-14. Model predictions for convulsions in female F344 rats exposed to 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

RDX by gavage on GDs 6−19 (Cholakis et al., 1980); BMR = 1% ER

Modela

Goodness of fit BMD1Pct

(mg/kg-d)

BMDL1Pct

p-value AIC (mg/kg-d) Basis for model selection

Gamma 0.989 42.003 0.866 0.149 The Quantal-Linear model is 

selected based on lowest BMDL 

(BMDLs differed by more than 

threefold). 

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

a

Selected model in bold; scaled residuals for selected model for doses 0, 0.2, 2, and 20 mg/kg-day were 0.00, 

−0.52, −1.03, and 0.49, respectively. The BMD10 and BMDL10 for the selected model were 1.88 and 

1.29 mg/kg-day, respectively. b

The Multistage 3° model may appear equivalent to the Multistage 2° model; however, differences exist in digits 

not displayed in the table.

BMR = 1% ER; dose shown in mg/kg-day.

Figure D-12. 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 GDs 6−19 (Cholakis et al., 1980). 

 0

 0.2

 0.4

 0.6

 0.8

 0 5 10 15 20

Fraction 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

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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Quantal Linear Model using Weibull Model (Version: 2.16; Date: 2/28/2013) 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

The form of the probability function is: P[response] = background + 

(1-background)*[1-EXP(-slope*dose)]

Benchmark Dose Computation

BMR = 1% ER

BMD = 0.179224

BMDL at the 95% confidence level = 0.122966

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0.0384615

Slope 0.056077 0.0588587

Power Not applicable 1

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.6686

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Table D-15. Model predictions for combined incidence of convulsion and 

mortality in male and female F344 rats exposed to RDX by gavage for 90 days

(Crouse et al., 2006); BMR = 1% ER

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

Modela

Goodness of fit BMD1Pct

(mg/kg-d)

BMDL1Pct

p-value AIC (mg/kg-d) Basis for model selection

Gamma 0.245 99.260 3.73 2.10 The log-logistic and quantal-linear 

models did not achieve an 

adequate fit (goodness-of-fit 

p-value <0.10). The multistage 2° 

model was excluded from model 

selection because the residual in 

the lowest dose group, near the 

BMD, was above 1.5 in absolute 

value. Of the remaining models, 

the multistage 3° model was 

selected based on lowest BMDL 

(BMDLs differed by more than 

threefold). 

Dichotomous-Hill 0.436 98.317 5.22 3.04

Logistic 0.0859 102.17 1.81 0.846

LogLogistic 0.305 98.593 3.70 2.20

Probit 0.101 101.85 2.16 0.853

LogProbit 0.316 98.465 4.22 2.75

Weibull 0.152 101.16 2.45 1.24

Multistage 4°b 0.229 99.182 2.56 0.486

Multistage 3°c 0.229 99.182 2.56 0.486

Multistage 2° 0.165 102.01 1.22 0.470

Quantal-Linear 0.0052 113.90 0.144 0.113

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg-day were 0, 

−0.88, −0.14, −0.01, 1.92, −1.55, respectively. b

The Multistage 4° model may appear equivalent to the Multistage 3° model; however, differences exist in digits 

not displayed in the table.

c

The Multistage 3° model may appear equivalent to the Multistage 4° model; however, differences exist in digits 

not displayed in the table.

BMR = 1% ER; dose shown in mg/kg-day.

 0

 0.2

 0.4

 0.6

 0.8

 1

 0 2 4 6 8 10 12 14

Fraction Affected

dose

Multistage Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

13:21 01/06 2016

BMDL BMD

 

Multistage

Figure D-13. Plot of incidence rate by dose with fitted curve for Multistage 3° 

model for Model predictions for combined incidence of convulsion and 

mortality in male and female F344 rats exposed to RDX by gavage for 90 days

(Crouse et al., 2006). 

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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

Multistage Model. (Version: 3.4; Date: 05/02/2014) 

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 = 2.56012

BMDL at the 95% confidence level = 0.486284

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0

Beta(1) 0 0.0272036

Beta(2) 0 0.00626035

Beta(3) 0.000598962 0

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. p-value

Full model −44.71 6

Fitted model −48.59 1 7.76102 5 0.17

Reduced model −9.88 1 70.3406 5 <0.0001

AIC: = 99.1817

Goodness-of-Fit Table

Dose Est. Prob. Expected Observed Size Scaled residuals

0 0 0 0 20 0

4 0.0376 0.752 0 20 −0.88

8 0.2641 5.282 5 20 −0.14

10 0.4506 9.012 9 20 −0.01

12 0.6448 12.896 17 20 1.92

15 0.8675 17.351 15 20 −1.55

Chi^2 = 6.88 d.f. = 5 p-value = 0.2294

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1 Male Reproductive Effects

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

Table D-16 (and Figure D-14) presents the BMD modeling results for incidence of testicular 

degeneration for male B6C3F1 mice based on data from Lish et al. (1984), using a BMR of 10% ER. 

Table D-16. Model predictions for testicular degeneration in male B6C3F1

mice exposed to RDX by diet for 24 months (Lish et al., 1984); BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

Gammab

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-ay were 0.00, 

0.32, −0.61, 0.43, and −0.17, respectively. b

For the Gamma and Weibull models, the power parameter estimates were 1 (boundary of parameter space). The 

models in this row are 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. The models in this row may appear equivalent to the 

Gamma model; however, differences exist in digits not displayed in the table.

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

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1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

BMR = 10% ER; dose shown in mg/kg-day.

Figure D-14. Plot of incidence rate by dose, with fitted curve for selected 

model, for testicular degeneration in male B6C3F1 mice exposed to RDX by 

diet for 24 months (Lish et al., 1984). 

