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# TOXICOLOGICAL REVIEW

OF

BENZO[a]PYRENE

(CAS No. 50-32-8)



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

June 2011

NOTICE

This document is an Interagency Science Consultation draft. This information is distributed solely for the purpose of pre-dissemination peer re view 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 implic ations.

U.S. Environmental Protection Agency

Washington, DC

### ii DRAFT - DO NOT CITE OR QUOTE DISCLAIMER

This document is a preliminary draft for review pur poses only. This information is distributed solely for the purpose of pre-dissemina tion peer review under applicable information quality guidelines. It has not been formally disse minated by EPA. It does not represent and should not be construed to represent any Agency det ermination or policy. Mention of trade names or commercial products does not constitute en dorsement or recommendation for use.

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CONTENTS TOXICOLOGICAL REVIEW OF BENZO[a]PYRENE (CA S No. 50-32-8) 1

2

3 

### LIST OF TABLES ....................................

................................................... ................................ VII 4 

### LIST OF FIGURES ...................................

................................................... ................................. X 5 

### LIST OF ABBREVIATIONS AND ACRONYMS ................

................................................... . XII 6 

### FOREWORD ..........................................

................................................... ................................ XVI 7 

### AUTHORS, CONTRIBUTORS, AND REVIEWERS .............. .............................................. XVII 8 9 

### 1. INTRODUCTION ..................................

................................................... ................................ 1 10 11 

### 2. CHEMICAL AND PHYSICAL INFORMATION .............

................................................... ... 3 12 13 

### 3. TOXICOKINETICS ................................

................................................... ............................... 9 14 

### 3.1. ABSORPTION ..................................

................................................... ............................... 9 15 

### 3.1.1. Inhalation Exposure .......................

................................................... ............................ 9 16 

### 3.1.1.1. Inhalation Exposure in Humans

.................................................. ......................... 9 17 

### 3.1.1.2. Inhalation Exposure in Animals

................................................... ....................... 11 18 

### 3.1.2. Oral Exposure .............................

................................................... ............................. 17 19 

### 3.1.2.1. Oral Exposure in Humans

.................................................. ................................ 18 20 

### 3.1.2.2. Oral Exposure in Animals

.................................................. ................................. 18 21 

### 3.1.3. Dermal Exposure ...........................

................................................... .......................... 20 22 

### 3.1.3.1. Dermal Exposure in Humans

.................................................. ............................ 20 23 

### 3.1.3.2. Dermal Exposure in Animals

.................................................. ............................ 23 24 

### 3.1.4. Other Types of Exposure ...................

................................................... ...................... 25 25 

### 3.2. DISTRIBUTION ................................

................................................... ............................ 26 26 

### 3.2.1. Inhalation Exposure .......................

................................................... .......................... 26 27 

### 3.2.2. Oral Exposure .............................

................................................... ............................. 28 28 

### 3.2.3. Dermal and Other Exposures.................

................................................... .................. 29 29 

### 3.3. METABOLISM ..................................

................................................... ........................... 32 30 

### 3.3.1. Phase I Metabolism ........................

................................................... ......................... 34 31 

### 3.3.1.1. CYP450-dependent Reactions

.................................................. ........................... 34 32 

### 3.3.1.2. Non-CYP-related metabolic pathways

.................................................. .............. 38 33 

### 3.3.2. Phase II Metabolism .......................

................................................... ......................... 40 34 

### 3.3.3. Tissue-specific Metabolism ................

................................................... ..................... 43 35 

### 3.3.3.1. Respiratory Tract Tissues

.................................................. ................................. 43 36 

### 3.3.3.2. GI Tract and Liver Tissues

.................................................. ............................... 47 37 

### 3.3.3.3. Skin

.................................................. ................................................... ................. 48 38 

### 3.3.3.4. Reproductive Tissues and Fetal Metabolism

.................................................. .... 50 39 

### 3.3.3.5. Other Tissues

.................................................. ................................................... . 53 40 

### 3.4. ELIMINATION .................................

................................................... ............................ 53 41 

### 3.4.1. Inhalation Exposure .......................

................................................... .......................... 53 42 

### 3.4.2. Oral Exposure .............................

................................................... ............................. 56 43 

### 3.4.3. Dermal Exposure and Other Exposure Routes .

................................................... ....... 57 44 

### 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS ................. 59 45

46 

### 4. HAZARD IDENTIFICATION .........................

................................................... .................... 64 47 

### 4.1. HUMANS STUDIES ..............................

................................................... ....................... 64 48 

### 4.1.1. Sources of Human Exposure .................

................................................... .................. 64 49 

### 4.1.2. Biomonitoring of benzo[a]pyrene Exposure an d Effects In Humans ........................ 64 50

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4.1.2.1. Urinary Excretion of benzo[a]pyrene

.................................................. ............... 65 1 

### 4.1.2.2. Adduct Formation with DNA and Protein

.................................................. ........ 66 2 

### 4.1.2.2.1. DNA Adduct measures in blood

.................................................. ...................... 69 3 

### 4.1.2.2.2. Adduct measures in reproductive tissues

.................................................. ........ 72 4 

### 4.1.2.3. Benzo[a]pyrene -Induced Cytogenetic Damage

.................................................. 73 5 

### 4.1.3. Epidemiologic Findings in Humans ..........

................................................... .............. 75 6 

### 4.1.3.1. Molecular Epidemiology and Case-Control Cancer Stud ies .............................. 76 7 

### 4.1.3.2. Cohort Cancer Studies

.................................................. ...................................... 77 8 

### 4.1.3.3. Noncancer Disease Caused by benzo[a]pyrene

................................................. 81 9 

### 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN 10 

### ANIMALS—ORAL, INHALATION, AND DERMAL .............. .................................... 85 11 

### 4.2.1. Oral ......................................

................................................... .................................... 85 12 

### 4.2.1.1. Subchronic Studies

.................................................. ............................................ 85 13 

### 4.2.1.2. Chronic Studies and Cancer Bioassays

.................................................. ............ 93 14 

### 4.2.2. Inhalation ................................

................................................... ............................... 106 15 

### 4.2.2.1. Short-term and Subchronic Studies

.................................................. ................ 106 16 

### 4.2.2.2. Chronic Studies and Cancer Bioassays

.................................................. .......... 106 17 

### 4.2.3. Dermal Exposure ...........................

................................................... ........................ 108 18 

### 4.2.3.1. Skin-Tumor Initiation-Promotion Assays

.................................................. ....... 108 19 

### 4.2.3.2. Carcinogenicity Dermal Bioassays

................................................... ................ 109 20 

### 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL, INHA LATION, AND 21 

### DERMAL ............................................

................................................... ......................... 118 22 

### 4.3.1. Oral ......................................

................................................... .................................. 118 23 

### 4.3.2. Inhalation ................................

................................................... ............................... 124 24 

### 4.4. OTHER DURATION OR ENDPOINT-SPECIFIC STUDIES . ..................................... 129 25 

### 4.4.1. Acute Neurological Studies ................

................................................... ................... 129 26 

### 4.4.2. Immunological Studies .....................

................................................... ..................... 133 27 

### 4.4.2.1. Oral Exposure Immunological Studies

.................................................. ........... 133 28 

### 4.4.2.2. Inhalation Exposure Immunological Studies

.................................................. .. 134 29 

### 4.4.2.3. Other Exposure Route Immunological Studies

................................................. 134 30 

### 4.4.2.4. Other Exposure Route Developmental Immunotoxicy ...................................... 135 31 

### 4.4.3. Cancer Bioassays (Other Routes of Exposure)

.................................................. ...... 135 32 

### 4.4.4. Atherogenesis Studies .....................

................................................... ...................... 136 33 

### 4.4.5. Reproductive Studies (Other Routes of Exposu re) ............................................... .... 138 34 

### 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MOA ....... 139 35 

### 4.5.1. Genotoxicity ...............................

................................................... .......................... 140 36 

### 4.5.2. Metabolic pathways ........................

................................................... ....................... 161 37 

### 4.5.3. Mechanistic Studies- Mutagenesis and Tumor I nitiation ......................................... . 164 38 

### 4.5.3. Tumor Promotion and Progression ...........

................................................... ............. 166 39 

### 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS ........ ........................................... 171 40 

### 4.6.1. Oral ......................................

................................................... .................................. 171 41 

### 4.6.2. Inhalation .................................

................................................... ............................. 177 42 

### 4.6.3. Dermal ....................................

................................................... ............................... 180 43 

### 4.6.4. Mode-of-Action Information ................

................................................... ................. 180 44 

### 4.6.4.1. Forestomach Lesions from Oral Exposure

.................................................. ..... 180 45 

### 4.6.4.2. Immune System Effects

.................................................. .................................... 180 46 

### 4.6.4.3. Developmental and Reproductive Toxicity Effects ........................................... 182 47 

### 4.7. EVALUATION OF CARCINOGENICITY ...............

................................................... . 185 48 

### 4.7.1. Summary of Overall Weight-of-Evidence .....

................................................... ........ 185 49

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4.7.2. Synthesis of Human, Animal, and Other Suppo rting Evidence ............................... 185 1 

### 4.7.3. Mode-of-Action Information ................

................................................... ................. 187 2 

### 4.7.3.1. Hypothesized MOA

.................................................. ......................................... 187 3 

### 4.7.3.2. Experimental Support for the Hypothesized MOA ............................................ 188 4 

### 4.7.3.2. Other Possible MOAs

.................................................. ..................................... 190 5 

### 4.7.3.4. Conclusions About the Hypothesized Mode of Action ...................................... 191 6 

### 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES ..... .......................................... 193 7 

### 4.8.1. Possible Childhood Susceptibility .........

................................................... ................ 193 8 

### 4.8.2. Possible Gender Differences ...............

................................................... .................. 196 9 

### 4.8.3. Genetic Polymorphisms .....................

................................................... .................... 198 10 11 

### 5. DOSE-RESPONSE ASSESSMENTS .....................

................................................... ........... 201 12 

### 5.1. ORAL REFERENCE DOSE (RfD) ...................

................................................... .......... 201 13 

### 5.1.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification ... 201 14 

### 5.1.2. METHODS OF ANALYSIS .......................

................................................... ................. 205 15 

### 5.1.3. RfD Derivation—Including Application of Unc ertainty Factors (UFs)................... 209 16 

### 5.1.4. Previous RfD Assessment ...................

................................................... .................. 210 17 

### 5.2. INHALATION REFERENCE CONCENTRATION (RfC) .... ....................................... 210 18 

### 5.2.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification ... 210 19 

### 5.2.2. Methods of Analysis- Adjustment to a Human E quivalent Concentration (HEC) ... 212 20 

### 5.2.3. RfC Derivation- Including Application of Un certainty Factors (UFs) .................... 214 21 

### 5.2.4. Previous RfC Assessment....................

................................................... .................. 215 22 

### 5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION 23 

### REFERENCE CONCENTRATION ...........................

................................................... . 216 24 

### 5.4. CANCER ASSESSMENT ...........................

................................................... ................ 217 25 

### 5.4.1. Oral Exposure—Oral Slope Factor ...........

................................................... ............ 217 26 

### 5.4.1.1. Choice of Study/Data—with Rationale and Justificati on—Oral Exposure ...... 217 27 

### 5.4.1.2. Dose-response Data—Oral Exposure

................................................... ............ 218 28 

### 5.4.1.3. Dose Adjustments and Extrapolation Method(s)—Oral E xposure ................... 220 29 

### 5.4.1.4. Oral Slope Factor Derivation

.................................................. ......................... 223 30 

### 5.4.2. Inhalation Exposure—Inhalation Unit Risk ..

................................................... ........ 225 31 

### 5.4.2.1. Choice of Study/Data—with Rationale and Justificati on—Inhalation Exposure 32



### ................................................... ................................................... ................................... 225 33 

### 5.4.2.2. Dose-response Data—Inhalation Exposure

.................................................. ... 226 34 

### 5.4.2.3. Dose Adjustments and Extrapolation Method(s)—Inhala tion Exposure .......... 227 35 

### 5.4.2.4. Inhalation Unit Risk Derivation

................................................... ..................... 227 36 

### 5.4.3. Dermal Exposure—Dermal Slope Factor .......

................................................... ....... 227 37 

### 5.4.3.1. Choice of Study/Data—with Rationale and Justificati on—Dermal Exposure . 227 38 

### 5.4.3.2. Dose-response Data—Dermal Exposure

.................................................. ........ 228 39 

### 5.4.3.3. Dose Adjustments and Extrapolation Method(s)—Dermal Exposure .............. 234 40 

### 5.4.3.4. Dermal Slope Factor Derivation

.................................................. .................... 235 41 

### 5.4.3.5. Dermal Slope Factor Cross Species Scaling

.................................................. ... 236 42 

### 5.4.4. Application of Age-Dependent Adjustment Fac tors .............................................. .. 238 43 

### 5.4.5. Uncertainties in Cancer Risk Values .......

................................................... .............. 240 44 

### 5.4.5.1. Oral Slope Factor

.................................................. ........................................... 240 45 

### 5.4.5.2. Inhalation Unit Risk

.................................................. ........................................ 240 46 

### 5.4.5.3 Dermal Slope Factor

................................................... ....................................... 241 47 

### 5.5.5. Previous Cancer Assessment ................

................................................... ................. 242 48 49

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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE 1 

### RESPONSE ..........................................

................................................... .............................. 244 2 

### 6.1. HUMAN HAZARD POTENTIAL ......................

................................................... ........ 244 3 

### 6.2. DOSE RESPONSE ...............................

................................................... ....................... 244 4 

### 6.2.1. RfD .......................................

................................................... ................................. 244 5 

### 6.2.2. RfC .......................................

................................................... ................................. 246 6 

### 6.2.3. Cancer ....................................

................................................... ................................ 247 7 

### 6.2.3.1. Cancer—Oral .............................

................................................... ......................... 247 8 

### 6.2.3.2. Cancer—Inhalation........................

................................................... ..................... 248 9 

### 6.2.3.3. Cancer—Dermal ...........................

................................................... ...................... 248 10 11 

### 7. REFERENCES ....................................

................................................... ............................... 250 12 13 

### APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC COMMENTS 14 

### AND DISPOSITION ...................................

................................................... ....................... 287 15 

### APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR NO NCANCER .......... 288 16 

### APPENDIX C. ADDITIONAL CALCULATIONS FOR THE RFC .. ..................................... 327 17 

### APPENDIX D. TIME-TO-TUMOR MODELING FOR THE ORAL SL OPE FACTOR ........ 330 18 

### APPENDIX E. TIME-TO-TUMOR MODELING FOR THE INHALAT ION UNIT RISK .... 360 19 

### APPENDIX F. BENCHMARK MODELING FOR THE DERMAL SLOP E FACTOR ......... 367 20 

### APPENDIX G. ADDITIONAL INFORMATION IN SUPPORT OF T HE DERMAL SLOPE 21 

### FACTOR ............................................

................................................... ................................. 395 22 

### APPENDIX H. ALTERNATIVE APPROACHES FOR CROSS-SPECI ES SCALING OF THE 23 

### DERMAL SLOPE FACTOR ...............................

................................................... ............... 398 24

25

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Table 2-1. Physical properties and chemical identi ty of benzo[a]pyrene .............................. ........ 4 Table 2-2. Benzo[a]pyrene Concentrations in Air ..

................................................... .................... 5 Table 2-3. Benzo[a]pyrene Levels in Food .........

................................................... ........................ 6 Table 2-4. Levels of benzo[a]pyrene in Soil ......

................................................... ......................... 7 Table 3-1. Distribution of benzo[a]pyrene in selec ted tissues of male rats following i.v. dosing with 2 mg/kg.......................................

................................................... ................................. 30 Table 3-2. Species differences in tracheobronchial benzo[a]pyrene metabolism........................ 44 Table 3-3. Excretion of benzo[a]pyrene metabolites in several animal species ........................ .. 55 Table 3-4. Biliary excretion of benzo[a]pyrene met abolites in several species ....................... ... 56 Table 4-1. Studies of PAH-DNA adducts in human pop ulations or tissues exposed to PAHs ... 67 Table 4-2. Exposure-related effects in male Wistar rats exposed to benzo[a]pyrene by gavage 5 days/week for 5 weeks .............................

................................................... ........................... 87 Table 4-3. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene by gavage 5 days/week for 5 weeks .............................

................................................... ........................... 90 Table 4-4. Means ± SD a 

### for liver and thymus weights in Wistar rats expose d to benzo[a]pyrene by gavage 5 days/week for 90 days .................

................................................... .................... 92 Table 4-5. Incidences of exposure-related neoplasm s in Wistar rats treated by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks ........

................................................... ............. 95 Table 4-6. Incidences of alimentary tract tumors i n Sprague-Dawley rats chronically exposed to benzo[a]pyrene in the diet or by gavage in caffeine solution ......................................... ....... 98 Table 4-7. Incidence of nonneoplastic and neoplast ic lesions in female B6C3F 1 

### mice fed benzo[a]pyrene in the diet for up to 2 years ......

................................................... ................ 100 Table 4-8. Tumor incidence in oral exposure rodent cancer bioassays with limitations for describing dose-response relationships for lifetime exposure to benzo[a]pyrene................ 102 Table 4-9. Incidence of respiratory and upper dige stive tract tumors in male hamsters treated for life with benzo[a]pyrene by inhalation.............

................................................... ................. 107 Table 4-10. Numbers of animals with pharynx and la rynx tumors in male hamsters exposed by inhalation to benzo[a]pyrene for life .............

................................................... .................... 108 Table 4-11. Skin tumor incidence and time of appea rance in male C57L mice dermally exposed to benzo[a]pyrene for up to 103 weeks .............

................................................... ................ 109 Table 4-12. Skin tumor incidence and time of appea rance in male SWR, C3HeB, and A/He mice dermally exposed to benzo[a]pyrene for life or unti l a skin tumor was detected ................ 110 Table 4-13. Tumor incidence in female Swiss mice d ermally exposed to benzo[a]pyrene for up to 93 weeks .......................................

................................................... ................................. 111 Table 4-14. Skin tumor incidence in female NMRI an d Swiss mice dermally exposed to benzo[a]pyrene ....................................

................................................... .............................. 112 Table 4-15. Skin tumor incidence in female NMRI mi ce dermally exposed to benzo[a]pyrene

.................................................. ................................................... ......................................... 114 Table 4-16. Skin tumor incidence in female NMRI mi ce dermally exposed to benzo[a]pyrene

.................................................. ................................................... ......................................... 114 Table 4-17. Skin tumor incidence and time of appea rance in female CFLP mice dermally exposed to benzo[a]pyrene for 104 weeks ...........

................................................... ............. 115 Table 4-18. Skin tumor incidence in female NMRI mi ce dermally exposed to benzo[a]pyrene for life...............................................

................................................... ....................................... 116 Table 4-19. Skin tumor incidence in male C3H /HeJ mice dermally exposed to benzo[a]pyrene for 24 months .....................................

................................................... ............................... 117

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Table 4-20. Means ± SD for ovary weight in female SD-rats ........................................... ........ 119 Table 4-21. Reproductive effects in male and femal e CD-1 F1 mice exposed in utero to benzo[a]pyrene ....................................

................................................... .............................. 121 Table 4-22. Effect of prenatal exposure to benzo[a ]pyrene on indices of reproductive performance in F1 female NMRI mice ................

................................................... ............. 123 Table 4-23. Pregnancy outcomes in female F344 rats treated with benzo[a]pyrene on GDs 11–21 by inhalation .....................................

................................................... ................................. 125 Table 4-24. In vitro genotoxicity studies of benzo [a]pyrene in non-mammallian cells ............ 141 Table 4- 25. In vitro genotoxicity studies of benz o[a]pyrene in mammalian cells .................... 143 Table 4-26. In vivo genotoxicity studies of benzo [a]pyrene ......................................... ........... 148 Table 4-27. NOAELS and LOAELs for noncancer effect s in animals repeatedly exposed to benzo[a]pyrene by the oral route ..................

................................................... ..................... 172 Table 4-28. NOAELS and LOAELs for noncancer effect s in animals repeatedly exposed to benzo[a]pyrene by the inhalation route ............

................................................... ................. 179 Table 5-1. Means ± SD for ovary weight in female S D-rats ............................................ ......... 204 Table 5-2. Summary of BMDs and BMDLs for modeled no ncancer effects following oral

exposure ..........................................

................................................... .................................. 207 Table 5-3. Pregnancy outcomes in female F344 rats treated with benzo[a]pyrene on GDs 11–21 by inhalation .....................................

................................................... ................................. 212 Table 5-4. Incidence data for tumors in Wistar rat s exposed to benzo[a]pyrene by gavage, 5 days/week for 104 weeks ...........................

................................................... ....................... 220 Table 5-5. Incidence data for tumors in female B6C 3F 1 

### mice exposed to benzo[a]pyrene in the diet for 104 weeks ................................

................................................... ............................. 220 Table 5-6. Human equivalent PODs and oral slope fa ctors derived from multistage-Weibull modeling of tumor incidence data at multiple tissue sites in Wistar rats and B6C3F 1 

### mice exposed to benzo[a]pyrene orally for 2 years ......

................................................... ............. 223 Table 5-7. Incidence of tumors in male hamsters ex posed by inhalation to benzo[a]pyrene for life...............................................

................................................... ....................................... 226 Table 5-8. Skin tumor incidence, benign or maligna nt, in C57L male mice dermally exposed to benzo[a]pyrene ....................................

................................................... .............................. 232 Table 5-9. Skin tumor incidence, benign or maligna nt in female Swiss or NMRI mice dermally exposed to benzo[a]pyrene .........................

................................................... ....................... 233 Table 5-10. Skin tumor incidence, benign or malign ant, in female CFLP mice dermally exposed to benzo[a]pyrene .................................

................................................... ............................. 234 Table 5-11. Skin tumor incidence, benign or malig nant, in male C3H /HeJ mice dermally exposed to benzo[a]pyrene(Sivak et al., 1997) .....

................................................... ............ 234 Table 5.13. Application of ADAFs to benzo[a]pyrene cancer risk following a lifetime (70-year) oral exposure .....................................

................................................... ................................ 239 Table 5-14. Application of ADAFs to benzo[a]pyrene cancer risk following a lifetime (70-year) inhalation exposure ...............................

................................................... ............................ 239 Table 5-15. Application of ADAFs to benzo[a]pyrene cancer risk following a lifetime (70-year) dermal exposure ...................................

................................................... ............................. 240 Table B-1. Liver weight (±SD) a 

### in male F344 rats administered benzo[a]pyrene by g avage for 90 days............................................

................................................... ................................... 288 Table B-2. BMD modeling results for increased live r weight in male rats, with BMR=10%... 288 Table B-3. Means ± SD a 

### for thymus weight in male Wistar rats exposed to b enzo[a]pyrene by gavage 5 days/week for 90 days ....................

................................................... .................... 292 Table B-4. Model predictions for decreased thymus weight in male Wistar rats—90 days ..... 292 Table B-5. Means ± SD a 

### for thymus weight in female Wistar rats exposed to benzo[a]pyrene by

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gavage 5 days/week for 90 days ....................

................................................... .................... 297 Table B-6. Model predictions for decreased thymus weight in female Wistar rats—90 days .. 297 Table B-7. Means ± SD for thymus weight in male Wi star rats exposed to benzo[a]pyrene by gavage 5 days/week for 35 days ....................

................................................... .................... 302 Table B-9. Exposure-related effects in male Wistar rats exposed to benzo[a]pyrene by gavage 5 days/week for 5 weeks .............................

................................................... ......................... 307 Table B-10. Model predictions for decreased spleen B-cells in male Wistar rats—35 days ..... 307 Table B-11. Dose-response data for forestomach hyp erplasia in Wistar rats and B6C3F 1 

### rats orally exposed to benzo[a]pyrene for 2 years ......

................................................... ............. 312 Table B-12. Summary of BMDs and BMDLs from the bes t fitting model forestomach hyperplasia—oral exposure .........................

................................................... ...................... 313 Table B-13. Model predictions for forestomach hype rplasia in male Wistar rats in a 2-year study

.................................................. ................................................... ......................................... 313 Table B-14. Model predictions for forestomach hype rplasia in female Wistar rats in a 2-year study .............................................

................................................... ..................................... 316 Table B-15. Model predictions for forestomach hype rplasia in female B6C3F1 mice in a 2-year study .............................................

................................................... ..................................... 319 Table B-16. Means ± SDa for ovary weight in female SD-rats .......................................... ....... 323 Table B-17. Model predictions for decreased ovary weight in female SD-rats—60 days ........ 323 Table D-1. Tumor incidence data, with time to death with tumor; male rats exposed by gavage to Benzo[a]pyrene (Kroese et al., 2001)...............

................................................... ................. 330 Table D-2. Tumor incidence data, with time to death with tumor; female rats exposed by gavage to Benzo[a]pyrene (Kroese et al., 2001) ...........

................................................... ................ 332 Table D-3. Tumor incidence data, with time to death with tumor; female mice exposed to Benzo[a]pyrene via diet (Beland and Culp, 1998) ...

................................................... ......... 334 Table D-4. Derivation of HEDs to use for BMD model ing of Wistar rat tumor incidence data from Kroese et al. (2001) .........................

................................................... ......................... 336 Table D-5. Derivation of HEDs for BMD modeling of B6C3F1 female mouse tumor incidence data from Beland and Culp (1998) ..................

................................................... .................. 336 Table D-6. Summary of Model Selection and Modeling Results for best-fitting multistageWeibull models, using time-to-tumor data for rats ( Kroese et al., 2001) ............................ 3 37 Table F-1. Summary of model selection and modeling results for best-fitting multistage models, for multiple data sets of skin tumors in mice follo wing dermal benzo[a]pyrene exposure . 367 Table G-1: Exposure methods for selected lifetime dermal exposure mouse cancer bioassays for benzo[a]pyrene-induced skin tumors ................

................................................... ................ 396 Table H-1. Alternative approaches to cross-species scaling .......................................... ............ 401

### x DRAFT - DO NOT CITE OR QUOTE LIST OF FIGURES

Figure 2-1. Structural formula of benzo[a]pyrene.

................................................... ..................... 3 Figure 3-1. Metabolic pathways for benzo[a]pyrene.

.................................................. ................ 33 Figure 3-2. The stereospecific activation of benzo [a]pyrene. ........................................ .............. 35 Figure 4-1. Interaction of PAHs with the AhR. ....

................................................... .................. 168 Figure 4-2. NOAELs and LOAELs for selected noncanc er effects from repeated oral exposure to benzo[a]pyrene. ................................

................................................... ............................. 175 Figure 4-3. Proposed principal pathways and key ev ents in the benzo[a]pyrene carcinogenic MOA................................................

................................................... .................................. 188 Figure B-1. Fit of polynomial model to data on inc reased liver weight in male Wistar rats—90 days...............................................

................................................... ..................................... 289 Figure B-2. Fit of linear model (nonconstant varia nce) to data on decreased thymus weight in male Wistar rats—90 days. .........................

................................................... ...................... 293 Figure B-3. Fit of linear model (constant variance ) to data on decreased thymus weight in female Wistar rats—90 days. .......................

................................................... ..................... 298 Table B-8. Model predictions for decreased thymus weight in male Wistar rats—35 days ..... 302 Figure B-4. Fit of linear model (constant variance ) to data on decreased thymus weight in male Wistar rats—35 days. ..............................

................................................... .......................... 303 Figure B-5. Fit of linear model to data on decreas ed spleen B-cells in male Wistar rats—35 days...............................................

................................................... ..................................... 308 Figure B-6. Fit of log logistic model to data on f orestomach hyperplasia in male Wistar rats in a 2-year study. .....................................

................................................... ................................. 314 Figure B-7. Fit of log logistic model to data on f orestomach hyperplasia in female Wistar rats in a 2-year study. ...................................

................................................... ................................ 317 Figure B-8. Fit of log logistic model to data on f orestomach hyperplasia in female B6C3F 1 

### mice in a 2-year study. ................................

................................................... ............................... 320 Figure B-9. Fit of linear/polynomial (1°) model to data on decreased ovary weight ................ 32 4 Figure C-1. Human Fractional Deposition...........

................................................... ................... 327 Figure C-2. Rat Fractional Deposition ............

................................................... ........................ 329 Figure F-1. Fit of multistage model to skin tumors in C57L mice exposed dermally to benzo[a]pyrene (Poel, 1959); graph and model output

. ................................................. ...... 368 Figure F-2. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to benzo[a]pyrene (Roe et al., 1970); graph and model output. ........................................... .... 370 Figure F-3. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph and mo del output. ....................................... . 372 Figure F-4. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph and mo del output. ....................................... . 374 Figure F-5. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to benzo[a]pyrene (Schmähl et al., 1977); graph and mo del output. ....................................... 376 Figure F-6. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to benzo[a]pyrene (Habs et al., 1980); graph and model output. .......................................... ... 378 Figure F-8. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to benzo[a]pyrene (Grimmer et al, 1983); graph and mod el output. ........................................ 383 Figure F-9. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and mo del output. ....................................... 385 Figure F-9. Fit of log-logistic model to skin tumo rs in female CFLP mice exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and mo del output. ....................................... 388

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Figure F-11. Fit of multistage model to skin tumor s in female CFLP mice exposed dermally to benzo[a]pyrene (Grimmer et al., 1984), highest dose dropped; graph and model output. ... 390 Figure F-12. Fit of multistage model to skin tumor s in female CFLP mice exposed dermally to benzo[a]pyrene (Sivak et al., 1997); graph and mod el output. ........................................ ... 393

### xii DRAFT - DO NOT CITE OR QUOTE LIST OF ABBREVIATIONS AND ACRONYMS

1-OH-Py 1-hydroxypyrene 3-MC 3-methylcholanthrene 8-OHdG 8-hydroxydeoxyguanosine ADAFs age-dependent adjustment factors AFC antibody forming cells Ah aryl hydrocarbon AHH aryl hydrocarbon hydroxylase AhR Ah receptor AhRE AhR-responsive element AhRR AhR repressor AIC Akaike’s Information Criterion AKR aldo-keto reductase ALT alanine aminotransferase Arnt Ah receptor nuclear translocator AST serum aspartate transaminase ATSDR Agency for Toxic Substances and Disease Registry AUC area under the curve benzo[a]pyrene benzo[a]pyrene BeP benzo[e]pyrene BMD benchmark dose BMDL benchmark dose, 95% lower bound BMDS Benchmark Dose Software BMR benchmark response BPDE benzo[a]pyrene-7,8-diol-9,10-epoxide BPQ benzo[a]pyrene-7,8-quinone BRCA breast cancer antigen BrdU bromodeoxyuridine BSM benzene-soluble matter BUN blood urea nitrogen CA chromosomal aberrations CASRN Chemical Abstracts Service Registry Number CAT chloramphenicol acetyltransferase CB carbon black CDK cyclin-dependent kinase CFU-GM colony forming unit-granulocyte macrophage CHL Chinese hamster lung cells CHO Chinese hamster ovary cells CI confidence interval CNS central nervous system Con A Concanavalin A CONSAAM Conversational SAAM COX cyclooxygenase CPDB Cancer Potency Database cSt centi-Stoke CTPV coal tar pitch volatiles CYP cytochrome

### xiii DRAFT - DO NOT CITE OR QUOTE

CYP450 cytochrome P450 dG deoxyguanosine dG-N 2 

### -BPDE 10 β -(deoxyguanosin-N 2 

### -yl)-7 β ,8 α ,9 α -trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene DHH dihydrodiol dehydrogenase DMBA 7,12-dimethylbenzanthracene DMSO dimethyl sulfoxide DNCB 2,4-dinitrochlorobenzene DRE dioxin-responsive element ED effective dose EGFR epidermal growth factor receptor EH epoxide hydrolase ELISA enzyme-linked immunosorbent assay EPC endothelial progenitor cells EpRE electrophile (or antioxidant) response element ER estrogen receptor EROD 7-ethoxyresorufin-O-deethylase ETS environmental tobacco smoke Fe 2 

### O 3 

### ferrous oxide FEL frank effect level Ga 2 

### O 3 

### gallium oxide GD gestational day GGT γ

-glutamyl transferase GI gastrointestinal GJIC gap junctional intercellular communication GLP good laboratory practice GM-CSF granulocyte-macrophage colony stimulating factor GNMT glycine N-methyltransferase GP glycophorin GSH reduced glutathione GST glutathione-S-transferase hAR human androgen receptor HED human equivalent dose HF human fibroblasts HFC high-frequency cells HL human lymphocytes HPLC high-performance liquid chromatography hprt hypoxanthine guanine phosphoribosyl transferase Hsp heat shock protein IARC International Agency for Research on Cancer IC 50 

### half maximal inhibitory concentration IFN interferon Ig immunoglobulin IGF insulin-like growth factor IHD ischemic heart disease IL interleukin

γ -INF gamma-interferon i.p. intraperitoneal IRIS Integrated Risk Information System

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i.v. intravenous KLH keyhole limpet hemocyanin ko knock-out LALN lung-associated lymph nodes LC-MS liquid chromatography-mass spectrometry LDH lactate dehydrogenase LH luteinizing hormone LOAEL lowest-observed-adverse-effect level LPS lipopolysaccharide MAP mitogen-activated protein MCHC mean cell hemoglobin concentration MCP monocyte-chemoattractant protein M-CSF macrophage colony stimulating factor MGP manufactured gas plant residue MLR mixed lymphocyte response MMAD mass median aerodynamic diameter MN micronucleus MOA mode of action MPO myeloperoxidase NADH nicotinamide adenine dinucleotide phosphate NAT N-acetyl transferase NER nucleotide excision repair NF naphthoflavone NK natural-killer NMDA N-methyl-D-aspartate NO nitrous oxide NOAEL no-observed-adverse-effect level NQO NADPH:quinone oxidoreductase NSAID non-steroidal anti-inflammatory drug NTP National Toxicology Program OR odds ratio PAH polycyclic aromatic hydrocarbon PBMC peripheral blood mononuclear cell PBPK physiologically-based pharmacokinetic PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PHA phytohemagglutinin PHS prostaglandin H synthase PMN polymorphonuclear leukocyte PND postnatal day

p.o. per os POD point of departure Py pyrene RBC red blood cell RfC reference concentration RfD reference dose RN reaction network ROS reactive oxygen species RR relative risk

### xv DRAFT - DO NOT CITE OR QUOTE

RT-PCR real-time or reverse transcriptase PCR

s.c. subcutaneous SAAM Simulation, Analysis and Modeling SAM S-adenosylmethionine SCC squamous cell carcinoma SCE sister chromatid exchanges SCE-H SCE heterogeneity index SD standard deviation SEM standard error of the mean SIR standardized incidence ratio SLRL sex-linked recessive lethal SMR standardized mortality ratio SNP single nucleotide polymorphisms SPF specific pathogen-free SRBC sheep red blood cell SSB single strand break TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TEF toxicity (or toxic) equivalency factor TGF transforming growth factor TK thymidine kinase TNF tumor necrosis factor TPA 12-O-tetradecanoylphorbol-13-acetate TWA time-weighted average UDP uridine diphosphate UDPGA UDP glucuronic acid UDS unscheduled DNA synthesis UF uncertainty factor UGT UDP-dependent glucuronosyltransferase Vmax maximum substrate turnover velocity vSMC vascular smooth muscle cell WBC white blood cells WHO World Health Organization WT wild type WTC World Trade Center XP xeroderma pigmentosum XPA xeroderma pigmentosum group A

### xvi DRAFT - DO NOT CITE OR QUOTE FOREWORD

The purpose of this Toxicological Review is to prov ide scientific support and rationale for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to benzo[a]pyrene (benzo[a]pyrene). It is not intende d to be a comprehensive treatise on the chemical or toxicological nature of benzo[a]pyrene. The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose Response, is to present the major conclusions reached in the derivation of the reference dose, reference concentration and cancer assessment, wher e applicable, and to characterize the overall confidence in the quantitative and qualitative aspe cts of hazard and dose response by addressing the quality of data and related uncertainties. The discussion is intended to convey the limitations of the assessment and to aid and guide the risk ass essor in the ensuing steps of the risk

assessment process. For other general information about this assessment or other questions relating to IRIS, the reader is referred to EPA’s IRIS Hotline at (20 2) 566-1676 (phone), (202) 566-1749 (fax), or hotline.iris@epa.gov (email address).

### xvii DRAFT - DO NOT CITE OR QUOTE AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGER

Kathleen Newhouse National Center for Environmental Assessment U.S. Environmental Protection Agency Washington, DC

CONTRIBUTORS

Christine Cai Gene Ching-Hung Hsu Glinda Cooper Martin Gehlhaus Scott Glaberman Karen Hogan Margaret Pratt John Schaum John Stanek Suryanarayana Vulimiri National Center for Environmental Assessment U.S. Environmental Protection Agency Washington, DC

CONTRACTING SUPPORT

Heather Carlson-Lynch Peter McClure Kelly Salinas Joe Santodonato Julie Stickney SRC, Inc. North Syracuse, NY

George Holdsworth Lutz W. Weber Oak Ridge Institute for Science and Education Oak Ridge, TN

Janusz Z. Byczkowski Consultant Fairborn, OH

Andrew Maier Lynne Haber Bernard Gadagbui

### xviii DRAFT - DO NOT CITE OR QUOTE

Toxicology Excellence for Risk Assessment Cincinnati, OH

Glenn Talaska University of Cincinnati Cincinnati, OH

REVIEWERS This document has been provided for review to EPA s cientists.

INTERNAL EPA REVIEWERS

Stephen Nesnow National Health and Environmental Effects Research Laboratory US Environmental Protection Agency Research Triangle Park, NC

Rita Schoeny Office of Water U.S. Environmental Protection Agency Washington, DC

### 1 DRAFT - DO NOT CITE OR QUOTE 1. INTRODUCTION 1

2 

### This document presents background information and j ustification for the Integrated Risk 3 

### Information System (IRIS) Summary of the hazard and dose-response assessment of 4 

### benzo[a]pyrene (benzo[a]pyrene). IRIS Summaries ma y include oral reference dose (RfD) and 5 

### inhalation reference concentration (RfC) values for chronic and other exposure durations, and a 6 

### carcinogenicity assessment. 7 

### The RfD and RfC, if derived, provide quantitative i nformation for use in risk assessments 8 

### for health effects known or assumed to be produced through a nonlinear (presumed threshold) 9 

### mode of action (MOA). The RfD (expressed in units of mg/kg-day) is defined as an estimate 10 

### (with uncertainty spanning perhaps an order of magn itude) of a daily exposure to the human 11 

### population (including sensitive subgroups) that is likely to be without an appreciable risk of 12 

### deleterious effects during a lifetime. The inhalat ion RfC (expressed in units of mg/m 3 

### ) is 13 

### analogous to the oral RfD, but provides a continuou s inhalation exposure estimate. The 14



### inhalation RfC considers toxic effects for both the respiratory system (portal-of-entry) and for 15 

### effects peripheral to the respiratory system (extra respiratory or systemic effects). Reference 16 

### values are generally derived for chronic exposures (up to a lifetime), but may also be derived for 17 

### acute ( ≤ 24 hours), short-term (>24 hours up to 30 days), an d subchronic (>30 days up to 10% of 18 

### lifetime) exposure durations, all of which are deri ved based on an assumption of continuous 19 

### exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are 20 

### derived for chronic exposure duration. 21 

### The carcinogenicity assessment provides information on the carcinogenic hazard 22 

### potential of the substance in question and quantita tive estimates of risk from oral and inhalation 23 

### exposure may be derived. The information includes a weight-of-evidence judgment of the 24 

### likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic 25 

### effects may be expressed. Quantitative risk estima tes may be derived from the application of a 26 

### low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on 27 

### the estimate of risk per mg/kg-day of oral exposure . Similarly, a plausible inhalation unit risk is 28 

### an upper bound on the estimate of risk per μ g/m 3 

### air breathed. 29 

### There is evidence in humans and animal studies, dem onstrating an increased incidence of 30 

### skin tumors with increasing dermal exposure to poly cyclic aromatic hydrocarbons (PAHs) 31 

### mixtures including benzo[a]pyrene or to benzo[a]pyr ene alone. Thus this assessment for 32 

### benzo[a]pyrene derives a dermal slope factor; a qua ntitative risk estimate that is a plausible 33 

### upper bound on the estimate of risk per μ g/day of dermal exposure. 34 

### Development of these hazard identification and dose -response assessments for 35 

### benzo[a]pyrene has followed the general guidelines for risk assessment as set forth by the 36 

### National Research Council (NRC, 1983). EPA Guideli nes and Risk Assessment Forum 37 

### Technical Panel Reports that may have been used in the development of this assessment include 38

### 2 DRAFT - DO NOT CITE OR QUOTE

the following: Guidelines for the Health Risk Assessment of Chemic al Mixtures (U.S. EPA, 1 

### 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Recommendations for 2 

### and Documentation of Biological Values for Use in R isk Assessment (U.S. EPA, 1988), 3 

### Guidelines for Developmental Toxicity Risk Assessme nt (U.S. EPA, 1991), Interim Policy for 4 

### Particle Size and Limit Concentration Issues in Inh alation Toxicity (U.S. EPA, 1994a), Methods 5 

### for Derivation of Inhalation Reference Concentratio ns and Application of Inhalation Dosimetry 6 

### (U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk A ssessment (U.S. EPA, 7 

### 1995), Guidelines for Reproductive Toxicity Risk Assessmen t (U.S. EPA, 1996), Guidelines for 8 

### Neurotoxicity Risk Assessment (U.S. EPA, 1998), Science Policy Council Handbook : Risk 9 

### Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. 10 

### EPA, 2000b), Supplementary Guidance for Conducting Health Risk A ssessment of Chemical 11



### Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference Concen tration 12



### Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a)

, 13 

### Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens 14 

### (U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A 15 

### Framework for Assessing Health Risks of Environment al Exposures to Children (U.S. EPA, 16 

### 2006b). 17 

### The literature search strategy employed for this co mpound was based on the Chemical 18 

### Abstracts Service Registry Number (CASRN) and at le ast one common name. Any pertinent 19 

### scientific information submitted by the public to t he IRIS Submission Desk was also considered 20 

### in the development of this document. The relevant literature was reviewed through December, 21



### 2010. 22

23

### 3 DRAFT - DO NOT CITE OR QUOTE Benzo[a]pyrene 

### 2. CHEMICAL AND PHYSICAL INFORMATION 1

2

3 

### Benzo[a]pyrene is a five-ring polycyclic aromatic h ydrocarbon (PAH) (Figure 2-1). It is 4 

### a pale yellow crystalline solid with a faint aromat ic odor. It is relatively insoluble in water and 5 

### has low volatility. Benzo[a]pyrene is released to the air from both natural and anthropogenic 6 

### sources and removed from the atmosphere by photoche mical oxidation; reaction with nitrogen 7 

### oxides, hydroxy and hydroperoxy radicals, ozone, su lfur oxides, and peroxyacetyl nitrate; and 8 

### dry deposition to land or water. In air, benzo[a]p yrene is predominantly adsorbed to particulates 9 

### but may also exist as a vapor at high temperatures (NLM, 2010). The structural formula is 10 

### presented in Figure 2-1. The physical and chemical properties of benzo[a]pyrene are shown in 11



### Table 2-1. 12

13

14

15

16

17

18

19

20

21

22

23 

### Figure 2-1. Structural formula of benzo[a]pyrene. 24

25 

### There is no known commercial use for benzo[a]pyrene and it is only produced as a 26 

### research chemical. It is found ubiquitously in the environment primarily as a result of 27 

### incomplete combustion emissions. It is released to the environment via both natural sources 28 

### (such as forest fires) and anthropogenic sources in cluding stoves/furnaces burning fossil fuels 29 

### (especially wood and coal), motor vehicle exhaust, cigarettes, and various industrial combustion 30 

### processes (ATSDR, 1995). Benzo[a]pyrene is also fo und in soot and coal tars. Mahler et al. 31 

### (2005) has reported that urban run-off from asphalt -paved car parks treated with coats of coal-tar 32 

### emulsion seal could account for the majority of PAH s in many watersheds (Mahler et al. , 2005). 33 

### Occupational exposure to PAHs occurs primarily thro ugh inhalation and skin contact during the 34 

### production and use of coal tar and coal tar-derived products, such as roofing tars, creoste and 35 

### asphalt (IARC, 2010). Chimney sweeping can result in exposure to benzo[a]pyrene 36 

### contaminated soot (ATSDR, 1995). As shown below in Table 2-2, benzo[a]pyrene exposure can 37 

### also occur to workers involved in the production of aluminum, coke, graphite, and silicon 38



### carbide. 39

40

### 4 DO NOT CITE OR QUOTE

Table 2-1. Physical properties and chemical identi ty of benzo[a]pyrene

CASRN 50-32-8 Synonyms Benzo[d,e,f]chrysene; 3,4-benzopyrene, 3,4- benzpyrene; benz[a]pyrene; benzo[a]pyrene; BP http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CHEM

Melting point 179–179.3°C O’Neil et al. (2001) Boiling point 310–312 ° C at 10 mm Hg O’Neil et al. (2001) Vapor pressure, at 20°C 5 × 10 - 7 mm Hg Verschueren (2001) Density 1.351 g/cm 3 IARC (1973) Flashpoint (open cup) No data Water solubility at 25°C 1.6–2.3 × 10 - 3 mg/L ATSDR (1995); Howard and Meylan (1997) Log K ow 6.04 Verschueren (2001)

Odor threshold No data Molecular weight 252.32 O’Neil et al. (2001)

Conversion factors a 1 ppm = 10.32 mg/m 3 Verschueren (2001) Empirical formula C 20 H 12 http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CHEM

a Calculated based on the ideal gas law, PV= n RT at 25°C: ppm = mg/m 3 × 24.45 ÷ molecular weight.

1

2 

### Inhalation 3

4 

### ATSDR (1995) reports average indoor concentrations of benzo[a]pyrene as 0.37 to 1.7 5 

### ng/m 3 

### for smokers and 0.27 to 0.58 ng/m 3 

### for non-smokers. Naumova et al. (2002) measured 6 

### PAHs in 55 nonsmoking residences in three urban are as during June 1999-May 2000. Mean 7 

### indoor benzo[a]pyrene levels ranged from 0.02 to 0. 078 ng/m 3 

### . They also reported outdoor 8 

### benzo[a]pyrene levels ranging from 0.025 to 0.14 ng /m 3 

### . They concluded that indoor levels of 9 

### the 5-7 ring PAHs (such as benzo[a]pyrene) were dom inated by outdoor sources and observed an 10 

### average indoor/outdoor ratio of approximately 0.7. Mitra and Wilson (1992) measured 11 

### benzo[a]pyrene air levels in Columbus, OH and found elevated indoor levels in homes with 12 

### smokers. They measured an average outdoor air conc entration of 1.38 ng/m 3 

### and indoor 13 

### concentrations of 0.07 ng/m 3 

### for homes with electrical utilities, 0.91 ng/m 3 

### for homes with gas 14 

### utilities, 0.80 ng/m 3 

### for homes with gas utilities and a fireplace, 2.75 ng/m 3 

### for homes with gas 15 

### utilities and smokers, and 1.82 ng/m 3 

### for homes with gas utilities, smokers, and a firep lace. 16 

### Mitra and Ray (1995) evaluated data on benzo[a]pyre ne air levels in Columbus, OH and reported 17 

### an average of 0.77 ng/m 3 

### inside homes and 0.23 ng/m 3 

### outdoors. Park et al. (2001) measured 18 

### ambient levels of benzo[a]pyrene in Seabrook, TX du ring 1995-1996. Based on continuous 19 

### measurements over this period, they found an averag e of 0.05 ng/m 3 

### (vapor plus particulate). 20 

### Parke et al. (2001) also reports average ambient ai r levels in ng/m 3 

### from other studies conducted 21 

### earlier as 1.0 for Chicago, 0.19 for Lake Michigan, 0.01 for Chesapeake Bay and 0.02 for Corpus 22 

### Christie, TX. Petry et al. (1996) conducted person al air sampling during 1992 at 5 workplaces in 23

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Switzerland: carbon anode production, graphite prod uction, silicon carbide production, bitumen 1 

### paving work, and metal recycling. These data are s ummarized in Table 2-2. 2 

### Santodonato et al. (1981) estimated adult daily int ake from inhalation as ranging from 9 3 

### to 43 ng/day. EC (2002) reported the following benz o[a]pyrene air levels in Europe during the 4 

### 1990’s: rural areas: 0.1 – 1 ng/m 3 

### and urban areas: 0.5 – 3 ng/m 3 

### . They estimated the mean 5



### intake via inhalation for an adult non-smoker as 20 ng/day. The data from exposure studies by 6 

### Naumova et al. (2002) suggest typical inhalation in takes may be lower (probably due in part to 7 

### the focus on nonsmoker residences). These data sug gest that air exposures are typically less than 8 

### 0.14 ng/m 3 

### which would result in 2 ng/day assuming a 13 m 3 

### /day inhalation rate (adult average 9 

### based on USEPA, 1997). 10

11 

### Table 2-2. Benzo[a]pyrene Concentrations in Air 12 

### Setting Year n Concentration (ng/m 3 

### ) reference

Outdoor - Urban Los Angeles, CA 1999-2000 19 0.065 Naumova et al., 2002 Houston, TX 1999-2000 21 0.025 Naumova et al., 2002 Elizabeth, NJ 1999-2000 15 0.14 Naumova et al., 20 02 Seabrook, TX 1995-1996 NA 0.05 Park et al. 2001 Columbus, OH 1986-1987 8 0.23 Mitra and Ray, 1995

Indoor Residential Los Angeles, CA 1999-2000 19 0.078 Naumova et al., 2002 Houston, TX 1999-2000 21 0.020 Naumova et al., 2002 Elizabeth, NJ 1999-2000 15 0.055 Naumova et al., 20 02 Columbus, OH 1986-1987 8 0.77 Mitra and Ray, 1995 Columbus, OH 10 0.07 - 2.75 Mitra and Wilson, 1992 Homes with smokers 0.37 – 1.7 ATSDR, 1995 Homes without smokers 0.27 – 0.58 ATSDR, 1995

Occupational: Aluminum production 30 - 530 ATSDR, 1995 Coke production 150 – 672 0 8000 ATSDR, 1995

Petry et al., 1996 Carbon anode production - Switzerland 1992 30 1100 Petry et al., 1996

Graphite production - Switzerland 1992 16 83 Petry et al., 1996

SiC production - Switzerland 1992 14 36 Petry et al ., 1996 Metal recovery - Switzerland 1992 5 14 Petry et al. , 1996 Bitumen paving - Switzerland 1992 9 10 Petry et al. , 1996 NA = Not Available 13

14 

### Airborne intake of benzo[a]pyrene in the environmen t predominantly occurs via 15 

### inhalation of insoluble carbonaceous particles (e.g . soot, diesel particles) to which organic 16 

### compounds, such as PAHs, are adsorbed. Reliable, q uantitative measurements of the percent 17

### 6 DO NOT CITE OR QUOTE

absorption of benzo[a]pyrene from insoluble particl es are not available; however, studies in 1 

### experimental animals indicate that benzo[a]pyrene i s readily absorbed from carbonaceous 2 

### particles following inhalation exposure (Gerde et a l., 2001; Hood et al., 2000). 3

4

5 

### Oral 6 

### The processing and cooking of foods is viewed as th e dominant pathway of PAH 7 

### contamination in foods (as reviewed by Bostrom, 200 2). Among the cooking methods that lead 8 

### to PAH contamination are the grilling, roasting and frying of meats. Raw meat, milk, poultry 9 

### and eggs will normally not contain high levels of P AH due to rapid metabolism of these 10 

### compounds in the species of origin. However, some marine organisms, such as mussels and 11



### lobsters are known to adsorb and accumulate PAH fro m water, which may be contaminated, for 12 

### example by oil spills. Vegetables and cereal grain s can become contaminated primarily through 13 

### aerial deposition of PAHs present in the atmosphere (Li 2009). 14 

### Kazerouni et al. (2001) measured benzo[a]pyrene in a variety of commonly consumed 15 

### foods collected from grocery stores and restaurants in Maryland (analyzed as a composite from 16 

### 4-6 samples of each food type). The foods were tes ted after various kinds of cooking. These 17 

### results are reported in Table 2-3. The concentrati ons were combined with food consumption 18



### data to estimate intake. The intakes of the 228 su bjects ranged from approximately 10 to 160 19 

### ng/d with about 30% in the 40 to 60 ng/day range. The largest contributions to total intake were 20 

### reported as bread cereal and grain (29%) and grille d/barbecued meats (21%). 21 

### Kishikawa et al. (2003) measured benzo[a]pyrene le vels in cow milk, infant formula and 22 

### human milk from Japan. They report the following m eans: cow milk - 0.03 ng/g (n=14), infant 23 

### formula – 0.05 ng/g (n=3) and human milk – 0.002 (n =51). 24 

### From the surveys conducted in six EU countries, the mean or national-averaged dietary 25



### intake of benzo[a]pyrene 

### for an adult person was estimated in the range 0.05 to 0.29 μ g/day 26 

### (European Commission[EC], 2002). In the UK, averag e intakes on a ng kg -1 

### day -1 

### basis were 27 

### estimated for the following age groups: adults – 1. 6, 15 to 18 years – 1.4, 11 to 14 years – 1.8, 7 28 

### to 10 years – 2.6, 4 to 6 years – 3.3 and toddlers 3.1 – 3.8. The major contributors were the oils 29 

### and fats group (50%), cereals (30%) and vegetables (8%) (EC, 2002). The contribution from 30 

### grilled foods appeared less important in Europe tha n the U.S. because grilled foods are consumed 31 

### less often (EC, 2002). 32

33 

### Table 2-3. Benzo[a]pyrene Levels in Food 34 

### Concentration (ng/g) Meat Fried or broiled beef 0.01 – 0.02 Grilled beef 0.09 – 4.9 Fried or broiled chicken 0.08 – 0.48 Grilled chicken 0.39 – 4.57

### 7 DO NOT CITE OR QUOTE

Fish 0.01 – 0.24 Smoked fish 0.1 Bread 0.1 Breakfast Cereals 0.02 – 0.3 Vegetable Oil 0.02 Eggs 0.03 Cheese <0.005 Butter <0.005 Milk 0.02 Fruit 0.01 – 0.17 Source: Kazerouni et al., 2001 1

2 

### Estimates of oral bioavailability from animal studi es range from about 30-70% (Ramesh 3 

### et al, 2001b; Cavret et al., 2003; Hecht et al., 19 79). Direct information regarding absorption of 4 

### benzo[a]pyrene in humans is limited. One study ind icated near 100% absorption of 5 

### benzo[a]pyrene in eight subjects exposed to benzo[a ]pyrene through the ingestion of charbroiled 6 

### meat (Hecht et al., 1979). Other dietary factors l ikely influence the oral absorption of 7 

### benzo[a]pyrene. In experimental animals, a high f at diet appears to increase absorption of 8 

### benzo[a]pyrene whereas a high fiber or protein rich diet appears to decrease absorption 9 

### (Kawamura et al., 1988; O’Neill et al., 1991; Mirvi sh et al., 1981). 10

11 

### Dermal 12

13 

### The general population can be exposed dermally to b enzo[a]pyrene when contacting soils 14 

### or materials which contain benzo[a]pyrene such as s oot or tar. Exposure can also occur via the 15 

### use of dermally applied pharmaceutical products whi ch contain coal tars, including formulations 16 

### used to treat conditions such as eczema and psorias is (IARC, 2010). 17 

### PAHs are commonly found in all types of soils. ATS DR (1995) reported benzo[a]pyrene 18 

### levels in soil for a variety of settings: 2-1,300 μ g/kg in rural areas, 4.6 – 900 μg/kg in 19 

### agricultural areas and 165-220 μg/kg in urban areas and 14,000-159,000 μg/kg at contaminated 20 

### sites (before remediation). The soil levels for al l land uses appear highly variable. The levels 21 

### are affected by proximity to roads/combustion sourc es, use of sewage sludge derived 22 

### amendments on agricultural lands, particle size and organic carbon content. Wilke (2000) 23 

### reports that PAH levels in soils have generally inc reased during the 1900’s and that sediment 24 

### studies suggest some declines may have occurred sin ce the 1970’s. An illustration of 25 

### benzo[a]pyrene levels in soil is presented in Table 2-4. 26

27 

### Table 2-4. Levels of benzo[a]pyrene in Soil 28 Reference Location Land Type Concentration Mean (μg/kg) Butler et al, 1984 UK Urban 1165 Vogt et al. 1987 Norway Industrial 321 Norway Rural 14 Yang et al. 1991 Australia Residential 363

### 8 DO NOT CITE OR QUOTE

Maliszewska, 1996 Poland Agricultural 22 Trapido, 1999 Estonia Urban 106 Estonia Urban 398 Estonia Urban 1113 Estonia Urban 1224 Estonia Rural 6.8 Estonia Rural 15 Estonia Rural 27 Estonia Rural 31 Nam et al., 2008 UK Rural 46 Norway Rural 5.3 Mielke et al. 2001 New Orleans Urban 276Nadal et al, 2004 Spain Industrialchemical 100

Spain Industrialpetrochemical 18

Spain Residential 56 Spain Rural 22 Maliszewska, 2009 Poland Agricultural 30 Wilkce, 2000 Various temperate Arable 18 Various temperate Grassland 19 Various temperate Forest 39 Various temperate Urban 350 Bangkok Urban-tropical 5.5 Brazil Forest-tropical 0.3

1

2 

### A number of studies have measured dermal absorption of benzo[a]pyrene from soil (Turkall et 3 

### al. 2008; Moody et al, 2007; Roy and Singh, 2001; R oy et al, 1998; Wester et al, 1990; Yang et 4 

### al, 1989). These studies utilize in vitro and in v ivo testing in a variety of animal species and in 5 

### vitro testing with human skin samples. . The abso rption percentages of benzo[a]pyrene from 6 

### soil, as tested in vitro for human skin, ranged fro m 0.9 to 15% (Moody et al., 2007; Roy et al., 7 

### 1998; Wester et al., 1990). However, major methodological differences between t hese studies 8 

### exist including whether the amount of benzo[a]pyren e left in the skin depot was included as part 9 

### of the absorbed fraction or whether only benzo[a]py rene or its metabolites passing into the 10 

### receptor fluid was quantified. 11 

### These studies of benzo[a]pyrene absorption from so il suggest that reduced absorption of 12 

### benzo[a]pyrene occurs with increasing organic carbo n content and clay content of the soils. 13 

### They also indicate that dermal absorption increases as soil aging decreases (ie. contact time 14 

### between soil and chemical). 15

16

### 9 DRAFT - DO NOT CITE OR QUOTE 3. TOXICOKINETICS 1

2

3 

### Benzo[a]pyrene is one of the most extensively studi ed PAH compounds. Numerous 4 

### primary reports and secondary reviews are present i n the scientific literature that describe the 5 

### toxicokinetics of benzo[a]pyrene following oral, in halation, and dermal exposures. 6 

### Benzo[a]pyrene is absorbed following exposure by in halation, oral, and dermal routes. The rate 7 

### and extent of absorption are dependent upon the exp osure medium. For example, bioavailability 8 

### of benzo[a]pyrene is dependent on vehicle character istics and adsorption to particles. The 9 

### presence of benzo[a]pyrene in body fat, blood, live r, and kidney and the presence of 10 

### benzo[a]pyrene metabolites in serum and excreta ind icate wide tissue distribution. 11 

### Benzo[a]pyrene metabolism occurs in essentially all tissues, with high metabolic capacity in the 12 

### liver and significant metabolism in tissues at the portal of entry (lung, skin, and gastrointestinal 13 

### [GI] tract) and in reproductive tissues. Stable me tabolic products identified in body tissues and 14 

### excreta are very diverse and include phenols, quino nes, and dihydrodiols. These classes of 15 

### metabolites are typically isolated as glucuronide o r sulfate ester conjugates in the excreta but can 16 

### also include glutathione conjugates formed from qui nones or intermediary epoxides. The 17 

### primary route of metabolite elimination is in the f eces, particularly following exposure by the 18 

### inhalation route. To a lesser degree benzo[a]pyren e metabolites are eliminated via urine. 19 

### Overall, benzo[a]pyrene is eliminated quickly with a biological half-life of several hours. 20

21



### 3.1. ABSORPTION 22 

### 3.1.1. Inhalation Exposure 23



### 3.1.1.1. Inhalation Exposure in Humans 24 

### The absorption of benzo[a]pyrene is frequently asse ssed by identification of 25 

### benzo[a]pyrene metabolites in the urine of people e xposed to emissions from combustion 26 

### processes. Because of the nature of these processe s, oral and dermal exposures are likely to 27 

### accompany exposure through the inhalation route, re ndering estimates of inhalation-only 28 

### exposure rather imprecise. For these reasons, quan titative estimates of absorption via the 29 

### respiratory tract cannot be derived from these stud ies. Nevertheless, the observation of 30 

### benzo[a]pyrene metabolites (as well as DNA adducts) in tissues and excreta of exposed humans 31 

### provides qualitative evidence for benzo[a]pyrene ab sorption, at least some of which is likely to 32 

### be via the respiratory tract. 33 

### Becher and Bjørseth (1983) studied urinary excretio n of 11 PAHs in highly exposed 34 

### Norwegian aluminum smelter workers (11 exposed: 7 smokers, 4 nonsmokers; 9 controls: 35 

### 5 smokers, 4 nonsmokers). The authors compared uri nary excretion of parent compound to that 36 

### of hydroxy-metabolites (determined as parent compou nd following a chemical reduction 37 

### procedure) and found that, in the case of benzo[a]p yrene, 92% of the total excreted were 38

### 10 DRAFT - DO NOT CITE OR QUOTE

metabolites. These values differed widely for the other 10 PAHs, several of which could be 1 

### detected only following the reduction procedure. E xposed nonsmoking workers excreted 0.104 2



### ± 0.154 μ g total benzo[a]pyrene per mmol creatinine (range: 0.002–0.37; parent compound plus 3 

### metabolites) in urine (0.0153 ± 0.016 without one e xtreme outlayer), while smoking workers 4



### excreted 0.025 ± 0.016 μ g/mmol creatinine (range: 0.01–0.063); the differe nce was not 5 

### significant. Total PAH excretion in the urine of a luminum plant workers was also higher in 6 

### nonsmokers than in smokers (6.61 ± 3.59 vs. 5.65 ± 2.31 μ g/mmol creatinine). Air 7 

### concentrations of PAHs in the aluminum reduction pl ant were typically 100 μ g/m 3 

### . The authors 8 

### concluded that neither high occupational exposure t o PAHs nor smoking status provided accurate 9 

### determinants for PAH body burdens and that interind ividual differences in absorption or 10 

### metabolism played a major role. 11 

### Grimmer et al. (1994) measured PAH metabolites in t he 24-hour urines of four coke oven 12 

### workers whose exposure to PAHs had been monitored w ith personal air samplers on 13 

### 4 consecutive workdays. They observed a correlatio n between benzo[a]pyrene amounts 14 

### extracted from the sampler filters and benzo[a]pyre ne-9,10-dihydrodiol concentrations in urine. 15 

### Urinary concentrations following similar levels of exposure, however, varied by a factor of about 16 

### 5 among the four workers, which the authors attribu ted to differences in genetically determined 17 

### metabolism. One of the central findings in that st udy was that only a very small fraction of the 18 

### inhaled benzo[a]pyrene (0.013%) was recovered from urine, suggesting poor pulmonary 19 

### absorption, poor metabolism, or that urine is not a major route for excretion of benzo[a]pyrene. 20 

### In the case of phenanthrene and pyrene (Py), percen tages recovered from urine were at least 21 

### fivefold higher. 22 

### Gündel et al. (2000) studied the urinary excretion of metabolites of eight PAHs, among 23 

### them benzo[a]pyrene, in 19 workers at a fireproof s tone manufacturing plant in Germany, and 24 

### provided concentrations in the air to which the wor kers were exposed. In the case of 25 

### benzo[a]pyrene, the median for personal air sampler s was 1.07 μ g/m 3 

### (range: 0.043–2.96), and 26 

### the median for stationary air sampling was 1.31 μ g/m 3 

### (range: 0.63–5.41). Other PAH air 27 

### concentrations ranged from 0.11 μ g/m 3 

### (dibenz[a,h]anthracene) to 4.85 μ g/m 3 

### (chrysene). The 28 

### median for urinary excretion of 3-OH-benzo[a]pyrene (the only benzo[a]pyrene metabolite 29 

### evaluated) was 1.58 ng/mmol creatinine (range: 0.3 4–22.6). This was by far the lowest level of 30 

### PAH metabolites found in the urine of exposed worke rs; for comparison, phenanthrene, which 31



### showed almost the same median concentration as benz o[a]pyrene in personal air samplers 32 

### (1.08 μ g/m 3 

### ), produced a total of 679 ng/mmol creatinine in th e form of metabolites (range: 33 

### 205–4,700). (The author’s values were given as μ g/g creatinine and recalculated using a mol. wt. 34 

### of 113.12 for creatinine. Values for phenanthrene metabolites were obtained by addition of 35 

### urinary concentrations of four metabolites.) The a uthors pointed out that they were not able to 36



### detect a correlation between the levels of individu al PAH exposures and urinary excretion of 37



### related metabolites. 38

### 11 DRAFT - DO NOT CITE OR QUOTE

Wu et al. (2002) found a statistically significant correlation between trans

- anti

1 

### benzo[a]pyrene-tetrol in the urine of coke oven wor kers and PAH concentrations in benzene 2 

### extracts obtained from personal air monitoring devi ces. These workers were exposed to a variety 3 

### of PAHs, including benzo[a]pyrene. The results wer e not influenced by smoking or alcohol 4 

### consumption habits. However, genetic factors had s ome influence on urinary trans

- anti

5 

### benzo[a]pyrene-tetrol levels (e.g., workers homozyg ous for the cytochrome (CYP)1A1 MspI 6 

### variant displayed 27% higher urinary tetrol levels than did workers heterozygous or wild type 7 

### [WT] for this variant). There was also a statistic ally significant correlation between urinary 8



### levels of trans

- anti -benzo[a]pyrene-tetrol and 1-hydroxypyrene (1-OH-Py ), a metabolite not 9 

### derived from benzo[a]pyrene, but from Py metabolism that is frequently used for assessment of 10 

### PAH exposure. 11 

### Hecht et al. (2003) attempted to establish a proced ure for the assessment of PAH 12 

### exposure by measurement of urinary metabolites of p henanthrene. They compared levels of 13 

### r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenan threne to those of 1-OH-Py or 14 

### r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenz o[a]pyrene ( trans-anti -benzo[a]pyrene tetrol) 15 

### in the urine of psoriasis patients treated with coa l tar, of coke oven workers, and of smoking or 16 

### nonsmoking unexposed control persons. They demonst rated statistically significant correlations 17 

### among all three metabolites in the urine of coke ov en workers but not in psoriasis patients, 18 

### despite the fact that the latter had 13- to 94-fold higher urinary metabolite concentrations. In 19 

### controls, only trans-anti -PheT vs. 1-OH-Py was assessed, and the correlation was statistically 20 

### significant. The authors emphasized that urinary c oncentrations of trans-anti -benzo[a]pyrene 21 

### tetrol were 8,000–19,000 times lower than those of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4- 22 

### tetrahydrophenanthrene. In a similar attempt, Arie se et al. (1994) tried to establish a correlation 23 

### between urinary 1-OH-Py and 3-OH-benzo[a]pyrene and found a significant correlation in 24 

### unexposed controls but not in exposed coke oven wor kers, who displayed significantly elevated 25 

### 1-OH-Py levels but no corresponding elevation of 3- OH-benzo[a]pyrene. 26 

### The available data from human exposure studies prov ide qualitative evidence of 27 

### benzo[a]pyrene absorption via the respiratory tract and also indicate that, in comparison with 28 

### other PAHs, benzo[a]pyrene is absorbed in an unpred ictable fashion. Most authors appear to 29 

### assume that, in occupational settings, exposure occ urs predominantly by the inhalation route. 30 

### Occupational inhalation exposure to air contaminate d with high levels of benzo[a]pyrene does 31 

### not necessarily result in correspondingly elevated excretion of benzo[a]pyrene metabolites. 32 

### These qualitative observations in humans are suppor ted by inhalation and instillation 33



### toxicokinetic studies in animals. 34

35



### 3.1.1.2. Inhalation Exposure in Animals 36 

### Gerde et al. (1993a) conducted a series of studies on the disposition of benzo[a]pyrene in 37 

### the respiratory tract of dogs. Seven-year-old fema le beagle dogs (n = 3) were exposed to a bolus 38

### 12 DRAFT - DO NOT CITE OR QUOTE

of 77 mg aerosolized benzo[a]pyrene crystals (parti cle size <1 μ m) injected directly into the 1 

### trachea of the animals during a single breath. Blo od levels of benzo[a]pyrene in the ascending 2 

### aorta and right atrium were monitored to evaluate t he rate of appearance of benzo[a]pyrene in the 3 

### systemic circulation. Benzo[a]pyrene concentration s in the blood built up rapidly and peaked at 4 

### 1.8 minutes. Over the 15-minute time period after dosing, approximately 92% of the 5 

### administered benzo[a]pyrene was cleared from the lu ngs. The half-life for lung clearance was 6 

### 2.4 minutes. Comparing lung clearance rates for be nzo[a]pyrene and the less lipophilic 7 

### phenanthrene, which is cleared more quickly, the au thors concluded that the clearance rate was 8 

### limited by diffusion through the alveolar septa for highly lipophilic compounds such as 9 

### benzo[a]pyrene. A half-life of 2 hours was estimat ed for clearance of absorbed benzo[a]pyrene 10 

### from the blood. These data demonstrate that absorp tion of benzo[a]pyrene from the pulmonary 11 

### portion of the respiratory tract of animals (as opp osed to the tracheal, bronchial, and bronchiolar 12 

### portions) is nearly complete and very rapid. 13 

### In a second study in this series, Gerde et al. (199 3b) determined the disposition of 14 

### benzo[a]pyrene in the conducting airways by instill ing benzo[a]pyrene (dissolved in 20 μ L saline 15 

### and administered as a mist) into a main stem bronch us or distal portion of the trachea of female 16 

### beagle dogs (n = 4). Benzo[a]pyrene retained in mu cus was determined from lavage samples to 17 

### assess the degree to which benzo[a]pyrene was trans ferred from the mucus into the bronchial 18 

### epithelium. Roughly 34% of the benzo[a]pyrene was retained in the mucus collected 1 minute 19 

### after instillation. Benzo[a]pyrene in the mucus cl eared at the same rate as inert particles (90 20 

### mm/minute), suggesting transport via the mucociliar y escalator. The estimated half-life for 21 

### clearance of benzo[a]pyrene from the mucus to the r espiratory epithelium was 9.5 minutes. 22 

### Therefore, the mucociliary escalator acted like a r ather shallow pool for benzo[a]pyrene. 23 

### Absorption into the airway walls was evaluated by i nstilling benzo[a]pyrene solutions into the 24 

### upper bronchial tree and measuring benzo[a]pyrene a nd metabolites in tissues. Approximately 25 

### 20% of the benzo[a]pyrene dose penetrated into the epithelium of the left and right bronchus, 26 

### respectively, within 45 minutes and cleared from th e main stem bronchi with a half-time of 1.4 27 

### hours. Benzo[a]pyrene metabolites were also measur ed in main stem airway segments (trachea 28 

### and bronchi were dissected into approximately 1-cm- long segments). The percent of 29 

### benzo[a]pyrene recovered as parent compound was 62 and 33% after 45 minutes and 1.5 hours, 30 

### respectively. The pattern of metabolites varied ac ross these segments. Benzo[a]pyrene tetrols 31 

### ranged from 9 to 37% and benzo[a]pyrene-9,10-diol r anged from 3 to 18% of the administered 32



### dose. About 8% of the administered dose was covale ntly bound to tissues. The authors 33 

### concluded that the significantly higher retention t ime in the bronchi, as compared to pulmonary 34 

### epithelium, made the conducting portion of the resp iratory tract a possible target of 35 

### benzo[a]pyrene toxicity. 36 

### In the third paper of the series, Gerde et al. (199 3c) used the results from the previous 37 

### two studies to evaluate benzo[a]pyrene dosimetry in the respiratory tract. The authors concluded 38

### 13 DRAFT - DO NOT CITE OR QUOTE

that benzo[a]pyrene uptake occurs via diffusion-lim ited transport through the lung epithelium. 1 

### Uptake by the alveolar epithelium took only a few m inutes while uptake by the airway 2 

### epithelium took hours, due to the much thicker air/ blood barrier in the conducting airways. This 3 

### would result in longer residence time in the respir atory tract tissues, where benzo[a]pyrene can 4 

### be metabolized to reactive metabolites, making the conducting airways an important target for 5 

### benzo[a]pyrene. 6 

### This series of studies by Gerde et al. (1993a, b, c ) assessed the absorption of 7 

### benzo[a]pyrene in the respiratory tract using an ae rosolized solution of benzo[a]pyrene in saline. 8 

### However, other studies in animals assessed the bioa vailability of benzo[a]pyrene in the lung 9 

### using benzo[a]pyrene adsorbed onto particle substra tes. Gerde et al. (2001) evaluated the 10 

### bioavailability of diesel soot-adsorbed benzo[a]pyr ene in three 1-year-old beagle dogs (n = 3). 11 

### Soot particles were denuded by toluene extraction a nd benzo[a]pyrene was adsorbed onto the 12 

### soot as a surface coating. The dogs were exposed t o a single, 220 mL bolus of aerosolized 13 

### benzo[a]pyrene-coated diesel soot (mass median aero dynamic diameter [MMAD] 1.3 ± 0.2 μ m), 14 

### followed by 90 mL of clean air to facilitate delive ry of benzo[a]pyrene to the alveolar region, 15 

### and arterial and venous blood samples were taken at intervals over a 1-hour period. In separate 16 

### tests, the amount of benzo[a]pyrene deposited was d etermined to be 36 ± 20 μ g (n = 6). The 17 

### concentration of benzo[a]pyrene in the blood peaked at about 2 minutes and the first half-life of 18 

### absorption was approximately 4 minutes. Although o ne dog received approximately seven times 19 

### the dose of benzo[a]pyrene than the other two, prob ably due to variability in the aerosol 20 

### generation technique, the fractional retention of b enzo[a]pyrene in the lung was similar in all 21 

### three dogs, indicating first-order absorption kinet ics. The initial absorption was rapid; <10% of 22 

### the dose remained in the lung after 30 minutes. Ho wever, there was a small fraction of 23 

### benzo[a]pyrene that remained adsorbed to the soot e ven after 5.6 months in the lungs, when the 24 

### fraction of material coating the particles had decr eased to approximately 16% of what would be a 25 

### monolayer of benzo[a]pyrene molecules deposited on the soot particles. The authors suggested 26 

### that the small portion of tightly adsorbed benzo[a] pyrene reflected limited high-energy binding 27 

### sites that cover only a fraction of the soot partic le surface. Benzo[a]pyrene was further released 28 

### from particles transported to the lymph nodes to ap proximately 10% of a monolayer coating, 29 

### which may reflect the more reactive chemical enviro nment provided by alveolar macrophages. 30 

### Only 30% of the benzo[a]pyrene that remained bound to particles was present as parent 31 

### compound. Based on these results, most of the adso rbed benzo[a]pyrene was readily released 32 

### from diesel soot into the systemic circulation, mos tly as parent benzo[a]pyrene (with only a 33 

### minor portion as metabolites), while a small fracti on was released from the soot at a much slower 34



### rate. 35 

### Ramesh et al. (2001a) conducted a toxicokinetic stu dy of inhaled benzo[a]pyrene in F344 36 

### rats. The rats were exposed for a single 4-hour pe riod via nose-only inhalation to aerosol 37 

### concentrations of 0.1, 1.0, or 2.5 mg/m 3 

### of benzo[a]pyrene adsorbed to carbon black (CB) 38

### 14 DRAFT - DO NOT CITE OR QUOTE

particles. The particle size distribution was mono disperse and largely in the respirable range (the 1 

### reported MMAD was 1.7 μ m with a geometric standard deviation [SD] of 0.085 μ m), suggesting 2 

### that the results reflect absorption from the entire lung, since particles of the size distribution use d 3 

### here are expected to be deposited in all respirator y tract regions. Plasma and lung tissue 4 

### concentrations of benzo[a]pyrene and metabolites we re evaluated at 30, 60, 120, and 240 5 

### minutes postexposure. The plasma benzo[a]pyrene co ncentration peaked at 1 hour postexposure, 6



### and 65% of the inhaled aerosol was cleared from the lung at 2 hours postexposure, presumably at 7 

### all dose levels, though this was not stated explici tly in the study. There was a significant 8 

### difference in the time course of plasma levels betw een male and female rats. Female plasma 9 

### benzo[a]pyrene levels were about one-third lower at 30 minutes, about 28% higher at 1 hour, and 10 

### marginally lower than the male levels at later time points. This study is limited by the fact that 11 

### administered aerosol concentration was reported ins tead of deposited dose, plasma samples were 12 

### not collected during the 4-hour exposure period, an d the study could not distinguish between 13 

### absorption from the respiratory tract and mucocilia ry clearance followed by absorption from the 14 

### gut. 15 

### Rapid absorption through the lungs was also shown f ollowing intratracheal 16



### administration of 1 μ g/kg body weight [ 3 

### H]-benzo[a]pyrene dissolved in triethylene glycol i n 17 

### male Sprague-Dawley rats (Weyand and Bevan, 1986). Elimination of radiolabel from the lung 18 

### was biphasic with half-lives of 5 and 116 minutes. The highest levels of liver radiolabel, 19 

### equivalent to 21% of the administered dose, were fo und within 10 minutes after exposure, 20 

### suggesting rapid absorption from the upper respirat ory tract. The authors noted that it was 21 

### unlikely that the appearance of radiolabel in organ s was due to GI tract absorption after 22 

### mucociliary clearance because the tracheal cannula was left in place for the entire experiment 23 

### and levels of radioactivity in the stomach increase d only slowly. Based on a comparison of 24 

### benzo[a]pyrene concentrations in the blood followin g intratracheal administration versus 25 

### intravenous (i.v.) dosing, the authors calculated t he pulmonary bioavailability of benzo[a]pyrene 26 

### as 57%. A significant degree of metabolism occurre d in the lungs (as measured by the 27 

### concentration of metabolites in lung), suggesting t hat benzo[a]pyrene absorption into the 28 

### systemic circulation is limited by first-pass metab olism in the lung. 29 

### Petridou-Fischer et al. (1988) applied 10 μ L aliquots of [ 14 

### C]-benzo[a]pyrene in a 30 

### gelatin:saline solution over a 2-hour period to the ethmoid and maxillary nasal turbinates of two 31 

### female cynomolgus monkeys and four male beagle dogs to assess differences in benzo[a]pyrene 32 

### disposition in portions of the nose. The dose of b enzo[a]pyrene was not provided, but using the 33 

### total radioactivity administered (93 μ Ci per animal) and the specific activity of the rad iolabeled 34 

### benzo[a]pyrene that was used (39 mCi/mmol), a total administered dose of 0.6 mg/animal can be 35 

### calculated. (98 μ Ci × 1 mmol/39 mCi × 1 mCi/1,000 μ Ci × 252 mg benzo[a]pyrene/mmol = 36 

### 0.6 mg/animal.) No radioactivity was found in bloo d (collected over 2 hours in dogs and 3 hours 37 

### in monkeys), and very little radioactivity was iden tified in excreta. Urinary excretion reached a 38

### 15 DRAFT - DO NOT CITE OR QUOTE

maximum of 0.69% of the administered dose in dogs a nd 0.07% in monkeys, while in feces a 1



### maximum of 6.42% of the administered dose was recov ered in dogs and 1.17% in monkeys over 2 

### a period of 48 hours. These results suggest only l imited systemic absorption of benzo[a]pyrene 3



### from the nasal turbinates under the test conditions used. The results of this study are in contrast 4



### to more traditional inhalation or intratracheal ins tillation experiments, which have demonstrated 5 

### significant absorption via other portions of the re spiratory tract following inhalation or 6 

### intratracheal instillation. Little radioactivity w as recovered from the mucus, blood, or excreta, 7 

### suggesting that benzo[a]pyrene and its metabolites were sequestered in the nasal tissues. 8 

### Several studies demonstrated rapid desorption of be nzo[a]pyrene bound to particles. 9 

### However, the adsorption matrix can impact the bioav ailability of inhaled benzo[a]pyrene. Leung 10 

### et al. (1988) reported that benzo[a]pyrene adsorbed on diesel soot particles and suspended in 11



### buffer was transferred to microsomes in vitro far l ess efficiently than free benzo[a]pyrene. The 12 

### authors concluded that benzo[a]pyrene transfer to m icrosomes depends on the lipid content of 13 

### the particles rather than on protein in the medium. Microsomes may enhance the slow transfer of 14 

### benzo[a]pyrene from particles, which may become an important source of exposure with long 15 

### retention times. No metabolism of benzo[a]pyrene a dsorbed to particles was detected in this 16 

### study, suggesting that particle-bound benzo[a]pyren e serves as a slow release source of 17 

### benzo[a]pyrene to the respiratory tract. These fin dings are consistent with the report by Gerde et 18 

### al. (2001) that a slow-release phase follows the in itial rapid desorption of benzo[a]pyrene from 19 

### diesel soot. Furthermore, Gerde and Scholander (19 89) found in an in vitro study that the release 20 

### from carrier particles was the rate-limiting step i n the absorption of benzo[a]pyrene by the 21 

### bronchial epithelium. 22 

### The absorption of inhaled benzo[a]pyrene may also b e affected by the size of the particle 23 

### to which it is adsorbed. Elimination of benzo[a]py rene from the lungs of mice was investigated 24 

### following intratracheal administration of benzo[a]p yrene crystals (0.5–1.0 μ m in size) or 25 

### benzo[a]pyrene-coated carbon particles (0.5–1.0 μ m or 15–30 μ m) (Creasia et al., 1976). 26 

### Approximately 50% of the benzo[a]pyrene crystals we re cleared within 1.5 hours and >95% 27



### were cleared within 24 hours of treatment. In cont rast, benzo[a]pyrene clearance was 28 

### approximately 50% after 36 hours following exposure to benzo[a]pyrene absorbed onto small 29 

### carbon particles. With larger carbon particles, de sorption was even slower, requiring 4–5 days to 30 

### release 50% of bound benzo[a]pyrene. The differenc e in absorption rate for small versus large 31 

### carbon particles suggests an influence of particle area surface on the rate of desorption. 32 

### The deposition, retention, and bioavailability of b enzo[a]pyrene as a pure aerosol or 33 

### adsorbed onto gallium oxide ( 67 

### Ga 2 

### O 3 

### ) particles was investigated by Sun et al. (1982). Male and 34 

### female F344 rats were exposed nose-only for 30 minu tes to atmospheres containing 0.6 mg/m 3 35 

### [ 3 

### H]-benzo[a]pyrene absorbed onto 67 

### Ga 2 

### O 3 

### or to 1.0 μ g/L neat (i.e., the pure chemical) [ 3 

### H]- 36 

### benzo[a]pyrene (MMADs were reported as approximatel y 0.1 μ m in both cases). Radiolabel was 37 

### detected in the esophagus, stomach, small and large intestines, cecum, liver, kidney and blood; 38

### 16 DRAFT - DO NOT CITE OR QUOTE

however, the time to reach peak tissue concentratio ns differed considerably between the two 1 

### exposure regimens. Based on the total amount excre ted, 22% of the inhaled dose of 2 

### benzo[a]pyrene on Ga 2 

### O 3 

### was released over 16 days, but only 10% of the inh aled dose of pure 3 

### benzo[a]pyrene was released. Since the amount excr eted can reflect differences in absorption, 4 

### i.e., uptake via pulmonary epithelium vs. ingestion of cleared particles, and hence alternative 5 

### tissue distribution and metabolism, this result can not be used quantitatively to estimate 6 

### bioavailability of inhaled benzo[a]pyrene. The stu dy established that benzo[a]pyrene adsorbed 7 

### to particles had a longer respiratory tract retenti on period. For benzo[a]pyrene coated on Ga 2 

### O 3 

### , 8 

### 1 day was required to clear 90% of the [ 3 

### H]-benzo[a]pyrene lung and trachea burdens that wer e 9 

### present 30 minutes after exposure. In contrast, on ly 1.5 and 4 hours were required to clear 90% 10 

### of pure benzo[a]pyrene burdens from the lung and tr achea, respectively. A different effect was 11 

### observed in the nose, where clearance of 90% benzo[ a]pyrene coated on Ga 2 

### O 3 

### required 7 hours 12 

### as compared to 20 hours for pure benzo[a]pyrene aer osol. Thereafter, clearance curves for 13 

### particle-bound and neat benzo[a]pyrene were similar ; the authors attributed this to the absence of 14 

### a mucociliary escalator in the nose. Inhalation of benzo[a]pyrene on Ga 2 

### O 3 

### also increased the 15 

### dose of the compound and its metabolites to the sto mach, liver, and kidneys, which the authors 16 

### suggest may have resulted from mucociliary clearanc e with subsequent ingestion. 17 

### benzo[a]pyrene absorption from the respiratory trac t may also be affected by the 18 

### characteristics of the vehicle. Following intratra cheal administration in hydrophilic triethylene 19 

### glycol, approximately 70% of the benzo[a]pyrene adm inistered was excreted within 6 hours by 20 

### male Sprague-Dawley rats (Bevan and Ulman, 1991). In contrast, 58.4% and 56.2% of 21 

### administered benzo[a]pyrene were excreted within a 6-hour period when the lipophilic solvents 22 

### ethyl laurate and tricaprylin, respectively, were t he vehicles. 23 

### Pregnant Wistar rats were exposed head-only for 95 minutes on gestational day (GD) 17 24 

### to 200, 350, 500, 650, or 800 mg/m 3 

### of a [ 3 

### H]-benzo[a]pyrene microcondensate generated from 25 

### heated pure material (Withey et al., 1993). Partic le sizes ranged from 0.61 to 0.88 μ m MMAD. 26 

### Immediately following exposure (no time estimate wa s provided), blood radiolabel 27 

### concentrations varied >eightfold over the fourfold dose range (2.66 ± 0.51 vs. 21.96 ± 1.37 μ g/g 28 

### at lowest and highest dose, respectively). Six hou rs after exposure, blood radiolabel 29



### concentrations had decreased two- to fourfold from the earlier observation but retained a >10- 30 

### fold difference over the dose range (0.74 ± 0.12 vs . 9.56 ± 2.1 μ g/g). Therefore, the difference in 31 

### the ratio of blood levels to dose range was not lik ely due to an initial rapid phase of 32 

### benzo[a]pyrene absorption only at high exposures. However, the study authors did not provide 33 

### an explanation for this finding. Radiolabel was de tected at 0 and 6 hours in all maternal tissues 34 

### and in fetuses examined, indicating that systemic a bsorption had occurred. 35 

### Hood et al. (2000) exposed male and timed pregnant female Sprague-Dawley rats to 36



### 100 μ g/m 3 

### benzo[a]pyrene-CB aerosol (nose only for 4 hours o n GD 15), and collected blood 37 

### was analyzed at 30, 60, 120, 180, and 240 minutes f or concentrations of benzo[a]pyrene. The 38

### 17 DRAFT - DO NOT CITE OR QUOTE

benzo[a]pyrene aerosol particle distribution was tr imodal with a significant portion of particles 1



### <1 μ m in size. The particle size distribution was expe cted to result in deposition across all 2 

### regions of the respiratory tract, including the pul monary region. Following exposure, blood 3 

### benzo[a]pyrene levels peaked at 30 minutes (the fir st time point reported), with females 4 

### exhibiting approximately a 1.7-fold higher peak con centration than males. At later time points, 5 

### the female benzo[a]pyrene blood concentrations were similar to those observed in males. By 6 

### 240 minutes postexposure benzo[a]pyrene in blood ha d diminished to <5% of the peak level. 7 

### The authors did not report a mass balance to allow for the determination of the percentage of 8 

### dose that was absorbed. Furthermore, benzo[a]pyren e metabolites were not measured. 9 

### Nevertheless, the appearance of benzo[a]pyrene in t he blood at the earliest time point measured 10 

### is consistent with the conclusion that benzo[a]pyre ne is rapidly absorbed from the respiratory 11 

### tract. Although the peak came earlier than in the Ramesh et al. (2001a) study, the same trend of 12 

### gender differences was observed, with females displ aying higher peak blood levels than males. 13 

### In summary, although quantitative estimates of huma n lung absorption are not available, 14 

### existing toxicity and biological monitoring studies suggest that benzo[a]pyrene is absorbed in the 15 

### respiratory tract, albeit rather poorly, following inhalation exposure of humans. The evidence 16 

### suggests, however, that in humans it is difficult t o establish a relationship between 17 

### benzo[a]pyrene exposure and urinary excretion of it s metabolites due to large interindividual 18 

### variation, most likely the result of different gene tic makeups and varying background exposures. 19 

### Numerous controlled studies indicate that benzo[a]p yrene is well absorbed in animals following 20 

### inhalation or intratracheal instillation. In gener al, the animal studies show that benzo[a]pyrene is 21 

### absorbed rapidly (within minutes) and extensively. The rate of absorption varies across regions 22 

### of the respiratory tract, with more rapid absorptio n in the pulmonary regions and slower 23 

### absorption in the conducting airways and nose. In some studies in rats, blood benzo[a]pyrene 24 

### peak levels at early exposure time points differed between females and males, but leveled out at 25 

### later time points. Quantitative estimates of benzo [a]pyrene absorption from the respiratory tract 26



### are difficult to derive because the contribution of absorption from the GI tract following 27 

### mucociliary clearance and metabolism of benzo[a]pyr ene in the respiratory tract itself are often 28 

### difficult to determine. Another complication in in terpreting these studies is that benzo[a]pyrene 29 

### absorption from the lung depends on the characteris tics of the exposure vehicle or the nature of 30 

### the particle to which benzo[a]pyrene is adsorbed. In general the data indicate that 31 

### benzo[a]pyrene is released to a greater extent from hydrophilic vehicles than from lipophilic 32 

### solvents; particle-bound benzo[a]pyrene is released more slowly than the neat compound; and 33 

### desorption from large particles is slower than from smaller particles. Because much of the 34 

### environmental benzo[a]pyrene is adsorbed onto parti cles of other materials, the effect of the 35 

### carrier particle is highly relevant to environmenta l exposures. 36

37 

### 3.1.2. Oral Exposure 38

### 18 DRAFT - DO NOT CITE OR QUOTE

3.1.2.1. Oral Exposure in Humans 1 

### In a study with eight volunteers who ingested broil ed meat containing approximately 2



### 8.6 μ g of benzo[a]pyrene, the concentration of benzo[a]p yrene in feces was below detection 3 

### limits (<0.1 μ g/person) (Hecht et al., 1979). Although the analy tical method used in this study 4 

### assessed only parent compound, not fecal metabolite s, the result can be interpreted as indicating 5 

### that the ingested benzo[a]pyrene was absorbed compl etely from the GI tract. In addition, studies 6



### were conducted to assess DNA adduct levels or benzo [a]pyrene metabolites in humans exposed 7 

### to PAHs by the oral route. In general, these human dietary studies are not adequate to develop 8 

### quantitative estimates of oral bioavailability; in one case no measurable relationship between 9 

### benzo[a]pyrene intake and internal dose measure was found (Scherer et al., 2000). 10

11



### 3.1.2.2. Oral Exposure in Animals 12 

### The bioavailability of benzo[a]pyrene was evaluated in F344 rats dosed by gavage with 13 

### 100 mg/kg benzo[a]pyrene dissolved in peanut oil an d sacrificed 0, 0.5, 1.0, 2.0, 4.0, 8.0, 24, 48, 14 

### or 72 hours after dosing (Ramesh et al., 2001b). B lood, liver, reproductive tissues, urine and 15 

### feces were analyzed for benzo[a]pyrene and metaboli tes. Plasma benzo[a]pyrene levels peaked 16 

### at 8 hours and fecal levels at 2.4 hours postexposu re. Lipophilic metabolites of benzo[a]pyrene 17 

### peaked at 2 hours in liver, 8 hours in feces, 24 ho urs in blood and lung, and 48 hours 18 

### postexposure in urine. Water-soluble metabolites r eached their maxima at 4 hours in liver and 19 

### lung, 8 hours in blood and feces, and 48 hours in u rine. Some of the disposition patterns 20 

### displayed minor peaks at earlier time points. Base d on comparison with plasma levels following 21 

### i.v. injection, the oral bioavailability was estima ted by the authors as approximately 40%; 22 

### however, the details of this determination were not presented. 23 

### Foth et al. (1988) conducted several experiments to determine the oral bioavailability of 24 

### benzo[a]pyrene. In male Sprague Dawley rats, the a reas under the blood concentration-time 25 

### curve (AUCs) following oral versus i.v. bolus doses of benzo[a]pyrene (dissolved in Krebs 26 

### Ringer buffer with 4% bovine serum albumin) were co mpared. The oral bioavailability was 27



### estimated as 7.8 and 11.5% for doses of 3.2 and 4.0 nmol [ 3 

### H]-benzo[a]pyrene per rat 28 

### (approximately 1.8–2.7 and 2.2–3.4 μ g/kg, respectively, calculated as 3.2–4 nmol/rat × 29



### 0.252 μ g/nmol ÷ (0.30–0.46) kg body weight as stated by the author s). This result may reflect 30 

### limited systemic absorption of benzo[a]pyrene at lo w doses. In contrast to this result, analysis of 31 

### benzo[a]pyrene concentrations in arterial blood and bile after continuous intraduodenal infusion 32 

### of radiolabeled benzo[a]pyrene (in the same vehicle as above) showed that approximately 40% 33 

### of the administered dose was absorbed by the duoden um over a 240-minute period (Foth et al., 34 

### 1988). In a similar experimental design, bile- and duodenum-cannulated male Sprague-Dawley 35 

### rats were given [ 3 

### H]-benzo[a]pyrene in corn oil with and without exog enous bile (Rahman et al., 36 

### 1986). The absorption of benzo[a]pyrene was estima ted from the cumulative recovery of 37 

### radioactivity in the bile and urine over 24 hours. This study showed that absorption of 38

### 19 DRAFT - DO NOT CITE OR QUOTE

benzo[a]pyrene was enhanced by bile as absorption i n the presence of endogenous bile only was 1 

### 22.9% of that when exogenous bile was administered. 2 

### Cavret et al. (2003) reported that absorption of be nzo[a]pyrene (as measured by the 3 

### percentage of orally administered radioactivity app earing in the portal blood in 24 hours) was 4 

### 30.5% in pigs fed 1 L of 4% fat milk containing 235 μ g/L [ 14 

### C]-benzo[a]pyrene (this 5 

### corresponds to an oral dose of approximately 6 μ g/kg based on the reported body weight of 40 6 

### kg). The level of radioactivity increased rapidly between 1 and 6 hours, with maximum uptake 7 

### between 3 and 6 hours after dosing. 8 

### In male F344 rats administered [ 14 

### C]-benzo[a]pyrene in peanut oil via gavage at doses 9



### from 0.04 to 4.0 μ mol/rat (approximately 0.03–0.04 mg/kg and 3.4–4 mg /kg, respectively, 10



### calculated as 0.04–4 μ mol/rat × 0.252 mg/ μ mol ÷ 0.25–0.30 kg body weight as stated by the 11 

### authors) approximately 85% of the radiolabel was re covered in the feces and 1–3% in the urine 12 

### after 168 hours (Hecht et al., 1979). Because radi olabel in feces may represent unabsorbed as 13 

### well as absorbed parent compound that is subsequent ly eliminated via biliary excretion, 14 

### bioavailability cannot be estimated from this study . However, the percent of radioactivity 15 

### recovered as parent benzo[a]pyrene was small (rangi ng from 6 to 13% of the administered 16 

### radioactivity), suggesting that a minimum of 73% of the administered dose was absorbed (i.e., 17 

### total in urine + total in feces – feces as benzo[a] pyrene). 18 

### Dietary matrices may have an impact on the absorpti on of benzo[a]pyrene from the GI 19 

### tract. For example, intestinal absorption of benzo [a]pyrene was enhanced in rats when the 20 

### compound was solubilized in lipophilic compounds su ch as triolein, soybean oil, and high-fat 21 

### diets, as compared with fiber- or protein-rich diet s (O’Neill et al., 1991; Kawamura et al., 1988). 22 

### This may be relevant for the absorption of benzo[a] pyrene from charbroiled meats and other fatty 23



### foods. 24 

### O’Neill et al. (1990) assessed the intestinal absor ption of [ 14 

### C]-benzo[a]pyrene given to 25 

### rats by gavage in olive oil with regular rat chow o r with low-fat diets high or low in either fiber 26 

### or beef protein (used to represent human diets). B enzo[a]pyrene and its metabolites were 27 

### recovered from feces, where they had been trapped b y microcapsules given by gavage 2 hours 28 

### prior to benzo[a]pyrene. Benzo[a]pyrene was absorb ed from rat chow differently than from 29 

### representative human diets. Dietary fiber decrease d the availability of benzo[a]pyrene, as 30 

### evidenced by the appearance of lower metabolite amo unts in the GI tract, while the beef31 

### enriched diet affected absorption to result in incr eased formation of 1,6- and 3,6-benzo[a]pyrene 32 

### diones. Urinary excretion of benzo[a]pyrene was de creased in rats given the high fiber diet but 33



### not the beef-enriched diet. The total amount of be nzo[a]pyrene excreted in feces and the 34 

### feces/urine ratio were increased by the high-fiber diet but not by the beef-enriched diet. These 35 

### results indicated that bioavailability of benzo[a]p yrene from the GI tract is affected by the type 36 

### of diet and that bioavailability studies in animals , using typical laboratory animal chow, may not 37 

### appropriately model the situation with varied human diets. 38

### 20 DRAFT - DO NOT CITE OR QUOTE

The potential influence of diet on PAH bioavailabil ity was investigated also by Wu et al. 1 

### (1994). Female mice were fed either gel or powder diets containing coal tar with detectable 2 

### levels of benzo[a]pyrene, Py, and other PAHs. Urin e samples were collected on the first, 3 

### seventh, and fourteenth day of treatment. Measurem ent of the amount of the Py metabolite 1- 4 

### OH-Py in the urine showed that the diet matrix did not influence the bioavailability of the PAHs. 5 

### Stavric and Klassen (1994) administered radiolabele d benzo[a]pyrene dissolved in various 6 

### vehicles (water, corn oil, liquid paraffin, or 50% ethanol) by gavage and monitored intestinal 7 

### absorption in bile-cannulated rats. The animals we re fed diets with or without added carbon 8 

### particles and typical food components, such as quer cetin or chlorogenic acid. They observed that 9 

### aqueous vehicles, quercetin, chlorogenic acid, or c arbon particles reduced biliary excretion of 10 

### benzo[a]pyrene, while lipid media such as corn oil increased it strongly. The authors postulated 11 

### that absorption of benzo[a]pyrene from food was aff ected by its solubility in the vehicle, by 12 

### physical adsorption, and/or by adduction of benzo[a ]pyrene to certain food ingredients. On the 13 

### other hand, Mirvish et al. (1981) observed that var ying the corn oil content of a synthetic diet 14 

### containing 100 μ g/g benzo[a]pyrene had little influence on fecal ex cretion of unmetabolized 15 

### benzo[a]pyrene. However, addition of 5% wheat bran to the synthetic diet, or using standard lab 16 

### chow, increased the fecal excretion of parent compo und 13-fold. They suggested that the 17 

### insoluble dietary fiber sequestered benzo[a]pyrene in the GI tract. 18 

### In summary, absorption of ingested benzo[a]pyrene w as demonstrated qualitatively in 19 

### exposed humans by the excretion of metabolites or t he presence of DNA adducts. However, 20



### these studies are not sufficient to determine the r ate and extent of absorption from the GI tract in 21 

### humans. Animal studies have produced variable resu lts, in part due to different study designs. 22 

### Standard approaches in animal studies suggest that the oral bioavailability ranges from 10 to 23 

### 40%. However, some studies have indicated that the standard diets of laboratory animals may 24 

### not model the human oral exposure to benzo[a]pyrene appropriately. No data on species or 25 

### gender-based differences in absorption were identif ied. 26

27 

### 3.1.3. Dermal Exposure 28 

### Several studies in humans and experimental animals have investigated the dermal 29 

### absorption of benzo[a]pyrene. Benzo[a]pyrene metab olites or DNA adducts were measured in 30 

### humans exposed dermally to benzo[a]pyrene-containin g mixtures in biological monitoring 31 

### studies. These studies provide only qualitative su pport for assessing the rate and degree of 32 

### dermal absorption of benzo[a]pyrene through human s kin. However, some studies provide 33 

### quantitative information on the degree of benzo[a]p yrene absorption through the skin in 34 

### volunteers or in explanted viable skin samples from tissue donors. 35

36



### 3.1.3.1. Dermal Exposure in Humans 37

### 21 DRAFT - DO NOT CITE OR QUOTE

Van Rooij et al. (1993) demonstrated differences in absorption rates of PAHs in coal tar 1