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% ER

BMD = 55.9784

BMDL at the 95% confidence level = 16.2787

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0 0

Intercept −2.0054E+00 −1.9976E+00

Slope 0.179828 0.172286

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0 20 40 60 80 100

Fraction 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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Analysis of Deviance Table

2 

3 

4 

5 

6 

7 

Model Log (likelihood) Number of parameters Deviance Test d.f. 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

AIC: = 97.5635

Goodness-of-Fit Table

Dose Est. prob. Expected Observed Size Scaled 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 = 0.8759

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

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1 Kidney Effects

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

Table D-17 (and Figure D-15) presents the BMD model results for incidence of suppurative 

inflammation of the prostate in male F344 rats based on data from Levine et al. (1983b), using a 

BMR of 10% ER.

Table D-17. Model predictions for prostate suppurative inflammation in male 

F344 rats exposed to RDX by diet for 24 months (Levine et al., 1983b); 

BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

Gammab

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

Weibullg 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-day were 

−0.289, 0.172, 0.846, −1.298, and 0.819, respectively. b

The Gamma model had a power parameter estimate of 1 (boundary of parameter space). The Multistage 2°, 3°, 

and 4° models had b2, b3, and b4 coefficients of 0 (boundary of parameter space). The models in this row are 

equivalent to the Quantal-Linear model. c

The Weibull model may appear equivalent to the Quantal-Linear model; however, differences exist in digits not 

displayed in the table.

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

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Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

BMR = 10% ER; dose shown in mg/kg-day.

Figure D-15. 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., 1983b). 

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% ER

BMD = 1.67454

BMDL at the 95% confidence level = 0.468688

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0.0452202 0.037037

Intercept −1.4970E+00 −1.3564E+00

Slope 0.417872 0.36341

 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 40

Fraction 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

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1 Analysis of Deviance Table

2 

3 

4 

5 

6 

7

8 

9 

10

11

12

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.2035

D.1.3. Mortality: Dose-Response Analysis and BMD Modeling Documentation

This appendix also presents a quantitative dose-response analysis of mortality incidence 

from studies identified in Section 2.1.6 (see Table D-18). 

Table D-18. Mortality data selected for dose-response modeling for RDX

Reference Species/sex Dose

Incidence / Total (%) or

Mean ± SD (no. of animals)

Lish et al. (1984)

(mortality at 11 wks)

Male B6C3F1

mouse

0 mg/kg-d 

1.5

7 

35

175/100

1 / 85 (1%)

0 / 85 (0%)

0 / 85 (0%)

0 / 85 (0%)

30 / 85 (35%)

Lish et al. (1984)

(mortality at 11 wks)

Female 

B6C3F1 mouse

0 mg/kg-d 

1.5

7 

35

175/100

0 / 85 (1%)

0 / 85 (0%)

0 / 85 (0%)

0 / 85 (0%)

36 / 85 (42%)

Levine et al. (1981b)a Female F344 

rat

0 mg/kg-d 

10

30

100

300

600

0 / 30 (0%)

1 / 10 (10%)

0 / 10 (0%)

5 / 10 (50%)

10 / 10 (100%)

10 / 10 (100%)

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Reference Species/sex Dose

Incidence / Total (%) or

Mean ± SD (no. of animals)

Male F344 rat 0 mg/kg-d

10

30

100

300

600

0 / 30 (0%)

0 / 10 (0%)

0 / 10 (0%)

8 / 10 (80%)

10 / 10 (100%)

10 / 10 (100%)

Male and 

female F344 

rat, combined

0 mg/kg-d

10

30

100

300

600

0 / 60 (0%)

1 / 20 (5%)

0 / 20 (0%)

13 / 20 (65%)

20 / 20 (100%)

20 / 20 (100%)

von Oettingen et al. 

(1949)

Rats, 

sex/strain not 

specified

0 mg/kg-d

15

25

50

0 / 20 (0%)

0 / 19 (0%)b

8 / 20 (40%)

8 / 20 (40%)

Cholakis et al. (1980)

(2-generation study)

Female CD rat 0 mg/kg-d

5

16

50

0 / 22 (0%)

0 / 22 (0%)

0 / 22 (0%)

6 / 22 (27%)

Levine et al. (1983b)

(mortality at 13 wks)

Male F344 rat 0 mg/kg-d

0.3

1.5

8

40

0 / 75 (0%)

0 / 75 (0%)

0 / 75 (0%)

0 / 75 (0%)

0 / 75 (0%)

Female F344 

rat

0 mg/kg-d

0.3

1.5

8

40

0 / 75 (0%)

0 / 75 (0%)

0 / 75 (0%)

0 / 75 (0%)

0 / 75 (0%)

Cholakis et al. (1980)

(13-week study)

Male F344 rat 0 mg/kg-d

10

14

20

28

40

0 / 10 (0%)

0 / 10 (0%)

0 / 10 (0%)

0 / 10 (0%)

0 / 10 (0%)

0 / 10 (0%)

Female F344 

rat

0 mg/kg-d

10

14

20

28

40

0 / 9 (0%)c

0 / 10 (0%)

0 / 10 (0%)

0 / 10 (0%)

0 / 10 (0%)

0 / 10 (0%)

(Crouse et al. (2006)) Female F344 

rat

0 mg/kg-d

4

8

0 / 10 (0%)

0 / 10 (0%)

1 / 10 (20%)

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Reference Species/sex Dose

Incidence / Total (%) or

Mean ± SD (no. of animals)

10

12

15

2 / 10 (20%)

5 / 10 (50%)

4 / 10 (40%)

Male F344 rat 0 mg/kg-d

4 

8 

10

12

15

0 / 10 (0%)

0 / 10 (0%)

1 / 10 (10%)

3 / 10 (30%)

2 / 10 (20%)

3 / 10 (30%)

Male and 

female F344 

rat, combined

0 mg/kg-d

4 

8 

10

12

15

0 / 20 (0%)

0 / 20 (0%)

2 / 20 (10%)

5 / 20 (25%)

7 / 20 (35%)

7 / 20 (35%)

Cholakis et al. (1980)

(gestational exposure)

Female F344 

rats

(gestational 

exposure)

0 mg/kg-d

0.2

2 

20

0 / 24 (0%)

0 / 24 (0%)

0 / 24 (0%)

5 / 24 (21%)

Angerhofer et al. (1986) Female SD 

rate

(gestational 

exposure)

0 mg/kg-d

2 

6 

20

0 / 39 (0%)

1 / 40 (3%)

1 / 40 (3%)

16 / 51 (31%)

Cholakis et al. (1980) Female New 

Zealand white 

rabbit 

(gestational 

exposure)

0 mg/kg-d

0.2

2 

20

0 / 11 (0%)

0 / 11 (0%)

0 / 11 (0%)

0 / 12 (0%)

a

For Levine et al. (1981a) and 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 each of these endpoints prior to modeling. b

For von Oettingen et al. (1949), one mortality was reported in the 15 mg/kg-d dose group. However, this 

mortality was most likely not related to RDX, so the animal that died was excluded. 

c

For Cholakis et al. (1980), one accidental death was reported in the 0 mg/kg-d dose group. The animal that died 

was excluded. 

1 

2 

3 

4 

5 

6 

7 

8 

Tables D-19 to D-22 present the BMD modeling results for incidence of mortality from 

Crouse et al. (2006), von Oettingen et al. (1949), Levine et al. (1983b), and Angerhofer et al. (1986). 

The following datasets were not modeled because each had either no response or a positive 

response only in the highest dose group: 11-week mortality from Lish et al. (1984), both male (one 

death in control group) and female; 13-week mortality data from Levine et al. (1983b), both male 

and female; mortality in female CD rats and male and female F344 rats from Cholakis et al. (1980); 

and mortality in female F344 rats during gestational exposure from Cholakis et al. (1980). 

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1 Table D-19. Model predictions for combined mortality in male and female 

2 

3 

 4 

5 

6 

7 

8 

9 

F344 rats exposed to RDX by diet for 13 weeks (Levine et al., 1981); BMR = 1% 

ER 

Modela

Goodness of fit

BMD1Pct

(mg/kg-day)

BMDL1Pct

p-value AIC (mg/kg-day) Basis for model selection

Gamma 0.401 41.100 49.8 12.7 The quantal-linear model was 

excluded because the fit has a 

residual below -2 at 4 mg/kg-d. 

The multistage 4° model was 

selected as the representative 

multistage model based on 

lowest AIC. From among the 

multistage 4° and non-multistage 

models, the multistage 4° model 

was selected based on lowest 

BMDL (BMDLs differed by more 

than threefold). 

Logistic 0.346 41.429 18.3 8.25

LogLogistic 0.257 43.098 73.2 16.9

Probit 0.328 41.727 15.0 6.82

LogProbit 0.257 43.098 58.6 19.5

Weibull 0.257 43.101 56.6b 6.51b

Multistage 2° 0.424 42.942 7.72 2.01

Quantal-Linear 0.139 50.257 1.12 0.818

Multistage 3° 0.503 41.520 7.71 2.08

Multistage 4° 0.535 40.935 7.85 2.15

Multistage 5° 0.371 42.928 7.86 2.15

a

Selected model in bold; scaled residuals for selected model for doses 0, 10, 30, 100, 300, and 600 mg/kg/day 

were 0.00, 1.48, -0.97, 0.05, 0.00, and 0.00, respectively. The BMD10 and BMDL10 estimates for the selected 

model were 47.2 and 22.2 mg/kg-d, respectively. b

The parameter convergence parameter was increased to 2e-08 to obtain convergence.

Figure D-16. Plot of incidence rate by dose, with the fitted curve of the 

multistage 2° model, for combined mortality in male and female F344 rats 

exposed to RDX by diet for 13 weeks (Levine et al., 1981); BMR = 1% ER.

 0

 0.2

 0.4

 0.6

 0.8

 1

 0 100 200 300 400 500 600

Fraction Affected

dose

Multistage Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

13:54 02/27 2014

BMDL BMD

 

Multistage

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1 Multistage Model. (Version: 3.3; Date: 02/28/2013)

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

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 = 7.85287

BMDL at the 95% confidence level = 2.15059

Parameter Estimates

Variable Estimate Default Initial 

Parameter Values

Background 0 0

Beta(1) 0.00127544 1.9710E+17

Beta(2) 0 0

Beta(3) 0 0

Beta(4) 9.0721E-09 0

Analysis of Deviance Table

Model Log(likelihood

)

# Param's Deviance Test d.f. p-value

Full model -16.9192 6

Fitted model -18.4677 2 3.09685 4 0.5418

Reduced 

model

-102.298 1 170.758 5 <.0001

AIC: = 40.9353

Goodness of Fit Table

Dose Est. Prob. Expected Observed Size Scaled Resid

0 0 0 0 60 0

10 0.0128 0.255 1 20 1.484

30 0.0446 0.892 0 20 -0.966

100 0.6447 12.894 13 20 0.05

300 1 20 20 20 0

600 1 20 20 20 0

Chi^2 = 3.14 d.f = 4 P-value = 0.5352

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1 Table D-20. Summary of BMD modeling results for model predictions for 

2 

3 

4 

5 

 6 

7 

8 

9 

10

11

mortality (number found dead) in rats exposed to RDX in the diet for 13 

weeks (von Oettingen et al., 1949)

Modela Goodness of fit BMD1Pct

(mg/kg-day)

BMDL1Pct

(mg/kg-day)

Basis for model selection

p-value AIC

Gamma 0.0341 66.088 3.14 0.648 All the models besides 

dichotomous-Hill had goodnessof-fit p-values less than 0.10 and 

thus did not provide an adequate 

fit to the data. For the 

dichotomous-Hill model, the 

slope parameter acheived the 

BMDS internal upper bound (18), 

so the results from this model 

were not reliable. No model was 

selected. 