### ointment at various skin sites in volunteers. A do se of 2.5 mg/cm 2 

### of coal tar ointment, which 2



### consisted of 10% coal tar in a vehicle of zinc oxid e paste, was applied to 24 cm 2 

### of skin at the 3 

### forehead, shoulder, volar forearm, palmar side of t he hand, groin, or ankle and allowed to stand 4 

### for 45 minutes before removing the residue. Dermal absorption of PAH was determined through 5 

### measuring the disappearance of PAH fluorescence fro m the skin surface. Absorption rate 6 

### constants ranged from 0.036 to 0.135/hour, across a pplication sites, suggesting that 20–56% of 7



### the dose would be absorbed within 6 hours. A 69% d ifference in dermal absorption rates 8 

### between anatomical sites was reported, while only a 7% difference between individual 9



### volunteers was observed. The skin on the shoulder absorbed the greatest dose of PAH, followed 10 

### by the forehead, forearm, and groin, with the ankle s and palms absorbing the least amount. 11 

### There was, however, no correlation between anatomic al site of exposure and excretion of 1-OH12 

### Py in urine. The authors estimated that 0.3–1.4% o f the PAH dose (assessed as 1-OH-Py) 13 

### became systemically available, although systemic me asurement of benzo[a]pyrene or its 14 

### metabolites was not reported. 15 

### Quantitative comparisons of dermal penetration of h uman skin and other mammalian 16 

### species have been conducted. Kao et al. (1985) com pared benzo[a]pyrene permeation through 17 

### skin in short-term organ cultures using skin harves ted from mice, rats, rabbits, guinea pigs, 18 

### marmosets, and human donors. Two test systems were used, a dynamic one where skin samples 19 

### were held on top of chambers flushed with fresh org an culture medium at a constant rate and a 20 

### static one where skin samples were incubated on a f ilter disk in a culture dish over culture 21



### medium at 36°C. The culture medium was a modified minimal essential medium with Earle’s 22 

### salts and 10% fetal calf serum. In addition, all e xperiments were conducted with fresh, 23 

### metabolically viable skin and with metabolically no nviable skin (previously frozen or poisoned 24 

### with cyanide). Using the static system, [ 3 

### H]-benzo[a]pyrene dissolved in acetone was applied to 25 

### full-thickness skin samples (2.5 μ g/cm 2 

### ) and medium collected after 24 hours to be assayed for 26 

### [ 3 

### H]-benzo[a]pyrene and metabolites. The overall pen etration rate of benzo[a]pyrene was about 27



### 2.6% of the dose in 24 hours from viable human skin and only about 0.5% from nonviable skin. 28 

### After 24 hours penetration through viable skin, 52% of the radioactivity in medium consisted of 29 

### water-soluble benzo[a]pyrene metabolites and 18% wa s parent compound. By contrast, after 30 

### penetrating through nonviable skin, 90% of the reco vered radioactivity was parent compound, 31 

### and the ratio of water- to lipid-soluble metabolite s was much lower. Results in marmoset, rat, 32



### and rabbit were similar. Skin from the mouse allow ed more than 10% of the dose to penetrate, 33 

### while that of guinea pig let only a negligible perc entage of the dose penetrate. In all species, 34 

### metabolism was an important determinant of permeati on, with very low rates observed in 35



### nonviable skin. 36 

### Using the dynamic test system, Kao et al. (1985) st udied the influence of dermal 37 

### metabolism on the rate of penetration of benzo[a]py rene in more detail. Comparing rat to mouse 38

### 22 DRAFT - DO NOT CITE OR QUOTE

skin they again found a 10-fold difference in the a mount penetrating in 16 hours through viable 1 

### skin, approximately 0.7% of the dose in rat skin vs . approximately 7% in murine skin, but most 2 

### of this species difference disappeared when previou sly frozen skin was used or potassium 3 

### cyanide was added to the medium to destroy metaboli c capability. An additional factor was 4 

### responsiveness to aryl hydrocarbon (Ah)-receptor ag onists: dermal penetration in responsive 5 

### mice given an inducing dose of 3-methylcholanthrene (3-MC) was more than twice that in 6 

### noninduced Ah-responsive mice, and the latter was s imilar to nonresponsive mice (see Section 7 

### 3.3 for further discussion of Ah-responsiveness). These results show that functional enzyme 8 

### systems facilitate penetration of benzo[a]pyrene or its metabolites. 9 

### Wester et al. (1990) compared skin penetration of [ 14 

### C]-benzo[a]pyrene dissolved in 10 

### acetone versus benzo[a]pyrene adsorbed onto soil us ing skin from human cadavers. Skin was 11



### dermatomed to 500 μ m and stored in medium at 4°C to preserve viability . The authors used a 12 

### dynamic system with undiluted human serum as the re ceptor fluid and 24-hour exposure. 13 

### Radiolabeled benzo[a]pyrene was applied to the skin , and concentrations of radioactivity in the 14 

### receptor fluid, skin, and surface skin wash were de termined. For benzo[a]pyrene applied in 15 

### acetone, 23.7% of the applied dose was recovered in the skin, 0.09% was recovered in the 16 

### receptor fluid, and 53.0% was recovered in the surf ace wash. The amount of radiolabel 17 

### recovered from exposure to benzo[a]pyrene in soil w as 1.4% of the dose in skin and 0.01% in 18 

### receptor fluid, indicating that the exposure matrix greatly impacted dermal absorption. The 19 

### considerable difference in penetration through huma n skin observed in this study (only 0.09% of 20 

### the dose per 24 hours) and that of Kao et al. (1985 ) (2.6% of the dose) is at least in part a result 21 

### of differences in experimental design, such as full thickness (approximately 1.5 mm) vs. 22 

### dermatomed (0.5 mm) skin and synthetic receptor flu id with 10% serum vs. pure human serum. 23 

### Together, these studies suggest that benzo[a]pyrene is absorbed into human skin but does not 24 

### penetrate through the skin rapidly. 25 

### In a second part of the same study, Wester et al. ( 1990) also evaluated the dermal 26 

### penetration of benzo[a]pyrene in female rhesus monk eys in vivo. Radiolabeled benzo[a]pyrene 27 

### was applied to a 12 cm 2 

### area of the abdominal skin that was protected by a nonocclusive cover. 28



### The material was maintained on the skin for 24 hour s, after which time the skin area was 29



### washed. Urine was collected from the animals durin g the initial 24-hour exposure period and 6 30 

### days after. Skin penetration was determined as the ratio of urinary radiolabel for topically 31 

### exposed animals compared to monkeys administered th e same dose by i.v. injection. When 32 

### benzo[a]pyrene was dissolved in acetone, 51% of the applied dose was absorbed as compared to 33 

### 13.2% when benzo[a]pyrene was applied in soil. The se results further stress the fact that the 34 

### dermal absorption of benzo[a]pyrene depends on the vehicle of administration (pure substance 35 

### vs. contaminated environmental material). 36 

### Also using human tissues, van der Bijl and van Eyk (1999) compared the mean flux of 37 

### benzo[a]pyrene across vaginal and buccal mucosa sam ples from human donors. No significant 38

### 23 DRAFT - DO NOT CITE OR QUOTE

difference between the two tissue types was observe d, and steady-state flux was very low, 1 

### approximately 0.01% of the dose per cm 2 

### /minute (400 cpm/cm 2 

### /minute in a figure in the paper, 2 

### or 4,000 dpm [assuming 10% [ 3 

### H]-liquid scintillation counting efficiency] out of a 17.33 μ Ci 3 

### dose, or 3.8 × 10 7 

### dpm, equals about 0.01% of the dose/cm 2 

### /minute). These results indicate that 4 

### benzo[a]pyrene can be absorbed from and metabolized by the female reproductive tract. 5 

### However, the results cannot be compared to those ob tained with human skin. First, there was the 6 

### difference in absorbing surface, namely, the quasi- dry, mainly lipophilic stratum corneum vs. 7 

### fresh epithelium. Second, there were also fundamen tal differences in experimental design: 8 

### although van der Bijl and van Eyk (1999) used a dyn amic system, the tissues had been frozen 9 

### previously, benzo[a]pyrene was administered in aque ous solution in a large compartment on top 10 

### of the tissue sample, receptor fluid was a simple b uffer, and incubation was performed at 20°C. 11 

### Potter et al. (1999) also demonstrated that the exp osure matrix can affect the dermal 12 

### absorption of benzo[a]pyrene in mice in vivo as wel l as in human skin in vitro. In particular, the 13 

### uptake of benzo[a]pyrene decreased as the viscosity of the oil product used as vehicle increased. 14 

### Although mouse skin absorbed more radiolabel than h uman skin, the trend of decreasing 15 

### absorption with increasing viscosity found in the m ouse was also present in human skin. 16 

### Vehicles tested included mineral oils (32–198 centi -Stoke [cSt], measure of viscosity), residual 17 

### aromatic extracts (5,160–5,400 cSt), and bitumens ( 0.65–69 × 10 6 

### cSt). Most likely, differences 18 

### in diffusion coefficients for benzo[a]pyrene in the various vehicles would adequately describe 19 

### the differences in dermal absorption. 20

21



### 3.1.3.2. Dermal Exposure in Animals 22 

### Yang et al. (1989) compared dermal absorption of be nzo[a]pyrene in crude oil adsorbed 23 

### to soil (particle size of 150 μ m or less) versus benzo[a]pyrene in the crude oil a lone. In the in 24 

### vivo portion of the study, test materials spiked wi th 100 ppm [ 3 

### H]-benzo[a]pyrene were placed 25



### over a 7-cm 2 

### portion of shaved dorsal skin of female Sprague-Da wley rats. Urine and feces were 26 

### collected daily over 4 days, and at the end of the experiment animals were sacrificed and 27 

### radioactivity in tissues was determined. The total percent of the absorbed dose recovered in 28 

### excreta was greater for benzo[a]pyrene in crude oil (35.3%) than oil adsorbed to soil 29 

### (approximately 9.2%). While the latter number refe rs to soil applied to the skin as a monolayer, 30 

### when six times the amount of soil, and thus a sixfo ld dose, was applied, the absolute amount of 31 

### benzo[a]pyrene absorbed remained virtually the same . The authors also found that using viable 32 

### skin samples in vitro gave almost identical results to those obtained in vivo. 33 

### Ng et al. (1992) examined the percutaneous absorpti on of radiolabeled benzo[a]pyrene in 34 

### the hairless guinea pig. A single dose of 28 μ g benzo[a]pyrene dissolved in 50 μ L acetone was 35 

### applied to 4 cm 2 

### of dorsal skin and covered with a protective pad f or 24 hours. Urine and feces 36 

### were collected at 6 and 12 hours and then daily for the next 7 days postexposure. Approximately 37



### 34% of the radiolabel was absorbed and eliminated i n 24 hours. Most excretion had occurred by 38

### 24 DRAFT - DO NOT CITE OR QUOTE

day 3 and then continued at a lower rate to reach a bout 73% by day 7. Benzo[a]pyrene in the 1 

### skin wash accounted for 11% of the applied dose. C omparison with the amount excreted 2 

### following intramuscular injection (which reached ab out 85% in 7 days) suggested that the dermal 3 

### bioavailability of benzo[a]pyrene was high (about 8 5%). Ng et al. (1992) noted that, in an in 4 

### vitro study, only metabolites were present in the r eceptor solution, indicating that metabolism in 5 

### the skin preceded systemic absorption. This is con sistent with the findings of Kao et al. (1985) 6 

### that dermal absorption may be aided by a high metab olic capacity of the skin but leaves open the 7 

### question how much of the reactive metabolites escap e from the dermal compartment. 8 

### In an earlier study, Kao et al. (1984) used viable mouse skin to study the dose 9 

### dependence of dermal absorption of benzo[a]pyrene. They administered 1, 2, 4, and 6 μ g 10 

### benzo[a]pyrene per cm 2 

### full-thickness, shaved skin incubated at 36°C for 24 hours. The 11 

### percentage of absorbed benzo[a]pyrene dose decrease d with increasing dose: 24, 17, 12, and 7% 12 

### with 1, 2, 4, and 6 μ g/cm 2 

### , respectively. The authors also found that, with doses of 4 and 13



### 6 μ g/cm 2 

### , the total amount of penetrated benzo[a]pyrene-der ived radioactivity remained similar, 14 

### indicating saturation of the skin’s absorption and metabolic capacity. When the donor mice were 15 

### pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce cytochrome P450 16 

### (CYP450) enzymes in the skin, amounts of penetrated benzo[a]pyrene increased to 38, 33, 30, 17 

### and 19% of the dose, respectively. In addition to the increased rate of absorption, a shift in 18 

### metabolic pattern was observed among the benzo[a]py rene metabolites in the medium. CYP450 19 

### induction caused a large increase in metabolite con jugates at the expense of polar metabolites 20 

### and benzo[a]pyrene diols, and a strong increase in covalent binding of benzo[a]pyrene to skin 21



### DNA. 22 

### Kao et al. (1988) also investigated the influence o f skin hair on the rate of benzo[a]pyrene 23 

### penetration. Using the same dynamic system, six st rains of haired and two strains of hairless 24



### mice were administered a dose of 1 μ g/cm 2 

### benzo[a]pyrene. After 16 hours of incubation, 25 

### between 4.4 and 9.4% of the dose had penetrated thr ough viable skin of haired mice but only 2– 26 

### 2.9% of the dose through skin of hairless mice. Si nce the authors found that permeation of 27 

### testosterone, which was used as a comparison compou nd with similar physiochemical properties 28 

### as benzo[a]pyrene, was the same in haired and hairl ess mice, 70% of the dose, they concluded 29 

### that skin appendages have considerable metabolic ca pacity for benzo[a]pyrene. Extending these 30 

### findings to humans implies that in the assessment o f dermal absorption of benzo[a]pyrene, 31 

### presence of dense hair may play an additional role. 32 

### Morse and Carlson (1985) compared in vivo dermal ab sorption of benzo[a]pyrene in 33 

### BALB/c and SENCAR mice to find an explanation for t he higher sensitivity of the latter strain 34 

### toward benzo[a]pyrene-induced skin tumors. Animals were treated with 50 mg/kg 35 

### benzo[a]pyrene dissolved in acetone. Levels of ben zo[a]pyrene in skin of BALB/c mice did not 36 

### reach higher peak levels, but stayed up to three ti mes higher than in SENCAR mice between 6 37 

### and 24 hours postexposure. The levels of benzo[a]p yrene-derived radioactivity in liver, lung, 38

### 25 DRAFT - DO NOT CITE OR QUOTE

and stomach also peaked at comparable levels, but s tayed at up to twice the levels in SENCAR 1 

### mice between 6 and 48 hours posttreatment, compared with BALB/c mice. There were no strain 2 

### differences in these tissues when benzo[a]pyrene wa s administered orally. The metabolic 3 

### capacity of the skin was assessed by measuring DNA binding of benzo[a]pyrene in skin. 4 

### Six hours after exposure DNA-binding of benzo[a]pyr ene was higher in SENCAR than in 5 

### BALB/c mice, but by 24 hours after topical administ rations, DNA adducts were higher in 6 

### BALB/c than in SENCAR mice, possibly indicating dif ferences in metabolism. Kao et al. 7 

### (1988) had included the same two strains of mice in one of their studies and found comparable 8 

### results, with 9.2% of the dose penetrating over 16 hours in BALB/c and 4.4% in SENCAR mice. 9 

### However, the results of Morse and Carlson (1985) di d not provide the expected toxicokinetic 10 

### explanation for the difference in skin tumor sensit ivity between the two mouse strains. 11 

### In summary, due to the use of coal tar products for medicinal purposes, quantitative data 12 

### on dermal absorption of benzo[a]pyrene in humans ar e more abundant than for other routes of 13 

### exposure. The animal-to-human differences are sign ificant. Generally, mice have greater 14 

### absorption than humans, followed by rats and rabbit s. The 24-hour penetration values range 15 

### from 1 to 3% in viable human skin; much greater amo unts of material are absorbed into the skin 16 

### but do not readily permeate through it. Dermal abs orption of PAH is strongly dependent on 17 

### anatomical site (69% difference across six sites), while inter-individual variation is much smaller 18 

### (7% difference across nine volunteers). Furthermor e, dermal absorption is highly dependent on 19 

### underlying metabolic activity of the skin. The veh icle of exposure also impacts dermal 20 

### absorption, with vehicles that absorb benzo[a]pyren e, such as soil, or vehicles with low diffusion 21 

### coefficients (high viscosity) decreasing the rate a nd degree of absorption. 22

23 

### 3.1.4. Other Types of Exposure 24 

### Ewing et al. (2006) used isolated, perfused rat lun gs and delivered benzo[a]pyrene coated 25 

### onto silica carrier particles (average size: 3.5 μ m) at mean doses of 2.2, 36, and 8,400 ng to each 26 

### lung within <1 minute. Perfusate was collected for 77 minutes thereafter. Lungs and perfusates 27 

### were analyzed for benzo[a]pyrene and metabolites. Absorption was strongly dose-dependent: at 28 

### the low and mid exposure levels benzo[a]pyrene conc entration increased rapidly in the perfusate 29 

### to reach a maximum within <5 minutes, then decrease d over the remaining observation period. 30 

### At the high dose, benzo[a]pyrene in the perfusate r eached the maximum at about 30 minutes 31 

### after exposure and stayed at a constant level from there on, i.e., the absorption of benzo[a]pyrene 32 

### proceeded at zero order until all deposited solid b enzo[a]pyrene was dissolved. The mass 33 

### balances for benzo[a]pyrene equivalents in lung vs. perfusate were lung ca. one-third perfusate at 34 

### the low dose, lung = perfusate at the mid dose, and lung about twice that of perfusate at the high 35 

### dose. At the low exposure level metabolism was app arently able to convert most of the parent 36 

### compound, while at the highest exposure level most of the absorbed benzo[a]pyrene remained 37 

### unmetabolized even at the end of the experiment. T he results suggest that, at low doses (2.2 38

### 26 DRAFT - DO NOT CITE OR QUOTE

ng/lung) benzo[a]pyrene is absorbed very efficientl y in rat lung (two-thirds absorbed in 1 

### 77 minutes), while at higher doses the rate of abso rption decreases markedly, either because of 2 

### diffusion limitation, or by saturation of local met abolism. 3

4



### 3.2. DISTRIBUTION 5 

### No adequate quantitative studies of benzo[a]pyrene tissue distribution in exposed humans 6 

### were identified. Obana et al. (1981) observed low levels of benzo[a]pyrene in liver and fat 7 

### tissues from autopsy samples. However, prior expos ure histories were not available for the 8 

### donors. Nevertheless, the identification of benzo[ a]pyrene metabolites or DNA adducts in 9 

### tissues and excreta of PAH-exposed populations sugg est that benzo[a]pyrene is widely 10



### distributed. 11

12 

### 3.2.1. Inhalation Exposure 13 

### Numerous studies evaluated the disposition of benzo [a]pyrene or its metabolites 14 

### following inhalation or instillation in the respira tory tract in animals. The distribution of 15 

### benzo[a]pyrene following inhalation exposure was sh own to be similar in various species. Male 16 

### and female F344 rats were exposed nose-only for 30 minutes to atmospheres containing 17 

### 0.6 mg/m 3 

### [ 3 

### H]-benzo[a]pyrene adsorbed onto 67 

### Ga 2 

### O 3 

### or to 1.0 mg/m 3 

### neat [ 3 

### H]- 18 

### benzo[a]pyrene (Sun et al., 1982). There were qual itative differences in time and amount of 19 

### absorption between the exposure regimens. With eit her exposure regimen, high tissue levels of 20 

### radiolabel were found in the small and large intest ines and cecum, followed by liver, kidney, and 21 

### blood. Levels in the upper GI tract (esophagus and stomach) differed, with significant levels of 22 

### radioactivity in the stomach following exposure to [ 3 

### H]-benzo[a]pyrene adsorbed onto 67 

### Ga 2 

### O 3 23 

### but only minimal levels in the stomach following ex posure to neat benzo[a]pyrene aerosol. 24 

### Twelve hours after exposure, the highest tissue con centrations of radiolabel were found in the 25 

### lower GI tract for both regimes. The differences i n distribution of radiolabel likely reflect the 26



### relative contribution of two mechanisms for the del ivery of benzo[a]pyrene-related radioactivity: 27 

### for particle-adsorbed benzo[a]pyrene, mucociliary c learance followed by ingestion may be a 28 

### significant factor, while aerosolized benzo[a]pyren e is likely to be more readily absorbed in the 29 

### respiratory tract only. 30



### Other inhalation studies measured levels of adminis tered radioactivity as benzo[a]pyrene 31 

### parent compound or metabolites in whole blood, plas ma or lung, but not in any other tissues 32 

### (Ramesh et al., 2001a; Gerde et al., 1993a, b). Th e rapid appearance of benzo[a]pyrene and 33



### metabolites in the blood is consistent with the con clusion that benzo[a]pyrene is readily 34 

### bioavailable following exposure by the inhalation r oute. The degree to which absorbed 35 

### benzo[a]pyrene or metabolites was delivered to targ et tissues was not determined in these 36



### studies. 37

### 27 DRAFT - DO NOT CITE OR QUOTE

Weyand and Bevan (1986) examined benzo[a]pyrene dis position and metabolism in male 1 

### Sprague-Dawley rats following intratracheal instill ation of 1 μ g/kg [ 3 

### H]-benzo[a]pyrene 2 

### dissolved in triethylene glycol. The amount of rad ioactivity in various organs was determined at 3



### timed intervals between 5 and 360 minutes after dos ing. Peak levels of radioactivity as the 4 

### percentage of the administered dose per organ/tissu e as well as time profiles were as follows: 5

6



### • Early peak levels: lungs (59.5% at 5 minutes, decl ining 10-fold over 360 minutes); blood 7 

### (3–4% between 5 and 15 minutes, declining only twof old by 360 minutes); spleen (0.5% 8 

### at 5 minutes, declining very slowly); liver (20.8% at 10 minutes, gradually declining); 9 

### heart/thymus (1.6% at 10 minutes, barely declining from 0.5% after 30 minutes). 10

11



### • Medium term peak levels: carcass (27.1% at 60 minu tes, mostly stable at around 12 

### approximately 25% between 10 and 90 minutes and at approximately around 22% 13 

### thereafter); kidney (2.4% at 90 minutes but rather stable at approximately around 2% 14 

### between 10 and 360 minutes); testes (1.3% at 90 min utes but rather stable at 15 

### approximately around 1% between 15 and 360 minutes)

. 16

17



### • Late peak levels: stomach (6.9% at 120 minutes, de creasing slowly); intestinal contents 18 

### (44.7% at 360 minutes, increasing over time period) ; intestines (14.9% at 360 minutes, 19 

### continually increasing). 20

21 

### These profiles are consistent with rapid uptake and delivery of benzo[a]pyrene to well22 

### perfused tissues, followed by clearance from the ti ssues via metabolism and excretion 23 

### (particularly in the feces). The profile of radioa ctivity appearing in the GI tract suggests partial 24 

### removal of benzo[a]pyrene from the lungs via mucoci liary escalator. Total recovery of 25



### radiolabel at 5 minutes was 96% of the dose and 104 % at 360 minutes, indicating complete 26 

### recovery from the tissues. 27 

### Weyand and Bevan (1986) also investigated benzo[a]p yrene metabolites in tissues. 28 

### Quinones were at highest concentrations in both lun g and liver 5 minutes after instillation, 29 

### accounting for 12 and 7% of radiolabeled extractabl e material, respectively. Benzo[a]pyrene 30 

### disposition was also investigated in male rats with and without biliary cannulas. Distribution 31 

### patterns among organs were similar, though the amou nt excreted in bile and intestinal contents 32



### was 77% of the administered dose in cannulated rats and 53% in animals that were not 33



### cannulated. The intestinal contents carried lower fractions of the administered dose as thioether 34 

### and glucuronic acid conjugates than the bile, indic ating enterohepatic recirculation of 35 

### benzo[a]pyrene metabolites. 36 

### A comparative intratracheal instillation study with Sprague-Dawley rats, Gunn rats, 37 

### guinea pigs, and hamsters gave results (Weyand and Bevan, 1987) qualitatively similar to those 38 

### reported by Weyand and Bevan (1986). Doses of 0.16 or 350 μ g [ 3 

### H]-benzo[a]pyrene per animal 39 

### were administered intratracheally, and the distribu tion of benzo[a]pyrene-derived radioactivity 40



### was determined in various tissues. Relative amount s of radiolabel recovered per gram of tissue 41

### 28 DRAFT - DO NOT CITE OR QUOTE

at 6 hours were: lung (1.7%) > kidney (0.76%) ≈ liver (0.67%) > testes (0.21%) = spleen 1 

### (0.21%) = heart (0.21%) ≈ GI tract (0.19%) > stomach (0.13%) > carcass (0.07 1%). This pattern 2 

### of distribution was qualitatively similar among all species tested at both doses, but the relative 3 

### values differed between species. For example, in S prague-Dawley rats the liver burden 4 

### represented 0.67% of the recovered radiolabel/gram of tissue, while this value was 2.16% for 5 

### hamsters, 0.35% for guinea pigs, and 1.02% for Gunn rats after the 0.16 μ g/animal dose. The 6 

### study did not include the intestine or its contents , where likely the majority of radioactivity was. 7 

### When benzo[a]pyrene was instilled intratracheally i nto mice, Schnizlein et al. (1987) found that 8 

### radioactivity in the lung declined steadily through out the 144-hour investigation period, while 9 

### levels in liver, spleen, and intestine peaked at 8 hours after dosing, decreased slowly until 10 

### 24 hours, and declined rapidly thereafter. It was also noted that as the lung burden of the 11 

### radiolabel decreased, radioactivity increased in lu ng-associated lymph nodes (LALN) over the 6- 12 

### day study, suggesting distribution of benzo[a]pyren e or its metabolites via the lymph. 13 

### Bevan and Ulman (1991) administered 1 μ g/kg [ 3 

### H]-benzo[a]pyrene intratracheally to 14 

### male Sprague-Dawley rats in three different liquid vehicles. At 6 hours after dosing, 56.2, 58.4, 15 

### and 70.5% of the dose delivered from tricaprylin, e thyl laurate, and triethylene glycol, 16 

### respectively, were recovered from bile. Recovery f rom whole lung after 6 hours was 13.0% of 17 

### the administered dose for tricaprylin and 15.6% for ethyl laurate but only 2.6% for triethylene 18 

### glycol, indicating that pulmonary absorption of ben zo[a]pyrene was more efficient from a less 19 

### hydrophobic vehicle than from a highly hydrophobic one. Among the other organs, kidney and 20 

### liver retained rather high levels of radioactivity (around 2 and 5%, respectively, for the whole 21 

### organs). Two percent or less of the administered d ose was recovered from intestine and its 22 

### contents in this study. 23 

### Pregnant Wistar rats were exposed for 95 minutes on GDs 17 to 200, 350, 500, 650, or 24 

### 800 mg/m 3 

### [ 3 

### H]-benzo[a]pyrene generated as a microcondensate fr om heated pure material 25 

### (Withey et al., 1993). Immediately (time not speci fied) following exposure, the ranking of 26 

### benzo[a]pyrene concentrations was maternal lung > b lood > liver > kidney > fat > fetus. When 27 

### total metabolites (as measured by detection of radi olabel) were measured immediately following 28 

### dosing, the ranking was maternal lung > blood > liv er > kidney > fetus > fat. Six hours after 29 

### exposure, benzo[a]pyrene concentrations were fat > lung > kidney > liver > blood > fetus, while 30 

### total metabolite concentrations were lung = fat > k idney > liver = blood > fetus. Concentrations 31 

### of benzo[a]pyrene and metabolites in the GI tract w ere not reported. This study is consistent 32 

### with other studies in showing wide tissue distribut ion of benzo[a]pyrene. In addition, the results 33 

### also demonstrated placental transfer of benzo[a]pyr ene and its metabolites. 34

35 

### 3.2.2. Oral Exposure 36 

### Saunders et al. (2002) evaluated the neurotoxicity of benzo[a]pyrene in male F344 rats 37 

### following a single gavage dose in peanut oil at dos es of 0, 25, 50, 100, or 200 mg/kg body 38

### 29 DRAFT - DO NOT CITE OR QUOTE

weight (10/sex/dose). Benzo[a]pyrene and metabolit e concentrations were monitored for up to 1 

### 96 hours after administration in plasma, cerebellum , and cerebral cortex. Unmetabolized 2 

### benzo[a]pyrene was observed in brain tissue only at the two highest doses, peaking at 2 to 3 

### 4 hours and then gradually decreasing. Total metab olite concentrations in plasma as well as 4 

### brain tissue peaked at 2 hours, remained at similar levels until 6 hours and then gradually 5 

### decreased. By 96 hours after dosing benzo[a]pyrene or its metabolites had dropped to trace 6



### levels. The distribution of metabolites shifted ov er the observation period, with diol metabolites 7 

### (4,5-, 7,8-, and 9,10-benzo[a]pyrene diols) predomi nating for the first 12 hours and hydroxy 8 

### metabolites (3-OH, 9-OH-benzo[a]pyrene) predominati ng at later time points. The distribution 9 

### of metabolites was similar in plasma and brain. 10 

### Neubert and Tapken (1988) administered a single 12 mg/kg oral dose of [ 14 

### C]- 11 

### benzo[a]pyrene to groups of five pregnant NMRI:Han- mice on GDs 11, 12, and 13 to determine 12 

### whether placental transfer occurred. Six, 24, and 48 hours after treatment radiolabel was found 13 

### in the maternal lung, liver, and kidney (between 5 and 17% of the dose were recovered per gram 14 

### tissue at 6 hours post dosing, decreasing to 0.5–1. 3% by 48 hours). Radiolabel was also found in 15 

### the placenta and embryonic liver at one to two orde rs of magnitude less than that found in 16



### maternal tissues. Similar results were found with five pregnant albino rats that received a single 17 

### oral dose of 200 mg/kg benzo[a]pyrene in sunflower oil (Shendrikova and Aleksandrov, 1974). 18 

### Three hours after treatment, fetal levels of radiol abel were approximately 10% of the maternal 19



### concentration. 20 

### Taken together it is apparent that, in rats, benzo[ a]pyrene can penetrate the blood barrier, 21 

### and in pregnant mice or rats it crosses the placent al barrier and reaches the fetus. Otherwise, 22 

### distribution data on benzo[a]pyrene following oral administration are insufficient to establish a 23 

### cogent picture of organ or tissue distribution. 24

25 

### 3.2.3. Dermal and Other Exposures 26



### Some studies have evaluated the distribution of ben zo[a]pyrene and its metabolites 27 

### following dermal or other dose routes. Morse and C arlson (1985) sought to determine whether 28 

### differences in toxicokinetic parameters could expla in the difference in tumor response between 29 

### SENCAR (high susceptibility) and BALB/c mice (low s usceptibility). The mice were 30 

### administered radiolabeled benzo[a]pyrene via dermal application or orally, and the time course 31 

### of radioactivity levels was assessed in several org ans. Following topical application of 32 

### benzo[a]pyrene, organ levels of radioactivity were generally 1.5–4 times higher in SENCAR 33 

### than in BALB/c mice. Radioactivity levels were liv er > lung ≈ stomach, with an approximately 34



### twofold difference. DNA adduct levels were liver > lung > stomach, with a threefold difference. 35 

### Following oral dosing, tissue radioactivity levels differed little between the two strains and 36 

### essentially followed the same pattern of distributi on. However, DNA adduct levels following 37 

### oral dosing were much higher than after topical adm inistration, approximately 6 times higher in 38

### 30 DRAFT - DO NOT CITE OR QUOTE

stomach and liver and 10 times higher in lung. RNA and protein binding showed patterns 1 

### similar to DNA binding. This study seems to indica te that the route of administration exerts little 2 

### influence on the tissue distribution of benzo[a]pyr ene. 3 

### Moir et al. (1998) also measured the toxicokinetics of benzo[a]pyrene in male Wistar rats 4 

### dosed i.v. with 2, 6, or 15 mg/kg of [ 14 

### C]-benzo[a]pyrene dissolved in an emulphor/saline 5 

### emulsion. The concentrations of both benzo[a]pyren e-derived radioactivity and parent 6 

### compound were determined in blood, adipose tissue, lung, liver, kidney, heart, spleen, brain, and 7



### testes as well as in urine and feces. The concentr ations in all tissues examined except lung 8 

### appeared to follow a similar pattern of smoothly in creasing and decreasing curves, while the lung 9 

### data were rather erratic (this pattern may reflect temporary trapping of lipid vesicles from the 10 

### benzo[a]pyrene emulsion in the fine lung vessels). The authors also extracted selected tissues 11 

### and determined parent compound levels by high-perfo rmance liquid chromatography (HPLC). 12 

### Peak concentrations of [ 14 

### C]-benzo[a]pyrene equivalents and of parent compoun d and time to 13 

### peak for the 2 mg/kg dose group are given in Table 3-1. Similar patterns were observed at 14 

### higher doses, with notable exceptions noted below. 15

16 

### Table 3-1. Distribution of benzo[a]pyrene in selec ted tissues of male rats

following i.v. dosing with 2 mg/kg

Tissue/organ a Blood Adipose b Kidney c Liver Lung d

Total tissue radioactivity Peak level ( μ g/g tissue) 4.27 ± 0.25 2.31 ± 0.87 8.94 ± 1.21 20. 55 ± 2.20 40.54 ± 4.85 Peak time (min) 5 120 5 and 20 5 5 and 120 Parent compound Peak level ( μ g/g tissue) 2.64 ± 0.84 3.96 ± 1.92 7.68 ± 1.23 11. 33 ± 4.66 5.17 ± 1.15 Peak time (min) 5 120 5 to 20 5 5

a Mean ± SD, n = 3–4.

b Adipose tissue levels showed broad maxima between 2 0 and 480 min; the highest values are shown.

c Total radioactivity levels in kidney changed little between 30 and 480 min.

d Parent compound lung had another maximum of 14.65 ± 1.67 μ g/g at 120 min; a second maximum at this time point was observed at the higher doses, too, but th ose did not exceed the value of the first maximum a t 5 min.

Source: Moir et al. (1998).

17 

### At all exposure levels blood maintained the lowest initial concentrations (with some 18 

### exceptions in white adipose tissue discussed below) . Half-lives for benzo[a]pyrene parent 19 

### compound in blood after the 2 and 6 μ g/kg doses were 36 and 25 minutes, respectively, wh ile no 20 

### parent compound could be detected at time points >4 80 minutes. Following the 15 μ g/kg dose, 21 

### blood showed an initial benzo[a]pyrene decline simi lar to the lower doses, but benzo[a]pyrene 22 

### could be recovered up to the end of the study at 32 hours post dosing, and with these additional 23 

### data a second elimination phase half-life for benzo [a]pyrene was estimated at 408 minutes. The 24

### 31 DRAFT - DO NOT CITE OR QUOTE

final half-lives for elimination of benzo[a]pyrene “metabolites” (total benzo[a]pyrene-derived 1 

### radioactivity minus parent compound) from blood wer e 666, 848, and 179 minutes for the 2, 6, 2 

### and 15 mg/kg doses, respectively. It is fair to sp eculate that the second phase of elimination is 3 

### the result of redistribution of parent compound (an d subsequent metabolism) from a large, deep 4 

### compartment such as adipose tissue and/or enterohep atic circulation. 5 

### Similar to the results in the lung, erratic pattern s of radioactivity occurred in adipose 6 

### tissue. Peaks and dips in radioactivity levels in lung occurred roughly with a pattern opposite to 7 

### that in adipose tissue. This might suggest occasio nal redistribution of benzo[a]pyrene parent 8 

### compound between a pool of vesicles trapped in the lung and adipose tissue. Data derived from 9



### these tissues should therefore be viewed with cauti on. Moir et al. (1998) noted that, as a general 10 

### rule, increasing tissue radioactivity levels follow ing administration of [ 14 

### C]-benzo[a]pyrene 11



### reflected metabolite accumulation. Kinetics models were fit to the data to derive rate constants 12 

### for clearance of benzo[a]pyrene. Many organs displ ayed rapid uptake phases for 13 

### benzo[a]pyrene: liver (uptake complete at the firs t time point, 5 minutes) kidney, brain, testis, 14 

### and adipose tissue, which was followed by a bi-expo nential decline (except in adipose tissue). 15 

### Half-lives for benzo[a]pyrene parent compound elimi nation changed with the dose administered 16 

### (half-life in minutes at doses of 2, 6, and 15 μ g/kg, respectively): liver (28, 163, and 281), 17 

### kidney (498, 456, and 389), and adipose tissue (239 , 945, and 781). The following organs were 18



### evaluated for the 2 μ g/kg dose only: brain (2,326 minutes), heart (25 m inutes), and spleen (38 19 

### minutes); the latter, most likely due to nondetecta ble radioactivity levels at later time points, 20 

### reflect the initial elimination phase only. Elimin ation half-lives of benzo[a]pyrene metabolites in 21 

### liver and several other organs were in the range of 10–15 hours and independent of dose, 22 

### suggesting first-order elimination. Overall, the r esults of Moir et al. (1998) suggest a pattern 23



### consistent with initial wide distribution determine d by blood flow, with lipophilicity and rates of 24 

### metabolism contributing to temporal patterns of org an levels after the initial distribution period. 25



### Some studies showed that reactive metabolites of be nzo[a]pyrene are transported in the 26 

### blood and may be distributed to tissues incapable o f benzo[a]pyrene metabolism. Ginsberg and 27 

### Atherholt (1989) evaluated DNA adduct formation aft er intraperitoneal (i.p.) administration of 28 

### benzo[a]pyrene in mice. Serum of benzo[a]pyrene-tr eated mice incubated with splenocytes or 29 

### salmon sperm DNA resulted in adduct formation sugge sting that reactive benzo[a]pyrene 30 

### metabolites were systemically distributed and avail able for interaction with target tissues. DNA 31 

### adduct levels formed in vivo were highest in liver, lung, and spleen, with levels in kidney and 32 

### stomach significantly lower. 33 

### Taken together, the limited human data and few toxi cokinetic studies in animals 34 

### demonstrate that benzo[a]pyrene and its metabolites are widely distributed throughout the body. 35 

### Inhalation of particle-bound or pure benzo[a]pyrene results in significant levels of 36 

### benzo[a]pyrene and metabolites in numerous tissues. Distribution of inhaled benzo[a]pyrene and 37



### its metabolites to the GI tract is a result of both mucociliary clearance of particulates from the 38

### 32 DRAFT - DO NOT CITE OR QUOTE

lung and of biliary excretion following metabolism. Following absorption, benzo[a]pyrene and 1 

### metabolites are initially found predominantly in th e highly perfused tissues, such as the lung, 2 

### liver, and kidneys. Lower amounts are distributed to other tissues, including the male 3 

### reproductive organs, central nervous system, and ad ipose tissue. Despite its high lipophilicity 4 

### benzo[a]pyrene has no specific affinity for lipid-r ich tissues, most likely because of its rapid 5 

### metabolism to more hydrophilic compounds. There is some indication that the distribution of 6 

### benzo[a]pyrene is not much influenced by the route of administration. Reactive benzo[a]pyrene 7



### metabolites are also distributed via blood where th ey may form protein adducts or reach tissues 8 

### themselves unable to form reactive benzo[a]pyrene m etabolites. Benzo[a]pyrene or metabolites 9



### are also transferred to the fetus at concentrations one to two orders of magnitude lower than 10 

### those found in maternal tissues (Withey et al., 199 3; Neubert and Tapkin, 1988; Shendrikov and 11 

### Aleksandrov, 1974). 12

13



### 3.3. METABOLISM 14 

### The metabolism of benzo[a]pyrene is a critical aspe ct of the assessment of its potential 15 

### toxicity because for many endpoints reactive metabo lites are likely to contribute to the toxic 16 

### response. Numerous reviews on the metabolism of be nzo[a]pyrene are available (Miller and 17 

### Ramos, 2001; WHO, 1998; ATSDR, 1995; Conney et al., 1994; Grover, 1986; Levin et al., 18 

### 1982; Gelboin, 1980). Key concepts have been adapt ed largely from these reviews and 19 

### supplemented with recent findings. 20 

### benzo[a]pyrene is metabolized extensively by Phase I reactions to form numerous 21 

### oxidative or reactive metabolites that are targets for further metabolism through diverse phase II 22 

### reactions. Many of the enzymes involved in benzo[a ]pyrene metabolism are isoenzymes within 23 

### gene families, the members of which have varying me tabolic specificities. Many of these 24 

### enzymes are encoded by genes that show functional p olymorphism. Many of the critical 25 

### enzymes are inducible by a variety of agents, inclu ding benzo[a]pyrene itself, and, therefore, 26 

### studies that evaluate benzo[a]pyrene kinetics follo wing single short-term exposures may not be 27 

### representative of the kinetics of benzo[a]pyrene fo llowing longer-term exposure conditions or 28 

### exposure to mixtures. Metabolism of benzo[a]pyrene is species-, strain-, and organ-system29 

### specific. There are age- and gender-related differ ences in the expression of many of the key 30 

### enzymes that must be considered. 31 

### The metabolism of benzo[a]pyrene has been extensive ly studied using both in vivo and in 32 

### vitro models, and a schematic representation of met abolic pathways is provided in Figure 3-1. 33 

### Only Phase I reaction products are shown. Phase II reactions include glutathione conjugation of 34 

### diol epoxides, sulfation and glucuronidation of phe nols, and reduction of quinones by 35 

### NADPH:quinone oxidoreductase (NQO)with subsequent c onjugation. 361

2 

### Source: Miller and Ramos (2001). 3

4 

### Figure 3- 1. Metabolic pathways for 5

6 

### Some enzymes involved in the metabolism of 7 

### (although benzo[a]pyrene itself is a relatively weak inducer compared to oth er environmental 8 

### pollutants, such as certain dioxins and polychlorin ated biphenyls). The cellular mechanisms 9 

### underlying the inducibility of these enzymes, as we ll as the relative p 10 

### has been reviewed in detail (Miller and Ramos, 2001 ; Whitlock, 1999; Nebert et al., 1993). 11 

### Inducibility of genes that metabolize 12 

### genetic locus that imparts inducibility is designat ed the Ah locus, so named for the enzyme 13 

### activity aryl hydrocarbon hydroxylase (AHH) (now kn own to be catalyzed by enzyme activities 14 

### from the CYP1 family. The Ah gene encodes a cytosolic receptor that regulates th e inducible 15



### 33 DRAFT - DO NOT CITE OR QUOTE

Source: Miller and Ramos (2001).

1. Metabolic pathways for benzo[a]pyrene.

Some enzymes involved in the metabolism of benzo[a]pyrene are highly inducible

itself is a relatively weak inducer compared to oth er environmental pollutants, such as certain dioxins and polychlorin ated biphenyls). The cellular mechanisms underlying the inducibility of these enzymes, as we ll as the relative p otency of various inducers, has been reviewed in detail (Miller and Ramos, 2001 ; Whitlock, 1999; Nebert et al., 1993). Inducibility of genes that metabolize benzo[a]pyrene is termed genetic responsiveness and the genetic locus that imparts inducibility is designat ed the Ah locus, so named for the enzyme activity aryl hydrocarbon hydroxylase (AHH) (now kn own to be catalyzed by enzyme activities gene encodes a cytosolic receptor that regulates th e inducible

DO NOT CITE OR QUOTE are highly inducible itself is a relatively weak inducer compared to oth er environmental pollutants, such as certain dioxins and polychlorin ated biphenyls). The cellular mechanisms otency of various inducers, has been reviewed in detail (Miller and Ramos, 2001 ; Whitlock, 1999; Nebert et al., 1993). is termed genetic responsiveness and the genetic locus that imparts inducibility is designat ed the Ah locus, so named for the enzyme activity aryl hydrocarbon hydroxylase (AHH) (now kn own to be catalyzed by enzyme activities gene encodes a cytosolic receptor that regulates th e inducible

### 34 DRAFT - DO NOT CITE OR QUOTE

expression of genes that encode Phase I (CYP1A and CYP1B isoforms) enzymes and may 1 

### interact coordinately in regulating genes that enco de isoforms of the Phase II enzymes uridine 2 

### diphosphate (UDP)-dependent glucuronosyltransferase s [UGTs], GSTs, NQO1) that metabolize 3 

### the products of Phase I metabolism. The role of th ese enzyme systems in benzo[a]pyrene 4



### metabolism is discussed below. Due to the inducibi lity of benzo[a]pyrene metabolism, 5 

### interpretation of toxicity studies should consider whether the studies were conducted in species 6 

### and strains that have inducible metabolism, whether duration of exposure was sufficient to 7 

### induce benzo[a]pyrene metabolism, and whether or no t known inducers were administered. 8

9



### 3.3.1. Phase I Metabolism 10



### 3.3.1.1. CYP450-dependent Reactions 11 

### Phase I reactions are catalyzed primarily by the mi xed function oxidase system of 12 

### enzymes associated with CYP450 to form arene oxides . This initial phase I metabolic reaction 13 

### of benzo[a]pyrene is carried out primarily by the i nducible activities of CYP1A1 or CYP1B1 and 14 

### the constitutively expressed and somewhat inducible CYP1A2, depending on the tissue. Other 15 

### CYP isoforms may also catalyze the initial oxidatio n reactions. NAPDH:CYP reductase is an 16 

### important cofactor for this reaction as a supplier of redox equivalents (Byczkowski and Gessner, 17 

### 1989). In addition to the CYP450 enzymes there are also a number of oxidoreductases that are 18 

### not typically considered Phase I enzymes, yet can p lay an important role in the oxidative 19 

### metabolism of benzo[a]pyrene (see below). 20 

### The isomerization of the arene oxides to their resp ective phenol metabolites is thought to 21 

### be a nonenzymatic process; however, physical/chemic al studies have shown that rearrangement 22 

### is susceptible to catalysis by amines (Johnson and Bruice, 1975). This would suggest that 23 

### rearrangements in vivo could be catalyzed by the am ino or sulfhydryl groups of proteins. 24 

### Typically, a single phenolic isomer tends to be pro duced and the direction of regioselective ring 25 

### opening is predictable based on the relative stabil ity of the two possible cationic intermediates 26 

### (Fu et al., 1978). In accordance with these predic tions, exclusively 3-OH-benzo[a]pyrene but not 27 

### the other possible phenol metabolite, 2-OH-benzo[a] pyrene, is formed from the 2,3-oxide of 28 

### benzo[a]pyrene (Yang et al., 1977). The other mono phenol metabolites of benzo[a]pyrene 29 

### include 1-, 6-, 7-, and 9-OH-benzo[a]pyrene. Benzo [a]pyrene epoxide formation may yield both 30 

### phenols and dihydrodiols. Arene oxides that are po or substrates for epoxide hydrolase (EH) or 31 

### the less stable ones that rearrange rapidly, such a s benzo[a]pyrene 2,3-oxide, are less likely to 32 

### yield dihydrodiols. 33 

### The arene oxides can be hydrolyzed by EH to form di hydrodiols (Oesch, 1980). The 34 

### dihydrodiols may be further metabolized by CYPs to form diol epoxides, which are the DNA35 

### reactive metabolites that have been the subject of most studies. In particular, much of the study 36 

### of oxidative products of benzo[a]pyrene metabolism has been done for the 7,8-oxide, since it is a 37 

### precursor to the potent DNA-binding metabolites. T he metabolism of benzo[a]pyrene, as well as 38

### 35 DRAFT - DO NOT CITE OR QUOTE

of PAHs in general, proceeds with a high degree of stereoselectivity. Since most aromatic bonds 1 

### are prochiral, their epoxidation catalyzed by CYPs often results in optically active products. 2 

### Liver microsomes from rats stereospecifically oxidi ze the 7,8-bond of benzo[a]pyrene to yield 3 

### almost exclusively the (+)-benzo[a]pyrene-(7,8)-oxi de (see Figure 3-2). Each enantiomer of the 4 

### 7,8-oxide is stereospecifically converted by EH to a different dihydrodiol via attack of water at 5 

### the 8-position. The (+)-benzo[a]pyrene-7,8-oxide g ives rise to the (-)-benzo[a]pyrene-7,8- 6 

### dihydrodiol, while the (-)-benzo[a]pyrene-7,8-oxide yields the (+)-benzo[a]pyrene-7,8-di7



### hydrodiol. 8

9 

### Source: Grover (1986). 10

11 

### Figure 3-2. The stereospecific activation of benzo [a]pyrene. 12

13 

### Further metabolism of the (-)-benzo[a]pyrene-7,8-di hydrodiol enantiomer by rat CYP 14 

### enzymes preferentially yields (+)-benzo[a]pyrene-7R ,8S-diol-9S,10R-epoxide [(+)-anti-BPDE], 15 

### which is believed to be the most potent carcinogen among the four stereoisomers (Figure 3-2). 16



### Formation of these stereoisomers does not occur at equimolar ratios, and the ratios differ 17 

### between biological systems. For example, in a stud y with rabbit livers, purified microsomes 18 

### oxidized the (-)-benzo[a]pyrene-7,8-dihydrodiol to isomeric diol epoxides in a ratio ranging from 19 

### 1.8:1 to 11:1 in favor of the (+)-anti-BPDE isomer (Deutsch et al., 1979). 20 

### Another important factor in evaluating variability in the metabolic activation of 21 

### benzo[a]pyrene is the degree to which functional po lymorphisms plays a role. Schwarz et al. 22 

### (2001) used recombinant CYP1A1 allelic variants to determine catalytic activity in vitro. 23 

### Catalytic activity of the variant allele 1A1.4 was 70%, and that of variant allele 1A1.2 was only 24 1

2

3

4

5 6 7 8 9 10 11 12 O

O HO

OH

HO

OH Mixed Function Oxidase System (+)-BP-7,8-oxide

(-)-BP-7,8-oxide Epoxide Hydrolase

Epoxide Hydrolase (-)-BP-7,8-diol

(+)-BP-7,8-diol HO

OH (+)-BP-7R,8S-diol-9S,10R-epoxide (+) anti BPDE

HO

OH

HO OH

HO

OH (-)-BP-7R,8S-diol-9R,10S-epoxide (-) syn BPDE

(+)-BP-7S,8R-diol-9S,10R-epoxide (+) syn BPDE

(-)-BP-7S,8R-diol-9R,10S-epoxide (-) anti BPDE

O O O

Mixed Function Oxidase System

Mixed Function Oxidase System

O

### 36 DRAFT - DO NOT CITE OR QUOTE

50% of the WT allele, CYP1A1.1. Km values were gen erally lower for variants than for the 1 

### WT. Each variant produced BPDE, with the activity of CYP1A1.1 > 1.2 > 1.4. The formation 2 

### of diol epoxides was stereospecific, with the allel ic variants producing about three times the 3 

### amount of (±)-anti-BPDE isomers, the suspected ulti mate carcinogens, as compared to the 4 

### noncarcinogenic stereoisomers (±)-syn-BPDE. Wu et al. (2002) found no relationship between 5 

### benzo[a]pyrene metabolite formation and the CYP1A1 MspI polymorphism. The identification 6 

### and characterization of CYP polymorphisms has been the subject of numerous reviews (e.g., 7 

### Wormhoudt et al., 1999). 8 

### Several studies have attempted to clarify the quest ion of which CYP isozyme is 9 

### predominantly responsible for the metabolism of ben zo[a]pyrene. The studies used knock-out 10 

### (ko) animals in which either one of the isozymes in question, CYP1A1 1A2, or 1B1, or the Ah 11 

### receptor (AhR) had been removed or inactivated (CYP 1A1 and 1B1 levels respond to AhR 12 

### induction, while 1A2 is expressed constitutively). Kleiner et al. (2004) measured DNA adduct 13 

### formation in the epidermis of 1B1 –/– 

### , 1A2 –/– 

### , AhR –/– 

### , and WT mice. Six [ 3 

### H]-PAHs were 14 

### administered topically in one dose of 10–2000 nmol and animals were sacrificed after 24 hours. 15 

### Absence of CYP1A2 had very little effect on benzo[a ]pyrene adduct formation; in 1B1 –/– 

### mice 16 

### adducts were about 150% of WT (not significant), wh ile in AhR –/– 

### they were only 27% of WT. 17 

### These findings differed considerably with other PAH s. The benzo[a]pyrene-DNA adduct was 18 

### identified as being derived from (+)-anti-BPDE. Th e authors concluded that 1A1 was the 19 

### primary CYP to metabolize benzo[a]pyrene, and that 1B1 rather serves detoxification. 20 

### Sagredo et al. (2006) conducted a similar experimen t, but with various types of AhR 21



### knock out mice. AhR +/+



### , +/– 

### , and –/– 

### mice were treated once with 100 mg/kg benzo[a]pyre ne by 22 

### gavage. Twenty-four hours after treatment gene exp ression for CYP1A1, 1B1, and AhR was 23 

### measured in lung, liver, spleen, kidney, heart, and blood by real-time or reverse transcriptase 24 

### PCR (RT-PCR). CYP1A1 expression was increased foll owing benzo[a]pyrene treatment in +/+ 25



### and +/– 

### mice (generally higher in heterozygotes), but –/– 

### mice expressed no 1A1. There was a low 26 

### level of basic 1B1 expression in all three genotype s that was inducible by benzo[a]pyrene in 27 

### lung, but not in liver in AhR +/+ 

### and +/– 

### , but not at all in either tissue of AhR –/– 

### mice. Expression 28



### of 1A1 was 25–40 times that of 1B1. There was an A hR gene-dose-response relationship for the 29 

### basal CYP1A1 expression, i.e., +/+ > +/– > –/–, but no such dependence was seen for 1B1. 30 

### Protein adduct levels were spleen > liver > lung > heart > plasma > kidney. The tissue levels of 31 

### adducts showed an inverse relationship with AhR gen e dose, i.e., –/– > +/– > +/+. Similarly, the 32 

### levels of unmetabolized benzo[a]pyrene and of free benzo[a]pyrene-tetrol metabolites were 33 

### higher in all organs of AhR

– 

### mice, as compared with AhR + 

### mice. Also, in the livers of AhR

– 34 

### mice the levels of the less carcinogenic tetrol II were much higher than those of the tetrol I, 35 

### opposite to the situation in AhR + 

### mice. The authors suggested that the high levels of free 36 

### benzo[a]pyrene metabolites in AhR –/– 

### mice were the result of delayed bioactivation, and that a 37 

### powerful AhR-independent pathway for benzo[a]pyrene metabolism must exist. The authors 38

### 37 DRAFT - DO NOT CITE OR QUOTE

explained the very high levels of benzo[a]pyrene ad ducts in organs outside the liver as the result 1 

### of slow detoxification of the agent in the liver of AhR

– 

### mice, allowing high concentrations of the 2 

### parent compound to reach distant tissues. These fi ndings establish important roles in 3 

### benzo[a]pyrene metabolism for both CYP1A1 and 1B1, but they do not clarify which enzyme is 4 

### responsible for biological activation, and which fo r detoxification. 5 

### Uno et al. (2006) investigated the finding that CYP 1A1 (1A1 –/– 

### ) knock out mice are more 6



### sensitive than WT animals to the toxic effects of o rally administered benzo[a]pyrene. They 7 

### produced a series of C57BL/6-based single- or doubl e knock out mice, 1A2 –/– 

### , 1B1 –/– 

### , 1A1/1B1

– 8

/– 

### and 1A2/1B1 –/– 

### . Benzo[a]pyrene was administered in the feed at 1 .25, 12.5, or 125 mg/kg for 9 

### 18 days (this dose is well tolerated by WT C57BL/6 mice for 1 year, but lethal within 30 days to 10



### the 1A1 –/– 

### type). Steady-state blood levels of benzo[a]pyren e, reached within 5 days of 11 

### treatment, were ~25 times higher in 1A1 –/– 

### and ~75 times higher in 1A1/1B1 –/– 

### than in WT mice, 12 

### while in the other knock out types clearance differ ed little from that in WT animals. 13



### Pretreatment of the animals with TCDD to induce the CYP1 enzyme family resulted in decreased 14 

### benzo[a]pyrene peak blood levels and AUCs in WT, bu t doubled peak blood levels and vastly 15



### increased AUCs in the two 1A1 –/– 

### types. A lower-than-WT benzo[a]pyrene clearance w as 16 

### observed only in the two 1A1 –/– 

### types. DNA adduct levels, measured by [ 32 

### P]-postlabeling in 17 

### liver, spleen, and bone marrow, were highest in the 1A1 –/– 

### mice at the higher doses, and in the 18



### 1A1/1B1 –/– 

### mice at the mid dose only. Only 1A1 –/– 

### mice, but not the other genotypes, showed 19 

### signs of severe toxicity. In conclusion, the autho rs of the study painted a rather complex picture 20 

### of how the three CYP1 family enzymes affect the tox icokinetics of benzo[a]pyrene. 21 

### Detoxification of benzo[a]pyrene is mostly achieved by 1A1, probably not only in liver, but also 22 

### in the intestine. Second, in spleen and bone marro w 1B1 brings about metabolic activation of 23 

### benzo[a]pyrene, which, in the absence of 1A1, resul ts in damage to the immune system. The 24 

### authors suggested that tissue-specific expression o f 1A1 and 1B1, respectively, determine an 25 

### organism’s susceptibility to benzo[a]pyrene toxicit y and, possibly, carcinogenicity. 26 

### Van Lipzig et al. (2005) conducted experiments conc erning potential estrogenic activity 27 

### of mono- and dihydroxylated metabolites of benzo[a] pyrene. Estrogen receptor (ER) affinity 28 

### and estrogenic activity were tested in T47D human b reast adenocarcinoma cells (for 29 

### experimental details see Section 4.5.2). Benzo[a]p yrene metabolite mixtures were generated 30 

### using β -naphthoflavone ( β -NF)-induced rat liver microsomes. Several hydroxy lated 31 

### benzo[a]pyrene metabolites had measurable estrogeni c activity. In an attempt to identify 32 

### enzymes involved in benzo[a]pyrene hydroxylation, t he researchers added a specific CYP1A2 33



### inhibitor to the metabolic activation mixture and f ound a ~15-fold increased estrogenic activity 34 

### of the benzo[a]pyrene metabolite mix. They suggest ed that 1A2 inhibition drove the metabolism 35 

### of benzo[a]pyrene towards formation of more estroge nic metabolites. These findings impart a 36 

### role to CYP1A2 in the metabolism of benzo[a]pyrene. 37

### 38 DRAFT - DO NOT CITE OR QUOTE

To summarize, there is experimental evidence to sug gest that three members of the CYP1 1 

### family, 1A1, 1A2, and 1B1, contribute to the metabo lism of benzo[a]pyrene. However, the 2 

### available data do not attribute precise roles to ei ther of these enzymes. There is evidence that 3 

### their expressions, and thus activities, are organ- or tissue-specific and that local isozyme 4 

### activities determine not only the ratio of toxifica tion vs. detoxification, but also the pattern of 5 

### highly toxic vs. less toxic metabolites. 6

7



### 3.3.1.2. Non-CYP-related metabolic pathways 8 

### Other bioactivation processes independent of CYP-me diated diolepoxide formation have 9 

### been demonstrated for benzo[a]pyrene. One-electron oxidation (via CYPs or peroxidases) can 10 

### generate radical cations, which, in turn, can gener ate benzo[a]pyrene quinones (see bottom 11 

### portion of Figure 3-1). These metabolites may gene rate DNA damage through redox cycling or 12 

### the formation of depurinating adducts (McCoull et a l., 1999; Cavalieri et al., 1990). Kim et al. 13 

### (2000) treated male Sprague-Dawley rats with 20 mg/ rat benzo[a]pyrene by i.p. injection and 14 

### reported that the pattern of lipid peroxidation and increase in antioxidant enzymes correlated 15 

### with formation of quinone metabolites of benzo[a]py rene. Direct i.v. injection of 16 

### benzo[a]pyrene and a series of metabolites confirme d the quinone metabolites indeed to be 17 

### associated with the observed increase in lipid pero xidation. Secondary oxidation of 6-OH18 

### benzo[a]pyrene can generate hydroquinone and quinon e metabolites at the 1,6-, 3,6-, or 6,12- 19 

### positions. Quinone formation may be catalyzed by d ihydrodiol dehydrogenases (DHHs), such as 20 

### aldo-keto reductase (AKR) (Penning et al., 1999). Tsuruda et al. (2001) showed in vitro that the 21 

### expression of rat DHH in human breast cancer MCF-7 cells generated 7,8-benzo[a]pyrene-diones 22 

### from benzo[a]pyrene-7,8-diol. 23 

### Mallet et al. (1991) treated tetradecanoylphorbol a cetate-stimulated human 24 

### polymorphonuclear leukocytes (PMNs) with (±)-trans- 7,8-dihydroxy-7,8-dihydro25 

### benzo[a]pyrene. They found that the cells were abl e to transform the benzo[a]pyrene-diol into 26 

### the diolepoxide and tetrols with a stereochemical anti/syn ratio of six. The kinetics of the 27 

### reaction suggested that hydrogen peroxide or a ferr yl-oxygen-transfer were involved. Because 28 

### myeloperoxidase (MPO) uses hydrogen peroxide in its reaction, the authors inhibited this 29 

### enzyme specifically with azide and found that the f ormation of tetrols from benzo[a]pyrene-diol 30 

### was reduced. Thus, MPO is able to execute the meta bolic activation of benzo[a]pyrene. 31 

### Byczkowski and Kulkarni (1990) observed that benzo[ a]pyrene diol can be cooxygenated 32 

### during lipid peroxidation to form the diolepoxide. To reduce interference from CYP-catalyzed 33 

### reactions they used term human placental microsomes , which are low in CYP450. Peroxidative 34 

### conditions were created by a redox cycling system c omprised of partially chelated ferrous ions 35 

### and NADPH:CYP450 oxidase; this system quadrupled th e formation of malonic dialdehyde (a 36 

### measure of lipid peroxidation) compared with placen tal microsomes alone. The peroxidative 37 

### system increased overall metabolism of benzo[a]pyre ne and benzo[a]pyrene-7,8-diol by 27– 38

### 39 DRAFT - DO NOT CITE OR QUOTE

28%, compared with placental microsomes alone. The amounts of individual metabolites were 1 

### changed to various extents in the presence of perox idative conditions; the most striking 2



### observation was the more than doubled formation of trans-anti-benzo[a]pyrene-tetrol (the 3 

### proximate carcinogen) and an almost tripled binding to protein. There was a highly significant 4 

### correlation (

p < 0.0005) between malondialdehyde and trans-anti-b enzo[a]pyrene-tetrol 5 

### formation. The authors pointed out that by means o f this metabolic pathway the human fetus 6 

### could be exposed to BPDE despite the absence of pro nounced CYP450 activity in term placenta. 7 

### Similarly, a redox cycling system based on vanadate -IV ions was able to increase formation of 8 

### trans-anti-benzo[a]pyrene-tetrol five- to sixfold i n the presence of term human placental 9 

### microsomes (Byczkowski and Kulkarni, 1992). 10 

### Flowers et al. (1996) investigated the role of radi cal and reactive oxygen species (ROS) 11 

### formation in benzo[a]pyrene-quinone-induced redox c ycling, a reaction involving DHH, which 12 

### can oxidize BP-diol to benzo[a]pyrene-7,8-dione (BP Q). BPQ is mutagenic and genotoxic. In 13 

### isolated rat hepatocytes BPQ was incorporated coval ently into DNA (30 ± 17 adducts/10 6 

### base 14 

### pairs) while extensive DNA fragmentation took place . DNA fragmentation was also observed in 15 

### hepatocytes treated with BP-diol; the effect was pa rtially abolished when an inhibitor of DHH 16 

### was added to the reaction. Hepatocytes treated wit h either BP-diol or BPQ produced superoxide 17 

### anion radical, formation of which could be blocked by DHH inhibitors. In an in vitro experiment 18 

### it was shown that BPQ at 0.05–10 μ M caused DNA strand scission in the presence of NAD PH 19



### and CuCl 2 

### , suggesting that redox-cycling took place. DNA st rand scission was prevented by 20 

### catalase and hydroxyl radical scavengers but not by superoxide dismutase. The authors 21 

### concluded that DHH metabolizes (+/-)-anti-BPDE to B PQ, which in turn causes extensive DNA 22 

### fragmentation via the generation of ROS. 23 

### In a subsequent study, Flowers et al. (1997) provid ed more detail on the redox cycling 24 

### reaction of BPQ. They showed that the reaction req uired the presence of NADPH (1 mM) and 25



### Cu 2+ 

### (10 μ M). During the reaction superoxide anion radicals, benzo[a]pyrene semiquinone 26 

### radicals, hydroxyl radicals, and H 2 

### O 2 

### were formed. Hydroxyl radical scavengers, such as 27 

### mannitol, sodium benzoate, or formic acid prevented the redox cycling (as assessed by DNA 28 

### strand scission), as did the Cu + 

### chelators bathocuproine or neocuproine. The resul ts were 29 

### interpreted as indicating that redox cycling of ben zo[a]pyrene quinone involves a Cu + 

### -catalyzed 30



### Fenton reaction. 31 

### Other peroxidases such as prostaglandin H synthase (PHS) may also generate radical 32 

### cations from benzo[a]pyrene (Parman and Wells, 2002 ). Marnett (1990) reviewed the role of 33 

### PHS in benzo[a]pyrene metabolism. Peroxy radicals epoxidize the procarcinogen 34 

### benzo[a]pyrene-7,8-diol via PHS to the epoxide BPDE . Stereochemical experiments have 35 

### allowed the distinction between peroxide-mediated a nd CYP450-mediated epoxide formation 36 

### from benzo[a]pyrene. Thus, peroxy radical-dependen t epoxidation of benzo[a]pyrene-7,8-diol 37 

### occurs in rat liver microsomes, mouse skin homogena tes, cultured fibroblasts, cultured hamster 38

### 40 DRAFT - DO NOT CITE OR QUOTE

trachea, and freshly isolated mouse epidermal cells . Peroxy radical-generated metabolites are 1 

### predominant in uninduced animals, while in ß-NF-ind uced animals the CYP450-produced 2 

### metabolites prevail. There are other pathways by w hich peroxides can oxidize benzo[a]pyrene3 

### 7,8-diol because inhibition of PGH synthase with no n-steroidal anti-inflammatory drug 4 

### (NSAIDs) does not prevent BPDE formation. 5 

### Redox-active quinones are formed through the oxidat ive metabolism of benzo[a]pyrene, 6 

### particularly at the 6-position (see Figure 3-1). N QO1 is an important enzyme for the 7 

### detoxification of reactive quinones. Joseph and Ja iswal (1998) reported that NQO1 expression 8 

### inhibited the formation of benzo[a]pyrene-quinone a dducts with DNA and mutagenicity in vitro. 9 

### There are also potential ring opening mechanisms fo r benzo[a]pyrene. Stansbury et al. (2000) 10 

### used activated polymorphonuclear monocytes or a rec onstituted in vitro system to generate a 11 

### ring-opening dialdehyde metabolite from the benzo[a ]pyrene-7,8-diol-9,10-epoxide (BPDE) that 12 

### can react with DNA to generate unique DNA adducts t hat are different from the ones formed by 13



### BPDE. 14 

### Nordling et al. (2002) identified a novel benzo[a]p yrene metabolite (7-oxo15 

### benz[d]anthracene-3,4-dicarboxylic acid anhydride) in the urine of benzo[a]pyrene-treated rats. 16 

### Follow-up in vitro experiments with this compound f ound it to be weakly mutagenic in 17 

### salmonella strain TA102, able to induce DNA stand b reaks in HT-29 cells, capable of inducing 18 

### cytotoxicity via an apoptotic mechanism, and increa sing gene expression through a 19 

### cyclooxygenase (COX)-2 promoter in HCT 116 cells. These results demonstrate that novel 20 

### benzo[a]pyrene metabolites have toxicological prope rties distinct from those of the better studied 21



### BPDE. 22 

### The roles of PHS-2 (now mostly called COX-2), MPO, NQO1, and other enzymes in the 23 

### oxidative metabolism of benzo[a]pyrene may be cruci al, but not enough data are as yet available 24 

### to attempt a quantitative comparison with the CYP-m ediated pathways. This may be of some 25 

### importance in the assessment of cancer risks becaus e not only CYP450 isozymes, but also MPO 26 

### and NQO1 exhibit gene polymorphism. This is an are a where much more research is necessary. 27

28



### 3.3.2. Phase II Metabolism 29 

### The reactive products of phase I metabolism are sub ject to the action of several phase II 30 

### conjugation and detoxification enzyme systems that display preferential activity for specific 31 

### oxidation products of benzo[a]pyrene. These phase II reactions play a critical role in protecting 32 

### cellular macromolecules from binding with reactive benzo[a]pyrene diolepoxides, radical 33 

### cations, or ROS. Therefore, the balance between Ph ase I activation of benzo[a]pyrene and its 34 

### metabolites and detoxification by Phase II processe s is an important determinant of toxicity. 35 

### The diol epoxides formed from benzo[a]pyrene metabo lism are not usually found as 36 

### urinary metabolites, rather they are detected as ad ducts of nucleic acids or proteins if not further 37 

### metabolized. Detoxification of the diol epoxide me tabolites of benzo[a]pyrene is through 38

### 41 DRAFT - DO NOT CITE OR QUOTE

rearrangement to form tetrols or via conjugation wi th glutathione. Early work by Gozukara et al. 1 

### (1981) demonstrated that exogenous EH added to benz o[a]pyrene-treated human monocytes 2 

### reduced DNA binding, suggesting a role for this enz yme in the inactivation of diol epoxides. 3 

### Furthermore, nonenzymatic hydration of diol epoxide s proceeds rapidly in aqueous media in the 4 

### absence of EH to yield tetrol products via cis or t rans addition. A second mechanism for the 5 

### detoxification of reactive diol epoxides formed fro m benzo[a]pyrene is through glutathione 6 

### (GSH) conjugation. This Phase II reaction is catal yzed by GSTs. GSTs are a family of enzymes 7 

### with varying substrate specificity and distribution among tissues (reviewed in Hayes and 8 

### Strange, 2000; Eaton and Bammler, 1999; Hayes and P ulford, 1995). Primary isoforms of 9 

### relevance for conjugation of BPDE include the α , μ , π , and θ isoforms (GSTA, GSTM, GSTP, 10 

### GSTT, respectively). 11 

### Numerous studies using human GSTs expressed in mamm alian cell lines have 12 

### demonstrated the ability of GST to metabolize benzo [a]pyrene diol epoxides. For example, Dreij 13 

### et al. (2002) demonstrated that GST isozymes, inclu ding alpha class GSTA1-1, GSTM1-1, and 14 

### GSTP1-1 isoforms, had significant catalytic activit y toward benzo[a]pyrene-derived diol 15 

### epoxides. Robertson et al. (1986) incubated isolat ed human GST isoforms with GSH and 16 

### (±)anti-BPDE to assess differences in their catalyt ic properties. Maximum substrate turnover 17 

### velocity (V max 

### ) values for α , μ , and π were 38, 570, and 825 nmol mg -1 

### minute -1 

### , and K m 

### values 18 

### were 28, 27, and 54 μ M, respectively. Rojas et al. (1998) reported that no BPDE adducts were 19 

### formed in GSTM1-positive cells, but adducts were pr esent in GSTM1-negative cells. This body 20 

### of in vitro studies suggests that GST is an importa nt detoxification mechanism for 21 

### benzo[a]pyrene-derived epoxides. This compelling e vidence for a role of GSTs in protecting 22 

### against reactive benzo[a]pyrene metabolites has tri ggered several molecular epidemiology 23 

### studies. However, recent studies on the impact of polymorphism on adduct levels in PAH24 

### exposed human populations did not succeed in showin g clear relationships between CYP1A1, 25 

### EH, or GSTM1 polymorphisms and DNA (Hemminki et al. , 1997) or blood protein adduct 26 

### formation (Pastorelli et al., 1998). 27 

### Conjugation with glucuronide is another important d etoxification mechanism for 28 

### oxidative benzo[a]pyrene metabolites. Most of the phenolic metabolites of benzo[a]pyrene are 29 

### further metabolized by glucuronidation or sulfation , and significant portions of total metabolites 30



### in excreta or tissues can be recovered in this form . Bevan and Sadler (1992) administered a 31 

### single 2 μ g/kg benzo[a]pyrene dose by intratracheal instillat ion to male Sprague-Dawley rats and 32 

### assessed the benzo[a]pyrene metabolite profiles in bile after 6 hours. Identified metabolites were 33 

### 31.2% quinol diglucuronides, 30.4% thioether conjug ates, 17.8% monoglucuronide, 6.2% sulfate 34 

### conjugates, and 14.4% unconjugated metabolites. 35 

### The UGTs are a family of enzymes that catalyze the conjugation of UDP-glucuronide 36 

### with endogenous substrates (e.g., bilirubin) as wel l as xenobiotics (reviewed in Guillemette, 37 

### 2003). UGT isoforms as well as their allelic varia nts show different patterns of tissue 38

### 42 DRAFT - DO NOT CITE OR QUOTE

distribution and catalytic activity toward benzo[a] pyrene-derived phenols and diols. For 1 

### example, Fang and Lazarus (2004) assessed the abili ty of the allelic variants UGT1A1 and 1A9 2 

### in human liver to catalyze glucuronidation of benzo [a]pyrene-7,8-diol. Microsomes from 3 

### subjects homozygous for the UGT1A1*28 variant (pres ent in approximately 12% of the 4 

### Caucasian population) had approximately twofold low er UGT1A1 protein levels than liver 5



### microsomes from individuals with the WT allele. Ad dition of a UGT1A9 inhibitor to the 6 

### incubation decreased benzo[a]pyrene-glucuronide for mation three- to sixfold, suggesting that 7 

### UGT1A9 also has significant catalytic activity towa rd benzo[a]pyrene-7,8-diols. With UGT1A9 8 

### activity blocked, glucuronide formation in UGT1A1*2 8 homozygotes was significantly lower 9 

### than in WT and heterozygous individuals. The appar ent Km for this reaction did not differ 10 

### among microsomes from allelic variants. 11 

### UGT activity also shows significant interindividual variability. Hu and Wells (2004) 12 

### evaluated glucuronidation of benzo[a]pyrene metabol ites in human lymphocytes (HL) in vitro. 13 

### The degree to which glucuronide conjugates were for med varied over 200-fold (percent of 14 

### metabolites as glucuronide conjugates ranged from 0 .01 to 5% of total benzo[a]pyrene 15 

### metabolites). Incubation of lymphocytes with benzo [a]pyrene, benzo[a]pyrene-7,8-diol, or 16 

### benzo[a]pyrene-4,5-diol resulted in covalent bindin g to protein with, in the case of 17 

### benzo[a]pyrene, a 220-fold inter-individual variabi lity. Addition of the UGT substrate, UDP 18 

### glucuronic acid (UDPGA), lowered the inter-individu al variability to 143-fold. For 19 

### benzo[a]pyrene or its diols there was a statistical ly highly significant relationship between 20 

### increase in glucuronidation and decrease in protein binding. Cytotoxicity also was inversely 21 

### correlated to conjugation of diols and diones, sugg esting that glucuronidation is an important 22 

### pathway for protection from chemically reactive ben zo[a]pyrene metabolites. 23 

### Sulfation, normally a defoliation process, can prod uce a DNA-damaging intermediate in 24 

### the case of benzo[a]pyrene. It was shown that in r at or mouse liver cytosolic sulfotransferase (in 25 

### the presence of 3'-phosphoadenosine 5'-phosphosulfa te) catalyzes formation of sulfates of 26 

### benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol, benzo[a]py rene-7,8-dihydrodiol, and benzo[a]pyrene27 

### 7,8,9,10-tetrol. All three sulfates were tested fo r their ability to bind to DNA, but only the 28 

### benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol-sulfate for med DNA adducts (Surh and Tannenbaum, 29 

### 1995). 30 

### Although not specific for benzo[a]pyrene, there is now considerable evidence that genetic 31 

### polymorphisms of the GST, UGT, and EH genes impart an added risk to humans for developing 32 

### cancer. Of some significance to the assessment of benzo[a]pyrene may be that smoking, in 33 

### combination with genetic polymorphism at several ge ne loci (for detail, see Section 4.8.3), 34 

### increases the risk for bladder cancer (Moore et al. , 2004; Choi et al., 2003; Park et al., 2003) and 35 

### lung cancer (Alexandrie et al., 2004; Lin et al., 2 003). Also, Leng et al. (2004), according to the 36 

### English abstract of a paper in Chinese, showed that coke oven workers (who are exposed to 37 

### PAHs, including benzo[a]pyrene) homozygous at the P 187S site of the NQO1 gene or carrying 38

### 43 DRAFT - DO NOT CITE OR QUOTE

the null variant of the GSTM1 gene had a significan tly increased risk of chromosomal damage in 1 

### peripheral blood lymphocytes, while the risk was mu ch lower than controls in subjects with a 2 

### variant allele at the H113Y site of the EH gene. 3

4 

### 3.3.3. Tissue-specific Metabolism 5



### 3.3.3.1. Respiratory Tract Tissues 6 

### benzo[a]pyrene treatment has been associated with t he induction of respiratory tract 7 

### tumors. This finding is consistent with the abilit y of the lung to metabolize benzo[a]pyrene, 8



### which has been demonstrated in numerous studies. E wing et al. (2006) investigated the 9 

### hypothesis that lung cancer following PAH induction may be a result of slow absorption and 10 

### extensive metabolism in the thick respiratory epith elia. The researchers used isolated, perfused 11 

### rat lung to investigate these processes. Benzo[a]p yrene was coated onto 3.5 μ m silica carrier 12 

### particles at concentrations to deliver an average o f 2.2, 36, and 8,400 ng to each lung within <1 13



### minute. Perfusate was collected for 77 minutes the reafter. Both perfusates and lungs were 14 

### analyzed for benzo[a]pyrene and metabolites. Absor ption and metabolism were both strongly 15 

### dose-dependent: at the low and mid exposure levels benzo[a]pyrene concentration increased 16 

### rapidly in the perfusate to a maximum after <5 minu tes, then decreased over the remaining 17 

### observation period. At the high dose, benzo[a]pyre ne in perfusate reached the maximum at 18 

### about 30 minutes after exposure and stayed at a con stant level from there on, i.e., the absorption 19 

### of benzo[a]pyrene proceeded at zero order until all deposited solid benzo[a]pyrene was 20 

### dissolved. The mass balances for benzo[a]pyrene eq uivalents in lung vs. perfusate were lung ca. 21 

### one-third perfusate at the low dose, lung = perfusa te at the mid dose, and lung about twice that of 22 

### perfusate at the high dose. At the low exposure le vel metabolism was apparently able to convert 23 

### most of the parent compound, while at the highest e xposure level most of the absorbed 24 

### benzo[a]pyrene remained unmetabolized even at the e nd of the experiment. The authors pointed 25 

### out that these findings may explain why many attemp ts have failed to inducer lung cancer in 26 

### animals using high-dose particle inhalation protoco ls. The results further confirm that 27 

### benzo[a]pyrene metabolism is organ specific. 28 

### Autrup et al. (1980) compared the metabolic capacit y of tracheobronchial tissues in 29 

### culture among several species, including humans, mi ce, rats, hamsters and bovines. Results from 30 

### this study are summarized in Table 3-2. Benzo[a]py rene was metabolized extensively in tissues 31 

### from all species tested, with lower amounts of meta bolites identified in rats and nonresponsive 32 

### mice. Patterns of metabolism differed among the sp ecies but showed formation of a complex 33 

### array of metabolites, including phenols, diols, tet rols, and quinones. Data summarized from the 34 

### study suggest that under the conditions tested: (1 ) upper respiratory tract tissues for all species 35 

### were able to metabolize benzo[a]pyrene, (2) the deg ree of phase II conjugation products was 36 

### greatest in humans, followed by hamsters, Ah-respon sive mice, bovines, rats, and Ah37 

### nonresponsive mice, (3) multiple phase II conjugati on pathways were operative in tissues of all 38

### 44 DRAFT - DO NOT CITE OR QUOTE

species, although the relative proportions of conju gate formation varied, and (4) conducting 1 

### airway tissues from all species were able to metabo lize benzo[a]pyrene into DNA-reactive 2 

### metabolites, with DNA binding greatest in hamster t rachea followed by human and bovine 3



### bronchus. 4 

### DNA binding in human, rat, and mouse tissue was sim ilar but considerably higher in 5 

### hamster (Table 3-2). The results were quite variab le among individuals, a 33-fold difference in 6 

### human bronchus, a fivefold variation in human trach ea, and a threefold difference in bovine 7 

### bronchus, but minimal variation among individuals o f the laboratory animal species. Overall, 8 

### these results show that human lung tissue metaboliz es benzo[a]pyrene in a manner that is 9 

### qualitatively similar to that of species that are s usceptible to lung tumors, although some 10 

### quantitative differences in specific metabolic path ways are observed (Autrup et al., 1980). 11

12 

### Table 3-2. Species differences in tracheobronchial benzo[a]pyrene

metabolism

Species Total

metabolites a Ratio organic/water

soluble metabolites Percent water soluble metabolites as

sulfate esters, glucuronides, and glutathione conjugates DNA binding b

C57B1/6N mouse 1.00 ± 0.25 1.4 31/30/39 10

DBA/2N mouse 0.40 ± 0.13 0.5 28/27/44 10

CD rat 0.65 ± 0.10 0.5 31/13/56 10 Syrian golden hamsters 1.34 ± 0.13 1.2 20/17/63 26

Bovine bronchus 0.93 ± 0.12 0.12 24/29/38 16

Human trachea 1.09 ± 0.48 2.5 56/12/32 11

Human - main-stem bronchus 1.33 ± 0.72 1.7 44/7/51 16

Human secondary and tertiary bronchus 1.75 ± 0.82 2.3 44/6/51 16

a Mean ± SD in pmol/ μ g DNA.

b Mean ± SD in pmol/mg DNA; results are from trachea in mouse, rat, and hamster.

Source: Autrup et al. (1980).

13 

### In vitro studies with human bronchial epithelial an d lung tissue showed that 14 

### benzo[a]pyrene is metabolized to the 7,8- and 9,10- diols and, to a lesser extent, to the 4,5-diol 15 

### and 3-OH metabolites (Autrup et al., 1978; Cohen et al., 1976). The metabolites were identified 16 

### as glutathione and sulfate conjugates; no glucuroni de metabolites were found. The ability of 17 

### human tissues to metabolize benzo[a]pyrene has also been demonstrated in lung-derived cell 18 

### lines. Kiefer et al. (1988) demonstrated that benz o[a]pyrene was metabolized in vitro in the 19 

### human lung cancer cell line NCI-H322. These cells were also able to form benzo[a]pyrene-7,8- 20 

### diol, suggesting that human lung cells are able to generate carcinogenic metabolites of 21 

### benzo[a]pyrene. Approximately 30% of the detected metabolites were water-soluble, about 30% 22

### 45 DRAFT - DO NOT CITE OR QUOTE

of which were glutathione conjugates. Sulfates, bu t not glucuronide conjugates, were also 1



### detected. 2 

### A complement of enzymes for the oxidative metabolis m of benzo[a]pyrene in the lungs 3



### has been identified in both humans and animal tissu es, and these activities are inducible with 4 

### prior exposure. Wei et al. (2001) evaluated CYP1A1 levels in fresh lung tissue from nine human 5 

### donors. CYP1A1 and CYP1A2 were present at variable levels in lung tissues based on mRNA, 6 

### protein levels, enzyme activity, and ability of S9 fractions to induce mutagenicity in an Ames 7 

### assay. CYP1B1 was not identified. The authors emp hasized that, in contrast to some previous 8 

### studies, they were able to identify CYP1A2 in human lung (Wei et al., 2001). In a subsequent 9 

### study, Wei et al. (2002) lung tissue from 27 human donors was evaluated for CYP1A status. 10 

### CYP1A1 and CYP1A2 transcripts were present at varia ble levels in nearly all samples and were 11 

### inducible by benzo[a]pyrene treatment. Mean induci ble CYP1A2 levels were roughly four times 12 

### lower than CYP1A1. Microsomes prepared from these tissues pretreated with benzo[a]pyrene 13



### resulted in a threefold increase in DNA adduct form ation, while pretreatment with the potent 14 

### AhR ligand TCDD increased benzo[a]pyrene-DNA adduct formation to 24-fold over controls. 15 

### This result shows that that CYP1A activity was high ly inducible via the AhR pathway in human 16 

### lung tissues. 17 

### These results using human donor tissues are consist ent with the body of literature 18 

### demonstrating the induction of benzo[a]pyrene metab olism in lungs of rodents. For example, 19 

### Vainio et al. (1976) compared the metabolism of int ratracheally-instilled benzo[a]pyrene in the 20 

### isolated perfused rat lung of both control and rats induced with 3-MC. Pretreated rat lungs had 21 

### increased covalent binding of [ 3 

### H]-benzo[a]pyrene to lung tissue, decreased appeara nce of 22 

### unmetabolized benzo[a]pyrene in perfusion liquid, a nd increased formation of water soluble 23 

### metabolites. Bompart and Clamens (1990) assessed A HH activity in male Sprague-Dawley rats 24 

### given 2 mg/kg benzo[a]pyrene by i.p. injection week ly for 30 weeks. Every third week, five 25 

### animals were sacrificed and lung and liver microsom es were prepared for determination of AHH 26 

### activity (as measured by formation of the 3-OH meta bolite of benzo[a]pyrene). Control levels of 27 

### AHH in lung were much lower than in liver. AHH act ivity in the lung increased with repeated 28 

### benzo[a]pyrene doses until approximately week 15, w hen it reached levels approximately 29 

### eightfold over controls. Even after this induction , lung AHH activity was still approximately 30- 30 

### fold lower than in liver. Benzo[a]pyrene treatment s had no effect on AHH activity in the liver in 31 

### this study. 32 

### Petridou-Fischer et al. (1988) instilled radiolabel ed benzo[a]pyrene into the nasal 33 

### turbinates (ethmoid and maxillary) of monkeys and d ogs. Metabolic activity in these tissues was 34 

### demonstrated by the formation of diverse metabolic products (phenols, diols, tetrols, and 35 

### quinones). No region-specific metabolism was ident ified. Even though ethmoid versus 36 

### maxillary turbinates contain different CYP activiti es, the pattern of metabolites was qualitatively 37

### 46 DRAFT - DO NOT CITE OR QUOTE

similar. Differences were not compared quantitativ ely due to the small number of animals used 1 

### (four dogs and two monkeys). 2 

### Bond et al. (1988) showed differential levels of Ph ase I and Phase II activities in 3 

### respiratory tract regions of dogs. Benzo[a]pyrene metabolism was greater in nasal tissue than in 4 

### lung tissue. Metabolic activities towards benzo[a] pyrene in various areas of the upper 5 

### respiratory tract ranged from 5 to 15 pmol/mg prote in/minute; however, in the ethmoid turbinates 6 

### they reached approximately 45 pmol/mg-minute. The was no difference in the regional patterns 7 

### of metabolite formation. Although CYP isozyme acti vity was similar in liver and lung, 8 

### benzo[a]pyrene metabolism in respiratory tract tiss ues was about one-tenth that in liver (one9 

### third for the ethmoid turbinates). EH activity was highest in the lower generations of the 10 

### conducting airways, followed by liver then nasal ti ssues. GST activity was highest in liver, 11 

### followed by nasal tissues. UGT activity was more e venly distributed among lung regions, and 12



### was similar to levels in the liver. These data sho w that in dogs the nasal region and lungs have 13 

### greater metabolic capability for benzo[a]pyrene and its metabolites than the conducting airways, 14 

### and levels of metabolism are generally similar to t hose observed in the liver. 15 

### Dahl et al. (1985) evaluated benzo[a]pyrene metabol ism in the respiratory tract tissues 16 

### from Syrian hamsters. All regions of the respirato ry tract had metabolic activity as assessed by 17 

### the formation of benzo[a]pyrene metabolites. Activ ity was highest in the nasal tissues on a per 18 

### gram tissue basis, with similar activities observed in esophagus, forestomach, trachea, larynx, 19 

### and lungs. Total metabolism on a per organ basis w as highest for the lung and trachea. Similar 20 

### results were obtained for lung and nasal tissue of rats that had inhaled benzo[a]pyrene (Wolff et 21 

### al., 1989). 22 

### Persson et al. (2002) showed that [ 3 

### H]-benzo[a]pyrene instilled nasally in female 23 

### Sprague- Dawley rats was taken up in nasal structur es (sustenacular cells and Bowman’s glands). 24 

### Transport of benzo[a]pyrene or metabolites (as dete rmined by radiography) via axons of 25 

### olfactory neurons to the olfactory bulb was identif ied, indicating uptake into nasal structures and 26 

### transport to central nervous system (CNS) structure s via neurons. 27 

### Weyand and Bevan (1986) examined benzo[a]pyrene dis position and metabolism in male 28 

### Sprague-Dawley rats following intratracheal instill ation of 1 μ g/kg body weight [ 3 

### H]- 29 

### benzo[a]pyrene. The overall concentration of benzo [a]pyrene metabolites in lung and liver 30 

### decreased over the 360-minute period, with a shift from predominately lipid-soluble metabolites 31 

### to an increasing component of water-soluble metabol ites at later times. In the lung, 32 

### benzo[a]pyrene metabolites represented 47.7% of the administered dose in the organic and 33 

### 11.9% in the aqueous fraction, respectively, at 5 m inutes, while at 360 minutes the corresponding 34 

### fractions were 2.16 and 1.83%, respectively. The m etabolite profile determined in the organic 35 

### phase at 360 minutes was as follows: conjugates or polyhydroxylated compounds, 16.3%; 9,10- 36 

### diol, 4.95%; 4,5-diol, 2.60%; 7,8-diol, 3.11%; 1,6- quinone, 2.99%; 3,6-quinone, 4.09%; 6,12- 37 

### quinone, 2.23%; 9-OH, 2.26%; 3-OH, 4.59%; and benzo [a]pyrene, 20.0%. Over the 360-minute 38

### 47 DRAFT - DO NOT CITE OR QUOTE

period, notable shifts in the relative proportion o f benzo[a]pyrene and metabolite levels in the 1 

### lung indicated a net increase in the contribution o f conjugates and tetrols, stable to decreasing 2 

### levels of quinones, increasing levels of diols and phenols, and decreasing levels of parent 3 

### benzo[a]pyrene. A similar pattern was observed for concentrations of benzo[a]pyrene 4 

### metabolites in the liver, except that quinone level s also increased in this tissue over the 6-hour 5 

### period. This identification of significant levels of quinone metabolites early in the lungs is 6 

### consistent with in vitro studies, such as that of A utrup et al. (1980) in which rat lung had a high 7 

### capacity to form quinones originating from the oxid ation of benzo[a]pyrene at the 6 position. 8



### Most of the metabolism studies have focused on the role of the CYP1 isoforms on 9 

### benzo[a]pyrene metabolism in the lung. However, ot her CYPs may also be important for 10 

### benzo[a]pyrene metabolism among species. For examp le, Shultz et al. (2001) reported that 11 

### benzo[a]pyrene was metabolized using recombinant CY P2F2 from mouse lung in a cell-free in 12 

### vitro assay, although metabolic capability was less than that of other isoforms. CYP2F2 13 

### expression in mouse lung airways was greater than t hat in tracheal parenchyma, showing region14 

### specific metabolic differences. 15

16



### 3.3.3.2. GI Tract and Liver Tissues 17 

### Fontana et al. (1999) reported that in healthy volu nteers fed diets enriched with char18 

### grilled meat, CYP1A1 and 1A2 activities were induce d in the liver and CYP1A1 protein levels 19 

### were increased in small intestine biopsies. No cha nge in CYP3A4 or 3A5 levels was observed. 20 

### DNA adducts in peripheral blood cells were inversel y correlated to CYP1A levels in enterocytes 21 

### and CYP1A2 levels in liver. These findings point t o the presence of AhR ligands, such as 22 

### benzo[a]pyrene, in char-grilled meat and lend furth er support to the complexity of 23 

### benzo[a]pyrene metabolism by CYP isozymes. 24 

### In a human hepatoma cell line (HepG2) incubated wit h [ 3 

### H]-benzo[a]pyrene, 25 

### radiolabeled metabolites were recovered primarily i n the medium (88.4% of the radioactive 26 

### material) (Diamond et al., 1980). Sixty-four perce nt of the metabolites were unidentified water27



### soluble metabolites. Chloroform extractable metabo lites (36% of the radiolabel) included 7,8- 28 

### diol, 9,10-diol, quinones, 3-OH (16% combined) meta bolites, and unmetabolized benzo[a]pyrene 29 

### (20%). The cell lysate contained the same metaboli tes, but the proportions of the 3-OH 30 

### metabolite and parent compound were relatively high er. Enzymatic treatment for conjugate 31 

### formation did not change recovery of radioactivity, suggesting that at least this tumor cell line 32 

### did not extensively form phenol products. The auth ors noted that the HepG2 cell line did not 33 

### utilize the major phenol detoxification pathway of rodent cell cultures. 34 

### Similar results were obtained with human hepatocyte s in culture (Monteith et al., 1987). 35 

### Following incubation for 24 hours, the primary meta bolites of [ 3 

### H]-benzo[a]pyrene were 36 

### unidentified highly polar, water-soluble conjugates . The next four most prevalent metabolites 37 

### consisted of 3-OH-benzo[a]pyrene and the 4,5-, 9,10 - and 7,8-diols. As the dose of 38

### 48 DRAFT - DO NOT CITE OR QUOTE

benzo[a]pyrene was increased from 10 to 100 μ mol, the amount of metabolites increased in a 1 

### linear fashion, suggesting that the capacity of hum an hepatocytes to metabolize benzo[a]pyrene 2 

### was not saturated at benzo[a]pyrene concentrations up to 100 μ mol. 3-MC induced rat 3 

### microsomes convert benzo[a]pyrene to the BPDE appro ximately 10 times faster than 4 

### microsomes from uninduced rats, likely through the induction of CYP1A1 (Keller et al., 1987). 5 

### The rate-limiting step appeared to be competition f or CYP1A1 binding sites between parent 6 

### benzo[a]pyrene and the benzo[a]pyrene-7,8-diol. 7 

### The metabolism of benzo[a]pyrene in the GI tract an d liver has been studied. Zheng et al. 8 

### (2002) determined the expression of UGT isozyme lev els and activity toward benzo[a]pyrene 9 

### from human tissue samples of the liver, lung, and r egions of the aerodigestive tract (tongue, 10 

### tonsil, floor of the mouth, larynx, esophagus). Gl ucuronidation of phenolic benzo[a]pyrene 11 

### metabolites was detected in all aerodigestive tract tissues examined and, of the isoforms tested, 12 

### activity was identified for UGT1A7, 1A8, and 1A10. No UGT expression or glucuronidation 13 

### activity was detected in lung tissue. 14 

### Bentsen-Farmen et al. (1999a) measured CYP1A1 induc tion and DNA adduct levels in 15 

### Wistar rats given i.p. doses of 3-MC and followed b y a single dose of benzo[a]pyrene, or a single 16 

### benzo[a]pyrene dose without pretreatment. 3-MC pre treatment increased CYP1A1 activity in 17



### the liver and DNA adduct levels in both liver and l ung, with significant correlation to CYP1A1 18 

### activity in lung but not in liver. The results ind icate the difficulty in using DNA adducts as a 19 

### biomarker in short-term exposures. The authors fur ther reported that the study results were 20 

### highly dependent on the analytical technique used. 21 

### Ramesh et al. (2001b) exposed F344 rats for up to 9 0 days to benzo[a]pyrene in the diet 22 

### at doses of 0, 5, 50, or 100 mg/kg-day. AHH activi ty (as measured by formation of the 3-OH23 

### benzo[a]pyrene) in the liver was increased in both males and females (approximately twofold 24 

### higher in females at the end of the study) in a dos e- and duration-dependent manner. 25 

### Granberg et al. (2000) treated female NMRI mice wit h [ 3 

### H]-benzo[a]pyrene by i.v. 26 

### injection and identified tissue-bound radiolabel in the lung, liver, and cardiovascular endothelial 27 

### cells. The levels of tissue-bound radioactivity we re correlated to CYP1A1, as determined by 28 

### 7-ethoxyresorufin-O-deethylase (EROD) activity. Pr etreatment with the AhR ligand β -NF 29 

### increased tissue binding in lung and heart cells. 30

31



### 3.3.3.3. Skin 32 

### Indirect evidence for metabolism of benzo[a]pyrene in skin is plentiful and includes 33 

### numerous studies of dermally exposed humans or anim als with subsequent detection of 34 

### benzo[a]pyrene metabolites in tissues or excreta. These types of studies provide qualitative 35



### evidence for dermal metabolism but are confounded b y potential contribution of metabolism in 36 

### other tissues following systemic circulation. Howe ver, direct measurements of benzo[a]pyrene 37 

### metabolism have been made in in vitro models using human skin or skin cells. 38

### 49 DRAFT - DO NOT CITE OR QUOTE

Hall and Grover (1988) also showed that human skin samples can metabolize 1 

### benzo[a]pyrene. In skin samples from 11 subjects, the majority of benzo[a]pyrene-7,8-diol 2 

### formed from benzo[a]pyrene was recovered as the (-) enantiomer. However, the stereospecific 3 

### formation of tetrol metabolites was highly variable , suggesting significant inter-individual 4 

### variability in the relative formation of the DNA re active epoxide metabolites from 5 

### benzo[a]pyrene. Bowman et al. (1997) assessed leve ls of the 7,8,9,10-tetrol metabolite in the 6 

### urine of 43 psoriasis patients treated with coal ta r medication. Urinary benzo[a]pyrene 7 

### metabolites were detected in 40% of patients vs. 10 % of matched controls. Amounts of 8 

### metabolites detected were highly variable, but prec ise measures of applied dose were not 9



### available. These studies show that human skin can metabolize benzo[a]pyrene to diverse 10 

### products, the metabolism of benzo[a]pyrene is induc ible and variable with regard to 11 

### stereospecific formation of downstream metabolites, and there may be significant interindividual 12



### variation. 13 

### Merk et al. (1987) used hair follicles from volunte ers to study benzo[a]pyrene 14 

### metabolism and the effect of coal-tar-containing sh ampoos. Hair follicles were able to form 15 

### numerous metabolites of benzo[a]pyrene. The result s with coal-tar-exposed individuals showed 16 

### that AHH activity was inducible in these cells and inhibition of CYP activity decreased the 17 

### metabolism of benzo[a]pyrene and the formation of D NA binding activity. Alexandrov et al. 18 

### (1990) demonstrated that hair follicles from human subjects (10 healthy female smokers and 10 19 

### healthy female nonsmokers) were able to generate be nzo[a]pyrene tetrols from diol epoxide 20 

### precursors. There was stereospecific metabolism wi th most of the tetrol formed from (-)- 21 

### benzo[a]pyrene-7,8-diol consistent with extensive f ormation of ( ± )-anti-BPDE rather than the 22 

### ( ± )-syn-BPDE metabolite. Agarwal et al. (1991) repor ted that human melanocytes treated in 23 

### culture formed diverse benzo[a]pyrene metabolites, including dihydrodiols, hydroxyl 24 

### compounds, quinones, and their glucuronide and sulf ate conjugates. 25 

### Kao et al. (1985) investigated the metabolism of be nzo[a]pyrene in skin from several 26 

### species, including humans. In the case of human sk in, TCDD induction could not be studied, but 27 

### the authors investigated the influence of metabolic viability of the skin on metabolism. Skin 28 

### samples were treated with 2 μ g/cm 2 

### [ 14 

### C]-benzo[a]pyrene in acetone and were incubated for 29



### 24 hours. Medium under the skin was then extracted with ethyl acetate but was not subjected to 30 

### hydrolysis to identify conjugate formation. Fifty- two percent of the radioactivity in culture 31 

### medium below viable human skin was composed of wate r-soluble metabolites, 8% were lipid32 

### soluble polar metabolites, 17% were diols, 1.2% wer e monophenols, 2.5% were quinones, and 33 

### 18% were parent compound. By contrast, previously frozen, nonviable skin allowed mostly 34 

### parent compound to pass (50% of radioactivity in th e medium) and considerably smaller portions 35 

### of water-soluble and polar metabolites and diols to pass, but, surprisingly, relatively increased 36 

### portions of monophenols and quinines to pass. Resu lts for skin from other species—marmoset, 37 

### rabbit, rat, mouse—were similar: water-soluble met abolites varied between 55 and 77% of the 38

### 50 DRAFT - DO NOT CITE OR QUOTE

radioactivity in the medium; polar metabolites, 5.8 –13.2%; diols, 7.9–15.2%; monophenols, 0.7– 1 