Dichotomous-Hill 0.984 57.888 17.2 10.9

Logistic 0.0044 70.074 3.40 2.08

LogLogistic 0.0397 65.853 3.39 0.529

Probit 0.0056 69.283 3.28 1.94

LogProbit 0.0426 65.464 5.67 0.409

Weibull 0.0349 66.233 2.30 0.641

Multistage 3°a 0.0351 66.517 1.22 0.628

Multistage 2°b 0.0351 66.517 1.22 0.628

Quantal-Linear 0.0995 64.639 0.919 0.623

a The Multistage 3° model may appear equivalent to the Multistage 2° model, however differences exist in digits 

not displayed in the table.

b The Multistage 2° model may appear equivalent to the Multistage 3° model, however differences exist in digits 

not displayed in the table.

Figure D-17. Plot of incidence rate by dose with fitted curve for DichotomousHill model for Model predictions for mortality (number found dead) in rats 

exposed to RDX in the diet for 13 weeks (von Oettingen et al., 1949); dose 

shown in mg/kg-day.

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 0

 0.1

 0.2

 0.3

 0.4

 0.5

 0.6

 0.7

 0 10 20 30 40 50

Fraction Affected

dose

Dichotomous-Hill Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

14:56 02/04 2016

BMDL BMD

 

Dichotomous-Hill

OTE
Supplemental Information―Hexahydro-1,3,5-trinitro-1,3,5-triazine

1 Table D-21. Model predictions for combined mortality (number found dead) 

2 

3 

in male and female F344 rats exposed to RDX by gavage for 90 days (Crouse et 

al., 2006); BMR= 1% ER 

Modela Goodness of fit BMD1Pct

(mg/kg-day)

BMDL1Pct

(mg/kg-day)

Basis for model selection

p-value AIC

Gamma 0.794 93.263 3.46 0.840 The multistage 2° model was 

selected as the representative 

multistage model based on 

lowest AIC. From among the 

multistage 2° and non-multistage 

models, the multistage 2° model 

was selected based on lowest 

BMDL (BMDLs differed by more 

than threefold). 

Logistic 0.474 95.709 2.11 1.11

LogLogistic 0.794 93.332 3.17 0.872

Probit 0.574 94.797 2.40 1.07

LogProbit 0.854 92.832 3.96 1.48

Weibull 0.743 93.698 2.76 0.641

Multistage 2° 0.858 91.926 2.11 0.463

Quantal-Linear 0.535 95.345 0.405 0.288

Multistage 5°b 0.731 93.851 2.42 0.433

Multistage 4°c 0.731 93.851 2.42 0.433

Multistage 3° 0.731 93.851 2.42 0.439

Dichotomous-Hill 0.998 93.343 5.96 1.95

a

Selected model in bold; scaled residuals for selected model for doses 0, 4, 8, 10, 12, and 15 mg/kg/day were 

0.00, -0.86, -0.46, 0.53, 0.72, and 0.45, respectively. The BMD10 and BMDL10 for the selected model were 6.82 

and 4.41 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.

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1 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

Figure D-18. Plot of incidence rate by dose, with the fitted curve of the 

multistage 2° model, for mortality in male and female F344 rats exposed to 

RDX by gavage for 90 days (Crouse et al., 2006). BMR = 1% ER; 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...)]

 0

 0.1

 0.2

 0.3

 0.4

 0.5

 0.6

 0 2 4 6 8 10 12 14

Fraction Affected

dose

Multistage Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

14:12 02/27 2014

BMDL BMD

 

Multistage

Benchmark Dose Computation.

BMR = 1% Extra risk

BMD = 2.10625

BMDL at the 95% confidence level = 0.462994

Parameter Estimates

Variable Estimate Default Initial 

Parameter Values

Background 0 0

Beta(1) 0 0.0134587

Beta(2) 0.00226548 0.00141278

Analysis of Deviance Table

Model Log(likelihood

)

# Param's Deviance Test d.f. p-value

Full model -43.6462 6

Fitted model -44.963 1 2.63354 5 0.7563

Reduced 

model

-55.6472 1 24.0019 5 0.0002169

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1 AIC: = 91.926

2 

3

4 

5 

6 

7 

8 

9 

10

Goodness of Fit Table

Dose Est. Prob. Expected Observed Size Scaled Resid

0 0 0 0 20 0

4 0.0356 0.712 0 20 -0.859

8 0.135 2.699 2 20 -0.458

10 0.2027 4.054 5 20 0.526

12 0.2784 5.567 7 20 0.715

15 0.3993 7.987 7 20 -0.451

Chi^2 = 1.94 d.f = 5 P-value = 0.8576

Table D-22. Model predictions for mortality in female Sprague-Dawley rats 

exposed to RDX by gavage on gestation days 6–15 (Angerhofer, 1986); BMR = 

1% ER 

Modela Goodness of fit BMD1Pct

(mg/kg-day)

BMDL1Pct

(mg/kg-day)

Basis for model selection

p-value AIC

Gamma 0.314 89.496 5.15 0.538 The multistage 3° model was 

selected based on lowest AIC. 

From among the multistage 3° 

and non-multistage models, the 

multistage 3° model was selected 

based on lowest BMDL (BMDLs 

differed by more than threefold). 

Logistic 0.667 87.213 3.88 2.16

LogLogistic 0.312 89.473 4.88 0.560

Probit 0.643 87.196 3.37 1.87

LogProbit 0.319 89.522 5.58 0.885

Weibull 0.309 89.458 4.62 0.541

Quantal-Linear 0.450 87.502 0.652 0.452

Multistage 3° 0.655 86.906 1.68 0.588

Multistage 2° 0.554 87.291 1.78 0.555

a

Selected model in bold; scaled residuals for selected model for doses 0, 2, 6, and 20 mg/kg-d were 0.00, 0.76, -

0.52, and 0.04, respectively. The BMD10 and BMDL10 for the selected model were 10.9 and 6.09 mg/kg-d, 

respectively. 