### 1.8%; quinones, 0.6–2.1%; and parent compound, 0% ( mouse), to 6.7% (rabbit). Metabolites 2 

### found below nonviable skin from animal species were not reported. 3



### Animal studies are consistent with the metabolism d ata in human skin. Ng et al. (1992) 4 

### used an in vitro culture system with skin from fema le hairless guinea pigs and concluded that the 5 

### degree of penetration of benzo[a]pyrene was depende nt on metabolism, since the collection of 6 

### the administered radioactivity was much greater for viable than nonviable skin. At the lowest 7 

### dose tested (32.1 nmol/cm 2 

### ), 37% of the applied radioactivity was recovered i n the receptor fluid 8 

### within 24 hours, 84% of which were metabolites, inc luding OH derivatives, dihydrodiol diones, 9 

### and conjugates. The tetrol metabolite of the DNA-r eactive epoxide accounted for 2.56% of the 10 

### administered dose. MacNicoll et al. (1980) assesse d metabolism of [ 3 

### H]-benzo[a]pyrene in 11 

### Swiss mouse skin maintained in short-term organ cul ture. After incubation for 24 hours, 12 

### radioactivity derived from benzo[a]pyrene consisted of 3 benzo[a]pyrene-equivalents of lipid13 

### soluble metabolites, 147 benzo[a]pyrene-equivalents water-soluble metabolites, and 5 14 

### benzo[a]pyrene-equivalents bound to skin. The high proportion of water-soluble metabolites 15 

### indicates that benzo[a]pyrene was readily metaboliz ed. 16 

### Kao et al. (1984) evaluated metabolism of benzo[a]p yrene as a factor in the dermal 17 

### penetration of benzo[a]pyrene in mouse skin. Skin samples formed predominantly polar 18 

### metabolites and diols (approximately 20% of the rad ioactivity each), with small shares 19 

### (approximately 1% of the penetrated material) of co njugates, monophenols, and quinones. 20 

### Parent compound was recovered at about 1.5%. TCDD induction changed the portion of 21 

### conjugates to almost 8%, at the expense of polar me tabolites, diols, and parent compound, but 22 

### did not affect monophenol or quinone formation. In vestigation of the radioactivity left in the 23 

### skin revealed that it was mostly parent compound (a lmost 50% of the dose in uninduced and a 24 

### little more than 30% in induced skin), with about 1 2–16% water-soluble metabolites and polar 25 

### metabolites, diols, monophenols, and quinones decre asing in that order from ~5% of the dose to 26



### <2%. 27

28



### 3.3.3.4. Reproductive Tissues and Fetal Metabolism 29 

### Several toxicity studies have demonstrated the abil ity of benzo[a]pyrene to impair 30 

### reproductive function in male and female rodents as well as induce developmental toxicity. 31 

### Therefore, the ability of benzo[a]pyrene to be meta bolized in tissues that affect reproduction or 32 

### development is of interest. Williams et al. (2000) reported the presence of CYP1A1, CYP1A2, 33 

### and CYP1B1 transcripts in prostate tissue from huma n donors exposed in short-term organ 34 

### culture and the activation of benzo[a]pyrene as ind icated by DNA adduct formation. Primary 35 

### human prostate cells have also been demonstrated to metabolize benzo[a]pyrene in vitro (Martin 36 

### et al., 2002). 37

### 51 DRAFT - DO NOT CITE OR QUOTE

Bao et al. (2002) exposed a human endometrium epith elial cell line, which expresses low 1 

### constitutional levels of EROD activity (used as a m easure of CYP1 activity), to benzo[a]pyrene 2 

### (1 mM) in vitro and observed a 12-fold induction of CYP1A1. Specific inhibitors of CYP1B1 3 

### and CYP1A2 had no effect on EROD activity, suggesti ng little metabolic contribution from these 4 

### two isoforms in endometrial tissues, while the CYP1 A1 inhibitor α -NF (100 nM) inhibited 5 

### EROD activity more than 60%. Melikian et al. (1999 ) measured DNA adducts in cervical 6 

### epithelial and stromal tissues in smokers and nonsm okers. Increased levels of adducts in the 7 

### tissues of smokers suggested that delivery of benzo [a]pyrene to the cervical tissues with 8 

### subsequent metabolism may occur, although transport of reactive metabolites generated in other 9 

### tissues was not specifically ruled out. 10 

### Ramesh et al. (2003, available only as abstract) in vestigated benzo[a]pyrene metabolism 11 

### in multiple organ systems in rats exposed by nose-o nly inhalation to 75 μ g/m 3 

### benzo[a]pyrene 12 

### adsorbed on CB 4 hours/day for 60 days. AHH activi ty and benzo[a]pyrene metabolism were 13 

### reported for various tissues. A diverse spectrum o f metabolites was identified, including 14 

### dihydrodiols and 3- and 9-monophenols. The concent rations of BPDE were highest in testes, 15 

### providing mechanistic support for previously observ ed effects of benzo[a]pyrene on male 16 

### reproductive parameters in animal toxicity studies. 17 

### Exposure to reactive benzo[a]pyrene metabolites may be a concern both in utero and 18 

### during lactation. Wu et al. (2003) measured the ge neration of benzo[a]pyrene metabolites in F1 19 

### generation pups and determined the mRNA development profile for the AhR in the absence and 20 

### presence of subacute exposure concentrations of ben zo[a]pyrene in preweaning rats. Pregnant 21 

### F344 rats were exposed to 25, 75, or 100 μ g/m 3 

### benzo[a]pyrene aerosols via nose-only 22 

### inhalation, 4 hours/day for 10 days (GDs 11–21). B enzo[a]pyrene metabolites and mRNA and 23 

### protein expression profiles of AhR and CYP1A1 were analyzed in the cerebral cortex, 24 

### hippocampus, liver, and plasma. Plasma and cerebra l cortex benzo[a]pyrene levels on postnatal 25 

### day (PND) 0 changed with the dose. Benzo[a]pyrene decreased steadily with time in these 26 

### tissues, always reflecting the administered dose, r eaching nondetectable levels by PND 30. In 27 

### plasma, diols represented approximately 60% of the total metabolites over the period of 28 

### 20 PNDs. In the cerebral cortex, diols represented close to 80% of metabolites soon after 29 

### parturition, decreasing to approximately 40% by PND 20. Benzo[a]pyrene-7,8-diol represented 30 

### a maximum of approximately 25% of the recovered met abolites in plasma and approximately 31



### 30% of recovered metabolites in the cerebral cortex

. 32 

### There was a statistically significant ( p < 0.05) up-regulation of AhR mRNA, a subsequent 33 

### induction of CYP1A1 mRNA, and a significant increas e in CYP1A1 protein levels in pup livers 34



### at 100 μ g/m 3 

### compared to unexposed controls (data were presente d only for PND 60 and the 35



### 100 μ g/m 3 

### concentration). The AhR mRNA expression profile i n the developing cerebral cortex 36 

### and hippocampus indicated up-regulation of AhR duri ng the first 3 postnatal weeks at all 37 

### concentrations, although these differences were not statistically significant due to large 38

### 52 DRAFT - DO NOT CITE OR QUOTE

individual variation. At the high concentration, A hR mRNA abundance was more than twice 1 

### that of controls by PND 30. However, this up-regul ation of AhR mRNA was not accompanied 2 

### by a concomitant up-regulation of CYP1A1 mRNA in th e CNS. In fact, expression of CYP1A1 3 

### in these tissues was very low or absent during the 1-month postnatal period after exposure to 4



### 100 μ g/m 3 

### of benzo[a]pyrene (the only dose tested). Based o n these findings, the authors 5 

### suggested that the up-regulation of the AhR may inc rease the potential for benzo[a]pyrene 6 

### neurotoxicity via the activation of CYP450 in liver and the subsequent deposition of toxic 7 

### metabolites in the developing CNS. The results do not suggest that benzo[a]pyrene metabolism 8 

### by CYP1A1 is induced in the developing CNS. 9 

### Pregnant Swiss Webster mice were administered radio labeled benzo[a]pyrene by i.v. 10 

### injection, and maternal and fetal levels of benzo[a ]pyrene and radiolabel were determined. Fetal 11



### tissue levels of radiolabel increased while materna l tissues decreased. The level of 12 

### benzo[a]pyrene decreased in fetal tissue during thi s period, suggesting the accumulation of 13 

### benzo[a]pyrene metabolites. Increasing the capacit y of maternal plasma to bind benzo[a]pyrene 14 

### by administering a benzo[a]pyrene antiserum decreas ed fetal accumulation of radiolabel, 15 

### suggesting that bioavailability had decreased (McCa be and Flynn, 1990). Other investigators 16 

### have also demonstrated the ability of benzo[a]pyren e to be transported to the fetus (Neubert and 17 

### Tapken, 1988; Shendrikova and Aleksandrov, 1974). Martin et al. (2000) exposed exfoliated 18 

### cells from breast milk of human donors to benzo[a]p yrene in vitro. Treated cells showed 19 

### increased DNA single strand breaks (SSB) relative t o controls, indicating that these cells can 20 

### activate benzo[a]pyrene. Taken together, the resul ts from these various experiments suggest that 21 

### placental and lactational transfer of benzo[a]pyren e and active metabolites may be of concern. 22 

### The developmental expression patterns of metabolizi ng enzymes can be an important 23 

### determinant of childhood susceptibility. Numerous in vivo mechanistic tumor screening assays 24 

### have been conducted in newborn mice (see Section 4. 4.1 on mechanistic cancer studies), and the 25 

### observed increases in tumors provide evidence for t he ability of young animals to metabolize 26 

### benzo[a]pyrene to DNA reactive metabolites. Meliki an et al. (1989) administered [ 3 

### H]- 27 

### benzo[a]pyrene or [ 3 

### H]-BPDE via i.p. injection to CD-1 mice on PND 1, 8 , or 15 and formation 28 

### of benzo[a]pyrene metabolites was determined in lun g and liver over time periods ranging from 29



### 2 minutes to 24 hours after the last dose. In the lung, metabolites included diones, quinones, and 30 

### phenols, and metabolite levels in the lung were hig her on day 1 than on days 8 or 15. In liver, a 31 

### different spectrum of metabolites was observed, dom inated by unidentified polar metabolites. 32 

### The percentage of radiolabel as metabolites was inc reased on days 8 and 15, perhaps reflecting 33 

### greater inducibility of benzo[a]pyrene metabolism i n liver versus the lung. Formation of 34 

### glucuronide or sulfate conjugates was greater than glutathione conjugates in both tissues. The 35 

### total amount of benzo[a]pyrene metabolized by Phase II enzymes was greater in liver than in 36 

### lung on day 1 but was similar on days 8 and 15. Ag e-dependent expression of genes that encode 37

### 53 DRAFT - DO NOT CITE OR QUOTE

metabolizing enzymes has been the subject of review s (e.g., Cresteil, 1998) that are addressed in 1



### Section 4.4. 2

3



### 3.3.3.5. Other Tissues 4 

### Moore et al. (1982) exposed organ cultures of human and rat bladder tissues to [ 3 

### H]- 5 

### benzo[a]pyrene for 24 hours and evaluated metabolit e profiles. Benzo[a]pyrene was metabolized 6 

### by bladder tissues of both species. Total mean amo unt of metabolites formed was higher in 7 

### human than rat bladders (twofold). A similar spect rum of diverse metabolites was generated, 8 

### although relative proportions varied. Formation of benzo[a]pyrene-7,8-diol was similar in both 9 

### species and similar levels of DNA binding was obser ved. These results indicate that bladder 10 

### tissues have metabolic capability for benzo[a]pyren e and that the metabolic capacity is similar 11



### between humans and rats. 12 

### Several other target tissues for benzo[a]pyrene tox icity have been found to have the 13 

### capacity to metabolize benzo[a]pyrene. For example , Moorthy et al. (2003) treated mouse aortic 14 

### smooth muscle cells with benzo[a]pyrene, 3-OH-benzo [a]pyrene, or benzo[a]pyrene-3,6-quinone 15



### in vitro. Several DNA adducts were identified that were attributed to the 3-OH and BPQ 16 

### metabolites. Benzo[a]pyrene treatment increased CY P1B1 but not CYP1A1 in these cells. The 17 

### authors suggest that CYP1B1 activates benzo[a]pyren e to 3-OH and BPQ metabolites, which 18 

### induce the DNA damage responsible for changes that are important precursors for 19



### atherosclerosis. 20

21



### 3.4. ELIMINATION 22 

### 3.4.1. Inhalation Exposure 23 

### Human studies of benzo[a]pyrene exposure have gener ally been limited to individuals 24 

### exposed to coke oven emissions, coal tars, or other products containing a mixture of PAHs and 25 

### are of limited value in assessing urinary benzo[a]p yrene exposure biomarkers. Numerous 26 

### studies have evaluated 1-OH pyrene in urine as a ge neral marker for PAH exposure, but, because 27 

### 1-OH pyrene is not a metabolite of benzo[a]pyrene, these data are not directly useful in 28 

### evaluating the toxicokinetics of benzo[a]pyrene. S ome studies have unsuccessfully attempted to 29 

### quantify exposure to benzo[a]pyrene via measurement of parent compound or its metabolites. 30 

### Bentsen-Farmen et al. (1999b) compared air concentr ations of PAHs to PAH metabolites in the 31 

### urine of 17 electrode paste plant workers and detec ted 1-OH-Py but no benzo[a]pyrene 32 

### metabolites despite the fact that benzo[a]pyrene in personal air samples showed mean exposure 33



### levels of 0.3 μ g/m 3 

### . Waidyanatha et al. (2003) also attempted to meas ure PAH exposure via 34 

### urinary metabolite levels of coke oven workers; sev eral PAHs but no benzo[a]pyrene were 35 

### detected. In other cases, exposure and urinary met abolites were positively correlated (Hecht et 36 

### al., 2003; Wu et al., 2002; Gündel et al., 2000; Gr immer et al., 1993; Becher and Bjørseth, 1983). 37

### 54 DRAFT - DO NOT CITE OR QUOTE

Wu et al. (2002) reported a correlation between ben zo[a]pyrene-tetrols and total PAH exposure 1



### in coke oven workers. 2 

### Several studies evaluated the elimination of benzo[ a]pyrene in animals following 3 

### exposure via the respiratory tract. Petridou-Fisch er et al. (1988) applied 10 μ L aliquots of [ 14 

### C]- 4 

### benzo[a]pyrene in a gelatin:saline solution over a 2-hour period to the ethmoid and maxillary 5 

### nasal turbinates of monkeys and dogs to assess diff erences in benzo[a]pyrene disposition in 6 

### portions of the nose. Urine levels reached a maxim um of 0.69% of the administered dose in 7 

### dogs and 0.07% in monkeys, while fecal levels reach ed a maximum of 6.42% of the dose in dogs 8 

### and 1.17% in monkeys over 48 hours. The pattern of metabolites in the excreta was consistent 9 

### with results from other respiratory tract depositio n studies, showing that benzo[a]pyrene is 10 

### excreted preferentially in the feces. Wolff et al. (1989) investigated the effects of nose-only 11 

### exposure of male and female F344 rats to unlabeled benzo[a]pyrene for 4 weeks, followed by a 12 

### single exposure to [ 14 

### C]-benzo[a]pyrene. Four weeks after treatment with the radiolabel, the rats 13 

### had eliminated approximately 96% of the total dose in the feces. The mean half-life was 14



### calculated as 22 hours for feces and 28 hours for u rine. Sun et al. (1982) exposed male and 15 

### female F344 rats via nose-only inhalation for 30 mi nutes to atmospheres containing 0.6 μ g/L 16 

### [ 3 

### H]-benzo[a]pyrene absorbed onto 67Ga2O3 or to 1.0 μ g/L neat [ 3 

### H]-benzo[a]pyrene and 17 

### measured levels of radioactivity in tissues and exc reta. With either exposure, benzo[a]pyrene 18 

### excretion declined rapidly over the first 3 days af ter exposure, with little additional excretion 19 

### occurring by 5 days postexposure. Excretion in fec es was much greater than in urine for either 20 

### exposure regimen. Following exposure of rats to ne at benzo[a]pyrene, feces accounted for 86% 21 

### of total excreted radioactivity and urine for 14%. Metabolite profiles were not evaluated. Wang 22 

### et al. (2003) exposed B6C3F 1 

### mice for 10 days to asphalt fumes in an inhalation chamber at a 23 

### concentration of approximately 180 mg/m 3 

### ; the benzo[a]pyrene content of the fumes was not 24 

### reported. Benzo[a]pyrene metabolites measured in t he urine of exposed mice were in the ng/100 25 

### mL range and were identified at the following mass ratios (with benzo[a]pyrene arbitrarily set as 26 

### 1): 7,8,9,10-benzo[a]pyrene-tetrol, 14; benzo[a]py rene-7,8-diol epoxides, 17; benzo[a]pyrene27 

### 7,8-diol, 3.6; 3-OH-benzo[a]pyrene, 10. 28 

### In a study investigating the disposition of benzo[a ]pyrene, 1 μ g/kg of the [ 3 

### H]-labeled 29 

### compound dissolved in triethylene glycol was instil led into the trachea of male Sprague-Dawley 30 

### rats (Weyand and Bevan, 1986). Approximately 60% o f the administered dose was associated 31 

### with the intestine and intestinal contents, and 2.2 % of the radiolabel was recovered in the urine 32 

### 6 hours after dosing. The relative contribution of water- and lipid-soluble metabolites in the 33



### intestinal contents was 18.2 and 26.5% of the admin istered dose, respectively. The amounts of 34 

### metabolites determined in the organic phase 6 hours after the end of exposure were as follows 35 

### (percent of total in the organic phase): conjugate s or polyhydroxylated compounds (24.1%), 36 

### 9,10-diol (7.75%), 4,5-diol (8.43%), 7,8-diol (5.73 %), 1,6-quinone (7.13%), 3,6-quinone 37 

### (7.85%), 6,12-quinone (7.10%), 9-OH (2.24%), 3-OH ( 4.66%), benzo[a]pyrene (1.55%). The 38

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total amounts of metabolites in feces and urine wer e not reported in this study, and, due to 1 

### enterohepatic circulation, the intestinal content c oncentrations may not reflect the final amounts 2



### of each metabolite in the excreta. Bevan and Sadle r (1992) administered a single intratracheal 3 

### instillation of benzo[a]pyrene (2 μ g/kg) to male Sprague-Dawley rats and assessed meta bolite 4 

### profiles in bile after 6 hours. Relative metabolit e levels were 31.2% diglucuronides, 30.4% 5 

### thioether conjugates, 17.8% monoglucuronides, 6.2% sulfate conjugates, and 14.4% 6 

### unconjugated metabolites. 7 

### Weyand and Bevan (1987) studied the differences in benzo[a]pyrene elimination among 8 

### rats, hamsters, and guinea pigs. Male Sprague-Dawl ey and Gunn rats (the latter a strain 9 

### genetically deficient in bilirubin glucuronidation) , male Syrian golden hamsters, and male 10 

### Dunkin-Hartley guinea pigs were given single intrat racheal doses of 0.16 μ g or 350 μ g [ 3 

### H]- 11 

### benzo[a]pyrene per animal. Tissue levels and the a mount of radiolabel excreted into the urine 12 

### and bile 6 hours after treatment are given in Table 3-3. Urinary excretion of benzo[a]pyrene 13 

### metabolites amounted only to a small fraction of bi liary excretion in all tested species. The 14 

### results suggested that the metabolic capacity for b enzo[a]pyrene became saturated in guinea pigs 15 

### and Sprague-Dawley and Gunn rats but not in hamster s at the 350 μ g/animal dose (note: rats 16 

### weighed 200–250 grams, average high dose 1.56 mg/kg ; hamsters 100–140 grams, 2.92 mg/kg; 17 

### and guinea pigs 600–850 grams, 0.48 mg/kg), indicat ing that hamsters, who received about 6 18 

### times the dose of guinea pigs, command a metabolic system that handles benzo[a]pyrene very 19 

### well. Moir et al. (1998) administered benzo[a]pyre ne i.v. to Wistar rats and recovered, at 8 hours 20 

### after a 2 mg/kg dose, 4.27% of the dose in urine bu t only 0.06% in feces, confirming the fact of 21 

### enterohepatic circulation of benzo[a]pyrene metabol ite conjugates (Hirom et al., 1983; Weyand 22 

### and Bevan, 1986). 23

24 

### Table 3-3. Excretion of benzo[a]pyrene metabolites in several animal species

Dose Route of

excretion Gunn rat a Sprague-Dawley

rat a Syrian golden

hamster a Dunkin-Hartley

guinea pig a

0.16 μ g/animal Urine 0.98 ± 0.16 2.21 ± 1.1 3.74 ± 1.2 2.22 ± 1.2

Bile 59.4 ± 1.07 70.3 ± 2.0 54.6 ± 3.6 71.7 ± 1.9

350 μ g/animal Urine 1.44 ± 1.82 1.68 ± 0.6 2.53 ± 0.4 1.27 ± 0.4

Bile 30.8 ± 1.51 55.0 ± 2.0 52.9 ± 2.7 47.9 ± 4.9

a Values are percent of the applied dose at 6 hours a fter intratracheal instillation.

Source: Weyand and Bevan (1987).

25 

### The pattern of metabolites in bile was also reporte d; results are compiled in Table 3-4. 26 

### At each dose thioether conjugates predominated but to a varying extent in each species. Guinea 27 

### pigs evidently used preferentially thioether conjug ation for biliary benzo[a]pyrene elimination, 28 

### with the other conjugates (or nonconjugated metabol ites) making up a small portion of biliary 29

### 56 DRAFT - DO NOT CITE OR QUOTE

excretion. At the high dose a shift in the relativ e proportion of metabolites was observed, with 1 

### thioethers decreasing, glucuronides and sulfates in creasing, and total nonconjugated remaining 2 

### unchanged in both rat strains and hamster. The dat a also indicate that glucuronidation and 3 

### sulfation became saturated at the high dose only in guinea pigs, while thioether formation 4 

### capacity became overwhelmed by the high dose in all species. Glucuronide levels in the bile of 5 

### Gunn rats were about one-half those of Sprague-Dawl ey rats; this strain also excreted fewer 6 

### nonconjugated metabolites and appeared to have a ge nerally lower metabolic capacity for 7 

### benzo[a]pyrene. 8

9 

### Table 3-4. Biliary excretion of benzo[a]pyrene met abolites in several

species

Metabolite group Dose ( μ g/animal) Gunn rat a SpragueDawley rat a Syrian golden

hamster a Dunkin- Hartley

guinea pig a

Nonconjugated 0.16 4 11.5 4.1 1.7

350 2 8.3 4.2 1.3

Glucuronides 0.16 7 12.9 11.9 4.5

350 9 18.7 18.1 0.2

Sulfate conjugates 0.16 8 3 3.7 1.9

350 8 5.9 6.9 1.1

Thioether conjugates 0.16 40 42.9 33.8 54.1

350 12 22.1 23.7 45.4

a Values are percent of the applied dose at 6 hours a fter intratracheal instillation.

Source: Weyand and Bevan (1987).

10 

### Following intratracheal administration of 1 μ g/kg body weight [ 3 

### H]-benzo[a]pyrene to 11 

### male Sprague-Dawley rats in hydrophilic triethylene glycol, 70.5% of the administered 12 

### benzo[a]pyrene was excreted into bile within 6 hour s (Bevan and Ulman, 1991). In contrast, 13 

### benzo[a]pyrene excretion was 58.4 and 56.2% in the same time period when the lipophilic 14 

### solvents ethyl laurate and tricaprylin, respectivel y, were the vehicles. Benzo[a]pyrene (in 15 

### triethylene glycol) excretion in bile was described as biphasic with half-lives of 31 and 100 16 

### minutes, respectively. Individual metabolite conce ntrations were not monitored. 17

18 

### 3.4.2. Oral Exposure 19 

### Only limited data were identified on the eliminatio n of benzo[a]pyrene following 20 

### exposure by the oral route. Although the dietary/o ral route is likely to be the predominant route 21 

### of exposure for the general population not occupati onally exposed, it has received very little 22 

### attention (Stavric and Klassen, 1994). In the only study investigating this issue in humans, the 23 

### concentration of benzo[a]pyrene was below detection limits (<0.1 μ g/person) in the feces of 24

### 57 DRAFT - DO NOT CITE OR QUOTE

eight volunteers who had ingested broiled meat cont aining approximately 8.6 μ g of 1 

### benzo[a]pyrene (Hecht et al., 1979). 2 

### Ramesh et al. (2001b) evaluated benzo[a]pyrene disp osition in F344 rats dosed via 3 

### gavage with 100 mg/kg benzo[a]pyrene dissolved in p eanut oil. Recovery of unmetabolized 4 

### benzo[a]pyrene from feces reached a maximum of 35% of the dose at 24 hours but was very low 5 

### at 48 hours and thereafter. Lipid-soluble metaboli tes in feces reached approximately 28% of all 6 

### metabolites by 8 hours after dosing then declined s harply. Lipid-soluble metabolites in urine 7 

### reached 35–40% of total by 48 hours then declined t o approximately 10% by 72 hours. Diol 8 

### metabolites were most numerous in feces, while phen ols predominated in urine. 9 

### Hecht et al. (1979) conducted a study of fecal excr etion of benzo[a]pyrene and its 10



### metabolites in rats. In male F344 rats administere d [ 14 

### C]-benzo[a]pyrene via gavage (0.04, 0.4, 11



### or 4 μ mol/animal), approximately 85% of radiolabel was re covered in feces, and 1–3% in urine 12 

### after 168 hours. The portion of benzo[a]pyrene rec overed from feces as parent compound 13 

### ranged from 13 to 6% of the administered dose withi n 48 hours. In rats fed charcoal-broiled 14 

### hamburger containing 52.7 μ g benzo[a]pyrene/kg meat, 11% of the benzo[a]pyrene was excreted 15 

### unchanged in feces. 16

17 

### 3.4.3. Dermal Exposure and Other Exposure Routes 18 

### Bowman et al. (1997) detected benzo[a]pyrene-tetrol s in 40% of the urine samples from 19 

### psoriasis patients treated with coal tar medication , as compared to only 10% of those of controls. 20 

### No specific measures of applied dose were available

. 21 

### In a dermal absorption study, Yang et al. (1989) ev aluated the recovery of [ 3 

### H]- 22 

### benzo[a]pyrene, 100 ppm in crude oil applied topica lly, in urine and feces of female Sprague23 

### Dawley rats. Total recovery of applied radioactivi ty over 96 hours was 5.3% in urine and 27.5% 24



### in feces. Individual metabolite concentrations wer e not measured. Ng et al. (1992) examined the 25 

### percutaneous absorption of radiolabeled benzo[a]pyr ene in the hairless guinea pig. Following a 26 

### single application of 28 μ g benzo[a]pyrene (dissolved in 50 μ L acetone applied to 4 cm 2 

### of 27 

### dorsal skin), approximately 34% of the administered radiolabel was eliminated within 24 hours. 28 

### Most excretion had occurred by day 3 and continued slowly to reach 73% by day 7. Relative 29 

### amounts of benzo[a]pyrene or metabolites in urine v ersus feces were not reported. 30 

### Moir et al. (1998) dosed male Wistar rats i.v. with 2, 6, or 15 mg/kg of [ 14 

### C]- 31 

### benzo[a]pyrene and examined excretion in urine and feces over 32 hours. At 8 hours after 32 

### injection, urinary excretion was 4.3, 2.6, and 3.2% of the dose, while fecal excretion was only 33 

### 0.06, 5.6, and 0.43% of the 2, 6, and 15 mg/kg dose s, an indication of enterohepatic circulation. 34 

### The amount of radioactivity excreted in the urine a fter 32 hours was similar in each dose group 35 

### (6–7% of the administered dose). However, the prop ortion of the administered dose in the feces 36 

### was dose-dependent. At the low dose (2 mg/kg) feca l excretion accounted for 26% of the dose, 37

### 58 DRAFT - DO NOT CITE OR QUOTE

while at the mid- and high-doses, fecal excretion a ccounted for 56 and 50% of the dose, 1 

### respectively. No measurement of specific metabolit es or whole body clearance was conducted. 2 

### Following i.v. administration of 3 μ mol [ 14 

### C]-benzo[a]pyrene to male New Zealand white 3 

### rabbits, approximately 30% of the dose was recovere d in the bile and 12% in the urine within 4 

### 6 hours after treatment (Chipman et al., 1982). Ex cretion in the bile was biphasic, with estimated 5 

### half-lives of 0.27 and 4.62 hours. Further analysi s of metabolite profiles in bile and urine were 6



### conducted. Treatment of bile or urine with β -glucuronidase or aryl sulfatase increased the 7 

### amount of radioactivity recovered in ethyl acetate extracts, suggesting that sulfate and 8 

### glucuronide conjugation are important contributors to benzo[a]pyrene metabolites in excreta. 9 

### Analysis of these extracts revealed the primary met abolite as 9,10-diol, with lower amounts of 10 

### numerous other metabolites identified (diols, quino nes, and monophenols). 11 

### Likhachev et al. (1992) measured the excretion of b enzo[a]pyrene-7,8-diol and 3-OH12 

### benzo[a]pyrene in L10 rats given a single i.p. dose of 200 mg/kg benzo[a]pyrene. Urine and 13 

### feces were collected over a 15-day period and then again at 30 days after exposure for another 5 14 

### days. Both metabolites were excreted in feces at t wo to three times the amount excreted in urine. 15 

### Metabolites in urine and feces decreased steadily o ver the 15-day postexposure period, but no 16 

### more metabolites were detected at 30 days. A compa rative study of metabolism in male Macaca 17 

### fascicularis monkeys (five animals) and male L10 rats (four ani mals) was also performed. Both 18 

### species were given a single i.p. dose of 100 mg/kg benzo[a]pyrene, and levels of benzo[a]pyrene 19 

### metabolites in feces were evaluated over 8 days. T otal benzo[a]pyrene metabolites were 20 

### significantly lower in monkeys than in rats, but th e results were hampered by infrequent feces 21 

### collection in monkeys. Monkeys had an approximatel y fourfold higher benzo[a]pyrene-7,8-diol 22 

### to 3-OH-benzo[a]pyrene ratio in feces than rats. T ogether these data suggest that monkeys have 23 

### lower excretion of benzo[a]pyrene (and perhaps lowe r metabolism), and formation of the 24 

### proximate carcinogenic metabolite is higher than in rats. 25 

### In another part of this study (Likhachev et al., 19 92), benzo[a]pyrene metabolism and 26 

### excretion were assessed following multiple exposure s. L10 rats were given i.p. injections of 27 

### 1 mg/kg benzo[a]pyrene every 11th day for 10 treatm ents. Benzo[a]pyrene metabolites were 28 

### measured in excreta for 8 days after 1, 5, and 10 t reatments. After the single dose, excretion in 29 

### feces was favored over urine, and benzo[a]pyrene-7, 8-diol was the predominant metabolite. The 30



### rate of metabolite excretion was decreased consider ably after 5 and 10 doses, respectively. The 31 

### authors attributed this finding to age-related chan ges in metabolic capacity or “exhaustion of 32 

### enzymes.” They also compared metabolite profiles f or individual rats with their tumor responses 33 

### and based on the results suggested a link between t umor latency and elimination of 34 

### benzo[a]pyrene metabolites (although small sample s ize and variability limited the power of the 35 

### analysis). A direct correlation between benzo[a]py rene-7,8-diol excreted in urine and tumor 36 

### latency was observed. It was postulated that highe r excretion of reactive benzo[a]pyrene 37

### 59 DRAFT - DO NOT CITE OR QUOTE

metabolites caused fewer of these metabolites to bi nd to DNA targets and thus increased tumor 1 

### latency. 2 

### In addition to benzo[a]pyrene and its metabolites, adducts of benzo[a]pyrene with 3



### nucleotides have also been identified in feces and urine of animals but only as a small fraction of 4 

### the administered dose. Autrup and Seremet (1986) a dministered i.p. doses of 0, 10, 50, or 5



### 100 μ g/kg tritiated benzo[a]pyrene to male Wistar rats a nd collected urine over 24-hour periods 6 

### for 72 hours. The level of BPDE adducts with guani ne detected in urine was dose-dependent. 7 

### The authors reported that, at the high dose, 0.15% of the administered benzo[a]pyrene dose was 8



### excreted in the urine as this adduct within 48 hour s. Rogan et al. (1990, only study abstract was 9 

### available for review) reported that 0.02% of a benz o[a]pyrene dose was excreted as an adduct 10 

### with guanine in urine and feces over 5 days. 11 

### Overall, the data on benzo[a]pyrene elimination in humans are too limited to estimate 12 

### quantitative rates of elimination. The situation i s further complicated by the existence of 13 

### multiple pathways in which many of the key enzymes exhibit polymorphisms. In the context of 14 

### biomonitoring studies, benzo[a]pyrene metabolites h ave been detected in the urine of exposed 15 

### humans, but the fecal excretion has not been invest igated in any detail. The animal data are 16 

### consistent in showing that feces are the primary ro ute of elimination of benzo[a]pyrene, while 17 

### urinary excretion plays a lesser role. A diverse a rray of metabolites, as well as parent 18 

### benzo[a]pyrene, is found in the excreta. Enterohep atic circulation of benzo[a]pyrene metabolite 19 

### conjugates has been demonstrated in animals and may exist in humans as well, but its impact on 20 

### the toxicokinetics of benzo[a]pyrene is not underst ood. 21 

### Similar considerations apply to the toxicokinetics of benzo[a]pyrene as a whole. A few 22 

### animal studies that have attempted comprehensive as sessments of benzo[a]pyrene metabolism. 23 

### Although a lot is known from ex vivo studies about which humans tissue can metabolize 24 

### benzo[a]pyrene to what extent, comprehensive studie s to estimate overall metabolic capacities or 25



### the balance between Phase I and Phase II metabolism in humans have not been conducted. 26 

### There are also no comprehensive data on the tissue distribution of benzo[a]pyrene in humans, not 27 

### counting a number of studies that have reported pos t mortem levels in a few tissues in the 28 

### absence of any exposure assessments. Thus, despite the fact the humans are universally exposed 29 

### to benzo[a]pyrene, there is a great need for more k nowledge concerning its toxicokinetics. 30

31 

### 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS 32 

### Several toxicokinetic or pharmacokinetic models of benzo[a]pyrene have been developed 33 

### for rodents (rat and hamster), but none has been ca librated in humans. Bevan and Weyand 34 

### (1988) performed compartmental pharmacokinetic anal ysis of distribution of radioactivity in 35 

### male Sprague-Dawley rats, following the intratrache al instillation of benzo[a]pyrene to normal 36 

### and bile duct-cannulated animals (Weyand and Bevan, 1987, 1986). The authors used the 37 

### Simulation, Analysis and Modeling (SAAM) and Conver sational SAAM (CONSAAM) 38

### 60 DRAFT - DO NOT CITE OR QUOTE

computer programs to model the disposition of label ed benzo[a]pyrene and its metabolites. A 1 

### good fit to the experimental data was achieved with 10 assumed compartments corresponding to 2 

### lung, blood, liver, other tissues (reflecting the s um of radioactivity in kidney, stomach, testis, 3 

### spleen, heart, and thymus), carcass (reflecting the sum of radioactivity in skin, fat, bones, 4 

### muscle, and blood in those organs), and several add itional hypothetical compartments that linked 5 

### the central compartment with intestines (including their contents) and urine. Enterohepatic 6



### circulation and intestinal secretion were both incl uded in the model. The model allowed 7 

### calculation of linear rate constants for moving rad ioactivity among compartments (in minute -1 

### ), 8 

### representing the probability per unit time that rad ioactivity from one compartment would be 9 

### transferred to another. The model adequately simul ated disposition of benzo[a]pyrene and its 10 

### metabolites, measured as total radioactivity in blo od, organs and excreta, under the assumption 11 

### that the kinetics of benzo[a]pyrene and its metabol ites are the same. The authors emphasized 12 

### differences between their approach in building the implicit SAAM model and the approach used 13 

### to build explicit PBPK models. Roth and Vinegar (1 990) reviewed the capacity of the lung to 14 

### impact the disposition of chemicals and used benzo[ a]pyrene as a case study. A PBPK model 15 

### was presented based on data from Wiersma and Roth ( 1983a, b) and was evaluated against tissue 16 

### concentration data from Schlede et al. (1970). The model was structured with compartments for 17 

### arterial blood, venous blood, lung, liver, fat, and slowly as well as rapidly perfused tissues. 18 

### Metabolism in liver and lung was estimated using ki netic data from control rats and rats 19 

### pretreated with 3-MC to induce benzo[a]pyrene metab olism. Benzo[a]pyrene binding was 20 

### accounted for in blood, liver, and lung, but only o ne binding component was used, which, as the 21 

### authors suggested, resulted in a rather poor fit fo r liver. The model was built based on blood and 22



### tissue concentrations measured over 5 hours in rats given a dose of 117 nmol/kg by i.v. injection 23



### into the arterial circulation or the venous blood s upply of the liver. The model was tested against 24 

### the data of Schlede et al. (1970). The number of d ata points was limited (six time points for the 25 

### venous circulation, liver, and fat and only a singl e time point for the lung). The model predicted 26 

### the data of Schlede et al. (1970) reasonably well, although deviations were apparent for several 27 

### compartments. Most notably, the model overpredicte d concentrations in the induced lung, 28 

### uninduced and induced liver, and induced fat compar tments. The results of PBPK simulations 29 

### showed that induction of metabolizing enzymes incre ased the amount of benzo[a]pyrene cleared 30 

### by the lungs relative to the liver. 31 

### An interesting result of the Roth and Vinegar (1990 ) modeling study was that even 32 

### though the metabolic clearance of benzo[a]pyrene in the lungs was low in comparison to the 33 

### liver, under simulated pathological conditions of r educed hepatic blood flow, a substantial 34 

### metabolic clearance was carried on by well perfused lungs. This illustrates that changes in tissue 35 

### perfusion can shift the organ pattern of benzo[a]py rene metabolic clearance in vivo, and 36 

### consequently, might also shift the organ pattern of benzo[a]pyrene-induced disease, such as 37 

### cancer. The authors emphasized that their PBPK mod el with the results of simulations should 38

### 61 DRAFT - DO NOT CITE OR QUOTE

not be taken as a definite model for benzo[a]pyrene but that it could be used for designing further 1 

### studies of benzo[a]pyrene metabolic clearance. The y also suggested that it would be appropriate 2 

### to incorporate into the PBPK model a description of the appearance of various metabolites of 3 

### benzo[a]pyrene. 4 

### Moir et al. (1998) conducted a pharmacokinetic stud y on benzo[a]pyrene to obtain data 5 

### for model development. Rats were injected with [ 14 

### C]-benzo[a]pyrene at doses of 2, 6, or 6 

### 15 mg/kg and blood, liver, fat, and richly perfused tissue were sampled at 15 time points from 5 7 

### minutes to 32 hours after dosing. Moir (1999) then described a model for lung, liver, fat, richly 8 

### and slowly perfused tissues, and venous blood, with saturable metabolism occurring in the liver. 9 

### The fat and richly perfused tissues were modeled as diffusion-limited, while the other tissues 10 

### were flow-limited. The model was developed using t he 15 mg/kg dose group data from the Moir 11 

### et al. (1998) study and validated using the 2 and 6 mg/kg data. The model predicted the blood 12 

### benzo[a]pyrene concentrations well, although it ove restimated the 6 mg/kg results at longer 13 

### times (>100 minutes). The fat and richly perfused tissue benzo[a]pyrene levels were reproduced 14 

### by the model fairly well at the two highest dose le vels but underestimated at the 2 mg/kg dose. 15 

### The model produced a poor fit to the liver data, un derestimating benzo[a]pyrene tissue levels at 16 

### the two lowest doses. At the high dose the model o verestimated the liver benzo[a]pyrene 17



### concentration at times <200 minutes but afterwards underestimated them increasingly more. It 18 

### appeared that the model could not accommodate a slo w elimination phase at times beyond 200 19 

### minutes for the mid and high dose levels. The mode l simulations were also compared to data of 20 

### Schlede et al. (1970) who had injected rats with 0. 056 mg/kg body weight of benzo[a]pyrene. 21 

### Again the model predicted blood and fat benzo[a]pyr ene concentrations quite well but 22 

### underestimated liver benzo[a]pyrene concentrations. 23 

### Moir (1999) suggested that the poor prediction of f at benzo[a]pyrene concentrations after 24 

### injection of 2 mg/kg benzo[a]pyrene in their own st udy (Moir et al., 1998) was due to analytical 25 

### error, causing the model to fail. This explanation is credible because the measured fat 26 

### benzo[a]pyrene concentrations following injection o f 2 mg/kg benzo[a]pyrene were 27 

### indistinguishable from those obtained following inj ection of 6 mg/kg benzo[a]pyrene (Moir et 28 

### al., 1998). The author also speculated that the po or fit to liver benzo[a]pyrene concentration data 29 

### was due to binding of benzo[a]pyrene to receptors i n the liver. The model predicted blood and 30 

### fat benzo[a]pyrene concentrations fairly accurately over a wide range of doses (0.056– 31 

### 15 mg/kg), but it was limited by the failure to acc urately predict liver benzo[a]pyrene 32 

### concentrations. The model included only one satura ble metabolic pathway, and only parent 33



### chemical concentrations were used to establish the model. Metabolites were not modeled.An 34 

### attempt to scale the rodent PBPK model to humans, r elevant to risk assessment of oral exposures 35 

### to benzo[a]pyrene, was presented by Zeilmaker et al . (1999a, b). The PBPK model for 36 

### benzo[a]pyrene was derived from an earlier model fo r TCDD in rats (Zeilmaker and van 37 

### Eijkeren, 1997). The structure of the mainly perfu sion-limited PBPK model included 38

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compartments for blood, adipose tissue (with diffus ion limitation), slowly and richly perfused 1 

### tissues, and the liver (Figure 3-3). However, ther e was no separate compartment for the lung. 2 

### The liver compartment featured the AhR-dependent CY P450 induction mechanism and DNA 3 

### adduct formation as a marker for formation of genot oxic benzo[a]pyrene metabolites. It was 4



### assumed that DNA adduct formation and the bulk benz o[a]pyrene metabolism were mediated by 5 

### two different metabolic pathways. The model was ex perimentally calibrated in rats with the data 6



### for EROD and formation of DNA adducts in the liver after i.v. administration of a single dose 7 

### and per oral (p.o.) administration of a single or r epeated doses of benzo[a]pyrene (Zeilmaker et 8 

### al., 1999a). 9 

### In order to scale this rat PBPK model to human, Zei lmaker et al. (1999b) assumed 10 

### identical values for several parameters in rats and humans, respectively: benzo[a]pyrene tissue 11 

### partition coefficients, AhR concentration in liver, rate constant for the decay of the 12 

### benzo[a]pyrene-CYP450 complex, half-life of the CYP 450 protein, fraction and rate of GI 13 

### absorption of benzo[a]pyrene, and rates of formatio n and repair of DNA adducts in liver. The 14 

### basal CYP450 activity in humans was assumed to be l ower than that in rat liver (ranging from 15 

### almost absent up to equal to that in the rat). The mechanism of AhR-dependent induction of 16 

### CYP450 dominated the simulated benzo[a]pyrene-DNA a dduct formation in the liver. The 17



### results of PBPK model simulations indicated that th e same dose of benzo[a]pyrene administered 18 

### to rats or humans might produce one order of magnit ude higher accumulation of DNA adducts in 19 

### human liver when compared with the rat (Zeilmaker e t al., 1999b). 20 

### Even though the model of Zeilmaker et al. (1999b) r epresents a major improvement in 21 

### predictive modeling of benzo[a]pyrene toxicokinetic s, the results of modeling, which included 22 

### interspecies extrapolation, bear significant uncert ainties. As emphasized by the authors, the 23 

### conversion of benzo[a]pyrene to its mutagenic and c arcinogenic metabolites could not be 24 

### explicitly modeled in human liver because no suitab le experimental data were available. 25 

### According to the authors, to improve the model woul d require direct measurements of basal 26



### activities of CYP1A1 and CYP1A2 and formation of be nzo[a]pyrene-DNA adducts in human 27 

### liver. Moreover, despite the prior results of the study by Roth and Vinegar (1990), the metabolic 28 

### clearance of benzo[a]pyrene in the lungs was not ad dressed. Also, the toxicokinetic modeling by 29 

### Zeilmaker et al. (1999b) addressed only one pathway of benzo[a]pyrene metabolic activation, a 30 

### single target organ (the liver), and one route of a dministration (oral). 31 

### For modeling and predicting of the health outcomes of exposures to benzo[a]pyrene, a 32 

### mechanistically accurate PBPK model needs to follow , over time and in several target organs, 33 

### the rate of accumulation of benzo[a]pyrene-DNA addu cts and/or the distribution and fate of 34 

### benzo[a]pyrene metabolites (e.g., BPDE) that bind t o DNA and other macromolecules. 35 

### Alternatively, a stable derivative of the "ultimate ly carcinogenic" metabolite (e.g., 36 

### benzo[a]pyrene trans-anti-tetrol) may be used as an internal dose surrogate. Calibration of such 37 

### a model requires quantitation of these benzo[a]pyre ne metabolites in biological samples, which, 38

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in turn, would require refined and sufficiently sen sitive analytical methods. Therefore, while the 1 

### metabolic pattern of benzo[a]pyrene has been relati vely well characterized qualitatively in 2 

### animals, both in vitro and in vivo, the quantitativ e kinetic relationships between overlapping and 3 

### sometimes competing metabolic reactions in potentia l target organs, essential for meaningful 4 

### PBPK modeling, are yet not well defined. 5 

### Furthermore, the potential for scaling this model t o humans without complete 6 

### reparameterization is questionable. According to L iao (2004), the recombinant human CYP1A1 7 

### has a higher activity for the formation of benzo[a] pyrene-hydroxy metabolites and a lower 8 

### activity for the formation of benzo[a]pyrene-diones , when compared with recombinant rat 9 

### CYP1A1. The RN-PBPK model overestimated the benzo[ a]pyrene concentration in the liver, 10 

### but after 4 hours, it underestimated benzo[a]pyrene concentration in fat. In contrast to the 11 

### findings of Zeilmaker et al. (1999a), the adipose t issue compartment was modeled only as 12 

### perfusion-limited. Tissue-blood partition coeffici ents for benzo[a]pyrene and its metabolites 13



### used in the RN-PBPK model were estimated but not va lidated experimentally or otherwise. 14 

### Also, no adjustment was made for using in vitro enz yme activities, especially when measured in 15 

### recombinant proteins as was done in the calibration of this model, although they typically differ 16



### from those in vivo. 17 

### The published PBPK models for benzo[a]pyrene were e valuated to determine whether the 18 

### existing models could be used to extrapolate from r ats to humans or for a route to route 19 

### extrapolation from oral exposure to the inhalation route. The focus was concentrated on models 20



### for the inhalation route since several well conduct ed studies by the oral route exist from which to 21 

### derive toxicity values. No full PBPK model for the inhalation route was identified. It was 22 

### concluded that at present, none of the published mo dels allow for computation of the resulting 23 

### internal doses used in the only cancer bioassay ava ilable for the inhalation route (Thyssen et al. 24 

### 1981), nor is there a model for humans that simulat es the typical inhalation exposure to 25 

### benzo[a]pyrene on poorly soluble carbonaceous parti cles. 26

### 64 DRAFT - DO NOT CITE OR QUOTE 4. HAZARD IDENTIFICATION 1

2

3 

### 4.1. HUMANS STUDIES 4 

### 4.1.1. Sources of Human Exposure 5 

### Although it has no commercial production or signifi cant uses, benzo[a]pyrene is 6 

### nevertheless a ubiquitous environmental contaminant resulting from the incomplete combustion 7 

### of organic matter. Essentially all humans experien ce repeated exposure to benzo[a]pyrene, 8 

### which can begin in utero and continue throughout li fe. The magnitude of benzo[a]pyrene 9 

### exposure depends on several factors related to life style (e.g., diet, tobacco smoking), occupation, 10 

### and living conditions (e.g., urban versus rural set ting, domestic heating and cooking methods). 11 

### A distinguishing feature concerning benzo[a]pyrene exposure is that environmental sources 12 

### always occur as complex mixtures, which may consist of numerous PAHs, including 13 

### heterocyclic and nonheterocyclic forms as well as a za arenes and nitro-substituted PAHs 14 

### (reviewed in Bostrom et al., 2002; WHO, 1998; ATSDR , 1995; IARC, 1973). Many of these 15 

### complex mixture components are carcinogens, and som e can exceed the carcinogenic potency of 16 

### benzo[a]pyrene, as observed in animal bioassays. 17 

### With the exception of certain occupational exposure sources such as aluminum 18 

### production and the conversion of coal to coke and c oal tar, the major contributor to total PAH 19 

### (and thus benzo[a]pyrene) exposure in nonsmokers is the diet (Cogliano et al., 2008; Straif et al., 20 

### 2005; Bostrom et al., 2002; Cenni et al., 1993; And ersson et al., 1983; Bjorseth et al., 1978a, b). 21 

### In particular, charbroiled and grilled meats, and c ertain grain products, are important 22 

### determinants of PAH exposure in most populations. In very limited populations receiving 23



### clinical coal tar treatment of the skin for conditi ons such as psoriasis, acute dermal exposure to 24 

### benzo[a]pyrene may greatly exceed that received fro m both occupational and nonoccupational 25 

### sources (Godschalk et al., 2001; Pavanello et al., 1999). 26

27 

### 4.1.2. Biomonitoring of Benzo[a]pyrene Exposure an d Effects In Humans 28 

### Quantitative exposure assessment becomes very chall enging for complex mixture 29 

### components such as benzo[a]pyrene, where individual exposures vary depending on background 30 

### concentrations in the environment, lifestyle factor s, and occupation. An alternative to measuring 31 

### concentrations in various environmental media, huma n biomonitoring focuses on biomarkers of 32 

### internal dose which may also serve as mechanistic i ndicators of early biological response 33 

### following exposure to a genotoxic agent. Carcinoge n exposure biomarkers are generated by 34 

### uptake and metabolic processes which may result eit her in detoxification and excretion or in 35



### bioactivation to reactive forms that can bind coval ently with DNA and other macromolecules. 36 

### Following exposure to complex PAH mixtures, biomoni toring of benzo[a]pyrene uptake can 37 

### involve evaluation of urinary metabolites of benzo[ a]pyrene or surrogates, DNA adducts in 38

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peripheral blood cells such as leukocytes and lymph ocytes, protein adducts in hemoglobin and 1 

### serum albumin, and cytogenetic damage in lymphocyte s (Gyorffy et al., 2008; Godschalk et al., 2 

### 2003; Bostrom et al., 2002; Scherer et al., 2000). Biomarkers offer advantages over traditional 3 

### benzo[a]pyrene exposure concentration monitoring be cause they can be quantitative indicators of 4 

### an individual’s environmental exposure and internal carcinogen dose irrespective of time, route 5 

### of exposure, and inter-individual metabolic differe nces. Associations between increased cancer 6 

### risk and levels of specific biomarkers, such as ben zo[a]pyrene-DNA adducts in target or 7 

### surrogate tissues, also provide important mechanist ic information in studies of human disease 8 

### etiology. 9 

### Considerable progress has been achieved in the appl ication of biomarker methods for 10 

### human biomonitoring of carcinogens (Vineis and Pere ra, 2007; Perera and Weinstein, 2000; 11 

### Poirier et al., 2000). However, further validation is needed before using biomarkers to routinely 12 

### predict disease risk. Successful application of bi omarkers in benzo[a]pyrene exposure 13 

### assessment currently depends on several assumptions : 1) urinary metabolites, whether derived 14 

### from benzo[a]pyrene or surrogate PAH compounds such as Py or phenanthrene, provide 15 

### information about recent benzo[a]pyrene exposures; 2) DNA and protein adduct measurements 16 

### in easily accessible tissues serve as surrogate bio markers of long-term benzo[a]pyrene exposure 17 

### and internal dose for less accessible target tiss ues (e.g., lung); 3) and cytogenetic biomarkers 18 

### reflect early biological effects that correlate wit h relevant preclinical events occurring at target 19 

### sites, and with biomarkers of biologically effectiv e benzo[a]pyrene dose. Evidence is 20 

### accumulating to support these assumptions for carci nogenic PAHs, in general, and for 21 

### benzo[a]pyrene, in particular, although conflicting and variable results among some studies limit 22 

### their application for dose-response assessment. Im portant issues to address include differences 23 

### in biomarker assay sensitivity and specificity, acq uired and inherited variations in PAH 24 

### bioactivation, detoxification, and DNA repair, and uncertain carcinogen intake(Gyorffy et al., 25 

### 2008; Divi et al., 2002; Poirier et al., 2000; Sche rer et al., 2000; Santella et al., 1994). 26

27



### 4.1.2.1. Urinary Excretion of Benzo[a]pyrene 28 

### Based on animal studies, urinary excretion of benzo [a]pyrene is a minor pathway of 29 

### elimination compared with elimination in the feces. The hydroxylated metabolites of 30 

### benzo[a]pyrene, 3-hydroxy-benzo[a]pyrene (3-OH-benz o[a]pyrene) and 9-hydroxy31 

### benzo[a]pyrene, have been identified in the urine o f humans exposed to PAH, although they are 32 

### often not detectable in human urine despite known e xposure to benzo[a]pyrene (Rossella et al., 33 

### 2009; Hecht, 2002; Bentsen-Farmen et al., 1999b). In numerous human studies, the 34 

### hydroxylated metabolite of Py, 1-OH-Py, is typicall y used as a surrogate indictor of internal 35 

### exposure to carcinogenic PAH mixtures based on the assumption that levels of 1-OH-Py in urine 36 

### correlate with exposure to individual PAH compounds of higher molecular weight, including 37 

### benzo[a]pyrene. Although urinary 1-OH-Py appears s uitable as a sensitive biomarker for total 38

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PAH exposure, results are variable for the correlat ion of 1-OH-Py with the benzo[a]pyrene 1 

### metabolite, 3-OH-benzo[a]pyrene, in urine (Forster et al., 2008; Godschalk et al., 1998a). In a 2 

### study of urinary PAHs and their hydroxylated metabo lites in 55 coke oven workers, 1-OH-Py 3 

### was found in 100% of the urine samples, whereas 3-O H-benzo[a]pyrene was always below the 4 

### quantification limit (Rossella et al., 2009). In a ddition, reported correlations between urinary 1- 5 

### OH-Py levels and PAH-DNA adducts in white blood cel ls (WBCs) are conflicting, although in 6 

### one study, 3-OH-benzo[a]pyrene levels in urine were significantly correlated with specific 7 

### benzo[a]pyrene diol epoxide adducts in skin DNA fol lowing dermal application of coal tar 8 

### ointments (Gyorffy et al., 2008; Godschalk et al., 1998a). 9 

### Buckley et al. (1995) found that excretion of benzo [a]pyrene metabolites in residents 10 

### (non-smokers not employed in high PAH occupational environments) of Phillipsburg, New 11 

### Jersey, correlated better with ingestion of benzo[a ]pyrene from food rather than with 12 

### environmental inhalation exposures. Benzo[a]pyrene metabolites in urine were measured 13 

### following “reverse metabolism,” a procedure that in volves enzymatic hydrolysis of 14 

### benzo[a]pyrene conjugates followed by chemical conv ersion of all hydroxylated benzo[a]pyrene 15 

### metabolites back to the parent compound for subsequ ent thin-layer chromatography and 16 

### scanning spectrofluorometry. Buckley et al. (1995) estimated pulmonary uptake at 11 and 2.3 ng 17 

### benzo[a]pyrene/person/day for winter and summer, re spectively, based on 24-hour personal air 18 

### measurements. The median intake of benzo[a]pyrene from the diet was estimated at 176 ng/day. 19 

### The median urinary excretion of benzo[a]pyrene and metabolites was 121 and 129 ng/day in 20 

### winter and summer, respectively. Based on multiple regression analyses of estimated inhaled 21 

### and ingested doses, change in urinary excretion of benzo[a]pyrene was only marginally 22 

### predictive of benzo[a]pyrene exposure. Most of the variation in urinary benzo[a]pyrene 23 

### excretion was explained by the ingested dose. Thes e results confirm an earlier study of 24 

### occupationally exposed (aluminum plant) workers usi ng the same reverse metabolism procedure 25 

### to measure urinary PAH elimination (Becher and Bjør seth, 1983). Although a significant 26 

### difference in urinary PAH excretion was seen in non occupationallly exposed smokers versus 27 

### nonsmokers, PAH metabolite levels in urine of alumi num plant workers did not reflect the large 28 

### difference in inhalation exposure relative to contr ols (Becher and Bjørseth, 1983). 29 

### Gündel et al. (2000) investigated PAH exposure and urinary metabolite excretion in 30 

### 19 workers from a fireproof stone factory. Along w ith other PAH metabolites in urine, they 31 

### measured 3-OH-benzo[a]pyrene levels following airbo rne exposures to benzo[a]pyrene ranging 32



### from 0.043 to 5.41 μ g/m 3 

### based on personal and stationary air sampling. Th e study authors 33 

### failed to identify a correlation between benzo[a]py rene inhalation exposure concentration and 34 

### urinary 3-OH-benzo[a]pyrene excretion, nor between 1-OH-Py and 3-OH-benzo[a]pyrene levels 35



### in urine. 36

37



### 4.1.2.2. Adduct Formation with DNA and Protein 38

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A large body of literature supports DNA and protein adducts as biomarkers of a 1 

### biologically effective dose (ED) for exposures to s ome DNA reactive human carcinogens 2 

### (Gyorffy et al., 2008; Vineis and Perera, 2007; Hec ht, 2004; Godschalk et al., 2003; Bostrom et 3 

### al., 2002; Perera and Weinstein, 2000; Poirier et a l., 2000). DNA adduct formation is considered 4 

### to be a necessary early event in tumor formation fo r many such compounds, particularly large, 5 

### bulky carcinogens such as benzo[a]pyrene and PAHs i n general. In PAH exposed populations, 6 

### there is evidence demonstrating the formation of DN A adducts with tobacco-derived PAH 7 

### including benzo[a]pyrene, not only sites directly e xposed, but also in distant organs and the 8 

### peripheral blood (see Table 4-1). 9

10 

### Table 4-1. Studies of PAH-DNA adducts in human pop ulations or tissues 11 

### exposed to PAHs 12 13 Reference Study description Arnould et al. (1998) DNA adducts from leukocytes f rom heavy smokers Arnould et al. (1999) DNA adducts in leukocytes fro m workers from plant producing carbon electrodes Arnould et al. (2000) DNA adducts in leukocytes of coke oven workers Assennato et al. (1993) DNA adducts in peripheral b lood leukocytes in coke oven workers Bartsch et al. (1999) PAH DNA adduct levels in lung parenchyma of coke oven workers and smokers Bartsch et al. (1999) DNA adduct formation in smoke rs, tobacco chewers, coke oven workers Bhattacharya et al. (2003) benzo[a]pyrene-DNA adduc ts in human urine Casale et al. (2001) benzo[a]pyrene adducts in urin e of cigarette smokers and women exposed to household smoke Galati et al. (2001) DNA adducts in sera of humans exposed to PAHs Gallagher et al. (1993) DNA adducts in blood cells, placental syncytial nuclei, placental tissue homogenates, and lung cells following exposure to c igarette or coal smoke Godschalk et al. (1998a) DNA adducts in alveolar ma crophages and subpopulations of white blood cells in smokers Godschalk et al. (1998b) DNA adducts in biopsies of treated skin and in WBCs along with levels of urinary 1-OH-pyrene in psoriasis patients being tre ated with coal tar Hemminki et al. (1997 DNA adduct formation in expo sed humans Izzotti et al. (1991, 1992) DNA adducts in pulmonar y alveolar macrophages following exposure to cigarette smoke Li et al. (2001) DNA adduct levels in human periphe ral blood lymphocytes of SCC patients and controls Li et al. (2002) BPDE-DNA adducts in human breast t issues Lodovici et al. (1998) benzo[a]pyrene levels in aut optic lungs of smokers and nonsmokers Lodovici et al. (1999) DNA adduct formation in huma n white blood cells from smokers and nonsmokers Mancini et al. (1999) DNA adducts in cervical cells from cigarette smokers Melikian et al. (1999) DNA adducts in epithelial an d stromal cervical tissue samples from women smokers and self-reported nonsmokers after hysterec tomy for nonmalignant conditions Paleologo et al. (1992) BPDE-DNA adducts in the whi te blood cells of patients treated with coal tar preparations Pavanello et al. (1999) DNA adducts in mononuclear white blood cells of coke oven workers and chimney sweeps Rojas et al. (2000) PAH DNA adduct levels in lung p arenchyma of coke oven workers and smokers Scherer et al. (2000) benzo[a]pyrene adducts of hem oglobin and albumin in smokers and

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Reference Study description nonsmokers Schoket et al. (1993) DNA adducts in lymphocytes in aluminum plant workers Schwartz et al. (2003) DNA analysis (content, damag e, cell cycle, and apoptosis) in smokers and nonsmokers Shinozaki et al. (1999) DNA adduct formation in agi ng smokers and non-smokers Van Delft et al. (1998) WBC-DNA adducts in workers in a carbon electrode manufacturing facility Wiencke et al. (1990) benzo[a]pyrene DNA adducts an d SCEs in human lymphocytes Zenzes et al. (1999a) DNA adducts in sperm cells fr om smokers and nonsmokers Zenzes et al. (1999b) DNA adducts in embryos from s moking couples Zhang et al. (1995) DNA adduct levels in oral mucos a cells from nonsmokers and smokers

### 69 DRAFT - DO NOT CITE OR QUOTE

1 

### Measurement of DNA adducts in target tissues (e.g., lung) can be difficult and invasive; 2 

### however, readily accessible nucleated tissue (e.g., WBCs, sperm cells, cervical cells) and 3 

### proteins (e.g., hemoglobin and albumin in blood) ca n serve as surrogate biomarkers of exposure. 4 

### DNA adducts in WBCs reflect exposure over a relativ ely long period, and are indicative of the 5 

### individual’s metabolic and DNA repair capability, b oth of which are genetically influenced. 6 

### Although protein adducts are not thought to be mech anistic intermediates in PAH-initiated 7 

### carcinogenesis, they offer several advantages as bi omarkers. For example, hemoglobin and 8 

### albumin are much more abundant than DNA; their addu cts are not subject to removal by 9 

### enzymatic repair, and they can integrate exposures over the protein lifespan of days to weeks. 10 

### In humans, PAH albumin and hemoglobin adducts have often been used to investigate 11 

### internal dosimetry of direct tobacco and environmen tal tobacco smoke (ETS) exposures. 12 

### Elevated benzo[a]pyrene adducts are often reported in smokers versus nonsmokers and in 13 

### children exposed to ETS from their smoking mothers (Hecht, 2004; Philips, 2002). 14 

### Benzo[a]pyrene-protein adduct levels in smokers are typically increased twofold compared with 15 

### nonsmokers (Scherer et al., 2000; Sherson et al., 1 990). 16

17



### 4.1.2.2.1. DNA adduct measures in blood 18 

### Major advances in methodology have greatly increase d the sensitivity and specificity 19 

### (Himmelstein et al., 2009; Jarabek et al., 2009) of DNA adducts measurements. The various 20 

### techniques utilized include immunoassays and immuno histochemistry, [ 32 

### P]- and [ 33 

### P]- 21 

### postlabelling of modified nucleotides paired with t hin layer or high performance liquid 22 

### chromatography (TLC and HPLC, respectively), fluore scence and phosphorescence 23 

### spectroscopy, electrochemical detection, and mass s pectrometry (Arlt et al., 2007; Poirier et al., 24 

### 2000). [ 32 

### P]-postlabelling assays and immunological methods a re the most commonly used. The 25 

### immunoassays employ an antiserum generated against benzo[a]pyrene-DNA adducts. In 26 

### addition to DNA adducts formed by benzo[a]pyrene, t he antibody cross reacts with DNA adducts 27 

### formed by other carcinogenic polycyclic aromatic hy drocarbons (PAHs) (Weston, et al, 1989) 28 

### but has no affinity for DNA or PAHs alone. DNA add ucts detected in non-laboratory samples 29 

### using this technique are therefore referred to as “ PAH-DNA” adducts. The [ 32 

### P]-postlabelling 30 

### method is highly sensitive in detecting bulky DNA a dducts, including but not limited to 31 

### benzo[a]pyrene- and PAH-DNA adducts, enabling quant itation down to 1 DNA adduct/10 9 32 

### normal nucleotides, and achieving high resolution o f individual adducts particularly when 33 

### combined with HPLC. Unless specific chromatographi c standards are available to assist in 34 

### identification, adducts detected using this techniq ue are referred to in general as “bulky DNA 35 

### adducts”. Accelerator mass spectrometry is a relat ively new method for adduct determination 36 

### that has demonstrated even greater sensitivity. Mo st studies do not identify the specific DNA 37 

### adduct structures, but instead report total “PAH-DN A,” “bulky-DNA,” “hydrophobic-DNA,” or 38

### 70 DRAFT - DO NOT CITE OR QUOTE

“aromatic-DNA” adducts (Alexandrov et al., 2002). Further investigations are needed to 1



### examine correlations between DNA adduct levels dete rmined by different methods, and 2 

### correlations between DNA adducts and urinary PAH me tabolites. 3 

### benzo[a]pyrene-DNA and PAH-DNA adducts have been me asured in WBCs in several 4 

### PAH-exposed human groups and found to be associated with, and predictive of, elevated cancer 5 

### risks (Vineis and Perera, 2007; Pavanello et al., 1 999). Coke oven workers experience especially 6 

### high exposures to PAHs and demonstrate increased lu ng cancer rates. Pavanello and coworkers 7 

### (2005) studied a group of 67 highly exposed coke ov en workers for genetic factors that can 8 

### modulate individual responses to carcinogenic PAHs. Levels of BPDE-DNA adducts in 9 

### mononuclear WBCs (lymphocyte plus monocyte fraction ) were associated with workplace PAH 10 

### exposure as indicated by urinary 1-OH-Py excretion. The authors concluded that the elevated 11 

### levels of BPDE-DNA adducts reflected both exposure and individual variation in expression of 12 

### genes involved with glutathione conjugation activit y and DNA excision repair capacity. 13 

### In a similar study, Pavanello et al. (2006) screene d 585 Caucasian municipal workers 14 

### (52% males, 20–62 years old) from northeast Italy f or BPDE-DNA adduct formation in 15 

### peripheral lymphocytes. Forty-two percent of the pa rticipants had elevated anti-BPDE-DNA 16 

### adduct levels, defined as >0.5 adducts/10 8 

### nucleotides (mean, 1.28 ± 2.80 adducts/10 8 17 

### nucleotides). Comparison of adduct levels with que stionnaire responses indicated that smoking, 18 

### frequent consumption of PAH-rich meals (>52 times/y ear vs. <52 times/year), and long time 19 

### periods spent outdoors (>4 hours/day vs. <4 hours/d ay) were risk factors as all increased BPDE20 

### DNA adduct levels significantly. Exposure to indoo r combustion sources (use of fireplace, coal21 

### or wood stove >5 times/year) significantly increase d the frequency of subjects positive for 22 

### BPDE-DNA adducts. Exposure to heavy traffic did no t alter lymphocyte BPDE-DNA adduct 23 

### levels. Smoking and high-PAH diets were associated with increased BPDE-DNA adduct levels. 24 

### In nonsmokers, high-PAH diets and extended time spe nt indoors associated with increased 25 

### BPDE-DNA adduct formation, while in smokers, person al cigarette smoking was the only factor 26 

### that was positively correlated with adduct levels. These results demonstrate the potential utility 27



### of BPDE-DNA adduct measurement as a biomarker of PA H exposure not only in heavily PAH28 

### exposed occupations and tobacco smokers, but also i n the general population, including 29



### nonsmokers. 30 

### Several studies investigated the level of DNA adduc ts in the WBCs of foundry workers 31 

### (Perera et al., 1988) or coke oven workers (Arnould et al., 2000; Assennato et al., 1993a, b) using 32 

### an immunoassay. The antibody’s cross-reactivity wi th adducts originating from PAHs other than 33 

### benzo[a]pyrene generally results in higher reported adduct levels than the [ 32 

### P]-postlabelling 34 

### method which can isolate benzo[a]pyrene-DNA adducts . All three studies involved several 35 

### levels of potential benzo[a]pyrene exposure as asse ssed by environmental sampling: 0.02, 5, 25, 36



### and 45 μ g/m 3 

### (Arnould et al., 2000); <0.05, 0.05–0.2, and >0.2 μ g/m 3 

### (Perera et al., 1988); and 37



### 0.03–12.6 μ g/m 3 

### (Assennato et al., 1993a, b). Adduct levels range d from 0.05 to 7 fmol PAH- 38

### 71 DRAFT - DO NOT CITE OR QUOTE

DNA per μ g DNA, and variation within exposure groups was hig h; however, all three studies 1 

### reported statistically significant correlations bet ween exposure and adduct levels. However, 2 

### there are a large number of studies, many of them c onducted by the same laboratories cited here, 3 

### that did not report positive correlations between benzo[a]pyrene exposure and DNA adduct 4 

### formation in people exposed occupationally or to ci garette smoke (e.g., van Delft et al., 2001; 5 

### Arnould et al., 1999; Pan et al., 1998; Peluso et a l., 1998; Lewtas et al., 1997; Assennato et al., 6 

### 1993a, b; Kriek et al., 1993; Paleologo et al., 199 2; Herbert et al., 1990). These studies illustrate 7 

### that attempting to correlate DNA adducts with benzo [a]pyrene exposure by a single route of 8 

### exposure (e.g., inhalation), or by occupation alone , may produce highly variable or misleading 9



### results. 10 

### Perera et al. (2005a) measured BPDE-DNA adduct leve ls in maternal and umbilical cord 11 

### blood obtained following normal delivery from 329 n onsmoking pregnant women exposed to 12 

### emissions from fires during the 4 weeks following t he collapse of the World Trade Center 13 

### (WTC) building in New York City on 09/11/2001. BPD E-DNA adduct levels were highest in 14 

### study participants who lived within 1 mile of the W TC, with inverse correlation between cord 15 

### blood levels and distance from WTC. For the group of participants that resided within 1 mile of 16 

### the WTC, maternal levels were 0.30 ± 0.16 adducts/1 0 8 

### nucleotides, umbilical cord levels were 17 

### 0.28 ± 0.08; for the group employed within 1 mile o f WTC, the corresponding values were 0.25 18 

### ± 0.11 and 0.24 ± 0.12, and for the unexposed refer ent group, the values were 0.22 ± 0.10 and 19 

### 0.23 ± 0.10. Differences between referent and expo sed subjects were marginally significant (0.1 20



### > p > 0.05); however, the trend for adducts in materna l blood decreasing with increasing distance 21 

### from the WTC was statistically significant ( p = 0.02) as was the finding that the percentage of 22 

### participants with detectable BPDE-DNA adducts incre ased significantly (trend p = 0.05) with 23 

### increasing time and proximity to WTC–from 52.6% (re ference group) to 66.7% (employed 24 

### group) to 80.0% (resident group). 25 

### Tuntawiroon et al. (2007) evaluated airborne PAH co ncentrations and internal biomarker 26 

### levels in 115 Thai school boys (8–12 years old) att ending schools adjacent to high-density traffic 27 

### areas in Bangkok (high exposure group), compared wi th 69 boys (9–13 years old) attending 28 

### schools located in a provincial area (low exposure group). Ambient air concentrations (roadside 29 

### and school areas) and personal breathing zone air c oncentrations of 10 particulate PAHs were 30 

### measured in Bangkok and in the provincial location. Peripheral blood samples were collected 31 

### from the school boys for [ 32 

### P]-postlabeling determination of bulky DNA adducts in lymphocytes. 32 

### Based on toxic equivalency factors (TEFs, Nisbet an d LaGoy, 1992) for PAH, benzo[a]pyrene 33 

### equivalent exposures from personal breathing zone m easurements in Bangkok children were 34 

### about 3.5-fold greater than in rural provincial chi ldren ( p < 0.001). Interestingly, although bulky 35 

### DNA adducts were increased fivefold in Bangkok chil dren compared with provincial children ( p 36 

### < 0.001), adduct levels were negatively correlated with total PAH and benzo[a]pyrene equivalent 37



### exposures. 38

### 72 DRAFT - DO NOT CITE OR QUOTE

1 

### 4.1.2.2.2. Adduct measures in reproductive tissues 2

3 

### Zenzes et al. (1998) used immunostaining to detect PAH-DNA adducts in ovarian 4 

### granulosa-lutein cells from women undergoing proced ures for reproductive assistance. The 32 5 

### women in the sample were separated by smoking statu s and consisted of 14 active smokers (1-20 6 

### cigarettes per day), 7 passive smokers (nonsmoker b ut husband smoked), and 11 non-smokers 7 

### with a non-smoking partner. The collected cells wer e fixed and stained with the anti-BP-DNA 8 

### antibody which is known to cross-react with several carcinogenic PAH-DNA adducts. In this 9 

### assay, darker nuclear staining results from a great er concentration of PAH-DNA adducts and is 10 

### assigned a higher score. The proportion of nuclei exhibiting respective intensities of staining 11 

### were combined to obtain an overall intensity score for the sample. The observed staining 12 

### intensity was related to smoking status, with a mea n ± SE of 1.91 ± 0.10 among active smokers, 13 

### 1.21 ± 0.20 among passive smokers, and 0.62 ± 0.17 among nonsmokers (p < 0.0001). The 14 

### authors concluded that smoking-related seminal DNA adducts could be a potential source of 15 

### transmissible zygotic DNA damage. 16 

### In another study using the same immunostaining assa y, Zenzes et al. (1999a) investigated 17



### the occurrence of PAH-DNA adducts in semen in relat ion to tobacco use. The study included 23 18 

### men (11 smokers and 12 nonsmokers), mean age 37 yea rs, recruited through couples attending 19 

### an in vitro fertilization – embryo transfer clinic in Toronto. Among smokers, the mean amount 20 

### smoked was 20.6 cigarettes per day. The PAH-DNA ad duct staining intensity was higher in the 21 

### sperm samples from smokers (mean ± SE, 1.73 ± 0.09) when compared with nonsmokers (0.93 ± 22 

### 0.10) (p < 0.0001); 5.7% and 30.9% of the sperm fro m smokers and nonsmokers, respectively, 23 

### exhibited negative staining and 21.2% and 3.8%, res pectively, exhibited strong staining. The 24 

### authors concluded that smoking-related DNA adducts in semen could potentially be a source of 25 

### zygotic DNA damage. 26 

### A related study examined the presence of PAH-DNA ad ducts in 112 blastomere cells 27 

### from 22 pre-implantation embryos available through an in vitro fertilization – embryo transfer 28 

### clinic (Zenzes et al., 1999b). The donated embryos were grouped with respect to the maternal 29 

### and paternal smoking status (n = 8 both parents smo ked, n = 12 father smoked but mother did not 30 

### smoke, and n=7 neither parent smoked). Five of th e embryos (4 from the father-only smoking 31 

### and 1 from a non-smoking couple) were very fragment ed and were not used in the analysis of 32 

### PAH-DNA staining intensity. Among the smoking pare nts, women smoked less per day than 33 

### men (mean ± SE, 13.0 ± 3.0 and 16.8 ± 1.5 cigarett es per day for women and men, respectively). 34 

### The intensity score was higher for embryos with at least one parent who smoked (1.40 ± 0.28) 35 

### compared with embryos from nonsmoking parents (0.38 ± 0.14) (p = 0.015), but there was little 36 

### difference between embryos with two compared with o ne smoking parent (1.34 ± 0.30 and 1.48 37 

### ± 0.55 for both smokers and one smoker, respective ly). Similar results were seen using the 38

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proportion of embryos exhibiting any staining rathe r than a mean staining measure, and with the 1 

### proportion of blastomere cells exhibiting staining. Intensity score was correlated with the 2 

### amount smoked by the father (p = 0.020) but not by the mother. Analysis of the presence of 3 

### PAH-DNA adducts in sperm cells also revealed an inc reased proportion and staining intensity in 4 

### samples from smokers compared with nonsmokers (p < 0.0001 for categorical analysis of 5 

### negative, weak, moderate and strong staining intens ity). The authors noted that the demonstrated 6 

### presence of these adducts in the embryos, reflectin g most strongly a paternal origin, may affect 7 

### the viability of the pregnancy. 8

9



### 4.1.2.3. Benzo[a]pyrene-Induced Cytogenetic Damage 10 

### Many studies measure cytogenetic damage as biomarke rs of early biological effects 11 

### which also reflect exposure to genotoxic chemicals. Standard cytogenetic end points include 12 

### chromosomal aberration (CA), sister chromatid excha nge (SCE), micronucleus (MN) formation, 13 

### hypoxanthine guanine phosphoribosyl transferase (hp rt) mutation frequency, and GPA mutation 14 

### frequency (Gyorffy et al., 2008). These biomarkers are often incorporated in multi-endpoint 15 

### studies with other biomarkers of exposure. Because they indicate related but different endpoints, 16



### there is often a lack of correlation between the di fferent categories of biomarkers. 17 

### Merlo et al. (1997) evaluated DNA adduct formation (measured by [ 32 

### P]-postlabelling) 18 

### and micronuclei in WBCs of 94 traffic policemen vs. 52 residents from the metropolitan area of 19 

### Genoa, Italy. All study subjects wore personal air samplers for 5 hours of one work shift, and 20 

### levels of benzo[a]pyrene and other PAHs. Policemen were exposed to 4.55 ng 21 

### benzo[a]pyrene/m 3 

### air, compared with urban residents who were expose d to 0.15 ng/m 3 

### . DNA 22 

### adduct levels in policemen were 35% higher than in urban residents ( p = 0.007), but micronuclei 23 

### in urban residents were 20% higher than in policeme n ( p = 0.02). Linear regressions of DNA 24 

### adducts and MN incidence, respectively, vs. benzo[a ]pyrene exposure levels did not reveal 25 

### significant correlations. 26 

### Perera and coworkers assessed DNA damage in Finnish iron foundry workers in two 27 

### separate studies and using three methodologies. Ba sed on results from personal sampling and 28 

### stationary monitoring in both studies, three levels of benzo[a]pyrene air concentrations were 29 

### defined: low (<5 ng/m 3 

### benzo[a]pyrene), medium (5–12 ng/m 3 

### ), and high (>12 ng/m 3 

### ). (Perera 30 

### et al., 1994, 1993). In the first study, involving 48 workers, several biomarkers were analyzed 31 

### for dose-response and interindividual variability ( Perera et al., 1993). PAH-DNA adducts were 32 

### determined in WBCs using an immunoassay as describe d in Section 4.1.2.2.1 and enzyme-linked 33 

### immunosorbent assay (ELISA) with fluorescence detec tion. Mutations at the hprt locus were 34 

### also measured in WBC DNA. The latter assay is base d on the fact that each cell contains only 35 

### one copy of the hprt gene, which is located on the X-chromosome. While male cells have only 36 

### one X-chromosome, female cells inactivate one of th e two X-chromosomes at random. The gene 37 

### is highly sensitive to mutations such that in the e vent of a crucial mutation in the gene, enzyme 38

### 74 DRAFT - DO NOT CITE OR QUOTE

activity disappears completely from the cell. In a ddition, mutations at the GPA gene locus were 1 

### measured in red blood cells (RBCs). The GPA mutati on frequency was not correlated with 2 

### either benzo[a]pyrene exposure or PAH-DNA adduct fo rmation. However, both PAH-DNA 3 

### adduct levels and hprt mutation frequency increased with increasing benzo[a]pyrene exposure. 4 

### In addition, there was a highly significant correla tion between incidence of hprt mutations and 5 

### PAH-DNA adduct levels ( p = 0.004). 6 

### In a second study, Perera et al. (1994) surveyed 64 iron foundry workers with 7 

### assessments conducted in 2 successive years; 24 of the workers provided blood samples in both 8 

### years. Exposure to benzo[a]pyrene, collected by pe rsonal and area sampling in the 1st year of 9 

### the study, ranged from <5 to 60 ng/m 3 

### and was estimated to have decreased by 40% in the 2 nd 10 

### year. The levels of PAH-DNA adducts were roughly 5 0% lower in the 2nd year, presumably 11 

### reflecting decreased exposure. The longer-lived hp rt mutations were not as strongly influenced 12 

### by the decreasing exposure to benzo[a]pyrene. Stud y subjects who did not have detectable levels 13 

### of DNA adducts were excluded from the study. As in the previous study, a strong correlation 14 

### between DNA adduct levels and incidence of hprt mut ations was observed (Perera et al., 1993). 15 

### Kalina et al. (1998) studied several cytogenetic ma rkers in 64 coke oven workers and 16 

### 34 controls employed at other locations within the same plant. Airborne benzo[a]pyrene and 17 

### seven other carcinogenic PAHs were collected by per sonal air samplers, which showed ambient 18 

### benzo[a]pyrene concentrations ranging widely from 0 .002 to 50 μ g/m 3 

### in coke oven workers and 19



### from 0.002 to 0.063 μ g/m 3 

### in controls. CAs, SCEs, high-frequency cells (HFC s), and SCE 20 

### heterogeneity index (SCE-H) were all significantly increased with benzo[a]pyrene exposure. 21 

### Except for increases in HFCs, no effect of smoking was observed. Consistent with studies of 22 

### PAH-DNA adduct formation, reduced cytogenetic respo nse at high exposure levels produced a 23 

### nonlinear dose-response relationship. The authors also evaluated the potential influence of 24 

### polymorphisms in enzymes involved in the metabolism of benzo[a]pyrene. Glutathione S25 

### transferase M1 (GSTM-1) and N-acetyl transferase (N AT)-2 polymorphisms were studied and 26 

### no evidence of the two gene polymorphisms having an y influence on the incidence of 27 

### cytogenetic damage was found. 28 

### Motykiewicz et al. (1998) conducted a similar study of genotoxicity associated with 29 

### benzo[a]pyrene exposure in 67 female residents of a highly polluted industrial urban area of 30 

### Upper Silesia, Poland, and compared the results to those obtained from 72 female residents of 31 

### another urban but less polluted area in the same pr ovince of Poland. Urinary mutagenicity and 32 

### 1-OH-Py levels, PAH-DNA adducts in oral mucosa cell s (detected by immunoperoxidase 33 

### staining), SCE, HFC, CA, bleomycin sensitivity, and GSTM-1 and CYP1A1 polymorphisms in 34 

### blood lymphocytes were investigated. High volume a ir samplers and gas chromatography were 35 

### used to quantify ambient benzo[a]pyrene levels whic h during the summer were 3.7 ng/m 3 

### in the 36 

### polluted area and 0.6 ng/m 3 

### in the control area. During winter, levels rose t o 43.4 and 7.2 ng/m 3 37 

### in the two areas, respectively. The cytogenetic bi omarkers (CA and SCE/HFC), urinary 38

### 75 DRAFT - DO NOT CITE OR QUOTE

mutagenicity, and urinary 1-OH-Py excretion were si gnificantly increased in females from the 1 

### polluted area, and differences appeared to be more pronounced during winter time. PAH-DNA 2 

### adduct levels were significantly increased in the s tudy population, when compared to the 3 

### controls, only in the winter season. No difference in sensitivity to bleomycin-induced 4 

### lymphocyte chromatid breaks was seen between the tw o populations. As with the study by 5 

### Kalina et al. (1998), genetic polymorphisms assumed to affect the metabolic transformation of 6 

### benzo[a]pyrene were not associated with any differe nce in the incidence of DNA damage. 7 

### In a study of Thai school boys in urban (Bangkok) a nd rural areas, bulky (including but 8 

### not limited to BPDE-type) DNA adduct levels were me asured in lymphocytes along with DNA 9 

### single strand breaks (SSBs), using the comet assay, and DNA repair capacity (Tuntawiroon et al., 10 

### 2007). Ambient air and personal breathing zone mea surements indicated that Bangkok school 11 

### children experienced significantly higher exposures to benzo[a]pyrene and total PAHs. A 12 

### significantly higher level of SSBs (tail length 1.9 3 ± 0.09 vs. 1.28 ± 0.12 μ m, +51%; p < 0.001) 13 

### was observed in Bangkok school children when compar ed with rural children, and this parameter 14 

### was significantly associated with DNA adduct levels . A significantly reduced DNA repair 15 

### capacity (0.45 ± 0.01 vs. 0.26 ± 0.01 γ -radiation-induced deletions per metaphase, -42%; 16



### p < 0.001) was also observed in the city school chil dren, again significantly associated with 17 

### DNA adduct levels. It was not evident why higher e nvironmental PAH exposure would be 18 

### associated with lowered DNA repair capacity. Howev er, because the personal breathing zone 19



### PAH levels and DNA adduct levels were not associate d with each other, it is conceivable that the 20 

### city school children had a priori lower DNA repair capacities that contributed significantly to the 21 

### high adduct levels. The authors considered genetic differences between the two study 22 

### populations as a possible reason for this observati on. 23

24 

### 4.1.3. Epidemiologic Findings in Humans 25



### The association between human cancer and contact wi th PAH-containing substances, 26 

### such as soot, coal tar, and pitch, has been widely recognized since the early 1900s (Bostrom et 27 

### al., 2002). Although numerous epidemiology studies establish an unequivocal association 28 

### between PAH exposure and human cancer, defining the causative role for benzo[a]pyrene and 29 

### other specific PAHs remains a challenge. In essent ially all reported studies, either the 30 

### benzo[a]pyrene exposure and/or internal dose are no t known, or the benzo[a]pyrene carcinogenic 31 

### effect cannot be distinguished from the effects of other PAH and non-PAH carcinogens. 32 

### Nevertheless, three types of investigations provide support for the involvement of 33 

### benzo[a]pyrene in some human cancers: molecular ep idemiology studies; population- and 34 

### hospital-based case-control studies; and occupation al cohort studies. In some cohort studies, 35 

### benzo[a]pyrene exposure concentrations were measure d and thus provide a means to link 36 

### exposure intensity with observed cancer rates. In case-control studies, by their nature, 37 

### benzo[a]pyrene and total PAH doses can only be esti mated. 38

### 76 DRAFT - DO NOT CITE OR QUOTE

1



### 4.1.3.1. Molecular Epidemiology and Case-Control Cancer Stud ies 2 

### Defective DNA repair capacity leading to genomic in stability and, ultimately, increased 3 

### cancer risk is well documented (Wu et al., 2007, 20 05). Moreover, sensitivity to mutagen4 

### induced DNA damage is highly heritable and thus rep resents an important factor that determines 5 

### individual cancer susceptibility. Based on studies comparing monozygotic and dizygotic twins, 6 

### the genetic contribution to BPDE mutagenic sensitiv ity was estimated to be 48.0% (Wu et al., 7 

### 2007). BPDE has been used as an etiologically rele vant mutagen in case-control studies to 8 

### examine the association between elevated lung and b ladder cancer risk and individual sensitivity 9 

### to BPDE-induced DNA damage. Mutagen sensitivity is determined by quantifying chromatid 10 

### breaks or DNA adducts in phytohemagglutinin-stimula ted peripheral blood lymphocytes as an 11 

### indirect measure of DNA repair capacity. 12 

### In a hospital-based case-control study involving 22 1 lung cancer cases and 229 healthy 13 

### controls, DNA adducts were measured in stimulated p eripheral blood lymphocytes after 14 

### incubation with BPDE in vitro (Li et al., 2001). L ung cells from cancer cases showed consistent 15 

### statistically significant elevations in induced BPD E-DNA adducts, compared with controls, 16 

### regardless of subgroup by age, sex, ethnicity, smok ing history, weight loss, or family history of 17 

### cancer. The BPDE-induced DNA adduct levels, when g rouped by quartile using the levels in 18 

### controls as cutoff points, were significantly dose- related with lung cancer risk (odds ratios [ORs] 19 

### 1.11, 1.62, and 3.23; trend test, p < 0.001). In a related hospital-based case-contro l study 20 

### involving 155 lung cancer patients and 153 healthy controls, stimulated peripheral blood 21 

### lymphocytes were exposed to BPDE in vitro (Wu et al ., 2005). DNA damage/repair was 22 

### evaluated using the comet assay, and impacts on cel l cycle checkpoints measured using a 23 

### fluorescence-activated cell-sorting method. The lu ng cancer cases exhibited significantly higher 24 

### levels of BPDE-induced DNA damage than the controls ( p < 0.001), with lung cancer risk 25 

### positively associated with increasing levels of DNA damage when grouped in quartiles (trend 26



### test, p < 0.001). In addition, lung cancer patients demon strated significantly shorter cell cycle 27 

### delays in response to BPDE exposure, which correlat ed with increased DNA damage. 28 

### Sensitivity to BPDE-induced DNA damage in bladder c ancer patients supports the results 29 

### observed in lung cancer cases. In a hospital-based case-control study involving 203 bladder 30 

### cancer patients and 198 healthy controls, BPDE-indu ced DNA damage was specifically 31 

### evaluated at the chromosome 9p21 locus in stimulate d peripheral blood lymphocytes (Gu et al., 32 

### 2008). Deletions of 9p21, which includes critical components of cell cycle control pathways, are 33 

### associated with a variety of cancers. After adjust ing for age, sex, ethnicity, and smoking status, 34 

### individuals with high BPDE-induced damage at 9p21 w ere significantly associated with 35 

### increased bladder cancer risk (OR 5.28; 95% confide nce interval [CI] 3.26–8.59). 36 

### Categorization of patients into tertiles for BPDE s ensitivity relative to controls demonstrated a 37 

### dose-related association between BPDE-induced 9p21 damage and bladder cancer risk. 38

### 77 DRAFT - DO NOT CITE OR QUOTE

Collectively, the results of molecular epidemiology studies with lung and bladder cancer patients 1 

### indicate that individuals with a defective ability to repair BPDE-DNA adducts are at increased 2 

### risk for cancer and, moreover, that specific genes linked to tumorigenesis pathways may be 3 

### molecular targets for benzo[a]pyrene and other carc inogens. 4 

### Due to the importance of the diet as a benzo[a]pyre ne exposure source, several 5 

### population- and hospital-based case-control studies have investigated the implied association 6 

### between dietary intake of benzo[a]pyrene and risk f or several tumor types. In a study involving 7 

### 193 pancreatic cancer cases and 674 controls (Ander son et al., 2005), another involving 8 

### 626 pancreatic cancer cases and 530 controls (Li et al., 2007), and a third involving 9 

### 146 colorectal adenoma cases and 228 controls (Sinh a et al., 2005), dietary intake of 10 

### benzo[a]pyrene was estimated using food frequency q uestionnaires. In all studies, the primary 11 

### focus was on estimated intake of benzo[a]pyrene (an d other carcinogens) derived from cooked 12 

### meat. Overall, cases when compared with controls h ad higher intakes of benzo[a]pyrene and 13 

### other food carcinogens, leading to the conclusion t hat benzo[a]pyrene plays a role in the etiology 14 

### of these tumors in humans. In a supportive follow- up case-control study of colorectal adenomas, 15 

### increased leukocyte PAH-DNA adducts were measured i n cases when compared with controls, 16 

### using a method that recognizes BPDE and several oth er PAHs bound to DNA (Gunter et al., 17 

### 2007). 18

19



### 4.1.3.2. Cohort Cancer Studies 20 

### Epidemiologic studies of workers in PAH-related occ upations indicate increased human 21 

### cancer risks associated with iron and steel product ion, roofing, carbon black production, and 22 

### exposure to diesel exhaust (Bosetti et al., 2007). Exposure to benzo[a]pyrene is only one of 23 

### numerous contributors to the cancer risk from compl ex PAH-containing mixtures that occur in 24 

### the workplace. Although some occupational cohort s tudies report measured or estimated 25 

### inhalation exposure concentrations for benzo[a]pyre ne, none report biomarkers of internal 26 

### benzo[a]pyrene dose in study subjects (reviewed in Bosetti et al., 2007; Armstrong et al., 2004). 27 

### Several of these cohort studies (summarized below) demonstrate a positive exposure-response 28 

### relationship with cumulative PAH exposure using ben zo[a]pyrene—or a proxy such as benzene29 

### soluble matter (BSM) that can be converted to benzo [a]pyrene—as an indicator substance. 30 

### These studies provide insight and support for the c ausative role of benzo[a]pyrene in human 31



### cancer. 32

33

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4.1.3.2.1. Cancer incidence in aluminum and electrode produc tion plants. 1 

### Exposure to benzo[a]pyrene and BSM in aluminum smel ter workers is strongly 2 

### associated with bladder cancer and weakly associate d with lung cancer (Boffetta et al., 1997; 3 

### Tremblay et al., 1995; Armstrong et al., 1994; Gibb s, 1985; Theriault et al., 1984). In an 4 

### analysis of pooled data from nine cohorts of alumin um production workers, 688 respiratory tract 5 

### cancer cases were observed versus 674.1 expected (p ooled RR 1.03; CI 0.96–1.11) (Bosetti et al., 6 

### 2007). A total of 196 bladder cancer cases were ob served in eight of the cohorts, compared with 7 

### 155.7 expected (pooled RR 1.29; CI 1.12–1.49). Bas ed on estimated airborne benzo[a]pyrene 8 

### exposures from a meta-analysis of eight cohort stud ies, the predicted lung cancer relative risk 9 

### (RR) per 100 μ g/m 3 

### -years of cumulative benzo[a]pyrene exposure was 1. 16 (95% CI 1.05–1.28) 10 

### (Armstrong et al., 2004). 11 

### Spinelli et al. (2006) reported a 14-year update to a previously published historical cohort 12 

### study (Spinelli et al., 1991) of Canadian aluminum reduction plant workers. The results 13 

### confirmed and extended the findings from the earlie r epidemiology study. The study surveyed a 14 

### total of 6,423 workers with ≥ 3 years of employment at an aluminum reduction plan t in British 15 

### Columbia, Canada, between the years 1954 and 1997, and evaluated all types of cancers. The 16 

### focus was on cumulative exposure to coal tar pitch volatiles, measured as BSM and as 17 

### benzo[a]pyrene. Benzo[a]pyrene exposure categories were determined from the range of 18 

### predicted exposures over time from statistical expo sure models. There were 662 cancer cases, of 19 

### which approximately 98% had confirmed diagnoses. T he overall cancer mortality rate 20 

### (standardized mortality ratio [SMR] 0.97; CI 0.87–1 .08) and cancer incidence rate (standardized 21 

### incidence ratio [SIR] 1.00; CI 0.92–1.08) were not different from that of the British Columbia 22 

### general population. However, this study identified significantly increased incidence rates for 23 

### cancers of the bladder (SIR 1.80; CI 1.45–2.21) and the stomach (SIR 1.46; CI 1.01–2.04). The 24 

### lung cancer incidence rate was only slightly higher than expected (SIR = 1.10; CI 0.93–1.30). 25 

### Significant dose-response associations with cumulat ive benzo[a]pyrene exposure were seen for 26 

### bladder cancer (

p trend <0.001), stomach cancer ( p trend <0.05), lung cancer ( p trend <0.001), 27 

### non-Hodgkin lymphoma ( p trend <0.001), and kidney cancer ( p trend <0.01), although the 28



### overall incidence rates for the latter three cancer types were not significantly elevated versus the 29 

### general population. Similar cancer risk results we re obtained using BSM as the exposure 30 

### measure; the cumulative benzo[a]pyrene and BSM expo sures were highly correlated (r = 0.94). 31 

### In several occupational cohort studies of workers i n Norwegian aluminum production 32 

### plants, personal and stationary airborne PAH measur ements were performed. 33 

### In a study covering 11,103 workers and 272,554 pers on × years of PAH exposure, cancer 34 

### incidence was evaluated in six Norwegian aluminum s melters (Romundstad et al., 2000a, b). 35 

### Reported estimates of PAH exposure concentrations r eached a maximum of 3,400 μ g/m 3 

### PAH 36 

### (680 μ g/m 3 

### benzo[a]pyrene). The overall number of cancers ob served in this study did not differ 37 

### significantly from control values (SIR 1.03; CI 1.0 –1.1). The data from this study showed 38

### 79 DRAFT - DO NOT CITE OR QUOTE

significantly increased incidences for cancer of th e bladder (SIR 1.3; CI 1.1–1.5), and elevated, 1 

### but not significant, SIRs for larynx (SIR 1.3; CI 0 .8–1.9), thyroid (SIR 1.4; CI 0.7–2.5), and 2 

### multiple myeloma (SIR 1.4; CI 0.9–1.9). Incidence rates for bladder, lung, pancreas, and kidney 3 

### cancer (the latter three with SIRs close to unity) were subjected to a cumulative exposure4 

### response analysis. The incidence rate for bladder cancer showed a trend with increasing 5 

### cumulative exposure and with increasing lag times ( up to 30 years) at the highest exposure level. 6 

### The incidence of both lung and bladder cancers was greatly increased in smokers. The authors 7 

### reported that using local county rates rather than national cancer incidence rates as controls 8 

### increased the SIR for lung cancer (SIR 1.4; CI 1.2– 1.6) to a statistically significant level. 9

10



### 4.1.3.2.2. Cancer incidence in coke oven, coal gasification, and iron and steel foundry 11



### workers. 12 

### An increased risk of death from lung and bladder ca ncer is reported in some studies 13 

### involving coke oven, coal gasification, and iron an d steel foundry workers (Bostrom et al., 2002; 14 

### Boffetta et al., 1997). An especially consistent r isk of lung lung cancer across occupations is 15 

### noted when cumulative exposure is taken into consid eration (e.g. RR of 1.16 per 100 unity-yrs 16 

### for aluminum smeleter workers, 1.17 for coke oven w orkers, and 1.15 for coal gasificiation 17 

### workers). In an analysis of pooled data from 10 co horts of coke production workers, 762 lung 18 

### cancer cases were observed versus 512.1 expected (p ooled RR 1.58; CI 1.47–1.69) (Bosetti et al., 19 

### 2007). Significant variations in risk estimates am ong the studies were reported, particularly in 20 

### the large cohorts (RRs of 1.1, 1.2, 2.0, and 2.6). There was no evidence for increased bladder 21 

### cancer risk in the coke production workers. Based on estimated airborne benzo[a]pyrene 22 

### exposures from a meta-analysis of 10 cohort studies , the predicted lung cancer RR per 23



### 100 μ g/m 3 

### -years of cumulative benzo[a]pyrene exposure was 1. 17 (95% CI 1.12–1.22) 24 

### (Armstrong et al., 2004). 25 

### A meta-analysis of data from five cohorts of gasifi cation workers reported 251 deaths 26 

### from respiratory tract cancer, compared with 104.7 expected (pooled RR 2.58; 95% CI 2.28– 27 

### 2.92) (Bosetti et al., 2007). Pooled data from thr ee of the cohorts indicated 18 deaths from 28 

### urinary tract cancers, versus 6.0 expected (pooled RR 3.27; 95% CI 2.06–5.19). Based on 29 

### estimated airborne benzo[a]pyrene exposures from a meta-analysis of four gas worker cohort 30 

### studies, the predicted lung cancer RR per 100 μ g/m 3 

### -years of cumulative benzo[a]pyrene 31 

### exposure was 1.15 (95% CI 1.11–1.20) (Armstrong et al., 2004). 32 

### Increased risks were reported in iron and steel fou ndry workers for cancers of the 33 

### respiratory tract, bladder and kidney. In an analy sis of pooled data from 10 cohorts, 34 

### 1,004 respiratory tract cancer cases were observed versus 726.0 expected (pooled RR 1.40; CI 35 

### 1.31–1.49) (Bosetti et al., 2007). A total of 99 b ladder cancer cases were observed in seven of 36 

### the cohorts, compared with 83.0 expected (pooled RR 1.29; CI 1.06–1.57). For kidney cancer, 37

### 80 DRAFT - DO NOT CITE OR QUOTE

40 cases were observed compared with 31.0 expected based on four studies (pooled RR 1.30; 1 

### 95% CI 0.95–1.77). 2 

### Xu et al. (1996) conducted a nested case-control st udy, surveying the cancer incidence 3 

### among 196,993 active or retired workers from the An shan Chinese iron and steel production 4 

### complex. A large number of historical benzo[a]pyre ne measurements (1956–1995) were 5 

### available. The study included 610 cases of lung ca ncer and 292 cases of stomach cancer, with 6 

### 959 matched controls from the workforce. After adj usting for nonoccupational risk factors such 7 

### as smoking and diet, significantly elevated risks f or lung cancer and stomach cancer were 8 

### identified for subjects employed for 15 or more yea rs, with ORs varying among job categories. 9 

### For either type of cancer, highest risks were seen among coke oven workers: lung cancer, OR = 10 

### 3.4 (CI 1.4–8.5); stomach cancer, OR = 5.4 (CI 1.8– 16.0). 11 

### There were significant trends for long-term cumulat ive benzo[a]pyrene exposure vs. lung 12 

### cancer (

p = 0.004) or stomach cancer ( p = 0.016) incidence. For cumulative total 13 

### benzo[a]pyrene exposures of < 0.84, 0.85-1.96, 1.97 -3.2 and ≥ 3.2 the ORs for lung cancer were 14 

### 1.1 (CI 0.8-1.7), 1.6 (CI 1.2-2.3), 1.6 (1.1-2.3) a nd 1.8 (CI 1.2-2.5). For cumulative total 15 

### benzo[a]pyrene exposures of < 0.84, 0.85-1.96, 1.97 -3.2 and ≥ 3.2 the ORs for stomach cancer 16 

### were 0.9 (CI 0.5-1.5), 1.7 (CI 1.1-2.6), 1.3 (0.8-2 .1) and 1.7 (CI 1.1-2.7). However, the 17 

### investigators noted that additional workplace air c ontaminants were measured, which might have 18 

### influenced the outcome. Of these, asbestos, silica , quartz, and iron oxide-containing dusts may 19 

### have been confounders. For lung cancers, cumulativ e exposures to total dust and silica dust both 20 

### showed significant dose-response trends ( p = 0.001 and 0.007, respectively), while for stomac h 21 

### cancer, only cumulative total dust exposure showed a marginally significant trend ( p = 0.061). 22 

### For cumulative total dust exposures of < 69, 69-279 , 280-882 and ≥ 883 mg/m 3 

### the ORs for lung 23 

### cancer were 1.4 (CI 1.2-1.9), 1.2 (CI 1.0-2.19), 1. 4 (CI 1.0-2.0) and 1.9 (CI 1.3-2.5), 24 

### respectively. For cumulative silica dust exposures of < 3.7, 3.7-10.39, 10.4-27.71 and ≥ 27.72 25 

### mg/m 3 

### the ORs for lung cancer were 1.7 (CI 1.2-2.4), 1.5 (CI 1.0-2.1), 1.5 (CI 1.0-2.1) and 1.8 26 

### (CI 1.2-2.5), respectively. For cumulative total d ust exposures of < 69, 69-279, 280-882 and ≥ 27 

### 883 mg/m 3 

### ORs for stomach cancer were 1.3 (CI 0.8-2.1), 14 ( CI 0.9-2.2), 12 (CI 0.8-1.9) and 28 

### 1.6 (CI 1.1-2.5), respectively. 29 

### Exposure-response data from studies of coke oven wo rkers in the United States have 30 

### often been used to derive quantitative risk estimat es for PAH mixtures, and for benzo[a]pyrene 31 

### as an indicator substance (Bostrom et al., 2002). However, there are numerous studies of coke 32 

### oven worker cohorts that do not provide estimates o f benzo[a]pyrene exposure. An overview of 33



### the results of these and other studies can be obtai ned from the review of Boffetta et al. (1997). 34

35



### 4.1.3.2.3. Cancer incidence in asphalt workers and roofers. 36 

### These groups encompass different types of work (asp halt paving vs. roofing) and also 37 

### different types of historical exposure that have ch anged from using PAH-rich coal tar pitch to the 38

### 81 DRAFT - DO NOT CITE OR QUOTE

use of bitumen or asphalt, both of which are rather low in PAHs due to their source (crude oil 1 

### refinery) and a special purification process. Incr eased risks for lung cancer were reported in 2 

### large cohorts of asphalt workers and roofers; evide nce for increased bladder cancer risk is weak 3 

### (Burstyn et al., 2007; Partanen and Boffetta, 1994; Chiazze et al., 1991; Hansen, 1991, 1989; 4 

### Hammond et al., 1976). In an analysis of pooled da ta from two cohorts of asphalt workers, 822 5 

### lung cancer cases were observed versus 730.7 expect ed (pooled RR 1.14; CI 1.07–1.22) (Bosetti 6 

### et al., 2007). In two cohorts of roofers, analysis of pooled data indicated that 138 lung cancer 7 

### cases were observed, compared with 91.9 expected (p ooled RR 1.51; CI 1.28–1.78) (Bosetti et 8 

### al., 2007). 9

10



### 4.1.3.3. Noncancer Disease Caused by Benzo[a]pyrene 11 

### Because accumulating evidence indicates that PAH ex posure is a risk factor for ischemic 12 

### heart disease (IHD), Burstyn et al. (2005) investig ated 418 cases of fatal IHD in a cohort of 13 

### 12,367 asphalt paving workers exposed to low-level PAH from bitumen and coal tar. The 14 

### follow-up started in 1953 and ended in 2000, with a n average exposure of 17 ± 9 years 15 

### (minimum: one work season), resulting in 193,889 p erson-years of observation. Previous 16 

### analyses of this cohort indicated no association be tween PAH exposure and excess mortality 17 

### from cancer or by all causes. Quantitative estimat es of exposure to benzo[a]pyrene were 18 

### obtained for paving operations on the basis of prev iously available personal exposure 19 

### measurements from workers in the asphalt industry ( but not necessarily from cohort members). 20 

### Exposures were calculated as average (0–68 [referen ce], 68–105, 106–146, 147–272, and 273+ 21 

### ng/m 3 

### ) and cumulative (0–189 [reference], 189–501, 502–9 31, 932–2,012, and 2,013+ ng/m 3



### - 22 

### years), respectively. 23 

### Cumulative and average exposure indices for benzo[a ]pyrene were positively associated 24 

### with mortality from IHD; the highest RR coincided w ith an average exposure to benzo[a]pyrene 25 

### of 273 ng/m 3 

### or higher (RR = 1.64; CI = 1.13–2.28). A similar risk was observed for the highest 26 

### cumulative benzo[a]pyrene exposure group (>2,013 ng /m 3 

### -years) (RR = 1.58; CI = 0.98–2.55). 27 

### Length of employment had no influence on this resul t. A dose-response was evident for IHD, 28 

### but not for other types of cardiovascular disease. The RR remained elevated even with 29 

### adjustment for smoking as a confounder; the RR was 1.24 under the extreme assumption of 0% 30 

### never smokers, 30% former smokers, and 70% current smokers in the highest-exposed group. 31 

### The authors discussed the possibility of bias becau se some of their study subjects might have 32 

### been exposed to other IHD-causing factors that were not controlled for in their study, or might 33



### have been misclassified. 34 

### An occupational study of Canadian aluminum smelter workers investigated the effect of 35 

### benzo[a]pyrene exposure on cardiopulmonary mortalit y (Friesen et al., 2010). Adjusted internal 36 

### comparisons for smoking were conducted using Cox re gression for male subjects (n = 6,423). 37 

### Ischemic heart disease (IHD) was associated with cu mulative benzo[a]pyrene exposure with a 38

### 82 DRAFT - DO NOT CITE OR QUOTE

hazard ratio of 1.62 (95% CI 1.06-2.46) in the high est benzo[a]pyrene exposure category. For 1 

### active employment, the hazard ratio for IHD was 2.3 9 (95% CI 0.95-6.05) in the highest 2 

### cumulative benzo[a]pyrene exposure category. 3 

### Other studies have reported potential prenatal effe cts and associated birth outcomes 4 

### induced by inhalation exposure to PAHs, including b enzo[a]pyrene. Perera et al. (2005a) studied 5 

### 329 nonsmoking pregnant women (30 ± 5 years old) po ssibly exposed to PAHs from fires during 6



### the 4 weeks after 09/11/2001. Maternal and umbilic al cord blood levels of benzo[a]pyrene 7 

### (BPDE)-DNA adducts were highest in study participan ts who lived within 1 mile of the WTC, 8



### with an inverse correlation between cord blood leve ls and distance from the WTC. Neither cord 9 

### blood adduct level nor ETS alone was positively cor related with adverse birth outcomes. 10 

### However, the interaction between ETS exposure and c ord blood adducts was significantly 11 

### associated with reduced birth weight and head circu mference. Among babies exposed to ETS in 12



### utero, a doubling of cord blood benzo[a]pyrene-DNA adducts was associated with an 8% 13 

### decrease in birth weight ( p = 0.03) and a 3% decrease in head circumference ( p = 0.04). 14 

### Perera et al. (2005b) compared various exposures—ET S, nutrition, pesticides, material 15 

### hardship—with birth outcomes (length, head circumfe rence, cognitive development). ETS 16 

### exposure and intake of PAH-rich foods by pregnant w omen were determined by questionnaire. 17 

### Levels of benzo[a]pyrene(BPDE)-DNA adducts were det ermined in umbilical cord blood 18 

### collected at delivery. The study population consis ted of Dominican or African-American 19 

### nonsmoking pregnant women (n = 529; 24 ± 5 years ol d) free of diabetes, hypertension, HIV, 20 

### and drug or alcohol abuse. Benzo[a]pyrene adducts, ETS, and dietary PAHs were not 21 

### significantly correlated with each other. However, the interaction between benzo[a]pyrene-DNA 22 

### adducts and ETS exposure was significantly associat ed with reduced birth weights (-6.8%; p = 23 

### 0.03) and reduced head circumference (-2.9%; p = 0.04). 24 

### Tang et al. (2006) measured benzo[a]pyrene(BPDE)-DN A adducts in maternal and 25 

### umbilical cord blood obtained at delivery from a co hort of 150 nonsmoking women and their 26 

### newborns in China. Exposure assessment was related to the seasonal operation of a local, coal27 

### fired power plant; however, airborne PAH concentrat ions were not measured. Dietary PAH 28



### intake was not included as a covariate because it d id not significantly contribute to the final 29 

### models, but ETS, sex, and maternal height and weigh t were considered as covariates. DNA 30 

### adduct levels were compared to several birth outcom es and physical development parameters, 31 

### such as gestational age at birth; infant sex, birth weight, length, head circumference, and 32 

### malformations; maternal height and pregnancy weight total weight gain; complications of 33 

### pregnancy and delivery; and medications used during pregnancy. 34 

### High cord blood adduct levels were significantly a ssociated with reduced infant/child 35 

### weight at 18 months ( β = -0.048, p = 0.03), 24 months ( β = -0.041, p = 0.027), and 30 months of 36 

### age ( β = -0.040, p = 0.049); decreased birth head circumference was ma rginally associated with 37 

### DNA adduct levels ( β = -0.011, p = 0.057). Maternal adduct levels were correlated n either with 38

### 83 DRAFT - DO NOT CITE OR QUOTE

cord blood adduct levels nor with fetal and child g rowth. Among female infants, cord blood 1 

### adduct levels were significantly associated with sm aller birth head circumference ( p = 0.022) and 2 

### with lower weight at 18 months ( p = 0.014), 24 months (

p = 0.012), and 30 months of age ( p = 3 

### 0.033), and with decreased body length at 18 months of age ( p = 0.033). Among male infants, 4 

### the corresponding associations were also inverse bu t statistically not significant. 5



### Considerable evidence of a deleterious effect of sm oking on male and female fertility has 6 

### accumulated from epidemiological studies of time to pregnancy, ovulatory disorders, semen 7 

### quality, and spontaneous abortion (reviewed in Wayl en et al., 2009; Cooper and Moley, 2008; 8 

### Soares and Melo, 2008). In addition, the effect of smoking, particularly during the time of the 9 

### perimenopausal transition, on acceleration of ovari an senescence (menopause) has also been 10 

### established (Midgette and Baron, 1990). More limit ed data is available pertaining specifically to 11 

### measures of benzo[a]pyrene and reproductive outcome s. 12 

### Neal et al. (2008, 2007) examined levels of benzo[ a]pyrene and other PAHs in follicular 13 

### fluid and serum sample from 36 women undergoing in vitro fertilization at a clinic in Toronto, 14 

### and compared the successful conception rate in rela tion to benzo[a]pyrene levels. The women 15 

### were classified by smoking status, with 19 who were current cigarette smokers, 7 with passive or 16 

### sidestream smoke exposure (i.e., non-smoker with a partner who smoked) and 10 non-smoked 17 

### exposed. An early follicular phase blood sample an d follicular fluid sample from the follicle at 18 

### the time of ovum retrieval were collected and analy zed for the presence of benzo[a]pyrene, 19 

### acenapthelene, phenanthrene, pyrene and chrysene us ing GC/MS (detection limit 5 pg/ml). The 20 

### frequency of non-dectable levels of serum benzo[a]p yrene was highest in the non-smoking group 21 

### (60.0%, 14.3%, and 21.0% below detection limit in n on-smoking, sidestream smoke, and active 22 

### smoking groups, respectively). A similar pattern w as seen with follicular fluid benzo[a]pyrene 23 

### (30.0%, 14.3%, and 10.5% below detection limit limi t in non-smoking, sidestream smoke, and 24 

### active smoking groups, respectively). In the analy ses comparing mean values across groups, an 25 

### assigned value of 0 was used for non-detectable sam ples. Follicular fluid benzo[a]pyrene levels 26 

### were higher in the active smoking group (mean ± SE, 1.32 ± 0.68 ng/ml) than in the sidestream 27 

### (0.05 ± 0.01 ng/ml) or non-smoking (0.03 ± 0.01 ng/ ml) groups (p = 0.04). The between-group 28 

### differences in serum benzo[a]pyrene levels were not statistically significant (0.22 ± 0.15, 0.98 ± 29 

### 0.56, and 0.40 ± 0.13 ng/ml in non-smoking, sidestr eam smoke, and active smoking groups, 30 

### respectively), and there were no differences in rel ation to smoking status. Among active 31 

### smokers, the number of cigarettes smoked per day wa s strongly correlated with follicular fluid 32 

### benzo[a]pyrene levels (r= 0.7, p < 0.01). Follicul ar fluid benzo[a]pyrene levels were 33 

### significantly higher among the women who did not co nceive (1.79 ng/ml ± 0.86) compared with 34 

### women who did get pregnant (mean approximately, 0.1 0 ng/ml, as estimated from graph) (p < 35 

### 0.001), but serum levels of benzo[a]pyrene were not associated with successful conception. 36 

### A small case-control study conducted between August 2005 and February 2006 in 37 

### Lucknow city (Uttar Pradesh), India examined PAH co ncentrations in placental tissues (Singh et 38

### 84 DRAFT - DO NOT CITE OR QUOTE

al., 2008) in relation to risk of preterm birth. Th e study included 29 cases (delivery between 28 1 

### and < 36 weeks gestation) and 31 term delivery cont rols. Demographic data smoking history, 2 

### reproductive history and other information were col lected by interview, and a 10 g sample of 3 

### placental tissue was collected form all participant s. Concentration of specific PAHs in placental 4 

### tissue was determined using HPLC. In addition to b enzo[a]pyrene, the PAHs assayed were 5 

### naphthalene, acenapththylene, phenanthrene, fluoren e, anthracene, benzo(a)anthracene, 6 

### fluoranthene, pyrene, beno(k)fluoranthene, benzo(b) fluoranthene, benzo(g,h,i)perylene, and 7 

### dibenzo(a,h)anthracene. PAH exposure in this popul ation was from environmental sources and 8 

### from cooking. The age of study participants ranged from 20 to 35 years. There was little 9 

### difference in birthweight between cases and control s (mean 2.77 and 2.75 in the case and control 10 

### groups, respectively). Placental benzo[a]pyrene le vels were lower than the levels of the other 11 

### PAHs detected (mean 8.83 ppb in controls for benzo [a]pyrene compared with 25-30 ppb for 12 

### anthracene, beno(k)fluoranthene, benzo(b)fluoranthe ne, and dibenzo(a,h)anthracene, 59 ppb for 13 

### acenaphthylene, and 200 – 380 naphthalene, phenanth rene, fluoranthene, and pyrene; non14 

### detectable levels of fluorine, benzo(a)anthracene, and benzo(g,h,i)perylene were found). There 15 

### was little difference in benzo[a]pyrene levels betw een cases (mean ± SE 13.85 ± 7.06 ppb) and 16 

### controls (8.83 ± 5.84 ppb), but elevated levels of fluoranthene (325.91 ± 45.14 and 208.6 ± 17 

### 21.93 ppb in cases and controls, p < 0.05) and benz o(b)fluoranthene (61.91 ± 12.43 and 23.84 ± 18 

### 7.01 ppb in cases and controls, p < 0.05) were seen

. 19 

### Wu et al. (2010) conducted a study of benzo[a]pyren e-DNA adduct levels in relation to 20 

### risk of fetal death in Tianjin, China. This case-c ontrol study included women who experienced a 21 

### missed abortion before 14 weeks gestational age, th at is a fetal death that remained in utero and 22 

### so required surgical intervention. Cases were matc hed by age and gravidity to controls (women 23 

### undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded 24 

### women who smoked, women with chronic disease and pr egnancy complications, and women 25 

### with occupational exposures to PAHs. Residency wit hin Tianjin for at least one year was also an 26 

### eligibility criterion. The participation rate was high: 81 of 84 eligible cases participanted and 81 27 

### of 89 eligible controls participated. Data pertaini ng to demographic characteristics, reproductive 28 

### history, and factors relating to potential PAH expo sure were collected using a structured 29 

### interview, and samples from the aborted tissue were obtained. In two of the four hospitals used 30 

### in the study, blood samples from the women (n=51 ca ses and 51 controls) were also collected. 31 

### The presence of benzo[a]pyrene-BPDE adducts was ass essed in the blood and tissue samples 32 

### using HPLC. There was no correlation between blood and aborted tissue levels of 33 

### benzo[a]pyrene adducts (r = –0.12 for the 102 blood -tissue pairs, r = –0.02 for the 51 case paris 34 

### and r = –0.21 for the 51 control pairs). (The authors noted that there was little difference 35 

### between women with and without blood samples in ter ms of the interview- based measures 36



### collected or in terms of the DNA-adduct levels in a borted tissue.) benzo[a]pyrene-adduct levels 37 

### were similar but slightly lower in the aborted tiss ue of cases compared with controls (mean ± SD 38

### 85 DRAFT - DO NOT CITE OR QUOTE

4.8 ± 6.0 in cases and 6.0 ± 7.4 in controls, p = 0 .29). In the blood samples, however, 1 

### benzo[a]pyrene-adduct levels were higher in cases ( 6.0 ± 4.7 and 2.7 ± 2.2 in cases and controls, 2 

### respectively, p < 0.001). In logistic regression a nalyses using a continuous adduct measure, the 3 

### OR was 1.35 (95% CI 1.11 – 1.64) per adduct/10 8 

### nucleotide. These results adjusted for 4 

### education and household income, but were very simil ar to the unadjusted results. Categorizing 5 

### exposure at the median value resulted in an adjuste d OR of 4.27 (95% CI 1.41 – 12.99) in the 6 

### high compared with low benzo[a]pyrene-adduct group. There was no relation between 7 

### benzo[a]pyrene-adduct levels in the aborted tissue and missed abortion in the logistic regression 8 

### analyses using either the continuous (adjusted OR 0 .97, 95% CI 0.93 – 1.02) or dichotomous 9 

### exposure measure (adjusted OR 0.76, 95% CI 0.37 – 1 .54). Associations between missed 10 

### abortion and several interview-based measures of po tential PAH exposure were also seen: 11 

### adjusted OR 3.07 (95% CI 1.31 – 7.16) for traffic c ongestion near residence, 3.52 (95% CI 1.44 12 

### – 8.57) for commuting by walking, 3.78 (95% CI 1.11 – 12.87) for routinely cooked during 13 

### pregnancy, and 3.21 (95% CI 0.98 – 10.48) for indus trial site or stack near residence, but there 14 

### was no association with other types of commuting (e .g., by bike, car, or bus). 15

16

17



### 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN 18 

### ANIMALS—ORAL, INHALATION, AND DERMAL 19



### 4.2.1. Oral 20



### 4.2.1.1. Subchronic Studies 21

22 

### De Jong et al. (1999) 35-day rat study 23 

### De Jong et al. (1999) treated male Wistar rats (eig ht/dose group) with benzo[a]pyrene 24 

### (98.6% purity) dissolved in soybean oil by gavage 5 days/week for 35 days at doses of 0, 3, 10, 25 

### 30, or 90 mg/kg-day (adjusted doses: 0, 2.14, 7.14 , 21.4, and 64.3 mg/kg-day). At the end of the 26 

### exposure period, rats were necropsied, organ weight s were determined, and major organs and 27 

### tissues were prepared for histological examination (adrenals, brain, bone marrow, colon, caecum, 28 

### jejunum, heart, kidney, liver, lung, lymph nodes, e sophagus, pituitary, spleen, stomach, testis, 29 

### and thymus). Blood was collected for examination o f hematological endpoints, but there was no 30 

### indication that serum biochemical parameters were a nalyzed. Immune parameters included 31 

### determinations of serum immunoglobulin levels (IgG, IgM, IgE, and IgA), relative spleen cell 32 

### distribution, and spontaneous cytotoxicity of splee n cell populations determined in a natural33 

### killer (NK) cell assay. 34 

### Body weight gain was decreased beginning at week 2 at the high dose of 90 mg/kg-day; 35 

### there was no effect at lower doses (De Jong et al., 1999). Hematology revealed a dose-related 36 

### decrease in RBC count, hemoglobin, and hematocrit a t ≥ 10 mg/kg-day (Table 4-2). A minimal 37 

### but significant increase in mean cell volume and a decrease in mean cell hemoglobin 38

### 86 DRAFT - DO NOT CITE OR QUOTE

concentration were noted at 90 mg/kg-day, and may i ndicate dose-related toxicity for the RBCs 1 

### and/or RBC precursors in the bone marrow. A decrea se in WBCs, attributed to a decrease in the 2 

### number of lymphocytes (approximately 50%) and eosin ophils (approximately 90%), was 3 

### observed at 90 mg/kg-day; however, there was no eff ect on the number of neutrophils or 4 

### monocytes. A decrease in the cell number in the bo ne marrow observed in the 90 mg/kg-day 5 

### dose group was consistent with the observed decreas e in the RBC and WBC counts at this dose 6 

### level. In the 90 mg/kg-day dose group, brain, hear t, kidney, and lymph node weights were 7 

### decreased and liver weight was increased (Table 4-2 ). Decreases in heart weight at 3 mg/kg-day 8 

### and in kidney weight at 3 and 30 mg/kg-day were als o observed, but these changes did not show 9 

### dose-dependent responses. Dose-related decreases i n thymus weight were statistically 10 

### significant at ≥ 10 mg/kg-day (Table 4-2). 11

12

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Table 4-2. Exposure-related effects in male Wistar rats exposed to benzo[a]pyrene by gavage 5 days/week for 5 weeks

Effect Dose (mg/kg-d)

0 3 10 30 90 Hematologic effects (mean ± SD; n = 7–8) White blood cells (10 9 /L) Red blood cells (10 9 /L) Hemoglobin (mmol/L) Hematocrit (L/L) 14.96 ± 1.9 8.7 ± 0.2 10.5 ± 0.2 0.5 ± 0.01 13.84 ± 3.0

8.6 ± 0.2 10.4 ± 0.3 0.5 ± 0.01 13.69 ± 1.8 a

8.3 ± 0.2 9.8 ± 0.2 a 0.47 ± 0.01 a 13.58 ± 2.9 a

7.8 ± 0.4 9.5 ± 0.4 a 0.46 ± 0.02 a 8.53 ± 1.1 a

7.1 ± 0.4 a 8.6 ± 0.6 a 0.43 ± 0.02 a Serum immunoglobulin levels (mean ± SD; n = 7–8) IgM IgG IgA IgE 100 ± 13 100 ± 40 100 ± 28 100 ± 65 87 ± 16

141 ± 106 73 ± 29 50 ± 20 86 ± 31

104 ± 28 78 ± 67 228 ± 351 67 ± 16 a

106 ± 19 72 ± 22 145 ± 176 81 ± 26

99 ± 29 39 ± 19 a 75 ± 55 Cellularity (mean ± SD; n = 7–8) Spleen (cell number ×10 7 ) Bone marrow (G/L) 59 ± 15 31 ± 7 71 ± 14

36 ± 5 59 ± 13

31 ± 8 63 ± 10

27 ± 8 41 ± 10 a

19 ± 4 a Spleen cell distribution (%) B cells T cells Th cells Ts cells 39± 4 40 ± 9 23 ± 7 24 ± 5 36 ± 2

48 ± 12 26 ± 7 26 ± 6 34 ± 3 a

40 ± 9 24 ± 5 24 ± 7 32 ± 4 a

36 ± 2 22 ± 4 19 ± 2 23 ± 4 a

44 ± 6 26 ± 4 27 ± 5 Body (g) and organ (mg) weights (means; n = 7–8) Body weight Brain Heart Kidney Liver Thymus Spleen Mandibular lymph nodes Mesenteric lymph nodes Popliteal lymph nodes 305 1,858 1,030 1,986 10,565 517 ± 47 551 152 165 19 282 a

1,864 934 a 1,761 a 9,567 472 ± 90 590 123 148 18 300

1,859 1,000 1,899 11,250 438 ± 64 a 538 160 130 a 19 293

1,784 967 1,790 a 11,118 388 ± 71 a 596 141 158 17 250 a

1,743 a 863 a 1,626 a 12,107 a 198 ± 65 a 505 89 a 107 a 10 a Thymus cortex surface area (% of total surface area of thymus; mean ± SD; n = 6–8) 77.9 ± 3.8 74.4 ± 2.2 79.2 ± 5.9 75.8 ± 4.0 68.9 ± 5.2 a

a Significantly (

p < 0.05) different from control mean. For body wei ght and organ weight means, SDs were only reported for thymus weights.

Source: De Jong et al. (1999).

1 

### Statistically significant reductions were also obse rved in the relative cortex surface area 2 

### of the thymus and thymic medullar weight at 90 mg/k g-day, but there was no difference in cell 3 

### proliferation between treated and control animals u sing the proliferating cell nuclear antigen 4 

### (PCNA) technique. Changes in the following immune parameters were noted: dose-related and 5 

### statistically significant decrease in the relative number of B cells in the spleen at 10 (13%), 30 6 

### (18%), and 90 mg/kg-day (41%); significant decrease s in absolute number of cells harvested in 7

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the spleen (31%), in the number of B cells in the s pleen (61%), and NK cell activity in the spleen 1 

### (E:T ratio was 40.9 ± 28.4% that of the controls) a t 90 mg/kg-day; and a decrease in serum IgM 2 

### (33%) and IgA (61%) in rats treated with 30 and 90 mg/kg-day, respectively. The decrease in 3 

### the spleen cell count was attributed by the study a uthors to the decreased B cells and suggested a 4 

### possible selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The study 5 

### authors considered the decrease in IgA and IgM to be due to impaired production of antibodies, 6 

### suggesting a role of thymus toxicity in the decreas ed (T-cell dependent) antibody production. In 7 

### addition to the effects on the thymus and spleen, h istopathologic examination revealed treatment8 

### related lesions only in the liver and forestomach a t the two highest dose levels, but the incidence 9 

### data for these lesions were not reported by De Jong et al. (1999). Increased incidence for 10 

### forestomach basal cell hyperplasia ( p < 0.05 by Fisher’s exact test) was reported at 30 and 11 

### 90 mg/kg-day, and increased incidence for oval cell hyperplasia in the liver was reported at 12 

### 90 mg/kg-day (

p < 0.01, Fisher’s exact test). The results indicat e that 3 mg/kg-day was a no13 

### observed-adverse-effect level (NOAEL) for effects o n hematological parameters (decrease in 14 

### RBC count, hemoglobin, and hematocrit) and immune p arameters (decreased thymus weight and 15 

### percent of B cells in the spleen) noted in Wistar r ats at 10 mg/kg-day (the lowest-observed16 

### adverse-effect level [LOAEL]) and above. Lesions o f the liver (oval cell hyperplasia) and 17 

### forestomach (basal cell hyperplasia) occurred at do ses ≥ 30 mg/kg-day. 18

19 

### Knuckles et al. (2001) 90-day rat study 20 

### Knuckles et al. (2001) exposed male and female F344 rats (20/sex/dose group) to 21 

### benzo[a]pyrene (98% purity) at doses of 0, 5, 50, o r 100 mg/kg-day in the diet for 90 days. Food 22 

### consumption and body weight were monitored, and the concentration of benzo[a]pyrene in the 23 

### food was adjusted every 3–4 days to maintain the ta rget dose. The authors indicated that actual 24 

### intake of benzo[a]pyrene by the rats was within 10% of the calculated intake, and the nominal 25



### doses were not corrected to actual doses. Hematolo gy and serum chemistry parameters were 26 

### evaluated. Urinalysis was also performed. Animals were examined for gross pathology, and 27 

### histopathology was performed on selected organs (st omach, liver, kidney, testes, and ovaries). 28 

### Statistically significant decreases in RBC counts a nd hematocrit level (decreases as much as 10 29 

### and 12%, respectively) were observed in males at do ses ≥ 50 mg/kg-day and in females at 30 

### 100 mg/kg-day. A maximum 12% decrease (statistical ly significant) in hemoglobin level was 31 

### noted in both sexes at 100 mg/kg-day. Blood chemis try analysis showed a significant increase in 32 

### blood urea nitrogen (BUN) only in high-dose (100 mg /kg-day) males. Histopathology 33 

### examination revealed an apparent increase in the in cidence of abnormal tubular casts in the 34 

### kidney in males at 5 mg/kg-day (40%), 50 mg/kg-day (80%) and 100 mg/kg-day (100%), 35 

### compared to 10% in the controls. Only 10% of the females showed si gnificant kidney tubular 36 

### changes at the two high dose levels compared to zer o animals in the female control group. The 37 

### casts were described as molds of distal nephron lum en and were considered by the study authors 38

### 89 DRAFT - DO NOT CITE OR QUOTE

to be indicative of renal dysfunction. From this s tudy, male F344 rats appeared to be affected 1 

### more severely by benzo[a]pyrene treatment than the female rats. However the statistical 2 

### significance of the kidney lesions are unclear. Se veral reporting gaps and inconsistencies 3 

### regarding the reporting of kidney abnormalities in Knuckles et al (2001) make interpretation of 4 

### the results difficult. Results of histopathologica l kidney abnormalities (characterized primarily 5 

### as kidney casts) were presented graphically and the data were not presented numerically in this 6 

### report. No indication was given in the graph that any groups are statistically different than 7 

### controls, though visual examination of the magnitud e of response and error bars appears to 8 

### indicate a four fold increase in kidney casts in ma les compared to the control group (40 9 

### compared to 10%). The figure legend reports the da ta as “percentage incidence of abnormal 10 

### kidney tissues” and reports values are mean plus or minus standard deviation. However, text 11



### under the materials and methods section states that for histopathological data, Fisher’s Exact Test 12 

### was used. This would involve the pairwise comparis on of incidence and not means. There are 13 

### additional internal inconsistencies in the data pre sented. Data appear to indicate that incidences 14 

### for males are as follows: control: 10%, 5mg/kg-day: 40%, 50 mg/kg-day: 80% and 100 mg/kg15 

### day: 100%, however, these incidences are inconsiste nt with the size of the study groups which 16 

### were reported as 6-8 animals per group. The study authors were contacted but did not respond to 17 

### EPA’s request for clarification of study design and /or results. Due to issues of data reporting, a 18



### LOAEL could not be established for the increased in cidence of kidney lesions. Based on the 19 

### statistically significant hematological effects inc luding decreases in RBC counts, hematocrit, and 20 

### BUN, the NOAEL in males was 5 mg/kg-day and the LOA EL was 50 mg/kg-day, based on in 21 

### F344 rats. No exposure-related histological lesion s were identified in the stomach, liver, testes, 22 

### or ovaries in this study. 23

24 

### Kroese et al. (2001) 5-week rat study 25 

### In a range-finding study, Wistar (specific pathogen -free [SPF] Riv:TOX) rats 26 

### (10/sex/dose group) were administered benzo[a]pyren e (97.7% purity) dissolved in soybean oil 27 

### by gavage at dose levels of 0, 1.5, 5, 15, or 50 mg /kg body weight, 5 days/week for 5 weeks 28 

### (Kroese et al., 2001). Behavior, clinical symptoms , body weight, and food and water 29 

### consumption were monitored. None of the animals di ed during the treatment period. Animals 30



### were sacrificed 24 hours after the last dose. Urin e and blood were collected for standard 31 

### urinalysis and hematology and clinical chemistry ev aluation. Liver enzyme induction was 32 

### monitored based on EROD activity in plasma. Animal s were subjected to macroscopic 33 

### examination, and organ weights were recorded. The esophagus, stomach, duodenum, liver, 34 

### kidneys, spleen, thymus, lung, and mammary gland (f emales only) from the highest-dose and 35 

### control animals were evaluated for histopathology. Intermediate dose-groups were examined if 36 

### abnormalities were observed in the higher-dose grou ps. 37

### 90 DRAFT - DO NOT CITE OR QUOTE

A significant, but not dose-dependent, increase in food consumption in males at 1



### ≥ 1.5 mg/kg-day and a decrease in females at ≥ 5 mg/kg-day was observed (Kroese et al., 2001). 2 

### Water consumption was statistically significantly a ltered in males only, a decrease at 1.5, 5, and 3 

### 15 mg/kg-day and an increase at 50 mg/kg-day. Orga n weights of lung, spleen, kidneys, 4 

### adrenals, and ovaries were not affected by treatmen t. There was a dose-related, statistically 5 

### significant decrease in thymus weight in males at 1 5 and 20 mg/kg-day (decreased by 28 and 6 

### 33%, respectively) and a significant decrease in fe males at 50 mg/kg-day (decreased by 17%) 7 

### (Table 4-3). In both sexes, liver weight was stati stically significantly increased only at 8 

### 50 mg/kg-day by about 18% (Table 4-3). 9

10 

### Table 4-3. Exposure-related effects in Wistar rats exposed to

benzo[a]pyrene by gavage 5 days/week for 5 weeks

Organ Dose (mg/kg)

0 1.5 5 15 50 Liver weight (g; mean ± SD) Males Females 6.10 ± 0.26 4.28 ± 0.11 6.19 ± 0.19

4.40 ± 0.73 6.13 ± 0.10

4.37 ± 0.11 6.30 ± 0.14

4.67 ± 0.17 7.20 ± 0.18 a

5.03 ± 0.15 a Thymus weight (mg; mean ± SD) Males Females 471 ± 19 326 ± 12 434 ± 20

367 ± 23 418 ± 26

351 ± 25 342 ± 20 a

317 ± 30 317 ± 21 a

271 ± 16 a Basal cell hyperplasia of the forestomach (incidence with slight severity) Males Females 1/10 0/10 1/10

1/10 4/10

1/10 3/10

3/10 a 7/10

7/10 a

a Significantly (

p < 0.05) different from control mean; n = 10/sex/gr oup.

Source: Kroese et al. (2001).

11 

### Hematological evaluation revealed only statisticall y nonsignificant, small dose-related 12 

### decreases in hemoglobin in both sexes, and RBC coun ts in males. Clinical chemistry analysis 13 

### showed a small, but statistically significant, incr ease in creatinine levels in males only at 14 

### 1.5 mg/kg-day, but this effect was not dose-depende nt. A dose-dependent induction of liver 15 

### microsomal EROD activity was observed, with a five- fold induction at 1.5 mg/kg-day compared 16 

### to controls, reaching 36-fold in males at 50 mg/kg- day; the fold induction in females at the top 17 

### dose was less than in males. At necropsy, signific ant, dose-dependent macroscopic findings 18



### were not observed. 19 

### Histopathology examination revealed a statistically significant increase in basal cell 20 

### hyperplasia in the forestomach of females at doses ≥ 15 mg/kg-day (Kroese et al., 2001). The 21 

### induction of liver microsomal EROD was not accompan ied by any adverse histopathologic 22 

### findings in the liver at the highest dose, 50 mg/kg -day, so the livers from intermediate-dose 23

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groups were, therefore, not examined. An increased incidence of brown pigmentation of red 1 

### pulp (hemosiderin) in the thymus was observed in tr eated animals of both sexes. However, this 2 

### tissue was not examined in intermediate-dose groups . This range-finding 5-week study 3 

### identified a NOAEL of 5 mg/kg-day and a LOAEL of 15 mg/kg-day, based on decreased thymus 4 

### weight and forestomach hyperplasia in Wistar rats. 5

6 

### Kroese et al. (2001) 90-day rat study 7 

### Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (10/sex/dose group) to 8 

### benzo[a]pyrene (98.6% purity, dissolved in soybean oil) by gavage at 0, 3, 10, or 30 mg/kg body 9 

### weight, 5 days/week for 90 days. The rats were exa mined daily for behavior and clinical 10 

### symptoms and by palpation. Food and water consumpt ion, body weights, morbidity, and 11 

### mortality were monitored. At the end of the exposu re period, rats were subjected to macroscopic 12 

### examination and organ weights were recorded. Blood was collected for hematology and serum 13 

### chemistry evaluation and urine was collected for ur inalysis. All gross abnormalities, particularly 14 

### masses and lesions suspected of being tumors were a lso evaluated. The liver, stomach, 15 

### esophagus, thymus, lung, spleen, and mesenteric lym ph node were examined histopathologically. 16 

### In addition, cell proliferation in forestomach epit helium was measured as the prevalence of S17 

### phase epithelial cells displaying bromodeoxyuridine (BrdU) incorporation. 18



### There were no obvious effects on behavior of the an imals, and no difference was 19 

### observed in survival or food consumption between ex posed animals and controls (Kroese et al. 20 

### 2001). Higher water consumption and slightly lower body weights than the controls were 21 

### observed in males but not females at the high dose of 30 mg/kg-day. Hematological 22 

### investigations showed only nonsignificant, small do se-related decreases in RBC count and 23 

### hemoglobin level in both sexes. Clinical chemistry evaluation did not show any treatment24 

### related group differences or dose-response relation ships for alanine aminotransferase (ALT), 25 

### serum aspartate transaminase (AST), lactate dehydro genase (LDH), or creatinine, but a small 26



### dose-related decrease in γ -glutamyl transferase (GGT) activity was observed i n males only. 27 

### Urinalysis revealed an increase in urine volume in males at 30 mg/kg-day, which was not dose 28 

### related. At the highest dose, both sexes showed in creased levels of urinary creatinine and a dose29 

### related increase in urinary protein. However, no f urther investigation was conducted to 30 

### determine the underlying mechanisms for these chang es. At necropsy, reddish to brown/gray 31 

### discoloration of the mandibular lymph nodes was con sistently noted in most rats; occasional 32 

### discoloration was also observed in other regional l ymph nodes (axillary). Statistically significant 33 

### increases in liver weight were observed at 10 and 3 0 mg/kg-day in males only (15 and 29% ) and 34 

### a decrease in thymus weight in both sexes only at 3 0 mg/kg-day (17 and 33% decrease in 35 

### females and males, respectively, compared with cont rols) (Table 4-4). At 10 mg/kg-day, thymus 36 

### weight in males was decreased by 15%, but the decre ase did not reach statistical significance. 37

38

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Table 4-4. Means ± SD a 

### for liver and thymus weights in Wistar rats expose d to benzo[a]pyrene by gavage 5 days/week for 90 days

Organ Dose (mg/kg-d)

0 3 10 30 Liver weight (g) Males Females 7.49 ± 0.97 5.54 ± 0.70 8.00 ± 0.85

5.42 ± 0.76 8.62 ± 1.30 b

5.76 ± 0.71 9.67 ± 1.17 b

6.48 ± 0.78 b

Thymus weight (mg) Males Females 380 ± 60 320 ± 60 380 ± 110

310 ± 50 330 ± 60

300 ± 40 270 ± 40 b

230 ± 30 b

a Reported as SE, but judged to be SD (and confirmed by study authors).

b Significantly (

p < 0.05) different from control mean; student t-tes t (unpaired, two-tailed); n = 10/sex/group.

Source: Kroese et al. (2001).

1 

### Histopathologic examination revealed what was chara cterized by Kroese et al. (2001) as 2 

### basal cell disturbance in the epithelium of the for estomach in males ( p < 0.05) and females 3 

### ( p < 0.01) at 30 mg/kg-day. The basal cell disturban ce was characterized by increased number of 4 

### basal cells, mitotic figures, and remnants of necro tic cells; occasionally early nodule 5 

### development; infiltration by inflammatory cells (ma inly histiocytes); and capillary hyperemia, 6 

### often in combination with the previous changes (Kro ese et al., 2001). Incidences for these 7 

### lesions (also described as “slight basal cell hyper plasia”) in the 0, 3, 10, and 30-mg/kg-day 8 

### groups were 0/10, 2/10, 3/10, and 7/10 in female ra ts and 2/10, 0/10, 6/10, and 7/10 for male rats. 9 

### Nodular hyperplasia was noted in one animal of eith er sex at 30 mg/kg-day. A significant 10 

### ( p < 0.05) increase in proliferation of forestomach ep ithelial cells was detected at doses 11



### ≥ 10 mg/kg-day by morphometric of analysis of nuclei with BrdU incorporation. The mean 12 

### numbers of BrdU-staining nuclei per unit surface ar ea of the underlying lamina muscularis 13 

### mucosa were increased by about two- and three- to f our-fold at 10 and 30 mg/kg-day, 14 

### respectively, compared with controls. A reduction of thymus weight and increase in the 15 

### incidence of thymus atrophy (the report described t he atrophy as slight, but did not specify the 16 

### full severity scale used in the pathology examinati on) was observed in males only at 30 mg/kg17 

### day (

p < 0.01 compared with controls). Incidences for th ymus atrophy for the control through 18 

### high-dose groups were 0/10, 0/10, 0/10, and 3/10 fo r females and 0/10, 2/10, 1/10, and 6/10 for 19 

### males. No significant differences were observed in the lungs of control and treated animals. In 20 

### the esophagus, degeneration and regeneration of mus cle fibers and focal inflammation of the 21 

### muscular wall were judged to be a result of the gav age dosing rather than of benzo[a]pyrene 22



### treatment. 23 

### The target organs of benzo[a]pyrene toxicity in thi s 90-day dietary study of Wistar rats 24 

### were the forestomach, thymus, and liver. The LOAEL for forestomach hyperplasia, decreased 25

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thymus weight, thymus atrophy, and increased liver weight was 30 mg/kg-day and the NOAEL 1 

### was 10 mg/kg-day. 2

3



### 4.2.1.2. Chronic Studies and Cancer Bioassays 4

5 

### Kroese et al. (2001) 2-year rat study 6 

### Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (52/sex/dose group) to 7 

### benzo[a]pyrene (98.6% purity) in soybean oil by gav age at nominal doses of 0, 3, 10, or 8 

### 30 mg/kg-day, 5 days/week, for 104 weeks. Mean ach ieved dose levels were 0, 2.9, 9.6, and 9 

### 29 mg/kg-day. Additional rats (6/sex/group) were s acrificed after 4 and 5 months of exposure 10 

### for analysis of DNA adduct formation in blood and m ajor organs and tissues. The rats were 11 

### 6 weeks old at the start of exposure. The rats wer e examined daily for behavior and clinical 12 

### symptoms and by palpation. Food and water consumpt ion, body weights, morbidity, and 13 

### mortality were monitored during the study. Complet e necropsy was performed on all animals 14 

### that died during the course of the study, were foun d moribund, or at terminal sacrifice (organ 15 

### weight measurement was not mentioned in the report by Kroese et al., 2001). The organs and 16 

### tissues collected and prepared for microscopic exam ination included: brain, pituitary, heart, 17 

### thyroid, salivary glands, lungs, stomach, oesophagu s, duodenum, jejunum, ileum, caecum, colon, 18 

### rectum, thymus, kidneys, urinary bladder, spleen, l ymph nodes, liver pancreas, adrenals, sciatic 19 

### nerve, nasal cavity, femur, skin including mammary tissue, ovaries/uterus, and testis/accessory 20 

### sex glands. Some of these tissues were examined on ly when gross abnormalities were detected. 21 

### All gross abnormalities, particularly masses and le sions that appeared to be tumors, were also 22



### examined. 23 

### At 104 weeks, survival in the control group was 65% (males) and 50% (females), 24 

### whereas mortality in the 30 mg/kg-day dose group wa s 100% after about week 70. At 80 weeks, 25 

### survival percentages were about 90, 85 and 75% in f emale rats in the 0, 3, and 10 mg/kg-day 26 

### groups, respectively; in males, respective survival percentages were about 95, 90, and 85% at 27



### 80 weeks. Survival of 50% of animals occurred at 1 04, 104, about 90, and 60 weeks for control 28 

### through high-dose females; for males, the respectiv e times associated with 65% survival were 29 

### 104, 104, 104, and about 60 weeks. The high mortal ity rate in high-dose rats was attributed to 30 

### liver or forestomach tumor development, not to nonc ancer systemic effects. After 20 weeks, 31 

### body weight was decreased (compared with controls b y >10%) in 30-mg/kg-day males, but not 32 

### in females. This decrease was accompanied by a dec rease in food consumption. Body weights 33 

### and food consumption were not adversely affected in the other dose groups compared to 34 

### controls. In males, there was a dose-dependent inc rease in water consumption starting at week 35 

### 13, but benzo[a]pyrene treatment had no significant effects on water consumption in females. 36 

### Tumors were detected at significantly elevated inci dences at several tissue sites in female 37



### and male rats at doses ≥ 10 and ≥ 3 mg/kg-day, respectively (Table 4-4; Kroese et al. , 2001). The 38

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tissue sites with the highest incidences of tumors were the liver (hepatocellular adenoma and 1 

### carcinoma) and forestomach (squamous cell papilloma and carcinoma) in both sexes (Table 4-4). 2



### The first liver tumors were detected in week 35 in high-dose male rats. Liver tumors were 3 

### described as complex, with a considerable proportio n (59/150 tumors) metastasizing to the lungs. 4 

### At the highest dose level, 95% of rats with liver t umors had malignant carcinomas (95/100; 5 

### Table 4-4). Forestomach tumors were associated wit h the basal cell proliferation observed 6 

### (without diffuse hyperplasia) in the forestomach of rats in the preliminary range-finding and 7 

### 90-day exposure studies described previously in Sec tion 4.2.1. At the highest dose level, 59% of 8 

### rats with forestomach tumors had malignant carcinom as (60/102; Table 4-4). Other tissue sites 9 

### with distinctly elevated incidences of tumors in th e 30 mg/kg-day dose group included: the oral 10 

### cavity (papilloma and squamous cell carcinoma [SCC] ) in both sexes, and the jejunum 11 

### (adenocarcinoma), kidney (cortical adenoma) and ski n (basal cell adenoma and carcinoma) in 12 

### male rats (Table 4-4). In addition, auditory canal tumors (carcinoma or squamous cell papilloma 13 

### originating from pilo-sebaceous units including the Zymbal’s gland) were also detected in both 14 

### sexes at 30 mg/kg-day, but auditory canal tissue wa s not histologically examined in the lower 15 

### dose groups and the controls (Table 4-4). Gross ex amination revealed auditory canal tumors 16 

### only in the high-dose group. 17

18

19

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Table 4-5. Incidences of exposure-related neoplasm s in Wistar rats treated by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks

Dose (mg/kg-d)

0 3 10 30 b

Site Females a Oral cavity Papilloma SCC Basal cell adenoma Sebaceous cell carcinoma 0/19 1/19 0/19 0/19 0/21

0/21 0/21 0/21 0/9

0/9 1/9 0/9 9/31 c

9/31 c 4/31 1/31 Oesophagus Sarcoma undifferentiated Rhabdomyosarcoma Fibrosarcoma 0/52 0/52 0/52 0/52

1/52 0/52 2/52

4/52 3/52 0/52

0/52 0/52

Forestomach Squamous cell papilloma SCC 1/52 0/52 3/51

3/51 20/51 c

10/51 c 25/52 c

25/52 c

Liver Hepatocellular adenoma Hepatocellular carcinoma Cholangiocarcinoma Anaplastic carcinoma 0/52 0/52 0/52 0/52 2/52

0/52 0/52 0/52 7/52 c

32/52 c 1/52 1/52 1/52

50/52 c 0/52 0/52 Auditory canal Benign tumor Squamous cell papilloma Carcinoma 0/0 0/0 0/0 0/0

0/1 0/1 0/0

0/0 0/0 1/20

1/20 13/20 c

Males a Oral cavity Papilloma Squamouse cell carcinoma Basal cell adenoma Sebaceous cell carcinoma 0/24 1/24 0/24 0/24 0/24

0/24 0/24 0/24 2/37

5/37 0/37 0/37 10/38 c

11/38 c 2/38 2/38

Forestomach Squamous cell papilloma SCC 0/52 0/52 7/52 c

1/52 18/52 c

25/52 c 17/52 c

35/52 c Jejunum Adenocarcinoma 0/51 0/50 1/51 8/49 c

Liver Hepatocellular adenoma Hepatocellular carcinoma Cholangiocarcinoma 0/52 0/52 0/52 3/52

1/52 0/52 15/52 c

23/52 c 0/52 4/52

45/52 c 1/52 Kidney Cortical adenoma Adenocarcinoma Urothelial carcinoma 0/52 0/52 0/52 0/52

0/52 0/52 7/52 c

2/52 0/52 8/52 c

0/52 3/52 Auditory canal Benign Squamous cell papilloma Carcinoma Sebaceous cell adenoma 0/1 0/1 0/1 0/1 0/0

0/0 0/0 0/0 1/7

0/7 2/7 0/7 0/33

4/33 19/33 c 1/33

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Table 4-5. Incidences of exposure-related neoplasm s in Wistar rats treated by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks

Dose (mg/kg-d)

0 3 10 30 b Skin and mammary Basal cell adenoma Basal cell carcinoma SCC Keratoacanthoma Trichoepithelioma Fibrosarcoma Fibrous histiocytoma (malignant) 2/52 1/52 0/52 1/52 0/52 0/52 0/52 0/52

1/52 1/52 0/52 1/52 3/52 0/52 1/52

0/52 1/52 1/52 2/52 5/52 1/52 10/51 c

4/51 5/51 4/51 8/51 c 0/51 1/52

a Incidences are for number of rats with tumors compa red with number of tissues examined histologically. Auditory canal and oral cavity tissues were only examined hi stologically when abnormalities were observed upon macroscopic examination.

b This group had significantly decreased survival.

c Statistically significant difference ( p ≤ 0.01), Fisher’s exact test; analysis of auditory c anal tumor incidence was based on assumption of n = 52 and no tumors in the controls.

Source: Kroese et al. (2001).

1 

### Kroese et al. (2001) did not systematically investi gate nonneoplastic lesions detected in 2 

### rats sacrificed during the 2-year study, because th e focus was to identify and quantitate tumor 3 

### occurrence. However, incidences were reported for nonneoplastic lesions in tissues or organs in 4 

### which tumors were detected (i.e., oral cavity, oeso phagus, forestomach, jejunum, liver, kidney, 5 

### skin, mammary, and auditory canal). The reported n onneoplastic lesions associated with 6 

### exposure were the forestomach basal cell hyperplasi a and clear cell foci of cellular alteration in 7



### the liver. Incidences for forestomach basal cell h yperplasia in the control through high-dose 8 

### groups were: 1/52, 8/51, 13/51, and 2/52 for femal es and 2/50, 8/52, 8/52, and 0/52 in males. 9 

### Incidences for hepatic clear cell foci of cellular alteration were 22/52, 33/52, 4/52, and 2/52 for 10 

### females and 8/52, 22/52, 1/52, and 1/52 for males. These results indicate that the lowest dose 11 

### group, 3 mg/kg-day, was a LOAEL for increased incid ence of forestomach hyperplasia and 12 

### hepatic histological changes in male and female Wis tar rats exposed by gavage to 13 

### benzo[a]pyrene for up to 104 weeks. The lack of an increase in incidence of these nonneoplastic 14



### lesions in the forestomach and liver at the interme diate and high doses (compared with controls) 15 

### may be associated with increased incidences of fore stomach and liver tumors at these dose levels 16 

### (see Table 4-4). 17 

### As an adjunct study to the 2-year gavage study with Wistar rats, Kroese et al. (2001) 18 

### sacrificed additional rats (6/sex/group) after 4 an d 5 months of exposure (0, 1, 3, 10, or 19 

### 30 mg/kg-day) for analysis of DNA adduct formation in WBCs and major organs and tissues. 20 

### Additional rats (6/sex/time period) were exposed to 0.1 mg/kg-day benzo[a]pyrene for 4 and 21 

### 5 months for analysis of DNA adduct formation. Usi ng the [ 32 

### P]-postlabeling technique, five 22

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benzo[a]pyrene-DNA adducts were identified in all o f the examined tissues at 4 months (WBCs, 1 

### liver, kidney, heart, lung, skin, forestomach, glan dular stomach, brain). Only one of these 2 

### adducts (adduct 2) was identified based on co-chrom atography with a standard. This adduct, 3



### identified as dG-N 2 

### -BPDE, was the predominant adduct in all organs of female rats exposed to 4 

### 10 mg/kg-day, except the liver and kidney, in which another adduct (unidentified adduct 4) was 5 

### predominant. Levels of total adducts (number of be nzo[a]pyrene-DNA adducts per 10 10 6 

### nucleotides) in examined tissues (from the single 1 0 mg/kg-day female rat) showed the following 7 

### order: liver > heart > kidney > lung > skin > fore stomach ≈ WBCs > brain. Mean values for 8 

### female levels of total benzo[a]pyrene-DNA adducts ( number per 10 10 

### nucleotides) in four organs 9 

### showed the same order, regardless of exposure group : liver > lung > forestomach ≈ WBCs; 10 

### comparable data for males were not reported). Mean total benzo[a]pyrene-DNA adduct levels in 11



### livers increased in both sexes from about 100 adduc ts per 10 10 

### nucleotides at 0.1 mg/kg-day to 12 

### about 70,000 adducts per 10 10 

### nucleotides at 30 mg/kg-day. In summary, these re sults suggest 13 

### that total benzo[a]pyrene-DNA adduct levels in tiss ues at 4 months were not independently 14 

### associated with the carcinogenic responses noted af ter 2 years of exposure to benzo[a]pyrene. 15 

### The liver showed the highest total DNA adduct level s and a carcinogenic response, but total 16 

### DNA adduct levels in heart, kidney, and lung (in wh ich no carcinogenic responses were 17 

### detected) were higher than levels in forestomach an d skin (in which carcinogenic responses were 18 

### detected). 19

20 

### Brune et al. (1981) 2-year rat study 21 

### Groups of Sprague-Dawley rats (32/sex/dose) were fe d diets delivering a daily dose of 22 

### 0.15 mg benzo[a]pyrene/kg body weight every 9 th 

### day or 5 times/week (Brune et al., 1981). 23 

### Other groups (32/ sex/dose) were given gavage doses of 0.15 mg benzo[a]pyrene (in aqueous 24 

### 1.5% caffeine solution)/kg every 9 th 

### day, every 3 rd 

### day, or 5 times/week. The study included an 25 

### untreated control group (to compare with the dietar y exposed groups) and a gavage vehicle 26 

### control group (each with 32 rats/sex). Rats were t reated until moribundity or death occurred, 27 

### with average annual doses are reported in Table 4-2 (mg/kg-year, calculated by Brune et al. 28 

### [1981]). The following tissues were prepared for h istopathological examination: tongue, larynx, 29 

### lung, heart, trachea, esophagus, stomach, small int estine, colon, rectum, spleen, liver, urinary 30 

### bladder, kidney, adrenal gland, and any tissues sho wing tumors or other gross changes. Survival 31 

### was similar among the groups, with the exception th at the highest gavage-exposure group 32 

### showed a decreased median time of survival (Table 4 -5). Increased incidences of portal-of-entry 33 

### tumors (forestomach, esophagus, and larynx) were ob served in all of the gavage-exposed groups 34 

### and in the highest dietary exposure group (Table 4- 5). Following dietary administration, all 35 

### observed tumors were papillomas. Following gavage administration, two malignant forestomach 36 

### tumors were found (one each in the mid- and high-do se groups) and the remaining tumors were 37 

### benign. The data in Table 4-5 show that the carcin ogenic response to benzo[a]pyrene was 38

### 98 DRAFT - DO NOT CITE OR QUOTE

stronger with the gavage protocol compared with die tary exposure, and that no distinct difference 1 

### in response was apparent between the sexes. Tumors at distant sites (mammary gland, kidney, 2 

### pancreas, lung, urinary bladder, testes, hematopoie tic, and soft tissue) were not considered 3 

### treatment-related as they were also observed at sim ilar rates in the control group (data not 4 

### provided). The study report did not address noncan cer systemic effects. 5

6 

### Table 4-6. Incidences of alimentary tract tumors i n Sprague-Dawley rats

chronically exposed to benzo[a]pyrene in the diet o r by gavage in caffeine solution

Average annual dose (mg/kg-yr) Estimated average daily dose a (mg/kg-d) Forestomach tumors b Total alimentary tract

tumors c (larynx, esophagus, forestomach) Median survival time (wks) benzo[a]pyrene by gavage in 1.5% caffeine solution 0 0 3/64 (4.7%) 6/64 (9.4%) 102 6 0.016 12/64 (18.8%) d 13/64 (20.3%) 112 18 0.049 26/64 (40.1%) e 26/64 (40.6%) 113 39 0.107 14/64 (21.9%) e 14/64 (21.9%) 87 benzo[a]pyrene in diet 0 0 2/64 (3.1%) 3/64 (4.7%) 129 6 0.016 1/64 (1.6%) 3/64 (4.7%) 128 39 0.107 9/64 (14.1%) d 10/64 (15.6%) 131

a Average annual dose divided by 365 d.

b No sex-specific forestomach tumor incidence data we re reported by Brune et al. (1981).

c Sex-specific incidences for total alimentary tract tumors were reported as follows: Gavage (control – high dose): Male: 6/ 32, 7/32, 15/32, 8/32 Female: 0/32, 6/32, 11/32, 6/32 Diet (control – high dose): Male: 3/32, 3/32, 8/32 Female: 0/32, 0/32, 2/32

d Significantly (

p < 0.1) different from control using a modified χ 2 test that accounted for group differences in survival time.

e Significantly (

p < 0.05) different from control using a modified χ 2 test that accounted for group differences in survival time.

Source: Brune et al. (1981).

7 

### Beland and Culp (1998; Culp et al., 1998) 2-year mo use study 8 

### In the other modern cancer bioassay with benzo[a]py rene, female B6C3F 1 

### mice (48/dose 9 

### group) were administered benzo[a]pyrene (98.5% puri ty) at concentrations of 0 (acetone 10 

### vehicle), 5, 25, or 100 ppm in the diet for 2 years (Beland and Culp, 1998; Culp et al., 1998). 11 

### This study was designed to compare the carcinogenic ity of coal tar mixtures with that of 12 

### benzo[a]pyrene and included groups of mice fed diet s containing one of several concentrations 13 

### of two coal tar mixtures. Benzo[a]pyrene was disso lved in acetone before mixing with the feed. 14 

### Control mice received only acetone-treated feed. F emale mice were chosen because they have a 15 

### lower background incidence of lung tumors than male B6C3F 1 

### mice. Culp et al. (1998) reported 16

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that the average daily intakes of benzo[a]pyrene in the 25- and 100-ppm groups were 104 and 1



### 430 μ g/day, but did not report intakes for the 5-ppm gro up. Based on the assumption that daily 2 

### benzo[a]pyrene intake at 5 ppm was one-fifth of the 25-ppm intake (about 21 μ g/day), average 3 

### daily doses for the three benzo[a]pyrene groups are estimated at 0.7, 3.3, and 16.5 mg/kg-day. 4 

### Estimated doses were calculated using time-weighted average (TWA) body weights of 0.032 kg 5 

### for the control, 5- and 25-ppm groups and 0.026 kg for the 100-ppm group (estimated from 6 

### graphically presented data). Food consumption, bod y weights, morbidity, and mortality were 7 

### monitored at intervals, and lung, kidneys, and live r were weighed at sacrifice. Necropsy was 8 

### performed on all mice that died during the experime nt or survived to the end of the study period. 9 

### Limited histopathologic examinations (liver, lung, small intestine, stomach, tongue, esophagus) 10 

### were performed on all control and high-dose mice an d on all mice that died during the 11 

### experimental period, regardless of treatment group. In addition, all gross lesions found in mice 12 

### of the low- and mid- dose groups were examined hist opathologically. 13 

### None of the mice administered 100 ppm benzo[a]pyren e survived to the end of the study, 14 

### and morbidity/mortality was 100% by week 78. Decre ased survival was also observed at 25 ppm 15 

### with only 27% survival at 104 weeks, compared with 56 and 60%, in the 5-ppm and control 16 

### groups, respectively. In the mid- and high-dose gr oup, 60% of mice were alive at about 90 and 17 

### 60 weeks, respectively. Early deaths in exposed mi ce were attributed to tumor formation rather 18 

### than other causes of systemic toxicity. Food consu mption was not statistically different in 19 

### benzo[a]pyrene-exposed and control mice. Body weig hts of mice fed 100 ppm were similar to 20 

### those of the other treated and control groups up to week 46, and after approximately 52 weeks, 21 

### body weights were reduced in 100-ppm mice compared with controls. Body weights for the 5- 22 

### and 25-ppm groups were similar to controls througho ut the treatment period. Compared with the 23 

### control group, no differences in liver, kidney, or lung weights were evident in any of the treated 24 

### groups (other organ weights were not measured). 25 

### Papillomas and/or carcinomas of the forestomach, es ophagus, tongue, and larynx at 26 

### elevated incidences occurred in groups of mice expo sed to 25 or 100 ppm, but no exposure27 

### related tumors occurred in the liver or lung (Table 4-6; Beland and Culp [1998]; Culp et al. 28 

### [1998]). The forestomach was the most sensitive ti ssue, and demonstrated the highest tumor 29 

### incidence among the examined tissues and was the on ly tissue with an elevated incidence of 30 

### tumors at 25 ppm (Table 4-6). In addition, most of the forestomach tumors in the exposed 31 

### groups were carcinomas, as 1, 31, and 45 mice had f orestomach carcinomas in the 5-, 25-, and 32 

### 100-ppm groups respectively. Nonneoplastic lesions were also found in the forestomach at 33 

### significantly (

p < 0.05) elevated incidences: hyperplasia at ≥ 5 ppm and hyperkeratosis at ≥ 25 34 

### ppm (Table 4-6). The esophagus was the only other examined tissue showing elevated incidence 35 

### of a nonneoplastic lesion (basal cell hyperplasia, see Table 4-6). Tumors (papillomas and 36 

### carcinonas) were also significantly elevated in the esophagus and tongue at 100 ppm (Table 4-6). 37 

### Esophogeal carcinomas were detected in 1 mouse at 2 5 ppm and in 11 mice at 100 ppm. Tongue 38

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carcinomas were detected in seven 100-ppm mice; the remaining tongue tumors were 1 

### papillomas. Although incidences of tumors of the l arynx were not significantly elevated in any 2 

### of the exposed groups, a significant dose-related t rend was apparent (Table 4-6). 3

4 

### Table 4-7. Incidence of nonneoplastic and neoplast ic lesions in female

B6C3F 1 

### mice fed benzo[a]pyrene in the diet for up to 2 ye ars

Tissue and lesion Incidence (%) benzo[a]pyrene concentration (ppm) in diet a

0 5 25 100 Average daily doses (mg/kg-d)

0 0.7 3.3 16.5 Liver (hepatocellular adenoma) 2/48 (2) 7/48

(15) 5/47

(11) 0/45

(0) Lung (alveolar/bronchiolar adenoma and/or carcinoma ) 5/48 (10) 0/48

(0) 4/45

(9) 0/48

(0) Forestomach (papilloma and/or carcinoma) b 1/48 (2) 3/47

(6) 36/46 b

(78) 46/47 b

(98) Forestomach (hyperplasia) b 13/48 (27) 23/47 a

(49) 33/46 b

(72) 37/47 b

(79) Forestomach (hyperkeratosis) b 13/48 (27) 22/47

(47) 33/46 b

(72) 38/47 b

(81) Esophagus (papilloma and/or carcinoma) b 0/48 (0) 0/48

(0) 2/45

(0) 27/46 b

(59) Esophagus (basal cell hyperplasia) b 1/48 (2) 0/48

(0) 5/45

(11) 30/46 b

(65) Tongue (papilloma and/or carcinoma) b 0/49 (0) 0/48

(0) 2/46

(4) 23/48 b

(48) Larynx (papilloma and/or carcinoma) b 0/35 (0) 0/35

(0) 3/34

(9) 5/38

(13)

a Significant (

p < 0.05) dose-related trend calculated for incidenc es of these lesions.

b Significantly different from control incidence ( p < 0.05); using a modified Bonferonni procedure for multiple comparisons to the same control.

Source: Beland and Culp (1998); Culp et al. (1998)

. 5 

### Neal and Rigdon (1967; Rigdon and Neal 1969, 1966) mouse study 6 

### Neal and Rigdon (1967) fed BAP (purity not reported ) at concentrations of 0, 1, 10, 20, 7 

### 30, 40, 45, 50, 100 and 250 ppm in the diets of mal e and female CFW-Swiss mice. 8 

### Corresponding doses (in mg/kg-day) were calculated 1 

### as 0, 0.2, 1.8, 3.6, 5.3, 7.1, 8, 8.9, 17.8, 9 

### 44.4 mg/kg-day. The age of the mice ranged from 17- 180 days old and the treatment time from 10 

### 1-197 days; the size of the treated groups ranged f rom 9 to 73. There were 289 mice (number of 11

1 Calculation: mg/kg-day = (ppm in feed x kg food/da y)/kg body weight. Reference food consumption rate s of 0.0062 kg/day (males) and 0.0056 kg/day (females) a nd reference body weights of 0.0356 kg (males) and 0.0305 (females) were used (U.S. EPA, 1988) and resulting doses were averaged between males and females.

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mice/sex not stated) in the control group. No fores tomach tumors were reported in the 0-, 0.2- 1 

### and 1.8 mg/kg-day dose groups. The incidence of for estomach tumors in the 20-, 30-, 40-, 45-, 2 

### 50-, 100- and 250-ppm dose groups (3.6, 5.3, 7.1, 8 , 8.9, 17.8, 44.4 mg/kg-day) were 1/23, 0/37, 3 

### 1/40, 4/40, 23/34, 19/23 and 66/73, respectively. 4

5 

### Other oral exposure cancer bioassays in mice 6 

### Numerous other oral exposure cancer bioassays in mi ce have limitations which restrict 7 

### their usefulness for characterizing dose-response r elationships between chronic-duration oral 8 

### exposure to benzo[a]pyrene and noncancer effects or cancer, but collectively, they provide strong 9 

### evidence that oral exposure to benzo[a]pyrene can c ause portal-of-entry site tumors (see Table 4- 10 

### 8 for references). 11

12102 DRAFT - DO NOT CITE OR QUOTE1Table 4-8. Tumor incidence in oral exposure rodentcancer bioassays with limitations for describing dose-responserelationships for lifetime exposure to benzo[a]pyreneSpecies/strainExposure Results Comments ReferenceMouse/HaICRGroups of nine mice (9 wks old) were fedbenzo[a]pyrene in the diet (0, 0.2, or0.3 mg/g diet) for 12 wks and sacrificed.Estimated doses were 0, 27.3, or41 mg/kg-d.Incidence with forestomach tumors:Control 0/9Low 6/9High 9/9Less than lifetime exposure duration;glandular stomach, lung, and livers fromcontrol and exposed mice showed notumors.Triolo et al., 1977Mouse/HaICR Groups of 12–20 mice (10 wks old) werefed benzo[a]pyrene in the diet (0.1, 0.3,or 1.0 mg/g diet) for 12–20 wks.Estimated doses were 14.3, 42.0, or192 mg/kg-d.Incidence with forestomach tumors:Low 11/20 (18 wks)Mid 13/19 (20 wks)High 12/12 (12 wks)Less than lifetime exposure duration; onlystomachs were examined for tumors;tumors found only in forestomach.Wattenberg, 1972Mouse/CD-1 20 female mice (9 wks old) were given1 mg benzo[a]pyrene by gavage 2times/wk for 4 wks and observed for19 wks. Estimated dose is 33 mg/kg-d,using an average body weight of 0.030 kgfrom reported data.Incidence with forestomach tumors:Exposed 17/20 (85%)Controls 0/24Less than lifetime exposure duration; onlystomach were examined for tumors; tumorsfound only in forestomach.El-Bayoumy, 1985Mouse/BALB 25 mice (8 wks old) were given 0.5 mgbenzo[a]pyrene 2 times/wk for 15 wks.5/25 mice had squamous carcinomas ofthe forestomach; tumors were detected 28–65 wks after treatment.Less than lifetime exposure duration; thefollowing details were not reported:inclusion of controls, methods for detectingtumors, and body weight data.Biancifiori et al.,1967Mouse/C3H 19 mice (about 3 mo old) were given0.3 mL of 0.5% benzo[a]pyrene inpolyethylene glycol-400 by gavage,once/d for 3 d.By 30 wks, 7/10 mice had papillomas; nocarcinomas were evidentLess than lifetime exposure duration. Berenblum andHaran, 1955Mouse/albino Groups of 17–18 mice were given singledoses of benzo[a]pyrene and allowed tosurvive until terminal sacrifice at 569 d.Incidence of mice (that survived at least to60 d) with forestomach papillomas:Dose (μg) IncidenceExperiment 1 Experiment 2Control 0/17 0/1812.5 3/17 2/1850 0/17 1/17200 8/17 ------aLess than lifetime exposure duration; GItract examined for tumors with hand lens;body weight data not reported.Field and Roe, 1965103 DRAFT - DO NOT CITE OR QUOTETable 4-8. Tumor incidence in oral exposure rodentcancer bioassays with limitations for describing dose-responserelationships for lifetime exposure to benzo[a]pyreneSpecies/strainExposure Results Comments ReferenceMouse/HaICR 20 mice (9 wks old) were givenbenzo[a]pyrene in the diet (0.3 mgbenzo[a]pyrene/g diet) for 6 wks andsacrificed after 20 wks in the study.8/20 exposed mice had forestomachtumors.Less than lifetime exposure duration; onlystomachs were examined for tumors;tumors found only in forestomach; nononexposed controls were mentioned.Wattenberg, 1974Mouse/A/HeJ 12 female mice (9 wks old) were givenstandard diet for 25 d, and 3 mgbenzo[a]pyreneby gastric intubation on d7 and 21 of the study. Mice were killedat 31 wks of age and examined for lungtumors.12/12 exposed mice had lung tumors Less than lifetime exposure duration; onlylungs examined for tumors; no nonexposedcontrols were mentioned.Wattenberg, 1974Mouse/white Groups of 16–30 mice were givenbenzo[a]pyrene in triethylene glycol(0.001–10 mg) wkly for 10 wks andobserved until 19 mo.Tumors in stomach antrumDose (mg) – Carcinoma - Papilloma0.001 0/16 0/160.01– 0/26 2/260.1 0/24 5/241.0 11/30 12/3010 16/27 7/27Less than lifetime exposure duration. Fedorenko etal.,1967,as cited in U.S.EPA, 1991aMouse/albino Groups of about 160 female mice (70 dof age; strain unknown) were given 0 or8 mg benzo[a]pyrene mixed in the dietover a period of 14 mo.Gastric tumors were observed at thefollowing incidence:Control 0/1588 mg benzo[a]pyrene total 13/160Close to lifetime exposure duration; dailydose levels and methods of detectingtumors were not clearly reported.Chouroulinkov et al.,1967Mouse/A/J Groups 40 female mice (8 wks old) weregiven 0 or 0.25 mg benzo[a]pyrene (in2% emulphor) by gavage 3 times/wk for8 wks. Mice were killed at 9 mo of ageand examined for lung or forestomachtumors.Incidence for mice surviving at 9 mo ofage:Lung tumorsControl 11/38Exposed 22/36Forestomach tumorsControl 0/38Exposed 33/36Less than lifetime duration of exposure;only lungs and GI tract were examined fortumors.Robinson et al., 1987Mouse/SwissalbinoGroups of mice (9–14 wks old) weregiven single doses of 0 or 0.05 mgbenzo[a]pyrene in polyethylene glycol400 by gavage. Surviving mice werekilled at 18 mo of age and examined formacroscopic tumors.Forestomach tumor incidence:Dose (μg) – Carcinoma – Papilloma0 0/65 2/6550 1/61 20/61Less than lifetime duration of exposure;exposure-related tumors only found inforestomach.Roe et al., 1970104 DRAFT - DO NOT CITE OR QUOTETable 4-8. Tumor incidence in oral exposure rodentcancer bioassays with limitations for describing dose-responserelationships for lifetime exposure to benzo[a]pyreneSpecies/strainExposure Results Comments ReferenceMouse/ICR Groups of 20 or 24 mice (71 d old) weregiven 1.5 mg benzo[a]pyrene by gavage2 times/wk for 4 wks; terminal sacrificewas at 211 d of age. Estimated dose wasabout 50 mg benzo[a]pyrene/kg, using anaverage body weight of 0.03 kg duringexposure from reported data.Incidence of mice with forestomachneoplasms.Experiment 1 23/24Experiment 2 19/20Less than lifetime duration of exposure;only stomachs were examined for tumors;tumors found only in forestomach;nonexposed controls were not mentioned.Benjamin et al., 1988Mouse/CFW Groups of mice (mixed sex) were fedbenzo[a]pyrene in the diet (dissolved inbenzene and mixed with diet) at 0, 1, 10,20, 30, 40, 45, 50, 100, or 250 ppm in thediet.ppm Exposure Forestomach tumor(d) incidence1 110 0/2510 110 0/2420 110 1/2330 110 0/3740 110 1/4045 110 4/4050 152 24/34100 110 19/23250 118 66/73Less than lifetime exposure duration; novehicle control group; animals ranged fromthree wks to 6 mo old at the start of dosing;only alimentary tract was examined fortumors; (see also Rigdon and Neal, 1969,1967, 1966).Neil and Rigdon,1967Rat/ SpragueDawleyGroups of Sprague-Dawley rats(32/sex/dose) were fed diets delivering adaily dose of 0.15 mg benzo[a]pyrene/kgbody weight every 9thday or5 times/week (Brune et al., 1981). Othergroups (32/ sex/dose) were given gavagedoses of 0.15 mg benzo[a]pyrene (inaqueous 1.5% caffeine solution)/kg every9thday, every 3rdday, or 5 times/week.Dose larynx, esopogus, and forestomach(gavage) tumors0 6/640.016 13/640.049 26/640.107 14/64(diet)0 3/640.016 3/640.107 10/64Doses are annual averages. Non-standardtreatment protocol involved animals beingtreated for 5 days a week or fewer;relatively high control incidence comparedto other gavage studies;Brune et al., 1981Mouse/A/J Groups of female mice were fedbenzo[a]pyrene in the diet at 0, 16, or 98ppm for 260 d. Average intakes ofbenzo[a]pyrene were 0, 40.6, and256.6μg/mouse/d. Estimated doses are0, 1.6, and 9.9 mg/kg-d using a chronicreference body weight value of 0.026 kg(U.S. EPA, 1988).Incidence of mice surviving to 260 d:Lung tumorsControl 4/2116 ppm 9/2598 ppm 14/27Forestomach tumorsControl 0/2116 ppm 5/2598 ppm 27/27Close to lifetime exposure duration; A/Jstrain of mice particularly sensitive tochemically induced cancer; only lungs andstomachs were examined for tumors.Weyand et al., 1995105 DRAFT - DO NOT CITE OR QUOTEa---- = not evaluated1

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4.2.2. Inhalation 1



### 4.2.2.1. Short-term and Subchronic Studies 2 

### Wolff et al. (1989) exposed groups of 40 male and 4 0 female F344/Crl rats, via nose 3 

### only, to 7.5 mg benzo[a]pyrene/m 3 

### for 2 hours/day, 5 days/week for 4 weeks (correspo nding to a 4 

### TWA of 0.45 mg/m 3 

### ). Rats were 10–11 weeks old at the beginning of t he experiment. 5 

### Benzo[a]pyrene (>98% pure) aerosols were formed by heating and then condensing the 6 

### vaporized benzo[a]pyrene. The particle MMAD was 0. 21 μ m. Subgroups of these animals 7 

### (six/sex/dose) were exposed for 4 days or 6 months after the end of the 4-week exposure, 8 

### respectively, to radiolabeled aluminosilicate parti cles. Lung injury was assessed by analyzing 9 

### clearance of radiolabeled aluminosilicate particles and via histopathologic evaluations. Body 10 

### and lung weights, measured in subgroups from 1 day to 12 months after the exposure did not 11



### differ between controls and treated animals. Radio labeled particle clearance did not differ 12 

### between the control and treated groups, and there w ere no significant lung lesions. This study 13 

### identified a NOAEL for lung effects of 0.45 mg/m 3 

### -day for a short-term exposure. 14

15 

### 4.2.2.2. Chronic Studies and Cancer Bioassays 16 

### Thyssen et al. (1981) conducted an inhalation study in which male Syrian golden 17 

### hamsters were exposed to benzo[a]pyrene for their n atural lifetime. Groups of 20–30 male 18 

### Syrian golden hamsters (8 weeks old) were exposed b y nose-only inhalation to NaCl aerosols 19 

### (controls; 240 μ g NaCl/m 3 

### ) or benzo[a]pyrene condensed onto NaCl aerosols at three nominal 20 

### concentrations of 2, 10, or 50 mg benzo[a]pyrene/m 3 

### for 3–4.5 hours/day, 5 days/week for 1– 21 

### 41 weeks, followed by 3 hours/day, 7 days/week for the remainder of study (until hamsters died 22 

### or became moribund). Thyssen et al. (1981) reporte d average measured benzo[a]pyrene 23 

### concentrations to be 0, 2.2, 9.5, or 46.5 mg/m 3 

### . More than 99% of the particles were between 0.2 24



### and 0.5 μ m in diameter, and over 80% had diameters between 0 .2 and 0.3 μ m. The particle 25 

### analysis of the aerosols was not reported to modern standards (MMAD and geometric SD were 26 

### not reported). Each group initially consisted of 2 4 hamsters; final group sizes were larger as 27 

### animals dying during the first 12 months of the stu dy were replaced. 28 

### Survival was similar in the control, low-dose, and mid-dose groups, but was significantly 29 

### decreased in the high-dose group. Average survival times in the control, low-, mid-, and high30 

### dose groups were 96.4 ± 27.6, 95.2 ± 29.1, 96.4 ± 2 7.8, and 59.5 ± 15.2 weeks, respectively. 31



### After the 60 th 

### week, body weights decreased and mortality increas ed steeply in the highest dose 32 

### group. Histologic examination of organs (a complet e list of organs examined histologically was 33 

### not reported by Thyssen et al. [1981]) revealed a d ose-related increase in tumors in the upper 34 

### respiratory tract, including the nasal cavity, phar ynx, larynx, and trachea and in the digestive 35 

### tract in the mid- and high-dose groups (Table 4-8). A statistical analysis was not included in the 36 

### Thyssen et al. (1981) report. No lung tumors were observed. Squamous cell tumors in the 37 

### esophagus and forestomach were also observed in the high-dose group, presumably as a 38

### 107 DRAFT - DO NOT CITE OR QUOTE

consequence of mucociliary particle clearance. Tum ors were detected in other sites, but none of 1 

### these appeared to be related to exposure. The resu lts indicated that the pharynx and larynx, 2 

### including the epiglottis, were the main cancer targ ets (Table 4-8). 3

4 

### Table 4-9. Incidence of respiratory and upper dige stive tract tumors in male

hamsters treated for life with benzo[a]pyrene by in halation

Tumor site Reported benzo[a]pyrene concentration (mg/m 3 )

0 a 2 b 10 50

Tumor incidence (latency in wks c ) Nasal cavity 0 0 3/26 (116 ± 1.5) 1/25 (79) Larynx 0 0 8/26 (107.1 ± 15.5) 13/25 (67.6 ± 12.1) Trachea 0 0 1/26 (115) 3/25 (63.3 ± 33.3) Lung 0 0 0 0 Pharynx 0 0 6/26 (97.2 ± 16.9) 14/25 (67.5 ± 12.2) Esophagus 0 0 0 2/25 (70, 79) Forestomach 0 0 1/26 (119) 1/25 (72)

a Effective number of animals in control group: n = 27.

b Effective number of animals in 2 mg/m 3 dose group: n = 27.

c Mean ± SD.

Source: Thyssen et al. (1981).

5 

### Under contract to the U.S. EPA, Clement Associates (1990) obtained the individual 6 

### animal data (including individual animal pathology reports, time-to-death data, and exposure 7 

### chamber monitoring data) collected by Thyssen et al . (1981). Re-analysis of the original data 8 

### revealed several errors and omissions in the publis hed report. The actual exposure protocol was 9 

### as follows: 4.5 hours/day 5 days/week on weeks 1–1 2, 3 hours/day 5 days/week on weeks 13– 10 

### 29, 3.7 hours/day 5 days/week on week 30, 3 hours/d ay 5 days/week on weeks 31–41, and 11 

### 3 hours/day 7 days/week for the reminder of the exp eriment. In addition, actual exposure 12 

### concentrations varied widely from week to week. Be cause different animals were started at 13 

### different times, each individual animal had an expo sure history somewhat different than others in 14 

### the same exposure group. In order to deal with thi s problem, Clement Associates (1990) used 15 

### the orignial individual animal data to calculate av erage continuous lifetime exposures for each 16 

### individual hamster. Group averages of individual a verage continuous lifetime exposure 17 

### concentrations were 0, 0.25, 1.01, and 4.29 mg/m 3 

### for the control through high-exposure groups. 18 

### For this assessment, the individual animal patholog y reports prepared by Thyssen et al. 19 

### (1981) and obtained by Clement Associates (1990) we re examined to independently assess the 20 

### numbers of hamsters with tumors in the larynx, phar ynx, and nose in each group. Table 4-9 21 

### presents the number of animals with tumors in the l arynx and pharynx and the numbers of 22 

### animals in each exposure group. Numbers of animals with either laryngeal or pharyngeal tumors 23

### 108 DRAFT - DO NOT CITE OR QUOTE

are also noted in Table 4-10, since these two types of tumors arise in close anatomical proximity 1 

### from similar cell types. Examination of the indivi dual animal pathology reports also showed that 2 

### all of the nasal, forestomach, esophageal, and trac heal tumors occurred in animals that also had 3 

### either laryngeal or pharyngeal tumors, except for t wo animals in the mid-dose group that 4 

### displayed nasal tumors (one malignant and one benig n) without displaying tumors in the pharynx 5 

### or larynx. 6

7 

### Table 4-10. Number of animals with pharynx and lar ynx tumors in male

hamsters exposed by inhalation to benzo[a]pyrene fo r life

Average continuous benzo[a]pyrene concentration a (mg/m 3 ) Number of

hamsters in

group b Larynx b Pharynx b Larynx or pharynx, combined c

Malignant All Malignant All Malignant All

Control 27 0 0 0 0 0 0

0.25 27 0 0 0 0 0 0

1.01 26 8 11 7 9 11 16

4.29 34 9 12 17 18 17 18

a As calculated by Clement Associates (1990) from air monitoring data collected by Thyssen and colleague s. b As counted from information in Table D-1 in Appendi x D, which was obtained from examination of individ ual animal pathology reports prepared by Thyssen and co lleagues and obtained by Clement Associates.

c As counted from information in Table D-1 in Appendi x D. Nasal, forestomach, esophageal, and tracheal tumors occurred in hamsters that also had tumors in the la rynx or pharynx, except for two animals in the mid- dose group that displayed nasal tumors (one malignant and one benign) without displaying tumors in the pharynx or larynx.

8 

### Several studies have investigated the carcinogenici ty of benzo[a]pyrene in hamsters 9 

### exposed by intratracheal instillation. Single-dose studies verified that benzo[a]pyrene is 10 

### tumorigenic, but do not provide data useful charact erizing dose-response relationships because of 11 

### their design (Kobayashi, 1975; Reznik-Schuller and Mohr, 1974; Henry et al., 1973; Mohr, 1971; 12 

### Saffiotti et al., 1968; Gross et al., 1965; Herrold and Dunham, 1962). One multiple-dose study, 13 

### which utilized very low doses (0.005, 0.02, and 0.0 4 mg, once every 2 weeks), failed to find any 14 

### tumorigenic response (Kunstler, 1983). Tumorigenic responses (mostly in the respiratory tract) 15 

### were found at higher dosage levels (0.25–2 mg benzo [a]pyrene once per week for 30–52 weeks) 16 

### in four multiple-dose studies (Feron and Kruysse, 1 978; Ketkar et al., 1978; Feron et al., 1973; 17 

### Saffiotti et al., 1972). These studies identify th e respiratory tract as a cancer target with exposur e 18 

### to benzo[a]pyrene by intratracheal instillation and provide supporting evidence for the 19 

### carcinogenicity of benzo[a]pyrene at portal-of-entr y sites. 20

21 

### 4.2.3. Dermal Exposure 22



### 4.2.3.1. Skin-Tumor Initiation-Promotion Assays 23

### 109 DRAFT - DO NOT CITE OR QUOTE

Results from numerous studies indicate that acute d ermal exposure to benzo[a]pyrene 1 

### induces skin tumors in mice when followed by repeat ed exposure to a potent tumor promoter 2 

### (Weyand et al., 1992; Cavalieri et al., 1991, 1981; Rice et al., 1985; El-Bayoumy et al., 1982; 3 

### LaVoie et al., 1982; Raveh et al., 1982; Slaga et a l., 1980, 1978; Wood et al., 1980; Hoffmann et 4 

### al., 1972). The typical exposure protocol in these studies involved the application of a single 5 

### dose of benzo[a]pyrene (typically ≥ 20 nmol per mouse) to dorsal skin of mice followed by 6 

### repeated exposure to a potent tumor promoter, such as 12- O -tetradecanoylphorbol-13-acetate 7 

### (TPA). 8

9



### 4.2.3.2. Carcinogenicity Dermal Bioassays 10

11 

### Poel (1959) 12 

### Poel (1959) applied benzo[a]pyrene in toluene to s haved interscapular skin of groups of 13 

### 13–56 male C57L mice at doses of 0, 0.15, 0.38, 0.7 5, 3.8, 19, 94, 188, 376, or 752 μ g, 14 

### 3 times/week for up to 103 weeks or until the appea rance of a tumor by gross examination 15 

### (3 times weekly). Some organs (not further specifi ed) and interscapular skin in sacrificed mice 16 

### were examined histologically. With increasing dose level, the incidence of mice with skin 17 

### tumors increased and the time of tumor appearance d ecreased (see Table 4-10). Doses >3.8 μ g 18 

### were associated with 100% mortality after increasin gly shorter exposure periods, none greater 19 

### than 44 weeks. Poel (1959) did not mention the app earance of exposure-related tumors in tissues 20 

### other than interscapular skin. 21

22 

### Table 4-11. Skin tumor incidence and time of appea rance in male C57L

mice dermally exposed to benzo[a]pyrene for up to 1 03 weeks

Dose ( μ g) a Incidence of mice with

gross skin tumors Time of first tumor appearance (wks) Incidence of mice

with epidermoid carcinoma b Length of exposure

period(wks) 0 (Toluene) 0/33 (0%) − 0/33 (0%) 92 0.15 5/55 (9%) 42–44 c 0/55 (0%) 98 0.38 11/55 (20%) 24 2/55 (4%) 103 0.75 7/56 (13%) 36 4/56 (7%) 94 3.8 41/49 (84%) 21–25 32/49 (65%) 82 19 38/38 (100%) 11–21 37/38 (97%) 25-44 c 94 35/35 (100%) 8–19 35/35 (100%) 22-43 188 12/14 (86%) 9–18 10/14 (71%) 20-35 376 14/14 (100%) 4–15 12/14 (86%) 19-35 752 13/13 (100%) 5–13 13/13 (100%) 19-30

a Indicated doses were applied to interscapular skin 3 times/wk for up to 103 wks or until time of appea rance of a grossly detected skin tumor.

b Carcinomas were histologically confirmed.

### 110 DRAFT - DO NOT CITE OR QUOTE c Ranges reflect differing information in Tables 4 an d 6 of Poel (1959).

Source: Poel (1959).

1 

### Poel (1960) applied benzo[a]pyrene in a toluene veh icle to shaved interscapular skin of 2 

### groups of 14–25 male SWR, C3HeB, or A/He mice 3 tim es/week at doses of 0, 0.15, 0.38, 0.75, 3 

### 3.8, 19.0, 94.0, or 470 μ g benzo[a]pyrene per application, until mice died o r a skin tumor was 4 

### observed. Time ranges for tumor observations were provided, but not times of death for mice 5 

### without tumors, so it was not possible to evaluate differential mortality among all dose groups or 6 

### the length of exposure for mice without tumors. Wi th increasing dose level, the incidence of 7



### mice with skin tumors increased and the time of tum or appearance decreased (Table 4-11). The 8



### lowest dose level did not induce an increased incid ence of mice with skin tumors in any strain, 9 

### but strain differences in susceptibility were evide nt at higher dose levels. SWR and C3HeB mice 10



### showed skin tumors at doses ≥ 0.38 μ g benzo[a]pyrene, whereas AH/e mice showed tumors a t 11



### doses ≥ 19 μ g benzo[a]pyrene (Table 4-11). Except for metastas es of the skin tumors to lymph 12 

### nodes and lung, Poel (1960) did not mention the app earance of exposure-related tumors in 13 

### tissues other than interscapular skin. 14

15 

### Table 4-12. Skin tumor incidence and time of appea rance in male SWR,

C3HeB, and A/He mice dermally exposed to benzo[a]py rene for life or until a skin tumor was detected

Dose ( μ g) a SWR mice C3HeB mice A/He mice

Tumor incidence b Time of first

tumor

appearance (weeks) Tumor

incidence b Time of first

tumor

appearance (weeks) Tumor

incidence b Time of fist

tumor

appearance (weeks) 0 (Toluene) 0/20 (0%) 0/17 (0%) — 0/17 (0%) — 0.15 0/25 (0%) — 0/19 (0%) — 0/18 (0%) — 0.38 2/22 (9%) 55–55 3/17 (18%) 81–93 0/19 (0%) — 0.75 15/18 (83%) 25–72 4/17 (24%) 51–93 0/17 (0%) — 3.8 12/17 (70%) 25–51 11/18 (61%) (35–73 0/17 (0%) — 19.0 16/16 (100%) 12–28 17/17 (100%) 13–32 21/23 (9 1%) 21–40 94.0 16/17 (94%) 9–17 18/18 (100%) 10–22 11/16 (69 %) 14–31 470.0 14/14 (100%) 5–11 17/17 (100%) 4–19 17/17 (10 0%) 4–21

a Indicated doses were applied 3times/week for life o r until a skin tumor was detected. Mice were 10–14 wks old at initial exposure.

b Incidence of mice exposed 10 or more wks with a ski n tumor.

Source: Poel (1960).

16

17

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Roe et al. (1970) treated groups of 50 female Swiss mice with 0 (acetone vehicle), 0.1, 1 

### 0.3, 1, 3, or 9 μ g benzo[a]pyrene applied to the shaved dorsal skin 3 times/week for up to 2 

### 93 weeks; all surviving mice were killed and examin ed for tumors during the following 3 weeks. 3 

### The dorsal skin of an additional control group was shaved periodically but was not treated with 4 

### the vehicle. Mice were examined every 2 weeks for the development of skin tumors at the site of 5 

### application. Histologic examinations included: (1 ) all skin tumors thought to be possibly 6 

### malignant; (2) lesions of other tissues thought to be neoplastic; and (3) limited nonneoplastic 7 

### lesions in other tissues. As shown in Table 4-13, markedly elevated incidences of mice with skin 8 

### tumors were only found in the two highest dose grou ps (3 or 9 μ g ), compared with no skin 9 

### tumors in the control groups. Malignant skin tumor s (defined as tumors with invasion or 10 

### penetration of the panniculus carnosus muscle) were detected in 4/41 and 31/40 mice in the 11



### 3- and 9- μ g groups, respectively, surviving to at least 300 days. Malignant lymphomas were 12 

### detected in all groups, but the numbers of cases we re not elevated compared with expected 13 

### numbers after adjustment for survival differences. Lung tumors were likewise detected in 14 

### control and exposed groups at incidences that were not statistically different. 15

16



### Table 4-13. Tumor incidence in female Swiss mice d ermally exposed to

benzo[a]pyrene for up to 93 weeks

Dose (μg) a Cumulative number of mice with skin tumor/survivors Skin tumor incidence b Malignant

Lymphoma incidence c Lung

Tumor incidence c 200 d 300 d 400 d 500 d 600 d 700 d No treatment 0/48 0/43 0/40 0/31 0/21 0/0 0/43 (0%) 19/44 (43%) 12/41 (29%) Acetone 0/49 0/47 0/45 0/37 0/23 0/0 0/47 (0%) 12/4 7 (26%) 10/46 (22%) 0.1 0/45 1/42 1/35 1/31 1/22 1/0 1/42 (2%) 11/43 (2 6%) 10/40 (25%) 0.3 0/46 0/42 0/37 0/30 0/19 0/0 0/42 (0%) 10/43 (2 3%) 13/43 (30%) 1 0/48 0/43 0/37 1/30 1/18 1/0 1/43 (2%) 16/44 (36% ) 15/43 (35%) 3 0/47 0/41 1/37 7/35 8/24 8/0 8/41 (20%) 23/42 (55 %) 12/40 (30%) 9 0/46 4/40 21/32 28/21 33/8 34/0 34/46 (74%) 9/40 (23%) 5/40 (13%)

a Doses were applied 3 times/wk for up to 93 wks to s haved dorsal skin.

b Numerator: number of mice detected with a skin tum or. Denominator: number of mice surviving to 300 d for all groups except the highest dose group. For the high est dose group (in which skin tumors were first det ected between 200 and 300 d), the number of mice surviving to 200 d was used as the denominator.

c Numerator : number of mice detected with specified tumor. Denominator: number of mice surviving to 3 00 d unless a tumor was detected earlier, in which case the number dying before 300 d without a tumor was s ubtracted from the number of animals reported to have been ex amined.

Source: Roe et al. (1970).

17 

### Schmidt et al. (1973) dermally administered benzo[a ]pyrene in acetone to female NMRI 18 

### mice (100/group) and female Swiss mice. Benzo[a]py rene was applied to the shaved dorsal skin 19 

### twice weekly with doses of 0, 0.05, 0.2, 0.8, or 2 μ g until spontaneous death occurred or until an 20



### advanced carcinoma was observed. Skin carcinomas w ere identified by the presence of crater- 21

### 112 DRAFT - DO NOT CITE OR QUOTE

shaped ulcerations, infiltrative growth and the beg inning of physical wasting (i.e., cachexia). 1 

### Necropsy was performed for all animals, and histopa thological examination of the dermal site of 2 

### application and any other tissues with gross abnorm alities was conducted. Skin tumors were 3 

### observed at the two highest doses in both strains o f female mice (see Table 4-14), with induction 4 

### periods of 53.0 and 75.8 weeks for the 0.8 and 2.0 μ g NMRI mice and 57.8 and 60.7 weeks for 5 

### the Swiss mice, respectively. The authors indicate d that the latency period for tumor formation 6 

### was highly variable and significant differences amo ng exposure groups could not be identified, 7 

### but no further timing information was available, in cluding overall survival. Carcinoma was the 8 

### primary tumor type seen after lifetime application of benzo[a]pyrene to mouse skin. 9

10



### Table 4-14. Skin tumor incidence in female NMRI an d Swiss mice dermally

exposed to benzo[a]pyrene

Dose ( μ g) a,b Skin tumor incidence (all types) Incidence of papilloma Incidence of carcinoma

Female NMRI mice 0 (Acetone) 0/100 (0%) 0/100 (0%) 0/100 (0%) 0.05 0/100 (0%) 0/100 (0%) 0/100 (0%) 0.2 0/100 (0%) 0/100 (0%) 0/100 (0%) 0.8 2/100 (2%) 0/100 (0%) 2/100 (2%) 2 30/100 (30%) 2/100 (2%) 28/100 (28%)

Female Swiss mice 0 (Acetone) 0/80 (0%) 0/80 (0%) 0/80 (0%) 0.05 0/80 (0%) 0/80 (0%) 0/80 (0%) 0.2 0/80 (0%) 0/80 (0%) 0/80 (0%) 0.8 5/80 (6%) 0/80 (0%) 5/80 (6%) 2 45/80 (56%) 3/80 (4%) 42/80 (52%)

a Mice were exposed until natural death or until they developed a carcinoma at the site of application.

b Indicated doses were applied 2 times/wk to shaved s kin of the back.

Source: Schmidt et al. (1973).

11 

### Schmähl et al. (1977) applied benzo[a]pyrene 2 time s/week to the shaved dorsal skin of 12 

### female NMRI mice (100/group) at doses of 0, 1, 1.7, or 3 μ g in 20 μ L acetone. The authors 13 

### reported that animals were observed until natural d eath or until they developed a carcinoma at 14 

### the site of application. The effective numbers of animals at risk was about 80% of the nominal 15 

### group sizes, which the authors attributed to autoly is; no information was provided concerning 16 

### when tumors appeared in the relevant groups, how lo ng treatment lasted in each group, or any 17 

### times of death. Necropsy was performed on all mice and the skin of the back, as well as any 18 

### organs that exhibited macroscopic changes, were exa mined histopathologically. The incidence 19 

### of all types of skin tumors was increased in a dose related manner compared to controls (see 20 

### Table 4-15). Carcinoma was the primary tumor type observed following chronic dermal 21

### 113 DRAFT - DO NOT CITE OR QUOTE

exposure to benzo[a]pyrene, and skin papillomas occ urred infrequently. Dermal sarcoma was not 1



### observed. 2

3

### 114 DRAFT - DO NOT CITE OR QUOTE

Table 4-15. Skin tumor incidence in female NMRI mi ce dermally exposed to benzo[a]pyrene

Dose ( μ g) a,b Skin tumor incidence

(all types) Incidence of papilloma Incidence of car cinoma 0 1/81 (1%) 0/81 (0%) 0/81 (0%) 1 11/77 (14%) 1/77 (1%) 10/77 (13%) 1.7 25/88 (28%) 0/88 (0%) 25/88 (28%) 3 45/81 (56%) 2/81 (3%) 43/81 (53%)

a Mice were exposed until natural death or until they developed a carcinoma at the site of application.

b Indicated doses were applied 2 times/wk to shaved s kin of the back.

Source: Schmähl et al. (1977).