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1 

2 

3 

4

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

Figure D-19. Plot of incidence rate by dose, with the fitted curve of the 

multistage 3° model, for mortality in female Sprague-Dawley rats exposed to 

RDX by gavage on gestation days 6–15 (Angerhofer, 1986); BMR = 1% ER.

 0

 0.1

 0.2

 0.3

 0.4

 0.5

 0 5 10 15 20

Fraction Affected

dose

Multistage Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM

10:18 05/22 2014

BMDL BMD

 

Multistage

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 = 1.68097

BMDL at the 95% confidence level = 0.587568

Parameter Estimates

Variable Estimate Default Initial 

Parameter Values

Background 0 0.00807857

Beta(1) 0.00588873 0.00216407

Beta(2) 0 0

Beta(3) 0.0000319123 0.0000406218

Analysis of Deviance Table

Model Log(likelihood

)

# Param's Deviance Test d.f. p-value

Full model -41.0771 4

Fitted model -41.4531 2 0.752152 2 0.6866

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Reduced 

model

-57.4292 1 32.7043 3 <.0001

1 

2 

3 

4

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

AIC: = 86.9063

Goodness of Fit Table

Dose Est. Prob. Expected Observed Size Scaled Resid

0 0 0 0 39 0

2 0.012 0.478 1 40 0.759

6 0.0413 1.654 1 40 -0.519

20 0.3114 15.881 16 51 0.036

Chi^2 = 0.85 d.f = 2 P-value = 0.6549

D.2. BENCHMARK DOSE MODELING SUMMARY FOR CANCER ENDPOINTS

The cancer endpoints in the mouse that were selected for dose-response modeling are 

presented in Table D-23. For each endpoint, the doses and tumor incidence data used for the 

modeling are presented.

Table D-23. 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 B6C3F1

mouse

0

1.5

7 

35

107

1/67 (1%)a

4/62 (6%)

5/63 (8%)

10 /64 (16%)

4/31 (13%)

Alveolar/bronchiolar adenomas or 

carcinomas

Lish et al. (1984)

Female B6C3F1

mouse

0

1.5

7 

35

107

7/65 (11%)

3/62 (5%)

8/64 (13%)

12/64 (19%)

7/31 (23%)

a

For female mouse hepatocellular tumors from Lish et al. (1984), tumor incidence and totals are those reported in 

the Pathology Working Group (PWG) reevaluation (Parker et al., 2006). 

D.2.1. Evaluation of Model Fit and Model Selection for Mouse Tumor Data

First, to determine whether a time-to-tumor analysis was warranted, the survival curves 

were compared across dose groups for female mice in Lish et al. (1984) in the study to determine 

whether time of death should be incorporated in the dose-response analysis of tumors. A log-rank 

test on the Kaplan-Meier survival curves per dose was used to do the comparison, excluding 

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1 animals that died prior to week 11 when the dose was reduced in the high-dose group to 

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

100 mg/kg-day. The test yielded a nonsignificant result (p = 0.51), so a time-to-tumor analysis was 

not necessary for this study. 

Therefore, non-time-dependent dose-response analyses were conducted using standard 

BMDS models. For each tumor type, BMDS multistage-cancer models6 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 (χ2 p-value <0.057 indicates lack of fit). Other factors were used to assess model 

fit, including scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the 

vicinity of the BMR. The BMDL estimate 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” 

(i.e., differed by threefold or less), then the model selected was the one that yielded the lowest AIC 

value. If the BMDL estimates were not sufficiently close, then 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 

present if either the hepatocellular or alveolar/bronchiolar tumor (or both) was present, and not 

present otherwise. The two endpoints were assumed to be independent.

D.2.2. Modeling Results for Mouse Tumor Data

The BMD modeling results for mouse tumor data sets are provided in Tables D-24 to D-28

(and Figures D-20 to D-24). 

Mouse Tumor Data—BMD Modeling Results

Table D-24. Model predictions for combined alveolar/bronchiolar adenoma 

and carcinoma in female B6C3F1 mice exposed to RDX by diet for 24 months 

(Lish et al., 1984); BMR = 10% ER

Modela

Goodness of fit BMD10Pct 

(mg/kg-d)

BMDL10Pct 

p-value AIC (mg/kg-d) Basis for model selection

Multistage 1°b

Multistage 2°

Multistage 3°

Multistage 4°

0.417 218.68 52.8 27.7 All of the models reduced to the 

Multistage 1° model, so this model 

was selected. 

a

Selected model in bold. Scaled residuals for the selected model for doses 0, 1.5, 7, 35, and 107 mg/kg-day 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 row reduced to the Multistage 1° model.

6The coefficients of the multistage-cancer models were restricted to be nonnegative (beta values ≥0). 7A significance level of 0.05 from U.S. EPA (2012b) is used for selecting cancer models because the model 

family (multistage) is selected a priori. 

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

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1 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

22

BMR = 10% ER; dose shown in mg/kg-day.

Figure D-20. Plot of incidence rate by dose, with the fitted curve for the 

selected model, for combined alveolar/bronchiolar adenoma and carcinoma 

in female B6C3F1 mice exposed to RDX by diet for 24 months (Lish et al., 

1984). 