1 

### Habs et al. (1980) applied benzo[a]pyrene to the sh aved interscapular skin of female 2 

### NMRI mice (40/group) at doses of 0, 1.7, 2.8 or 4.6 μ g in 20 μ L acetone twice weekly, from 3 

### 10 weeks of age until natural death or gross observ ation of infiltrative tumor growth. Latency of 4 

### tumors, either as time of first appearance or avera ge time of appearance of tumors, was not 5 

### reported. Necropsy was performed on all animals, a nd the dorsal skin, as well as any organs 6 

### showing gross alterations at autopsy, was prepared for histopathological examination. Age7 

### standardized mortality rates, using the total popul ation of the experiment as the standard 8 

### population, were used to adjust tumor incidence fin dings in the study. Benzo[a]pyrene 9 

### application was associated with a statistically sig nificant increase in the incidence of skin tumors 10 

### at each dose level (see Table 4-17). 11

12



### Table 4-16. Skin tumor incidence in female NMRI mi ce dermally exposed to

benzo[a]pyrene

Dose ( μ g) a,b Skin tumor incidence Age-standardized tumor incide nce c 0 (acetone) 0/35 (0%) 0% 1.7 8/34 (24%) 24.8% 2.8 24/35 (68%) 89.3% 4.6 22/36 (61%) 91.7%

a Mice were exposed until natural death or until they developed a carcinoma at the site of application.

b Indicated doses were applied 2 times/wk to shaved s kin of the back.

c Mortality data of the total study population were u sed to derive the age-standardized tumor incidence.

Source: Habs et al. (1980).

13 

### Grimmer et al. (1983) and Grimmer et al. (1984) app lied benzo[a]pyrene (in 0.1 mL of a 14 

### 1:3 solution of acetone:dimethyl sulfoxide [DMSO]) to the interscapular skin of female CFLP 15 

### mice (65–80/group) 2 times/week for 104 weeks. Dos es were 0, 3.9, 7.7 and 15.4 μ g in the 1983 16

### 115 DRAFT - DO NOT CITE OR QUOTE

experiment, and 0, 3.4, 6.7, and 13.5 μ g in the 1984 experiment. Mice were observed until 1 

### spontaneous death, unless an advanced tumor was obs erved or if animals were found moribund. 2 

### Survival information was not provided; incidences r eflect the number of animals placed on 3 

### study. Necropsy was performed on all mice. Histop athological examination of the skin and any 4 

### other organ showing gross abnormalities was perform ed. Chronic dermal exposure to 5 

### benzo[a]pyrene produced a dose-related increase in skin tumor incidence and a decrease in tumor 6 

### latency (see Table 4-17). Carcinoma was the primar y tumor type observed and a dose-response 7 

### relationship was evident for carcinoma formation an d incidence of all types of skin tumors. 8

9 

### Table 4-17. Skin tumor incidence and time of appea rance in female CFLP

mice dermally exposed to benzo[a]pyrene for 104 wee ks

Dose ( μ g) a Skin tumor incidence

(all types) Incidence of papilloma Incidence of

carcinoma Tumor appearance in weeks Grimmer et al. (1983) 0 (1:3 Solution of acetone:DMSO) 0/80 (0%) 0/80 (0%) 0/80 (0%) —

3.9 22/65 (34%) 7/65 (11%) 15/65 (23%) 74.6 ± 16.78 b

7.7 39/64 (61%) 5/64 (8%) 34/64 (53%) 60.9 ± 13.90 15.4 56/64 (88%) 2/64 (3%) 54/64 (84%) 44.1 ± 7.66 Grimmer et al. (1984). 0 (1:3 Solution of acetone:DMSO) 0/65 (0%) 0/65 (0%) 0/65 (0%) —

3.4 43/64 (67%) 6/64 (9%) 37/64 (58%) 61 (53–65) c 6.7 53/65 (82%) 8/65 (12%) 45/65 (69%) 47 (43–50) 13.5 57/65 (88%) 4/65 (6%) 53/65 (82%) 35 (32–36)

a Indicated doses were applied twice/week to shaved s kin of the back.

b Mean ± SD.

c Median with 95% confidence interval.



### Habs et al. (1984) applied benzo[a]pyrene (in 0.01 mL acetone) to the shaved 10 

### interscapular skin of female NMRI mice at doses of 0, 2 or 4 μ g, 2 times/week for life. Animals 11 

### were observed twice daily until spontaneous death, unless an invasive tumor was observed. All 12 

### animals were necropsied and histopathological exami nation was performed on the dorsal skin 13 

### and any other organ with gross abnormalities. Chro nic dermal exposure to benzo[a]pyrene did 14 

### not affect body weight gain, but appeared to reduce survival at the highest dose with mean 15 

### survival times of 691, 648, and 528 days for the 0, 2, and 4 μ g/day groups, respectively. The 16 

### total length of exposure for each group was not rep orted, but can be inferred from the survival 17 

### data. Latency also was not reported. Benzo[a]pyre ne application resulted in a dose-related 18



### increase the incidence of total skin tumors and ski n carcinomas (see Table 4-18). Hematopoietic 19 

### tumors (at 6/20, 3/20, and 3/20) and lung adenomas (at 2/20, 1/20, and 0/20) were observed in 20

### 116 DRAFT - DO NOT CITE OR QUOTE

the controls and in the benzo[a]pyrene treatment gr oups, but did not appear to be treatment 1 

### related according to the study authors. 2

3



### Table 4-18. Skin tumor incidence in female NMRI mi ce dermally exposed to

benzo[a]pyrene for life

Dose ( μ g) a,b Skin tumor incidence

(all types) Incidence of

papilloma Incidence of

carcinoma Mean survival time,

days (95% confidence interval) 0 (Acetone) 0/20 (0%) 0/20 (0%) 0/20 (0%) 691 (600- 763) 2 9/20 (45%) 2/20 (10%) 7/20 (35%) 648 (440-729) 4 17/20 (85%) 0/20 (0%) 17/20 (85%) 528 (480-555)

a Mice were exposed until natural death or until they developed an invasive tumor at the site of applica tion.

b Indicated doses were applied 2 times/wk to shaved i nterscapular skin.

Source: Habs et al. (1984).

4 

### Higginbotham et al. (1993) 5 

### Groups of 23–27 female Ah-receptor-responsive Swis s mice were treated on a shaved 6 

### area of dorsal skin with 0, 1, 4, or 8 nmol (0, 0.2 5, 1, or 2 μ g/treatment) benzo[a]pyrene (>99% 7 

### pure) in acetone 2 times weekly for 40 weeks (Higgi nbotham et al., 1993). Surviving animals 8 

### were sacrificed 8 weeks later. Complete necropsies were performed, and tissues from the treated 9 

### area, lung, liver, kidney, spleen, urinary bladder, ovary, and uterus were harvested for 10 

### histopathologic examination. Histopathologic exami nation was performed on tissues from the 11 

### treated area, lungs, liver, kidneys, spleen, urinar y bladder, uterus, and ovaries, as well as any 12 

### other grossly abnormal tissue. Lung adenomas occur red in each group (1/27, 2/24, 1/23, 1/23), 13 

### and other tumors were noted in isolated mice (i.e., malignant lymphoma (spleen) in one low-dose 14 

### and one mid-dose mouse; malignant lymphoma with mid dle organ involvement in one high-dose 15 

### mouse, and hemangioma (liver) in one mid-dose mouse ) and were not considered dose related. 16 

### In addition, benzo[a]pyrene showed no skin tumors u nder the conditions of this bioassay. 17

18 

### Sivak et al. (1997) 19 

### Sivak et al. (1997) designed a study to compare the carcinogenicity of condensed asphalt 20 

### fumes (including benzo[a]pyrene and other PAHs) wit h several doses of benzo[a]pyrene alone. 21 

### For the purposes of this assessment, the exposure g roups exposed to PAH mixtures are not 22 

### discussed. Groups of 30 male C3H /HeJ mice were tr eated dermally twice/week to 0, 0.0001, 23 

### 0.001, or 0.01% (0, 0.05, 0.5, or 5 μ g) benzo[a]pyrene in a 50 μl volume of 24 

### cyclohexanone/acetone (1:1) for 104 weeks beginning at 8 weeks of age. All mice were 25 

### necropsied, and skin samples from all as well as an y grossly observed lesions were subjected to 26 

### histopathological examination. The incidence of sk in tumors and mean survival times for each 27

### 117 DRAFT - DO NOT CITE OR QUOTE

group are shown in Table 4-19. All high dose mice died before the final sacrifice. The extent of 1 

### deaths prior to one year in each group was not prov ided, so that the reported incidence may 2 

### underestimate the tumor rate of animals exposed lon g enough to develop tumors. However, the 3 

### crude skin tumor rates show an increasing trend in incidence. 4

5



### Table 4-19. Skin tumor incidence in male C3H /HeJ mice dermally exposed

to benzo[a]pyrene for 24 months

Dose ( μ g) a Skin tumor incidence (all types) No. died before final sacrifice Mean survival time, days 0 cyclohexanone/acetone (1:1) 0/30 (0%) 19 607

0.05 0/30 (0%) 15 630 0.5 5/30 (20%) 15 666 5.0 27/30 (90%) 30 449

a Indicated doses were applied twice/week to shaved d orsal skin.

Source: Sivak et al. (1997).

6

7 

### Albert et al. (1991) 8 

### To examine dose-response relationships and the time course of benzo[a]pyrene-induced 9 

### skin damage, DNA adduct formation, and tumor format ion, groups of 43–85 female Harlan mice 10 

### were treated dermally with 0, 16, 32, or 64 μ g of benzo[a]pyrene in 50 μ L of acetone once per 11 

### week for 29 weeks (Albert et al., 1991). Interscap ular skin of each mouse was clipped 3 days 12 

### before the first application and every 2 weeks ther eafter. Additional groups of mice were treated 13 

### for 9 weeks with 0, 8, 16, 32, or 64 μ g radiolabeled benzo[a]pyrene to determine benzo[a] pyrene 14 

### diolepoxide-DNA (BPDE-DNA) adduct formation in the epidermis at several time points (1, 2, 15 

### 4, and 9 weeks). Tumor formation was monitored onl y in the skin. 16 

### No tumors were present in vehicle-treated or untrea ted control mice. In exposed groups, 17 

### incidences of mice with skin tumors were not report ed, but time-course data for cumulative 18 

### number of tumors per mouse, corrected for deaths fr om nontumor causes, were reported. 19 

### Tumors began appearing after 12–14 weeks of exposur e for the mid- and high-dose groups and 20 

### at 18 weeks for the low-dose group. At study termi nation (35 weeks after start of exposure), the 21 

### mean number of tumors per mouse was approximately o ne per mouse in the low- and mid-dose 22 

### groups and eight per mouse in the high-dose group; indicating that most, if not all, mice in each 23 

### exposure group developed skin tumors and that the t umorigenic response was greatest in the 24 

### highest dose group. The majority of tumors were in itially benign, with an average time of 25 

### 8 weeks for progression from benign papillomas to m alignant carcinomas. Epidermal damage 26 

### occurred in a dose-related manner (more severe in t he high-dose group than in the low- and mid27 

### dose groups) and included statistically significant increases (compared with controls) in: [ 3 

### H]- 28 

### thymidine labeling and mitotic indices; incidence o f pyknotic and dark cells (signs of apoptosis); 29

### 118 DRAFT - DO NOT CITE OR QUOTE

and epidermal thickness. Only a minor expansion of the epidermal cell population was observed. 1 

### In the high-dose group, indices of epidermal damage increased to a plateau by 2 weeks of 2 

### exposure. The early time course of epidermal damag e indices was not described in the low- and 3 

### mid-dose groups, since data for these endpoints wer e only collected at 20, 24, and 30 weeks of 4 

### exposure. An increased level of BPDE-DNA adducts, compared with controls, was apparent in 5 

### all exposed groups after 4 weeks of exposure in the following order: 64>32>16>8 μ g/week. The 6 

### time-course data indicate that benzo[a]pyrene-induc ed increases in epidermal damage indices 7 

### and BPDE-DNA adducts preceded the appearance of ski n tumors. 8

9 

### 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL, INHA LATION, AND 10



### DERMAL 11 

### As discussed in Section 4.1.4.3, several studies of human cohorts have examined possible 12 

### associations between lower body weights or head cir cumference in newborns or infants with 13 

### benzo[a]pyrene-DNA adducts levels in cord blood and exposure to ETS (Tang et al., 2006; 14 

### Perera et al., 2005a, b). Available studies of re productive or developmental endpoints in 15 

### animals exposed orally or by inhalation to benzo[a] pyrene are reviewed as follows in this 16 

### section. No studies that evaluated reproductive or developmental effects following dermal 17 

### exposure were identified. 18

19



### 4.3.1. Oral 20 

### Mohamed et al. (2010) investigated multi-generation al effects in male mice following 21 

### exposure of six-week old C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day 22 

### benzo[a]pyrene for 6 weeks by daily gavage. Follow ing final treatment, male mice were allowed 23 

### to stabilize for one week prior to being mated with two untreated female mice to produce an F1 24 

### generation. Male mice were sacrificed one week af ter mating. F1 males were also mated with 25



### untreated female mice as were F2 males. The mice o f the F1, F2, and F3 generations were not 26 

### exposed to benzo[a]pyrene. The F0, F1, F2 and F3 m ice were all sacrificed at the same age (14 27 

### weeks) and endpoints including testis histology, sp erm count, sperm motility, and in vitro sperm 28 

### penetration (of hamster oocytes) were evaluated. T hese endpoints were analyzed statistically 29 

### using ANOVA and Tukey’s honest significance test an d results were reported graphically as 30



### means +/- SD. 31 

### Testicular atrophy was observed in the benzo[a]pyre ne treatment groups, but was not 32 

### statistically different than controls. Statistical ly significant reductions were observed in 33 

### epididymal sperm counts of F0 and F1 generations tr eated with the high or low dose of 34 

### benzo[a]pyrene. For F0 and F1 generations, epididy mal sperm counts were reduced 35 

### approximately 50% and 70%, respectively, in the low and high dose groups. Additionally, sperm 36 

### motility was statistically significantly decreased in the high dose in the F0 and F1 generations. 37 

### Sperm parameters of the F3 generation were not stat istically different from controls. An in vitro 38

### 119 DRAFT - DO NOT CITE OR QUOTE

sperm penetration assay revealed statistically sign ificantly reduced fertilization in F0 and F1 1 

### generations of the low and high dose groups. Howev er, the value of this in vitro test is limited as 2 

### it bypasses essential components of the intact anim al system (US EPA, 1996). Based on 3 

### decreased epididymal sperm counts of F0 and F1 gene rations, a LOAEL of 1 mg/kg-day was 4 

### established from this study (no NOAEL was identifie d). 5 

### Xu et al., (2010) treated female Sprague-Dawley rat s (6/group) to 0 (corn oil only), 5, or 6 

### 10 mg/kg-day benzo[a]pyrene by gavage every other d ay for a duration of 60 days. This resulted 7 

### in time weighted average doses of 0, 2.5, and 5 mg/ kg-day over the study period of 60 days. 8 

### Endpoints examined included ovary weight, estrous c ycle, 17B-estradiol blood level, and ovarian 9 

### follicle populations (including primordial, primary , secondary, atretic, and corpora leutea). 10 

### Animals were observed daily for any clinical signs of toxicity and following sacrifice, gross 11 

### pathological examinations were made and any finding s were recorded. All animals survived to 12 

### necropsy. A difference in clinical signs was not o bserved for the treated groups and body 13 

### weights were not statistically different in treated animals (though they appear to be depressed 6% 14 

### at the high dose). Absolute ovary weight was stati stically significantly reduced in the both the 15 

### low and high dose groups, 11 and 15% respectively ( see Table 4-20). Animals treated with the 16 

### high dose were noted to have a statistically signif icantly prolonged duration of the estrous cycle 17 

### and non-estrus phase compared to controls. Animals in the high dose group also had statistically 18 

### significantly depressed levels of estradiol (by app roximately 25%) and decreased numbers of 19 

### primordial follicles (by approximately 20%). This study also indicated a strong apoptotic 20 

### response of ovarian granulosa cells as visualized t hrough TUNEL labeling, however, the 21 

### strongest response was seen at the low dose; decrea sed apoptosis was also observed at the high 22 

### dose. Based on decreased ovary weight following 60 day oral exposure to benzo[a]pyrene, a 23 

### LOAEL of 2.5 mg/kg-day was established from this st udy (no NOAEL was identified). 24

25



### Table 4-20. Means ± SD for ovary weight in female SD-rats

Dose (mg/kg-d) a

0 2.5 5

Ovary weight (g) 0.160 ± 0.0146 0.143 ± 0.0098 b 0.136 ± 0.0098 b

Body weight (g) 261.67 ± 12.0 249.17 ± 11.2 247.25 ± 11.2

a TWA doses over the 60 day study period

b Statistically different from controls (p < 0.05) u sing one-way ANOVA

Source: Xu et al. (2010).

26

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Zheng et al., (2010) treated male Sprague-Dawley ra ts to 0 (corn oil only), 1, or 5 mg/kg1 

### day benzo[a]pyrene by daily gavage for a duration o f 30 (8/group) or 90 days (8/group). At 2 

### necropsy, the left testis of each animal was collec ted and weighed. Testes testosterone 3 

### concentrations were determined by radioimmunassay a nd results were expressed as ng/g testis 4 

### and reported graphically. Testicular testosterone was statistically significantly decreased in the 5 

### high dose group approximately 15% following 90 days of exposure. The low dose group also 6 

### appeared to have a similar average depression of te stosterone levels; however, the change did not 7 

### reach statistical significance. Testosterone level s measured in animals sacrificed following 30 8 

### days of benzo[a]pyrene exposure were not statistica lly different than controls. Based on 9 

### decreased testicular testosterone levels following 90 day oral exposure to benzo[a]pyrene, a 10 

### LOAEL of 5 mg/kg-day and a NOAEL of 1 mg/kg-day wer e identified. 11 

### McCallister et al., (2008) administered 0 or 300 ug /kg benzo[a]pyrene by oral gavage in 12 

### peanut oil to pregnant Long Evans rats (n= 5 or 6) on GDs 14 to 17. At this exposure level, no 13 

### significant changes were see in number of pups per litter, pup growth, or liver to body weight 14 

### ratios in control compared to benzo[a]pyrene expose d offspring. Treatment related differences 15 

### in brain to body weight ratios were observed only o n PND 15 and PND 30. Decreases in 16 

### cerebrocortical mRNA expression of the glutamatergi c NMDA receptor subunit was 17 

### significantly reduced (50%) in treated offspring co mpared to controls. In addition, in utero 18 

### exposed offpring exhibited decreased evoked cortica l neuronal activity in the barrel field cortex 19



### when tested at PN 90-120. 20 

### Rigdon and Neal (1965) administered diets containin g 1,000 ppm benzo[a]pyrene to 21 

### pregnant mice (nine/group) on GDs 10–21 or 5–21. T he pups were reported as appearing 22 

### generally normal at birth, but cannibalism was elev ated in the exposed groups. These results 23 

### contrast with an earlier study (Rigdon and Rennels, 1964) in which rats (strain not specified) 24 

### were fed diets containing benzo[a]pyrene at 1,000 p pm for approximately 28 days prior to 25 

### mating and during gestation. In the earlier study, five of eight treated females mated with 26 

### untreated males became pregnant, but only one deliv ered live young. The treated dam that 27 

### delivered had two live and two stillborn pups; one dead pup was grossly malformed. In the 28 

### remaining treated females, vaginal bleeding was obs erved on GDs 23 or 24. In the inverse 29 

### experimental design, three of six controls mated to benzo[a]pyrene-treated males became 30 

### pregnant and delivered live young. Visceral and sk eletal examinations of the pups were not 31 

### conducted. These studies were limited by the small numbers of animals, minimal evaluation of 32 

### the pups, lack of details on days of treatment (foo d consumption, weight gain), and the 33



### occurrence of cannibalism. 34

35 

### Reproductive effects of in utero exposure via oral route 36 

### MacKenzie and Angevine (1981) conducted a two-gener ation reproductive and 37 

### developmental toxicity study for benzo[a]pyrene in CD-1 mice. Benzo[a]pyrene was 38

### 121 DRAFT - DO NOT CITE OR QUOTE

administered by gavage in 0.2 mL of corn oil to groups of 30 or 60 pregnan t (the F0 generation) 1 

### mice at doses of 0, 10, 40, or 160 mg/kg-day on GDs 7–16 only. Therefore, unlike the standard 2 

### two-generation study, F1 animals were exposed only in utero. F1 offspring were evaluated for 3 

### postnatal development and reproductive function as follows. F1 pups (four/sex when possible) 4



### were allowed to remain with their mothers until wea ning on PND 20. Crossover mating studies 5 

### were then conducted. Beginning at 7 weeks of age, each F1 male mouse (n = 20–45/group) was 6 

### allowed to mate with two untreated virgin females f or 5-day periods for 25 days (for a total 7 

### exposure of 10 untreated females/F1 male), after wh ich time the males were separated from the 8 

### females. Fourteen days after separation from the m ales, (i.e., on days 14–19 of gestation), the 9 

### females were sacrificed and the numbers of implants , fetuses, and resorptions were recorded. 10 

### The F2 fetuses were then examined for gross abnorma lities. Similarly, each F1 female mouse (n 11 

### = 20–55/group), beginning at 6 weeks of age, was pa ired with an untreated male for a period of 6 12 

### months. Males were replaced if the females failed to produce a litter during the first 30-day 13 

### period. All F2 young were examined for gross abnor malities on day 1 of life and their weights 14 

### were recorded on day 4 of age. This F2 group was s acrificed on day 20 postpartum, while the 15



### F1 female was left with a male until the conclusion of the study. At 6 weeks of age, gonads of 16 

### groups of 10 male and 10 female F1 mice exposed to 0, 10, or 40 mg/kg-day benzo[a]pyrene in 17 

### utero were subjected to gross pathology and histolo gic examinations. 18 

### No maternal toxicity was observed. The number of F 0 females with viable litters at 19 

### parturition at the highest dose was statistically s ignificantly reduced by about 35% (Table 4-21), 20 

### but progeny were normal by gross observation. Part urition rates of the low- and mid-dose 21 

### groups were unaffected by treatment, and litter siz es of all treated groups were similar to the 22 

### control group throughout lactation. However, body weights of the F1 pups in the mid-dose and 23 

### high-dose groups were statistically significantly d ecreased on PND 20, by 7 and 13%, 24 

### respectively, and in all treated pups on PND 42, 6, 6, and 10% for the low, mid, and high dose, 25 

### respectively (Table 4-21). The number of F1 pups s urviving to PNDs 20 and 42 was 26 

### significantly reduced at the high dose ( p < 0.01), by 8 and 16%, respectively. When F1 male s 27



### were bred to untreated females and F1 females were mated with untreated males, a marked dose28 

### related decrease in fertility of > 30% was observed in both sexes, starting at the lowest exposure. 29 

### There were no treatment-associated gross abnormalit ies or differences in body weights in the F2 30 

### offspring. 31

32 

### Table 4-21. Reproductive effects in male and femal e CD-1 F1 mice exposed

in utero to benzo[a]pyrene

Effect Dose (mg/kg-d) a

0 10 40 160

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F0 mice with viable litters at parturition Mean ± SEM pup weight (g) at PND 20 Mean ± SEM pup weight (g) at PND 42 F1 male fertility index c F1 female fertility index d 46/60 (77%) 11.2 ± 0.1 29.9 ± 0.2 80.4 100.0 21/30 (70%)

11.6 ± 0.1 28.2 ± 0.3 b

52.0 b

65.7 b 44/60 (73%)

10.4 ± 0.1 b

28.0 ± 0.2 b

4.7 b

0.0 b 13/30 (43%) b

9.7 ± 0.2 b

26.8 ± 0.4 b

0.0 b

0.0 b

a Pregnant F0 mice were administered daily doses of b enzo[a]pyrene in corn oil on GDs 7–16.

b Significantly (

p < 0.05) different from control by unspecified test s.

c Beginning at 7 wks of age, each F1 male mouse (20–4 5/group) was exposed to 10 untreated females over a period of 25 d. Index = (females pregnant/females exposed to males) × 100.

d Beginning at 6 wks of age, each F1 female mouse (20 –55/group) was cohabitated with an untreated male f or a period of 6 mo.

Source: MacKenzie and Angevine (1981).

1 

### Exposure to benzo[a]pyrene caused a marked dose-rel ated decrease in the size of the 2 

### gonads. In F1 males, testes weights were statistic ally significantly reduced. Testes from animals 3 

### exposed in utero to 10 and 40 mg/kg-day weighed app roximately 60 and 18%, respectively, of 4 

### the weight of testes from the control animals (no F 2 offspring were produced in the high dose 5 

### group). This was confirmed by histopathologic obse rvation of atrophic seminiferous tubules in 6 

### the 40 mg/kg-day group that were smaller than those of controls and were empty except for a 7 

### basal layer of cells. The number of interstitial c ells in the testes was also increased in this group

. 8 

### Males from the 10 mg/kg-day group showed limited te sticular damage; although all exhibited 9 

### evidence of tubular injury, each animal had some se miniferous tubules that displayed active 10 

### spermatogenesis. Ovarian tissue was absent or redu ced in F1 females such that organ weights 11 

### were not possible to obtain. Examination of availa ble tissue in these females revealed 12 

### hypoplastic ovaries with few follicles and corpora lutea (10 mg/kg-day) or with no evidence of 13 

### folliculogenesis (40 mg/kg-day). Ovarian tissue wa s not examined in highest-dose females. 14 

### The LOAEL in this study was 10 mg/kg-day, based on decreases in mean pup weight 15 

### (<5%) at PND 42 of F1 offspring of dams treated wit h 10, 40, or 160 mg/kg-day benzo[a]pyrene, 16 

### marked decreases in the reproductive capacity (as m easured by fertility index) of both male and 17 

### female F1 offspring exposed at all three treatment levels of benzo[a]pyrene (by approximately 18 

### 30% in males and females), decreased litter size (b y about 20%) in offspring of F1 dams, and 19



### also the dramatic decrease in size and alteration i n anatomy of the gonads of both male and 20 

### female F1 mice exposed to 10 and 40 mg/kg-day benzo [a]pyrene in utero. A NOAEL was not 21



### identified. 22 

### In another reproductive and developmental toxicity study, benzo[a]pyrene was 23 

### administered by gavage in corn oil to nine female N MRI mice at a dose of 10 mg/kg-day on GDs 24 

### 7–16; a group of nine controls received corn oil (K ristensen et al., 1995). Body weights were 25 

### monitored. F0 females were kept with their offspri ng until after weaning (21 days after 26 

### delivery). At 6 weeks of age, one F1 female from e ach litter (n = 9) was caged with an untreated 27 

### male. The F2 offspring were inspected for gross de formities at birth, weight and sex were 28 

### recorded 2 days after birth, and the pups were sacr ificed. The F1 females were sacrificed after 6 29

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months of continuous breeding. The effects of benz o[a]pyrene treatment on fertility, ovary 1 

### weights, follicles, and corpora lutea were evaluate d. F0 females showed no signs of general 2 

### toxicity, and there was no effect on their fertilit y. F1 females had statistically significantly lowe r 3 

### median numbers of offspring, number of litters, and litter sizes and a statistically significantly 4 

### greater median number of days between litters as co mpared with the controls (Table 4-22). At 5 

### necropsy, the F1 females from treated F0 females ha d statistically significantly reduced ovary 6 

### weights; histologic examination of the ovaries reve aled decreased numbers of small, medium, or 7 

### large follicles and corpora lutea (Table 4-22). On ly one dose group was used in this study, with 8 

### decreased F1 female fertility observed following in utero exposure at the LOAEL of 10 mg/kg9 

### day; no NOAEL was identified. 10

11 

### Table 4-22. Effect of prenatal exposure to benzo[a ]pyrene on indices of

reproductive performance in F1 female NMRI mice

Endpoint (median with range in parentheses) Control a benzo[a]pyrene exposed a (10 mg/kg-d) Number of F2 offspring Number of F2 litters F2 litter size (number of pups per litter) Number of d between F2 litters 92 (26–121) 8 (3–8) 11.5 (6–15) 20.5 (20–21) 22 b (0–86) 3 b (0–8) 8 b (3–11) 21 b (20–23) F1 ovary weight (mg) Number of small follicles Number of medium follicles Number of large follicles Number of corpora lutea 13 (13–20) 44 (1–137) 9 (5–25) 14 (6–23) 16 (6–35) 9 b (7–13) 0 b (0–68) 0 b (0–57) 0 b (0–19) 0 b (0–14)

a Groups of nine female NMRI F0 mice were administere d 0 or 10 mg benzo[a]pyrene/kg by gavage in corn oi l on GDs 7–16. One F1 female from each litter was conti nuously bred with an untreated male for 6 mo.

b Significantly (

p < 0.05) different from control group by Wilcoxon r ank sum test or Kruskall-Wallis two-tailed test.

Source: Kristensen et al. (1995).

12 

### Reproductive effects in adults and repeated oral ex posure 13 

### Rigdon and Neal (1965) conducted a series of experi ments to assess the reproductive 14 

### effects of orally administered benzo[a]pyrene to Ah -responsive white Swiss mice. Female 15 

### animals (number not stated) were administered benzo [a]pyrene at 250, 500, or 1,000 ppm in the 16 

### feed before or during a 5-day mating period. Based on the initial body weight, the doses can be 17 

### estimated as 32, 56, and 122 mg/kg-day, respectivel y. No effect on fertility was observed at any 18 

### treatment dose, even when animals were fed 1,000 pp m benzo[a]pyrene for 20 days prior to 19 

### mating, but interpretation of this finding was marr ed by large variability in numbers of pregnant 20



### females and litter sizes for both treated and contr ol mice. In separate experiments, the fertility of 21 

### five male mice/group was not affected by exposure t o 1,000 ppm in food for up to 30 days prior 22 

### to mating with untreated females. Histologic exami nations showed that male mice fed 500 ppm 23 

### benzo[a]pyrene for 30 days had spermatozoa present in their testes; further details were not 24

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provided. The only treatment-related effect was a lack of weight gain related to feed 1 

### unpalatabilty. While this study suggests that prem ating exposure of male or female mice to 2 

### doses up to 122 mg/kg-day for 20 days may not affec t fertility, the sample sizes were too small 3 

### and study designs were too inconsistent to provide reliable NOAELs and LOAELs for 4 

### reproductive/developmental toxicity. 5 

### In an earlier study (Rigdon and Rennels, 1964) rats (strain not specified) were fed diets 6 

### containing benzo[a]pyrene at 1,000 ppm for approxim ately 28 days prior to mating and during 7 

### gestation. In this study, five of eight treated fe males mated with untreated males became 8 

### pregnant, but only one delivered live young. The t reated dam that delivered had two live and 9 

### two stillborn pups; one dead pup was grossly malfor med. In the remaining treated females, 10 

### vaginal bleeding was observed on GDs 23 or 24. In the inverse experimental design, three of six 11 

### controls mated to benzo[a]pyrene-treated males beca me pregnant and delivered live young. 12 

### Visceral and skeletal examinations of the pups were not conducted. These studies are 13 

### insufficiently reported and of insufficient design (e.g., inadequate numbers of animals for 14 

### statistical analysis) to provide reliable NOAELs or LOAELs for reproductive effects from 15 

### repeated oral exposure to benzo[a]pyrene. 16

17 

### Immunosuppression effects and in utero exposure via oral route 18 

### No studies were found that examined immune system e ndpoints following in utero 19 

### exposure via the oral route. The abstract of a rep ort by Holladay and Smith (1994) referred to 20 

### gavage dosing in a study of immune endpoints in fet uses of pregnant mice exposed to 21 

### benzo[a]pyrene on GDs 13–17, but the methods sectio n of the report described an i.p. injection 22 

### procedure in detail (see Section 4.4.2). 23

24



### 4.3.2. Inhalation 25

26

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Reproductive toxicity and in utero exposure via inh alation 1 

### Archibong et al. (2002) evaluated the effect of exp osure to inhaled benzo[a]pyrene on 2 

### fetal survival and luteal maintenance in timed-preg nant F344 rats. Prior to exposure on GD 8, 3 

### laparotomy was performed to determine the number of implantation sites, and confirmed 4 

### pregnant rats were divided into three groups, consi sting of rats that had four to six, seven to nine, 5 

### or more than nine conceptuses in utero. Rats in th ese groups were then assigned randomly to the 6 

### treatment groups or control groups to ensure a simi lar distribution of litter sizes. Animals 7 

### (10/group) were exposed to benzo[a]pyrene:CB aeroso ls at concentrations of 25, 75, or 8



### 100 μ g/m 3 

### via nose-only inhalation, 4 hours/day on GDs 11–20 . Control animals were either 9 

### sham-exposed to CB or remained entirely unexposed. Results of particle size analysis of 10 

### generated aerosols were reported by several other r eports from this laboratory (Inyang et al., 11 

### 2003; Ramesh et al., 2001a; Hood et al., 2000). Ae rosols showed a trimodal distribution with 12 

### averages of 95% cumulative mass with diameters <15. 85 μ m; 89% <10 μ m; 55% <2.5 μ m; and 13



### 38% <1 μ m (Inyang et al., 2003). Ramesh et al. (2001a) rep orted that the (MMAD ± GSD) for 14



### the 55% mass fraction with diameters <2.5 μ m was 1.7 ± 0.085. Progesterone, estradiol-17 β

, 15 

### and prolactin concentrations were determined in pla sma collected on GDs 15 and 17. Fetal 16 

### survival was calculated as the total number of pups divided by the number of all implantation 17 

### sites determined on GD 8. Individual pup weights a nd crown-rump length per litter per 18 

### treatment were determined on PND 4 (PND 0 = day of parturition). 19 

### Archibong et al. (2002) reported that exposure of r ats to benzo[a]pyrene caused 20 

### biologically and statistically significant ( p ≤ 0.05) reductions in fetal survival compared with t he 21 

### two control groups; fetal survival rates were 78.3, 38.0, and 33.8% per litter at 25, 75, and 22



### 100 μ g/m 3 

### , respectively, and 96.7% with CB or 98.8% per litt er in untreated controls (see Table 23 

### 4-23). Consequently, the number of pups per litter was also decreased in a concentration24 

### dependent manner. The decrease was ~50% at 75 μ g/m 3 

### and ~65% at 100 μ g/m 3 

### , compared with 25 

### sham-exposed and unexposed control groups. No effe cts on hormone levels were observed on 26 

### GDs 15 or 17 at the low-dose. Biologically signifi cant decreases in mean pup weights 27 

### (expressed as g per litter) of >5% were observed at doses ≥ 75 μ g/m 3 

### (14 and 16% decreases at 75 28



### and 100 μ g/m 3 

### , respectively, p < 0.05). Exposure to benzo[a]pyrene did not affec t crown-rump 29 

### length (see Table 4-22). 30

31 

### Table 4-23. Pregnancy outcomes in female F344 rats treated with

benzo[a]pyrene on GDs 11–21 by inhalation

Parameter a Administered concentration of benzo[a]pyrene (μg/m 3 ) 0 (unexposed control) 0

(carbon black) 25 75 100 Implantation sites 8.6 ± 0.2 8.8 ± 0.1 8.8 ± 0.5 9. 0 ± 0.2 8.8 ± 0.1 Pups per litter 8.5 ± 0.2 8.7 ± 0.2 7.4 ± 0.5 b 4.2 ± 0.1 b 3.0 ± 0.2 b

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Survival (litter %) 98.9 ± 1.1 96.7 ± 1.7 78.3 ± 4. 1 b 38.0 ± 2.1 b 33.8 ± 1.3 b

Pup weight (g/litter) 10.6 ± 0.1 8.8 ± 0.1 10.5 ± 0 .2 9.1 ± 0.2 b 8.9 ± 0.1 b

Crown-rump length (mm/litter) 29.4 ± 0.6 29.3 ± 0.5 28.0 ± 0.6 27.3 ± 0.7 27.9 ± 0.7

aValues presented as means ± SEM. bSignificantly different from controls at p < 0.05 by one-tailed post-hoc t-testing following ANOVA.

Source: Archibong et al. (2002).

1 

### benzo[a]pyrene exposure at 75 μ g/m 3 

### caused a statistically significant decrease in pla sma 2 

### progesterone, estradiol, and prolactin on GD 17; th ese levels were not determined in the rats 3 

### exposed to 100 μ g/m 3 

### (Archibong et al., 2002). Plasma prolactin is an indirect measure of the 4 

### activity of decidual luteotropin, a prolactin-like hormone whose activity is necessary for luteal 5 

### maintenance during pregnancy in rats. Control leve ls of prolactin increased from GDs 15 to 17, 6 

### but this increase did not occur in the rats exposed to 75 μ g/m 3 

### . Although the progesterone 7



### concentration at 75 μ g/m 3 

### was significantly lower than in controls on GD 17, the authors thought 8 

### that the circulating levels should have been suffic ient to maintain pregnancy; thus, the increased 9 

### loss of fetuses was thought to be caused by the low er prolactin levels rather than progesterone 10 

### deficiency. The reduced circulating levels of prog esterone and estradiol-17 β among 11 

### benzo[a]pyrene-treated rats were thought to be a re sult of limited decidual luteotropic support for 12 

### the corpora lutea. The authors proposed the follow ing mechanism for the effects of 13 

### benzo[a]pyrene on fertility: benzo[a]pyrene or its metabolites decreased prolactin and decidual 14 

### luteotropin levels, compromising the luteotropic su pport for the corpora lutea and thereby 15 

### decreasing the plasma levels of progesterone and es tradiol-17 β . The low estradiol-17 β may 16 

### decrease uterine levels of progesterone receptors, thereby resulting in fetal mortality. Based on 17 

### biologically and statistically significant decrease s in pups/litter and percent fetal survival/per 18 

### litter, the LOAEL was 25 μ g/m 3 

### ; no NOAEL was identified. 19

20 

### Neurotoxicity and in utero exposure via inhalation 21 

### To evaluate the effects of benzo[a]pyrene on the de veloping central nervous system, 22 

### Wormley et al. (2004) exposed timed-pregnant F344 r ats (10/group) to benzo[a]pyrene:CB 23 

### aerosols by nose-only inhalation on GDs 11–21 for 4 hours/day at a concentration of 100 μ g/m 3



### . 24 

### Results of particle size analysis of genenerated ae rosols were reported by other reports from this 25 

### laboratory (Ramesh et al., 2001a; Hood et al., 2000 ). Particle size analysis of a 100- μ g/m 3 26 

### aerosol showed a trimodal distribution with average s of 95% cumulative mass with diameters 27



### <15.85 μ m; 90% <10 μ m; 67.5% <2.5 μ m; and 66.2% <1 μ m; the MMAD ± GSD for the latter 28



### fraction was 0.4 ± 0.02 μ m (Hood et al., 2000). Dams were maintained to ter m and pups were 29 

### weaned on PND 30. Benzo[a]pyrene reduced the numbe r of live pups to one-third of control 30 

### values without affecting the number of implantation sites. During PNDs 60–70 electrical 31 

### stimulation and evoked field potential responses we re recorded via electrodes implanted into the 32

### 127 DRAFT - DO NOT CITE OR QUOTE

brains of the animals. Direct stimulation of perfo rant paths in the entorhinal region revealed a 1 

### diminution in long-term potentiation of population spikes across the perforant path-granular cell 2 

### synapses in the dentate gyrus of the hippocampus of F1 generation benzo[a]pyrene-exposed 3 

### animals; responses in exposed offspring were about 25% weaker than in control offspring. 4 

### Additionally, NMDA receptor subunit 1 protein (impo rtant for synaptic functioning) was down5 

### regulated in the hippocampus of benzo[a]pyrene expo sed F1 pups. The authors interpreted their 6 

### results as suggesting that gestational exposure to benzo[a]pyrene inhalation attenuates the 7 

### capacity for long-term potentiation (a cellular cor relate of learning and memory) in the F1 8 

### generation. 9 

### In a later study by this same group of investigator s, Wu et al. (2003) evaluated the 10 

### generation of benzo[a]pyrene metabolites in F1 gene ration pups, as well as the developmental 11 

### profile for AhR and mRNA. In this study, confirmed pregnant F344 rats were exposed to 12 

### benzo[a]pyrene:CB aerosols at 25, 75, or 100 μ g/m 3 

### via nose-only inhalation, 4 hours/day, for 13 

### 10 days (GDs 11–21). Control animals were exposed to CB (sham) to control for inert carrier 14 

### effects or they remained untreated. Each benzo[a]p yrene concentration had its own set of 15 

### controls (CB and untreated). Two randomly selected pups were sacrificed on each of PND 0, 3, 16 

### 5, 10, 15, 20, and 30. Body, brain, and liver weig hts were recorded. Benzo[a]pyrene metabolites 17 

### were analyzed in the cerebral cortex, hippocampus, liver, and plasma. A dose-related increase in 18 

### plasma and cortex benzo[a]pyrene metabolite concent rations in pups was observed. 19 

### Dihydrodiols (4,5-; 7,8-; 9,10-) dominated the meta bolite distribution profile up to PND 15 and 20 

### the hydroxy (3-OH-benzo[a]pyrene; 9-OH-benzo[a]pyre ne) metabolites after PND 15 at 21



### 100 μ g/m 3 

### (the only exposure concentration reported). Resul ts indicated a dose-related decrease 22 

### in the ratio of the total number of pups born per l itter to the total number of implantation sites per 23 

### litter. The number of resorptions at 75 and 100 μ g/m 3 

### , but not at 25 μ g/m 3 

### , was statistically 24 

### significantly increased compared with controls. 25

26 

### Adult male reproductive effects and repeated inhala tion exposure 27 

### Inyang et al. (2003) evaluated the effect of sub-ac ute exposure to inhaled benzo[a]pyrene 28 

### on testicular steroidogenesis and epididymal functi on in rats. Male F344 rats (10/group), 29 

### 13 weeks of age, were exposed to benzo[a]pyrene:CB aerosols at 25, 75, or 100 μ g/m 3 

### via nose30 

### only inhalation, 4 hours/day for 10 days. Control animals were either exposed to CB (sham) to 31 

### control for exposure to the inert carrier, or they remained untreated. Each benzo[a]pyrene 32 

### concentration had its own set of controls (CB and u ntreated). Aerosols showed a trimodal 33 

### distribution with averages of 95% cumulative mass < 15.85 μ m; 89% <10 μ m; 55% <2.5 μ m; and 34



### 38% <1 μ m (Inyang et al., 2003); an earlier report from thi s laboratory indicated that the 55% 35 

### mass fraction had a MMAD ± GSD of 1.7 ± 0.085 (Rame sh et al., 2001a). Blood samples were 36 

### collected at 0, 24, 48, and 72 hours after cessatio n of exposure to assess the effect of 37 

### benzo[a]pyrene on systemic concentrations of testos terone and luteinizing hormone (LH), 38

### 128 DRAFT - DO NOT CITE OR QUOTE

hormones that regulate testosterone synthesis. Rep roductive endpoints such as testis weight and 1 

### motility and density of stored (epididymal) sperm w ere evaluated. 2 

### Regardless of the exposure concentration, inhaled b enzo[a]pyrene did not affect testis 3 

### weight or the density of stored sperm compared with controls. However, inhaled benzo[a]pyrene 4 

### caused a concentration-dependent reduction in the p rogressive motility of stored sperm. 5 

### Progressive motility was similar at 75 and 100 μ g/m 3 

### , but these values were significantly lower 6 

### ( p < 0.05) than in any other group. The reduction in sperm motility post-cessation of exposure 7 

### was thought to be the result of benzo[a]pyrene limi ting epididymal function. Benzo[a]pyrene 8 

### exposure to 75 μ g/m 3 

### caused a decrease in circulating concentrations of testosterone compared 9 

### with controls from the time of cessation of exposur e (time 0) to 48 hours post-termination of 10 

### exposure (

p < 0.05). However, the decrease was followed by a compensatory increase in 11 

### testosterone concentration at 72 hours post-cessati on of exposure. Exposure to 75 μ g/m 3 

### caused 12 

### a nonsignificant increase in plasma LH concentratio ns at the end of exposure compared with 13 

### controls, which increased further and turned signif icant ( p < 0.05) for the remaining time of the 14 

### study period. The decreased plasma concentration o f testosterone, accompanied by an increased 15 

### plasma LH level, was thought to indicate that benzo [a]pyrene did not have a direct effect on LH. 16



### The authors also noted that the decreased circulati ng testosterone may have been secondary to 17 

### induction of liver CYP450 enzymes by benzo[a]pyrene . The authors concluded that subacute 18 

### exposure to benzo[a]pyrene contributed to impaired testicular endocrine function that ultimately 19 

### impaired epididymal function. Based on this study, the NOAEL was 25 μ g/m 3 

### and the LOAEL 20



### was 75 μ g/m 3 

### , based on a statistically significant reduction in the progressive motility of stored 21 

### sperm and impairment of testicular function with 10 days of exposure at 75 μ g/m 3



### . 22 

### In a follow up study with longer exposure duration, adult male F344 rats (10 per group) 23 

### were exposed to benzo[a]pyrene:CB aerosols at 75 μ g/m 3 

### via nose-only inhalation, 4 hours/day 24 

### for 60 days (Archibong et al., 2008; Ramesh et al., 2008). Rats in the control group were 25 

### subjected to the nose-only restraint, but were not exposed to the CB carrier. Blood samples were 26 

### collected at 0, 24, 48, and 72 hours after exposure terminated, and the animals sacrificed for 27 

### tissue analyses following the last blood sampling. Data were analyzed statistically for 28 

### benzo[a]pyrene effects on weekly body weights, tota l plasma testosterone and LH 29 

### concentrations, testis weights, density of stored s permatozoa, sperm morphological forms and 30 

### motility, benzo[a]pyrene metabolite concentrations and AHH activity, and morphometric 31 

### assessments of testicular histologies. Relative to controls, the results indicated 34% reduced 32 

### testis weight (

p < 0.025), reduced daily sperm production ( p < 0.025) and reduced intratesticular 33 

### testosterone concentrations ( p < 0.025). Plasma testosterone concentrations were reduced to 34



### about one-third of the level in controls on the las t day of exposure (day 60) and at 24, 48, and 72 35 

### hours later (

p < 0.05). However, plasma LH concentrations in ben zo[a]pyrene exposed rats were 36 

### elevated throughout the blood sampling time periods compared with controls ( p < 0.05). In 37 

### testis, lung, and liver, AHH activity, and benzo[a] pyrene-7,8-dihydrodiol (precursor to the DNA- 38

### 129 DRAFT - DO NOT CITE OR QUOTE

reactive BPDE) and benzo[a]pyrene-3,6-dione metabol ites were significantly ( p < 0.05) elevated 1 

### relative to controls. Progressive motility and mea n density of stored spermatozoa were 2 

### significantly reduced ( p < 0.05). Weekly body weight gains were not affect ed by benzo[a]pyrene 3 

### exposure. These results indicate that 60-day expos ure of adult male rats to benzo[a]pyrene:CB 4



### aerosols at 75 μ g/m 3 

### produced decreased testis weight; decreased intrat esticular and plasma 5 

### testosterone concentrations; and decreased sperm pr oduction, motility, and density. 6

7



### 4.4. OTHER DURATION OR ENDPOINT-SPECIFIC STUDIES 8 

### 4.4.1. Acute Neurological Studies 9 

### Saunders et al. (2001) administered benzo[a]pyrene (>97% pure in peanut oil) to F344 10 

### rats (10/sex/dose group) via a single gavage dose o f 0, 12.5, 25, 50, 100, or 200 mg/kg. Separate 11 

### groups of animals were used for motor activity asse ssment and functional observational battery. 12 

### Motor activity (horizontal, vertical, total distanc e, and stereotypic activity) was measured over 13 

### 2-hour intervals during the nocturnal phase (12 hou rs) for 5 consecutive days after treatment 14 

### (day 1). The functional observational battery, con sisting of 29 tests assessing autonomic, 15 

### neuromuscular, CNS excitability, CNS activity, sens orimotor, and physiological activity, was 16 

### administered before dosing and at 2, 4, 6, 12, 24, 48, 72, and 96 hours after dosing. Body weight 17



### was measured after the functional observational bat tery. 18 

### In both sexes of rat, body weight gain was signific antly reduced on day 4 and/or 5 at 19



### doses ≥ 25 mg/kg (Saunders et al., 2001). Body weight gain s were comparable to controls in the 20 

### 25 and 50 mg/kg groups by day 6. Higher doses of b enzo[a]pyrene resulted in prolonged 21 

### reductions in body weight gain, with reductions of 21–26% in both sexes on day 9 after 22 

### treatment. Weight gain had returned to control lev els after 2 weeks postdosing. At doses of 23 

### 50 mg/kg and higher, all measures of motor activity were significantly depressed in both sexes 24 

### beginning 2 hours after dosing and persisting throu gh the 12-hour post-dosing measurement. At 25 

### the 25 mg/kg dose, significant changes in motor act ivity were not observed until 4–6 hours after 26 

### treatment. When motor activity was measured over 2 4-hour intervals, significant depression of 27 

### motor activity (all measures) was observed at all d oses on day 1 post-treatment and at ≥ 50 mg/kg 28 

### on day 2. The results of the functional observatio nal battery showed significant effects on 29 

### neuromuscular endpoints (decreased mobility and gri p strength, abnormal gait, loss of righting 30 

### reflex), autonomic endpoints (increased defecation and urination), and sensorimotor endpoints 31 

### (decreased response to sound, touch, and pain) at a ll doses and in both sexes. Effects on most 32 

### parameters peaked at 6 hours post-dosing, with retu rn to control levels by 72 hours post-dosing. 33 

### The severity of effects on the FOB tests was greate r in males than in females. This study 34 

### identified a LOAEL of 25 mg/kg benzo[a]pyrene for a cute neurotoxicity; the NOAEL is 35 

### 12.5 mg/kg. 36 

### In a study with nearly identical design, Saunders e t al. (2002) treated male F344 rats 37 

### (10/dose) with single gavage doses of 0, 25, 50, 10 0, or 200 mg/kg benzo[a]pyrene (>97% pure, 38

### 130 DRAFT - DO NOT CITE OR QUOTE

in peanut oil). As in the study by Saunders et al. (2001), separate groups of animals were used 1 

### for motor activity assessment and functional observ ational battery (using the same endpoints 2 

### measured at the same time intervals); in addition, a third group of animals was treated at the 3 

### same doses and used for measurement of benzo[a]pyre ne and its metabolites in plasma and brain 4 

### tissue. At 2, 4, 6, 12, 24, 48, 72, and 96 hours a fter dosing, rats in the metabolism groups were 5 

### sacrificed for analysis of benzo[a]pyrene and its m etabolites in plasma and brain tissue. Dose6 

### and time-dependent effects on locomotor activity we re observed in all treated groups. In 7 

### measurements conducted over the 12 hours on day 1 a fter treatment, significant ( p < 0.05 relative 8 

### to vehicle controls) reductions in total distance t raveled (considered by the authors to represent 9 

### the most accurate measure of locomotor activity) we re observed at doses ≥ 50 mg/kg beginning at 10 

### 2–4 hours post dosing and persisting up to the 12-h our time point. When assessed on a daily 11 

### basis over the 5 posttreatment days, total distance traveled was significantly lower than controls 12 

### in all treated groups on day 1 and in groups expose d to ≥ 50 mg/kg on day 2. Significant ( p < 13 

### 0.05) dose-dependent effects on neuromuscular, sens orimotor, and autonomic parameters were 14 

### observed with benzo[a]pyrene treatment. Significan t increases in the severity of abnormal gait 15 

### and impaired sound and tail pinch responses, as wel l as decreased forelimb grip strength, 16 

### occurred in all dose groups; the severity of effect s peaked at 4 or 6 hours after treatment. 17 

### Increased severity of landing foot splay was observ ed at doses ≥ 50 mg/kg, also peaking in 18 

### severity at 6 hours after treatment. Effects on th is endpoint persisted from 2 to 24 hours post 19 

### treatment in the two high dose groups. Autonomic e ffects, consisting of increased frequency of 20 

### urination and defecation, occurred at doses ≥ 50 mg/kg; in the two high dose groups, these effect s 21 

### began 4 hours after dosing and persisted through 24 hours. The onset and duration of the effects 22 

### observed in this study corresponded well with the p lasma and brain tissue concentrations of 23 

### benzo[a]pyrene and its metabolites. In particular, levels of benzo[a]pyrene metabolites in brain 24 

### tissue peaked between 2 and 6 hours after dosing, a nd plasma levels peaked at 6 hours after 25 

### dosing, in all treated groups. By 72 hours after d osing, metabolite levels had returned to 26 

### baseline. Unmetabolized benzo[a]pyrene was detecte d in brain tissue only in the two highest 27 

### dose groups; levels in both brain tissue and plasma peaked between 2 and 6 hours after dosing. 28 

### Analysis of specific metabolite levels over time sh owed that the diol metabolites comprised a 29 

### larger percentage of the total metabolites at earli er time points (up to 12 hours after dosing), 30 

### while the hydroxyl metabolites predominated at late r times. The authors postulated that the 31 

### observed toxic effects were associated with the pro duction of benzo[a]pyrene diol metabolites 32 

### and/or related generation of ROS rather than an eff ect of the parent compound or hydroxyl 33 

### metabolites. The LOAEL in this study was 25 mg/kg based on suppression of locomotor activity 34 

### and evidence of impairment in the functional observ ational battery. 35 

### In a follow-up study, Saunders et al. (2006) attemp ted to correlate neurobehavioral 36 

### changes with levels of benzo[a]pyrene metabolites, antioxidant enzyme levels, and measures of 37 

### lipid peroxidation in selected brain regions. Sing le oral doses of 0, 25, 50, 100, or 200 mg/kg 38

### 131 DRAFT - DO NOT CITE OR QUOTE

benzo[a]pyrene (97% pure, in peanut oil) were admin istered by gavage to groups of 10 male 1 

### F344 rats. Motor activity, measured as total dista nce traveled over a 2-hour interval during the 2 

### nocturnal phase, was assessed at 0, 2, 4, 6, 12, 24 , 48, 72, and 96 hours post treatment. 3 

### Additional groups of animals were exposed to the sa me doses and sacrificed for collection of 4 

### blood and specific brain tissues (hippocampus, stri atum) at the same time points; benzo[a]pyrene 5 

### and its metabolites were measured by reverse phase HPLC in blood and brain tissues. In 6 

### addition, the activities of superoxide dismutase, c atalase, glutathione peroxidase, and levels of 7 

### malondialdehyde in striatum and hippocampus were de termined at 6 and 96 hours after 8 

### treatment. The authors reported that motor activit y was significantly ( p < 0.01) suppressed as 9 

### early as 2 hours after treatment and remained suppr essed through the 48-hour time point in the 10 

### groups exposed to 50 and 200 mg/kg benzo[a]pyrene; however, the data were not reported, and it 11 

### was not clear whether the 100 mg/kg dose group was affected. The authors indicated that the 12 

### maximum suppression occurred at 12 hours, when moto r activity was 72% lower than controls; 13 

### however, the affected dose group(s) was not reporte d. Data were reported graphically for the 14 

### 25 mg/kg group only; based on the graph, a signific ant ( p < 0.05) suppression of motor activity 15 

### occurred by 4 hours after treatment and persisted t hrough the 12-hour measurement. Activity 16 

### had returned to control levels by 72 or 96 hours in all dose groups. As in the study reported by 17 

### Saunders et al. (2002), the levels of benzo[a]pyren e metabolites in the plasma and brain 18



### correlated with the onset and duration of behaviora l effects; metabolite concentrations peaked 19 

### between 2 and 6 hours after treatment, when suppres sion of motor activity occurred. In addition, 20 

### the toxification/detoxification ratio of benzo[a]py rene metabolites (measured as the ratio of 7,8- 21 

### dihydrodiol 9,10-epoxide to 3[OH]benzo[a]pyrene) in plasma, cortex, cerebellum, hippocampus, 22 

### and striatum was higher (between 2 and 7) for the f irst 6 hours, indicating higher levels of the 23 

### more toxic epoxide metabolite. From 24 to 96 hours after treatment, the ratio was <1, indicating 24 

### that the hydroxyl form predominated. Measurement o f antioxidant enzyme levels in the striatum 25 

### and hippocampus at 6 and 96 hours after dosing show ed significant dose-related decreases in the 26 

### activities of superoxide dismutase and glutathione peroxidase, but enhanced catalase activity and 27 

### increased lipid peroxidation products in the striat um and hippocampus. The authors suggested 28 

### that benzo[a]pyrene-induced acute neurobehavioral e ffects may be associated with oxidative 29 

### stress resulting from generation of ROS and inhibit ion of brain antioxidants. 30 

### Evidence of neurotoxicity has also been reported in studies of acute exposure to 31 

### benzo[a]pyrene administered via parenteral routes. As part of a study of the neurotoxicity of 32 

### motorcycle exhaust, Liu et al. (2002) administered benzo[a]pyrene dissolved in corn oil via i.p. 33 

### injection to ICR mice (sex not specified; 4–6/group ) at doses of 0, 50, or 100 mg/kg-day for 34 

### 3 consecutive days. One day after the last treatme nt, motor nerve conduction velocity was 35



### measured in the tails. The mice were then sacrific ed for removal of the sciatic nerve, which was 36 

### assayed for Na + 

### /K + 

### -ATPase activity. Although the methods section ind icated that 37 

### benzo[a]pyrene-treated animals were tested for rota rod performance, results of this evaluation 38

### 132 DRAFT - DO NOT CITE OR QUOTE

were not reported. Data on motor nerve conduction velocity and Na + 

### /K + 

### -ATPase activity were 1 

### presented graphically. Exposure to benzo[a]pyrene at resulted in significant ( p < 0.05) 2 

### depression of motor nerve conduction velocity (abou t 25 and 40% decrease from control for 50 3 

### and 100 mg/kg-day doses, respectively, based on vis ual inspection data presented graphically), 4



### and decreased the Na + 

### /K + 

### -ATPase activities of sciatic nerves (about 20 and 30% decreases from 5 

### control, respectively). 6 

### Grova et al. (2007) evaluated the effects on short- term exposure to benzo[a]pyrene on 7 

### learning, memory, locomotor activity, and motor coo rdination. Groups of 10 female Balb/c mice 8 

### were treated with i.p. injections of benzo[a]pyrene (>97% pure, in vegetable oil) at doses of 0, 9 

### 0.02, 0.2, 2, 20, or 200 mg/kg-day for 10 consecuti ve days. At the end of exposure, locomotor 10 

### activity was measured in open field activity, and m otor coordination was assessed. Memory and 11 

### learning were evaluated using the Y maze (measures spontaneous alternation behavior) and the 12 

### Morris water maze (measures learning as escape late ncy in repeated trials). Finally, the animals 13 

### were sacrificed for removal of the brain; expressio n of the NMDA R1 receptor (involved in 14 

### cognitive function) subunit gene was measured in ei ght brain regions (cerebral trunk, cerebellum, 15 

### mesencephalum, hippocampus, hypothalamus, thalamus, frontal cortex, and temporal cortex) by 16 

### quantitative real-time reverse transcription PCR as say. In contrast to oral studies of 17 

### benzo[a]pyrene exposure (Saunders et al., 2006, 200 2, 2001), injection of benzo[a]pyrene did not 18 

### affect locomotor activity at any dose in this study . At the lowest doses (0.02 and 0.2 mg/kg19 

### day), benzo[a]pyrene exposure resulted in reduction s in the percentage of spontaneous 20 

### alternation in the Y maze. However, at the higher doses ( ≥ 2 mg/kg-day), there was no difference 21 

### from controls. The authors attributed this finding to increased activity and arousal at the higher 22 

### doses, postulated to result from an anxiolytic effe ct of benzo[a]pyrene. In the 5 th 

### trial of the 23 

### water maze, all benzo[a]pyrene groups showed impair ment; the escape latency was significantly 24 

### higher than controls. In contrast, the higher dose s of benzo[a]pyrene resulted in significantly 25 

### reduced latency during the first trial. No differe nces from control were observed in the 2 nd 

### , 3 rd



### , 26



### and 4 th 

### trials, or 1 day after the last dose. Benzo[a]pyr ene exposure resulted in modulation of 27 

### NMDA-R1 subunit gene expression; expression was sig nificantly ( p < 0.05) increased in the 28 

### cerebellum, mesencephalus, and hippocampus, but dec reased in the frontal cortex and cerebral 29 

### trunk. Effects on gene expression in the cerebellu m, frontal cortex, and hippocampus occurred at 30 

### all doses, while gene expression in the cerebral tr unk was affected only at doses of ≥ 0.2 mg/kg31 

### day, and expression in the mesencephalus occurred o nly at doses of ≥ 2.0 mg/kg-day. 32 

### In a follow-up study, Grova et al. (2008) evaluated the effects of exposure to 33 

### benzo[a]pyrene on anxiety-related behaviors (perfor mance in elevated-plus maze and hole-board 34 

### apparatus). The animals, group sizes, and doses we re the same as in Grova et al. (2007), but 35 

### exposure occurred over 11 days. Behavioral tests w ere administered 30 minutes after the final 36 

### dose, and the animals were sacrificed 1 hour after the tests. Benzo[a]pyrene exposure at 20 and 37 

### 200 mg/kg-day resulted in reduction in anxiety-rela ted behavior as measured by the increased 38

### 133 DRAFT - DO NOT CITE OR QUOTE

number of head dippings in the hole-board apparatus . In the elevated-plus maze test, only the 1 

### highest dose resulted in a significant reduction in anxiety (as measured by higher percentage of 2 

### open arm entries and time spent in open arms). 3

4 

### 4.4.2. Immunological Studies 5 

### Immunological effects (e.g., decreased thymus weigh t, decreased number of B cells in 6 

### spleen) have been reported in Wistar rats repeatedl y exposed to benzo[a]pyrene doses ≥ 10– 7 

### 15 mg/kg-day in standard oral toxicity studies (Kro ese et al., 2001; De Jong et al., 1999). 8 

### Diminished immune responses elicited by the dermal sensitizer, 2,4-dinitrochlorobenzene 9 

### (DNCB) have been observed in C56BL/6 mice orally ex posed to 13 mg/kg-day benzo[a]pyrene, 10 

### 3 times/week for 4 weeks (van den Berg et al., 2005 ). No studies were located examining 11 

### immune system endpoints following inhalation exposu re of animals to benzo[a]pyrene. Results 12 

### from studies of immune system endpoints in mice fol lowing i.p., subcutaneous (s.c.), or 13 

### intratracheal instillation exposure are consistent with immune suppression at dose levels 14 

### generally ≥ 40 mg/kg-day. The available animal studies identif y immune suppression as a 15 

### potential hazard of repeated oral exposure to benzo [a]pyrene at doses ≥ 10–15 mg/kg-day. 16

17



### 4.4.2.1. Oral Exposure Immunological Studies 18 

### As discussed in Section 4.2.1.1, dose-related decre ases in thymus weight and relative 19 

### number of B cells in the spleen were observed in ma le Wistar rats administered gavage doses 20



### ≥ 10 mg/kg-day for 35 days (De Jong et al., 1999). A t higher doses ( ≥ 30 mg/kg-day), serum IgM 21 

### and IgA levels were decreased. At the highest dose tested (90 mg/kg-day), the relative cortex 22 

### surface area of thymus and thymic medullar weight w ere significantly reduced; NK cell activity 23 

### in the spleen was also reduced at this dose. No ef fects on the immune system were observed at 24 

### 3 mg/kg-day (De Jong et al., 1999). In two additio nal subchronic gavage studies, thymus weight 25



### was decreased in a dose-related manner in male Wist ar rats exposed to doses ≥ 15 mg/kg-day 26 

### (5 days/week) for at least 5 weeks and in females e xposed to doses of ≥ 30 mg/kg-day 27 

### (5 days/week) for 90 days (Kroese et al., 2001). N o other immune system parameters were 28 

### assessed in these studies. In an adaptation of the sensitization-specific murine local lymph node 29 

### assay for use in testing immune function, van den B erg et al. (2005) tested several 30 

### immunomodulating compounds, including benzo[a]pyren e, for effects on the T-cell-dependent 31 

### immune response induced by the contact sensitizer, DNCB. Groups of eight male and eight 32 

### female C56BL/6 mice were given gavage doses of 13 m g/kg-day benzo[a]pyrene (purity not 33 

### reported) 3 times/week for 4 weeks, followed by sen sitization with DNCB (0, 0.33, 0.66, or 1% 34 

### solutions in acetone:corn oil) applied topically to the backs of both ears for 3 consecutive days. 35 

### Three days after the last DNCB treatment, the lymph nodes under the application area were 36 

### excised, weighed, and homogenized for preparation o f cell suspensions. The lymph node cell 37 

### suspensions were tested for cell proliferation capa city via measurement of [ 3 

### H]-thymidine 38

### 134 DRAFT - DO NOT CITE OR QUOTE

incorporation. Releases of the cytokines interfero n (IFN)- γ and interleukin (IL)-4 following 1 

### concanavalin A stimulation were assayed by ELISA. At the highest concentration of the 2 

### sensitizer, benzo[a]pyrene treatment reduced [ 3 

### H]-thymidine incorporation into lymphocytes by 3 

### approximately 30% (based on visual inspection of da ta presented graphically; p = 0.008) 4 

### compared with untreated controls. In this treatmen t group, benzo[a]pyrene also reduced the 5



### release of IFN- γ (approximately 75% less than controls based on gra phical data, not significant) 6 

### and IL-4 (approximately 60% less than controls base d on graphical data, p < 0.001). The results 7 

### indicated that benzo[a]pyrene modulates the immune response elicited by the sensitizer DNCB. 8

9



### 4.4.2.2. Inhalation Exposure Immunological Studies 10 

### No studies were located that examined immune system endpoints following inhalation 11 

### exposure of animals to benzo[a]pyrene. 12

13



### 4.4.2.3. Other Exposure Route Immunological Studies 14 

### A number of studies have shown suppression of both humoral and cell-mediated immune 15 

### responses in mice following i.p., s.c., or intratra cheal administration. Dose-related decreases in 16 

### spleen and serum IgM levels after challenge by shee p red blood cells (SRBC) were reported in 17 

### rats (10, 40 mg/kg-day) and mice (5, 20, 40 mg/kg-d ay) following s.c. injection of 18 

### benzo[a]pyrene for 14 days (Temple et al. 1993). Re duced spleen cell response to SRBC and 19 

### lipopolysaccharides were observed in B6C3F 1 

### mice exposed to doses ≥ 40 mg/kg-day 20 

### benzo[a]pyrene by i.p. or s.c. injection for 4–14 d ays (Lyte and Bick, 1985; Dean et al., 1983; 21 

### Munson and White, 1983) or by intratracheal instill ation for 7 days (Schnizlein et al., 1987). 22



### B6C3F 1 

### mice exhibited dose-dependent decreased resistance to Streptococcus pneumonia or 23 

### Herpes simplex type 2 following s.c. injection of 5 , 20, or 40 mg/kg benzo[a]pyrene for 14 days 24 

### (Munson et al., 1985). Galvan et al. (2006) report ed that single i.p. injections of mice with 25 

### 50 mg/kg benzo[a]pyrene caused decreased pro/pre B- lymphocytes and neutrophils in bone 26 

### marrow, without affecting numbers of immature and m ature B-lymphocytes or GR-1+ myeloid 27 

### cells. Several i.p. injection studies reported imm une suppression effects in mice exposed to 28 

### benzo[a]pyrene in utero at doses ranging from 50 to 150 mg/kg; effects inc luded decreased 29 

### spleen or thymus weights, suppression of antibody f orming cells in response to sheep red blood 30 

### cells, decreased spleen, thymic, or bone marrow cel lularity, and disrupted T-cell development 31 

### (Rodriguez et al. 1999; Holladay and Smith, 1995; L ummus and Henningsen, 1995; Holladay 32 

### and Smith, 1994; Urso and Johnson 1988; Urso et al. , 1988; Urso and Gengozian, 1984, 1982, 33 

### 1980). 34



### In contrast to the studies that have shown decremen ts in immune response, 35 

### benzo[a]pyrene may also induce sensitization respon ses. Epicutaneous application of 36 

### benzo[a]pyrene (100 μ g benzo[a]pyrene to C3H/HeN mice followed by ear ch allenge with 20 μ g 37

### 135 DRAFT - DO NOT CITE OR QUOTE

benzo[a]pyrene 5 days later) produced a contact hyp ersensitivity (a significant ear swelling) 1 

### response (Klemme et al., 1987). 2

3



### 4.4.2.4. Other Exposure Route Developmental Immunotoxicy 4



### While there are no oral or inhalation studies of be nzo[a]pyrene on the developing 5 

### immune system, several i.p. injection studies indic ate that this is an area of concern for both cell6 

### mediated and humoral immune ontogeny. In terms of c ell-mediated effects, Urso and Gengozian 7 

### (1984) reported severe suppression of the mixed lym phocyte response and moderate suppression 8 

### of the graft-versus-host response in mice exposed in utero to 150 mg/kg from GD 11 to 17. Both 9 

### effects persisted until 18 mo of age. Holladay and Smith (1994) found that mice exposed to 0, 10 

### 50, 100, or 150 mg/kg from GD 13 to 17 exhibited se vere fetal thymic atrophy when examined 11 

### on GD 18. In the same study, expression data of cel l surface markers (e.g. CD4, CD8) indicate 12 

### that benzo[a]pyrene may inhibit and/or delay thymoc yte maturation, possibly contributing to the 13 

### observed thymic atrophy. Several other studies also show decreased thymocyte numbers and 14 

### disrupted T cell maturation after in utero exposure to benzo[a]pyrene (Rodriguez et al., 1999 ; 15 

### Holladay and Smith, 1995; Lummus and Henningsen, 19 95; Urso et al., 1992; Urso and Johnson, 16 

### 1987). 17 

### In addition to direct thymus effects, Holladay and Smith (1994) reported a large reduction 18 

### in total cellularity in the fetal liver, which is t he primary hematopoietic organ during gestation 19 

### and a major source of thymocyte precursors beginnin g around GD 10-11 in mice (Pennit and 20 

### Vaddeur, 1989; Landreth and Dodson, 2005). This was accompanied by decreased expression of 21 

### terminal deoxynucleotidyl transferase (TdT), an int racellular marker known to be present in 22 

### cortical thymocyte progenitors in the fetal liver ( Fine et al., 1990; Silverstone et al., 1976). This 23 

### data suggests that benzo[a]pyrene also disrupts liv er hematopoiesis during gestation and may 24 

### interfere with prolymphoid seeding of the thymus, p ossibly contributing to thymic atrophy and 25 

### cell-mediated immunosuppression. Rodriguez et al. ( 1999) assessed downstream affects of T cell 26 

### development by showing that CD4 + 

### T-cells were reduced in the spleen of 1-week old m ice 27 

### following in utero benzo[a]pyrene exposure. 28



### There is also some evidence of humoral immune disru ption by benzo[a]pyrene during 29 

### fetal life. In a series of related studies, mice ex posed to benzo[a]pyrene during mid (GD 11 to 30 

### 13) or late (GD 16-18) gestation or both (GD 11 to 17) exhibited severe suppression of the 31 

### plaque-forming cell response to sheep red blood cel ls from 1 wk up to 18 mo after birth (Urso 32 

### and Gengozian, 1984, 1982, 1980). In their analysis of fetal liver cells, Holladay and Smith 33 

### (1994) reported large decreases in expression of Td T and CD45R cellular markers, both of which 34 

### are present on pre-B lymphocytes

. 35

36 

### 4.4.3. Cancer Bioassays (Other Routes of Exposure) 37

### 136 DRAFT - DO NOT CITE OR QUOTE

Cancer bioassays following i.p. injection of mice w ith benzo[a]pyrene have consistently 1 

### found cancer responses. Newborn mouse bioassays in volving postnatal injections of 2 

### benzo[a]pyrene (generally in the dose range of 0.5– 1 μmol/mouse on PNDs 1, 8, and 15) 3 

### consistently found increases in liver or lung tumor s, either increases in incidence of animals with 4 

### tumors or increased numbers of tumors per animal (L aVoie et al., 1994, 1987; Busby et al., 1989, 5 

### 1984; Weyand and LaVoie, 1988; Wislocki et al., 198 6; Buening et al., 1978; Kapitulnik et al., 6 

### 1978). Likewise, i.p. injection of pregnant mice w ith benzo[a]pyrene (100–150 mg/kg) during 7 

### gestation induced increased incidences of offspring with liver or lung tumors, compared with 8 

### controls (Urso and Gengozian, 1984; Bulay and Watte nberg, 1971). A/J adult mice given single 9 

### i.p. injections of benzo[a]pyrene showed a dose-rel ated increase in the number of lung tumors 10 

### per mouse with doses ranging from about 5 to 200 mg /kg (Mass et al., 1993). 11 

### Tumorigenic responses to s.c. administered benzo[a] pyrene have been observed mostly at 12 

### the site of injection in studies with mice (Nikonov a, 1977; Pfeiffer, 1977; Homburger et al., 13 

### 1972; Roe and Walters, 1967; Grant and Roe, 1963; S teiner, 1955; Rask-Nielson, 1950; Pfeiffer 14 

### and Allen, 1948; Bryan and Shimkin, 1943; Barry et al., 1935). 15 

### Positive cancer responses from other routes of expo sure have included: (1) mammary 16 

### tumors in rats with intramammilary administration ( Cavalieri et al., 1991, 1988a, b, c); 17 

### (2) cervical tumors in mice with intravaginal appli cation (Naslund et al., 1987); (3) injection site 18 

### sarcomas with intramuscular injection (Sugiyama, 19 73); (4) respiratory tract tumors in hamsters 19 

### with intratracheal instillation (Henry et al., 1973 ); and (5) tracheal epithelial tumors in rats with 20 

### intratracheal implantation (Topping et al., 1981, N ettesheim et al., 1977). 21

22 

### 4.4.4. Atherogenesis Studies 23 

### Cigarette smoking (see Ramos and Moorthy, 2005; Mil ler and Ramos, 2001; Thirman et 24 

### al., 1994, for review) and, to a more limited degre e, occupational exposure to PAH mixtures 25 

### (Burstyn et al., 2005) have been identified as risk factors associated with the development of 26



### atherosclerotic vascular disease and increased risk for cardiovascular mortality. Based on results 27 

### from in vivo and in vitro animal studies, reactive metabolites of PAHs, including 28 

### benzo[a]pyrene, are thought to play a role in the p rogression of atherosclerosis leading to 29 

### hardening and thickening of the arteries (see Ramos and Moorthey, 2005; Miller and Ramos, 30 

### 2001 for review). For example, in vivo exposure of Sprague-Dawley rats to 10 mg/kg 31 

### benzo[a]pyrene i.p. injections (once/week for 8 wee ks) induced aortic wall lesions related to 32 

### atherosclerosis including loss of endothelial integ rity and increase of smooth muscle cell mass 33 

### (Zhang and Ramos, 1997). The molecular mechanisms responsible for PAH-induced vascular 34 

### injury and the development of atherosclerosis are n ot well established, but current hypotheses 35 

### include roles for cell proliferative responses to i njury of endothelial cells from reactive 36 

### metabolites (including ROS) and genomic alterations in smooth muscle cells from reactive 37

### 137 DRAFT - DO NOT CITE OR QUOTE

metabolites leading to transformed vasculature cell s and eventual plaque formation (Ramos and 1 

### Moorthy, 2005). 2 

### Although many studies have been conducted in animal systems to study the mechanisms 3 

### by which PAHs may participate in the initiation and promotion of atherosclerosis, no studies 4 

### were located that examined relationships between le vels of exposure to benzo[a]pyrene (via 5 

### environmentally relevant routes) and the developmen t of aortic wall lesions related to 6 

### atherosclerosis, with the exception of a series of experiments involving repeated exposure of 7 

### Apolipoprotein E knock out (ApoE-/-) mice to oral d oses of 5 mg/kg benzo[a]pyrene (Knaapen 8 

### et al., 2007; Curfs et al., 2005, 2004; Godschalk e t al., 2003). ApoE-/- mice develop 9 

### spontaneous atherosclerosis, which is thought to be due to enhanced oxidative stress from the 10 

### lack of ApoE (Godschalk et al., 2003). 11 

### Treatment of male ApoE-/- mice with gavage doses of 5 mg/kg-day benzo[a]pyrene for 12 

### 4 days produced increased levels of lipid peroxidat ion-derived DNA modifications (etheno-DNA 13 

### adducts) and BPDE-DNA adducts in aorta, compared wi th unexposed ApoE-/- controls 14 

### (Godschalk et al., 2003). Repeated exposure of mal e ApoE-/- mice to 5 mg/kg once a week for 15



### 12 or 24 weeks did not cause enhancement of the ini tiation of plaques in the aortic arch 16 

### (compared with unexposed ApoE-/- controls), but cau sed larger plaques with increased plaque 17 

### layering and number of lipid cores, and increased p laque content of T-lymphocytes, compared 18 

### with unexposed ApoE-/- controls (Curfs et al., 2004 ). In another study, gavage exposure of male 19 

### ApoE-/- mice with 5 mg/kg benzo[a]pyrene or 5 mg/kg benzo[e]pyrene (BeP) once per week for 20 

### 24 weeks similarly increased plaque size and T-lymp hocyte content (Curfs et al., 2005). In 21 

### addition, exposure to benzo[a]pyrene, and to a less er extent BeP, was associated with increased 22 

### transforming growth factor beta (TGF β 1) protein levels in plaque macrophages; TGF β 1 is 23 

### thought to play a role in the migration of T-lympho cytes. No exposure-related differences were 24 

### noted in the location or number of plaques, oxidati ve DNA damage (assessed by immunostaining 25 

### for 8-hydroxydeoxyguanosine, 8-OHdG), or apoptosis in the plaques (Curfs et al., 2005). As 26 

### expected, the lungs of benzo[a]pyrene-exposed mice showed several benzo[a]pyrene-DNA 27 

### adducts, which were not detectable in the lungs of BeP-exposed or control ApoE-/- mice (Curfs 28 

### et al., 2005). In another study, increased express ion of monocyte-chemoattractant protein-1 29 

### (MCP-1) was found in aortic tissue from male ApoE-/ - mice exposed to 5 mg/kg benzo[a]pyrene 30 

### once per week by gavage for 2 weeks; this protein i s thought to recruit monocytes into 31 

### atherosclerotic lesions (Knaapen et al., 2007). 32 

### In summary, the results of the studies with ApoE-/- mice indicate that repeated oral 33 

### exposure to 5 mg/kg gavage doses of benzo[a]pyrene enhance the progression of (but do not 34 

### initiate) atherosclerosis through a general local i nflammatory process. The involvement of PAH35 

### DNA adducts was not evident in these studies, as in dicated by observations that BeP, which does 36 

### not cause DNA adducts, elicited similar plaque resp onses in ApoE-/- mice as benzo[a]pyrene. 37 

### Although these results demonstrate that repeated or al exposure to 5 mg/kg benzo[a]pyrene can 38

### 138 DRAFT - DO NOT CITE OR QUOTE

enhance atherosclerosis in animals, the altered gen etic disposition of ApoE-/- mice limits their 1 

### usefulness in describing human-relevant dose-respon se relationships for oral exposure to 2 

### benzo[a]pyrene and atherosclerosis. 3

4 

### 4.4.5. Reproductive Studies (Other Routes of Exposu re) 5 

### Mattison et al. (1980) examined the response to i.p . exposure to a single dose of 6 

### benzo[a]pyrene in female DBa/2N mice (n=15 per dose group); effects on fertility, primordial 7 

### oocyte destruction, and response to pregnant mare’s serum gonadotropins were evaluated. In the 8 

### 10 week breeding study, dose groups included the ve hicle control (corn oil) and 10, 100, 200 and 9 

### 500 mg benzo[a]pyrene/kg. Complete infertility was seen in the 200 and 500 mg/day groups, 10 

### with decreased fertility seen in the 10 and 100 mg/ kg dose groups, too. The total number of pups 11 

### born was 137, 91, 28, 0 and 0 and the mean number o f pups per mouse per week was 0.91, 0.61, 12 

### 0.20, 0.0, 0.0 in the 0, 10, 100, 200 and 500 mg/kg dose groups, respectively (p < 0.05 for 13 

### comparison of 0 to 10 mg/kg groups and 20 to 100 mg /kg groups). In a parallel study using a 14 

### single i.p. dose administered 21 days before sacrif ice, the percent of primordial oocytes 15 

### destroyed (compared with controls) was 0, 18, 19, 5 6, 88, 100% for doses of 0, 5, 10, 50, 100, 16 

### and 200 mg benzo[a]pyrene/kg. The differences at d oses ≥ 50 mg/kg were statistically 17 

### significant (p < 0.05) compared with controls. The results from these studies were used to 18



### calculated an ED 50 

### (i.e., dose producing a reduction in fertility or number of oocytes) of 25.5 19 

### mg/kg for fertility reduction and 24.5 mg/kg for pr imordial oocyte destruction. There was no 20 

### effect of benzo[a]pyrene exposure on ovary weight o r response to pregnant mare’s serum 21 

### gonadotropin, indicating that the effect of exposur e did not involve ovulation inhibition. 22 

### Another acute exposure study examined the effect of benzo[a]pyrene exposure on 23 

### ovulatory response (as determined by number of corp ora lutea) in female C57BL/6N mice 24 

### (Swartz and Mattison, 1985). Benzo[a]pyrene was gi ven as a control dose (corn oil vehicle), and 25 

### 1, 5, 50, 100, 500 mg/kg i.p., 20 animlals were inc luded per dose group and 5 were sacrificed at 26 

### weekly intervals. Ovaries were removed and serial sections were examined for histological 27 

### changes and counts of corpora lutea. There was a 3 5% mortality rate in the 500 mg/kg group, 28 

### but no evidence of treatment-related mortality in t he other groups. Mean number of corpora 29



### lutera in controls varied between 5.5 and 10.0 for the samples taken at 1, 2, 3, and 4 weeks post30 

### dose administration, with no time-related trend of increasing or decreasing number. The 1 mg/kg 31 

### dose group exhibited no decrease in number of corpo ra lutea compared with controls at any time 32 

### period (mean count varying between 6.2 and 7.2). T he number of corpora lutea was decreased (p 33 

### < 0.05) at all doses ≥ 5.0 mg/kg at 1 week post-administration (mean 0.0, 2.0, 0.0, 0.0 and 0.0 for 34 

### 5.0, 10, 50, 100 and 500 mg/kg compared with 6.8 an d 10.0 in the control and 1 mg/kg groups, 35 

### respectively); at 2 weeks decreases were seen at ≥ 50 mg/kg and at weeks 3 and 4 a decrease was 36 

### seen only at doses ≥ 100 mg/kg. Thus in addition to the destruction of primordial follicles seen 37

### 139 DRAFT - DO NOT CITE OR QUOTE

in Mattison et al. (1980), this study also demonstr ated an inhibition of ovulation by 1 

### benzo[a]pyrene that was time- and dose-related. 2 

### Miller et al. (1992) used the protocol described by Swartz and Mattison (1985) to further 3 

### examine the ovarian effects of acute benzo[a]pyrene exposure. As in the previous experiment, 4 

### doses of 1, 5, 10, 50, 100 and 500 mg/kg i.p. (with corn oil vehicle control) were administered to 5 

### female C57BL/6N mice (20 per group; 5 sacrificed at 1, 2, 3, and 4 weeks post-dose 6 

### administration). In addition to counts of corpora lutea, total ovarian volume and total and 7 

### individual corpora lutea volume were measured. The results pertaining to dose- and time8 

### dependent descreases in corpora lutea matched the r esults seen in Swartz and Mattison, 1985), 9



### and similar trends were seen in the total ovarian v olume and total corpora luteal volume 10 

### measures. However, volume of individual corpora lu tea was increased in the treated animals 11 

### compared with controls at weeks 2, 3 and 4 post-tre atment. The authors note that the recovery 12 

### seen in the effect on corpora lutea number by 2 wee ks post-treatment at the lower doses may 13 

### reflect an effect specifically on antral follicles, whereas the longer recover period at higher doses 14 

### indicates an additional effect on growing follicles

. 15 

### Borman et al. (2000) compared the ovarian effects o f benzo[a]pyrene, two other PAHs 16 

### (9,10-dimethylbenzanthracene and 3-methylcholanthre ne in female B6C3F 1 

### mice and Fischer 17 

### 344 rats); the ovotoxic 4-vinylcyclohexene (VHC)and its diepoxide metabolite (4- 18 

### vinylcyclohexene diexpoxide, VHD) were also include d to allow calculation of an “ovotoxic 19 

### index.” as positive controls. Doses of 0.0, 0.0075 , 0.015, 0.075, 0.15, 0.75, 3.5, 7.5 and 15 20 

### mg/kg in the mouse, and an additional dose of 60 mg /kg for the rat were administered i.p. 21 

### (sesame oil vehicle) daily for 15 days (6-7 animals per treatment group). The size of the ovaries 22 

### and the number of primordial, primary and secondary (containing an oocyte) follicles was 23 

### determined was determined after sacrifice (4 hours after the last dose administration). The 24



### ovotoxic index was defined as the lowest dose that resulted in a 50% loss (ED 50 

### ) of primordial 25 

### follicles. For benzo[a]pyrene in mice, the ED 50 

### was 3 mg/kg (0.012 mmol/kg) for primordial 26 

### follicles, a 50% loss in primary follicles was seen at 7.5 mg/kg (0.03 mmol/kg), but this level of 27 

### loss of secondary follicles was not seen even at t he highest dose used. The ED 50 

### for primordial 28 

### follicle loss was 0.02 mg/kg for dimethylbenzanthra cene and 0.045 mg/kg for 3- 29 

### methylcholanthrene. In rats, the 60 mg/kg (0.24 mm ol/kg) dose of benzo[a]pyrene resulted in a 30 

### 50% loss of primary and secondary follicles, but a much smaller decrease in primordial follicles 31 

### was seen (approximately 75% of control counts in th e 15 and 60 mg/kg groups). 32

33



### 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MOA 34 

### benzo[a]pyrene is a complete carcinogen in that it both initiates and promotes tumor 35 

### formation. Several mechanistic processes have been associated with benzo[a]pyrene 36 

### carcinogenicity, including oxidative metabolism, wh ich gives rise to reactive intermediates (see 37 

### Section 3.3), and formation of DNA adducts, both of which can lead to genotoxicity and 38

### 140 DRAFT - DO NOT CITE OR QUOTE

mutations in specific cancer-related genes. The ab ility of benzo[a]pyrene to function as a tumor 1 

### promoter may be related to cytotoxicity, AhR affini ty, and upregulation of genes related to 2 

### biotransformation, growth, and differentiation. 3 

### The following sections discuss mechanistic evidence for possible key events in the MOA 4 

### for cancer (a topic that is further discussed in Se ction 4.7.3). Information regarding MOAs for 5



### noncancer effects noted above is discussed in Secti on 4.6.3. 6

7



### 4.5.1. Genotoxicity 8 

### The ability of benzo[a]pyrene to cause mutations an d other forms of DNA damage in 9



### both in vivo and in vitro studies is well documented (see Tables 4-24, 4-25 and 4-26). With 10 

### metabolic activation (inclusion of S9) benzo[a]pyre ne is consistently mutagenic in the 11 

### prokaryotic Salmonella/ Ames and E. coli assays (Table 4-24). A rare exception was observe d, in 12 

### which benzo[a]pyrene did not induce mitotic recombi nation in eukaryotic S. cerevisiae 13 

### regardless of the presence of S9. In mammalian in vitro studies, benzo[a]pyrene is consistently 14 

### mutagenic, clastogenic and induces cell transformat ion both with and without metabolic 15 

### activation (Table 4-25). Cytogenetic damage in the form of chromosomal aberrations, 16 

### micronuclei, sister chromatid exchanges and aneuplo idy are commonplace following 17 

### benzo[a]pyrene exposure as are DNA adduct formation , single strand breaks, and induction of 18 

### DNA repair and unscheduled DNA synthesis. The in v itro mammalian cell assays were 19 

### conducted in various test systems, including human cell lines. 20 

### In in vivo studies, benzo[a]pyrene consistently te sted positive in multiple species and 21



### strains and under various test conditions in the fo llowing assays: cell transformation, 22 

### chromosomal aberrations, DNA adducts, DNA strand br eaks, micronuclei formation, gene 23 

### mutations (H-ras, K-ras, p53, lacZ

, Hprt ), sister chromatid exchanges, sperm abnormality, a nd 24 

### unscheduled synthesis. Negative results were nomin ally interspersed throughout the in vivo 25 

### mammalian assays, except for consistently negative results observed for unscheduled DNA 26 

### synthesis. 27 

### In human in vivo studies, exposures were to mixed PAHs through cigarette smoke or 28 

### occupational exposure. In a subset of these studie s, benzo[a]pyrene-specific DNA adducts have 29 

### been detected, and it has been demonstrated qualita tively that benzo[a]pyrene metabolites 30 

### damage DNA in exposed humans (see Table 4-26). 31

32141 DRAFT - DO NOT CITE OR QUOTETable 4-24. In vitro genotoxicity studies of benzo[a]pyrene in non-mammallian cellsResultReference+S9 – S9Endpoint/Test System: prokaryotic cellsForward mutationS. typhimurium TM677+ – Rastetter et al. (1982)S. typhimurium TM677+ ND Babson et al. (1986)Reverse mutationS. typhimurium TA1537; TA1538+ – Ames et al. (1973)S. typhimurium TA1535– – Ames et al. (1973)S. typhimurium TA98; TA1538+ ND Ames et al. (1975)S. typhimurium TA1537; TA1538+ – Glatt et al. (1975)S. typhimurium TA 1535– – Glatt et al. (1975)S. typhimurium TA98; TA100; TA1538+ ND McCann et al. (1975)S. typhimurium TA 1535– ND McCann et al. (1975)S. typhimurium TA1538+ ND Egert and Greim (1976)S. typhimurium TA1537+ ND Oesch et al. (1976)S. typhimurium TA1538, TA98+ – Wood et al. (1976)S. typhimurium TA98; TA100; TA1537+ – Epler et al. (1977)S. typhimurium TA1535– – Epler et al. (1977)S. typhimurium TA98; TA100+ – Obermeier and Frohberg (1977)S. typhimurium TA100+ ND Tang and Friedman (1977)S. typhimurium TA98+ – Pitts et al. (1978)S. typhimurium TA100+ ND Bruce and Heddle (1979)S. typhimurium TA98, TA100+ ND LaVoie et al. (1979)S. typhimurium TA1538+ – Rosenkranz and Poirier (1979)S. typhimurium TA98, TA100+ – Simmon et al. (1979a)S. typhimurium TA98+ ND Hermann (1981)S. typhimurium TA98, TA100+ ND Alfheim and Randahl (1984)S. typhimurium TA100+ ND Norpoth et al. (1984)S. typhimurium TA98, TA100, TA 1538ND – Glatt et al. (1985)S. typhimurium TA97, TA98, TA100+ – Sakai et al. (1985)142 DRAFT - DO NOT CITE OR QUOTES. typhimurium TA100+ – Carver et al. (1986)S. typhimurium TA98+ – Alzieu et al. (1987)S. typhimurium TA97, TA98, TA100, TA1537+ – Glatt et al. (1987)S. typhimurium TA97, TA98, TA100+ ND Marino (1987)S. typhimurium TA100+ ND Pahlman and Pelkonen (1987)S. typhimurium TA 98, TA100+ – Prasanna et al. (1987)S. typhimurium TA98+ ND Ampy et al. (1988)S. typhimurium TA98, TA100+ ND Bos et al. (1988)S. typhimurium TA98+ ND Lee and Lin (1988)S. typhimurium TA100+ ND Phillipson and Ioannides (1989)S. typhimurium TA98+ ND Antignac et al. (1990)S. typhimurium TA98– ND Gao et al. (1991)S. typhimurium TA98+ ND Balansky et al. (1994)S. typhimurium TA100– ND Balansky et al. (1994)DNA damageE. coli/pol A+ – Rosenkranz and Poirier (1979)E. coli/differential killing test+ – Tweats (1981)E. coliWP2-WP100/rec-assay+ ND Mamber et al. (1983)E. coli/SOS chromotest Pq37+ – Mersch-Sundermann et al. (1992)Endpoint/Test System: Non-mammalian eukaryotesMitotic recombinationS. cerevisiae D4-RDIIND – Siebert et al. (1981)S. cerevisiae D3– – Simmon (1979b)143 DRAFT - DO NOT CITE OR QUOTE12Table 4- 25. In vitro genotoxicity studies of benzo[a]pyrene in mammalian cellsAssay/Test SystemResultReference+S9 – S9Forward MutationHuman AHH-1 lymphoblastoid cellsND + Danheiser et al. (1989)Human lymphoblast (AHH-1) cells (Hprt)ND + Crespi et al. (1985)Human lymphoblastoid (AHH-1) cell lineND + Chen et al. (1996)Human fibroblast (MRC5CV1) cell line (Hprt)– ND Hanelt et al. (1997)Human lymphoblast (TK) cellsND + Barfknecht et al. (1982)Human lymphoblast (TK6) cells+ ND Crespi et al. (1985)Human embryonic epithelial (EUE) cellsND + Rocchi et al. (1980)Mouse L5178Y/HGPRT+ – Clive et al. (1979)Mouse lymphoma (L5178Y/TK+/-) cells+ – Clive et al. (1979)Mouse lymphoma (L5178Y/TK+/-) cells+ NDAmacher et al. (1980);Amacher and Turner (1980)Mouse lymphoma (L5178Y/TK+/-) cells+ – Amacher and Paillet (1983)Mouse lymphoma (L5178Y/TK+/-) cells+ ND Arce et al. (1987)Human HSC172 lung fibroblasts+ – Gupta and Goldstein (1981)Human Q3-wp normal lung keratinocytes+ ND Allen-Hofmann and Rheinwald (1984)Human SCC-13Y lung keratinocytesND + Allen-Hofmann and Rheinwald (1984)Chinese hamster ovary (CHO) cells (aprt)+ ND Yang et al. (1999)Chinese hamster ovary cells (5 marker loci)+ + Gupta and Singh (1982)Chinese hamster V79 cells (Co-cultured with irradiated HepG2 cells) + ND Diamond et al. (1980)Chinese hamster V79 lung epithelial cells+ ND Huberman (1976)Chinese hamster V79 lung epithelial cells+ ND Arce et al. (1987)Chinese hamster V79 lung epithelial cells+ ND O’Donovan (1990)Rat/Fischer, embryo cells/OuaRND + Mishra et al. (1978)DNA damageDNA adducts144 DRAFT - DO NOT CITE OR QUOTEHuman fibroblast (MRC5CV1) cell line+ ND Hanelt et al. (1997)Human peripheral blood lymphocytesND + Wienke et al. (1990)Hamster tracheal cellsND + Roggeband et al. (1994)Chinese hamster V79 lung epithelial cells+ ND Arce et al. (1987)Virus transformed Syrian hamster embryo and mouse C3H10T1/2 cells ND + Arce et al. (1987)Mouse lymphoma (L5178Y/TK+/-) cells+ ND Arce et al. (1987)Rat tracheal cellsND + Roggeband et al. (1994)DNA damage/single strand breaksHuman fibroblast (MRC5CV1) cell line+ ND Hanelt et al. (1997)Human hepatoma (HepG2) cell lineND + Tarantini et al. (2009)Human prostrate carcinoma (DU145) cell lineND + Nwagbara (2007)Mouse embryo fibroblast (C3H/10T1/2 CL 8) cellsND + Lubet et al. (1983)Rat C18 trachea epithelial cellsND +Cosma and Marchok, 1988;Cosma et al. (1988)Rat lymphocytesND + Gao et al. (1991)Unscheduled DNA synthesisHeLa cells+ ND Martin et al. (1978)Human fibroblasts+ ND Agrelo and Amos (1981)Human fibroblasts+ – Robinson and Mitchell (1981)Human HepG2[+] Valentin-Severin et al. (2004)Hamster Primary embryo cellsND + Casto et al. (1976)Hamster tracheal cellsND + Roggeband et al. (1994)Rat Hepatocytes[+] Michalopoulos et al. (1978)Rat tracheal cellsND – Roggeband et al. (1994)DNA repairHuman mammary epithelial cellsND + Leadon et al. (1988)Human skin fibroblastsND + Milo et al. (1978)Baby hamster kidney (BHK21/c13) cells[+] Feldman et al. (1978)145 DRAFT - DO NOT CITE OR QUOTEsecondary mouse embryo fibroblasts (C57BL/6) and human lymphocytes [+] Shinohara and Cerutti (1977)Rat/F344 hepatocytesND + Williams et al. (1982)Cytogenetic damageChromosomal aberrationsHuman blood cellsND + Salama et al. (2001)Human WI38 fibroblasts+ – Weinstein et al. (1977)Chinese hamster CHL+ – Matsuoka et al. (1979)Chinese hamster V79-4 lung epithelial cells– – Popescu et al. (1977)Mouse lymphoma (L5178Y/TK+/-) cells+ ND Arce et al. (1987)Rat Liver RL1 cells+ ND Dean (1981)MicronucleiHuman AHH-1 lymphoblastoid cellsND + Crofton-Sleigh et al. (1993)Human HepG2 liver cellsND + Wu et al. (2003b)Human lymphoblastoid (TK) cellsND + Fowler et al. (2010)Human MCL-5 lymphoblastoid cellsND + Crofton-Sleigh et al. (1993)Human peripheral blood lymphocytes+ ND Lo Jacono et al. (1992)Chinese hamster V79 cellsND + Whitwell et al. (2010)Chinese hamster V79-MZ cellsND + Matsuoka et al. (1999)Sister chromatid exchangesHuman C-HC-4 and C-HC-20 hepatoma cellsND + Abe et al. (1983a,b)Human diploid fibroblast (TIG-II) cell line+ + Huh et al. (1982)Human fibroblastsND + Juhl et al. (1978)Human blood cellsND + Salama et al. (2001)Human peripheral blood lymphocytesND + Rudiger et al. (1976)Human peripheral blood lymphocytesND + Craig-Holmes and Shaw (1977)Human peripheral blood lymphocytesND + Schoenwald et al. (1977)Human peripheral blood lymphocytesND + Wienke et al. (1990)Human peripheral blood lymphocytesND + Wienke et al. (1990)146 DRAFT - DO NOT CITE OR QUOTEHuman peripheral blood lymphocytes+ – Tohda et al. (1980)Human peripheral blood lymphocytes+ ND Lo Jacono et al. (1992)Chinese hamster Don-6 cellsND + Abe et al. (1983a,b)Chinese hamster V79 lung epithelial cells+ – Popescu et al. (1977)Chinese hamster V79 lung epithelial cells+ ND Mane et al. (1990)Chinese hamster V79 lung epithelial cells+ ND Wojciechowski et al. (1981)Chinese hamster V79 lung epithelial cells+ ND Arce et al. (1987)Chinese hamster V79 lung epithelial cellsND + Kulka et al. (1993)Chinese hamster ovary (CHO) cells+ – de Raat (1979)Chinese hamster ovary (CHO) cells+ – Husgafvel-Pursiainen et al. (1986)Chinese hamster ovary (CHO) cellsND + Wolff and Takehisa (1977)Chinese hamster ovary (CHO) cellsND + Pal et al. (1978)Hamster Chl cellsND + Shimizu et al. (1984)Rabbit peripheral blood lymphocytesND + Takehisa and Wolff (1978)Rat ascites hepatoma AH66-BND + Abe et al. (1983a,b)Rat esophageal tumor R1ND + Abe et al. (1983a,b)Rat hepatocyte (immortalized) cell lines (NRL cl-B,NRL cl-C and ARL) + ND Kulka et al. (1993)Rat hepatoma (Reuber H4-II-E) cellsND + Dean et al. (1983)Rat liver cell line ARL18ND + Tong et al. (1981)Rat pleural mesothelial cellsND + Achard et al. (1987)AneuploidyChinese hamster V79-MZ cellsND + Matsuoka et al. (1998)Cell transformationHuman BEAS-2B lung cells+ van Agen et al. (1997)Human breast epithelial (MCF-10F, MCF-7, T24) celllines ND + Calaf et al. (1993)Baby hamster kidney (BHK21/c13) cells+ ND Greb et al. (1980)Golden hamster embyro cells+ ND Mager et al. (1977)Syrian hamster embryo (SHE) cellsND + DiPaolo et al. (1969, 1971)Syrian hamster embryo cellsND + Dunkel et al. (1981)147 DRAFT - DO NOT CITE OR QUOTESyrian hamster embryo cells+ LeBoeuf et al. (1996)Syrian hamster embyro (SHE) cells/focus assayND + Casto et al. (1977)Fetal Syrain hamster lung (FSHL) cellsND + Emura et al. (1980, 1987)Virus infected rat embryo RLV/RE and RAT cells; mouse embryo AKR/Me cells; Syrainhamster embryo cells+ Heidelberger et al. (1983)Virus transformed Syrian hamster embryo and mouse C3H10T1/2 cells ND + Arce et al. (1987)Mouse C3H/10T1/2 embryo fibroblasts+ Nesnow et al. (2002, 1997)Mouse embryo fibroblast (C3H/10T1/2 CL 8) cellsND + Peterson et al. (1981)Mouse embryo fibroblast (C3H/10T1/2 CL 8) cellsND + Lubet et al. (1983)Mouse SHE cells; BALB/c-3t3 cells; C3H/10T1/2 cells; prostate cells + Heidelberger et al. (1983)Mouse BALB/c-3T3 cellsND + Dunkel et al. (1981)Mouse BALB/c-3T3 cells+ Matthews (1993)Mouse BALB/c-3T3 clone A31-1-1ND + Little and Vetroys (1988)Rat embyro cells/SA7 virus transformationND + DiPaolo and Casto (1976)Rat/Fischer, embryo cells (leukemia virus transformed) ND + Dunkel et al. (1981)Rat/Fischer, embryo cells/OuaRND + Mishra et al. (1978)Key: [+] = S9 status not given; “+” = positive; “-“ = negative; ND = not determined.12148 DRAFT - DO NOT CITE OR QUOTE1Table 4-26. In vivo genotoxicity studies of benzo[a]pyreneEndpoint Test System Test Conditions Results Dose Comment ReferenceMutationsHuman/Blood Tlymphocytes (smokersand nonsmokers);HPRTlocus mutation assayT-cells of lung cancer patients(smokers and nonsmokers fromlung cancer patients and populationcontrols with known smokingstatus) analyzed forHPRTlocusmutations.+Smokers andnonsmokersSplicing mutations, basepair substitutions, frameshiftand deletion mutationsobserved. Smokers andnonsmokers had GC-->TAtransversions (13% and 6%,respectively) and GC-->ATtransitions (24% and 35%,respectively) in HPRT geneconsistent with in vitromutagenicity ofbenzo[a]pyreneHackman et al.(2000)MutationsMouse/strains:T-stock,(SEC×C57BL)F1,(C3H×101)F1,(C3H×C57BL)F1forfemales; (101×C3H)F1or (C3H×101)F1formales; dominant-lethalmutation assay12-wk old males dosed withbenzo[a]pyrene i.p. and mated 3.5-6.5 days post-treatment with 12-wkold females from different stocks;sacrificed on days 12-15 aftervaginal plug was observed;females kept in a 5 hr-dark phaseto synchronize ovulation 5 wksbefore the start of the expt.;fertilized eggs collected from 9-11hrs after mating and first-cleavagemetaphase chromosomes prepared20 hrs after mating+500 mg/kgb.w.The % of dominant lethalmutations were in the orderof T-stock = (C3H×101)F1>(SEC×C57BL)F1>(C3H×C57BL)F1Generoso et al.(1979)Mutations, GCMice/strains: Malestocks: (101×C3H)F1;Female stocks (A):(101xC3H)F1, (B):(C3Hx101)F1, (C):(C3HxC57BL)F1,(D):(SECxC57BL)F1,(E):T-stock females;dominant lethalmutationsIn dominant lethal assay (DLA),12-wk-old males dosed i.p. withbenzo[a]pyrene and mated with 10-12 wk-old (#1) stock A females; or(#2) stock B females on the day ofdosing; or with (#3a) with stocksB, C and D females 3.5-7.5 dayspost-dosing, or with (#3b) withstocks B, C, D and E females 3.5-6.5 days post-dosing. Controlgroup mated at time correspondingto 1.5-4.5 days post-treatment inpositiveforDLA;negativefor HT500 mg/kgb.w.Dominant lethal effectswere observed in early tomiddle (4.5-5.5 and 6.5-7.5days post-treatment,respectively) spermatozoaand in preleptotenespermatocytes (32.5-33.5and 34.5-35.5 days posttreatment). In the HTA, nosignificant differencesobserved between treatedand control progeny.Generoso et al.(1982)149 DRAFT - DO NOT CITE OR QUOTEthe test groups.Mutations, GCMice/strains: Malestocks: (101×C3H)F1;Female stocks (A):(101xC3H)F1, (B):(C3Hx101)F1, (C):(C3HxC57BL)F1,(D):(SECxC57BL)F1,(E):T-stock females;heritable translocationsFor heritable translocation assay(HTA), males were mated withstocks B and D females 3.5-7.7days post-benzo[a]pyrenetreatment and male progenyscreened for translocationheterozygosity.–500 mg/kgb.w.No significant differencesobserved between treatedand control progeny.Generoso et al.(1982)Mutations, GC/spot testMouse/C57BL female ×T-strain male; somaticmutation assayMice mated for a 5-day period;101/4days post-appearance ofvaginal plug, females injected i.p.with benzo[a]pyrene or vehicle;offspring (pups) scored forsurvival, morphology and presenceof white near-midline ventral spots(WMVS) and recessive spots (RS).+100 and 500mg/kg b.w.Induced coat color mosaicsrepresent genetic changes(e.g. point mutations) insomatic cells. WMVS andRS represent melanocytecell killing andmutagenicity, respectively.Benzo[a]pyrene caused highincidence of RS but did notcorrelated with WMVS.Russell(1977)MutationsMouse/lacZtransgenic(MutaTMMouse)benzo[a]pyrene given orally incorn oil for 5 consecutive days;sacrificed 14 days after last dosing;Eleven organs analyzed forlacZMF+125mg/kg/dayHighest MF observed incolon followed by ileum >forestomach > bone marrow= spleen > glandularstomach > liver =lung>kidney = heartHakura et al.(1998)MutationsMouse/C57BL/6JDlb-1congenic;Dlb-1locusassayAnimals dosed i) i.p. with vehicleor benzo[a]pyrene 2, 4, or 6 dosesat 96 hr intervals; or ii) single doseof benzo[a]pyrene given i.p. or p.o.alone or 96 hours following asingle i.p. dosing with 10μg/kgTCDD+40 mg/kgb.w.benzo[a]pyrene caused adose-dependent increase inmutant frequency; i.p. routeshowed higher mutantfrequency than p.o. route;induction of mutations wereassociated with Ahresponsiveness.Brooks et al.(1999)150 DRAFT - DO NOT CITE OR QUOTEMutations (Hprtlocus)Mice/C57BL/6 (lacznegative andXPA+/+andXPA-/-); T lymphocytesGavage in corn oil 3 times/wk for0, 1, 5, 9, 13 wks; sacrificed 7 wksafter last treatment+ 13 mg/kgMutation sensitivity:XPA-/->XPA+/+Bol et al.(1998)MutationsMouse/Cockaynesyndrome-deficient(Csb-/-);heterozygous(Csb+/-)and wild type controls(Csb+/+); Hprtmutationfrequency assayCsb-/-/lacZ+/-andCsb+/-/lacZ+/-mice were dosed i.p. withbenzo[a]pyrene thrice a wk for 5,9, or 13 wks; ForHprtMF analysismice were sacrificed 3 wks afterlast treatment; spleenocytescollected; Forlac ZMF analysismice were sacrificed 3 days afterlast treatment and liver, lung andspleen collected.+ 13 mg/kglac ZMF detected in alltissues but no differencesbetween WT andCsb-/-mice;Hprtmutationssignificantly higher inCsb-/-mice than control mice.BPDE-dGuo adducts inHprt gene are preferentiallyremoved in WT mice thanCsb-/-mice.Wijnhoven et al.(2000)MutationsMouse/B6C3F1,forestomach H-ras, Kras&p53mutationsbenzo[a]pyrene given in feed in a2-year chronic feeding study;+5, 25, 100ppm68% K-ras (codons 12,13),10% H-ras (codon 13), 10%p53 mutations; all G-->TtransversionsCulp et al.(2000)MutationsMouse/lacZ/galE(MutaTMMouse); Skinpainting studyMice topically treated with a singledose or in five divided doses daily;sacrificed 7 or 21 days after thesingle or final treatment; DNAfrom skin, liver and lung analyzedfor mutations.+Skor –Li,Lu1.25 or 2.5mg/kg (25 or50μg/mouse)Skin showed significantdose- and time-dependentincrease in mutationfrequency; liver and lungshowed no mutations; MFfor single or multiple-doseregimens were similar.Dean et al.(1998)Mutations/ spottestMouse/T-strainbenzo[a]pyrene given to pregnantmice by gavage in 0.5 ml corn oilon GDs 5-10+10 mg/mouse(5 x 2 mg)Davidson andDawson (1976)Mutations (Hprtlocus)Mouse, 129/Ola (Wildtype); splenic TlymphocytesSingle i.p. injection followed bysacrifice 7 wks post-treatment+0, 50, 100,200, 400mg/kgdose-dependent increase inHprtMFBol et al.(1998)Mutations Mouse, A/J, maleSingle i.p. injection followed bysacrifice 28 days post-treatment+0, 0.05, 0.5, 550 mg/kgDose-dependent increase inlung tissue K-ras codon 12GT mutation frequencyMeng et al.(2010)Mutations/ geneMouse/CD-1; skinpapillomas (Ha-rasmutations)Female mice were initiatedtopically with a single dose ofbenzo[a]pyrene and 1 wk after+600nmol/mouseAbout 90% of papillomascontained Ha-ras mutations,all of them beingColapietro et al.(1993)151 DRAFT - DO NOT CITE OR QUOTEinitiation promoted twice weeklywith 5 nmol TPA for 14 wks. Onemonth after stopping TPAapplication, papillomas collectedand DNA from 10 individualpapillomas were analyzed for Haras mutations by PCR and directsequencing.transversions at codons 12(20% GGA-->GTA), 13(50% GGC-->GTC), and 61(20% CAA--> CTA).Mutation/Invivo-in vitroRat, WistarSingle dose by gavage; urine andfeces collected 0-24, 24-48 and 48-72 hrs post-treatment; urine andextracts of feces tested inS.typhimuriumTA100 strain with orwithout S9 mix andβ-glucuronidase+0, 1, 5, 10,100 mg/kgFecal extracts and urineshowed mutagenicity at andabove 1 and 10 mg/kg b.w.Benzo[a]pyrene,respectively. Highestmutagenic activity observedfor 0-24 hrs post-treatmentfor feces and 24-48 hrs posttreatment for urine withβ-glucuronidase±S9 mix.Willems et al(1991)Mutations, GC/geneD. melanogaster/ sexlinked recessive lethaltestBascmales exposed tobenzo[a]pyrene were mated withvirgin females ofBerlinK ormei9L1strains;±10 mMData inconclusive due tolow fertility rates ofmei-9L1females.Vogel et al.(1983)Mutations, GC/geneD. melanogaster/ sexlinked recessive lethaltestAdultBerlinmales treated orallywith benzo[a]pyrene+ 5 or 7.5 mM Low mutagenic activityVogel et al.(1983)Mutations, GC/geneD. melanogaster/BerlinK and Oregon-K strains;sex-linked recessivelethal testbenzo[a]pyrene dissolved inspecial fat and injected into theabdomen of flies.– 2 and 5 mM Negative at both dosesZijlstra andVogel(1984)Mutations, GC/geneD. melanogaster/ sexlinked recessive lethaltestMaleBerlin Klarvae treated withbenzo[a]pyrene for 9-11 days+ 0.1-4 mMThreefold enhancement inlethals in treated versuscontrolsVogel et al.(1983)152 DRAFT - DO NOT CITE OR QUOTEMutations, GC/geneD. melanogaster/CantonS (WT) males, FM6(homozygous for an Xchromosome) females;sex-linked recessivelethal testAdult male flies were fed on filterssoaked in benzo[a]pyrene for 48 or72 hrs; Treated and control malesmated with FM6 females, malestransferred to new groups offemales at intervals of 3, 2, 2, and3 days; four broods obtained; agroup of 100 daughters of eachmale were mated again; scored for% lethal– 250, 500 ppmAuthors report incompletedissolution ofbenzo[a]pyrene in DMSO asa possible cause of negativeresult.Valencia andHoutchens(1981)Mutation/geneD. melanogaster;somatic mutation - eyecolor mosaicism50 females and 20 females weremated in a culture bottle for 48 hrsallowing females to oviposit;adults then discarded and the eggsallowed to hatch; larvae fed onbenzo[a]pyrene deposited on foodsurface and the emerging adultmales scored for mosaicly coloredeye sectors;+ 1, 2, or 3 mMbenzo[a]pyrene waseffective as a mutagen; nodose-response observedFahmy andFahmy (1980)DNA adducts Human/white blood cellsWorkers were exposed for 6-8hrs/day for at least 4-6 monthsbefore blood collection; leukocyteDNA isolated, digested andbenzo[a]pyrene tetrols analyzed byHPLC with fluorescent detection(HPLC-FD). Low, medium, andhigh exposure groups correspondto < 0.15, 0.15 to 4, > 4 mg/m3 ofbenzo[a]pyrene, respectively.+< 0.15, 0.15to 4, > 4μg/m3ofbenzo[a]pyrenePAH exposure, CYP1A1status and smokingsignificantly affected DNAadduct levels, i.e.CYP1A1(*1/*2or*2A/*2a)>CYP1A1*1/*1;occupational >environmental exposure;smokers > nonsmokers;adducts increased with doseand duration of smokingRojas et al.(2000)DNA adducts Human/white blood cellsCoke oven workers were exposedto PAHs and benzo[a]pyrene-WBCDNA analyzed by HPLC-FD forBPDE-DNA adducts+0.14μg/m3BPDE-DNA adductsdetectable; no significantdifference between smokersand nonsmokers; nocorrelation with airbenzo[a]pyrene levels andadduct levelsMensing et al.(2005)153 DRAFT - DO NOT CITE OR QUOTEDNA adductsand MutationsMouse/C57BL/6lacZtransgenicMice dosed with single i.p.injection of benzo[a]pyrene inDMSO; sacrificed 1, 3, 5, 7, 14,21, and 28 days post-treatment;spleen, lung, liver, kidney andbrain collected, DNA isolated andanalyzed for mutations in laczreporter gene inE. coliand adductsby32P-postlabeling assay.+50 mg/kgb.w.BPDE-dG adduct levelspeaked between 5 and 7days post-treatment,followed by gradual decline;rate of removal highest inlung, liver and spleen andlowest in kidney and brain;mutant frequencies peakedbetween 7 and 14 days inlung, spleen, liver andkidney; brain was notsignificant at any time point.Boerrigter(1999)DNA adductsMice/(Ahr+/+,Ahr+/-,Ahr-/-)Gavage; sacrificed 24 hr post-treatment+100 mg/kgb.w.No induction of CYP inAhr-/-, but all alleles positivefor adduct formationSagredo et al2006DNA adductsMice/C57BL/6JCyp1a1(+/-) andCyp1a1(-/-)Single i.p. injection; sacrificed 24hrs post-treatment; liver DNAanalyzed by32P-postlabeling assay+500 mg/kgb.w.BPDE-DNA adduct levels4-fold higher inCyp1a1(-/-)mice thanCyp1a1(+/-) miceUno et al.(2001)DNA adducts Mouse/B6C3F1benzo[a]pyrene fed in diet for 4(100 ppm) or for 1, 2, 8, 16 and 32wks (5 ppm); sacrificed and liver,lungs, forestomach, small intestinecollected; DNA analyzed by32Ppostlabeling assay+5 ppm (32wks) and 100ppm (4 wks)Linear dose-response in 4-wk study; the 5 ppm groupsshowed a plateau after 4wks of feedingCulp et al.(2000)DNA adducts Mouse/Balb/c;Single i.p. injection; sacrificed 12hrs post-injection; liver andforestomach collected; DNAbinding of [3H]benzo[a]pyreneanalyzed by scintillation counting.+140μCi/100g b.w.Liver DNA had 3-foldhigher binding ofbenzo[a]pyrene than that offorestomachGangar et al.(2006)DNA adductsMice/BALB/cAnN(BALB), CBA/JN(CBA);32P-postlabelingassayAnimals dosed i.p. with or without24 hr pretreatment with TCDD+50 and 200mg/kgAdduct levels similar inboth strains dosed withbenzo[a]pyrene alone.TCDD pre-treatment had agreater suppressive effect onWu et al.(2008)154 DRAFT - DO NOT CITE OR QUOTEadduct formation in BALBrelative to CBA mice at lowdose but resulted in nosignificant difference inadduct levels at high dose.DNA adducts Mice/BALB/c, skin;Four doses of benzo[a]pyrenetopically applied to the shavedbacks of animals at 0, 6, 30 and 54hrs; sacrificed 1 day after lasttreatment; DNA analyzed by32Ppostlabeling assay+4 x 1.2μmol/animalFive adducts spots detectedReddy et al .(1984)DNA adductsMice/Swiss, epidermaland dermal skinSingle topical application onshaved backs; sacrificed 1, 3, and 7days post-treatment; epidermal anddermal cells separated; DNAisolated, digested with DNAseIand estimated DNA binding;adducts separated by HPLC.+250 nmol in150μlacetoneBoth cells positive forbenzo[a]pyrene adducts;epidermis>dermis; adductspersisted up to 7 days with agradual decline in levelsOueslati et al.(1992)DNA adductsRats/CD, PBLs, lungsand liverSingle i.p. injection; sacrificed 3days post-treatment; DNAanalyzed by Nuclease P1-endhanced32P-postlabeling assay.+2.5mg/animalBPDE-dG as major adductsand several minor adductsdetected in all tissuesRoss et al .(1991)DNA adductsRats/Sprague-Dawley,liverSingle i.p. injection followed bysacrifice at 4 hours post-treatment;liver DNA isolated and analyzedby32P-postlabeling assay.+100 mg/kgb.w.Two adduct spots detectedReddy et al .(1984)DNA adductsRat/Lewis; lung andliverAnimals received a single oraldose of benzo[a]pyrene intricaprylin; sacrificed 1, 2, 4, 11,and 21 days post-dosing; analyzedliver and lung DNA for BP-DNAadducts by32P-postlabeling assayand urine for 8-oxodG adducts byHPLC-ECD.+ 10 mg/kgBPDE-dG levels peaked 2days after exposure in bothtissues, higher in lungs thanliver at all time points,decline faster in liver thanlung; Increased 8-oxodGlevels in urine anddecreased levels in liver andlung.Briedé et al.(2004)155 DRAFT - DO NOT CITE OR QUOTEDNA adductsRats/F344;32P-postlabeling assaybenzo[a]pyrene given in the dietfor 30, 60, or 90 days; animalssacrificed and liver and lungisolated and DNA extracted andanalyzed for adducts.+0, 5, 50, 100mg/kgAdduct levels linear at lowand intermediate doses,nonlinear at high dose;Ramesh andKnuckels (2006)DNA adductsRats/Wistar; liver andPBL adductsSingle dose by gavage; sacrificed24 hrs post-dosing; PBL and liverDNA analyzed by32P-postlabelingfor BP-DNA adducts+0, 10, 100mg/kg b.w.At 100 mg dose total adductlevels in PBL were twofoldhigher than the levels inliver; adduct profilesdiffered between PBL andliverWillems et al(1991)DNA strandbreaksRats/Sprague-Dawley;Comet assayinstilled intratracheally with (i)single dose of benzo[a]pyrene inaqueous suspension; sacrificed at3, 24, 48 hrs post-treatment;alveolar macrophages, lung cells,lymphocytes, hepatocytes collected(ii) dose-response study andsacrificed at 24 hours posttreatment; lungs collected;Controls received normal salineinstillation; All cells analyzed bycomet assay.+Expt#1: 3 mgofbenzo[a]pyrene; Expt#2:dose-responsestudy with0.75, 1.5, 3mgbenzo[a]pyreneAll time points showedsignificant increase in SSB(Expt#1); A dose-responsein SSB observed (Expt#2)Garry et al.(2003a,b)DNA strandbreaksAquatic organisms:Carp(Cyprinus carpio),rainbow trout(Oncorhynchus mykiss),and clams (Spisulasachalinensis); CometassayAll organisms acclimatized intanks for 2 days, water changedevery 24 hrs; exposed tobenzo[a]pyrene in DMSO in atank; one third volume of tankcontents changed every 12 hrs;organisms sacrificed at 24, 48, 72,and 96 hrs post-treatment; cellsuspensions prepared from liver(carp and trout) or digestive gland(clam) for comet assay+0.05, 0.25,0.5 and 1ppmSignificant dose-responsefor strand breaks observed;carp and trout liver showedhighest response at 48 hrsand clam digestive glandshowed time-dependentincrease at highest conc.Kim and Hyun(2006)DNA strandbreaksRat, Brown NorwayUDS determined after 5 and 18 hrsof a single i.g. dosing– 62.5 mg/kg negative at both time pointsMullaart et al.(1989)156 DRAFT - DO NOT CITE OR QUOTEUnscheduledDNA synthesisRats/F344;Single i.p. injection ofbenzo[a]pyrene or DMSO;sacrificed at 2 or 12 hrs postexposure; liver isolated, hepatocytecultures were setup and incubatedwith 10 mCi/ml3H-thymidine for 4hrs; washed and autoradiographyperformed–100 mg/kgb.w.benzo[a]pyrene wasnegative at both time pointsMirsalis et al.(1982)UnscheduledDNA synthesisMouse/HOS:HR-1hairless; skinSingle topical application on twospots on the backs after strippingstratum corneum with adhesivetape to enhance penetration;sacrificed 24 hr post-treatment,skin isolated [3H]thymidine;cultured in ; epidermal UDSmeasured+0, 0.25, 0.5and 1% (w/v)in acetoneUDS index showed a dosedependent increase up to0.5% benzo[a]pyrene doseand then plateauedMori et al.(1999)UnscheduledDNA synthesisRat/Brown Norway;liverSingle intragastric injection;sacrificed at 5 and 18 hours postinjection–62.5 mg/kgb.w.benzo[a]pyrene wasnegative at both time pointsMullaart et al.(1989)UnscheduledDNA synthesisMouse/(C3Hf x 101)F1hybrid, germ cellsi.p. injection of benzo[a]pyrene;[3H]Thymidine injection later– 0.3 mL Concentration not specifiedSega(1979)UnscheduledDNA synthesisMouse, early spermatid i.p. injection –250-500mg/kg b.w.Reviewed by Sotomayorand Sega (2000)Sega(1982)ChromosomalaberrationsHamster/Chinese bonemarrowSingle, i.p. injection ofbenzo[a]pyrene dissolved intricapryline; animals sacrificed 24hours post-exposure+25, 50, 100mg/kg b.w.benzo[a]pyrene inducedCAs at 50 mg/kg/bw only,with negative responses atthe low and high doseBayer(1979)ChromosomalaberrationsMice/C57 (high AHHinducible) and DBA(low AHH inducible)strains; 11-day oldembryos; adult bonemarrowsStudy used 4 matings(female×male): C57×C57;DBA×DBA; C57×DBA;DBA×C57; Pregnant mice treatedorally on GD11 withbenzo[a]pyrene; sacrificed 15 hrspost-treatment; material liver, bone+ 150 mg/kgLevels of CAs: hybridembryos > homozygousDBA embryos >homozygous C57 embryos;Tissue AHH activity: C57mothers and their embryos >DBA females and theirAdler et al.(1989)157 DRAFT - DO NOT CITE OR QUOTEmarrow and placenta and embryoscollected; male mice dosedsimilarly and bone marrowscollected; individual embryo cellsuspensions and bone marrowpreparations scored for CAs.Tissue AHH activity measured.homozygous embryos. Noquantitative correlationbetween BP-induced CAsand AHH inducibility. Nodifferences in bone marrowmitotic index of males ofdifferent strains betweencontrol and treatmentgroups.ChromosomalaberrationsMouse/1C3F1 hybrid(101/E1xC31×E1)F1;CAs in bone marrowSingle dose by gavage; sacrificed30 hrs of post-dosing; bonemarrow from femur isolated andanalyzed for CAs+ 63 mg/kgSignificant increase in CAsin benzo[a]pyrene-treatedanimals compared tocontrols.Adler andIngwersen(1989)ChromosomalaberrationsRats/Wistar; PBLsSingle dose by gavage; sacrificed6, 24 and 48 hrs post-treatment;blood from abdominal aortacollected, whole blood cultures setup, CAs scored in 100 firstdivision PBLs per animal–0, 10, 100,200 mg/kgb.w.No difference betweencontrol and treatmentgroups at any dose or at anysampling time observedWillems et al(1991)MicronucleiHamster, Chinese, bonemarrowSingle, i.p. injection ofbenzo[a]pyrene dissolved intricaprylin; animals sacrificed 30hours post-exposure–100, 300, 500mg/kg b.w.Bayer(1979)Micronuclei Mice/B6C3F1 (hybrid);I.p. injection; several doses givento calculate LD50+232 mg/kg(LD50/7);259 mg/kg(LD50/4)Study conducted todetermine the toxicity ofbenzo[a]pyrene (LD50)Salamone et al.(1981)MicronucleiMouse/CD-1 and BDF1;bone marrowDosed orally once, twice or thriceat 24 hr intervals; sacrificed 24 hrsafter last treatment+250, 500,1000, 2000mg/kg b.w.significant increase at alldoses; no dose-response;double dosing at 500 mg/kgdose gave best responseShimada et al.(1990)MicronucleiMouse/CD-1 & BDF1,peripheral bloodreticulocytesGiven single i.p injection; tailblood collected at 24 hr intervalsfrom 0 to 72 hrs+62.5, 125,250, 500mg/kg b.w.maximum response seen at48 hrs post-treatmentShimada et al.(1992)MicronucleiRat/Sprague-Dawley,peripheral bloodreticulocytesGiven single i.p injection; tailblood collected at 24 hr intervalsfrom 0 to 96 hrs+62.5, 125,250, 500,1000 mg/kgb.w.maximum response seen at72 hrs post-treatmentShimada et al.(1992)158 DRAFT - DO NOT CITE OR QUOTEMicronucleiMouse/ICR [Hsd:(ICR)Br]benzo[a]pyrene was heated in oliveoil and given orally as a singledose; males, females and pregnantmothers used; pregnant mice dosedon GDs 16-17 and sacrificed onGDs 17-18; micronuclei evaluatedin adult bone marrow and fetalliver+150 mg/kgb.w.All groups significantlyhigher than controls forMN; Fetal liver moresensitive than any othergroupHarper et al.(1989)MicronucleiMouse/Swiss albino;bone marrowGiven orally in corn oil; sacrificed24 hr post-exposure+75 mg/kgb.w.Koraktar et al.(1993)MicronucleiMouse/Swiss; bonemarrow PCEGiven by gavage and sacrificed 36hrs post-treatment+75 mg/kgb.w.Rao and Nandan(1990)MicronucleiMice/CD-1 and MS/Aestrainsi.p. and p.o. administration +62.5, 125,250, 500mg/kggood dose response by bothroutes, strains; i.p. betterthan p.o.; MS/Ae strainmore sensitive than CD-1strainAwogi and Sato(1989)MicronucleiMouse/BDF1, bonemarrowMale and female mice aged 12-15wks given single i.p. injection ofbenzo[a]pyrene or corn oil;sacrificed 24, 48, and 72 hrs posttreatment; bone marrow smearsprepared, stained with MayGrunwald-Giemsa technique andscored for MN PCEs.+0, 25, 50, 60mg/kg b.w.Positive at all doses, timepoints and sexes tested.Dose-dependent increase inMN observed in both sexes;males responded better thanfemales; highest positiveresponse observed at 72 hrspost-injectionBalansky et al.(1994)MicronucleiMouse, HRA/Skhhairless, keratinocytesSingle topical application +0.5, 5, 50,100, 500mg/mouseHe and Baker(1991)MicronucleiMouse/HOS:HR-1,hairless; skinmicronucleiTopical application once daily for3 days; sacrificed 24 hrs after lasttreatment+0.4, 1, 2, 4mgNishikawa et al.(2005)MicronucleiMice/HR-1 hairless, skin(benzo[a]pyrene withslight radiation)+Exposure to sunlightsimulator to evaluatephotogenotoxicity andchemical exposureHara et al.(2007)MicronucleiRat, Sprague-Dawley,pulmonary alveolarmacrophagesi.t. instillation, once/day for 3 days +25 mg/kgb.w.De Flora et al.(1991)159 DRAFT - DO NOT CITE OR QUOTEMicronucleiRat, Sprague-Dawley,bone marrow cellsi.t. instillation, once/day for 3 days –25 mg/kgb.w.De Flora et al.(1991)MicronucleiFish (Carp, rainbowtrout, clams); Blood andhemolymph+0.05, 0.25,0.5 and 1ppmKim and Hyun(2006)SisterchromatidexchangesHamster/Chinese; SCEsin bone marrow8-12 wk-old animals dosed withtwo i.p. injections ofbenzo[a]pyrene given 24 hrs apart;animals sacrificed 24 hrs after lasttreatment, bone marrow fromfemur isolated and metaphasesanalyzed.+450 mg/kgb.w.Significant increase inmetaphase SCEs inbenzo[a]pyrene-treatedanimals compared tovehicle-treated controls.RoszinskyKocher et al.(1979)SisterchromatidexchangesHamster/Chinese,Animals implanted s.c. withbromodeoxyuridine (BrdU) tablet;2 hrs later given phorone (125 or250 mg/kg) i.p.; another 2 hrs laterdosed i.p. with benzo[a]pyrene; 24hrs post-BrdU dosing, animalsinjected with colchicine 10 mg/kgb.w., sacrificed 2 hrs later; bonemarrow from femur prepared forSCE assay+50 or 100mg/kg b.w.SCEs increased with lowdose of phoronesignificantly.Bayer et al.(1981)SisterchromatidexchangesHamster/Syrian, fetalliveri.p. injection to pregnant animalson GDs 11, 13 or 15; fetal liverSCEs were analyzed+50 and 125mg/kg b.w.Produced doubling of SCEfrequencyPereira et al.(1982)SisterchromatidexchangesHamster/Chinese, bonemarrowNA +2.5, 25, 40,50, 75, 100mg/kd b.w.Frequency of SCEsincreased≥40 mg/kg b.w.Bayer(1979)SisterchromatidexchangesMouse/DBA/2 &C57BL/6, bone marrowcellsTwo intragastric injections given;mice implanted with BrdU tablets,sacrificed on day 5, SCE estimated+10 or 100mg/kg b.w.SCEs and BP-DNA adductsin the order of C57Bl/6(AHH-inducible) < DBA/2(AHH-noninducible)Wielgosz et al.(1991)SisterchromatidexchangesMouse/DBA/2 &C57BL/6, spleeniclymphocytesTwo intragastric injections given;mice killed on 5th day and cellscultured for 48 hrs with BrdU.+10 or 100mg/kg b.w.SCEs and BP-DNA adductsin the order of C57Bl/6(AHH-inducible) < DBA/2(AHH-noninducible)Wielgosz et al.(1991)160 DRAFT - DO NOT CITE OR QUOTESisterchromatidexchangesRats/Wistar; PBLsSingle dose by gavage; sacrificed6, 24 and 48 hrs post-treatement;blood from abdominal aortacollected, whole blood cultures setup, SCEs scored in 50 seconddivision metaphases in PBLs peranimal+0, 10, 100,200 mg/kgLinear dose-response at anysampling time, however,significant at the highestdose only; no interactionbetween dose and samplingtimeWillems et al(1991)CelltransformationHamsters/LVG:LAKstrain (virus free);Transplacental hostmediated assayPregnant animals dosed i.p. withbenzo[a]pyrene on GD 10;sacrificed on GD 13, fetal cellcultures prepared, 10×106cells/plate; 5 days post-culturetrypsinized; subcultured every 4-6days thereafter and scored forplating efficiency andtransformation.+3 mg/100 gb.w.Quarles et al.(1979)8-oxodG, 8-oxodeoxyguanosine; AHH, aryl hydrocarbonhydroxylase; benzo[a]pyrene, benzo[a]pyrene; BPDE,benzo[a]pyrene diol epoxide; BrdU,bromodeoxyuridine; CAs, chromosomal aberrations; CSB, Cockayne syndrome; CYP, cytochrome P450; DLA, dominant lethal assay; DMSO,dimethylsulfoxide; ECD, electrochemical detection;FD, fluorescence detection; FM6, First Multiple No.6 is an X chromosome with a complex of inversions(tosuppress cross-over) and visible markers such as yellow body, white eyes and narrow eyes. GC, germlinecell; GD, gestational day; HPLC, high performanceliquid chromatography; HPRT, hypoxanthine-guanine phosphoribosyl transferase; HTA, heritable translocation assay; i.p., intraperitoneal; i.t., intratracheal; Li,Liver; Lu, Lung; MF, mutation frequency; PBL, peripheral blood lympbocytes; PCE, polychromatic erythrocytes; SCEs, sister chromatid exchanges; SFS,synchronous fluorescence spectrometry; Sk Skin TCDD, 2,3,7,8-tetrachlorodibenzodioxin; TPA, 12-tetradecanoyl-O-phorbol acetate; UDS, unscheduled DNAsynthesis; USERIA, ultrasensitive enzyme radioimmunoassay; WT, wild-type; XP, xeroderma pigmentosum;

### 161 DRAFT - DO NOT CITE OR QUOTE

1 

### 4.5.2. Metabolic pathways 2 

### Diol epoxide pathway 3 

### benzo[a]pyrene diol epoxide metabolites interact pr eferentially with the exocyclic amino 4 

### groups of deoxyguanine and deoxyadenine (Geacintov et al., 1997; Jerina et al., 1991). Adducts 5 

### may give rise to mutations unless these adducts are removed by DNA repair processes prior to 6 

### replication. The stereochemical nature of the diol epoxide metabolite (i.e., anti- vs. syn-diol 7 

### epoxides) affects the number and type of adducts an d mutation that occurs (Geacintov et al., 8 

### 1997). Transversion mutations (e.g., GC → TA or AT → TA) are the most common type of 9 

### mutation found in mammalian cells following diol ep oxide exposure (Bostrom et al., 2002). 10 

### Strong evidence for the association between benzo[a ]pyrene activation by the diol 11 

### epoxide pathway and key DNA-reactive and mutational events associated with tumor initiation 12 

### comes from the following observations: (1) (+)-ant i-BPDE is very reactive with guanine 13 

### residues in DNA (Koreeda et al., 1978; Jeffrey et a l., 1976); (2) (+)-anti-BPDE is more potent 14 

### than benzo[a]pyrene, benzo[a]pyrene phenols, and be nzo[a]pyrene diols in mutagenicity assays 15 

### in bacterial and mammalian cells (Malaveille et al. , 1977; Newbold and Brookes, 1976); 16 

### (3) When administered by ip injection to newborn mi ce, (+)-anti-BPDE is more potent than 17 

### benzo[a]pyrene phenols and benzo[a]pyrene diols and much more potent than benzo[a]pyrene 18 

### itself in lung tumorigenicity assays (Chang et al., 1987; Buening et al., 1978; Kapitulnik et al., 19 

### 1978); (4) (+)-anti-BPDE treatment resulted in ras gene codon 12 G → T point mutations, the 20 

### activation of the H-ras-1 proto-oncogene and transf ormation of NIH/3T3 cells (Marshall et al., 21 

### 1984); (5) (+)-anti-BPDE forms DNA adducts at speci fic “hotspots” in the p53 tumor suppressor 22 

### gene that are commonly mutated in lung and other ca ncer patients (Denissenko et al., 1996; 23 

### Puisieux et al., 1991); (6) lung tumors from nonsmo king patients who were chronically exposed 24 

### to smoky coal emissions contained mutated p53 and s howed a spectrum of mutations consistent 25 

### with (+)-anti-BPDE-associated mutations in the K-ra s oncogene (DeMarini et al., 2001); (7) 26



### elevated blood BPDE-DNA adducts have been observed in coke oven workers and chimney 27 

### sweeps, occupations associated with increased risks of cancer from PAH-containing complex 28 

### mixtures (Pavanello et al., 1999); (8) the spectrum of mutation in the K-ras, H-ras, and p53 genes 29 

### in forestomach tumors of mice fed benzo[a]pyrene in the diet for 2 years was consistent with (+)- 30 

### anti-BPDE DNA reactions (Culp et al., 2000); (9) K- ras mutations found in lung tumors from 31 

### A/J mice given single i.p. injections of benzo[a]py rene showed several guanine mutations at 32 

### codon 12, which are indicative of (+)-anti-BPDE DNA adduct formation (Ross and Nesnow, 33 

### 1999; Nesnow et al., 1998a, b, 1996, 1995; Mass et al., 1993); and (10) the major DNA adduct 34 

### formed in a murine embryonic fibroblast line transf ected with human p53 DNA and exposed to 35 

### 1uM benzo[a]pyrene for 96 hours was (+)-anti-BPDE-D NA. The concomitant spectrum of p53 36 

### mutations in the latter study had features similar to those found in human lung cancer: 37

### 162 DRAFT - DO NOT CITE OR QUOTE

predominance of G → T mutations, strand bias of transversions, and muta tion hot spots at codons 1 

### 157 to 158 (Liu et al., 2005). 2 

### As pointed out by Penning et al. (1999), the associ ation between BPDE-DNA adducts 3 

### and tumors from benzo[a]pyrene exposure is not enti rely specific given that dihydrodiol and diol 4 

### epoxides of benzo[a]pyrene are less potent tumorige nic agents in mouse skin than the parent 5 

### material (Slaga et al., 1977; Chouroulinkov et al., 1976) and oxidative damage to DNA has been 6 

### observed in rats treated with benzo[a]pyrene (Kim a nd Lee, 1997) and human mammary 7 

### epithelial cells exposed to benzo[a]pyrene (Leadon et al., 1988). In addition, although BPDE8



### DNA adduct levels in forestomach tissue were linear ly related to the amount of benzo[a]pyrene 9 

### consumed by mice in a 28-day study (Culp et al., 20 00, 1998, 1996a; Culp and Beland, 1994), 10 

### levels of BPDE-DNA adduct in lung and liver tissue (which did not develop tumors with 2 years 11 

### of exposure to benzo[a]pyrene in the diet) were sim ilar at 28 days to those in forestomach tumors 12 

### (Goldstein et al., 1998). These observations sugge st that BPDE-adduct levels alone are not the 13 

### only path to benzo[a]pyrene-induced tumors and prov ide indirect evidence for the other 14 

### mutagenic pathways to benzo[a]pyrene tumor initiati on. 15

16 

### Radical cation pathway 17



### Radical cation formation involves a one-electron ox idation by CYP or peroxidase 18 

### enzymes (i.e., horseradish peroxidase, prostaglandi n H synthetase) that produces electrophilic 19 

### radical cation intermediates (Cavalieri and Rogan, 1995, 1992). Radical cations can be further 20 

### metabolized to phenols and quinones (Cavalieri et a l., 1988d, e), or they can form unstable 21 

### adducts with DNA that ultimately result in depurina tion (Cavalieri et al., 2005, 1993; Rogan et 22 

### al., 1993). The predominant depurinating adducts o ccur at the N-3 and N-7 positions of adenine 23 

### and the C-8 and N-7 positions of guanine (Cavalieri and Rogan, 1995). 24 

### Abasic sites resulting from base depurination under go error-prone excision repair and can 25



### induce mutations such as those found in the H-ras o ncogene in mouse skin (Chakravarti et al., 26 

### 2000). One pathway to the formation of depurinatin g DNA adducts involves the formation of 27 

### DNA-reactive radical cations from benzo[a]pyrene vi a CYP peroxidases (Cavalieri and Rogan, 28 

### 1995). In mouse skin exposed to 200 nmol benzo[a]p yrene for 4 hours, a mix of stable and 29 

### depurinating DNA adducts was found: (+)-anti-BPDE DNA and depurinating adducts accounted 30 

### for 22 and 74% of the identified adducts, respectiv ely (Rogan et al., 1993). When mouse skin 31 

### was exposed to either the benzo[a]pyrene-7,8-diol o r BPDE, only stable BPDE-DNA adducts 32 

### were found (Rogan et al., 1993). In mouse skin tum ors induced by benzo[a]pyrene and 33 

### promoted by TPA, 7/13 examined tumors had H-ras onc ogene mutations attributed to apurinic 34 

### sites generated by loss of N-7 and C-8 guanine addu cts, and 2/13 tumors had H-ras mutations 35 

### attributed to loss of N-7 adenine adducts (Chakrava rti et al., 1995). Results from the Rogan et al. 36 

### (1993) and Chakravarti (1995) studies provide stron g in vivo evidence for the importance of both 37

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the diol epoxide and the radical cation pathways in the activation of benzo[a]pyrene to initiate 1 

### mouse skin tumors, possibly by inducing mutations i n critical genes. 2

3 

### o-Quinone/ROS pathway 4 

### The o-Quinone metabolites of PAHs are formed by enz ymatic dehydrogenation of 5 

### dihydrodiols (Bolton et al., 2000; Penning et al., 1999; Harvey, 1996; ATSDR, 1995). DHH 6 

### enzymes are members of the α -keto reductase gene superfamily. o-Quinone metabo lites are 7 

### potent cytotoxins, are weakly mutagenic, and are ca pable of producing a broad spectrum of DNA 8 

### damage. These metabolites can interact directly wi th DNA as well as result in the production of 9 

### ROS (i.e., hydroxyl and superoxide radicals) that m ay produce further cytotoxicity and DNA 10 

### damage. The DNA damage caused by o-quinones may in clude the formation of stable adducts 11 

### (Balu et al., 2004), N-7 depurinating adducts (McCo ull et al., 1999), oxidative base damage (i.e., 12 

### 8-oxo-2’-deoxyguanosine or 8-oxo-dG) (Park et al., 2006a), and strand scission (Flowers et al., 13 

### 1997). The ROS generated by the o-quinone metaboli tes of benzo[a]pyrene and other PAHs 14 

### have been shown to induce mutation in the p53 tumor suppressor gene using an in vitro yeast 15 

### reporter gene assay (Park et al., 2008; Shen et al. , 2006; Yu et al., 2002). 16 

### The o-quinone/ROS pathway also can produce depurinated DNA adducts fr om 17 

### benzo[a]pyrene metabolites (Jiang et al., 2007; 200 5). In this pathway, and in the presence of 18 

### NAD(P) + 

### , AKR oxidizes benzo[a]pyrene-7,8-diol to a ketol, which subsequently forms 19 

### benzo[a]pyrene-7-8-dione. This and other PAH o-qui nones react with DNA to form unstable, 20 

### depurinating DNA adducts. In the presence of cellu lar reducing equivalents, o-quinones can also 21 

### activate redox cycles which produce DNA-ROS (Pennin g et al., 1996). DNA damage in in vitro 22 

### systems following exposure to benzo[a]pyrene-7,8-di one or other o-quinone PAH derivatives 23 

### occurs through the AKR pathway and can involve the formation of stable DNA adducts (Balu et 24 

### al., 2004), N-7 depurinated DNA adducts (McCoull et al., 1999), DNA damage from ROS 25 

### (8-oxo-dG) (Park et al., 2006a) and strand scission (Flowers et al., 1997, 1996). 26 

### Benzo[a]pyrene-7,8-dione and other PAH o-quinones h ave been shown to induce mutations in 27 

### the p53 tumor suppressor gene using an in vitro yea st reporter gene assay (Park et al., 2008; Shen 28 

### et al., 2006; Yu et al., 2002). When the yeast wer e exposed to varying concentration of 29 

### benzo[a]pyrene-7,8-dione or (+)-anti-BPDE, levels o f 8-oxo-dG or (+)-anti-BPDE-DNA, 30 

### adducts were linearly related to p53 mutagenic freq uencies with similar slopes, suggesting that 31 

### these two types of DNA lesions were equipotent in p roducing p53 mutations in this system (Park 32 

### et al., 2008). When the p53 mutations were sorted into dominant and recessive mutants, the 33 

### dominant mutations clustered to p53 mutation hotspo ts observed in human lung cancer tissue 34 

### (Park, 2008). The combined results provide strong in vitro evidence for the potential for the o35 

### Quinone/ROS pathway to produce several DNA-damaging products from benz o[a]pyrene (e.g., 36 

### benzo[a]pyrene-7,8-dione and ROS) that lead to p53 mutations associated with human lung 37 

### cancer. In support of the operation of this pathwa y, and the other bioactivation pathways, in 38

### 164 DRAFT - DO NOT CITE OR QUOTE

humans, Jiang et al. (2007) used liquid chromatogra py-mass spectrometry (LC-MS) to provide 1 

### evidence for the formation of radical cations, diol epoxides, and o-quinones in cultured human 2 

### lung H358 cells following exposure to 4 μ M [ 3 

### H]-benzo[a]pyrene. 3

4

5 

### 4.5.3. Mechanistic Studies- Mutagenesis and Tumor I nitiation 6

7 

### Oncogene/tumor suppressor gene mutations (in vivo) 8 

### DeMarini et al. (2001) demonstrated mutations in th e p53 tumor suppressor gene and the 9 

### K-ras oncogene in the lung tumors of nonsmokers, wh ose tumors were associated with exposure 10 

### to smoky coal. Lung tumors were obtained from 24 n onsmoking women from China (age 30– 11 

### 63, mean age 48.5 ± 8.8 years) who used smoky coal in their homes without chimneys. 12



### Bronchioloalveolar adenocarcinoma and acinar adenoc arcinoma were observed in 54 and 46% of 13 

### the women studied, respectively. The observed muta tions in lung tumors were primarily G → T 14 

### transversions at either K-ras or p53. Mutation hot spots in the lung tumors examined 15 

### corresponded with hot spots for PAH adducts (codon 154), cigarette smoke associated mutations 16 

### (codon 249), and both of these events together (cod on 273). The mutation spectrum was 17 

### described as unique and consistent with exposure to PAHs in the absence of cigarette smoke. 18 

### Mutations in the K-ras, H-ras, and p53 genes were a ssessed in forestomach tumors 19 

### (n = 31) of mice fed benzo[a]pyrene in the diet (0, 5, 25, or 100 ppm) for 2 years (Culp et al., 20 

### 2000). Forestomach tumors had K-ras mutations (68% of tumors) that were G → T or C 21 

### transversions in codon 12 or 13. H-ras (codon 13) and p53 mutations characterized as G → T or 22



### C transversions were also each found in 10% of fore stomach tumors. 23 

### K-ras mutations were observed in A/J mouse lung tum ors (Nesnow et al., 1998a, b, 1996, 24 

### 1995; Mass et al., 1993). Benzo[a]pyrene was admin istered to male A/J mice (20/group) as a 25 

### single i.p. injection (0, 10, 50, 100, or 200 mg/kg in tricaprylin) and the presence of lung 26 

### adenomas were evaluated 8 months following injectio n. The number of lung adenomas/mouse 27 

### was significantly greater than control ( p < 0.05) for benzo[a]pyrene doses ≥ 50 mg/kg. Lung 28 

### tumor DNA was isolated and DNA sequence analysis of K-ras mutations was performed for 19 29 

### separate lung tumors. The DNA sequence analysis de monstrated several guanine mutations at 30 

### codon 12, including GGT → TGT (56% of tumors), GGT → GTT (25% of tumors), and 31



### GGT → GAT (19% of tumors). 32 

### H-ras mutations were studied in skin papillomas of SENCAR mice resulting from dermal 33 

### initiation by benzo[a]pyrene or benzo[a]pyrene-7,8- dihydrodiol (400 nmol) followed by TPA 34 

### promotion (Chakravarti et al., 2000, 1995). PCR am plification of the H-ras gene and sequencing 35 

### revealed that codon 13 (GGC to GTC) and codon 61 (C AA to CTA) mutations in papillomas 36 

### corresponded to the relative levels of depurinating adducts of guanine and adenine, despite the 37 

### formation of significant amounts of stable DNA addu cts. 38

### 165 DRAFT - DO NOT CITE OR QUOTE

1 

### DNA adducts in target tissues detected following ch emical exposure 2 

### DNA adducts of benzo[a]pyrene have been measured in target tissues of humans exposed 3 

### to PAH mixtures and experimental animals exposed to benzo[a]pyrene. Phillips et al. (2002) 4 

### provided a review of smoking-related DNA adducts de tected in human respiratory tract tissues. 5 

### BPDE-DNA adducts were detected in the lung, parench yma of cigarette smokers with lung 6 

### cancer (Godschalk et al., 2002; Bartsch et al., 199 9; Alexandrov et al., 1992). DNA was isolated 7 

### from the normal tissue (i.e., noncancerous) of the lung which was obtained during surgery. A 8 

### study using lung samples obtained on autopsy reveal ed that the average level of BPDE-DNA 9 

### adducts was higher in smokers (4.46 ± 5.76 per 10 8 

### bases) compared to ex-smokers (4.04 ± 2.37 10 

### per 10 8 

### bases) and nonsmokers (1.76 ± 1.69 per 10 8 

### bases) (Lodovici et al., 1998). 11 

### BPDE-DNA adducts were measured in skin biopsies of eczema patients treated with coal 12 

### tar preparations (Godschalk et al., 2001). Godscha lk et al. (1998a) performed a study examining 13 

### the level of DNA adducts in biopsies of treated ski n and in WBCs in psoriasis patients being 14 

### treated with coal tar. Urinary 1-OH-Py levels were serially monitored in all subjects. Skin 15 

### biopsies were taken before and after five treatment s, at which point the average number of DNA 16 

### adduct levels increased from 2.9 to 63.3 per 10 8 

### nucleotides. Total WBC DNA adducts increased 17 

### from 0.33 to 0.89 per 10 8 

### after five treatments, and then doubled to 1.59 pe r 10 8 

### when sampled 18 

### 1 week later. There was an increase in 1-OH-Py lev els from 0.75 to 186 μ g/L after one treatment 19



### and to 266 μ g/L after five treatments. One week later, mean 1- OH-Py levels were reduced to 20



### 2.4 μ g/L. Adduct levels in the skin increased over 20-f old with five treatments, while WBC 21 

### adduct levels approximately doubled over the same p eriod. 22



### DNA adduct levels were examined in the forestomach of groups of female B6C3F 1 

### mice 23 

### fed benzo[a]pyrene in the diet at concentrations of 5, 25, or 100 ppm for 28 days (Culp et al., 24 

### 2000, 1998, 1996a, b; Culp and Beland, 1994). [ 32 

### P]-postlabeling of forestomach DNA of 25 

### benzo[a]pyrene-treated mice revealed one major addu ct characterized as dG-N 2 

### -BPDE. There 26 

### was a linear relationship between the amount of ben zo[a]pyrene consumed and the concentration 27



### of dG-N 2 

### -BPDE in the forestomach of mice. For benzo[a]pyre ne, forestomach tumor incidence 28 

### increased sharply with adduct concentrations betwee n 50 and 140 fmol/mg DNA and in coal-tar 29 

### fed mice. Tumor incidence increased sharply with d G-N 2 

### -BPDE adduct levels between 20 and 30 

### 60 fmol/mg DNA. The same levels of adduct were pre sent in lung and liver of benzo[a]pyrene31 

### treated mice, although only the forestomach exhibit ed benzo[a]pyrene-induced tumors 32 

### (Goldstein et al., 1998). The presence of adducts in tumor-free tissue suggests that DNA adduct 33 

### levels alone are not necessarily predictors of tumo r outcome. 34 

### DNA adducts were identified and quantified in exper iments using the A/J mouse lung 35 

### model which results in lung adenomas in male A/J mi ce 8 months following a single i.p. 36 

### injection (Nesnow et al., 1998a, b, 1996, 1995; Ros s et al., 1995). Benzo[a]pyrene was 37 

### administered to male A/J mice (20-25/group) as a si ngle i.p. injection (0, 20, 50, or 100 mg/kg in 38

### 166 DRAFT - DO NOT CITE OR QUOTE

tricaprylin) and DNA was isolated from lung tissues at several time points between 1 and 21 1 

### days following injection. The primary DNA adduct i dentified in mouse lung tumors was a 2 

### benzo[a]pyrene bay region diol epoxide adduct of gu anine (7R, 8S, 9S-trihydroxy-10R-[2N-2’- 3 

### deoxyguanosyl]-7, 8, 9, 10-tetrahydro-benzo[a]pyren e). Two minor adducts were also observed 4 

### to result from the metabolism of 9-hydroxy-benzo[a] pyrene and trans-7,8-dihydroxy-7,8- 5 

### dihydro-benzo[a]pyrene. Quantitative analysis of D NA adducts by [ 32 

### P]-postlabeling illustrated 6 

### the importance of measuring DNA adduct levels over time. Total DNA adducts accumulated 7 

### rapidly between 3 and 9 days after exposure followe d by a gradual decrease. A time-integrated 8 

### DNA adduct level (TIDAL) was linearly related to th e administered dose of benzo[a]pyrene 9 

### (Ross et al., 1995). 10 

### Following i.p injection, benzo[a]pyrene also induce d DNA adducts in the lungs, liver, 11 

### and peripheral blood lymphocytes of rats (Ross et a l., 1991, 1990), the liver of Lewis rats 12 

### (Godschalk et al., 1998b), and lungs of BALB/c mice (Van Schooten et al., 1991). Qian et al. 13 

### (1998) treated male CD rats with intratracheal inst illation of fume condensates of roofing 14 

### asphalts and evaluated adducts in lung cells and WB Cs. Adducts were seen in the lungs but not 15 

### in WBCs, leading the authors to conclude that WBCs may not be a suitable surrogate for lung 16 

### cells. The adducts were not characterized, so the benzo[a]pyrene-specificity of the results cannot 17 

### be evaluated. Formation of DNA adducts from benzo[ a]pyrene metabolites also has been 18 

### observed in the lung and liver of male Sprague-Dawl ey rats after intratracheal administration of 19 

### benzo[a]pyrene (De Flora et al., 1991; Weyand and B evan, 1987). 20 

### DNA adducts have been reported in the lung and skin of dermally treated SENCAR mice 21 

### (Mukhtar et al., 1986), in the epidermis of Swiss m ice (Oueslati et al., 1992), and in the skin of 22 

### an unspecified strain of mice (Ingram et al., 2000) . When Talaska et al. (1996) compared the 23 

### dose-duration-response of benzo[a]pyrene-induced ad ducts in the skin, lung, and liver of Hsd 24 

### (ICR) BR mice treated dermally with 10, 25, or 50 n mol benzo[a]pyrene, accumulation of 25



### adducts was found to be linear with dose in the ski n and lung. In skin painting studies with 26 

### female SENCAR mice and various PAHs, Melendez-Colon et al. (1999) found that carcinogenic 27 

### potency correlated with DNA adduct levels in epider mal DNA rather than in the formation of 28 

### apurinic sites. Alexandrov and Rojas-Moreno (1990) found DNA adducts in epidermal 29 

### keratinocytes and dermal fibroblasts of Swiss mice treated dermally with benzo[a]pyrene but not 30 

### in similarly treated Wistar rats. BPDE-DNA adducts were measured in the lung, stomach, and 31 

### skin of male Lewis rats (15/group) following a sing le exposure to 10 mg/kg benzo[a]pyrene via 32 

### the intratracheal, gavage, and dermal routes, respe ctively (Godschalk et al., 2000). 33

34 

### 4.5.3. Tumor Promotion and Progression 35 

### benzo[a]pyrene has been shown to promote the growth of previously initiated cells, 36 

### resulting in the formation of tumors in the skin (s ee Section 4.2.3.1). The tumor promotion 37

### 167 DRAFT - DO NOT CITE OR QUOTE

properties of benzo[a]pyrene may be due to a compen satory response to cytotoxicity or via an 1 

### AhR-mediated effect on cell growth and differentiat ion. 2

3 

### Cytotoxicity and inflammatory response 4 

### The cytotoxicity of benzo[a]pyrene metabolites may contribute to tumor promotion via 5 

### inflammatory responses leading to cell proliferatio n (Burdick et al., 2003). Benzo[a]pyrene is 6 

### metabolized to o-quinones, which are cytotoxic, and can generate ROS (Bolton et al., 2000; 7 

### Penning, 1999). Benzo[a]pyrene o-quinones reduce t he viability and survival of rat and human 8 

### hepatoma cells (Flowers-Geary et al., 1996, 1993). Cytotoxicity was also induced by 9 

### benzo[a]pyrene and BPDE in a human prostate carcino ma cell line (Nwagbara et al., 2007). 10 

### Inflammatory responses to cytotoxicity may contribu te to the tumor promotion process. For 11 

### example, benzo[a]pyrene quinones (1,6-, 3,6-, and 6 ,12-benzo[a]pyrene-quinone) generated ROS 12 

### and increased cell proliferation by enhancing the e pidermal growth factor receptor 13 

### (EFGR)pathway in cultured breast epithelial cells ( Burdick et al., 2003). 14 

### Several studies have demonstrated that exposure to benzo[a]pyrene increases the 15 

### production of inflammatory cytokines which may cont ribute to cancer progression. Garçon et al. 16 

### (2001a, b) exposed Sprague-Dawley rats by inhalatio n to benzo[a]pyrene with or without ferrous 17 

### oxide (Fe 2 

### O 3 

### ) particles. They found that benzo[a]pyrene alone or in combination with Fe 2 

### O 3 18 

### particles elicited mRNA and protein synthesis of th e inflammatory cytokine, IL-1. Tamaki et al. 19 

### (2004) also demonstrated a benzo[a]pyrene-induced i ncrease in IL-1 expression in a human 20 

### fibroblast-like synoviocyte cell line (MH7A). Benz o[a]pyrene increases the expression of the 21 

### mRNA for CCL1, an inflammatory chemokine, in human macrophages (N’Diaye et al., 2006). 22 

### The benzo[a]pyrene-induced increase in CCL1 mRNA wa s inhibited by the potent AhR 23 

### antagonist 3 ′ -methoxy-4 ′ -nitroflavone. 24

25 

### AhR-mediated effects 26 

### The promotional effects of benzo[a]pyrene may also be related to AhR affinity and the 27 

### upregulation of genes related to biotransformation (i.e., induction of CYP1A1), growth, and 28 

### differentiation (Bostrom et al., 2002). Figure 4-1 illustrates the function of the AhR and depicts 29 

### the genes regulated by this receptor as belonging t o two major functional groups (i.e., induction 30 

### of metabolism or regulation cell differentiation an d proliferation). PAHs bind to the cytosolic 31 

### AhR in complex with heat shock protein 90 (Hsp90). The ligand-bound receptor is then 32 

### transported to nucleus in complex with the ARNT. T he AhR complex interacts with the Ah 33 

### responsive elements (AHRE) of the DNA to increase t he transcription of proteins associated with 34 

### induction of metabolism and regulation of cell diff erentiation and proliferation. 35

### 168 DRAFT - DO NOT CITE OR QUOTE AHR AHR Hsp90 Hsp90

AHR Hsp90

Hsp90 ARNT

ARNT Hsp90 Hsp90

AHR

ARNT AHRE DNA PAH

nucleus Enhanced specific mRNA production

Increased synthesis of PAH metabolizing enzymes Increased synthesis of proteins that regulate cell differentiation and proliferation

1



### AHRE DNA



### = Ah-responsive elements of DNA; Hsp90 = heat shoc k protein 90 2

3 

### Source: Okey et al. (1994). 4

5 

### Figure 4-1. Interaction of PAHs with the AhR. 6

7 

### Binding to the AhR induces enzymes that increase th e formation of reactive metabolites, 8 

### resulting in DNA binding and, eventually, tumor ini tiation. In addition, with persistent exposure, 9 

### the ligand-activated AhR triggers epithelial hyperp lasia, which provides the second step leading 10 

### from tumor initiation to promotion and progression (Nebert et al., 1993). Ma and Lu (2007) 11 

### reviewed several studies of benzo[a]pyrene toxicity and tumorigenicity in mouse strains with 12 

### high and low affinity AhRs. Disparities were obser ved in the tumor pattern and toxicity of Ah13 

### responsive (+/+ and +/-) and Ah-nonresponsive (-/-) mice. Ah-responsive mice were more 14 

### susceptible to toxicity and tumorigenicity in proxi mal target tissues such as the liver, lung, and 15 

### skin. For example, Shimizu et al. (2000) reported that AhR knock out mice (-/-), treated with 16 

### benzo[a]pyrene by s.c. injection or dermal painting , did not develop skin cancers at the treatment 17 

### site, while AhR-responsive (+/+) or heterozygous (+ /-) mice developed tumors within 18– 18 

### 25 weeks after treatment. Benzo[a]pyrene treatment increased CYP1A1 expression in the skin 19 

### and liver of AhR-positive mice (+/- or +/+), but CY P1A1 expression was not altered by 20 

### benzo[a]pyrene treatment in AhR knock out mice (-/- ). Talaska et al. (2006) also showed that 21 

### benzo[a]pyrene adduct levels in skin were reduced b y 50% in CYP1A2 knock out mice and by 22

### 169 DRAFT - DO NOT CITE OR QUOTE

90% in AhR knock out mice compared with WT C57Bl6/J mice following a single dermal 1 

### application of 33 mg/kg benzo[a]pyrene for 24 hours . Ma and Lu (2007) further noted that Ah2 

### nonresponsive mice were at greater risk of toxicity and tumorigenicity in remote organs, distant 3 

### from the site of exposure (i.e., bone marrow). As an example, Uno et al. (2006) showed that 4 

### benzo[a]pyrene (125 mg/kg/-day, p.o. for 18 days) c aused marked wasting, immunosuppression, 5 

### and bone marrow hypocellularity in CYP1A1 knock out mice, but not in WT mice. 6



### Some studies have demonstrated the formation of DNA adducts in the liver of AhR 7 

### knock out mice following i.p. or oral exposure to b enzo[a]pyrene (Sagredo et al., 2006; Uno et 8 

### al., 2006; Kondraganti et al., 2003). These findin gs suggest that there may be alternative (i.e., 9 

### non-AhR mediated) mechanisms of benzo[a]pyrene acti vation in the mouse liver. Sagredo et al. 10 

### (2006) studied the relationship between the AhR gen otype and CYP metabolism in different 11 

### organs of the mouse. AhR +/+



### , +/– 

### , and –/– 

### mice were treated once with 100 mg/kg benzo[a]pyre ne 12 

### by gavage. CYP1A1, CYP1B1, and AhR expression was evaluated in the lung, liver, spleen, 13 

### kidney, heart, and blood, via RT-PCR, 24 hours afte r treatment. CYP1A1 RNA was increased in 14 

### the lung and liver and CYP1B1 RNA was increased in the lung following benzo[a]pyrene 15



### treatment in AhR +/+ 

### and +/– 

### mice (generally higher in heterozygotes). Benzo[a ]pyrene treatment 16 

### did not induce CYP1A1 or CYP1B1 enzymes in AhR –/– 

### mice. The expression of CYP1A1 RNA, 17



### as standardized to β -actin expression, was generally about 40 times tha t of CYP1B1. The 18 

### concentration of benzo[a]pyrene metabolites and the levels of DNA and protein adducts were 19 

### increased in mice lacking the AhR, suggesting that there may be an AhR-independent pathway 20 

### for benzo[a]pyrene metabolism and activation. The high levels of benzo[a]pyrene DNA adducts 21 

### in organs other than the liver of AhR –/– 

### mice may be the result of slow detoxification of 22 

### benzo[a]pyrene in the liver, allowing high concentr ations of the parent compound to reach 23



### distant tissues. 24 

### Uno et al. (2006) also demonstrated a paradoxical i ncrease in liver DNA adducts in AhR 25 

### ko mice following oral exposure to benzo[a]pyrene. WT C57BL/6 mice and several knock out 26 

### mouse strains (CYP1A2 –/– 

### and CYP1B1 –/– 

### single ko, CYP1A1/1B1 –/– 

### and CYP1A2/1B1 –/– 27 

### double ko) were studied. Benzo[a]pyrene was admini stered in the feed at 1.25, 12.5, or 125 28 

### mg/kg for 18 days (this dose is well tolerated by W T C57BL/6 mice for 1 year, but lethal within 29 

### 30 days to the CYP1A1 –/– 

### mice). Steady-state blood levels of benzo[a]pyren e, reached within 5 30 

### days of treatment, were ~25 times higher in CYP1A1 –/– 

### and ~75 times higher in CYP1A1/1B1 –/– 31 

### than in WT mice, while clearance was similar to WT mice in the other knock out mouse strains. 32 

### DNA adduct levels, measured by [ 32 

### P]-postlabeling in liver, spleen, and bone marrow, were 33 

### highest in the CYP1A1 –/– 

### mice at the two higher doses, and in the CYP1A1/1B 1 –/– 

### mice at the 34 

### mid dose only. Adduct patterns, as revealed by 2-d imensional chromatography, differed 35 

### substantially between organs in the various knock o ut types. 36 

### Dertinger et al. (2001, 2000) demonstrated that AhR signaling may play a role in 37 

### cytogenetic damage caused by benzo[a]pyrene. The i n vivo formation of micronuclei in 38

### 170 DRAFT - DO NOT CITE OR QUOTE

peripheral blood reticulocytes of C57Bl/6J mice ind uced by a single i.p. injection of 1 

### benzo[a]pyrene (150 mg/kg) was eliminated by prior treatment with the potent AhR antagonist 2 

### 3'-methoxy-4'-nitroflavone. This antagonist also p rotected AhR null allele mice from 3 

### benzo[a]pyrene-induced increases in micronuclei for mation, suggesting that 3'-methoxy-4'- 4 

### nitroflavone may also act through a mechanism indep endent of the AhR (Dertinger et al., 2000). 5 

### Several in vitro studies have suggested that the Ah R plays a role in the disruption of cell 6 

### cycle control, possibly leading to cell proliferati on and tumor promotion following exposure to 7 

### benzo[a]pyrene (Andrysik et al., 2007; Chung et al. , 2007; Chen et al., 2003). Chung et al. 8 

### (2007) showed that benzo[a]pyrene-induced cytotoxic ity and apoptosis in mouse hepatoma 9 

### (Hepa1c1c7) cells occurred through a p53 and caspac e-dependent process requiring the AhR. 10 

### An accumulation of cells in the S-phase of the cell cycle (i.e., DNA synthesis and replication) 11 

### was also observed, suggesting that this process may be related to cell proliferation. Chen et al. 12 

### (2003) also demonstrated the importance of the AhR in benzo[a]pyrene-7,8-dihydrodiol- and 13 

### BPDE-induced apoptosis in human HepG2 cells. Both the dihydrodiol and BPDE affected Bcl2 14 

### (a member of a family of apoptosis suppressors) and activated caspase and p38 mitogen15 

### activated protein (MAP) kinases, both enzymes that promote apoptosis. When the experiments 16



### were conducted in a cell line that does not contain ARNT (see Figure 4-1), the dihydrodiol was 17 

### not able to initiate apoptotic event sequences, ind icating that activation to BPDE by CYP1A1 18 

### was required. BPDE did not induce apoptosis-relate d events in a p38-defective cell line, 19 

### illustrating the importance of MAP kinases in this process. In rat liver epithelial cells (WB-F344 20 

### cells), in vitro exposure to benzo[a]pyrene resulte d in apoptosis, a decrease in cell number, an 21 

### increase in the percentage of cells in S-phase (com parable to a proliferating population of WB22 

### F334 cells), and increased expression of cell cycle proteins (e.g., cyclin A) (Andrysik et al., 23 

### 2007). Benzo[a]pyrene-induced apoptosis was attenu ated in cells transfected with a dominant24 

### negative mutation of the AhR. 25

26 

### Inhibition of gap junctional intercellular communic ation 27 

### Gap junctions are channels between cells that allow substances of a molecular weight up 28 

### to roughly 1 kDa to pass from one cell to the other . This process of metabolic cooperation is 29 

### crucial for differentiation, proliferation, apoptos is, and cell death and consequently for the two 30 

### epigenetic steps of tumor formation, promotion, and progression. Chronic exposure to many 31 

### toxicants results in down-regulation of gap junctio ns. For tumor promoters, such as TPA or 32 

### TCDD, inhibition of intercellular communication is correlated with their promoting potency 33 

### (Sharovskaya et al., 2006; Yamasaki, 1990). 34 

### Blaha et al. (2002) surveyed the potency of 35 PAHs , including benzo[a]pyrene, to 35 

### inhibit gap junctional intercellular communication (GJIC). The scrape loading/dye transfer assay 36 

### was employed using a rat liver epithelial cell line that was incubated in vitro for 15, 30, or 60 37



### minutes with 50 μ M benzo[a]pyrene. After incubation, cells were was hed, and then a line was 38

### 171 DRAFT - DO NOT CITE OR QUOTE

scraped through the cells with a surgical blade. C ells were exposed to the fluorescent dye lucifer 1 

### yellow for 4 minutes and then fixed with formalin. Spread of the dye from the scrape line into 2 

### cells remote from the scrape was estimated under a fluorescence microscope. Benzo[a]pyrene 3 

### reduced spread of the dye after 30 minutes of expos ure (approximately 50% of control). 4 

### Recovery of GJIC was observed 60 minutes after expo sure. 5 

### Sharovskaya et al. (2006) studied the effects of ca rcinogenic and noncarcinogenic PAHs 6 

### on GJIC in HepG2 cells. Individual carcinogenic PA Hs inhibited GJIC in a temporary fashion 7 

### (70–100% within 24 hours), but removal of the PAH f rom culture reversed the effect. 8 

### Noncarcinogenic PAHs had very little effect on GJIC . Benzo[a]pyrene at 20 μ M inhibited GJIC 9 

### completely within 24 hours, while its noncarcinogen ic homolog, BeP, produced <20% inhibition. 10 

### The effect was not AhR-dependent, because benzo[a]p yrene inhibited GJIC in HepG2 cells to 11 

### the same extent as in hepatoma G27 cells, which exp ress neither CYP1A1 nor AhR. The authors 12 

### concluded that the effects of benzo[a]pyrene and Be P on GJIC were direct (i.e., not caused by 13 

### metabolites). 14

15



### 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 16



### 4.6.1. Oral 17 

### Numerous epidemiological studies are available whic h investigate associations between 18 

### PAH (including benzo[a]pyrene) dietary intake and c ancer incidence; however, no studies were 19 

### found which evaluate the contribution of benzo[a]py rene through dietary exposure in humans 20



### and noncancer health effects. Several studies in a nimal models are available evaluating the 21 

### sensitive noncancer effects following subchronic or chronic exposure to benzo[a]pyrene. The 22 

### types of effects observed following oral exposure w ere predominantly effects in the reproductive 23 

### and immune systems. Additionally, some minor hemat ological effects and kidney and 24



### forestomach effects were observed. Studies that id entified NOAELs and LOAELS for 25 

### noncancer effects in animals repeatedly exposed to benzo[a]pyrene by the oral route are 26



### summarized in Table 4-27. 27172 DRAFT - DO NOT CITE OR QUOTETable 4-27. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]pyrene by the oralrouteSpecies/sex Dose DurationNOAEL LOAELResponse at LOAEL Comments Referencemg/kg-dWistar rat/male and female0, 3, 10, or30 mg/kg-d,gavage, 5 d/wk2 yrs ND3↑Forestomach hyperplasia,↑liver clearcell foci of alterationHematological and organ weightvariables were not measured atterminal sacrifice.No exposure-related changes innoncancer histology in oral cavity,esophagus, forestomach, jejunum,liver, kidney, skin, mammary gland,or auditory canal (noncancer lesionswere only detected in tissues thatdeveloped tumors).↑Forestomach tumors in males at≥3 mg/kg-d.↑Forestomach, liver, and kidney(males only) tumors at 10 and30 mg/kg-d.Kroese et al.,2001B6C3F1mouse/femaleonlyEstimateddoses: 0, 0.7,3.3, or16.5 mg/kg-d indiet2 yrs ND0.73.30.73.316.5↑Forestomach hyperplasia↑Forestomach hyperkeratosis↑Esophagus (basal cell hyperplasia)No exposure-related changes inweight or histology of liver, kidney,or lung weight (other organs notmeasured).Hematologic variables were notexamined.↑Forestomach tumors at 3.3 and16.5 mg/kg-d;↑Esophagus and tongue tumors at16.5 mg/kg-d.Beland andCulp, 1998;Culp et al.,1998Wistar rat/male and female0, 3, 10, or30 mg/kg-d,5 d/wk90 d 1033010↓Thymus weight,↑liver weight,↑forestomach hyperplasia,↑slight thymicatrophy↑Forestomach epithelial cell proliferationindex (BrdU incorporation)No exposure-related changes inhematological variables or histologyof lung, spleen, or lymph node.Kroese et al.,2001F344 rat/male and female0, 5, 50, or100 mg/kg-d indiet90 d ND550550100↑renal tubular casts in males↓RBCs and hematocrit in males↓RBCs and hematocrit in females,↓hemoglobin in both sexes,↑liver:bodyweight ratio in malesNo exposure-related changes inother organ weights measured(stomach, testes, ovaries), or inhistology of stomach, liver, testes orovaries (other tissues were notexamined).Knuckles et al.,2001173 DRAFT - DO NOT CITE OR QUOTETable 4-27. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]pyrene by the oralrouteSpecies/sex Dose DurationNOAEL LOAELResponse at LOAEL Comments Referencemg/kg-dSD male rats 0, 1 or 5 mg/kgday by gavage90 d 1 5↓testicular testosterone Testosterone levels measured inanimals sacrificed after 30 d, werenot statistically different thancontrolsZheng et al.,2010SD female rats 0, 2.5 or 5mg/kg-day,gavage160 d ND2.52.55↓ovary weight↓estrogen and primordial follicles; alteredestrous cyclicityXu et al., 2010C57BL/6 malemice0, 1, or 10mg/kg-dayexposure to F0generation42 d ND1110↓epididymal sperm count in F0 and F1generations↓sperm motility in F0 miceMohamed etal., 2010Wistar rat/maleonly0, 3, 10, 30, or90 mg/kg-d,gavage, 5 d/wk35 d 31030103090↓RBCs, hemoglobin, and hematocrit,↓thymus weight,↓percent B cells inspleen↑Forestomach hyperplasia,↓serum IgMand IgA↑Liver oval cell hyperplasiaNo exposure-related histologicalchanges in adrenals, brain, bonemarrow, colon, caecum, jejunum,heart, kidney, lung, lymph nodes,esophagus, pituitary, spleen,stomach, testis, or thymus.De Jong et al.,1999Wistar rat/male and female0, 1.5, 5, 15, or50 mg/kg-d,gavage, 5 d/wk35 d 5151550↓Thymus weight,↑forestomachhyperplasia↑Liver weightNo exposure-related changes inhematological variables, weights ofkidney, spleen, lung, adrenals orovaries, or histology of liver,kidney, spleen, thymus, lung, ormammary gland.Kroese et al.,2001CD-1 mouse/ F0female;F1 male andfemale0,10, 40, or160 mg/kg-d,gavageGDs 7–16of F0pregnancy4010NDND160401010↓Number of F0 females with viable litters↓F1 body weight at PND 20↓F1 body weight at PND 42↓F1 male and F1female fertility indexBeginning at 6–7 wks of age, eachF1 male mouse (20–45/group) wasexposed to 10 untreated femalesover a period of 25 d. Beginning at6 wks of age, each F1 female mouse(20–55/group) was cohabitated withan untreated male for a period of6 mo.MacKenzie andAngevine,1981174 DRAFT - DO NOT CITE OR QUOTETable 4-27. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]pyrene by the oralrouteSpecies/sex Dose DurationNOAEL LOAELResponse at LOAEL Comments Referencemg/kg-dNMRI mouse/F0 female;F1 female0 or 10 mg/kg-dGDs 7–16of F0pregnancyND 10↓F1 female fertility (↓number of F2 littersand F2 litter size;↓ovary weight, and↓numbers of small, medium, or largefollicles and corpora lutea)Exposed F0 females showed nogross signs of toxicity and noeffects on fertility. One F1 femalefrom each litter was continuouslybred with an untreated male for6 mo.Kristensen etal., 1995ND = not determined1 Time weighted average dose ; animals treated by gavage, every other day to 0, 5, or 10 mg/kg-day1212(1) Kroese et al., 2001; (2) Beland and Culp, 1998;Culp et al., 1998; (3) De Jong et al., 1999; (4) Xu et al., 2010; (5) Zhe32010; (7) van den Berg et al., 2005; (8) MacKenzieand Angevine, 1981; (9) Kristensen et al.4hyperplasia; wt = weight.56Figure 4-2. NOAELs and LOAELs for selected noncancer effects from repeated oral exposure to7benzo[a]pyrene.8**0 5** **10 15** **20 25** **30 35** **FS hyp; rat; (1)** **FS hyp; mouse; (2)** **↓ thymus wt; rat; (1)** **↓ thymus wt; rat; (3)** **2 years**175 DRAFT -DO NOT CITE OR QUOTE(1) Kroese et al., 2001; (2) Beland and Culp, 1998;Culp et al., 1998; (3) De Jong et al., 1999; (4) Xu et al., 2010; (5) Zheng et al., 2010; (6) Mohamed et al.,2010; (7) van den Berg et al., 2005; (8) MacKenzieand Angevine, 1981; (9) Kristensen et al., 1995;↑= increased;↓= decreased; FS = forestomach; hyp =2. NOAELs and LOAELs for selected noncancer effects from repeated oral exposure to**↓ thymus wt; rat; (3)** **↓ B-cells in spleen; rat;** **(3) ↓ ovary wt;** **rat; (4) ↓ follicles** **and estrogen; rat;** **(4) ↓** **testosterone; rats; (5)** **↓ epididymal** **sperm count; mice; (6)** **↓ immune response;** **mouse; (7) 35-90 day** DO NOT CITE OR QUOTEng et al., 2010; (6) Mohamed et al.,= decreased; FS = forestomach; hyp =2. NOAELs and LOAELs for selected noncancer effects from repeated oral exposure to**↓ immune response;** **mouse; (7) ↓ F1 body** **weight and fertility;** **mouse; (8) ↓ F1** **fertility; mouse; (9)** **Gestational (days 7-16)**

### 176 DRAFT - DO NOT CITE OR QUOTE

The two oral chronic-duration studies identify fore stomach hyperplasia in rats (gavage 1 

### exposure) and mice (dietary exposure) as a sensitiv e effect—a LOAEL of 3 mg/kg-day for 2 

### forestomach hyperplasia in male and female Wistar r ats (Kroese et al., 2001) and a LOAEL of 3 

### 0.7 mg/kg-day for forestomach hyperplasia in female B6C3F 1 

### mice (Beland and Culp, 1998; 4 

### Culp et al., 1998). In both rats and mice, an incr easing incidence of animals with forestomach 5 

### tumors with increasing dose was also observed. 6



### Several immune related effects have been observed i n animals treated subchronicly with 7 

### benzo[a]pyrene, including decreased thymus weight, decreased % of B cells in the spleen, 8 

### decreased RBCs, and decreased serum immunoglobulins . LOAELs for decreased thymus 9 

### weights were 10 and 15 mg/kg-day in two different s tudies of Wistar rats exposed by gavage for 10 

### 35 days (Kroese et al., 2001; De Jong et al., 1999) and 30 mg/kg-day for Wistar rats exposed to 11 

### benzo[a]pyrene in the diet for 90 days (Kroese et a l., 2001). Thymus weights were not measured 12



### in the the available chronic studies. Decreased th ymus weight was accompanied by a decreased 13 

### percentage of B cells in spleen and decreased serum IgM and IgA in one 35-day Wistar rat study 14 

### (De Jong et al., 1999) and increased incidence of s light thymic atrophy in the 90-day Wistar rat 15 

### study (Kroese et al., 2001). Thymus atrophy, but n o histological thymus lesions, was noted in 16 

### the other 35-day study with Wistar rats (Kroese et al., 2001). Other support for immune effects 17 

### as a potential effect from repeated oral exposure t o benzo[a]pyrene is shown by decreased 18 

### immune responses in lymph nodes to the dermal sensi tizer, DNCB, in C56BL/6 mice given 13 19 

### mg/kg-day (LOAEL) 3 times/week for 4 weeks (van den Berg et al., 2005; see Section 4.4.2). 20



### Effects on RBC counts were also observed across the rat subchronic duration studies 21 

### (Table 4-25). LOAELs for decreased RBCs were 10 mg /kg-day in Wistar rats exposed by 22 

### gavage for 35 days (De Jong et al., 1999) and 50 mg /kg-day in male F344 rats exposed in the 23 

### diet for 90 days (Knuckles et al., 2001), but no si gnificant exposure-related changes in RBC 24



### counts were observed in Wistar rats in another 35 d ay study at doses up to 50 mg/kg-day (Kroese 25 

### et al., 2001) or at 30 mg/kg-day in Wistar rats exp osed in the diet for 90 days (Kroese et al., 26 

### 2001; see Table 4-26). When observed, the magnitud es of the decreases in RBC, hemoglobin, or 27 

### hematocrit were generally small: about 18% at 90 m g/kg-day and <10% at lower doses in Wistar 28 

### rats (De Jong et al., 1999) and about 10% in F344 r ats (Knuckles et al., 2001). Hematologic 29



### variables were not measured at the terminal sacrifi ces in the chronic duration studies in rats 30 

### (Kroese et al., 2001) or mice (Beland and Culp, 199 8; Culp et al., 1998). 31 

### Kidney effects characterized as increased incidence of renal tubular casts in male F344 32 

### rats were observed in a study by Knuckles et al 200 1). The most sensitive effect observed in this 33 

### study was an increase in abnormal tubular casts in the kidney in males at 5 mg/kg-day (40%), 34 

### 50 mg/kg-day (80%) and 100 mg/kg-day (100%), compar ed to 10% in the controls. In females, 35

### 177 DRAFT - DO NOT CITE OR QUOTE

only 10% showed significant kidney tubular changes at the two high dose levels compared to 1



### zero incidence in controls. 2 

### Reproductive and developmental effects following ge stational exposure to 3 

### benzo[a]pyrene have been observed in animal models. Decreased male reproductive endpoints 4 

### including decreased testicular testosterone, decrea sed epididymal sperm count, and decreased 5 

### sperm motility have been observed in rodents treate d subchronically (Mohamed et al., 2010; 6 

### Zheng et al., 2010). In addition, female reproduct ive endpoints including decreased ovary 7 

### weight, decreased estrogen, decreased primordial fo llicles, and estrus cyclicity have been 8 

### observed in female rats treated for 60 days (Xu et al., 2010). Impaired reproductive performance 9 

### in F1 mouse offspring (male and female) has been ob served following exposure of F0 mice to 10 

### oral doses as low as 10 mg/kg-day during GDs 7–16 ( Kristensen et al., 1995; MacKenzie and 11 

### Angevine, 1981). Effects observed included decreas ed ovary weight in F1 females and reduced 12 

### fertility as reflected by decreased mean number of F2 litters. F1 females had statistically 13 

### significantly lower median numbers of offspring, nu mber of litters, and litter sizes and a 14 

### statistically significantly greater median number o f days between litters as compared with the 15 

### controls (Kristensen et al., 1995). Another study of gestationally treated dams (GD 7-16) 16 

### identified statistically significant decrements in fertility, pup weight, and reproductive organ 17 

### weights and histology (MacKenzie and Angevine, 1981 ). These mouse developmental/ 18 

### reproductive toxicity studies observed effects at t he lowest dose tested (10 mg/kg-day). 19 

### Reductions in motor activity, decreased grip streng th, and decreased response to sound, touch, 20 

### and pain were observed in F344 rats following admin istration of single gavage doses of 21



### ≥ 25 mg/kg (Saunders et al., 2006, 2002, 2001; see Se ction 4.4.1), but similar evaluations of 22 

### neurological endpoints following repeated oral expo sure of animals to benzo[a]pyrene were not 23



### located. 24 

### Studies with ApoE-/- mice, which spontaneously deve lop atherosclerosis, show that 25 

### repeated oral exposure to 5 mg/kg gavage doses of b enzo[a]pyrene enhances the progression of 26 

### atherosclerosis through a general local inflammator y process (Knaapen et al., 2007; Curfs et al., 27 

### 2005, 2004; Godschalk et al., 2003; see Section 4.4 .4); however, available data are inadequate to 28 

### assess oral exposure dose-response relationships fo r benzo[a]pyrene-induced atherosclerosis in 29



### normal test animals. 30

31



### 4.6.2. Inhalation 32 

### Several epidemiological studies have associated inc reased occupational exposure of 33 

### benzo[a]pyrene with cardiac endpoints, specifically ischemic heart disease (Friesen et al., 2010; 34 

### Burstyn et al., 2005). Other studies have reported potential prenatal effects, birth outcomes, and 35 

### decreased fertility associated with increased expos ure to benzo[a]pyrene. Decreased head 36

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circumference, decreased birth weight, and decrease d postnatal weight have been reported (Tang 1 

### et al., 2006; Perera et al., 2005a,b;) in addition to increased risk of early fetal death (Wu et al., 2 

### 2010). Furthermore, elevated levels of benzo[a]pyr ene in follicular fluid have been associated 3 

### with reduced fertility (Neal et al., 2008). 4 

### In addition to epidemiological studies, several rep eated-exposure inhalation toxicity studies in 5 

### animals exist for benzo[a]pyrene (Archibong et al., 2002, 2008; Ramesh et al., 2008; Inyang et 6 

### al., 2003; Wormley et al., 2004). A lifetime-expos ure carcinogenicity study of Syrian golden 7 

### hamsters exposed to benzo[a]pyrene condensed onto N aCl aerosols at nominal concentrations of 8 

### 2, 10, or 50 mg/m 3 

### is available (Thyssen et al., 1981); however, nonc ancer effects were not 9



### evaluated. 10 

### Although no standard developmental toxicity studies are available for benzo[a]pyrene via 11 

### the inhalation route, decreased fetal survival and number of pups per litter were observed 12 

### following exposure of pregnant F344 rats to aerosol s of benzo[a]pyrene and CB at 13



### concentrations ≥ 25 μ g/m 3 

### on GDs 11–20 (Archibong et al., 2002). Decreased levels of plasma 14 

### progesterone, estradiol, and prolactin were observe d on GD 17 in dams exposed to 75 μ g/m 3 

### , but 15 

### not in those exposed to 25 μ g/m 3 

### (Archibong et al., 2002). Other rat studies from the same 16 

### laboratory have associated inhalation exposures to 100 μ g/m 3 

### benzo[a]pyrene:CB aerosols 17 

### during gestation with changes in electrophysiologic al variables in the hippocampus (Wormley et 18 

### al., 2004). Duration-dependent effects on male rep roductive endpoints including increased 19 

### luteinizing hormone, decreased circulating and intr atesticular concentrations of testosterone, 20 

### decreased testis weight, and decreased sperm motili ty have also been observed following 21 

### exposure of adult male F344 rats to benzo[a]pyrene: CB aerosols at 75 μ g/m 3 

### for 10 or 60 days 22 

### (Archibong et al., 2008; Ramesh et al., 2008; Inyan g et al., 2003). 23

24179 DRAFT - DO NOT CITE OR QUOTETable 4-28. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]pyrene by theinhalation routeSpecies/sex Dose DurationNOAEL LOAELResponse at LOAEL Comments Referenceμg/m3F344 rats 0, 25, 75, 100μg/m34hr/dGD 11-20 ND252575↓pups/litter, litter survival (%)↑resorptions↓plasma progesterone, estradiol, andprolactincarbon black used as carrier particleArchibong etal., 2002; Wuet al 2003F344 rats 0, 75 μg/m34hr/d60 d ND 75↑luteinizing hormone↓testosterone↓decreased testis weight↓sperm motilitycarbon black used as carrier particlein treatment group; controls notexposed to carbon blackArchibong etal., 2008F344 rats 0, 100 μg/m34hr/dGD ND100↓pups/litterelectrophysiological changes in thehippocampusWormley et al.,20041

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1



### 4.6.3. Dermal 2

3 

### Though numerous chronic cancer bioassays exist for benzo[a]pyrene by the dermal route, 4 

### noncancer effects were not reported in these studie s, nor are studies available evaluating 5 

### noncancer effects in humans exposed dermally to ben zo[a]pyrene. 6

7



### 4.6.4. Mode-of-Action Information 8



### 4.6.4.1. Forestomach Lesions from Oral Exposure 9 

### The development of forestomach hyperplasia in mice and rats from subchronic or chronic 10 

### oral exposures (by gavage and diet) to benzo[a]pyre ne is reasonably expected to involve a cell 11 

### proliferative response to cytotoxicity from reactiv e benzo[a]pyrene metabolic intermediates, 12 

### based on the extensive findings from research on th e bioactivation of benzo[a]pyrene and 13 

### carcinogenicity (see reviews on the bioactivation o f benzo[a]pyrene by Xu et al., 2009; Jiang et 14 

### al., 2007, 2005; Xue and Warshawsky, 2005; Penning et al., 1999; Harvey 1996; Cavalieri and 15 

### Rogan, 1995). Reactive intermediates that can reac t with cellular macromolecules and 16 

### potentially lead to cytotoxicity include BPDE, benz o[a]pyrene radical cations, benzo[a]pyrene o17 

### quinones, and ROS. Reactive benzo[a]pyrene metabol ites are also well known to reduce the 18 

### viability and survival of cultured cells involving mechanisms related to stimulation of apoptosis 19 

### (Andrysik et al., 2007; Chung et al., 2007; Nwagbar a et al., 2007; Chen et al., 2003; Jyonouchi et 20 

### al., 1999; Flowers-Geary et al., 1996, 1993). Mole cular details of cell proliferative responses to 21 

### cytotoxicity or apoptosis from benzo[a]pyrene metab olites are poorly understood, but Burdick et 22 

### al. (2006, 2003) provided evidence that benzo[a]pyr ene o-quinones could inhibit apoptosis and 23 

### increase cell proliferation in a model human mammar y epithelial cell system (MCF10A) via 24 

### activation of the epidermal growth factor receptor (EGFR) by ROS. The relationship of these 25 

### findings to development of benzo[a]pyrene-induced f orestomach hyperplasia is unknown. 26

27



### 4.6.4.2. Immune System Effects 28 

### Decreased thymus weight, decreased number of B cell s in spleen, and immune 29 

### suppression have been observed following oral, i.p. , s.c., or intratracheal instillation exposure to 30 

### benzo[a]pyrene. DeJong et al. (1999) and Kroese e t al. (2001) found decreased thymus weight 31 

### due to benzo[a]pyrene at oral doses ≥ 10 mg/kg body weight. In addition, several studies report 32 

### thymus effects at higher doses and/or by other rout es of exposure (e.g. Rodriguez et al., 1999; 33 

### Holladay and Smith, 1994). Reduced thymus size or weight have been noted to be among the 34 

### first indicators of immunotoxicity (Schuurman et al . 1992; Luster et al. 1988), and correlate well 35 

### with adverse histopathologic effects and the presen ce of lesions in the thymic cortex (Germolec 36 

### et al. 2004a, 2004b; Wachsmuth 1983). 37

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Interpretation of decreased thymus weight as an ad verse effect is supported by general 1 

### immunology literature as well as chemical-specific data. The thymic cortex is known to be a 2 

### major site of thymocyte proliferation and selection for maturation, and impairment can lead to 3 

### cell-mediated immune suppression (Kuper 2002, 1992; De Waal et al. 1997). Reduced thymus 4 

### weight is often attributed to decreased thymocyte p roliferation or increased thymocyte apoptosis 5 

### in the thymic cortex (Kamath et al., 1997; Vandebri el et al., 1999). DeJong et al. (1999) reported 6 

### a decrease in estimated thymic cortex weight at 10, 30, and 90 mg/kg benzo[a]pyrene, and 7 

### reduced medulla weight at 90 mg/kg, but did not cal culate the ratio between the two. This 8 

### suggests that both parts of the thymus were affecte d by benzo[a]pyrene, but that the cortex may 9 

### be more sensitive. De Jong also used immunohistoch emistry data to show that cell proliferation 10 

### was not affected, suggesting that thymic atrophy ma y be due to increased rates of thymocyte 11 

### apoptosis. However, a study using the murine LLNA showed that proliferation activity 12 

### decreased after a single 13 mg/ml oral dose of benz o[a]pyrene (van den Berg et al., 2005). 13 

### In addition to thymus effects, decreased B cell per centages in the spleen were observed at 14 

### 10, 30, and 90 mg/kg-day benzo[a]pyrene in a dose r esponse pattern. The absolute number of B 15 

### cells, however, was not significantly lower than co ntrol animals until 90 mg-kg/day. There is 16 

### also supportive evidence of humoral immune suppress ion at higher doses, as well as evidence of 17 

### overall toxicity of the bone marrow. This theory i s supported by the decrease in IgA and IgM 18 

### (incating T-cell dependant effects), and the dose-r elated toxicity of RBCs observed by Dejong et 19 

### al. (1999). 20 

### The MOA by which benzo[a]pyrene produces immune sys tem effects is not understood, 21



### but several in vitro studies have been conducted to investigate potential contributing 22 

### mechanisms. Benzo[a]pyrene induced myelotoxicity i n human cord blood cells (Carfi et al., 23 

### 2007) and mouse bone marrow cultures (Legraverend e t al., 1983), suppressed mouse B cell 24 

### lymphopoiesis (Hardin et al., 1992), and inhibited mitogen-induced proliferative responses of 25 

### mouse spleen cell cultures (Lee and Urso, 2007). B enzo[a]pyrene inhibition of the proliferative 26 

### responses of spleen cells to a mitogen was diminish ed by the presence of the AhR antagonist and 27 

### CYP inhibitor, α -NF, indicating the potential importance of benzo[a ]pyrene metabolites in the 28 

### immune suppression effect (Lee and Urso, 2007). Si milarly, the CYP1A1 inhibitor 1-(1- 29 

### propynyl)pyrene blocked B-cell growth inhibition by benzo[a]pyrene, but not through the 30 

### metabolite BPDE (Allan et al. 2006). 31 

### Carfi et al. (2007) described a series of in vitro assays designed to assess cytotoxicity, 32 

### myelotoxicity, cytokine release, and mitogen respon siveness in rat, mouse, and human cells (i.e., 33 

### peripheral lymphocytes and cord blood cells, and sp leen cells from rats and mice only). The 34 

### cytotoxicity half maximal inhibitory concentration (IC 50 

### ) value for benzo[a]pyrene was >200 μ M 35 

### in human, rat, and mouse cells. Benzo[a]pyrene pro duced myelotoxicity as evaluated by a dose36 

### related decrease in colony scoring for the colony forming unit-granulocyte macrophage (CFU37 

### GM) assay using human cord blood cells. Benzo[a]py rene reduced the release of specific 38

### 182 DRAFT - DO NOT CITE OR QUOTE

cytokines from HL following phytohemagglutinin (PHA ), gamma-interferon ( γ -INF), and 1 

### lipopolysaccharide (LPS) tumor necrosis factor (TNF

- α ) stimulation. Mitogen responsiveness in 2 

### rat and mouse spleen cells following stimulation wi th LPS or PHA was decreased by exposure to 3 

### benzo[a]pyrene. T-lymphocyte proliferation induced by anti-CD3 antibody was also inhibited by 4 

### benzo[a]pyrene in HL, but was not affected in mouse spleen cells at the highest concentration 5 

### (160 μ M). 6 

### Myelotoxicity was also observed in mouse bone marro w cultures exposed to 7 

### benzo[a]pyrene as evidenced by decreased cell survi val (Legraverend et al., 1983). The findings 8 

### in bone marrow cultures from Ah-responsive (C57BL/6 ) and Ah-nonresponsive (DBA/2) mice 9 

### suggest that AhR affinity may play a role in benzo[ a]pyrene-induced myelotoxicity. 10 

### Benzo[a]pyrene is more toxic to bone marrow cells f rom C57BL/6 mice in vitro compared to 11



### cells cultured from DBA/2 mice. 12 

### Hardin et al. (1992) treated cultured bone marrow c ells from DBA/2 and C57BL/6 mice 13 

### with benzo[a]pyrene at concentrations between 10 -4 

### and 10 -8 

### M. Benzo[a]pyrene suppressed B 14 

### cell lymphopoiesis in a dose-dependent manner even at the lowest concentration used. Bone 15 

### marrow cells from Ah-nonresponsive DBA/2 mice were less sensitive to the immunosuppressive 16 

### action of benzo[a]pyrene, compared with Ah-responsi ve C57BL/6 mice. The AhR antagonist 17



### and CYP450 inhibitor α -NF prevented benzo[a]pyrene-induced inhibition of B cell 18 

### lymphopoiesis from C57BL/6 mice in a concentration- dependent fashion. Benzo[a]pyrene also 19 

### induced apoptosis in cultured bone marrow cells. 20 

### Spleen cell cultures derived from C3H/HeJ and CBY/D 2 mice were exposed to benzo[a]pyrene 21 

### and assessed for T-lymphocyte proliferation in resp onse to mitogenic or antigenic stimulation 22 

### (Lee and Urso, 2007). Benzo[a]pyrene (10 μ M) produced an 80% decrease in the allogeneic 23 

### mixed lymphocyte response (MLR) assay, which is a m easure of the proliferative response to 24 

### antigenic stimulation. Benzo[a]pyrene (0.1, 1, and 10 μ M) also produced a dose-dependent 25 

### inhibition of the proliferative response to the mit ogen Concanavalin A (Con A). This inhibition 26 

### did not occur in spleen cells treated with the AhR antagonist and CYP450 inhibitor, α -NF. 27 

### BPDE-DNA adducts were detected in CH3 spleen cells cultured with 10 μ M benzo[a]pyrene in 28 

### the presence of Con A. 29

30



### 4.6.4.3. Developmental and Reproductive Toxicity Effects 31 

### Developmental and reproductive toxicity effects hav e been associated with oral and 32 

### inhalation exposure to benzo[a]pyrene. Impaired fe rtility, with associated lesions in ovarian 33 

### (decreased follicles) and testicular (atrophic semi niferous tubules) tissues, has been observed in 34 

### male and female F1 offspring following exposure of F0 female mice to 10 mg/kg-day 35 

### benzo[a]pyrene on GDs 7–16; a decrease in the numbe r of F0 females with viable litters was 36 

### observed at a higher dose level of 160 mg/kg-day (K ristensen et al., 1995; MacKenzie and 37 

### Angevine, 1981). Inhalation exposure of pregnant r ats to benzo[a]pyrene:CB aerosols during 38

### 183 DRAFT - DO NOT CITE OR QUOTE

gestation has also been associated with decreased f etal survival and number of pups per litter 1 