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% ER

BMD = 52.8078

BMDL at the 95% confidence level = 27.748

Benchmark dose upper bound (BMDU) at the 95% confidence level = 194.806

Taken together, (27.748, 194.806) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.00360387

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0.35

 0.4

 0.45

 0 20 40 60 80 100

Fraction 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

BMDL BMD

 

Multistage Cancer

Linear extrapolation

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0.093168 0.0998927

Beta(1) 0.00199517 0.00155773

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1 Analysis of Deviance Table

2

3 

4 

5 

6 

7 

8 

Model Log (likelihood) Number of parameters Deviance Test d.f. 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

AIC: = 218.682

Goodness-of-Fit Table

Dose Est. prob. 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 = 0.4168

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1 Table D-25. Model predictions for combined alveolar/bronchiolar adenoma 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

and carcinoma in female B6C3F1 mice exposed to RDX by diet for 24 months 

(Lish et al., 1984); BMR = 5% ER

Modela

Goodness of fit BMD5Pct

(mg/kg-d)

BMDL5Pct

p-value AIC (mg/kg-d) Basis for model selection

Multistage 1°b

Multistage 2°

Multistage 3°

Multistage 4°

0.417 218.68 25.7 13.5 All of the models reduced to the 

Multistage 1° model, so this model 

was selected.

a

Selected model in bold. Scaled residuals for the selected model for doses 0, 1.5, 7, 35, and 107 mg/kg-day were 

0.40, −0.40, −1.27, 0.50, 0.73, and −0.52, respectively. The BMD10 and BMDL10 for the selected model were 

52.8 and 27.7 mg/kg-day, respectively.

BMR = 5% ER; dose shown in mg/kg-day.

Figure D-21. Plot of incidence rate by dose, with fitted curve for selected 

model, for combined alveolar/bronchiolar adenoma and carcinoma in female 

B6C3F1 mice exposed to RDX by diet for 24 months (Lish et al., 1984). 

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0.35

 0.4

 0.45

 0 20 40 60 80 100

Fraction 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

BMDL BMD

 

Multistage Cancer

Linear extrapolation

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1 Multistage Cancer Model (Version: 1.10; Date: 02/28/2013) 

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% ER

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 initial parameter values

Background 0.093168 0.0998927

Beta(1) 0.00199517 0.00155773

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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

AIC: = 218.682

Goodness-of-Fit Table

Dose Est. prob. 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 = 0.4168

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1 Table D-26. Model predictions for combined hepatocellular adenoma and 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

carcinoma in female B6C3F1 mice exposed to RDX by diet for 24 months 

(Parker et al., 2006); BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

Multistage 1°b

Multistage 2°

Multistage 3°

Multistage 4°

0.160 164.06 64.2 32.6 All of the models reduced to the 

Multistage 1° model, so this model was 

selected.

a

Selected model in bold. Scaled residuals for the selected model for doses 0, 1.5, 7, 35, and 107 mg/kg-day were 

−1.37, 0.35, 0.54, 1.34, 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. 

BMR = 10% ER; dose shown in mg/kg-day.

Figure D-22. Plot of incidence rate by dose, with fitted curve for selected 

model, for combined hepatocellular adenoma and carcinoma in female B6C3F1

mice exposed to RDX by diet for 24 months (Parker et al., 2006). 

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0 20 40 60 80 100

Fraction 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

BMDL BMD

 

Multistage Cancer

Linear extrapolation

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1 Multistage Cancer Model (Version: 1.10; Date: 02/28/2013) 

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% ER

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 initial parameter values

Background 0.0520755 0.0658334

Beta(1) 0.00164105 0.000876864

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.16

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1 Table D-27. Model predictions for B6C3F1 female mouse combined 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

hepatocellular adenoma and carcinoma in mice exposed to RDX by diet for 

24 months (Parker et al., 2006); BMR = 5% ER

Modela

Goodness of fit BMD5Pct

(mg/kg-d)

BMDL5Pct

p-value AIC (mg/kg-d) Basis for model selection

Multistage 1°b

Multistage 2°

Multistage 3°

Multistage 4°

0.160 164.06 31.3 15.9 All of the models reduced to the 

Multistage 1° model, so this model was 

selected.

a

Selected model in bold; scaled residuals for selected model for doses 0, 1.5, 7, 35, and 107 mg/kg-day were −1.37, 

0.35, 0.54, 1.34, and −1.05, respectively. The BMD10 and BMDL10 for the selected model were 64.2 and 

32.6 mg/kg-day, 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.

BMR = 5% ER; dose shown in mg/kg-day.

Figure D-23. Plot of incidence rate by dose, with fitted curve for selected 

model, for B6C3F1 female mouse combined hepatocellular adenoma and 

carcinoma in mice exposed to RDX by diet for 24 months (Parker et al., 2006). 

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0 20 40 60 80 100

Fraction 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

BMDL BMD

 

Multistage Cancer

Linear extrapolation

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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 = 5% ER

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

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0.0520755 0.0658334

Beta(1) 0.00164105 0.000876864

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.16

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1 Combined results for presence of hepatocellular or alveolar/bronchiolar adenoma or 

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 B6C3F1 female mice exposed to RDX by diet for 24 months; BMR = 10% ER 

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 = 0.00566334

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1 Combined results for presence of hepatocellular or alveolar/bronchiolar adenoma or 

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

carcinoma in B6C3F1 female mice exposed to RDX by diet for 24 months; BMR = 5% ER

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 = 0.00581647

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1 Table D-28. Model predictions for combined hepatocellular adenoma and 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

carcinoma in female B6C3F1 mice exposed to RDX by diet for 24 months, using 

incidence frequencies from Parker et al. (2006) and sample sizes from Lish et 

al. (1984); BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

Multistage 1°b

Multistage 2°

Multistage 3°

Multistage 4°

0.171 163.98 64.8 32.8 All of the models reduced to the 

Multistage 1° model, so this model was 

selected. 

a

Selected model in bold. Scaled residuals for the selected model for doses 0, 1.5, 7, 35, and 107 mg/kg-day were -

1.34, 0.34, 0.49, 1.34, -1.03, 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. 

BMR = 10% ER, dose shown in mg/kg-day.

Figure D-24. Plot of incidence rate by dose with fitted curve for MultistageCancer 1° model for model predictions for combined hepatocellular adenoma 

and carcinoma in female B6C3F1 mice exposed to RDX by diet for 24 months, 

using incidence frequencies from Parker et al. (2006) and sample sizes from 

(Parker et al., 2006). 

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0 20 40 60 80 100

Fraction Affected

dose

Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

10:00 07/20 2015

BMDL BMD

 

Multistage Cancer

Linear extrapolation

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Multistage Model. (Version: 3.4; Date: 05/02/2014) 

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 = 64.7853

BMDL at the 95% confidence level = 32.7981

BMDU at the 95% confidence level = 291.495

Taken together, (32.7981, 291.495) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.00304896

1 

2 

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0.0525105 0.0656105

Beta(1) 0.0016263 0.000878945

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. p-value

Full model −77.2 5 

Fitted model −79.99 2 5.57217 3 0.13

Reduced model −82.43 1 10.462 4 0.03

AIC: = 163.978

Goodness-of-Fit Table

Dose Est. Prob. Expected Observed Size Scaled residuals

0 0.0525 3.413 1 65 −1.34

1.5 0.0548 3.399 4 62 0.34

7 0.0632 4.047 5 64 0.49

35 0.1049 6.716 10 64 1.34

107 0.2038 6.319 4 31 −1.03

Chi^2 = 5.02 d.f. = 3 p-value = 0.1706

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1 Combined results for presence of hepatocellular or alveolar/bronchiolar adenoma or 

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 B6C3F1 female mice exposed to RDX by diet for 24 months; for hepatocellular 

adenoma or carcinoma, the incidence frequencies from Parker et al. (2006) and the sample 

sizes from Lish et al. (1984) were used; BMR = 10% ER 

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 -187.33008597565913 

 Combined Log-likelihood Constant 166.068416550547 

 Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk 

Confidence level = 0.95

 BMD = 29.0933

 BMDL = 17.7048

Multistage Cancer Slope Factor = 0.00564818

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1 D.2.3. Dose-response Analysis and BMD Modeling Documentation for Other Tumor Data 

2

3 

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

Sets

This appendix also presents a quantitative dose-response analysis of incidence of liver 

carcinomas in male F344 rats (Levine et al., 1983b) and incidence of lung carcinomas in male 

B3C6F1 mice (Table D-29). The resulting candidate oral slope factors (OSFs) are presented for 

comparison with OSF estimates provided in Section 2.3.3 of the Toxicological Review. 

Table D-29. Liver carcinoma data from Levine et al. (1983b)

Endpoint and reference Species/sex Dose (mg/kg-d) Incidence/total

Alveolar/bronchiolar carcinomas

Lish et al. (1984)

Male B6C3F1

mouse

0

1.5

7 

35

107

3/63 (5%)

6/60 (10%)

3/62 (5%)

7/59 (12%)

5/27 (19%)

Hepatocellular carcinomas

Levine et al. (1983b)

Male F344 rat 0

0.3

1.5

8 

40

1/55 (2%)

0/55 (0%)

0/52 (0%)

2/55 (4%)

2/31a (6%)

a

The denominators listed in the table represent the number of animals that were alive 1 year after dosing began.

For male mice in Lish et al. (1984), a log-rank test on the Kaplan-Meier survival curves, 

stratified by dose, yielded a nonsignificant result (p-value ≥0.10), indicating that the survival curves

were similar across dose groups. Therefore, a time-to-tumor analysis was not necessary for 

hepatocellular carcinomas in Lish et al. (1984). A non-time-dependent dose-response analysis was 

conducted using BMDS multistage-cancer models, and the model selection procedures described in 

Section D.2.1 were used to select the appropriate models. Subsequently, the administered dose was 

converted to a human equivalent dose (HED) on the basis of (body weight)3/4 (U.S. EPA, 1992), as 

described in Section 2.3.2. The POD estimate for male mouse carcinomas and OSF calculated from 

this POD are provided in Table D-30; detailed BMD modeling results are provided in Table D-31

(and Figure D-25). 

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1 Table D-30. Model predictions and oral slope factor for alveolar/bronchiolar 

2 

3

4 

5 

6 

7 

8 

9 

10

11

12

13

14

15

16

17

18

19

20

21

carcinomas in male B6C3F1 mice exposed to RDX by diet for 2 years (Lish et al., 

1984) 

Tumor type

Selected 

model BMR

BMD,

mg/kg-d

BMDL,

mg/kg-d

POD = 

BMDL10-HEDa

mg/kg-d

Candidate OSFb

(mg/kg-d)-1

Alveolar/bronchiolar

carcinomas

Multistage 1° 10% ER 76.1 36.2 5.41 0.018

a

Based on allometric scaling of administered RDX dose; BMDL10-HED = BMDL10 × (BWa

1/4/BWh1/4), BWa = 0.035 kg, 

and BWh = 70 kg. b

Slope factor = BMR/BMDL10-HED, where BMR = 0.10 (10% ER).

Table D-31. Summary of BMD modeling results for model predictions for 

alveolar/bronchiolar carcinoma in male B6C3F1 mice exposed to RDX by diet 

for 2 years (Lish et al., 1984); BMR = 10% ER

Modela

Goodness of fit BMD10Pct

(mg/kg-d)

BMDL10Pct

p-value AIC (mg/kg-d) Basis for model selection

Multistage 1°b

Multistage 2°

Multistage 3°

Multistage 4°

0.561 162.00 76.1 36.2 All of the models reduced to the 

multistage 1° model, so this model was 

selected. 

a Selected model in bold; scaled residuals for selected model for doses 0, 1.5, 7, 35, and 107 mg/kg-day were 

−0.52, 1.08, −0.74, 0.27, and −0.1, 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.

BMR = 10% ER; dose shown in mg/kg-day.

Figure D-25. Plot of incidence rate by dose with fitted curve for MultistageCancer 1° model for Model predictions for alveolar/bronchiolar carcinoma in 

male B6C3F1 mice exposed to RDX by diet for 24 months (Lish et al., 1984). 