### (Archibong et al., 2002), decreased levels of plasm a progesterone, estradiol, and prolactin 2 

### (Archibong et al., 2002), changes in electrophysio logical variables in the hippocampus 3 

### (Wormley et al., 2004) and decreased cortical neuro n activity (McCallister et al., 2008). 4 

### Inhalation exposure of adult male rats to benzo[a]p yrene:CB aerosols (75 μ g/m 3 

### ) for 10 or 60 5 

### days caused decreased circulating and intratesticul ar concentrations of testosterone, decreased 6 

### testis weight, and decreased sperm motility (Archib ong et al., 2008; Ramesh et al., 2008; Inyang 7 

### et al., 2003). ). Acute i.p. exposure studies in a dult female DBa/2N or C57BL/6N mice 8 

### demonstrated a 50% destruction of primordial follic les with a single dose of 25 mg/kg (Mattison 9 

### et al., 1980) and a 15-day exposure of 3 mg/kg-day (Boorman et al., 2000). Other reproductive10



### related effects in these studies include decreased fertility (number of pups) (Mattison et al., 1980) 11 

### and ovulatory inhibition, as indicated by decreased number of corpora lutea, one week after a 12 

### dose of 5 mg/kg and 3-4 weeks after a dose of 100 mg/kg (Miller et al., 1992; Swartz and 13 

### Mattison, 1985). 14 

### In vivo studies have suggested that the mechanism o f decreased fertility in females and 15 

### decreased fetal survival may result from changes in circulating hormones (i.e., decreased 16 

### progesterone, estradiol-17 β , and prolactin levels) responsible for maintaining the uterine 17 

### environment in a state that can support embryonic a nd fetal development (Archibong et al., 2002, 18 

### see Section 4.3.2). Several in vitro studies have demonstrated low affinity binding of 19 

### benzo[a]pyrene to the estrogen receptor and alterat ion of estrogen-dependent gene expression 20 

### (Liu et al., 2006; Van Lipzig et al., 2005; Vondrac ek et al., 2002; Fertuck et al., 2001; Charles et 21 

### al., 2000); however, the role of these changes in b enzo[a]pyrene-induced reproductive toxicity is 22 

### unknown. Fertuck et al. (2001) showed in vitro effects of benzo[a]pyrene on estrogen-receptor23 

### mediated gene expression, but did not demonstrate e strogen-mediated uterotrophic effects 24 

### (increased uterine weight or lactoferrin mRNA expre ssion) in ovarectomized C57BL/J6 or 25 

### DBA/2 mice following in vivo administration of benz o[a]pyrene (0.1–10 mg/kg-day, p.o., for 3 26 

### consecutive days). 27 

### The mechanism(s) by which benzo[a]pyrene or its met abolites impair the development of 28 

### follicles in the ovary have been the focus of study for more than 30 years (Mattison and 29 

### Thorgeirsson, 1977). AHH is found in the ovary, an d inhibition of AHH activity reduces the 30 

### level of oocyte destruction seen with benzo[a]pyren e exposures in mice (Mattison and 31 

### Thorgeirsson, 1979). AHH activation is not suffici ent to explain the variation across strains in 32 

### oocyte destruction, however (Mattison and Nightinga le, 1980). Studies using intraovarian 33 

### injection of metabolites of benzo[a]pyrene indicate that the epoxide metabolite (+)-(7R,8S)-diol34 

### (9S,10R)-epoxide-2 is most strongly correlated with the oocyte counts in exposed mice 35 

### (Takizawa et al.,1984). 36 

### Most of the loss of oocytes that occurs in utero an d throughout the reproductive lifespan 37 

### in mice and in women occurs through programmed cell death (apoptosis), which is regulated by 38

### 184 DRAFT - DO NOT CITE OR QUOTE

the protein Bax. Activation of Bax leads to increased oocyte death, and PAHs (includi ng 1 

### benzo[a]pyrene) have been demonstrated to activate Bax gene transcription in mice (Matikainen 2 

### et al., 2002; 2001). However, an in vitro study of mouse ovarian cells obtained from 4-day old 3 

### pups did not show any evidence of increased markers of apoptosis with treatments of 4 

### benzo[a]pyrene concentrations of up to 1000 ng/ml f or 6 and for 24 hours (Tuttle et al., 2009). 5 

### Other mechanisms may be more relevant for the ovula tory inhibition effects seen with 6 

### benzo[a]pyrene exposures. In an intro experiment, Neal et al. (2007) demonstrated a dose7 

### dependent decrease in FSH-stimulated rat follicle g rowth, with 158, 99, 75, 38, 30 and 38% 8 

### change in follicle area for benzo[a]pyrene exposure concentrations of 0, 1.5, 5.0, 15, 45 and 135 9 

### ng /ml (p < 0.05 for all differences compared with controls). The authors noted that the lowest 10 

### dose at which this effect was seen, 1.5 ng/ml, was similar to the mean concentration of 11 

### benzo[a]pyrene seen in follicular fluid samples fro m women who smoked. 12 

### Several in vitro studies have investigated the poss ible mechanisms for impaired 13 

### spermatogenesis by benzo[a]pyrene including enhance ment of apoptosis of spermatogonia 14 

### (Revel et al., 2001), inhibition of spermatid meios is (Georgellis et al., 1990), Sertoli cell 15 

### cytotoxicity (Raychoudhury and Kubinski, 2003), and altered androgen hormone regulation 16 

### (Inyang et al., 2003; Vinggaard et al., 2000). 17 

### Revel et al. (2001) reported dose-related increases in apoptosis of spermatogonia 18



### harvested from the vas deferens of male BALB/c mice administered benzo[a]pyrene doses 19 

### ranging from 0.5 to 50 mg/kg via s.c. injection for 5 weeks. The competitive AhR inhibitor, 20 

### resveratrol (50 mg/kg, s.c.) given simultaneously w ith 5 mg/kg benzo[a]pyrene s.c. for 5 weeks, 21 

### suppressed both BPDE DNA adduct formation and apopt osis, suggesting a role for the AhR in 22 

### this benzo[a]pyrene-induced male reproductive toxic ity (Revel et al., 2001). 23 

### Georgellis et al. (1990) reported that concentratio ns of 0.1 μ M benzo[a]pyrene incubated 24 

### in vitro with seminiferous tubule segments from Spr ague-Dawley rats with microsome 25 

### preparations from the whole rat testes inhibited me iotic division of the spermatids and was 26 

### highly cytotoxic. 27 

### Raychoudhury and Kubinski (2003) isolated Sertoli c ells from CD rats and incubated the 28 

### cells in culture with benzo[a]pyrene. Benzo[a]pyre ne was cytotoxic to these cells at 50 and 29



### 100 μ g/mL. Treatment of the cells for 24 hours with 10 μ g/mL benzo[a]pyrene induced cell 30 

### killing through an apoptotic response (as measured by fluorescence labeling of apoptotic DNA 31 

### fragments). 32 

### Inyang et al. (2003) demonstrated that benzo[a]pyre ne inhalation altered circulating 33 

### levels or cellular responsiveness to androgenic hor mones such as testosterone (see Section 4.3.2). 34 

### Vinggaard et al., 2000) showed that benzo[a]pyrene antagonized the human androgen receptor 35 

### (hAR) in a sensitive reporter gene assay based on C HO cells transiently cotransfected with a 36 

### hAR vector and an MMTV-LUC vector (antiandrogen IC 50 

### of 3.9 μ M). 37

38

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4.7. EVALUATION OF CARCINOGENICITY 1 

### 4.7.1. Summary of Overall Weight-of-Evidence 2



### Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 3 

### benzo[a]pyrene is “carcinogenic to humans.” This c onclusion is based on evidence of 4 

### carcinogenicity in humans following exposure to dif ferent PAH mixtures containing 5 

### benzo[a]pyrene, extensive and consistent evidence o f carcinogenicity in laboratory animals 6 

### exposed to benzo[a]pyrene via all routes of adminis tration, and strong evidence that the 7 

### biological processes leading to benzo[a]pyrene carc inogenesis in laboratory animals are also 8 

### present in humans. Bioactivation of benzo[a]pyrene leads to the formation of DNA-reactive 9 

### metabolites which can produce mutations in key gene s, such as the p53 tumor suppressor gene 10 

### and the ras oncogene, leading to tumor formation (s ee Section 4.7.3.). 11

12 

### 4.7.2. Synthesis of Human, Animal, and Other Suppo rting Evidence 13 

### There is a large body of evidence for human carcino genicity for several PAH mixtures 14 

### containing benzo[a]pyrene, such as soot, coal tars, coal-tar pitch, mineral oils, and shale oils 15 

### (IARC, 2010; Baan et al., 2009; Straif et al., 2005 ). There is also evidence of carcinogenicity in 16 

### occupations involving exposure to PAH mixtures cont aining benzo[a]pyrene, such as aluminum 17 

### production, chimney sweeping, coal gasification, co al-tar distillation, coke production, iron and 18 

### steel founding, and paving and roofing with coal ta r pitch (IARC, 2010; Baan et al., 2009; Straif 19 

### et al., 2005). Increased cancer risks have been re ported among other occupations involving 20 

### exposure to PAH mixtures such as carbon black and d iesel exhaust (Bosetti et al., 2007; Straif et 21 

### al., 2005). Benzo[a]pyrene is also a notable const ituent of tobacco smoke (IARC 2004). An 22 

### increasing number of studies report exposure biomar kers such as benzo[a]pyrene- or PAH-DNA 23 

### adducts in white blood cells, and several cohort st udies (summarized in Section 4.1) demonstrate 24 

### a positive exposure-response relationship with cumu lative PAH exposure using 25 

### benzo[a]pyrene—or a proxy such as BSM that can be c onverted to benzo[a]pyrene—as an 26 

### indicator substance. Because benzo[a]pyrene is onl y one of many PAHs that could contribute to 27 

### these observed increases in cancer, the epidemiolog ic studies provide credible but limited 28 

### support for a causative role of benzo[a]pyrene in h uman cancer. 29 

### In laboratory animals (i.e., rats, mice, and hamste rs), exposure to benzo[a]pyrene via the 30 

### oral, inhalation, and dermal routes have been assoc iated with carcinogenic responses both 31 

### systemically and at the site of administration. Ch ronic oral exposure to benzo[a]pyrene was 32



### associated with forestomach and liver tumors in mal e and female Wistar rats (Kroese et al., 33 

### 2001), forestomach tumors in male and female Spragu e-Dawley rats (Brune et al., 1981), and 34 

### forestomach, esophagus, tongue, and larynx tumors i n female B6C3F 1 

### mice (Beland and Culp, 35 

### 1998; Culp et al., 1998). Auditory canal tumors we re also observed in male and female Wistar 36 

### rats (Kroese et al., 2001). Repeated or short-term oral exposure to benzo[a]pyrene was 37



### associated with forestomach tumors in more than 10 additional bioassays with several strains of 38

### 186 DRAFT - DO NOT CITE OR QUOTE

mice (see Table 4-4 in Section 4.2.1.2). Chronic i nhalation exposure to benzo[a]pyrene was 1 

### associated with tumors in the larynx and pharynx of male Syrian golden hamsters exposed to 2 

### benzo[a]pyrene:NaCl aerosols (Thyssen et al., 1981) . Intratracheal instillation of benzo[a]pyrene 3 

### was associated with respiratory tract tumors in mor e than 10 additional studies with hamsters 4 

### (see Section 4.2.2.2 for references). Chronic derm al application of benzo[a]pyrene (2–3 5 

### times/week) has been associated with mouse skin tum ors in 12 bioassays (see Section 4.2.3.2 for 6 

### references). Skin tumors in rats, rabbits, and gui nea pigs have also been associated with repeated 7 

### application of benzo[a]pyrene to skin in the absenc e of exogenous promoters (WHO, 1998; 8 

### ATSDR, 1995; IARC, 1983, 1973). When followed by r epeated exposure to a potent tumor 9 

### promoter, acute dermal exposure to benzo[a]pyrene i nduced skin tumors in numerous studies of 10 

### mice, indicating that benzo[a]pyrene is a strong tu mor-initiating agent in the mouse skin model 11 

### (see Section 4.2.3.1 for references). 12 

### Carcinogenic responses in animals exposed to benzo[ a]pyrene by other routes of 13 

### administration include: (1) liver or lung tumors i n newborn mice given acute postnatal i.p. 14 

### injections; (2) increased lung tumor multiplicity i n A/J adult mice given single i.p. injections; (3) 15 

### injection site tumors in mice following s.c. inject ion; (4) injection site sarcomas in mice 16 

### following intramuscular injection; (5) mammary tumo rs in rats with intramammilary 17 

### administration; (6) cervical tumors in mice with in travaginal application; and (7) tracheal tumors 18 

### in rats with intratracheal implantation (see Sectio n 4.4.3 for references). 19 

### Benzo[a]pyrene is classified as an alternant PAH, or a compound composed solely of 20 

### fused benzene rings. Nonalternant PAHs contain bot h benzene and five carbon rings. Among 21 

### alternant PAHs, important structural features relat ed to enhanced mutagenicity and 22 

### carcinogenicity include the presence of at least fo ur rings (Bostrom et al., 2002). The 23 

### carcinogenic activity of PAH compounds is influence d by specific structural features. Recently, 24 

### this knowledge has been exploited in an effort to d erive quantitative structure activity 25 

### relationship (QSAR) methods to evaluate the relatio nship between specific PAH structural 26 

### features and mechanistic events related to carcinog enesis (Bruce et al., 2008; Vijayalakshmi et 27 

### al., 2008). Alternant PAHs having four or more ben zene rings exhibit greater carcinogenic 28 

### potency than PAHs with two or three benzene rings ( Bostrom et al., 2002). The carcinogenic 29 

### activity of PAHs is also related to the specific ar rangement of the benzene rings. As a general 30 

### rule, PAHs with at least four rings and a classic b ay- or fjord-region (formed entirely by benzene 31 

### rings) may be characterized as containing structura l alerts for carcinogenesis. However, this 32 

### structural characterization is likely to be overly simplistic and other features may be important to 33 

### carcinogenesis. 34 

### As discussed in Section 4.5.1.1, several lines of e vidence related to tumor initiation 35 

### following mutagenicity are available for benzo[a]py rene including: (1) in vivo detection of 36 

### cancer-relevant oncogene/tumor suppressor gene muta tions in target tissue; (2) in vivo detection 37 

### of DNA adducts in target tissue; (3) in vivo DNA ad ducts, gene mutations, cytogenetic damage, 38

### 187 DRAFT - DO NOT CITE OR QUOTE

and other measures of primary DNA damage in non-tar get tissues; and (4) in vitro DNA adduct 1 

### formation, mutations, cytogenetic damage, and prima ry DNA damage in cells from target and 2 

### nontarget tissues. 3

4



### 4.7.3. Mode-of-Action Information 5



### 4.7.3.1. Hypothesized MOA 6 

### The carcinogenicity of benzo[a]pyrene, the most stu died and best characterized PAH, is 7 

### well documented (Xu et al., 2009; Jiang et al., 200 7, 2005; Xue and Warshawsky, 2005; Ramesh 8 

### et al., 2004; Bostrom et al., 2002; Penning et al., 1999; WHO, 1998; Harvey, 1996; ATSDR, 9 

### 1995; Cavalieri and Rogan, 1995; U.S. EPA, 1991b). EPA has concluded that benzo[a]pyrene 10 

### induces carcinogenicity via a mutagenic mode of act ion. Mutagenicity is a well-established 11 

### cause of carcinogenicity. This hypothesized mode of action is presumed to apply to all tumor 12 

### types and is relevant for all routes of exposure. The principal key events associated with the 13 

### mode of action for benzo[a]pyrene include: (1) bio activation of benzo[a]pyrene to reactive 14 

### metabolites (2) direct DNA damage by the reactive m etabolites, including the formation of DNA 15 

### adducts (3) formation and fixation of DNA mutations , particularly in tumor suppressor genes or 16 

### oncogenes and (4) clonal expansion of mutated cells . These events are depicted in Figure 4-3. 17

18

19

20 21 benzo[a]pyrene diol epoxide

radical cation

o

- quinone and ROS DNA Adducts

DNA Adducts

DNA Adducts and oxidative base damage Mutation ( Transversion K-Ras, H-Ras and p53 targets

)

Mutation (Depurination H-Ras target )

Mutation (Depurination)

, Oxidative damage and strand scission Proliferation of initiated cells Neoplasm

Inflammatory

response

Cytotoxicity HO

OH O

+ Exposure Metabolism

O

O Initiation Promotion Progression

Key Events in the MOA for benzo[a]pyrene Carcinogen icity

Binding to AhR

Upregulation of genes related to biotransformation, growth, and differentiation

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Figure 4-3. Proposed principal pathways and key ev ents in the 1 

### benzo[a]pyrene carcinogenic MOA. 2

3



### 4.7.3.2. Experimental Support for the Hypothesized MOA 4

5 

### Strength, consistency, specificity of association. There is an extensive database of in vitro and in 6 

### vivo studies demonstrating the genotoxicity and mut agenicity of benzo[a]pyrene following 7 

### metabolic activation (see Tables 4-24, 4-25 and 4-2 6). In vitro studies overwhelmingly support 8 

### the formation of DNA adducts, mutagenesis in bacter ia, yeast and mammalian cells, several 9 

### measures of cytogenetic damage (CA, SCE, MN), and D NA damage. In vivo systems in animal 10 

### models are predominantly positive for somatic mutat ions following benzo[a]pyrene exposure. 11 

### Additionally, some evidence exists that benzo[a]pyr ene can induce mutations in germ cells. 12 

### Benzo[a]pyrene is thought to be converted into reac tive intermediates via three principal 13 

### metabolic pathways: (1) activation to a reactive d iol epoxide via CYP1A1/1B1 and epoxide 14 

### hydrolase; (2) activation to a reactive radical cat ion via CYP peroxidases and (3) activation to a 15 

### reactive and redox active o-quinone metabolite via AKR1A1 and AKR1C1-1C4 (Xu et al., 2009; 16 

### Jiang et al., 2007, 2005; Xue and Warshawsky, 2005; Penning et al., 1999; Harvey 1996; 17 

### Cavalieri and Rogan, 1995). All three of these pat hways (discussed in detail in Sections 3.3. and 18 

### 4.5.2) lead to DNA damage including DNA adducts, de purination, and/or oxidative damage to 19 

### DNA. DNA damage, if not correctly repaired prior t o replication, can subsequently give rise to 20



### mutations. 21 

### Benzo[a]pyrene-DNA adducts, biomarkers of exposure and of effect, have been 22 

### extensively demonstrated with in vitro cell systems , in vivo animals studies, and in human target 23 

### tissues, including skin and lung (see Section 4.1.2 .). Specifically, elevated BPDE-DNA adducts 24 

### have been observed in coke oven workers and chimney sweepers, occupations associated with 25 

### increased risks of cancer from complex PAH-containi ng mixtures (Pavanello et al., 1999). 26



### BPDE-DNA adducts were also found to be elevated in the lungs of cigarette smokers with lung 27 

### cancer (Godschalk et al., 2002; Phillips et al., 20 02; Bartsch et al., 1999; Alexandrov et al., 28 

### 1992). Multiple epidemiological studies have indic ated that PAH exposed individuals who are 29 

### homozygous for a CYP1A1 polymorphism which increase s the inducability of this enzyme (thus 30 

### increasing the production of reactive diol epoxide metabolites) have increased levels of 31 

### benzo[a]pyrene-DNA adducts (Bartsch et al., 2006; A klillu et al., 2005; Alexandrov et al., 2002; 32 

### Perera and Weinstein, 2000). In addition, this po pulation of individuals also has a greater risk of 33 

### certain tumors, including those of the lung. 34 

### Mutations in the K-ras, H-ras, and p53 genes were a ssessed in forestomach tumors of 35 

### mice fed benzo[a]pyrene in the diet for 2 years (Cu lp et al., 2000). Forestomach tumors had K36 

### ras mutations (68% of tumors) that were G → T or C transversions in codon 12 or 13. H-ras 37 

### (codon 13) and p53 mutations characterized as G → T or C transversions were also found. 38

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K-ras mutations were observed in the A/J mouse lung tumor model following IP 1 

### treatment with benzo[a]pyrene, and were observed to be qualitatively different than K-ras 2 

### mutations in spontaneous lung tumors from control a nimals (Nesnow et al., 1996). Lung tumor 3 

### DNA was isolated and DNA sequence analysis of K-ras mutations was performed for 19 separate 4 

### lung tumors in the benzo[a]pyrene treated group and the control group. The DNA sequence 5 

### analysis demonstrated several guanine mutations at codon 12, which were different than the 6 

### spectrum of mutations found in untreated animals. S pecifically, the frequency of GGT → TGT 7 

### transversion mutations were significantly higher in the benzo[a]pyrene treated animals compared 8 

### to controls (56% vs. 0%) whereas GGT → GAT transition mutations were the predominant 9 

### mutation in lung tumors from control animals (58% v s. 19% in benzo[a]pyrene group). 10 

### Some human data exist which correlate the frequency of PAH-DNA adducts with gene 11 

### mutations in highly PAH-exposed populations. In a study of iron foundry workers (a high PAH 12 

### exposure population which has been demonstrated to have an increased risk of lung cancer 13 

### [Bosetti et al., 2007]), biological samples from wo rkers were analyzed for DNA adducts and 14 

### somatic gene mutations at the hprt locus (Perera et al.1994, 1993). A strong correlation between 15 

### PAH-DNA adduct levels and incidence of hprt mutatio ns was observed in individuals with 16 

### detectable levels of adducts, indicating that soma tic mutations were increased in parallel with 17 

### PAH-DNA adducts in workers exposed to PAHs. 18 

### Data in humans from a study by Marini et al. (2001) indicate that the types of mutations 19 

### commonly found in response to benzo[a]pyrene exposu re in in vitro and animal models are 20 

### similar to the spectrum of mutations in critical tu mor suppressor genes and/or oncogenes in 21 

### populations highly exposed to PAHs. DeMarini et al . (2001) demonstrated mutations in the p53 22 

### tumor suppressor gene and the K-ras oncogene in lun g tumors obtained from 24 nonsmoking 23 

### women from China, whose tumors were associated with exposure in their homes to smoky coal 24 

### from the use of stoves with no chimneys. The obser ved mutations in lung tumors were primarily 25



### G → T transversions at either K-ras or p53. Mutation h otspots in the lung tumors corresponded 26 

### with hot spots for PAH mutations (codon 154, codon 249, and codon 273). 27

28 

### Dose-response concordance and temporal relationship

. The metabolism of benzo[a]pyrene to 29 

### reactive metabolites is a necessary event which pre cedes mutagenesis. Mutation assays of 30 

### benzo[a]pyrene in salmonella typhimurium are overwhelmingly positive with the inclusion of S9 31 

### metabolic liver fractions, but are negative without the addition of the S9 metabolic enzymes (see 32 

### Table 4-24). 33 

### In mice, a dose-response and temporal relationship has been demonstrated between the 34



### formation of BPDE-DNA adducts and skin and forestom ach tumors. In a study using mice 35 

### treated dermally with benzo[a]pyrene once or twice per week for 15 weeks, a linear dose36 

### response of benzo[a]pyrene-induced adducts in the s kin, lung, and liver was observed (Talaska et 37 

### al. 1996). Another study examined the dose-respons e relationship and the time course of 38

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benzo[a]pyrene-induced skin damage, DNA adduct form ation, and tumor formation in female 1 

### mice. Mice were treated dermally with 0, 16, 32, o r 64 μ g of benzo[a]pyrene once per week for 2 

### 29 weeks (Albert et al., 1991). Indices of skin da mage and levels of BPDE-DNA adducts in skin 3 

### reached plateau levels in exposed groups by 2–4 wee ks of exposure. With increasing dose level, 4 

### levels of BPDE-DNA adducts (fmol/ μ g DNA) initially increased in a linear manner and b egan to 5 

### plateau at doses ≥ 32 μ g/week. Tumors began appearing after 12–14 weeks o f exposure for the 6 

### mid- and high-dose groups and at 18 weeks for the l ow-dose group. At study termination 7 

### (35 weeks after start of exposure), the mean number of tumors per mouse was approximately one 8 

### per mouse in the low- and mid-dose groups and eight per mouse in the high-dose group. The 9 

### time-course data indicate that benzo[a]pyrene-induc ed increases in BPDE-DNA adducts 10 

### preceded the appearance of skin tumors, consistent with the formation of DNA adducts as a 11 

### precursor event in benzo[a]pyrene induced skin tumo rs. 12 

### Culp et al. (1996a) compared dose-response relation ships for BPDE-DNA adducts and 13



### tumors in female B6C3F 1 

### mice exposed to benzo[a]pyrene in the diet at 0, 1 8.5, 90, or 350 14



### μ g/day for 28 days (to examine adducts) or 2 years ( to examine tumors). The benzo[a]pyrene 15 

### dose-tumor response data showed a sharp increase in forestomach tumor incidence between the 16



### 18.5 μ g/day group (6% incidence) and the 90 μ g/day group (78% incidence). The BPDE-DNA 17 

### adduct levels in forestomach showed a relatively li near dose-response throughout the 18 

### benzo[a]pyrene dose range tested. The appearance o f increased levels of BPDE-DNA adducts in 19 

### the target tissue at 28 days is temporally consiste nt with the contribution of these adducts to the 20 

### initiation of forestomach tumors. Furthermore, ab out 60% of the examined tumors had 21 

### mutations in the K-ras oncogene at codons 12 and 13 , which were G → T or G → C transversions 22 

### indicative of BPDE reactions with DNA (Culp et al., 1996a). 23

24 

### Biological plausibility and coherence. A mutagenic MOA for benzo[a]pyrene is supported b y a 25 

### large body of research over time with consistent ev idence of benzo[a]pyrene activation to 26 

### reactive metabolites leading to DNA-damage and muta tional events associated with tumor 27 

### initiation. Mutagenicity as a mode of action for c arcinogenicity in humans is generally accepted 28 

### and is a biologically plausible mechanism for tumor induction. The formation of DNA adducts 29 

### and oncogene/tumor suppressor mutations in organs t hat also displayed an increase in tumor 30



### incidence in rats and mice indicates coherence of t hese effects. Benzo[a]pyrene has been shown 31 

### to be mutagenic in vivo and in vitro, across specie s and tissue types



### . 32

33



### 4.7.3.2. Other Possible MOAs 34 

### In addition to mutagenicity, other MOAs which contr ibute to the carcinogenicity of 35 

### benzo[a]pyrene are possible, but are not as well st udied. The tumor promotion properties of 36 

### benzo[a]pyrene may involve cell proliferative respo nses to cytotoxicity or apoptosis from 37 

### benzo[a]pyrene metabolites, AhR-mediated effects on cell growth and differentiation, or anti- 38

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apoptotic signals elicited by metabolites (Burdick et al., 2006, 2003; Chen et al., 2003; Nebert et 1 

### al., 1993). Results from some studies indicate tha t exposure to benzo[a]pyrene or its metabolites 2 

### increases the production of inflammatory cytokines, such as IL-1, which may contribute to tumor 3 

### promotion (N’Diaye et al., 2006; Tamaki et al., 200 4; Garçon et al., 2001a, b). Benzo[a]pyrene 4 

### also has been shown to inhibit GJIC, a characterist ic associated with well-known tumor 5 

### promoters such as TPA (Sharovskaya et al., 2006; Bl aha et al., 2002). In summary, though there 6 

### are limited data which support other processes that may contribute to the carcinogenicity of 7 

### benzo[a]pyrene (inflammation, cytotoxicity, anti-ap optotic signaling, etc.); the available 8 

### evidence indicates that the primary mode of action for benzo[a]pyrene involves DNA reactivity 9 

### and mutagenicity leading to carcinogenesis. 10

11



### 4.7.3.4. Conclusions About the Hypothesized Mode of Action 12 

### The MOA of mutagenicity of benzo[a]pyrene through r eactive metabolites is extensively 13 

### supported by a large body of research. Mutations f rom DNA reactive benzo[a]pyrene 14 

### metabolites occur as early events in the carcinogen ic process and are not believed to be acquired 15 

### following cytoxicity or regenerative proliferation. Several lines of evidence relating to 16 

### mutagenicity and tumor initiation are available for benzo[a]pyrene including: in vitro evidence 17 

### of DNA adducts, mutations, cytogenetic damage, and primary DNA damage; in vivo DNA 18 

### adducts, gene mutations, cytogenetic damage, and ot her measures of primary DNA damage; 19 

### detection of DNA adducts in target tissue in vivo; and detection of cancer-relevant 20 

### oncogene/tumor suppressor gene mutations in target tissue in vivo. Taken together, these data 21 

### provide support for a mutagenic MOA for benzo[a]pyr ene-induced cancer. 22

23 

### Support for the hypothesized MOA in test animals 24 

### Benzo[a]pyrene induces gene mutations in a variety of in vivo and in vitro systems and 25 

### produces tumors in all animal species tested and al l routes of exposure. Strong, consistent 26 

### evidence indicates that the postulated key events: the metabolism benzo[a]pyrene to a DNA27 

### reactive intermediates, the formation of DNA adduct s, and the occurrence of subsequent 28 

### mutations in oncogenes and tumor suppressor genes o ccur in animal models. 29

30 

### Relevance of the hypothesized MOA to humans 31 

### Mutagenicity is a well-established cause of carcino genicity. A substantial database of 32 

### information on benzo[a]pyrene indicates that the po stulated key events: the metabolism of 33 

### benzo[a]pyrene to a DNA-reactive intermediates, the formation of DNA adducts, and the 34 

### formation of subsequent mutations in oncogenes and tumor suppressor genes all occur in human 35 

### tissues. The following lines of evidence from huma n studies provide support that the 36 

### hypothesized mutagenic MOA is relevant to humans: t he activation of benzo[a]pyrene to DNA 37 

### reactive metabolites occurs in humans in qualitativ ely and quantitatively similar manner 38

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compared to animals; DNA adducts specific to benzo[ a]pyrene have been found in a wide variety 1 

### of human tissues and are elevated in populations ex posed to high levels PAHs; increased DNA 2 

### mutations have been strongly associated with increa sing levels of PAH-DNA adducts in workers 3 

### occupationally exposed to PAHs; and the spectra of benzo[a]pyrene induced mutations in p53 4 

### tumor suppressor genes and ras oncogenes observed i n controlled in vitro cell systems and in 5 

### vivo animal studies are similar to the spectra of m utations in tumors from PAH-exposed humans. 6

7 

### Populations or lifestages particularly susceptible to the hypothesized MOA 8 

### The mutagenic mode of action is considered relevan t to all populations and lifestages. 9 

### The current understanding of biology of cancer indi cates that mutagenic chemicals, such as 10 

### benzo[a]pyrene, are expected to exhibit a greater e ffect in early life versus later life exposure 11 

### (U.S. EPA, 2005b; Vesselinovitch et al., 1979). Al though the developing fetus and infants may 12 

### have lower levels of some bioactivating enzymes tha n adults (e.g., CYP1A1/1B1), infants or 13 

### children are expected to be more susceptible to ben zo[a]pyrene-induced cancer at certain tissue 14 

### sites. Newborn or infant mice developed liver and lung tumors more readily than young adult 15 

### mice following acute i.p. exposures to benzo[a]pyre ne (Vesselinovitch et al., 1975; see Section 16 

### 4.8.1). These results indicate that exposure to be nzo[a]pyrene during early life stages presents 17 

### additional risk for cancer, compared with exposure during adulthood. The Supplemental 18



### Guidance for Assessing Susceptibility from Early-Life Exposu re to Carcinogens (U.S. EPA, 19 

### 2005b) recommends the application of age-dependent adjustment factors (ADAFs) for 20 

### carcinogens that act through a mutagenic mode of ac tion. Given the weight of the available 21 

### evidence, benzo[a]pyrene acts through a mutagenic m ode of carcinogenic action and the ADAFs 22 

### should be applied. 23 

### Population variability in metabolism and detoxifica tion of benzo[a]pyrene, in addition to 24 

### DNA repair capability, may affect cancer risk. Pol ymorphic variations in the human population 25 

### in CYP1A1, CYP1B1, and other CYPs have been implica ted as determinants of increased 26 

### individual lung cancer risk in some studies (Aklill u et al., 2005; Alexandrov et al., 2002; Perera 27 

### and Weinstein, 2000). The Phase II cytosolic GST, mediated by variants of the GSTM1 and 28 

### GSTT1 genes, prevents the formation of BPDE-DNA add ucts. Some evidence suggests that 29 

### humans lacking a functional GST gene have higher BP DE-DNA adduct levels and thus are at 30 

### greater risk for cancer (Vineis et al., 2007a; Pava nello et al., 2004; Perera and Weinstein, 2000; 31 

### Alexandrov et al., 2002). In addition, acquired d eficiencies or inherited gene polymorphisms 32 

### that affect the efficiency or fidelity of DNA repai r may also influence individual susceptibility to 33 

### cancer from environmental mutagens (Matullo et al., 2003; Shen et al., 2003; Cheng et al., 2000; 34 

### Perera and Weinstein, 2000; Wei et al., 2000; Amos et al., 1999). In general, however, available 35 

### support for the role of single polymorphisms in sig nificantly modulating human PAH cancer risk 36 

### is relatively weak or inconsistent. Combinations o f metabolic polymorphisms, on the other hand, 37

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may be critical determinants of cumulative DNA-dama ging dose, and thus susceptibility to 1 

### cancer from benzo[a]pyrene exposure. 2

3



### 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 4 

### 4.8.1. Possible Childhood Susceptibility 5 

### Increased childhood susceptibility to benzo[a]pyren e is supported by several lines of 6 

### evidence including epidemiological studies reportin g associations between adverse birth 7 

### outcomes and developmental effects and internal bio markers of exposure to benzo[a]pyrene, 8 

### presumably via exposure to complex PAH mixtures (Ta ng et al. 2008, 2006; Perera et al., 9 

### 2005a,b). The occurrence of BPDE-DNA in maternal a nd umbilical cord blood in conjunction 10 

### with exposure to ETS was associated with reduced bi rth weight and head circumference in 11 

### pregnant women living in the vicinity of fires from the 09/11/2001 disaster site in New York 12 

### City (Perera et al., 2005a). In other studies, ele vated levels of BPDE-DNA adducts in umbilical 13 

### cord blood were associated with: (1) reduced birth weights or reduced head circumference in the 14 

### offspring of 529 Dominican or African-American nons moking women (Perera et al., 2005b); and 15 

### (2) decreased body weight at 18, 24, and 30 months and deficits in several areas of development 16 

### as assessed by the Gesell Developmental Schedules a t 24 months in the offspring of nonsmoking 17 

### Chinese women living in the vicinity of a coal-fire d power plant (Tang et al., 2008, 2006). 18

19 

### Developmental neurotoxicity 20 

### Studies in humans and experimental animals indicate that exposure to PAHs in general, 21 

### and benzo[a]pyrene in particular, may impact neurol ogical development at relatively low 22 

### exposure levels. Observational studies in humans h ave suggested associations between 23 

### gestational exposure to PAHs and later measures of neurodevelopment (Perera et al., 2009; Tang 24 

### et al., 2008). An observational study of a Chinese population living in close proximity to a coal 25 

### fired power plant found increased levels of benzo[a ]pyrene-DNA adducts in cord blood were 26 

### associated with decreased developmental quotients i n offspring (Tang et al., 2008). In addition, 27 

### a study of pregnant women living or working near th e World Trade Center site in NYC found 28 

### high PAH exposure during pregnancy was associated w ith a reduction in verbal and full scale IQ 29 

### of offspring at 5 years of age (Perera et al., 2009 ). 30 

### A study in pregnant rats exposed by inhalation show ed a dose related increase in 31 

### benzo[a]pyrene metabolites in the cerebral cortex a nd hippocampus of pups, indicating the fetal 32 

### brain is exposed to benzo[a]pyrene and/or its metab olites following maternal inhalation exposure 33 

### (Wu et al., 2003). Another study which treated pr egnant rat dams to benzo[a]pyrene by 34 

### inhalation found a decrease in long term potentiati on (LTP) in the hippocampus of gestationally 35 

### treated pups compared with controls, indicating a p ossible effect on learning and memory in 36 

### benzo[a]pyrene exposed animals, though functional t ests were not conducted (Wormley et al., 37 

### 2004). Another study by the same group treated rat dams by gavage with low levels of 38

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benzo[a]pyrene (300 μg/kg) on GD 14-17 and observed benzo[a]pyrene metabolites in the brains 1 

### of pups and diminished cortical neuronal activity t o sensory input in exposed offspring compared 2 

### with controls (McCallister et al., 2008). In addit ion, a study of mice exposed to benzo[a]pyrene 3 

### lactationally observed statistically significant di fferences in performance in several neuromotor 4 

### and behavioral tests which indicated decreased righ ting reflex and disinhibition behavior 5 

### (Bouayed et al., 2009). 6

7 

### Reproductive effects 8 

### Epidemiological studies indicate that exposure to c omplex mixtures of PAHs, such as 9 

### through cigarette smoke, is associated with measure s of decreased fertility in humans (El Nemr 10 

### et al., 1998; Neal et al., 2005) and that prenatal exposure to cigarette smoking is associated with 11 

### reduced fertility of women later in life (Weinberg et al., 1989). A case-control study in a 12 

### Chinese population has also indicated that women wi th elevated levels of benzo[a]pyrene-DNA 13



### adducts in maternal blood were four times more like ly to have experienced a missed abortion 14 

### (Wu et al., 2010). 15 

### Oral multigenerational studies of benzo[a]pyrene ex posure in mice demonstrate effects 16 

### on fertility and the development of reproductive or gans in male and female offspring exposed to 17 

### benzo[a]pyrene during development at levels in whic h no overt toxicity or depression in fertility 18 

### is seen in the parental animals (Mackenzie and Ange vine 1981; Kristensen et al., 1995). 19 

### MacKenzie and Angevine (1981) exposed groups of mi ce to benzo[a]pyrene on GDs 7– 20 

### 16 and reproductive outcomes of the offspring were investigated. Fertility of the F1 generation 21 

### was decreased in a dose dependant manner. At matur ity, the fertility of these animals was tested. 22 

### The F1 male and F1 female fertility indices were si gnificantly decreased in each exposure group. 23 

### These reductions in fertility indices were associat ed with decreased testes and ovary weight in 24 

### the F1 animals. Male offspring showed histological damage of the seminiferous tubules and 25 

### female offspring had hypoplastic ovaries with few f ollicles and corpora lutea. Similar results 26 

### were reported in a study in which female mice were exposed by gavage to benzo[a]pyrene on 27 

### GDs 7–16 (Kristensen et al., 1995). F1 females had decreased mean ovary weight and reduced 28 

### fertility. At necropsy, the F1 females had reduced ovary weights with decreased numbers of 29 

### small, medium, or large follicles and corpora lutea . Inhalation exposure of pregnant female rats 30 

### to benzo[a]pyrene:CB aerosols during gestation has also been associated with decreased fetal 31 

### survival and number of pups per litter associated w ith decreased levels of plasma progesterone, 32 

### estradiol, and prolactin (Archibong et al., 2002). 33 

### These reductions in fertility observed in animal mo dels are supported by a large database 34 

### of animal studies in adult animals indicating that benzo[a]pyrene is ovotoxic with effects 35 

### including decreased ovary weight, decreased primord ial follicles, and reduced fertility (Mattison 36 

### et al., 1980; Swartz and Mattison 1985; Miller et a l., 1992; Borman et al, 2000). 37

38

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Developmental Immune effects 1 

### The severity and persistence of immune effects obse rved during in utero studies suggests 2 

### that immunotoxicity may be greater during gestation than adulthood (Dietert and Pieperbrink, 3 

### 2006; Holladay and Smialowicz, 2000). Urso and Geng ozian (1982) provide experimental 4 

### support demonstrating immunosuppression from benzo[ a]pyrene exposure during gestation was 5 

### greater than for mice exposed after birth to a 25-f old higher dose. There is also substantial 6 

### general literature indicating that disruption of th e immune system during certain critical periods 7 

### of development (e.g., initiation of hematopoiesis; migration of stem cells; expansion of 8 

### progenitor cells) may have significant and lasting impacts on lifetime immune function (e.g. 9 

### Burns-Naas et al., 2008; Dietert, 2008; Landreth et al., 2002; Dietert et al., 2000), as well as 10 

### more specific studies showing increased dose sensit ivity and disease persistence from 11 

### developmental versus adult chemical exposure (revie wed in Luebke et al., 2006). 12 

### Thymus toxicity is a sensitive and specific effect of benzo[a]pyrene and has been 13 

### observed in both prenatal and adult exposure studie s. The thymus serves as a major site of 14 

### thymocyte proliferation and selection for maturatio n, and impairment can lead to cell-mediated 15 

### immune suppression (Kuper 2002, 1992; De Waal et al ., 1997). The thymus is believed to be 16 

### critical for T lymphocyte production during early l ife and not in adulthood (Hakim et al., 2005; 17 

### Schonland et al., 2003; Petrie et al., 2002; Mackal l et al., 1995). Therefore, the decreases in 18 

### thymus weight observed in studies of adult animals exposed to benzo[a]pyrene suggest that 19 

### immunosuppression may be a heightened concern for i ndividuals developmentally exposed to 20 

### benzo[a]pyrene. 21

22



### Cancer 23 

### As mentioned above in section 4.7.3.4, investigatio ns in young animals exposed to 24 

### benzo[a]pyrene provide evidence that early life exp osure may present increased risk of cancer. 25 

### Comparisons of cancer responses in newborn (1 day o ld), infant (15 days old), and young adult 26 

### (42 days old) mice indicate that exposure to benzo[ a]pyrene during early life stages can present 27 

### additional risk for cancer, compared with exposure during young adulthood (Vesselinovitch et 28 

### al., 1975), but studies designed to compare risks o f cancer from early-life (including gestational 29 

### and pre-weaning) plus chronic adulthood exposures w ith risks from chronic adulthood exposure 30 

### alone were not located. Following i.p. injection o f single doses of 75 or 150 mg/kg 31 

### benzo[a]pyrene to newborn (1 day old), infant (15 d ays old), or young adult (42 days old), 32 

### newborn and infant mice more readily developed tumo rs than young adult mice in the liver and 33 

### lung, the most predominant tissue sites of cancer d evelopment under these exposure conditions 34 

### (Vesselinovitch et al., 1975). The benzo[a]pyrene- exposed groups also displayed increased 35 

### incidences of stomach and lymphoreticular tumors, b ut the data indicated that these tumors 36 

### developed more readily with exposure at 42 or 15 da ys, compared with exposure on PND 1 . 37

38

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4.8.2. Possible Gender Differences 1 

### Cheng et al. (2007) had conducted a study in which they found that lung tumor tissue 2 

### from nonsmoking females contained higher benzo[a]py rene-DNA adduct levels than that from 3 

### nonsmoking males. The adduct levels were associate d with CYP1A1 protein levels in the same 4 

### tissues. Female lung cancer tissue contains higher levels of DHH activity than male lung cancer 5 

### tissue. DHH is an enzyme that can divert benzo[a]p yrene-7,8-dihydrodiol into the quinone 6 

### pathway, thus preventing BPDE and DNA adduct format ion. It is highly expressed in liver, but 7 

### only weakly in lung. Cheng et al. (2007) decided t o investigate benzo[a]pyrene-DNA adduct 8 

### formation in several lung cancer cell lines to eluc idate the roles of CYP1A1 and DHH in this 9 

### process. They found that DNA adduct levels were in creased in cell lines that contain elevated 10



### CYP1A1 and DHH isoform 1 activities. When DHH1 act ivity was blocked in these cells, DNA 11 

### adduct levels were increased. Benzo[a]pyrene-DNA a dduct levels in 120 lung tumor samples 12 

### were associated with the protein levels of CYP1A1, but not DHH1. Comparing tumor tissues 13 

### from both genders lung cancer patients they observe d that a significantly higher percentage of 14 

### female lung tumors had measurable CYP1A1 levels, bu t were negative for DHH1, compared 15 

### with male tumors. The authors suggested that a gen der difference in DHH1 activity was in part 16 

### responsible for the increased lung tumor incidence in females. 17 

### Chang et al. (2007) conducted a study also based in the increased incidence of lung 18 

### cancer in human females, but focused on the benzo[a ]pyrene interaction with estrogen that result 19 

### in elevated COX-2 expression. COX-2 (aka PHS-2) ca n activate the procarcinogen 20 

### benzo[a]pyrene-7,8-dihydrodiol to BPDE. Human bron chial epithelial cells were treated with 21 

### benzo[a]pyrene and/or 17 β -estradiol. The combined, but not the individual t reatments induced 22 

### COX-2 expression. The authors considered their fin dings as mechanistic evidence towards 23 

### understanding the gender difference in susceptibili ty towards benzo[a]pyrene. 24 

### Taioli et al. (2007) reviewed the evidence for a co nnection between MPO polymorphism 25 

### and lung cancer (a more detailed overview of this s tudy is given in Section 4.8.3.3). MPO 26 

### converts benzo[a]pyrene metabolites into highly rea ctive epoxides and a known polymorphism 27 

### in its promoter region is said to afford some prote ction from lung cancer. Several genetic 28 

### variants of MPO are known, most of which result in deficiency of the enzyme. The MPO- G/G 29 

### genotype (WT) is said to be associated, among other s, with acute promyelocytic leukemia, 30 

### aerodigestive tract cancer, coronary artery disease , early-onset multiple sclerosis, and an 31



### increased incidence of Alzheimer disease. A multi- study analysis was conducted after several 32 

### epidemiologic studies had suggested an association between this gene polymorphism and lung 33



### cancer incidence. The data were stratified for eth nicity, age, gender, and smoking status but 34 

### neither age nor gender showed any association for M PO polymorphism and lung cancer risk. 35 

### The authors hypothesized that age- and gender-relat ed associations with MPO genotype and lung 36 

### cancer incidence, as had been observed in other stu dies, may be related to age- and gender37 

### dependent smoking habits rather than to the gene po lymorphism itself. 38

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Mammary epithelium and tissues from the female geni tal tract have the ability to activate 1 

### benzo[a]pyrene. Morris and Seifter (1992) made a s trong case for benzo[a]pyrene as a potential 2 

### causative agent in breast cancer, not only based on the anti-estrogenic action of benzo[a]pyrene 3 

### but also on its tendency to accumulate in adipose a nd hence breast tissue. Jeffy et al. (2002) also 4 

### pointed out that PAHs are risk factors for breast c ancer. Because the populations in the studies 5 

### presented in Section 4.1.4 were predominantly, if n ot exclusively males, data for breast or 6 

### cervical cancers in relation to benzo[a]pyrene expo sure are not available. 7 

### Gender differences in the response to benzo[a]pyren e have been demonstrated in 8 

### numerous animal studies (Knuckles et al., 2001; Kro ese et al., 2001; Ramesh et al., 2001a, 2000; 9 

### Hood et al., 2000; Rodriguez et al., 1997; Weyand e t al., 1994; Turusov et al., 1990; Weyand and 10 

### Bevan, 1987). The differences ranged from variatio ns in feed intake, with or without effects on 11 

### body weight, to differences in disposition, all the way to different susceptibility towards 12 

### benzo[a]pyrene-induced cancers. In some studies, f emales and males were dosed differently; in 13 

### general, females were more resistant to benzo[a]pyr ene toxicity than males. None of the studies 14 

### presented cogent explanations for the observed diff erences. 15 

### In the 2-year bioassay by Kroese et al. (2001), fem ale rats had fewer tumors of the 16 

### forestomach and auditory canal, but more tumors of the liver, compared with males. There was a 17 

### rather striking negative dose response for pituitar y tumors in females (eight to zero tumors from 18 

### control to highest dose), but not in males. Brune et al. (1981) used both sexes of animals in their 19 

### study, but did not report their findings for the se xes separately, allowing the conclusion that no 20



### obvious sex differences were observed. 21 

### Soyka (1980) found that prenatal treatment of mice with benzo[a]pyrene affected 22 

### response to an enzyme-inducing challenge with 3-MC in a gender-specific way when the animals 23 

### were 3 months old. Female offspring of benzo[a]pyr ene-treated mice had significantly elevated 24 

### hepatic microsomal aminopyrine demethylase activity , while CYP450 levels were significantly 25 

### lower in male offspring. 26 

### Sharma et al. (1997) specifically attempted to reso lve the gender difference in cancer 27 

### susceptibility of CD-1 mice. They focused on gluta thione S-transferase π (GSTP) because it 28 

### detoxifies BPDE. They noted that constitutive expr ession of GSTP in the liver of the male CD-1 29 

### mouse was higher than in the female and that GSTP a ctivity was much more inducible by the 30 

### antioxidant butylhydroxyanisole in the female than in the male mouse. They reported that 31 

### female mice were more susceptible to the carcinogen ic effect of benzo[a]pyrene than males, but 32 

### only females could be partially protected from benz o[a]pyrene-induced carcinogenesis by the co33 

### administration of butylhydroxyanisole. This is an indication that Phase II enzymes may play a 34 

### role in the gender difference towards benzo[a]pyren e toxicity. 35 

### Martin et al. (2004) used a transgenic mouse model to address the question of gender 36 

### differences. They used p53 heterozygous Tg.AC (v-H a-ras) mice, a strain possessing a 37 

### carcinogen-inducible ras oncogene, but only one fun ctional p53 tumor suppressor gene. The 38

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authors emphasized that these two mutations have be en observed frequently in human tumors. 1 

### Female and male animals received 20 mg/kg benzo[a]p yrene by gavage in corn oil twice weekly 2 

### for 10 weeks. Eighteen weeks after termination of dosing, tissues were collected for histologic 3 

### processing. There were evident differences in gend er response (percent of total, female 4 

### control/male control:female treated/male treated): mortality (60/60:70/40), thymus hyperplasia 5 

### (9/30:27/10), bladder papilloma (0/0:0/13), hepatoc yte vacuolization (54/92:86/55), 6 

### hematopoietic cell proliferation in spleen (57/8:82 /27), lymph node hyperplasia (0/0:0/33), and 7 

### malignant lymphoma (0/0:7/36). Neoplasia of the fo restomach did not show a gender difference. 8 

### The focus of this study was evaluation of feed modi fications in carcinogenesis studies (some of 9 

### the animals were given N-acetyl cysteine in the fee d); the authors did not speculate on the 10 

### reasons for the gender differences. Wei et al. (20 00) (see Section 4.1.4) observed that female 11 

### lung cancer patients displayed less DNA repair capa city than males. 12 

### benzo[a]pyrene has also been described as an anti-a ndrogen (see Sections 4.4.1.3 and 13 

### 4.5.3) in animal studies. The AhR mediates anti-es trogenic effects of its ligands (see Section 14 

### 4.6.3.2). The findings in animals—anti-estrogenic and potential protection from breast cancer— 15 

### are at odds with postulates made for humans that be nzo[a]pyrene may advance the development 16 

### of breast cancer (Jeffy et al., 2002; Morris and Se ifter, 1992). Li et al. (1999) reported that 41% 17 

### of the samples of noncancerous breast tissue from b reast cancer patients contained 18 

### benzo[a]pyrene-like DNA adducts, while no such addu cts were detected in tissues obtained from 19 

### breast reduction surgery patients. Gender-specific expression of glycine N-methyltransferase 20 

### (GNMT) has been shown for mice (Section 4.5.2) and might help to explain gender-related 21 

### effects, including cancer formation, in this specie s. Applicability to human populations, 22 

### however, has not been addressed. 23

24 

### 4.8.3. Genetic Polymorphisms 25 

### The metabolic formation and subsequent binding to c ritical positions in DNA of ultimate 26 

### carcinogenic forms of PAH are recognized as key mec hanistic events in tumorigenesis. 27 

### Increased PAH exposure concentrations are associate d with increased levels of DNA adducts and 28 

### other biomarkers in both target and surrogate tissu es. Observed biomarker levels vary 29 

### considerably even among persons with apparently com parable exposures (Garte et al., 2007; 30 

### Perera and Weinstein, 2000). While laboratory vari ation and uncertain exposure estimates 31 

### contribute to the differences, inter-individual var iation in PAH metabolism (activation and 32 

### detoxification) may be especially important. In pa rticular, heritable metabolic gene (single 33 

### nucleotide polymorphisms [SNPs]) appear in some cas es to influence individual susceptibility to 34 

### specific types of cancer, in addition to factors su ch as ethnicity, age, gender, nutrition, hormonal 35 

### and immune status, and preexisting health impairmen t. 36 

### In humans, benzo[a]pyrene is metabolized to the hig hly DNA-reactive BPDE by 37 

### microsomal CYP Phase I enzymes, mediated primarily by the CYP1A1 and CYP1B1 genes. 38

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Polymorphic variations in the human population that result in high inducibility of CYP1A1, 1 

### CYP1B1, and other CYPs have been implicated as dete rminants of increased lung cancer risk in 2 

### some studies (Aklillu et al., 2005; Alexandrov et a l., 2002; Perera and Weinstein, 2000). The 3 

### Phase II cytosolic GST, mediated by variants of the GSTM1 and GSTT1 genes, prevent the 4 

### formation of BPDE-DNA adducts. Some evidence sugge sts that humans lacking a functional 5 

### GST gene have higher PAH(BPDE)-DNA adduct levels an d thus be at greater risk for cancer 6 

### (Vineis et al., 2007a; Pavanello et al., 2004; Alex androv et al., 2002; Perera and Weinstein, 7 

### 2000). Similarly, acquired deficiencies or inherit ed gene polymorphisms that affect the 8 

### efficiency or fidelity of DNA repair may also influ ence individual susceptibility to cancer 9 

### (Matullo et al., 2003; Shen et al., 2003; Cheng et al., 2000; Perera and Weinstein, 2000; Wei et 10 

### al., 2000; Amos et al., 1999). In general, however , support for the role of SNP in significantly 11 

### modulating PAH cancer risk is relatively weak (i.e. , small increases in cancer risk) or 12 

### inconsistent, possibly due in part to small study s ize and the use of DNA-adduct detection 13 

### methods with low specificity (i.e., bulky DNA-adduc ts) and sensitivity. Combinations of 14 

### metabolic polymorphisms, on the other hand, are rec eiving increased attention as critical 15 

### determinants of cumulative DNA-damaging dose, and t hus individual cancer risk. 16 

### Following a report from Japan that the GSTM1 null g enotype combined with a mutated 17 

### CYP1A1 genotype was associated with increased lung cancer risk, Rojas and coworkers (2000) 18 

### measured specific BPDE-DNA adducts in leukocytes (H PLC with fluorometric detection) to 19 

### evaluate the impact of CYP1A1, GSTM1, and GSTT1 gen otype combinations. The human 20 

### subjects were 89 PAH-exposed coke oven workers (smo kers and nonsmokers) and 44 power 21 

### plant workers (all smokers) not occupationally expo sed to PAH. Increased adduct levels were 22 

### significantly correlated with CYP1A1 polymorphism, occupational PAH exposure, and smoking. 23 

### Combinations of genotypes were observed to have a s ignificant impact on BPDE-DNA adducts, 24 

### ranging from the absence of adducts in subjects wit h the active CYP1A1/GSTM1 genotypes to 25 

### the highest BPDE-DNA adduct level in the most susce ptible combination of mutated CYP1A1 26 

### with null GSTM1 genotype. The results provide mech anistic support for distinguishing high27 

### susceptibility benzo[a]pyrene-exposed subgroups, an d for understanding their association with 28



### increased cancer rates. 29 

### Pavanello et al. (2005) studied associations betwee n xeroderma pigmentosum 30 

### (XP)-linked gene polymorphisms, the GSTM1 polymorph ism, and bulky BPDE-type DNA 31 

### adduct formation in peripheral lymphocytes from 67 highly PAH-exposed male Polish coke oven 32 

### workers. The four XP genotypes studied impart low NER capacity, while the GSTM1 active or 33 

### null genotypes are associated with effective or ine ffective removal of biologically active 34 

### benzo[a]pyrene metabolites via GSH conjugation. Wo rkers were questioned for smoking habits, 35 

### charbroiled meat consumption, and other factors tha t might have affected their PAH exposure. 36 

### PAH exposure was assessed via urinary 1-OH-pyrene l evels. There was a statistically significant 37



### difference in the number of DNA adducts between the GSTM1 active and null carriers (3.37 ± 38

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2.20 vs. 6.73 ± 6.61 adducts per 10 8 

### nucleotides, respectively). For the DNA repair ge ne 1 

### polymorphisms, there was an increase in DNA adduct numbers from homozygous WT carriers to 2 

### low-DNA-repair homozygous variant carriers. This d ifference was statistically significant for 3 

### homozygous carriers of the XPC-PAT and XPA-A23G var iants, but not for the homozygous 4 

### XPD-Lys751Gln and XPD-Asp312Asn variants. Individu als with a combination of DNA repair5 

### unfavorable XPC or XPA genotypes and the GSTM1-null variant generally fell into the highest 6 

### tertile of DNA adduct numbers. Smoking status and diet did not influence urinary 1-OH-Py or 7 

### BPDE-DNA adduct levels. These results support the conclusion that certain gene polymorphism 8 

### combinations affecting DNA repair or detoxification capacities may increase health risks 9 

### resulting from PAH exposure. 10 

### Porter et al. (2005) also evaluated the influence o f XPA gene variants involved in NER 11 

### on BPDE-induced cytotoxicity. SV40-transformed hum an skin fibroblasts from an XP patient 12 

### with a nonsense XPA mutation were stably transfecte d with the WT XPA gene or either of two 13 

### rare XPA variants; the transfected genes could be o verinduced with ponasterone A. The WT 14 

### XPA and both variants had greatly improved survival compared to XPA-free cells. Survival was 15 

### even more improved by ponasterone A induction in th e variant, but not the WT cells. These 16 

### findings indicate that the polymorphic XPAs show gr eater ability to repair BPDE-induced DNA 17 

### damage, and thus may offer some protection from ben zo[a]pyrene-induced genotoxicity, while 18 

### the nonsense mutation is likely to increase genotox ic risk. 19

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1



### 5. DOSE-RESPONSE ASSESSMENTS 2

3

4 

### 5.1. ORAL REFERENCE DOSE (RfD) 5 

### 5.1.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification 6 

### There are limited data establishing associations be tween increased risk for noncancer 7 

### health effects in humans and exposure to benzo[a]py rene. Several epidemiology studies have 8 

### reported associations between adverse birth outcome s including reduced birth weight, postnatal 9 

### body weight, and head circumference and internal bi omarkers of exposure to benzo[a]pyrene 10 

### (BPDE-DNA adducts) via exposure to complex PAH mixt ures (Tang et al., 2008, 2006; Perera et 11 

### al., 2005a, b). However, extrapolations from these studies are complicated by the concomitant 12 

### exposure to multiple PAHs and other components in t he mixture. Thus, studies in humans were 13



### not selected to serve as the basis of the RfD. 14 

### The subchronic and chronic oral exposure animal dat abase includes a 2-year gavage 15 

### cancer bioassay with male and female Wistar rats (K roese et al. 2001), a 2-year dietary cancer 16 

### bioassay with female B6C3F 1 

### mice (Beland and Culp, 1998; Culp et al., 1998), a 90-day gavage 17 

### study with male and female Wistar rats (Kroese et a l., 2001), a 90-day dietary study with male 18 

### and female F344 rats (Knuckles et al., 2001), and a 35-day study in male Wistar rats evaluating 19 

### immune endpoints (De Jong et al., 1999). Also avai lable are five reproductive/developmental 20 

### toxicity studies in rodents examining reproductive endpoints in male Sprague-Dawley rats 21 

### (Zheng et al., 2010) and C57BL/6 mice (Mohamed et a l., 2010), in offspring of treated CD-1 and 22 

### NMRI female mice (Kristensen et al., 1995; MacKenzi e and Angevine, 1981), and in female 23 

### Sprague-Dawley rats (Xu et al., 2010). 24 

### Kroese et al. (2001) exposed Wistar rats to benzo[a ]pyrene in soybean oil by gavage at 25 

### doses of 0, 3, 10, or 30 mg/kg-day, 5 days/week, fo r 2 years. This study was primarily designed 26 

### as a cancer bioassay and did not evaluate other end points. An increase in the incidence of 27 

### animals with forestomach hyperplasia, compared with the control incidence, occurred at the low 28 

### and mid-dose but not the high-dose level; an dose-r elated, increased incidence of forestomach 29



### tumors was observed at doses ≥ 3 mg/kg-day. An increased incidence of hepatic cl ear cell foci 30



### of cellular alteration was also observed at the 3 m g/kg-day, but not at the 10 or 30 mg/kg-day. 31 

### At the two highest exposure levels, elevated incide nces of liver tumors were observed. 32



### Female B6C3F 1 

### mice were administered benzo[a]pyrene in the diet at average daily doses 33 

### of 0, 0.7, 3.3, and 16.5 mg/kg-day for 2 years (Bel and and Culp, 1998; Culp et al., 1998). An 34



### increase in the incidence of mice with forestomach hyperplasia, compared with the control 35 

### incidence, occurred at the lowest exposure level (2 3/47 at 0.7 mg/kg-day versus 13/48 in 36 

### controls). Similar to the rat bioassay (Kroese et al., 2001), forestomach hyperplasia was 37 

### observed with increasing incidence of animals with forestomach tumors (squamous cell 38

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papillomas or carcinomas) with increasing dose. N o other dose-related effects were reported in 1 

### this cancer bioassay. 2 

### A 90 day study also reported by Kroese et al. (2001 ) treated animals by gavage 5 3 

### days/week with 0, 3, 10, or 30 mg/kg benzo[a]pyrene in corn oil. The most sensitive effects 4 

### observed included increased liver weight and decrea sed thymus weight. Increases in liver weight 5 

### greater than 10% of controls were observed at 10 an d 30 mg/kg-day in males only, and were 6 

### statistically significant. However, there were no statistically significant elevations in liver 7 

### enzymes screened in serum (ALT, AST, LDH, and GGT). The biological significance of 8 

### increased liver weight in males in the absence of e levated liver enzymes in the serum is unclear. 9 

### A decrease in thymus weight was observed in both se xes at 30 mg/kg-day, at 17 and 33% in 10 

### females and males, respectively, compared with cont rols. At 10 mg/kg-day, thymus weight in 11 

### males was decreased by 15% (not statistically signi ficant). An increase in the incidence of 12 

### thymus atrophy was also observed in the 30 mg/kg-da y males that showed a reduction of thymus 13 

### weight. Incidences for thymus atrophy (categorized in severity as slight) for the control through 14 

### high-dose groups were 0/10, 0/10, 0/10, and 3/10 fo r females and 0/10, 2/10, 1/10, and 6/10 for 15 

### males. The thymus is an organ involved in the matu ration of immune cells especially early in 16 

### development. A change in thymus weight in the adul t animal may be accompanied by alterations 17 

### of the immune system in functional assays, but the significance of thymus weight changes alone 18



### is unknown. 19 

### Knuckles et al. (2001) exposed male and female F344 rats (6-8 per group) to 20 

### benzo[a]pyrene at doses of 0, 5, 50, or 100 mg/kg-d ay in the diet for 90 days. Statistically 21 

### significant decreases in RBC counts and hematocrit level (decreases as much as 10 and 12%, 22 

### respectively) were observed in males at doses ≥ 50 mg/kg-day and in females at 100 mg/kg-day. 23 

### The effect observed in this study at the lowest dos e was an increase in abnormal tubular casts in 24 

### the kidney in males in which increases were observe d at 5 mg/kg-day (40%), 50 mg/kg-day 25 

### (80%) and 100 mg/kg-day (100%), compared to 10% in the controls. In females, only 10% 26 

### showed significant kidney tubular changes at the tw o high dose levels compared to zero 27 

### incidence in controls. The incidences for kidney l esions were not provided; instead the data are 28 

### reported graphically as rounded percent incidences. Several reporting gaps in Knuckles et al 29 

### (2001) make interpretation of the results difficult . Specifically, the authors do not provide 30 

### statistical analysis of the renal endpoint nor do t hey provide the incidence data which would 31 

### allow for independent statistical analysis. The st udy author was contacted, but additional 32 

### clarification of the study data was not provided. Therefore, due to reporting gaps and resulting 33 

### reduced confidence, this study was not considered f urther in selecting the principal study. 34 

### De Jong et al. (1999) treated male Wistar rats (eig ht/dose group) with benzo[a]pyrene by 35 

### gavage 5 days/week for 35 days at doses of 0, 3, 10 , 30, and 90 mg/kg-day. Hematological and 36 

### immunological changes were reported. Small, but sta tistically significant, dose-related decreases 37 

### in RBC count (5%) and associated measures (hemoglob in, and hematocrit) were observed at 38

### 203 DRAFT - DO NOT CITE OR QUOTE

≥ 10 mg/kg-day. In addition, a dose-related and stat istically significant decrease in the relative 1 

### number of B cells (13%) in the spleen was observed at ≥ 10 mg/kg-day compared to controls. 2 

### Dose-related decreases in thymus weight were statis tically significant at ≥ 10 mg/kg-day. 3 

### Decreases in heart weight at 3 mg/kg-day and in kid ney weight at 3 and 30 mg/kg-day were also 4 

### observed, but these changes did not show dose-depen dent responses. At doses above 10 mg/kg5 

### day, significant decreases were observed in absolut e number of cells harvested in the spleen, in 6 

### the number of B cells in the spleen, and in NK cell activity in the spleen, as well as a decrease in 7 

### serum IgM and IgA in rats. 8 

### Zheng et al., (2010) treated male Sprague-Dawley ra ts (8/group) to 0, 1, or 5 mg/kg-day 9 

### benzo[a]pyrene by daily corn oil gavage for a durat ion of 30 or 90 days. Testicular testosterone 10 

### was statistically significantly decreased in the hi gh dose group (approximately 15%) following 11 

### 90 days of exposure. The low dose group also appea red to have a similar average depression of 12 

### testosterone levels; however, the change did not re ach statistical significance. 13 

### Mohamed et al. (2010) investigated multi-generation al effects in male mice following 14 

### exposure of six-week old C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day 15 

### benzo[a]pyrene for 6 weeks by daily corn oil gavage . Following final treatment, male mice were 16 

### mated with two untreated female mice to produce an F1 generation; F1 and F2 males were also 17



### mated with untreated female mice. The mice of the F1, F2, and F3 generations were not exposed 18 

### to benzo[a]pyrene. Statistically significant reduc tions of approximately 50% were observed in 19 

### epididymal sperm counts of F0 and F1 generations tr eated with the low dose of benzo[a]pyrene. 20 

### For F0 and F1 generations of the high dose group, e pididymal sperm counts were reduced 21 

### approximately 70%. Means and variances were not re ported but were presented graphically. 22 

### This study indicates that exposure to benzo[a]pyren e may have transgenerational effects on 23 

### sperm count. However, due to incomplete reporting, this study was not considered further for 24 

### selection as the principal study but was considered to be supportive of low dose male 25 

### reproductive effects following benzo[a]pyrene expos ure. 26 

### MacKenzie and Angevine (1981) exposed groups of 30 –60 female CD-1 mice to 0, 10, 27 

### 40, or 160 mg/kg-day benzo[a]pyrene on GDs 7–16. C rossover mating studies were then 28 

### conducted in which F1 offspring were mated continuo usly with untreated mice to determine 29 

### effects on fertility. Benzo[a]pyrene did not appea r to be overtly toxic to mothers or offspring. 30 

### However, statistically significant decreased pup we ight was observed at all dose levels at day 42. 31 

### At the lowest dose tested, pup weight was decreased 6% compared to control. At maturity, the 32 

### fertility of these animals was tested. The F1 male and F1 female fertility indices (i.e., percent of 33 

### mated animals that were pregnant) were significantl y decreased in each exposure group as 34 

### follows (control through high-dose groups): F1 male s: 80.4, 52.0, 4.7, and 0.0; F1 females 100, 35 

### 65.7, 0.0, and 0.0. After six months on the breedi ng study, 34% of the gestationally treated 36 

### females in the 10 mg/kg-day dose group failed to pr oduce any litters, and the the F1 females in 37 

### this dose group that did litter produced statistica lly significantly smaller litter sizes (19% 38

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reduction in mean litter size). These reductions i n fertility indices were associated with 1 

### decreased testes and ovary weight in the F1 animals . Testes weight was decreased 40 and 88% 2 

### at 10 and 40 mg/kg-day, respectively, and was assoc iated with histologic evidence of injury to 3 

### the seminiferous tubules. In female F1 animals, se vere reductions in ovarian tissues were 4 

### observed at all dose levels such that ovary weight measurements were difficult to obtain. 5



### Examination of available tissue in these females re vealed hypoplastic ovaries with few follicles 6 

### and corpora lutea (10 mg/kg-day) or with no evidenc e of folliculogenesis at the higher dose 7 

### (40 mg/kg-day). 8 

### Similar results were reported in a study in which g roups of nine NMRI F0 female mice 9 

### were exposed by gavage to 0 or 10 mg/kg-day benzo[a ]pyrene on GDs 7–16 (Kristensen et al., 10 

### 1995). F1 females were continuously bred with an u ntreated male for 6 months. F1 females had 11 

### decreased mean ovary weight (30% decreased, compare d with controls) and reduced fertility as 12 

### reflected by decreased mean number of F2 litters (t hree compared with eight for control F1 13 

### females). F1 females had statistically significant ly lower median numbers of offspring, number 14 

### of litters, and litter sizes and a statistically si gnificantly greater median number of days between 15 

### litters as compared with the controls. At necropsy , the F1 females had statistically significantly 16 

### reduced ovary weight with histologic examination re vealing decreased numbers of small, 17 

### medium, or large follicles and corpora lutea. 18 

### Xu et al., (2010) treated female Sprague-Dawley rat s (6/group) to 0, 5, or 10 mg/kg-day 19 

### benzo[a]pyrene by corn oil gavage every other day f or a duration of 60 days. This resulted in 20 

### time weighted average doses of 0, 2.5, and 5 mg/kg- day over the study period of 60 days. 21 

### Absolute ovary weight was statistically significant ly reduced in the both the low and high 22 

### benzo[a]pyrene dose groups (11 and 15%, respectivel y; see Table 5-1). Animals in the high dose 23 

### group also had statistically significantly depresse d levels of estradiol (by approximately 25%) 24 

### and decreased numbers of primordial follicles (by a pproximately 20%) compared to controls. 25 

### Statistically significantly altered estrus cyclicit y was also evident in the high dose of 26 

### benzo[a]pyrene. 27

28 

### Table 5-1. Means ± SD for ovary weight in female S D-rats

Dose (mg/kg-d) a

0 2.5 5

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Ovary weight (g) 0.160 ± 0.0146 0.143 ± 0.0098 b 0.136 ± 0.0098 b

Body weight (g) 261.67 ± 12.0 249.17 ± 11.2 247.25 ± 11.2

a TWA doses over the 60 day study period

b Statistically different from controls (p < 0.05) u sing one-way ANOVA

Source: Xu et al. (2010).

1 

### 5.1.2. Methods of Analysis 2 

### A number of noncancer effects observed following c hronic or subchronic administration 3 

### of benzo[a]pyrene were modeled with U.S. EPA’s Benc hmark Dose (BMD) Modeling Software 4 

### (BMDS) where data were amenable. These endpoints i ncluded increased liver weight, decreased 5 

### thymus weight, decreased percent of splenic B-cells , increased forestomach hyperplasia, and 6 

### decreased ovary weight (Xu et al., 2010; Kroese et al., 2001; De Jong et al., 1999). Zheng et al. 7 

### (2010) did not provide enough information (i.e., in cidences or means and variances) to allow for 8 

### BMD modeling. Data from other studies could not be modeled due study design utilizing only 9 

### one dose (Kristensen et al., 1995) or a highly elev ated magnitude of response at the lowest dose 10 

### (MacKenzie and Angevine 1981) in which extrapolatio n down to a suitable benchmark response 11 

### would be unsupported by the available models. 12



### In accordance with U.S. EPA’s Benchmark Dose Technical Guidance Document (U.S. 13 

### EPA, 2000b), the BMD and the 95% lower confidence l imit on the BMD (BMDL) were 14 

### estimated using a benchmark response (BMR) of 1 sta ndard deviation (SD) from the control 15



### mean for continous data or a BMR of 10% extra risk for dichotomous data in the absence of 16 

### information regarding what level of change is consi dered biologically significant, and also to 17 

### facilitate a consistent basis of comparison across endpoints and assessments. A summary of 18 

### modeling results for each endpoint is listed below in Table 5-2. Further details including the 19 

### output and graph for the best fit model can be foun d in Appendix B. In general, model fit was 20 

### assessed by a chi-square goodness-of-fit test (i.e. , models with p < 0.1 failed to meet the 21 

### goodness-of-fit criterion) and the Akaike Informati on Criterion (AIC) value (i.e., a measure of 22 

### the deviance of the model fit that allows for compa rison across models for a particular endpoint). 23 

### Of the models exhibiting adequate fit, the model yi elding the lowest AIC value was selected as 24 

### the best-fit model. (U.S. EPA, 2000b). 25 

### For the forestomach hyperplasia endpoint, all data sets provided adequate descriptions of 26 

### the dose-response relationship from chronic oral ex posure to benzo[a]pyrene, but at the highest 27 

### dose level for the rats (Kroese et al., 2001), the incidence of forestomach hyperplasia was not 28 

### increased relative to controls. It is possible tha t the forestomach hyperplasia observed following 29 

### benzo[a]pyrene exposure may be a precursor to the d evelopment of forestomach tumors, but 30 

### specific data supporting this conclusion are unavai lable. Regardless, the male and female data 31

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sets in rats (Kroese et al., 2001) were modeled wit hout the data from the highest dose group due 1 

### to the nonmontonic increase in response to increasi ng dose (Kroese et al., 2001). 2 

### Points of departure (PODs) for endpoints that were not amenable to BMD modeling were 3 

### identified using a NOAEL/LOAEL approach (Zheng et a l., 2010; Kristensen et al., 1995; 4 

### MacKenzie and Angevine, 1981). A LOAEL of 5 mg/kg- day was identified for Zheng et al. 5 

### (2010) for significantly descreased testicular test osterone. A LOAEL of 10 mg/kg-day was 6 

### identified as the POD for Mackenzie and Angevine fo r decreased postnatal body weight and 7 

### decreased fertility of male and female mice treated during gestation. A POD based on the 8 

### LOAEL of 10 mg/kg/day was established from Kristens en et al. (1995) based on decreased 9 

### fertility and decreased ovary weight in female mice treated during gestation. 10207 DRAFT - DO NOT CITE OR QUOTETable 5-2. Summary of BMDs and BMDLs for modeled noncancer effects following oral exposureEndpoint/data ExposuredurationBMR Fitted model Goodness-offitp-valueAIC BMD(mg/kg-d)BMDL(mg/kg-d)ReferenceIncreased Liver Weightin Male Wistar Mice90 d 10%Linear (1◦polynomial),Power0.58 49.51 8.11 5.8Kroese etal., 2001Polynomial (2◦) 0.74 50.53 4.53 2.29Hill 0.82 50.48 4.1 1.24Decreased ThymusWeight in Male WistarMice90 d 1 SDLinear , Polynomial(2◦), Power(nonconstant variance)0.23 380.71 16.40 11.30Kroese etal., 2001Hill (nonconstantvariance)NADecreased ThymusWeight in FemaleWistar rats90 d 1 SDLinear 0.81 349.12 10.52 7.64Kroese etal., 2001HillNAPolynomial (2◦) 0.77 350.80 13.29 7.77PowerNADecreased ThymusWeight in Male WistarMice35 d 1 SDLinear , Polynomial (2◦)0.52 381.41 14.41 11.58De Jong etal., 1999Hill 0.42 382.91 11.15 6.19PowerNADecreased Splenic Bcells in Male Wistar rats35 d 1 SDLinear, Polynomial (2◦),Power (constantvariance)0.21 145.28 15.58 12.43De Jong etal., 1999Hill (constant variance) 0.18 146.18 10.24 5.31Increased ForestomachHyperplasiaain MaleWistar Rats2 yrs 10%Log-logistic 0.13 112.27 5.31 2.39Kroese etal., 2001Gamma, Multistage,Weibull0.12 112.37 5.63 2.67Logistic 0.09 112.93 7.25 4.35LogProbit 0.06 113.88 8.36 4.52Probit 0.10 112.87 7.09 4.13Increased ForestomachHyperplasiaain Female2 yrs 10%Log-logistic 0.32 117.04 2.15 1.35Kroese etal., 2001Logistic 0.06 120.02 4.23 3.28208 DRAFT - DO NOT CITE OR QUOTEWistar RatsLogProbit 0.02 121.13 3.91 2.57Gamma, Multistage,Weibull0.24 117.42 2.40 1.59Probit 0.06 119.74 3.99 3.06Increased ForestomachHyperplasia in FemaleB6C3F1mice2 yrs 10%Log-logistic 0.21 193.3 0.329 0.115Beland andCulp, 1998Logistic 0.06 194.7 0.757 0.545LogProbit 0.29 192.1 0.670 0.448Gamma, Multistage,Weibull0.42 191.3 0.421 0.295Probit 0.03 196.6 0.946 0.711Decreased OvaryWeight in FemaleSprague-Dawley Rats60 d 1 SDLinear , Polynomial (1◦)0.39 -138.67 2.3 1.5Xu et al.,2010PowerNAaThe best fit of each model considered is summarized. For continuous models (linear, polynomial, power, Hill), if anadequate required including a variance model, onlythe results including modeled variance are summarized and the use ofnonconstant variance is indicated; otherwise constant variance was assumed. Details in Appendix B.bData for the high-dose group were excluded from themodeled dataset due to a decreased incidence judged to be due tocompeting effects masking the response.NA = not applicable, model failed

### 209 DRAFT - DO NOT CITE OR QUOTE

Several candidate principal studies (Kroese et al., 2001; De Jong et al., 1999; Zheng et 1 

### al., 2010; Kristensen et al., 1995; MacKenzie and A ngevine, 1981) reported liver, thymus, 2 

### immune, and reproductive effects at higher doses re lative to the ovary and forestomach effects 3 

### (reported by Kroese et al., 2001; Beland and Culp, 1998) and were considered less sensitive 4 

### measures of benzo[a]pyrene effects. Forestomach hy perplasia was not selected as the critical 5 

### effect, even though it was observed at lower doses compared with other effects, based on the 6 

### consideration that the reproductive and fertility e ffects, observed in animals and supported by 7 

### human data, appear to better characterize noncancer low dose effects of BaP. Specifically, the 8 

### Xu et al., (2010) study was chosen as the principal study and decreased ovarian weight as the 9



### critical effect for the derivation of the RfD. Thi s study identified biologically and statistically 10 

### significant decreases in ovary weight, estrogen, an d primordial follicles, and altered estrus 11 

### cycling in treated animals. These reductions in fe male reproductive parameters are supported by 12 

### a large database of animal studies indicating that benzo[a]pyrene is ovotoxic with effects 13 

### including decreased ovary weight, decreased primord ial follicles, and reduced fertility (Mattison 14 

### et al., 1980; MacKenzie and Angevine 1981; Swartz a nd Mattison 1985; Miller et al., 1992; 15 

### Kristensen et al., 1995; Borman et al, 2000). Addi tionally, studies indicate that exposure to 16 

### complex mixtures of PAHs, such as through cigarette smoke, is associated with measures of 17 

### decreased fertility in humans (El Nemr et al., 1998 ; Neal et al., 2005). Specific associations have 18 

### also been made between infertility and increased le vels of benzo[a]pyrene in follicular fluid in 19 

### women undergoing in vitro fertilization (Neal et al ., 2008). 20

21 

### 5.1.3. RfD Derivation—Including Application of Unc ertainty Factors (UFs) 22 

### Of the endpoints discussed in section 5.1.1., decre ased ovary weight in female rats 23 

### (BMDL 1SD 

### of 1.5 mg/kg-day) reported by Xu et al. (2010) was selected to serve as the critical 24 

### effect for the RfD. A total UF of 3000 was applied to the POD of 1.5 mg/kg-day to account for 25 

### several areas of uncertainty. 26



### An UF A



### of 10 was applied to account for toxicokinetic and toxicodynamic differences 27 

### associated with extrapolation from animals to human s. The available data do not provide 28 

### quantitative information on the difference in susce ptibility to benzo[a]pyrene between rats and 29



### humans. 30



### An UF H 

### of 10 was applied to account for variability in su sceptibility among members of 31 

### the human population (i.e., interindividual variabi lity). Insufficient information is available to 32 

### quantitatively estimate variability in human suscep tibility to benzo(a)pyrene. 33



### An UF S 

### of 10 was applied for the extrapolation of subchro nic-to-chronic exposure 34 

### duration. The 60-day study by Xu et al. (2010) fal ls well short of a lifetime duration. 35 

### Therefore, it is unknown whether effects would be m ore severe or would be observed at lower 36 

### doses with a longer exposure duration. 37

### 210 DRAFT - DO NOT CITE OR QUOTE

An UF L 

### of 1 was applied for LOAEL-to-NOAEL extrapolation because the current 1 

### approach is to address this factor as one of the co nsiderations in selecting a BMR for BMD 2 

### modeling. In this case, a BMR of a 1 SD change fro m the control mean in ovary weight was 3 

### selected under an assumption that it represents a m inimal biologically significant response level. 4



### An UF D 

### of 3 was applied to account for deficiencies in th e benzo[a]pyrene toxicity 5



### database. Limited observational studies in humans have suggested associations between 6 

### biomarkers of internal dose of benzo[a]pyrene and a dverse birth outcomes (including reduced 7 

### birth weight, postnatal body weight, head circumfer ence, and neurodevelopment) and decreased 8 

### fertility (Edwards et al., 2010; Neal et al., 2008; Tang et al., 2008, 2006; Perera et al., 2009; 9 

### 2005a, b). However, the likely contribution of mul tiple exposure routes in these studies make 10 

### extrapolation to exposure concentrations uncertain. Several animal studies exist for 11 

### benzo[a]pyrene to inform noncancer effects, includi ng subchronic oral toxicity studies in rats and 12 

### mice, and two developmental studies and several rep roductive studies in mice and rats. The lack 13 

### of a standard multigenerational study (specifically , one which includes exposure from pre-mating 14 

### to lactation) is a data gap, especially considering benzo[a]pyrene has been shown to affect 15 

### fertility in adult male and female animals by multi ple routes of exposure (Mohamed et al., 2010; 16 

### MacKenzie and Angevine 1981; Kristensen et al., 199 5; Archibong et al., 2008; Borman et al., 17 

### 2000; Swartz and Mattison 1985). In addition, the lack of a study examining functional 18 

### neurological endpoints following in utero exposure is also a data gap considering the available 19 

### epidemiological evidence showing the association of in utero PAH exposure and indicators of 20 

### decreased neurological development (Edwards et al., 2010, Perera et al., 2009, Tang et al., 2008). 21 

### Therefore, an UF of 3 was applied to the POD for th e lack of a standard multigenerational 22 

### reproductive toxicity study and a neurodevelopmenta l study. 23

24 

### The RfD for benzo[a]pyrene was calculated as follow s: 25

26



### RfD = BMDL 1SD 

### ÷ UF 27 

### = 1.5 mg/kg-day ÷ 3000 28 

### = 0.0005 mg/kg-day 29

30

31



### 5.1.4. Previous RfD Assessment 32 

### No RfD was derived in the previous IRIS assessment. 33

34 

### 5.2. INHALATION REFERENCE CONCENTRATION (RfC) 35 

### 5.2.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification 36 

### The only chronic inhalation study available for ben zo[a]pyrene was designed as a cancer 37 

### bioassay and did not report noncancer endpoints (Th yssen et al.1981). However, several repeat 38

### 211 DRAFT - DO NOT CITE OR QUOTE

dose reproductive and developmental toxicity studie s are available in which effects on fetal 1 

### survival and the male reproductive system have been observed. 2 

### Reproductive system variables were adversely impact ed in male F344 rats (10/group) 3 

### exposed to benzo[a]pyrene aerosols at 75 μg/m 3 

### for 60 days (Archibong et al., 2008; Ramesh et 4 

### al., 2008). The testes from exposed rats weighed 3 4% less than those from unexposed controls. 5 

### Treatment with benzo[a]pyrene was also associated w ith reductions (compared with control 6 

### values) in total tubular volume and weight (~20%) an d tubular length (~40%); total weight and 7 

### volume of interstitium per paired testis (12%); and percentage of progressively motile stored 8 

### spermatozoa, stored sperm density, and percentage o f morphologically normal sperm (~70– 9 

### 80%). Daily sperm production and levels of circula ting and intratesticular testosterone were also 10 

### decreased in treated rats (by about 50, 70, and 80% respectively), compared with controls. 11 

### Luteinizing hormone levels were 50–60% higher in tr eated rats than controls 72 hours after 12



### exposure. 13 

### In a developmental study, timed-pregnant F344 rats (10/group) were exposed to 14 

### benzo[a]pyrene on GDs 11–21 as a carbon black aeros ol at 100 μg/m 3 

### to assess neurological 15 

### endpoints in offspring during PNDs 60–70 (Wormley e t al., 2004). Pups of the F1 generation 16 

### were weaned on PND 30 and tested for long-term pote ntiation electrophysiological responses in 17 

### the hippocampus during PNDs 60–70. Although the nu mber of implantation sites in treated rats 18 

### was within 1% of unexposed controls, the percentage of pups born relative to recorded 19 

### implantation sites in each dam (the birth index) wa s reduced by 65% in treated rats compared 20 

### with unexposed controls. In addition, protein leve ls of NMDA receptor subunit 1 were down21 

### regulated (by 18% on PND 10 and 67% on PND 30) in t he hippocampus of benzo[a]pyrene22 

### exposed F1 pups, and the magnitude of the long-term potentiation response across the perforant 23 

### path-granular cells in the hippocampus of F1 rats w as consistently weaker than the response 24 

### observed for the controls (about 25% weaker), sugge sting that exposure to benzo[a]pyrene via 25 

### the inhalation route attenuates the capacity for lo ng-term potentiation in the F1 generation. 26 

### However, no functional tests to assess neurotoxicit y were conducted in this study. 27 

### In another developmental toxicity study, timed-preg nant F344 rats (10/group) were 28 

### exposed to benzo[a]pyrene aerosols at concentration s of 25, 75, or 100 μg/m 3 

### on GDs 11–20 and 29 

### evaluated for post-implantation fetal survival and hormone levels associated with pregnancy 30 

### (Archibong et al., 2002). The total number of impl antation sites in treated rats was within 5% of 31 

### the values obtained for sham-exposed and unexposed controls. However, dose-dependent trends 32 

### were observed for decreased numbers of pups per lit ter and percent fetal survival per litter with 33 

### increasing benzo[a]pyrene concentrations. The numb er of pups/litter was decreased by 34 

### approximately14, 50, and 65% at 25, 75, and 100 μg/ m 3 

### , respectively, compared with sham35 

### exposed and unexposed controls. Percent survival/w as similarly reduced by about 20, 60, and 36 

### 65%, respectively, at the same exposure concentrati ons. In addition, biologically significant 37 

### decreases in pup weights (presented as g/litter) we re observed at concentrations ≥ 75 μg/m 3 

### (14 38

### 212 DRAFT - DO NOT CITE OR QUOTE

and 16% decreases at 75 and 100 μ g/m 3 

### , respectively). Levels of plasma progesterone, es tradiol1



### 17 β , and prolactin on GD 17 were decreased by about 12 , 14, and 35%, respectively, at 25 μg/m 3 2 

### and 17, 60, and 70%, respectively, at 75 μg/m 3 

### compared with respective controls. 3 

### The study by Archibong et al. (2002) was selected a s the principal study as it observed 4 

### biologically significant effects at the lowest dose tested by the inhalation route. This study 5 

### indicates that the developing fetus is a sensitive target following inhalation exposure to 6 

### benzo[a]pyrene. A LOAEL of 25 μg/m 3 

### was identified based on exposure to benzo[a]pyrene on 7 

### GDs 11–20 that caused biologically significant redu ctions in fetal survival and body weight 8 

### decreases in the surviving pups (see Table 5-3). T he observed decrease in pup weight and fetal 9 

### survival were selected as critical effects as they are the most sensitive noncancer effects observed 10 

### following inhalation exposure to benzo[a]pyrene. T hough only a few studies exist which 11 

### evaluate benzo[a]pyrene by the inhalation route, ad ditional support for this endpoint can be 12 

### found from oral studies of benzo[a]pyrene. A devel opmental/reproductive study conducted via 13



### the oral route in mice observed decreased survival of litters, decreased pup weight, and decreased 14 

### reproductive organ weight following in utero exposu re to benzo[a]pyrene on GD 7-16 15 

### (MacKenzie and Angevine, 1981). 16

17 

### 5.2.2. Methods of Analysis- Adjustment to a Human E quivalent Concentration (HEC) 18 

### By definition, the RfC is intended to apply to cont inuous lifetime exposures for humans 19 

### (U.S. EPA, 1994). EPA recommends that adjusted con tinuous exposures be used for inhalation 20 

### developmental toxicity studies as well as for studi es of longer durations (U.S. EPA, 2002). The 21 

### LOAEL of 25 μg/m 3 

### based on decreased pup weight and fetal survival r eported in the 22 

### developmental study by Archibong et al. (2002) was selected to serve as the POD. 23

24 

### Table 5-3. Pregnancy outcomes in female F344 rats treated with

benzo[a]pyrene on GDs 11–21 by inhalation

Parameter a Administered concentration of benzo[a]pyrene (μg/m 3 ) 0 (unexposed control) 0

(carbon black) 25 75 100 Implantation sites 8.6 ± 0.2 8.8 ± 0.1 8.8 ± 0.5 9. 0 ± 0.2 8.8 ± 0.1 Pups per litter 8.5 ± 0.2 8.7 ± 0.2 7.4 ± 0.5 b 4.2 ± 0.1 b 3.0 ± 0.2 b

Survival (litter %) 98.9 ± 1.1 96.7 ± 1.7 78.3 ± 4. 1 b 38.0 ± 2.1 b 33.8 ± 1.3 b

Pup weight (g/litter) 10.6 ± 0.1 8.8 ± 0.1 10.5 ± 0 .2 9.1 ± 0.2 b 8.9 ± 0.1 b

Crown-rump length (mm/litter) 29.4 ± 0.6 29.3 ± 0.5 28.0 ± 0.6 27.3 ± 0.7 27.9 ± 0.7

a Values presented as means ± SEM.

b Significantly different from controls at p < 0.05 b y one-tailed post-hoc t-testing following ANOVA.

Source: Archibong et al. (2002).

25

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Data for decreased pup survival were not amenable t o BMD modeling due to the pattern 1 

### of variability (heterogeneous variances) in the dat a set. Therefore, the LOAEL from this study 2 

### was used as the POD. The LOAEL from this study is based on a 4 hour exposure of pregnant 3 

### rats to 25 μg/m 3 

### benzo[a]pyrene on GDs 11-20. This concentration w as adjusted to account for 4 

### the discontinuous daily exposure as follows: 5

6



### POD ADJ 

### = POD x hours exposed per day/24 hours 7 

### = LOAEL x (4 hr/24 hr) 8 

### = 25 μg/m 3 

### x 4/24 9 

### = 4.2 μg/m 3 10

11 

### The human equivalent concentration (HEC) was calcul ated from the POD ADJ 

### by 12 

### multiplying by a dosimetric adjustment factor (DAF) , which, in this case, was the regional 13 

### deposited dose ratio (RDDR ER 

### ) for extrarespiratory (i.e. systemic) effects as d escribed in 14 

### Methods for Derivation of Inhalation Reference Conc entrations and Application of Inhalation 15 

### Dosimetry (U.S. EPA, 1994b). The observed developmental effe cts are considered systemic in 16 

### nature (i.e., extrarespiratory) and the current nor malizing factor for extrarespiratory effects of 17 

### particles is body weight. In the case of benzo[a]p yrene, the RDDR ER 

### was calculated as follows: 18

19

H TOT A TOT

H E A E

A H ER 

## )

(F ) (F ) (V ) (V

BW BW RDDR ×

× = 20



### where: 21 

### BW = body weight (kg) 22 

### V E 

### = ventilation rate (L/min) 23 

### F TOT 

### = total fractional deposition 24

25 

### The total fractional deposition (F TOT 

### ) includes particle deposition in the nasal-pharyng eal 26 

### region, the tracheobronchial region, and the pulmon ary region. F TOT 

### for both animals and 27 

### humans was calculated using the Multi-Path Particle Dosimetry model, a computational model 28 

### that can be used for estimating human and rat airwa y particle deposition and clearance (MPPD; 29 

### Version 2.0 © 2006, publicly available through the Hamner Institute). The F TOT 

### was calculated 30 

### based on the average particle size of 1.7 ± 0.085 ( mass median aerodynamic diameter ± 31 

### geometric standard deviation) as reported in the de scription of particle generation methods in 32 

### Ramesh et al. (2000). For the model runs, the Yeh- Schum 5-lobe model was used for the human 33 

### and the asymmetric multiple path model was used for the rat (see Appendix C for MPPD model 34 

### output). Both models were run under nasal breathin g scenarios with the inhalability adjustment 35 

### selected. A geometric standard deviation (GSD) of 1 was used as the default by the model 36 

### because the reported GSD of 0.085 ≤ 1.05. 37

### 214 DRAFT - DO NOT CITE OR QUOTE

The human parameters used in the model for calculat ing F TOT 

### and in the subsequent 1



### calculation of the POD HEC 

### were as follows: human - BW, 70 kg; V E 

### , 13.8 L/min; breathing 2 

### frequency, 16 per minute; tidal volume, 860 mL; FRC (functional residual capacity), 3300 mL; 3 

### and URT (upper respiratory tract) volume, 50 mL. A lthough the most sensitive population in the 4 

### principal study is the developing fetus, the adult rat dams were exposed. Thus, adult human 5 

### parameters were used in the calculation of the HEC to extrapolate from a pregnant rat to a 6 

### pregnant human. The parameters used for the rat we re BW, 0.25 kg (based on the approximate 7 

### weight of a 100 day-old, female timed-pregnant Spra gue-Dawley rat); V E 

### , 0.18 L/min; breathing 8 

### frequency, 102 per minute; tidal volume, 1.8 mL; FR C (functional residual capacity), 4 mL; and 9 

### URT (upper respiratory tract) volume, 4.42 mL. All other parameters were set to the default 10 

### value (see Appendix C). 11 

### Under these conditions, the MPPD model calculated F TOT 

### values of 0.621 for the human 12 

### and 0.181 for the rat. Using the above equation, th e RDDR ER 

### was calculated to be 1.06. 13 

### From this, the POD HEC 

### was calculated as follows: 14

15



### POD HEC 

### = POD ADJ 

### × RDDR ER 16



### POD HEC 

### = 4.2 μg/m 3 

### × 1.1 17



### POD HEC 

### = 4.6 μg/m 3 18

19

20 

### 5.2.3. RfC Derivation- Including Application of Un certainty Factors (UFs) 21



### The critical effect for the derivation of the RfC w as identified as decreased fetal survival 22 

### and decreased pup weight associated with inhalation exposure to pregnant rats on GDs 11–20. 23 

### The LOAEL for decreased fetal survival was adjusted to a continuous human equivalent 24



### concentration and used as the POD for the derivatio n of the RfC. A total UF of 1000 was 25 

### applied to the POD HEC 

### to account for four main areas of uncertainty: 26



### A UF A



### of 3 was applied to account for uncertainties in e xtrapolating from rats to humans. 27 

### Application of a UF of 10 encompasses two areas of uncertainty: toxicokinetic and 28 

### toxicodynamic uncertainties. In this assessment, t he toxicokinetic component is mostly 29 

### addressed by the determination of a HEC as describe d in the RfC methodology (U.S. EPA, 30 

### 1994b). Therefore, a UF of 3 was applied to accou nt for the remaining toxicodynamic 31 

### uncertainties in the extrapolation from rats and hu mans. 32



### A UF H 

### of 10 was applied to account for variability in su sceptibility among members of 33 

### the human population (i.e., interindividual variabi lity). Insufficient information is available to 34 

### quantitatively estimate variability in human suscep tibility to benzo(a)pyrene. 35



### A UF L 

### of 10 was applied to account for the use of a LOAE L. A NOAEL was not 36 

### identified for decreased fetal survival observed by Archibong et al (2002). At the lowest dose, 37 

### benzo[a]pyrene treated dams gave birth to 15% fewer pups compared to dams treated with 38

### 215 DRAFT - DO NOT CITE OR QUOTE

vehicle alone (carbon black particles). Due to the lack of a NOAEL and the inability to model 1 

### the data set for decreased fetal survival, a UF of 10 was applied to extrapolate to a NOAEL. 2



### A UF S 

### of 1 was applied to account for extrapolation from subchronic to chronic exposure 3 

### because developmental toxicity resulting from a nar row period of exposure was used as the 4 

### critical effect. The developmental period is recog nized as a susceptible life stage when exposure 5 

### during a time window of development is more relevan t to the induction of developmental effects 6 

### than lifetime exposure (U.S. EPA, 1991a). 7



### A UF D 

### of 3 was applied to account for deficiencies in th e benzo[a]pyrene toxicity 8 

### database. One developmental study exists for benzo [a]pyrene by the inhalation route. The 9 

### developmental study by Archibong et al., 2002, whic h was used as the basis for the RfC, 10



### observed decreased fetal survival and decreased lit ter size following gestational treatment on 11



### GDs 11-20. Limited observational studies in humans have suggested associations between 12 

### biomarkers of internal doses of benzo[a]pyrene and adverse birth outcomes (including reduced 13 

### birth weight and postnatal weight, decreased head c ircumference, and impaired 14 

### neurodevelopment) and decreased fertility (Neal et al., 2008; Tang et al., 2008, 2006; Perera et 15 

### al., 2005a, b). A multigenerational reproductive s tudy examining these types of effects in 16



### animals does not exist for the inhalation route. H owever, oral multigenerational studies indicate 17 

### that effects on fertility would be expected in male and female offspring exposed to 18 

### benzo[a]pyrene during development (Mackenzie and An gevine 1981; Kristensen et al., 1995). In 19 

### addition, the lack of a study examining functional neurological endpoints following in utero 20 

### exposure is also a data gap considering the availab le epidemiological evidence showing the 21 

### association of in utero PAH exposure and indicators of decreased neurological development 22 

### (Edwards et al., 2010, Perera et al., 2009, Tang et al., 2008). Therefore, a UF of 3 was applied 23 

### to the POD for the lack of a multigenerational repr oductive toxicity study and a 24 

### neurodevelopmental study. 25

26

27 

### The RfC for benzo[a]pyrene was calculated as follow s: 28

29



### RfC = LOAEL ADJ[HEC] 

### ÷ UF 30 

### = 4.6 μg/m 3 

### -day ÷ 1000 31 

### = 4.6 x 10 -3 

### μg/m 3 

### -day or 5 x 10 -6 

### mg/m 3 32

33

34



### 5.2.4. Previous RfC Assessment 35

36 

### An RfC was not derived in the previous IRIS assessm ent. 37

38

39

### 216 DRAFT - DO NOT CITE OR QUOTE

5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION 1



### REFERENCE CONCENTRATION 2 

### The following discussion identifies uncertainties associated with the RfD and RfC for 3 

### benzo[a]pyrene. To derive the RfD, the UF approach (U.S. EPA, 2000, 1994b) was applied to a 4 

### POD based on decreased ovary weight in female rats exposed to benzo[a]pyrene. To derive the 5 

### RfC, this same approach was applied to a POD from a developmental study for the effect of 6 

### decreased fetal survival. Uncertainty factors were applied to the POD to account for 7 

### extrapolating from an animal bioassay to human expo sure, the likely existence of a diverse 8 

### population of varying susceptibilities, and for dat abase deficiencies. These extrapolations are 9 

### carried out with default approaches given the lack of data to inform individual steps. 10 

### The database for benzo[a]pyrene contains limited hu man data. The observation of effects 11 

### associated with benzo[a]pyrene exposure in humans i s complicated by several factors including 12 

### the existence of benzo[a]pyrene in the environment as one component of complex mixtures of 13 

### PAHs, exposure to benzo[a]pyrene by multiple routes of exposure, and the difficulty in obtaining 14 

### accurate exposure information. Data on the effects of benzo[a]pyrene alone are derived from a 15 

### large database of studies in animal models. The da tabase for oral benzo[a]pyrene exposure 16 

### includes two chronic bioassays in rats and mice, tw o developmental studies in mice, and several 17



### subchronic studies in rats. 18 

### Although the database is adequate for RfD derivatio n, there is uncertainty associated with 19 

### the database, because a NOAEL was not identified in the reproductive and developmental oral 20 

### toxicity studies, comprehensive two-generation repr oductive/developmental toxicity studies are 21 

### not available, and immune system endpoints affected in the subchronic-duration studies were not 22 

### evaluated in the chronic-duration toxicity studies. Additionally, the only available chronic 23 

### studies of oral exposure to benzo[a]pyrene focused primarily on neoplastic effects. These studies 24 

### identify forestomach hyperplasia as one of the more sensitive histological effects following 25 

### repeated oral exposure to benzo[a]pyrene. However, data from chronic cancer bioassays for 26 

### benzo[a]pyrene show no increase in this endpoint at the high dose in rats. An increased 27



### incidence of forestomach tumors is observed at simi lar doses; suggesting that this effect may be 28 

### pre-neoplastic in nature. 29 

### The only chronic inhalation study of benzo[a]pyrene was designed as a lifetime 30 

### carcinogenicity study and did not examine noncancer endpoints (Thyssen et al., 1981). However 31



### subchronic and short term inhalation studies are av ailable which examine developmental and 32 

### reproductive endpoints in rats. Developmental stud ies by the inhalation route identified 33 

### biologically significant reductions in the number o f pups/litter and percent fetal survival and 34 

### possible neurodevelopmental effects (e.g., diminish ed electrophysiological responses to stumuli 35 

### in the hippocampus) following gestational exposures . Additionally, a 60 day oral study in male 36 

### rats reported male reproductive effects (e.g., decr eased testes weight and sperm production and 37 

### motility), but provides limited information to char acterize dose-response relationships with 38

### 217 DRAFT - DO NOT CITE OR QUOTE

chronic exposure scenarios. One area of uncertaint y pertains to the lack of information regarding 1 

### fertility in animals exposed gestationally to benzo [a]pyrene, especially in light of developmental 2 

### studies by the oral route indicating reduced fertil ity in the F1 generation and decreased 3 

### reproductive organ weights. The database also lack s a multigenerational reproductive study via 4 

### the inhalation route. Areas of uncertainty include the lack of chronic inhalation studies focusing 5 

### on noncancer effects, limited data on dose-response relationships for impaired male or female 6 

### fertility with gestational exposure or across sever al generations, and limited data on immune 7 

### system endpoints with chronic exposure to benzo[a]p yrene. 8 

### The toxicokinetic and toxicodynamic differences for benzo[a]pyrene between the animal 9 

### species in which the POD was derived and humans are unknown. PBPK models can be useful 10 

### for the evaluation of interspecies toxicokinetics; however, the benzo[a]pyrene database lacks an 11 

### adequate model that would inform potential differen ces. There is some evidence from the oral 12 

### toxicity data that mice may be more susceptible tha n rats to some benzo[a]pyrene effects (such 13 

### as ovotocity [Borman et al., 2000]), though the und erlying mechanistic basis of this apparent 14 

### difference in not understood. Most importantly, it is unknown which animal species may be 15 

### more comparable to humans. 16

17

18



### 5.4. CANCER ASSESSMENT 19 

### As discussed in Section 4.7, benzo[a]pyrene is “car cinogenic to humans ” based on 20 

### evidence of carcinogenicity in humans exposed to di fferent PAH mixtures containing 21 

### benzo[a]pyrene, extensive and consistent evidence o f carcinogenicity in laboratory animals 22 

### exposed to benzo[a]pyrene via several routes of adm inistration, and extensive and consistent 23 

### evidence that the mode of action of carcinogenesis in laboratory animals also occurs in humans 24 

### exposed to PAH mixtures containing benzo[a]pyrene. 25

26 

### 5.4.1. Oral Exposure—Oral Slope Factor 27



### 5.4.1.1. Choice of Study/Data—with Rationale and Justificati on—Oral Exposure 28 

### Numerous cancer bioassays exist which identify tumo rs, primarily of the alimentary tract, 29 

### following oral exposure in rodents (see Table 4-8 f or references). These studies provide support 30 

### for the carcinogenic hazard for benzo[a]pyrene, how ever, are not suitable for dose-response 31 

### analysis due to limitations in study design, method s, and/or reporting. Specifically, several of 32 

### these studies 1) lack a vehicle control group 2) us e only one benzo[a]pyrene dose group or 3) use 33 

### a single one-time exposure to benzo[a]pyrene (Benja min et al., 1988; Robinson et al., 1987; El 34 

### Bayoumy, 1985; Wattenberg, 1974; Roe et al., 1970; Biancifiori et al. 1967; Chouroulinkov et 35 

### al., 1967; Field and Roe, 1970; Berenblum and Haran 1955). Of the controlled, multiple dose36 

### group, repeat-dosing studies that remain, most trea ted animals for less than a year, which is less 37

### 218 DRAFT - DO NOT CITE OR QUOTE

optimal for extrapolating to a lifetime exposure (W eyand et al., 1995; Triolo et al., 1977; 1 

### Fedorenko et al., 1967; Neal and Rigdon 1967). 2 

### Three 2-year oral bioassays remain which associate lifeftime benzo[a]pyrene exposure 3 

### with forestomach, liver, oral cavity, jejunum, kidn ey, auditory canal (Zymbal’s gland) tumors, 4 

### and skin or mammary gland tumors in male and female Wistar rats (Kroese et al., 2001); 5 

### forestomach tumors in male and female Sprague-Dawle y rats (Brune et al., 1981); and 6 

### forestomach, esophagus, tongue, and larynx tumors i n female B6C3F 1 

### mice (male mice were not 7 

### tested; Beland and Culp, 1998; Culp et al., 1998). Brune et al. (1981) dosed rats (32/sex/group) 8 

### with several concentration of benzo[a]pyrene dissol ved a 1.5% caffeine solution, sometimes as 9 

### infrequently as once every 9 th 

### day, for up to two years and observed increased fo restomach 10 

### tumors. This study was not selected for quantitati on due to the non-standard treatment protocol. 11 

### The rat bioassay by Kroese et al. (2001) and the mo use bioassay by Beland and Culp (1998) 12 

### were conducted in accordance to Good Laboratory Pra ctice (GLP) principles as established by 13 

### OECD. These studies included histological examinat ions for tumors in many different tissues, 14 

### contained three exposure levels and controls, conta ined adequate numbers of animals per dose 15 

### group (~50/sex/group), treated animals for two years or until death, and included detailed 16 

### reporting of methods and results (including individ ual animal data). 17 

### Therefore, the Kroese et al. (2001) and Beland a nd Culp s(1998) tudies were selected as 18 

### the best available studies for dose-response analys is and extrapolation to lifetime cancer risk 19 

### following oral exposure to benzo[a]pyrene. 20

21



### 5.4.1.2. Dose-response Data—Oral Exposure 22 

### Details of the rat (Kroese et al., 2001) and female mouse (Beland and Culp, 1998) study 23 

### designs are provided in Section 4.2.1.2. Dose-related, statistically significant increasing trends 24 

### in tumors were noted at the following sites: 25



### • Squamous cell carcinomas or papillomas of the fores tomach or oral cavity in male and 26 

### female rats; 27



### • Squamous cell carcinomas or papillomas of the fores tomach, tongue, larynx, or 28 

### esophagus in female mice; 29



### • Auditory canal carcinomas in male and female rats; 30



### • Kidney urothelial carcinomas in male rats; 31



### • Jejunum adenocarcinomas in female and male rats; 32



### • Hepatocellular adenomas or carcinomas in male and f emale rats; 33



### • Squamous cell carcinomas or basal cell tumors of th e skin or mammary gland in male 34



### rats. 35

36 

### These tumors were generally observed earlier during the study with increasing exposure 37 

### levels, and showed statistically significantly incr easing trends in incidence with increasing 38

### 219 DRAFT - DO NOT CITE OR QUOTE

exposure level (Cochran-Armitage trend test, p ≤ 0.001). These data are summarized in Tables 5- 1 

### 4 (male and female rats) and 5-5 (female mice). As recommended by the NTP (McConnell et al., 2 

### 1986), etiologically similar tumor types, i.e., ben ign and malignant tumors of the same cell type, 3 

### were combined for these tabulations when it was jud ged that the benign tumors could progress to 4 

### the malignant form, as outlined in the Cancer Guidelines (U.S. EPA, 2005a). In addition, when 5 

### one tumor type occurred across several functionally related tissues, as with squamous cell tumors 6 

### in the tongue, esophagus, larynx and forestomach, o r adenocarcinomas of the jejunum or 7 

### duodenum, these incidences were also aggregated as counts of tumor-bearing animals. 8 

### In the rat study (Kroese et al., 2001), the oral ca vity and auditory canal were examined 9 

### histologically only if a lesion or tumor was observ ed grossly at necropsy. Consequently, dose10 

### response analysis for these sites was not straightf orward. Use of the number of tissues examined 11 

### histologically as the number at risk would tend to overestimate the incidence, because the 12 

### unexamined animals were much less likely to have a tumor. On the other hand, use of all 13 

### animals in a group as the number at risk would tend to underestimate if any of the unexamined 14 

### animals had tumors which could only be detected mic roscopically. The oral cavity squamous 15



### cell tumors were combined with those in the foresto mach because both are part of the alimentary 16 

### tract, recognizing that there was some potential fo r underestimating this cancer risk. 17 

### The auditory canal tumors from the rat study were n ot considered for dose-response 18 

### separately or combined with another site. First, v ery few tissues were examined in the control 19 

### and lower dose groups (see Table 4-4). Also, the t umors were not clearly related to any other 20 

### site or incidence type, as they were described as a mixture of squamous and sebaceous cells 21 

### derived from pilosebaceous units. The tumors found were observed mainly in the high dose 22 

### groups and were highly coincident with the oral cav ity and forestomach tumors. That is, only 23 

### one mid-dose male with an auditory canal tumor did not also have a forestomach or oral cavity 24 

### squamous cell tumor. No low-dose male or female ra ts were found with auditory canal tumors. 25 

### While the investigators did not suggest that these tumors were metastases from other sites (in 26 

### which the auditory canal tumors could be reflection s of other tumor types), it is difficult to 27 

### conclude that they are independent on a purely stat istical basis without sufficient low-dose data. 28 

### Therefore dose-response analysis was not pursued fo r this site. 29

30

### 220 DRAFT - DO NOT CITE OR QUOTE

Table 5-4. Incidence data for tumors in Wistar rat s exposed to benzo[a]pyrene by gavage, 5 days/week for 104 weeks

Tumor site/type a Administered dose (mg/kg-d)

0 3 10 30

HED (mg/kg-d) b

Male rats 0 0.54 1.81 5.17 Forestomach or oral cavity: squamous cell papillom a or carcinoma 0/51 8/51 45/49 52/52

Hepatocellular adenoma or carcinoma 0/51 4/50 38/49 49/50 Jejunum/duodenum: adenocarcinoma 0/50 0/48 1/48 9/39 Kidney: urothelial carcinoma 0/52 0/52 0/52 3/52 Skin or mammary gland: Basal cell adenoma or carcinoma Squamous cell carcinoma 2/52 0/51 1/50

1/50 1/49

1/49 13/40

6/40

Female rats 0 0.49 1.62 4.85 Forestomach or oral cavity: squamous cell papilloma or carcinoma 1/52 6/50 30/47 50/51

Hepatocellular adenoma or carcinoma 0/52 1/50 39/4 7 51/51 Jejunum/duodenum: adenocarcinoma 0/50 0/46 0/45 4/42

a For each tissue site, the numerator of the tumor in cidence value is the number of animals bearing the specified tumors. The denominators are the number of animals examined histologically, minus the number of anima ls who died before the earlier of the first occurrence of the tumor type in each group or Week 52.

b HEDs for continuous exposure were calculated using the animal to human scaling factor for each dose gr oup × the administered dose × 5 d/7 d. Scaling factors used the form (TWA body weight/70) 0..25 , with the U.S. EPA (1988) reference body weight for humans (70 kg), and the T WA body weight for each dose group. See Table D- 4 for more information.