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 0.15

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 0.4

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Fraction Affected

dose

Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

18:19 01/08 2016

BMDL BMD

 

Multistage Cancer

Linear extrapolation

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

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Multistage Model. (Version: 3.4; Date: 05/02/2014) 

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 = 76.1197

BMDL at the 95% confidence level = 36.2316

BMDU at the 95% confidence level = 27443100

Taken together, (36.2316, 27443100) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.00276003

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0.0635642 0.0652915

Beta(1) 0.00138414 0.00131052

Analysis of Deviance Table

Model Log (likelihood) Number of parameters Deviance Test d.f. p-value

Full model −78 5

Fitted model −79 2 1.99294 3 0.57

Reduced model −81.08 1 6.15622 4 0.19

AIC: = 162.001

Goodness-of-Fit Table

Dose Est. Prob. Expected Observed Size Scaled residuals

0 0.0636 4.005 3 63 −0.52

1.5 0.0655 3.93 6 60 1.08

7 0.0726 4.501 3 62 −0.74

35 0.1078 6.363 7 59 0.27

107 0.1925 5.197 5 27 −0.1

Chi^2 = 2.06 d.f. = 3 p-value = 0.561

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curve than the other dose groups, indicating a substantial number of early deaths in the high-dose 

group. A log-rank test on the Kaplan-Meier survival curves, stratified by dose, yielded a significant 

result (p-value <0.001), in which case a time-to-tumor analysis is generally preferred. However, 

such an analysis was not possible because the data were insufficient to allow this analysis. 

Although tumor incidence was listed for each animal in the source article, 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. This prohibited

the matching of the times of death and the tumor incidence of the animals, thus prohibiting the use 

of a time-to-tumor analysis.

Therefore, a non-time-dependent dose-response analysis was conducted using BMDS 

multistage-cancer models. The model selection procedures described in Section D.2.1 were used to 

select the appropriate models. To account for the difference in the survival curves across the 

groups for rats, the number of animals alive at 12 months was used as the denominator in the 

analysis (denominators listed in Table D-28). 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 estimate the HED at the BMDL, HEDs based on both administered dose scaled by BW3/4

and physiologically based pharmacokinetic (PBPK) modeling were considered. Confidence in the 

revised rat PBPK model is relatively high (see Appendix C, Section C.1.5); however, the choice of an 

internal dose is not 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 administered dose scaled by BW3/4 and area under the curve (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 estimates for rat liver carcinomas and the OSFs calculated from these PODs are 

provided in Table D-32; detailed BMD modeling results are provided in Table D-33 (and Figure D26). Results based on two dose-metrics are presented: administered dose of RDX scaled by BW3/4

(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).

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1 Table D-32. Model predictions and oral slope factor for hepatocellular 

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carcinomas in male F344 rats administered RDX in the diet for 2 years (Levine 

et al., 1983b) 

Tumor type

Selected 

model BMR

BMD, 

mg/kg-d

BMDL, 

mg/kg-d

POD = 

BMDL05-HED,

mg/kg-d

Candidate OSFa

(mg/kg-d)-1

Hepatocellular carcinomas Multistage 1° 5% ER 28.5 11.8 2.88b, 5.75c 0.017b, 0.009c

a

Slope factor = BMR/BMDL05-HED, where BMR = 0.05 (5% ER). b

Based on allometric scaling of administered RDX dose; BMDL05-HED = BMDL05 × (BWa

1/4/BWh1/4), BWa = 0.25 kg, and 

BWh = 70 kg. c

Based on toxicological equivalence of PBPK model derived AUC of RDX blood concentration.

Table D-33. Model predictions for combined hepatocellular adenoma and 

carcinoma in F344 rats exposed to RDX by diet for 24 months (Levine et al., 

1983b); BMR = 5% ER

Modela

Goodness of fit BMD5Pct

(mg/kg-d)

BMDL5Pct

p-value AIC (mg/kg-d) Basis for model selection

Multistage 1°b

Multistage 2°

Multistage 3°

Multistage 4°

0.493 49.095 28.5 11.8 All of the models reduced to the 

Multistage 1° model, so this model

was selected.

a

Selected model in bold. Scaled residuals for the selected model for doses 0, 0.3, 1.5, 8, and 40 mg/kg-day 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.

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BMR = 5% ER; dose shown in mg/kg-day.

Figure D-26. 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., 1983b). 

Multistage Model (Version: 3.4; Date: 05/02/2014) 

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% ER

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

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 0.05

 0.1

 0.15

 0.2

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Fraction 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

BMDL BMD

 

Multistage Cancer

Linear extrapolation

Parameter Estimates

Variable Estimate Default initial parameter values

Background 0.00766363 0.00949438

Beta(1) 0.00180277 0.00149364

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1 Analysis of Deviance Table

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3 

4 

5 

6 

7 

8 

Model Log (likelihood) Number of parameters Deviance Test d.f. 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. prob. 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 = 0.493

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mammalian toxicological effects of RDX (twenty-four month chronic 

toxicity/carcinogenicity study of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in the 

Fischer 344 rat): Final report--Phase V. Volume 1. (ADA160774. IITRI-L6121-6. DAMD17-

79-C-9161). Chicago, IL: IIT Research Institute. 

http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA160774

Levine, BS; Lish, PM; Furedi, EM; Rac, VS; Sagartz, JM. (1983b). Determination of the chronic 

mammalian toxicological effects of RDX (twenty-four month chronic 

toxicity/carcinogenicity study of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in the 

Fischer 344 rat): Final report--phase V. Chicago, IL: IIT Research Institute. 

Levine, BS; Lish, PM; Furedi, EM; Rac, VS; Sagartz, JM. (1983c). Determination of the chronic 

mammalian toxicological effects of RDX (twenty-four month chronic 

toxicity/carcinogenicity study of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in the 

Fischer 344 rat): Final report--phase V. Volume 3. Chicago, IL: IIT Research Institute. 

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

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

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

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

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