Source: Kroese et al. (2001).

1

2



### Table 5-5. Incidence data for tumors in female B6C 3F 1 

### mice exposed to

benzo[a]pyrene in the diet for 104 weeks

Tumor site/type a Administered dose (mg/kg-d) b

0 0.7 3.3 16.5

HED (mg/kg-d) c

0 0.10 0.48 2.32 Forestomach, esophagus, tongue, larynx: squamous cell papilloma or carcinoma 1/48 3/48 38/46 46/47

a The numerator of the tumor incidence value is the number of animals bearing any of the listed tumors (see Table 4-6 in Section 4.2.1.2). The denominators are the number of tissues examined histologically, minus th e number of animals who died before the earlier of the first oc currence of the tumor type in each group or Week 52

. b Administered doses were calculated using TWA body w eight for mice and reported food intakes.

c HEDs were calculated using the animal to human scal ing factor for each dose group × the administered d ose. Scaling factors were calculated using U.S. EPA (198 8) reference body weights for humans (70 kg), and t he TWA body weight for each dose group: (TWA body weight/7 0) 0.25 × dose = HED. See Table D-5 for more information.

Source: Beland and Culp (1998).

3



### 5.4.1.3. Dose Adjustments and Extrapolation Method(s)—Oral E xposure 4

### 221 DRAFT - DO NOT CITE OR QUOTE

The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend 1 

### that the method used to characterize and quantify c ancer risk from a chemical is determined by 2



### what is known about the mode of action of the carci nogen and the shape of the cancer dose3 

### response curve. The dose response is assumed to be linear in the low dose range, when evidence 4 

### supports a mutagenic mode of action because of DNA reactivity, or if another mode of action 5 

### that is anticipated to be linear is applicable. In this assessment, EPA concluded that 6 

### benzo[a]pyrene causes cancer via a mutagenic MOA (a s discussed in Section 4.7.3). Thus, a 7 

### linear approach to low-dose extrapolation was used. 8 

### The high-dose groups of both the rat and mouse stud ies were dead or moribund by week 9 

### 79 for female mice, week 72 for female rats, and we ek 76 for male rats. Due to the occurrence of 10 

### multiple tumor types, earlier occurrence with incre asing exposure, and early termination of the 11 

### high-dose group in each study, methods that can ref lect the influence of competing risks and 12 

### intercurrent mortality on site-specific tumor incid ence rates are preferred. EPA has generally 13 

### used a model which incorporates the time at which d eath-with-tumor occurred as well as the 14 

### dose; the multistage-Weibull model is multistage in dose and Weibull in time, and has the form: 15

16 

### P(d, t) = 1 - exp[-(q 0 

### + q 1 

### d + q 2 

### d 2 

### + ... + q k 

### d

k 

### ) × (t ± t 0 

### ) z



### ]

, 17

18



### where P(d, t) represents the lifetime risk (probability) of canc er at dose d (i.e., human equivalent 19 

### exposure in this case) and age t (in bioassay weeks); parameters q i 

### ≥ 0, for i = 0, 1, ..., k; t is the 20 

### time at which the tumor was observed; and z is a parameter which characterizes the change in 21 

### response with age. The parameter t 0 

### represents the time between when a potentially fat al tumor 22 

### becomes observable and when it causes death, and is generally set to 0 either when all tumors are 23



### considered incidental or because of a lack of data to estimate the time reliably. The dose24 

### response analyses were conducted using the computer software program MultiStage-Weibull 25 

### (U.S. EPA, 2010), which is based on Weibull models drawn from Krewski et al. (1983). 26 

### Parameters were estimated using the method of maxim um likelihood. 27 

### Two general characteristics of the observed tumor types were considered prior to 28 

### modeling; allowance for different, although unident ified modes of action, and allowance for 29 

### relative severity of tumor types. First, etiologic ally different tumor types were not combined 30 

### across sites prior to modeling (that is, overall co unts of tumor-bearing animals were not 31 

### tabulated), in order to allow for the possibility t hat different tumor types could have different 32 

### dose-response relationships due to different under lying mechanisms or factors, such as latency. 33 

### Consequently, all of the tumor types listed separat ely in Tables 5-4 and 5-5 were also modeled 34 

### separately. 35 

### Additionally, the multistage-Weibull model can add ress relative severity of tumor types 36 

### by distinguishing between tumors as being either fa tal or incidental to the death of an animal, in 37 

### order to adjust partially for competing risks. Inc idental tumors are those tumors thought not to 38

### 222 DRAFT - DO NOT CITE OR QUOTE

have caused the death of an animal, while fatal tum ors are thought to have resulted in animal 1 

### death. Cause of death information for most early a nimals deaths was provided by the 2 

### investigators of both of the bioassays. In the rat study, tumors of the forestomach or liver were 3 

### the principal cause of death for most animals dying or sacrificed (due to moribundity) before the 4 

### end of the study, while tumors of the forestomach w ere the most common cause of early deaths 5 

### in the mouse study. 6 

### Adjustments for approximating human equivalent slop e factors applicable for continuous 7 

### exposure were applied prior to dose-response modeli ng. First, continuous daily exposure for the 8 

### gavage study in rats (Kroese et al, 2001) was estim ated by multiplying each administered dose 9 

### by (5 days)/(7 days) = 0.71, under the assumption o f equal cumulative exposure yielding 10 

### equivalent outcomes. Dosing was continuous in the mouse diet study (Beland and Culp, 1998), 11 

### so no continuous adjustment was necessary. Next, c onsistent with the Guidelines for 12 

### Carcinogen Risk Assessment (U.S. EPA, 2005a), an adjustment for cross-species scaling was 13 

### applied to address toxicological equivalence across species. Following EPA’s cross-species 14 

### scaling methodology, the time-weighted daily averag e doses were converted to human equivalent 15 

### doses on the basis of (body weight) 3/4 

### (U.S. EPA, 1992). This was accomplished by multip lying 16 

### administered doses by (animal body weight(kg)/70 kg ) 0.25 

### (U.S.EPA, 1992), where the animal 17 

### body weights were time-weighted averages from each group (see Tables D-4, D-5), and the U.S. 18 

### EPA (1988) reference body weight for humans is 70 k g. It was not necessary to adjust the 19 

### administered doses for lifetime equivalent exposure prior to modeling for the groups terminated 20 

### early, because the multistage-Weibull model charact erizes the tumor incidence as a function of 21 

### time, from which it provides an extrapolation to li fetime exposure. 22 

### The multistage-Weibull model was applied to the d atasets. Specific n-stage Weibull 23



### models were selected for each tumor dataset based o n the values of the log-likelihoods according 24 

### to the strategy used by EPA (U.S. EPA, 2002). If t wice the difference in log-likelihoods was less 25



### than a χ 2 

### with degrees of freedom equal to the difference in the number of stages included in the 26 

### models being compared, the models were considered c omparable and the most parsimonious 27 

### model (i.e., the lowest-stage model) was selected. This method generally led to the same 28 

### conclusion as selecting the model fit with the lowe st AIC. If a model with one more stage fitted 29 

### the low-dose data better than the most parsimonious model, then the model with one higher stage 30



### was selected. 31 

### PODs for estimating low-dose risk were identified at doses at the lower end of the 32 

### observed data, generally corresponding to 10% extra risk, where extra risk is defined as [P(d) - 33 

### P(0)]/[1 - P(0)]. The lifetime oral cancer slope f actor for humans is defined as the slope of the 34 

### line from the lower 95% bound on the exposure at th e POD to the control response (slope factor 35



### = 0.1/BMDL 10 

### ). This slope, a 95% upper confidence limit (UCL) represents a plausible upper 36



### bound on the true risk. 37

38

### 223 DRAFT - DO NOT CITE OR QUOTE

5.4.1.4. Oral Slope Factor Derivation 1



### The PODs estimated for each tumor site are summari zed in Table 5-5. Details of the 2 

### model selection process are provided in Table D-6. Using linear extrapolation from the 3



### BMDL 10 

### , human equivalent oral slope factors were derived for each gender/tumor site 4



### combination and are listed in Table 5-6. 5

6 

### Table 5-6. Human equivalent PODs and oral slope fa ctors derived from

multistage-Weibull modeling of tumor incidence data at multiple tissue sites in Wistar rats and B6C3F 1 

### mice exposed to benzo[a]pyrene orally for 2 years

Species Sex Tumor BMD 10 (mg/kg-d) BMDL 10

(mg/kg-d) Slope Factor a

(mg/kg-d) -1 Rats Male Forestomach, oral cavity: squamous cell

tumors 0.453 0.281 0.4

0.5 b Hepatocellular adenomas or carcinomas 0.651 0.449 0 .2 Jejunum/duodenum adenocarcinomas 3.03 2.38 0.04 Kidney: urothelial carcinomas 4.65 2.50 0.04 Skin, mammary: Basal cell tumors Squamous cell tumors 2.86 2.64 2.35

1.77 0.04

0.06 Female Forestomach, oral cavity: squamous cell

tumors 0.539 0.328 0.3

0.3 b Hepatocellular adenomas or carcinomas 0.575 0.507 0 .2 Jejunum/duodenum adenocarcinomas 3.43 1.95 0.05 Mice Female Forestomach, esophagus, tongue, larynx:

squamous cell tumors 0.127 0.071 1

a Human equivalent slope factor = 0.1/BMDL 10HED ; see Appendix D for details of modeling results.

b Estimates of risk of incurring at least one of the tumor types listed.

7 

### Oral slope factors derived from rat bioassay data v aried by gender and tumor site 8 

### (Table 5-6). Values ranged from 0.04 per mg/kg-day , based on kidney tumors in males, to 9 

### 0.4 per mg/kg-day, based on alimentary tract tumors in males. Slope factors based on liver 10 

### tumors in male and female rats (approximately 0.2 p er mg/kg-day) were only slightly lower than 11 

### slope factors based on alimentary tract tumors. Th e oral slope factor for female mice was 12 

### highest, at 1 per mg/kg-day for alimentary tract tu mors (Table 5-6), approximately fourfold 13 

### higher than the oral slope factor derived from the alimentary tract tumors in male rats. 14 

### Although the time-to-tumor modeling helps account f or competing risks associated with 15 

### decreased survival times and other tumors, consider ing the tumor sites individually still does not 16 

### convey the total amount of risk potentially arising from the sensitivity of multiple sites—that is, 17 

### the risk of developing any combination of the incre ased tumor types, not just the risk of 18 

### developing all simultaneously. One approach sugges ted in the Guidelines for Carcinogen Risk 19



### Assessment

(U.S. EPA, 2005a) would be to estimate cancer risk from tumor-bearing animals. 20 

### EPA traditionally used this approach until the Nati onal Resource Council (NRC) document 21 

### Science and Judgment (NRC, 1994) made a case that this approach would t end to underestimate 22 

### overall risk when tumor types occur in a statistica lly independent manner. In addition, 23

### 224 DRAFT - DO NOT CITE OR QUOTE

application of one model to a composite data set do es not accommodate biologically relevant 1 

### information that may vary across sites or may only be available for a subset of sites. For 2 

### instance, the time courses of the multiple tumor ty pes evaluated varied, as is suggested by the 3



### variation in estimates of z (see Table 5-6), from 1.5 (e.g., male rat skin or mammary gland basal 4 

### cell tumors), indicating relatively little effect o f age on tumor incidence, to 3.7 (e.g., male mouse 5 

### alimentary tract tumors), indicating a more rapidly increasing response with increasing age (in 6 

### addition to exposure level). The result of fitting a model with parameters which can reflect 7 

### underlying mechanisms, such as z in the multistage-Weibull model, would be difficul t to 8 

### interpret with composite data (i.e., counts of tumo r-bearing animals). A simpler model, such as 9 

### the multistage model, could be used for the composi te data but relevant biological information 10 

### would then be ignored. 11 

### Following the recommendations of the NRC (1994) reg arding combining risk estimates, 12



### statistical methods which can accommodate the under lying distribution of slope factors are 13 

### optimal, such as through maximum likelihood estimat ion or through bootstrapping or Bayesian 14 

### analysis. However, these methods have not yet been extended to models such as the multistage15 

### Weibull model. A method involving the assumption t hat the variability in the slope factors could 16 

### be characterized by a normal distribution is detail ed below (U.S. EPA, 2010). Using the results 17 

### in female rats to illustrate, the overall risk esti mate involved the following steps: 18

19 

### 1) It was assumed that the tumor groupings modeled abo ve were statistically 20 

### independent—that is, that the occurrence of a liver tumor was not dependent 21 

### upon whether there was a forestomach tumor. This a ssumption cannot currently 22 

### be verified, and if not correct could lead to an ov erestimate of risk from 23 

### summing across tumor sites. However, NRC (1994) ar gued that a general 24 

### assumption of statistical independence of tumor-typ e occurrences within 25 

### animals was not likely to introduce substantial err or in assessing carcinogenic 26 

### potency from rodent bioassay data. 27

28 

### 2) The models previously fitted to estimate the BMDs a nd BMDLs were used to 29 

### extrapolate to a lower level of risk (R), in order to reach the region of each 30 

### estimated dose-response function where the slope wa s reasonably constant and 31 

### upper bound estimation was still numerically stable . For these data, a 10 -3 

### risk 32 

### was generally the lowest risk necessary. The oral slope factor for each site was 33 

### then estimated by R/BMDL R 

### , as for the estimates for each tumor site above. 34

35 

### 3) The maximum likelihood estimates (MLE) of unit pote ncy (that is, risk per unit 36 

### of exposure) estimated by R/BMD R 

### , were summed across the alimentary tract, 37 

### liver, and jejunum/duodenum in female rats. 38

39 

### 4) An estimate of the 95% (one-sided) upper bound on t he summed oral slope 40 

### factor was calculated by assuming a normal distribu tion for the individual risk 41 

### estimates, and deriving the variance of the risk es timate for each tumor site from 42 

### its 95% upper confidence limit (UCL) according to t he formula: 43

### 225 DRAFT - DO NOT CITE OR QUOTE

1 

### 95% UCL = MLE + 1.645 × s.d., 2 

### rearranged to: 3 

### s.d. = (UCL – MLE ) / 1.645, 4

5 

### where 1.645 is the t-statistic corresponding to a o ne-sided 95% confidence 6 

### interval and >120 degrees of freedom, and the stand ard deviation (s.d.) is the 7 

### square root of the variance of the MLE. The varian ces (variance = s.d. 2 

### ) for each 8 

### site-specific estimate were summed across tumor sit es to obtain the variance of 9 

### the sum of the MLEs. The 95% UCL on the sum of MLE s was calculated from 10 

### the expression above for the UCL, using the varianc e of the sum of the MLE to 11 

### obtain the relevant s.d (s.d. = variance 1/2 

### ). 12

13 

### The resulting composite slope factor for all tumor types for male rats was 0.5 per mg/kg14 

### d, about 25% higher than the slope factor based on the most sensitive tumor site, oral cavity and 15 

### forestomach, while for female rats the composite sl ope factor was equivalent to that for the most 16 

### sensitive site (Table 5-6; see Table D-7 for detail s of the composite slope factor estimates). 17 

### The risk estimates from rats and mice spanned a nea rly five-fold range. As there are no 18 

### data to support any one result as most relevant for extrapolating to humans, the most sensitive 19 

### result was used to derive the oral slope factor. T he recommended slope factor for assessing 20 

### human cancer risk associated with chronic oral expo sure to benzo[a]pyrene is 1 per mg/kg-day, 21 

### based on the alimentary tract tumor response in fem ale B6C3F 1 

### mice. 22

23 

### 5.4.2. Inhalation Exposure—Inhalation Unit Risk 24



### 5.4.2.1. Choice of Study/Data—with Rationale and Justificati on—Inhalation Exposure 25 

### Inhalation exposure to benzo[a]pyrene was associate d with nasal adenocarcinomas and 26 

### squamous cell tumors in the larynx, pharynx, trache a, esophagus, and forestomach, of male 27 

### Syrian golden hamsters exposed to benzo[a]pyrene:Na Cl aerosols at concentrations of 10 or 50 28 

### mg/m 3 

### until natural death (up to 133 weeks) for 3–4.5 ho urs/day, 5-7 days/week (Thyssen et al., 29 

### 1981). Supportive evidence for the carcinogenicity of inhaled benzo[a]pyrene comes from 10 30 

### additional studies with hamsters exposed to benzo[a ]pyrene via intratracheal instillation (see 31 

### Section 4.2.2.2 for references). However, the use of intratracheal dosing alters the deposition, 32 

### clearance, and retention of substances and therefor e studies utilizing this exposure technique are 33 

### not as useful for the quantitative extrapolation of cancer risk from the inhalation of 34 

### benzo[a]pyrene in the environment (Driscoll et al., 2000). 35 

### The Thyssen et al. (1981) bioassay represents the o nly lifetime inhalation cancer bioassay 36 

### available for describing dose-response relationship s for cancer from inhaled benzo[a]pyrene. 37 

### Limitations of the study include the following: (1 ) only male animals were included; (2) particle 38 

### analysis of aerosols was not reported [i.e., MMAD a nd geometric SD were not reported], and 39 

### (3) benzo[a]pyrene exposure occurred through the in halation of hygroscopic particles 40 

### [benzo[a]pyrene was adsorbed onto NaCl aerosols] wh ich may have a different deposition than 41

### 226 DRAFT - DO NOT CITE OR QUOTE

benzo[a]pyrene adsorbed onto non-hygroscopic partic les in the environment. Strengths of the 1 

### study include exposure to hamsters for life, histol ogical tumor examination of organs, use of 2 

### multiple exposure groups, including approximately 3 0 male hamsters per group, and the 3 

### availability of individual animal pathology reports with time of death and tumor detection data. 4 

### Although the study has a few limitations, the stren gths of the study support use of the data to 5 

### derive an inhalation unit risk for benzo[a]pyrene. 6

7 

### 5.4.2.2. Dose-response Data—Inhalation Exposure 8 

### Survival was decreased relative to control only in the high-dose exposure group; mean 9 

### survival times in the 0, 2, and 10 mg/m 3 

### concentration groups were 96.4, 95.2, and 96.4 wee ks, 10 

### respectively, and 59.5 weeks in the 50 mg/m 3 

### group animals. Overall, tumors occurred earlier i n 11 

### the highest benzo[a]pyrene exposure group than in t he mid-exposure group. Increased 12 

### incidences of benign and malignant tumors of the la rynx, trachea, pharynx, esophagus and 13 

### forestomach were seen with increasing exposure conc entration. Benign tumors—papillomas, 14 

### polyps and papillary polyps—were considered by the study authors as early stages of the 15 

### squamous cell carcinomas in these tissues. 16 

### Nasal cavity tumors were also observed in the mid- and high-dose groups. Consideration 17 

### of early mortality (using the poly-3 approach; Bail er and Portier, 1988) suggested that an 18 

### increasing dose-response was consistent with these data. However trend testing was not 19 

### statistically significant (p=0.08), and the site wa s not considered further for unit risk derivation. 20 

### Table E-1 in Appendix E summarizes the individual a nimal tumor data, noting the presence or 21 

### absence of a tumor in these tissues, whether or not the tissue was available for examination by 22 

### the pathologist, and the time of death. A summary of the incidence of these tumors is provided 23



### in Table 5-7. 24

25



### Table 5-7. Incidence of tumors in male hamsters ex posed by inhalation to

benzo[a]pyrene for life Average continuous benzo[a]pyrene concentration a (mg/m 3 ) Number

of hamsters in group b Larynx Pharynx Trachea Esophagus Forestomach Any Tumor c Nasal

Cavity Tumors

Control 27 0 0 0 0 0 0 0

0.25 27 0 0 0 0 0 0 0

1.01 26 11 9 2 0 1 18 4

4.29 34 12 18 3 2 1 18 1

a Calculated from air monitoring data.

b Number of animals examined histologically, minus th e number of animals who died before the earlier of the first occurrence of the tumor type in each group or Week 52.

c Includes any animal with squamous cell carcinoma o f the larynx, pharynx, trachea, esophagus, or fores tomach.

Source: Thyssen et al. (1981) and a reanalysis of this data by Clement Associates (1990). See Append ix E for more detailed incidence data.

### 227 DRAFT - DO NOT CITE OR QUOTE

1



### 5.4.2.3. Dose Adjustments and Extrapolation Method(s)—Inhala tion Exposure 2 

### A toxicokinetic model to assist in cross species sc aling of benzo[a]pyrene inhalation 3 

### exposure was not available. In addition, default d osimetry adjustments utilized in the 4 

### benzo[a]pyrene RfC calculation could not be applied because aerosol particle distribution data 5



### were not available for the hamster inhalation bioas say by Thyssen et al. (1981). The carrier 6 

### particle used in Thyssen et al. (1981) was sodium c hloride, a soluble hygroscopic particle, and 7 

### the approaches presented in the RfC methodology gui delines (US EPA 1994b) were developed 8 

### for insoluble and nonhygroscopic particles. 9 

### The availability of the raw chamber air monitoring data and individual times on study 10 

### allowed the calculation of time-weighted average (T WA) continuous exposure rates for each 11 

### hamster. Group averages of individual TWA continuo us exposure concentrations were 0, 0.25, 12 

### 1.01, and 4.29 mg/m 3 

### , respectively, for the 0, 2, 10, and 50 mg/m 3 

### study concentrations. 13 

### A time-to-tumor dose-response model was fit to the time-weighted average exposure 14



### concentrations and the individual animal occurrence data for tumors in the larynx, pharynx, 15 

### trachea, esophagus, and forestomach (Table E-1 in A ppendix E) using the computer software 16 

### program multistage-Weibull (U.S. EPA, 2010) as desc ribed in Section 5.4.1.3. The 17 

### investigators did not determine cause of death for any of the animals. Since in the available oral 18 

### bioassays the investigators considered these same t umors to be fatal at least some of the time, 19 

### bounding estimates for the Thyssen et al. data were developed by treating the tumors alternately 20



### as either all incidental or all fatal. In either c ase, therefore, an estimate of t 0 

### (the time between a 21 

### tumor first becoming observable and causing death) could not be estimated. 22 

### Because benzo[a]pyrene is expected to cause cancer via a mutagenic MOA, a linear 23 

### approach to low dose extrapolation from the BMCL 10 

### was used (U.S. EPA, 2005a). 24

25



### 5.4.2.4. Inhalation Unit Risk Derivation 26 

### Modeling results are provided in Appendix E. The B MC (0.28 mg/m 3 

### ) and BMCL (0.20 27 

### mg/m 3 

### ) associated with an extra risk of 10% were calcula ted based on the occurrence of upper 28 

### respiratory and upper digestive tract tumors in mal e hamsters exposed to aerosols of 29 

### benzo[a]pyrene for 104 weeks using the multistage-W eibull model Using linear extrapolation 30



### from the BMCL 10 

### of 0.20 mg/m 3 

### , an inhalation unit risk of 0.5 per mg/m 3 

### or 5 x 10 -4 

### per μg/m 3 31



### was calculated. 32

33 

### 5.4.3. Dermal Exposure—Dermal Slope Factor 34



### 5.4.3.1. Choice of Study/Data—with Rationale and Justificati on—Dermal Exposure 35



### Skin cancer in humans has been documented to result from occupational exposure to 36 

### complex mixtures of PAHs including benzo[a]pyrene s uch as coal tar, coal tar pitches, non37 

### refined mineral oils, shale oils and soot (IARC, 20 10; Baan et al., 2009; Boffetta et al., 1997; 38

### 228 DRAFT - DO NOT CITE OR QUOTE

WHO, 1998; ATSDR, 1995), but no studies of human ex posures to benzo[a]pyrene alone are 1 

### known to exist. In animal models, numerous dose re sponse studies have demonstrated an 2 

### increased incidence of skin tumors with increasing dermal exposure to benzo[a]pyrene, in all 3 

### species tested (mice, rabbits, rats, and guinea pig s), though most benzo[a]pyrene chronic dermal 4 

### bioassays which provide quantitative information ha ve been conducted in mice. In addition, 5 

### mice appear to be the most sensitive laboratory mod el of carcinogenesis following dermal 6 

### benzo[a]pyrene exposure. Therefore, this analysis focuses on chronic carcinogenicity bioassays 7 

### in several strains of mice demonstrating increasing incidence of benign and malignant skin 8 

### tumors, and earlier occurrence of tumors with incre asing exposure, following repeated dermal 9 

### exposure to benzo[a]pyrene for the animals’ lifetim e. These studies involved 2- or 3-times/week 10 

### exposure protocols, at least two exposure levels pl us controls, and included histopathological 11 

### examinations of the skin and other tissues (Sivak e t al., 1997; Grimmer et al., 1984; 1983; Habs 12 

### et al., 1980; 1984; Schmähl et al., 1977; Schmidt e t al., 1973; Roe et al., 1970; Poel, 1960; 1959). 13 

### These data sets are described in greater detail in Section 4.2.3.2. 14 

### Because of the availability of the lifetime studies listed above, other carcinogenicity 15



### studies were not considered for this assessment. T he other studies included: 1) early “skin 16 

### painting” studies of benzo[a]pyrene carcinogenicity in mouse skin which did not report sufficient 17 

### information to estimate the doses applied (e.g., Wy nder and Hoffman 1959; Wynder et al, 1957); 18 

### 2) initiation-promotion studies utilizing acute dos ing of benzo[a]pyrene followed by repeated 19 

### exposure to a potent tumor promoter (sometimes benz o[a]pyrene at a lower dose than the 20 

### initiation step), because they are not as relevant for calculating risks from constant 21 

### benzo[a]pyrene exposure alone; 3) bioassays with on e benzo[a]pyrene dose level or with only 22 

### dose levels inducing 90–100% incidence of mice with tumors, because they provide relatively 23 

### little information about the shape of the dose-resp onse relationship (e.g., Wilson and Holland, 24 

### 1988); 4) studies with shorter exposure and observa tion periods (i.e., less than one year; Levin et 25 

### al., 1977; Nesnow et al, 1983; Albert et al., 1991; Emmett et al, 1981; Higginbotham et al., 1993) 26 

### which are less relevant for characterizing lifetime risk; and 5) studies involving vehicles 27 

### expected to interact with or enhance benzo[a]pyrene carcinogenicity (e.g., Bingham and Falk, 28 

### 1969) which precludes assessment of carcinogenic ri sks of benzo[a]pyrene alone. 29

30



### 5.4.3.2. Dose-response Data—Dermal Exposure 31 

### Several studies were considered for dose-response m odeling for derivation of the dermal 32 

### slope factor for benzo[a]pyrene, reflecting a relat ively large database. Study designs and the 33 

### extent of data reported varied across the studies, with no individual animal data available. All of 34 

### the studies identified in the previous section (Siv ak et al., 1997; Grimmer et al., 1984; 1983; 35 

### Habs et al., 1980; 1984; Schmähl et al., 1977; Schm idt et al., 1973; Roe et al., 1970; Poel, 1960; 36 

### 1959) were considered further in order to evaluate overall consistency of the available database. 37 

### These data sets are presented in Tables 5-8 through 5-12, and are grouped by study 38

### 229 DRAFT - DO NOT CITE OR QUOTE

characteristics such as mouse strain and vehicle in order to facilitate qualitative comparisons 1 

### where possible. 2 

### Each data set was examined for study design and bot h strengths and limitations that could 3 

### potentially impact the dose-response evaluation, in cluding the potential of early mortality to 4 

### impact the number at risk for developing tumors, an d the length of exposure. Although nearly all 5 

### studies reported lifetime exposures, this term most often indicated that exposure continued until 6 

### natural death, not a scheduled sacrifice at 104 wee ks. 7 

### The studies by Poel (1959, 1960) were conducted in male mice and used toluene as the 8 

### vehicle (see Tables 4-11, 4-12, respectively). In addition to a control group, the 1959 study 9 

### included nine dose groups of one mouse strain (C57L ) and the 1960 study included seven dose 10 

### groups of 3 other mouse strains. Both studies demo nstrated high mortality and tumor incidence 11 

### at higher exposure levels. As noted in Table 4-11, all C57L mice in dose groups with >3.8 12



### μ g/application died by Week 44 of the study (Poel, 1 959). Therefore, these five dose groups 13 

### were omitted prior to dose-response modeling becaus e of the relatively large uncertainty in 14 

### characterizing cancer risk in relation to lifetime exposure. Four dose groups in addition to 15 

### control remained. Among these groups mice survived and were exposed until weeks 83–103. 16 

### According to the lifespan ranges provided, at least one mouse in each dose group died before the 17 

### first appearance of tumor, but insufficient informa tion was available to determine how many; 18 

### consequently the incidence denominators were not ad justed. The dose-response data are 19



### summarized in Table 5-7. 20 

### For the Poel (1960) studies, all tumors in the high est three dose groups for each of the 21 

### three mouse strains had occurred by Week 40 (see Ta ble 4-12). While these observations 22 

### support concern for cancer risk, as noted above suc h results are relatively uncertain for 23 

### estimating lifetime cancer risk. In addition, ther e was no information indicating duration of 24 

### exposure for the mice without tumors; although expo sure was for lifetime, it might have been as 25 

### short as for the mice with tumors. Overall, these datasets did not provide sufficient information 26 

### to estimate the extent of exposure associated with the observed tumor incidence. Consequently 27 

### the experiments reported by Poel (1960) were not us ed for dose-response modeling. 28



### The studies listed in Table 5-9 all used acetone as the vehicle and either Swiss or NMRI, 29 

### female mice (Roe et al., 1970; Schmidt et al., 1973 ; Schmähl et al., 1977; Habs et al., 1980, 30 

### 1984). Roe et al. (1970) applied benzo[a]pyrene de rmally for 93 weeks or until natural death; 31 

### with the exception of the highest dose group, each group still had approximately 20 animals at 86 32 

### weeks (Table 4-14). The tumors were first observed in the lowest and highest dose groups 33 

### during the interval of weeks 29-43. Mice that died before week 29 were likely not at risk of 34 

### tumor development. However because tumor incidence and mortality were reported in 100-day 35 

### intervals, mice that had not been on study long eno ugh to develop tumors were not easily 36



### identifiable. Incidence denominators reflect the n umber of animals alive at Week 29, and thus 37

### 230 DRAFT - DO NOT CITE OR QUOTE

may tend to lead to underestimates of tumor risk if the number of animals at risk have been 1



### overestimated. 2 

### Schmidt et al. (1973) did not report survival infor mation, instead the authors provided 3 

### incidences based on the numbers of mice initially i ncluded in each dose group at the start of the 4 

### study. Overall latency was reported for the two hi gh dose groups in each series, but these data 5 

### only describe the survival of mice with tumors (ani mals were removed from study when a tumor 6 

### appeared). It is not clear how long exposures last ed overall in each dose group, or whether some 7 

### mice may have died on study from other causes befor e tumors appeared. While it is possible that 8 

### no mice died during the study, all of the other stu dies considered here demonstrate mortality. 9 

### However, the data were modeled as reported, recogni zing the possibility of underestimating risk 10 

### associated with incidences reported and lack of dur ation of exposure. 11 

### Schmähl et al. (1977) reported that reduced numbers of animals at risk (77−88 mice per 12 

### dose group compared with the initial group sizes of 100) resulted from varying rates of autolysis. 13 

### No other survival or latency information was provid ed, so all exposures were assumed to have 14 

### lasted for 104 weeks and were modeled as reported. Given the results of the other studies, it 15 

### seems possible that the numbers at risk in each gro up may be overestimated, which could lead to 16



### an underestimate of lifetime risk. 17 

### Habs et al. (1980) reported age-standardized skin t umor incidence rates, indicating earlier 18 

### mortality in the two highest dose groups (2.8 and 4 .6 μ g/application). These rates were used to 19 

### estimate the number at risk in the dose-response mo deling, by dividing the number of mice with 20 

### tumors by the age-standardized rates (see Table 5-9 ). Exposure lasted longer than 104 weeks in 21 

### the two lower exposure groups, at about 120 and 112 weeks, and until about 88 weeks in the 22 

### highest exposure group. Incidence in the two lower exposure groups may be higher than if the 23 

### exposure had lasted just 104 weeks. There was mort ality in the first 52 weeks of exposure, about 24 

### 10−15% in the three exposure groups, but because th ere was no information concerning when 25 

### tumors first appeared it is not possible to determi ne how much the early mortality may have 26 

### impacted the number of mice at risk in each group. 27 

### Habs et al. (1984) reported mean survival times (wi th 95% confidence intervals) for each 28 

### dose group. The confidence intervals supported the judgment that the control and lower dose 29 

### groups were treated for 104 weeks. The higher dose group (4 μ g/application) was probably 30 

### treated for less than 104 weeks, because the upper 95% confidence limit for the mean survival 31 

### was approximately 79 weeks (Table 4-20). However, since it was not possible to estimate a 32 

### more realistic duration for this group, an estimate of 104 weeks was used. 33 

### Grimmer et al. (1983 and 1984), studied female CFLP mice, using acetone:DMSO (1:3) 34 

### as the vehicle (see Table 5-10). Mean or median la tency times were reported (as well as 35 

### measures of variability), but no information concer ning overall length of exposure or survival 36



### was included in the results. The total of tumor-be aring mice and the reported percentages of 37 

### mice with any skin tumors was reported and varied a t most one animal from the number of 38

### 231 DRAFT - DO NOT CITE OR QUOTE

animals initially placed on study. The decreasing latency and variability and increasing tumor 1 

### incidence with increasing benzo[a]pyrene exposure s uggests that exposure probably did not last 2 

### for 104 weeks in at least the high dose group, but the available information did not provide 3 

### duration of exposure. The data reported were model ed under the assumption that at least some 4 

### animals in each group were treated and survived unt il Week 104. 5 

### The study listed in Table 5-11, Sivak et al. (1997) exposed male C3H /HeJ mice dermally 6 

### to benzo[a]pyrene in cyclohexanone/acetone (1:1) fo r 24 months, and reported mean survival 7 

### times for each group (see Table 4-21). All high do se mice died before the final sacrifice. From 8 

### the information provided it is apparent that the an imals in the control and lower two dose groups 9 

### survived until study termination at Week 104. The study authors did not report how long 10 

### treatment in the highest dose group lasted, but est imation of the figure from the publication 11 

### suggest that exposure duration was 74 weeks. The t umor incidences and estimated duration of 12 

### exposure for each dose group are presented in Table 5-11. 13

14

### 232 DRAFT - DO NOT CITE OR QUOTE

Table 5-8. Skin tumor incidence, benign or maligna nt, in C57L male mice dermally exposed to benzo[a]pyrene

Study Mouse

strain Dose ( μ g) a Average daily dose ( μ g/d) First

appearance of tumor (weeks) Length

of Exposure (weeks) Lifetime

Average Daily Dose b Skin tumor

incidence (all types) Poel (1959) C57L 0 (toluene) 0.15 0.38 0.75 3.8 0

0.06 0.16 0.32 1.63 —

42 24 36 21–25 92

98 103 94 82 0.00

0.05 0.16 0.24 0.80 0/33 (0%)

5/55 (9%) 11/55 (20%) 7/56 (13%) 41/49 (84%)

a Doses were applied to interscapular skin 3 times/wk for up to 103 weeks or until time of appearance of a grossly detected skin tumor. See Table 4-11 for data of fi ve highest dose groups (19-752 μ g ) in which all mice died by Week 44. These groups were not considered for dose -response modeling.

b See Section 5.4.3.3. for discussion of extrapolatio n to lifetime average daily doses.

1

2

### 233 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 5-9. Skin tumor incidence, benign or maligna nt in female Swiss or

NMRI mice dermally exposed to benzo[a]pyrene

Study Mouse

strain Dose ( μ g) Average daily dose ( μ g/d) First

appearance of tumor (weeks) Length of

exposure (weeks) Lifetime

average daily dose ( μ g/d) Skin tumor

incidence (all types)

Roe et al. (1970) a,b Swiss 0 (acetone) 0.1 0.3 1 3 9 0

0.04 0.13 0.43 1.29 3.86 —

29-43

— 57-71 43-57 29-43 93

93 93 93 93 93 0.00

0.03 0.09 0.31 0.92 2.76 0/49 (0%)

1/45 (2%) 0/46 (0%) 1/48 (2%) 8/47 (20%) 34/46 (74%)

Schmidt et al. (1973) c NMRI 0 (acetone) 0.05 0.2 0.8 2 0

0.01 0.06 0.23 0.57 —

—

— 53 e 76 e 104 d

104 104 104 104 0

0.01 0.06 0.23 0.57 0/100 (0%)

0/100 (0%) 0/100 (0%) 2/100 (2%) 30/100 (30%) Swiss 0 (acetone)

0.05 0.2 0.8 2 0

0.01 0.06 0.23 0.57 —

—

— 58 e 61 e 104

104 104 104 104 0

0.01 0.06 0.23 0.57 0/80 (0%)

0/80 (0%) 0/80 (0%) 5/80 (6%) 45/80 (56%)

Schmähl et al. (1977) c NMRI 0 (acetone) 1 1.7 3 0

0.29 0.49 0.86 —

NR NR NR 104

104 104 104 0

0.29 0.49 0.86 1/81 (1%)

11/77 (14%) 25/88 (28%) 45/81 (56%)

Habs et al. (1980) c NMRI 0 (acetone ) 1.7 2.6 4.6 0

0.49 0.74 1.31 —

NR NR NR 128

120 112 88 0

0.49 0.74 0.80 0/35 (0%% ) e

8/34 (24.8%) 24/27 (89.3%) 22/24 91.7%)

Habs et al. (1984) c NMRI 0 (acetone ) 2 4 0

0.57 1.14 —

NR NR 104

104 104 0

0.57 1.14 0/20 (0%)

9/20 (45%) 17/20 (85%)

a Doses were applied 3 times/week for up to 93 wks to shaved dorsal skin.

b Numerator: number of mice detected with a skin tum or. Denominator: number of mice surviving to 29 w eeks (200 days).

c Doses were applied 2 times/week to shaved skin of t he back. Mice were exposed until natural death or until they developed a carcinoma at the site of application.

d Exposure periods not reported were assumed to be 10 4 weeks; indicated in italics.

e Central tendency estimates; range or other variabil ity measure not reported.

f The percentages were reported by the authors as age -standardized tumor incidences, derived using morta lity data from the entire study population. The incidences r eflect reported counts of tumor-bearing animals and denominators estimated from the reported age-standa rdized rates. NR=not reported.

2

3

4

### 234 DRAFT - DO NOT CITE OR QUOTE

Table 5-10. Skin tumor incidence, benign or malign ant, in female CFLP mice dermally exposed to benzo[a]pyrene

Study Dose ( μ g) a Average daily dose ( μ g/d) Mean or

median time of tumor

appearance (weeks) Length of

exposure (weeks) d Lifetime

average daily dose ( μ g/d) Skin tumor

incidence (all types) e

Grimmer et al. (1983) 0 (1:3 acetone:DMSO) 3.9 7.7 15.4 0

1.1 2.2 4.4 

### — 74.6 ± 16.8 b

60.9 ± 13.9 44.1 ± 7.7 104

104 104 104 0

1.1 2.2 4.4 0/80 (0%)

22/65 (34%) 39/64 (61%) 56/64 (88%)

Grimmer et al. (1984) 0 (1:3 acetone:DMSO) 3.4 6.7 13.5 0

0.97 1.9 3.9 

### —

61 (53–65) c 47 (43–50) 35 (32–36) 104

104 104 104 0

0.97 1.9 3.9 0/80 (0%)

43/64 (67%) 53/65 (82%) 57/65 (88%)

a Indicated doses were applied twice/week to shaved s kin of the back for up to 104 weeks.

b Mean ± SD.

c Median and 95% confidence limit.

d Assumed exposure period is indicated in italics.

e Incidence denominators were calculated from reporte d tumor-bearing animals and reported percentages.

1 

### Table 5-11. Skin tumor incidence, benign or malig nant, in male C3H /HeJ

mice dermally exposed to benzo[a]pyrene(Sivak et al ., 1997)

Dose ( μ g) a Average daily dose ( μ g/d) First

appearance of tumor (weeks) Length of

exposure (weeks) b Lifetime

average daily dose ( μ g/d) Skin tumor

incidence (all types) 0 (1:1 cyclohexanone/acetone)

0.05 0.5 5.0 0 0.01 0.14 1.4 —

— NR ~43 104

104 104 74 0.0

0.01 0.14 0.51 0/30 (0%)

0/30 (0%) 5/30 (17%) 27/30 (90%)

a Indicated doses were applied twice/week to shaved d orsal skin.

b Assumed exposure period is indicated in italics. NR=not reported.

2

3



### 5.4.3.3. Dose Adjustments and Extrapolation Method(s)—Dermal Exposure 4 

### As with the oral and inhalation benzo[a]pyrene car cinogenicity data (see sections 5.4.1.3 5 

### and 5.4.2.3), benzo[a]pyrene’s dermal exposure carc inogenicity data were generally 6 

### characterized by earlier occurrence of tumors and i ncreased mortality with increasing exposure 7 

### level. However, individual animal data were not av ailable for any of the identified studies. 8 

### Therefore, time to tumor modeling was not possible. Each of the dermal data sets was modeled 9 

### using the multistage model, incorporating adjustmen ts for early mortality, when data were 10



### available. 11

### 235 DRAFT - DO NOT CITE OR QUOTE

1 

### First, for all studies, administered doses were con verted to average daily doses using the 2 

### equation: 3

4 

### Average dose/day = ( μ g/application) × (number of exposures/week ÷ 7 days /week). 5

6 

### Next, lifetime equivalent doses were estimated for study groups that were reported to end 7 

### before 104 weeks by multiplying the relevant averag e daily doses by (L e 

### /104) 3 

### , where L e 

### is the 8 

### length of exposure, based on observations that tumo r incidence tends to increase with age (Doll, 9 

### 1971). Note that exposure periods less than 52 wee ks would lead to a relatively large adjustment 10 

### [i.e., (52/104) 3 

### = 0.125, or an eightfold lower dose than administe red], reflecting considerable 11 

### uncertainty in lifetime equivalent dose estimates g enerated from relatively short studies. 12 

### The multistage-cancer model in the EPA BMDS (versio n 2.1) was fit to each data set in 13 

### Tables 5-8 through 5-11. The multistage model with the most parsimonious fit (fewest 14 

### parameters yielding an adequate fit) was selected t o calculate the potential POD from each data 15 

### set (see Appendix F for details). Because the mult istage model is preferred for cancer modeling, 16



### the conventional α -level of 0.05 was used to judge goodness-of-fit. If there was no adequate fit 17 

### using the multistage-cancer model, then other dicho tomous models were considered. If there 18 

### was still no adequate fit, high doses were dropped incrementally and the multistage-cancer model 19 

### was considered before attempting other models. BMD s and BMDLs associated with an extra 20 

### risk of 10% were calculated. Because benzo[a]pyren e is expected to cause cancer via a 21 

### mutagenic MOA, a linear approach to low dose extrap olation from the PODs (i.e., BMDL 10 

### ) was 22 

### used (U.S. EPA, 2005a) for candidate dermal slope f actors. 23

24



### 5.4.3.4. Dermal Slope Factor Derivation 25 

### Adequate model fits were found using the multistage model for all but one of the mouse 26



### skin tumor incidence data sets in Tables 5-8 to 5-1 1, as described in Appendix F. In one case, 27 

### the data from Grimmer et al. (1984) could not be ad equately fit by the multistage model initially, 28



### and the other dichotomous models available in BMDS were considered. Due to the supralinear 29 

### shape of the dose-response data, only the log-logis tic and dichotomous Hill models provided 30 

### adequate fits. Also due to the supralinear dose-re sponse shape, the point of departure for slope 31



### factor derivation was identified near the lowest re sponse of ~70%, in order to avoid excessive 32 

### extrapolation of the fitted model. 33 

### Dermal slope factors, calculated in units of risk p er (μg/day) using linear extrapolation 34



### from the BMDL 10 

### values, ranged from 0.25 to 1.8 per μg/day, a roug hly 7-fold range (see Table 35 

### 5-12). A number of differences among studies contr ibute to this range, including solvent choice, 36 

### sex and strain of mice studied, dose ranges and the level of detail reported. Mouse strains were 37 

### not repeated across sexes among these studies, so i t cannot be established whether male mice are 38

### 236 DRAFT - DO NOT CITE OR QUOTE

generally more or less sensitive than female mice t o benzo[a]pyrene dermal carcinogenicity, or 1



### whether Swiss or NRMI mice are more or less sensiti ve than other strains. In addition, different 2 

### solvents were used in the various studies with vary ing strain and sex combinations tested. For 3 

### example, toluene was used in one male study only an d all of the female studies used acetone. 4 

### Thus, any possible impact of the solvents used is n ot clear. The estimates derived from the two 5 

### studies in males were at the higher end of the rang e of slope factors derived, but the available 6



### information is too limited to conclude that males a re more sensitive than females. Also, as noted 7 

### earlier, incomplete mortality information in severa l of the female mouse studies (Schmidt et al., 8 

### 1973; Schmähl et al., 1977; Grimmer et al., 1983, 1 984; and Habs et al., 1980, 1984) suggests 9 

### that the derived dermal slope factors may underesti mate cancer risk. 10



### The BMDL 10 

### of 0.066 μg/animal-day, based on the tumor response in C3H/HeJ male 11 

### mice (Sivak et al., 1997), is recommended for devel oping a human dermal slope factor because it 12 

### is the lowest POD among studies with lower doses wh ere intercurrent mortality was less likely to 13 

### impact the number at risk and represented a chronic duration of exposure. 14

15



### Table 5-12. PODs derived from skin tumor incidence data in mice exposed to 16 

### benzo[a]pyrene by the dermal route of exposure a 17

18

Reference Mouse strain Solvent BMD 10 (μg/animal-d) BMDL 10

(μg/animal-d)

Male mice Sivak et al., 1997 C3H/HeJ Acetone/ cyclohexanone 0.12 0.066

Poel, 1959 C57L Toluene 0.12 0.077

Female mice Habs et al., 1984 NMRI Acetone 0.078 0.056 Grimmer et al., 1984 CFLP Acetone/DMSO 1.07 b 0.48 b

Schmahl et al., 1977 NMRI Acetone 0.23 0.15 Schmidt et al., 1973 Swiss Acetone 0.28 0.22 Grimmer et al., 1983 CFLP Acetone/DMSO 0.24 0.21 Habs et al., 1980 NMRI Acetone 0.29 0.22 Schmidt et al., 1973 NMRI Acetone 0.33 0.29 Roe et al., 1970 Swiss Acetone 0.69 0.39

a See Appendix F for details of modeling results. 19

b BMR=70% for this dataset, in order to avoid excessi ve extrapolation via the fitted model. 20 21 

### 5.4.3.5. Dermal Slope Factor Cross Species Scaling 22 

### Different methodologies have been established for i nterspecies scaling of points of 23 

### departure (PODs) used to derive oral slope factors and inhalation unit risks. Cross-species 24 

### adjustment of oral doses is based on allometric sca ling using the three-fourths power of body 25 

### weight. This adjustment accounts for more rapid di stribution, metabolism, and clearance in 26 

### small animals (US EPA 2005). Cross-species extrapo lation of inhalation exposures is based on 27 

### standard dosimetry models that consider factors suc h as solubility, reactivity, and persistence 28

### 237 DRAFT - DO NOT CITE OR QUOTE

(US EPA 1994). However, no established methodology exists to adjust for interspecies 1 

### differences in dermal toxicity at the point of cont act. Because there is no established 2 

### methodology for cross-species extrapolation of derm al toxicity, several alternative approaches 3 

### were evaluated (see Appendix H). Among the alterna tive described in Appendix H, cross4 

### species adjustment based on allometric scaling usin g body weight to the 3/4 power was selected. 5 

### Under this approach, rodents and humans exposed to the same daily dose of a carcinogen, 6 

### adjusted for BW 3/4 

### , would be expected to have equal lifetime risks of cancer. 7

8



### The POD M 

### derived from the mouse study by Sivak et al., (199 7) is adjusted to a human 9 

### equivalent dose (HED) as follows: 10

11



### POD HED 

### (μg/day) = POD M 

### (μg/day) × (BW H 

### / BW M 

### ) 3/4 12

13



### POD HED 

### (μg/day) = 0.066 μg/day × (70 kg / 0.035 kg) 3/4 14 

### = 19.7 μg/day 15

16

17 

### The resulting POD HED 

### is used to calculate the dermal slope factor for b enzo[a]pyrene: 18

19



### DSF = 0.1/ POD HED 20

21 

### DSF = 0.1/(19.7 μg/day) = 0.005 (μg/day) -1 22

23 

### Note that the DSF should only be used with lifetime human exposures < 20 μg/day, the 24 

### human equivalent of the bioassay POD, because above this level the dose-response relationship 25 

### may not be proportional to mass of the compound app lied. 26 

### Several assumptions are made in the use of this sca ling method. First, it is assumed that 27 

### the toxicokinetic processes in the skin will scale with interspecies differences in whole body 28 

### toxicokinetics. Secondly, it is assumed that the risk at low doses of benzo[a]pyrene is linear; 29 

### however, one study indicates that at high doses of benzo[a]pyrene, carcinogenic potency is 30 

### related to mass applied per unit skin and not to to tal mass (Davies 1967). However, this may be 31 

### due to promotional effects, such as inflammation, t hat are observed at high doses of 32 

### benzo[a]pyrene. 33 

### This slope factor has been developed for a local e ffect and it is not intended to estimate 34 

### systemic risk of cancer following dermal absorption of benzo[a]pyrene into the systemic 35 

### circulation. Although some information suggests th at benzo[a]pyrene metabolites can enter 36 

### systemic circulation following dermal exposure (God schalk et al 1998), lifetime skin cancer 37 

### bioassays which have included pathological examinat ion of other organs, have not found 38

### 238 DRAFT - DO NOT CITE OR QUOTE

elevated incidences of tumors at distal sites (Poel 1959; Roe et al., 1970; Schmidt et al., 1973; 1 

### Schmahl et al., 1977; Habs et al., 1980; Higginboth am et al.,1993). In addition, benzo[a]pyrene 2 

### tends to bind to targets within the skin rather tha n enter the plasma receptor fluid (a surrogate 3 

### measure of systemic absorption) in in vitro human s kin experiments. These data are consistent 4 

### with benzo[a]pyrene’s metabolism to reactive metabo lites within the viable layers of the skin 5 

### (Wester et al., 1990). Some studies indicate that the fraction of benzo[a]pyrene left within the 6 

### viable layers of the skin is a large portion of the applied dose (Moody et al., 2007; 1995). Taken 7 

### together, these data support the conclusion that th e risk of skin cancer following dermal exposure 8 

### likely outweighs cancer risks at distal organs. 9

10 

### 5.4.4. Application of Age-Dependent Adjustment Fac tors 11 

### Based on sufficient support in laboratory animals a nd relevance to humans (see Section 12 

### 4.7.3) benzo[a]pyrene is determined to be carcinoge nic by a mutagenic MOA. According to the 13 

### Supplemental Guidance for Assessing Susceptibility from Early Life Exposure to Carcinogens 14 

### (“ Supplemental Guidance ”) (U.S. EPA, 2005b), individuals exposed during ea rly life to 15 

### carcinogens with a mutagenic MOA are assumed to hav e increased risk for cancer. The oral 16 

### slope factor of 1.4 per mg/kg-day, inhalation unit risk of 0.5 per mg/m 3 

### , and dermal slope factor 17 

### of 0.0051 per μg/day for benzo[a]pyrene, calculated from data applicable to adult exposures, do 18 

### not reflect presumed early life susceptibility to t his chemical. Though some chemical specific 19 

### data exist for benzo[a]pyrene which demonstrate inc reased early life susceptibility to cancer 20 

### (Vesselinovitch et al. 1984), these data were not c onsidered sufficient to develop separate risk 21 

### estimates for childhood exposure, as they used acut e, i.p. exposures (U.S. EPA, 2005b). In the 22 

### absence of adequate chemical-specific data to evalu ate differences in age-specific susceptibility, 23 

### the Supplemental Guidance (U.S. EPA, 2005b) recomme nds that age-dependent adjustment 24 

### factors (ADAFs) be applied in estimating cancer ris k. 25



### The Supplemental Guidance (U.S. EPA, 2005b) establishes ADAFs for three speci fic age 26 

### groups. These ADAFs and their corresponding age gr oupings are: 10 for individuals exposed 27 

### <2 years, 3 for exposed individuals 2 to <16 years, and 1 for exposed individuals ≥ 16 years. The 28 

### 10- and 3-fold adjustments are combined with age sp ecific exposure estimates when estimating 29 

### cancer risks from early life (<16 years age) exposu res to benzo[a]pyrene To illustrate the use of 30



### the ADAFs established in the Supplemental Guidance (U.S. EPA, 2005b), sample calculations 31 

### are presented for three exposure duration scenarios , including full lifetime, assuming a constant 32 

### benzo[a]pyrene exposure of 0.001 mg/kg-day (Table 5 -13). 33 

### Calculations for the application of ADAFs to oral e xposures are presented in Table 5-13; 34 

### calculations for exposures by the inhalation and de rmal routes follow the same procedure (Table 35 

### 5-13 and 5-14). Exposure duration scenarios includ e full lifetime exposure (assuming a 70-year 36 

### lifespan), and two 30-year exposures at ages 0–30 a nd ages 20–50. Table 5-13 lists the four 37 

### factors (ADAFs, cancer risk estimate, assumed expos ure, and duration adjustment) that are 38

### 239 DRAFT - DO NOT CITE OR QUOTE

needed to calculate the partial cancer risk based o n the early age-specific group. The cancer risk 1 

### for each age group is the product of the four facto rs in columns 2–5. Therefore, the cancer risk 2 

### following daily benzo[a]pyrene oral exposure in the age group 0 to <2 years is the product of the 3



### values in columns 2–5 or 10 × 1× 0.001 × 2/70 = 4 × 10 -4 

### . The cancer risk for specific exposure 4



### duration scenarios that are listed in the last colu mn are added together to get the total risk. Thus, 5 

### a 70-year (lifetime) risk estimate for continuous e xposure to 0.001 mg/kg-day benzo[a]pyrene is 6



### 2 × 10 -3 

### , which is adjusted for early-life susceptibility a nd assumes a 70-year lifetime and 7 

### constant exposure across age groups. 8

9 

### Table 5.13. Application of ADAFs to benzo[a]pyrene cancer risk following a

lifetime (70-year) oral exposure

10 Age Group ADAF Unit risk Exposure Concentration Duration adjustment Cancer Risk

for Specific Exposure Duration Scenarios (per mg/kg-day) (mg/kg-day)

0–<2 yrs 10 1 0.001 2 yrs/70 yrs 0.0003 2–<16 yrs 3 1 0.001 14 yrs/70 yrs 0.0006

≥ 16 yrs 1 1 0.001 54 yrs/70 yrs 0.0007

Total Risk 0.002

11 

### In calculating the cancer risk for a 30-year consta nt exposure to benzo[a]pyrene at an 12 

### exposure level of 0.001 mg/kg-day from ages 0–30, t he duration adjustments would be 2/70, 13 

### 14/70, and 14/70, and the partial risks for the thr ee age groups would be 3 × 10 -4 

### , 6 × 10 -4 

### , and 14



### 2 × 10 -4 

### , which would result in a total risk estimate of 1 × 10 -3



### . 15 

### In calculating the cancer risk for a 30-year consta nt exposure to benzo[a]pyrene at an 16 

### exposure level of 0.001 mg/kg-day from ages 20–50, the duration adjustments would be 0/70, 17 

### 0/70, and 30/70. The partial risks for the three g roups are 0, 0, and 4 × 10 -4 

### , which would result 18



### in a total risk estimate of 4 × 10 -4



### . 19 

### Consistent with the approaches for the oral route o f exposure, the ADAFs should also be 20 

### applied when assessing cancer risks for subpopulati ons with early life exposures to 21 

### benzo[a]pyrene via the inhalation and dermal routes are presented in Tables 5-14 and 5-15. 22

23 

### Table 5-14. Application of ADAFs to benzo[a]pyrene cancer risk following a

lifetime (70-year) inhalation exposure

24

Age Group ADAF Unit risk Exposure Concentration Duration adjustment Cancer Risk

for Specific Exposure Duration Scenarios (per μg/m 3 ) (μg/m 3 )

### 240 DRAFT - DO NOT CITE OR QUOTE

0–<2 yrs 10 5 x 10 - 4 1 2 yrs/70 yrs 0.0001 2–<16 yrs 3 5 x 10 - 4 1 14 yrs/70 yrs 0.0003

≥ 16 yrs 1 5 x 10 - 4 1 54 yrs/70 yrs 0.0004

Total Risk 0.0008

1

2 

### Table 5-15. Application of ADAFs to benzo[a]pyrene cancer risk following a 3 

### lifetime (70-year) dermal exposure 4

5

Age Group ADAF Unit risk Exposure Concentration Duration adjustment Cancer Risk

for Specific Exposure Duration Scenarios (per μg/day) (μg/day)

0–<2 yrs 10 0.005 0.001 2 yrs/70 yrs 1 x 10 - 6 2–<16 yrs 3 0.005 0.001 14 yrs/70 yrs 3 x 10 - 6

≥ 16 yrs 1 0.005 0.001 54 yrs/70 yrs 4 x 10 - 6

Total Risk 8 x 10 - 5

6

7



### 5.4.5. Uncertainties in Cancer Risk Values 8



### 5.4.5.1. Oral Slope Factor 9 

### Uncertainty in the recommended oral slope factor is reflected in the range of slope factors 10 

### among tumors sites and species; the lowest and high est slope factors listed in Table 5-6 show 11 

### about a 35-fold difference. While the highest risk estimates were derived from the incidence 12 

### data for forestomach tumors in both rats and mice, the oral slope factor based on the mouse 13 

### forestomach data was about threefold higher than th e oral slope factor based on male rat data 14 

### (Table 5-6). These comparisons show that the selec tion of target organ, animal species, and 15 

### dosimetric extrapolation can impact the oral cancer risk estimate. However, all of the activation 16 

### pathways implicated in benzo[a]pyrene carcinogenici ty have been observed in human tissues and 17 

### associations have been made between the spectra of mutations in tumor tissues from 18 

### benzo[a]pyrene-exposed animals and humans exposed t o complex PAH mixtures containing 19 

### benzo[a]pyrene (see Section 4.7.3). 20

21



### 5.4.5.2. Inhalation Unit Risk 22 

### Only one animal cancer bioassay by the inhalation r oute is available which describes the 23 

### dose-response relationship for respiratory tract tu mors with chronic inhalation exposure to 24 

### benzo[a]pyrene (Thyssen et al., 1981). Although co rroborative information on dose-response 25 

### relationships in other animal species is lacking, t he findings for upper respiratory tract tumors are 26 

### consistent with findings in other hamster studies w ith intratracheal administration of 27 

### benzo[a]pyrene. This study is adequate for dose-re sponse analysis and derivation of an 28 

### inhalation unit risk estimate, but some associated uncertainty includes the inability to apply U.S. 29

### 241 DRAFT - DO NOT CITE OR QUOTE

EPA (1994b) dosimetry approaches to extrapolate inh aled doses from animals to humans, due to 1 

### the use of a soluble hygroscopic carrier particle ( NaCl) for the delivery of benzo[a]pyrene. One 2 

### likely consequence of the use of hygroscopic carrie r particles would be the growth of 3 

### benzo[a]pyrene-NaCl particles in the humid environm ent of the respiratory tract resulting in 4 

### increased particle diameter and resulting changes i n particle deposition, specifically, increased 5 

### impaction in the upper respiratory tract (Xu and Yu 1985; Ferron 1994; Asgharian 2004; 6 

### Varghese and Gangamma 2009). Exposure to benzo[a]p yrene in the environment predominantly 7 

### occurs via non-soluble, non-hygroscopic particles. The potential impact of differences in carrier 8 

### particle on the magnitude of the inhalation unit ri sk is unknown. 9

10



### 5.4.5.3 Dermal Slope Factor 11 

### Uncertainty in the recommended dermal slope factor is partly reflected in the range of 12 

### slope factors derived from the modeled mouse skin t umor data sets: the lowest and highest 13 

### dermal slope factors listed in Table 5-12 show a 7- fold difference (0.25-1.8 ug/day) in 14 

### magnitude. There is some indication that the recom mended dermal slope factor may 15 

### underestimate cancer risk, due to inadequate data t o take the observed decreasing tumor latency 16 

### with increasing exposure level into account. Relia nce on studies with the lowest exposure levels 17 

### where early mortality due to benzo[a]pyrene exposur e was low and exposures continued for 18 

### approximately 104 weeks may minimize this source of uncertainty. 19 

### Human dermal exposure to benzo[a]pyrene in the envi ronment likely occurs 20 

### predominantly through soil contact. The available mouse dermal bioassays of benzo[a]pyrene 21 

### relied on delivery of benzo[a]pyrene to the skin in a solvent solution (typically acetone or 22 

### toluene). The use of a volatile solvent likely re sults in a larger dose of benzo[a]pyrene available 23 

### for uptake into the skin (compared to soil). Relia nce on these studies may overestimate the risk 24 

### of skin tumors from benzo[a]pyrene contact through soil; however, cancer bioassays delivering 25 

### benzo[a]pyrene through a soil matrix are not availa ble. 26 

### There is uncertainty in extrapolating from the inte rmittent exposures in the mouse assays 27 

### to daily exposure scenarios. This assessment makes the assumption that risk is proportional to 28 

### total cumulative exposure. The extent to which thi s assumption under- or overestimates risk is 29



### unknown. 30



### The available data were not useful to determine whi ch animal species may be the best 31 

### surrogate for human dermal response to benzo[a]pyre ne. In extrapolation of the animal dermal 32 

### information to humans the inherent assumption is th at equal area of skin from a mouse or human 33 

### would have equal probability of developing a tumor upon benzo[a]pyrene exposure. 34 

### Qualitatively, the toxicokinetics and toxicodynamic s in mouse and human skin appear to be 35 

### similar (Knafla et al., 2010; Bickers et al., 1984) . Specifically, all of the activation pathways 36 

### implicated in benzo[a]pyrene carcinogenicity have b een observed in mouse and human skin and 37 

### associations have been made between the spectra of mutations in tumor tissues from 38

### 242 DRAFT - DO NOT CITE OR QUOTE

benzo[a]pyrene-exposed animals and humans exposed t o complex PAH mixtures containing 1 

### benzo[a]pyrene (see Section 4.7.3). 2 

### This dermal slope factor for benzo[a]pyrene is base d on skin cancer and it is not 3 

### developed to represent systemic cancer risk from de rmal exposure. It is unclear whether dermal 4 

### exposure to benzo[a]pyrene would result in elevated risk of systemic tumors. Some studies in 5 

### humans suggest that although the skin may be respon sible for a “first pass” metabolic effect, 6 

### benzo[a]pyrene-specific adduct levels have been det ected in WBC following dermal exposure to 7 

### benzo[a]pyrene, indicating that dermally applied be nzo[a]pyrene enters systemic circulation 8 

### (Godschalk et al., 1998). Although none of the lif etime dermal bioassays in mice, which 9 

### included macroscopic examination of internal organs , reported an elevation of systemic tumors 10 

### in benzo[a]pyrene-treated mice compared to controls (Poel 1959; Roe et al., 1970; Schmidt et al., 11 

### 1973; Schmahl et al., 1977; Habs et al., 1980; Higg inbotham et al.,1993), most of these studies 12 

### attempted to remove animals with grossly observed t umors of the skin from the study before the 13 

### death of the animal, possibly minimizing the develo pment of more distant tumors with longer 14 

### latency. The risk of benzo[a]pyrene-induced point of contact tumors in the skin likely competes 15 

### with systemic risk of tumors. Currently, the poten tial contribution of dermally absorbed 16 

### benzo[a]pyrene to systemic cancer risk is unclear. 17

18



### 5.5.5. Previous Cancer Assessment 19 

### The previous cancer assessment for benzo[a]pyrene w as posted on the IRIS database in 20 

### 1987. At that time, benzo[a]pyrene was classified as a probable human carcinogen (Group B2) 21 

### based on inadequate data in humans and sufficient d ata in animals via several routes of exposure. 22 

### An oral slope factor was derived from the geometric mean of four slope factor estimates based 23 

### on studies in Sprague-Dawley rats (Brune et al., 19 81) and CFW-Swiss mice (Neal and Rigdon, 24 

### 1967). Brune et al. (1981) administered 0.15 mg/kg benzo[a]pyrene in the diet every 9th day or 25 

### 5 days/week in a 1.5% caffeine solution until rats were moribund or dead. A single slope factor 26 

### estimate of 11.7 per mg/kg-day, based on a lineariz ed multistage model applied to the combined 27 

### incidence of forestomach, esophageal, and laryngeal tumors, was derived. In the Neal and 28 

### Rigdon (1967) bioassay, mice administered benzo[a]p yrene in the diet at concentrations ranging 29 

### from 1 to 250 ppm for up to 197 days developed sign ificantly increased incidences of 30 

### forestomach tumors. This study utilized mixed sex dose groups with mice from 3 weeks to 6 31 

### months old at the start of dosing. This study did not include concurrent controls. This 32 

### necessitated the use of historical controls (from S WR/J mice) for the incidence of forestomach 33 

### tumors from a study by (Rabstein et al., 1973). Th ree modeling procedures were used to derive 34



### risk estimates from these data. For one risk estim ate, Clement Associates (1990) fit a two-stage 35 

### response model, based on exposure-dependent changes in both transition rates and growth rates 36 

### of preneoplastic cells, to derive a value of 5.9 pe r mg/kg-day. In a U.S. EPA report (1991b), a 37 

### value of 9.0 per mg/kg-day, derived by linear extra polation from the 10% response point to the 38

### 243 DRAFT - DO NOT CITE OR QUOTE

background of an empirically fitted dose-response c urve, was identified. Finally, using a 1 

### Weibull-type model to reflect less-than-lifetime ex posure to benzo[a]pyrene, the same U.S. EPA 2 

### report (1991b) derived an upper-bound slope factor estimate of 4.5 per mg/kg-day. Since the 3 

### variance for the four slope factor estimates was lo w, and in order to consider all of the available 4 

### data, the geometric mean of these four estimates, 7 .3 per mg/kg-day, was recommended as the 5 

### oral slope factor. 6 

### An inhalation unit risk and dermal slope factor wer e not previously available on IRIS. 7

8

9

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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE 1



### RESPONSE 2

3

4 

### 6.1. HUMAN HAZARD POTENTIAL 5 

### benzo[a]pyrene is a five-ring nonsubstituted PAH, w hich is produced through natural and 6 

### anthropogenic processes involving the incomplete co mbustion or pyrrolysis of carbon-containing 7 

### materials. Benzo[a]pyrene exists in the environmen t in complex mixtures, which may consist of 8 

### numerous PAHs, including heterocyclic and nonhetero cyclic forms as well as aza arenes and 9 

### nitro-substituted PAHs. The magnitude of human exp osure to benzo[a]pyrene and other PAHs 10 

### depends on several factors related to lifestyle (e. g., diet, tobacco smoking), occupation, and 11 

### living conditions (e.g., urban versus rural setting , domestic heating, and cooking methods). 12 

### There are limited data establishing associations be tween increased risk for noncancer 13 

### health effects in humans and exposure to benzo[a]py rene. Several epidemiology studies have 14 

### reported associations between adverse birth outcome s including reduced birth weight, postnatal 15 

### body weight, and head circumference with internal b iomarkers of exposure to benzo[a]pyrene 16 

### (BPDE-DNA adducts) via exposure to complex PAH mixt ures (Tang et al., 2008, 2006; Perera et 17 

### al., 2005a, b). However, extrapolations from these studies are complicated by the concomitant 18 

### exposure to multiple PAHs and other components in t he mixture. 19 

### There is evidence of human carcinogenicity for seve ral PAH mixtures containing 20 

### benzo[a]pyrene, such as soot, coal tars, coal-tar p itch, mineral oils, and shale oils (IARC, 2010; 21 

### Baan et al., 2009; Straif et al., 2005). There is also evidence of carcinogenicity in occupations 22 

### involving exposure to PAH mixtures containing benzo [a]pyrene, such as aluminum production, 23 

### chimney sweeping, coal gasification, coal-tar disti llation, coke production, iron and steel 24 

### founding, and paving and roofing with coal tar pitc h (IARC, 2010; Baan et al., 2009; Straif et al., 25 

### 2005). Benzo[a]pyrene is also a notable constituen t of tobacco smoke (IARC 2004). An 26 

### increasing number of studies report exposure biomar kers such as benzo[a]pyrene- or PAH-DNA 27 

### adducts, and several cohort studies demonstrate a p ositive exposure-response relationship with 28 

### cumulative PAH exposure using benzo[a]pyrene as an indicator substance. Because 29 

### benzo[a]pyrene is only one of many PAHs that could contribute to these observed increases in 30 

### cancer, the epidemiologic studies provide credible but limited support for a causative role of 31 

### benzo[a]pyrene in human cancer. Studies in multiple species of laboratory animals indicate that 32 

### benzo[a]pyrene is carcinogenic by all routes of exp osure. 33

34



### 6.2. DOSE RESPONSE 35



### 6.2.1. RfD 36



### Limited human data are available to inform noncance r health effects following chronic 37 

### oral exposure to benzo[a]pyrene. Animal studies re porting effects of benzo[a]pyrene include 38

### 245 DRAFT - DO NOT CITE OR QUOTE

several chronic cancer bioassays (which report limi ted noncancer endpoints; Kroese et al. 2001; 1 

### Beland and Culp, 1998; Culp et al., 1998), several subchronic studies (Knuckles et al., 2001; De 2 

### Jong et al., 1999), developmental toxicity studies (Kristensen et al., 1995; MacKenzie and 3 

### Angevine, 1981) and several reproductive toxicity s tudies (Mohamed et al., 2010; Xu et al., 4 

### 2010; Zheng et al., 2010). 5 

### In consideration of the available studies reporting low-dose effects of chronic and 6 

### subchronic oral exposure to benzo(a)pyrene in anima ls, the Xu et al., (2010) study was chosen as 7 

### the principal study. This study identified biologi cally and statistically significant decrements in 8 

### ovary weight, number of primordial follicles, estro gen levels, and estrus cyclicity. These 9 

### reductions in female reproductive endpoints observe d in rats are supported by a large database of 10 

### animal studies indicating that benzo[a]pyrene, admi nistered by multiple routes of exposure, is 11 

### ovotoxic with effects including decreased ovary wei ght, decreased primordial follicles, and 12 

### reduced fertility (Mattison et al., 1980; MacKenzie and Angevine, 1981; Swartz and Mattison 13 

### 1985; Miller et al., 1992; Kristensen et al.,1995; Borman et al, 2000; Archibong et al., 2002). 14 

### Additionally, studies indicate that exposure to com plex mixtures of PAHs, such as through 15 

### cigarette smoke, is associated with measures of dec reased fertility in humans (El Nemr et al., 16 

### 1998; Neal et al., 2005). Specific associations ha ve also been made between infertility and 17 

### increased levels of benzo[a]pyrene in follicular fl uid in women undergoing in vitro fertilization 18 

### (Neal et al., 2008). 19 

### The RfD of 0.0005 mg/kg-day (0.5 μg/kg-day) was der ived using a BMDL 1SD 

### of 20 

### 1.5 mg/kg-day for reduced ovary weight in SD rats e xposed to benzo[a]pyrene via gavage for 60 21 

### days (Xu et al., 2010). To derive the RfD, this PO D was divided by a total UF of 3000 (factors 22 

### of 10 for animal-to-human extrapolation, human inte rindividual variability in susceptibility, and 23 

### subchronic to chronic extrapolation, and 3 for data base deficiencies). The default animal-to24 

### human extrapolation and human variability factors w ere applied because of the lack of 25 

### quantitative information to assess toxicokinetic or toxicodynamic differences between animals 26 

### and humans and the range of susceptibilities in hum an populations. A subchronic to chronic 27 

### extrapolation factor was applied because the POD wa s chosen from a study with a less than 28 

### lifetime exposure duration. In addition, a databas e uncertainty factor of 3 was applied to account 29 

### for deficiencies in the benzo[a]pyrene toxicity dat abase, primarily the lack of a standard 30 

### multigenerational reproductive study and the lack o f a neurodevelopmental study. 31



### The overall confidence in the RfD is low-to-medium. Confidence in the principal study 32 

### (Xu et al., 2010) is medium. The design, conduct, and reporting of this subchronic toxicity study 33 

### were adequate; however, the number of dose groups a nd number of animals per group were low. 34 

### Confidence in the database is low-to-medium primari ly due to the lack of a multigeneration 35 

### reproductive toxicity study (with exposure from pre -mating to sexual maturity) and the lack of a 36 

### neurodevelopmental study. Reflecting medium confid ence in the principal study and low-to37 

### medium confidence in the database, confidence in th e RfD is low-to-medium. 38

### 246 DRAFT - DO NOT CITE OR QUOTE

1 

### 6.2.2. RfC 2 

### The only chronic inhalation study available for ben zo[a]pyrene was designed as a cancer 3 

### bioassay and did not report noncancer endpoints (Th yssen et al.1981). However, several 4 

### repeated dose reproductive and developmental toxici ty studies are available in which effects on 5 

### fetal survival and the male reproductive system hav e been observed following inhalation 6



### exposure. 7 

### Archibong et al. (2002) was selected as the princip al study as it observed biologically 8 

### significant effects in F344 rats at the lowest dose tested by the inhalation route. This study 9 

### indicates that the developing fetus is a sensitive target following inhalation exposure to 10 

### benzo[a]pyrene. Exposure to benzo[a]pyrene at 25 μ g/m 3 

### on GDs 11–20 caused biologically 11 

### significant reductions in fetal survival and body w eight decreases in the surviving pups. The 12 

### observed decrease in pup weight and fetal survival were selected as critical effects as they are the 13 

### most sensitive noncancer effects observed following inhalation exposure to benzo[a]pyrene. 14 

### Additional support for this endpoint can be found f rom an oral study of benzo[a]pyrene in mice 15 

### which observed decreased survival of litters, decre ased pup weight, and decreased reproductive 16 

### organ weight following in utero exposure to benzo[a ]pyrene on GD 7-16 (MacKenzie and 17 

### Angevine, 1981). Though only a few studies exist t hat evaluate benzo[a]pyrene by the 18 

### inhalation route, the oral studies support the repr oductive and developmental effects observed in 19



### the available inhalation studies. 20



### The RfC of 5 x 10 -6 

### mg/m 3 

### -day was derived using a LOAEL ADJ[HEC] 

### of 4.6 μg/m 3 

### -day for 21 

### decrease in pup weight and fetal survival in F344 r ats exposed to benzo[a]pyrene aerosols on 22 

### GDs 11-20 (Archibong et al., 2002). To derive the RfC, the POD was divided by a total UF of 23 

### 1000 (factors of 3 for animal-to-human extrapolatio n, 10 for human interindividual variability in 24 

### susceptibility and LOAEL-to-NOAEL extrapolation, an d 3 for database deficiencies). The 25 

### default animal-to-human extrapolation and human var iability factors were applied because of the 26 

### lack of quantitative information to assess toxicody namic differences between animals and 27 

### humans, whereas the toxicokinetic component is addr essed by the determination of a HEC as 28 

### described in the RfC methodology (U.S. EPA, 1994b). The default human variability factor was 29 

### applied because of the lack of information regardin g the range of susceptibilities in human 30 

### populations. A LOAEL-to-NOAEL extrapolation factor was applied because a NOAEL was not 31 

### identified for decreased fetal survival observed by Archibong et al (2002). In addition, a 32 

### database uncertainty factor of 3 was applied to acc ount for deficiencies in the benzo[a]pyrene 33 

### toxicity database, primarily the lack of a standard multigenerational reproductive study. 34



### The overall confidence in the RfC is low-to-medium. Confidence in the principal study 35 

### (Archibong et al., 2002) is medium. The conduct, a nd reporting of this developmental dietary 36 

### study were adequate, however, a NOAEL was not ident ified. Confidence in the database is low37 

### to-medium due to the lack of a multigeneration repr oductive toxicity study, the lack of studies on 38

### 247 DRAFT - DO NOT CITE OR QUOTE

immune endpoints, and the lack of information regar ding subchronic and chronic inhalation 1 

### exposure. Reflecting medium confidence in the prin cipal study and low-to-medium confidence 2 

### in the database, confidence in the RfC is low-to-me dium. 3

4



### 6.2.3. Cancer 5



### Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 6 

### benzo[a]pyrene is “carcinogenic to humans” based on evidence of carcinogenicity in humans 7 

### exposed to different PAH mixtures containing benzo[ a]pyrene, extensive and consistent evidence 8 

### of carcinogenicity in laboratory animals exposed to benzo[a]pyrene via several routes of 9 

### administration, and strong evidence that mechanisms of carcinogenesis in laboratory animals 10 

### also occur in humans exposed to PAH mixtures contai ning benzo[a]pyrene. Strong evidence 11 

### links the metabolism of benzo[a]pyrene to DNA-react ive agents with key mutational events in 12 

### genes that can lead to tumor initiation. Specifica lly, the metabolic activation of benzo[a]pyrene 13 

### occurs in human tissues, and associations have been made between spectra of mutations in the 14 

### p53 tumor suppressor gene or ras oncogenes induced by benzo[a]pyrene metabolites and the 15 

### spectra of mutations in these genes in tumor tissue from benzo[a]pyrene-exposed animals and 16



### humans. 17 

### Several lines of evidence relating to mutagenicity and tumor initiation are available for 18 

### benzo[a]pyrene including: in vitro evidence of DNA adducts, mutations, cytogenetic damage, 19 

### and primary DNA damage; in vivo DNA adducts, gene m utations, cytogenetic damage, and other 20 

### measures of primary DNA damage; detection of DNA ad ducts in target tissue in vivo; and 21 

### detection of cancer-relevant oncogene/tumor suppres sor gene mutations in target tissue in vivo. 22 

### Taken together, these data provide support for a mu tagenic MOA for benzo[a]pyrene-induced 23 

### cancer. Because benzo[a]pyrene is expected to caus e cancer via a mutagenic MOA, a linear 24 

### approach to low-dose extrapolation was used in the derivation of the cancer risk estimates. 25 

### In the absence of appropriate benzo[a]pyrene-specif ic data to adjust cancer risk values for 26 

### early life exposure, ADAFs combined with age-specif ic exposure estimates should be applied to 27 

### the cancer risk values (oral slope factor, inhalati on unit risk, and dermal slope factor) when 28 

### assessing cancer risks for individuals exposed duri ng early life periods, as per U.S. EPA (2005b) 29 

### Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens

. 30

31



### 6.2.3.1. Cancer

— 

### Oral 32 

### Lifetime oral exposure to benzo[a]pyrene has been a ssociated with forestomach, liver, 33 

### oral cavity, jejunum or duodenum, and auditory cana l tumors in male and female Wistar rats 34 

### (Kroese et al., 2001), with forestomach tumors in m ale and female Sprague-Dawley rats (Brune 35 

### et al., 1981), and with forestomach, esophagus, ton gue, and larynx tumors in female B6C3F1 36 

### mice (male mice were not tested); (Beland and Culp, 1998; Culp et al., 1998). Less-than-lifetime 37 

### oral exposure to benzo[a]pyrene is also associated with forestomach tumors in more than 10 38

### 248 DRAFT - DO NOT CITE OR QUOTE

additional bioassays with several strains of mice ( see Section 4.2.1.2.). Both the rat bioassay by 1 

### Kroese et al. (2001) and the mouse bioassay by Bela nd and Culp (1998) included histological 2 

### examinations for tumors, three exposure levels and controls, and 50 animals per dose group. The 3 

### chronic studies by Kroese et al. and Beland and Cul p studies were therefore selected for dose4 

### response analysis. 5 

### EPA used the multistage-Weibull model in the deriva tion of the oral slope factor because 6 

### it incorporates the time at which death-with-tumor occurred and can account for differences in 7 

### mortality observed between the exposure groups in t he rat bioassay. Using linear extrapolation 8



### from the BMDL 10 

### , human equivalent oral slope factors were derived for each gender/tumor site 9 

### combination (slope factor = 0.1/BMDL 10 

### ). The oral slope factor of 1 per mg/kg-day is based on 10 

### the tumor response in the alimentary tract (foresto mach, esophagus, tongue, larynx) of female 11



### B6C3F 1 

### mice exposed to benzo[a]pyrene in the diet for 2 y ears (Beland and Culp, 1998). The 12 

### slope factor was derived by linear extrapolation fr om a human equivalent BMDL 10 

### of 0.07 13 

### mg/kg-day for forestomach, esophagus, tongue, and l arynx papillomas or carcinomas. The 14 

### recommended slope factor was selected as the factor with the highest value among a range of 15 

### slope factors derived from tumor responses at sever al sites in the 2-year male and female Wistar 16 

### rat bioassay by Kroese et al. (2001) and the 2-year female B6C3F 1 

### mouse bioassay by Beland 17 

### and Culp (1998). 18

19



### 6.2.3.2. Cancer

— 

### Inhalation 20 

### Inhalation exposure to benzo[a]pyrene was associate d with squamous cell neoplasia in 21 

### the larynx, pharynx, trachea, esophagus, and forest omach, of male Syrian golden hamsters 22 

### exposed to benzo[a]pyrene condensed onto NaCl parti cles (Thyssen et al., 1981). Supportive 23 

### evidence for the carcinogenicity of inhaled benzo[a ]pyrene comes from 10 additional studies 24 

### with hamsters exposed to benzo[a]pyrene via intratr acheal instillation (see Section 4.2.2.2 for 25 

### references). The Thyssen et al. (1981) bioassay re presents the best available data for describing 26 

### dose-response relationships for cancer from inhaled benzo[a]pyrene. 27 

### A time-to-tumor dose-response model was fit to the time-weighted average exposure 28



### concentrations and the individual animal occurrence data for tumors in the larynx, pharynx, 29 

### trachea, esophagus, and forestomach. The inhalatio n unit risk of 5 x 10 -4 

### per μg/m 3 

### was 30 

### calculated by linear extrapolation (slope factor = 0.1/BMDL 10 

### ) from a BMDL 10 

### of 0.20 mg/m 3 31 

### for the occurrence of upper respiratory and upper d igestive tract tumors in male hamsters 32 

### chronically exposed by inhalation to benzo[a]pyrene (Thyssen et al., 1981). 33

34



### 6.2.3.3. Cancer

— 

### Dermal 35



### Skin cancer in humans has been documented to result from occupational exposure to 36 

### complex mixtures of PAHs including benzo[a]pyrene s uch as coal tar, coal tar pitches, non37 

### refined mineral oils, shale oils and soot (Boffetta et al., 1997; WHO, 1998; ATSDR, 1995). No 38

### 249 DRAFT - DO NOT CITE OR QUOTE

human studies of exposure to benzo[a]pyrene alone a re known to exist. In animal models, 1 

### numerous dose response studies have demonstrated th e increased incidence of skin tumors with 2 

### increasing dermal exposure of benzo[a]pyrene, in al l species tested (mice, rabbits, rats, and 3 

### guinea pigs), though most benzo[a]pyrene chronic de rmal bioassays have been conducted in 4 

### mice. This analysis focuses on chronic carcinogeni city bioassays in several strains of mice 5 

### demonstrating increasing incidence of benign and ma lignant skin tumors following repeated 6 

### dermal exposure to benzo[a]pyrene for the animals’ lifetime. 7 

### As with the oral and inhalation benzo[a]pyrene carc inogenicity data (see Sections 5.4.1.3 8 

### and 5.4.2.3), benzo[a]pyrene’s dermal exposure carc inogenicity data were generally 9 

### characterized by earlier occurrence of tumors with increasing exposure and increased mortality 10 

### with increasing exposure level. Each of the dermal data sets was modeled using the multistage 11 

### model, incorporating adjustments for early mortalit y, when data were available, prior to 12 

### modeling. 13 

### The POD of 0.0066 μg/day, based on the tumor respon se in C3H/HeJ male mice (Sivak 14 

### et al., 1997), is recommended for developing a huma n dermal slope factor because it is the 15 

### highest POD among studies with low observed tumor r esponse to benzo[a]pyrene (20%). 16 

### Following the modeling, this POD from the Sivak et al. (1997) dataset in male C3H/HeJ mice 17 

### was adjusted by allometric scaling. The dermal slo pe factor of 0.005 per μg/day was calculated 18 

### by linear extrapolation (slope factor = 0.1/BMDL 10-HED 

### ) from the human equivalent POD (19.7 19 

### μg/day) for the occurrence of skin tumors in male m ice chronically exposed dermally to 20 

### benzo[a]pyrene (Sivak et al., 1997). 21 

### This dermal slope factor has been calculated based on the risk of skin tumors in mice 22 

### following dermal exposure to benzo[a]pyrene. As th is slope factor has been developed for a 23 

### local effect, it is not intended to estimate system ic risk of cancer following dermal absorption of 24 

### benzo[a]pyrene into the systemic circulation. 25

26

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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC 1



### COMMENTS AND DISPOSITION 2

3

4 

### [THIS PAGE INTENTIONALLY LEFT BLANK] 5

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APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR NO NCANCER 1

2 

### Increased liver weight (Kroese et al, 2001) male 3

4

5 

### Table B-1. Liver weight (±SD) a 

### in male F344 rats administered 6 

### benzo[a]pyrene by gavage for 90 days 7 8

Organ Dose (mg/kg-d)

0 3 10 30 Liver weight (g) Males 7.49 ± 0.97 8.00 ± 0.85 8.62 ± 1.30 b 9.67 ± 1.17 b

a Reported as SE, but judged to be SD (and confirmed by study authors).

b Significantly (

p < 0.05) different from control mean; student t-tes t (unpaired, two-tailed); n = 10/sex/group.

9 10

11

12 

### Table B-2. BMD modeling results for increased live r weight in male rats, 13 

### with BMR=10% 14 15

Study Endpoint Model AIC Goodness-offit p value BMD BMDL Kroese et al., 2001 Liver weight Linear (1o polynomial), Power 49.51 0.58 8.11 5.8

Polynomial (2°) a 50.53 0.74 4.53 2.29 Hill 50.48 0.82 4.1 1.24

16

a In order to consider apparent curvature in the dos e-response data, the polynomial coefficients were a llowed to be 17 negative; a satisfactory fit was achieved, with mon otonically increasing predictions within the observ ed data range. 18 Since the AIC was higher than for the linear model, the 2-degree polynomial was not selected as the be st fit. 19 20

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1 2 3 

### Figure B-1. Fit of polynomial model to data on inc reased liver weight in 4 

### male Wistar rats—90 days. 5 6 7 Model output: 8 9

=================================================== ================= 10 Polynomial Model. (Version: 2.13; Date: 04/0 8/2008) 11 Input Data File: C:\USEPA\BMDS21\Data\linLiver wtKroeseLinearDefault.(d) 12 Gnuplot Plotting File: C:\USEPA\BMDS21\Data\l inLiverwtKroeseLinearDefault.plt 13 Tue Jan 12 13:09:58 2010 14

================================================== ================== 15 16 BMDS Model Run 17

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 18 19 The form of the response function is: 20 21 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 22 23 24 Dependent variable = Liver_wt 25 Independent variable = Dose 26 rho is set to 0 27 Signs of the polynomial coefficients are not res tricted 28 A constant variance model is fit 29 30 Total number of dose groups = 4 31 Total number of records with missing values = 0 32 Maximum number of iterations = 250 33 Relative Function Convergence has been set to: 1 e-008 34 Parameter Convergence has been set to: 1e-008 35 36 37 38 Default Initial Parameter Values 39 alpha = 1.18058 40 6.5 7 7.5 8 8.5 9 9.5 10 10.5

0 5 10 15 20Mean Response

dose Linear Model with 0.95 Confidence Level

13:09 01/12 2010 BMD BMDL Linear

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rho = 0 Sp ecified 1 beta_0 = 7.71695 2 beta_1 = 0.0951703 3 4 5 Asymptotic Correlation Matrix of Paramet er Estimates 6 7 ( *** The model parameter(s) -rho 8 have been estimated at a boundary point, or have been specified by the user, 9 and do not appear in the correlati on matrix ) 10 11 alpha beta_0 beta_1 12 13 alpha 1 -3.6e-011 1e-010 14 15 beta_0 -3.6e-011 1 -0.68 16 17 beta_1 1e-010 -0.68 1 18 19 20 21 Parameter Estimate s 22 23 95.0% Wald Confidence Interval 24 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 25 alpha 1.09179 0.244132 0.613302 1.57028 26 beta_0 7.71695 0.224102 7.27772 8.15618 27 beta_1 0.0951703 0.0197929 0.0563769 0.133964 28 29 30 31 Table of Data and Estimated Values of Interest 32 33 Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled Res. 34

------ --- -------- -------- ---------- - ----------- ---------- 35 36

0 10 7.49 7.72 0.97 1.04 -0.687 37

2.1 10 8 7.92 0.85 1.04 0.252 38 7.1 10 8.62 8.39 1.3 1.04 0.688 39

21.4 10 9.67 9.75 1.17 1.04 -0.253 40 41 42 43 Model Descriptions for likelihoods calculated 44 45 46 Model A1: Yij = Mu(i) + e(ij) 47 Var{e(ij)} = Sigma^2 48 49 Model A2: Yij = Mu(i) + e(ij) 50 Var{e(ij)} = Sigma(i)^2 51 52 Model A3: Yij = Mu(i) + e(ij) 53 Var{e(ij)} = Sigma^2 54 Model A3 uses any fixed variance parameters th at 55 were specified by the user 56 57 Model R: Yi = Mu + e(i) 58 Var{e(i)} = Sigma^2 59 60 61 Likelihoods of Interest 62 63 Model Log(likelihood) # Param's AIC 64

A1 -21.212822 5 52.425644 65

A2 -20.156688 8 56.313377 66 A3 -21.212822 5 52.425644 67 fitted -21.756413 3 49.512826 68 R -30.879511 2 65.759022 69 70 71 Explanation of Tests 72 73 Test 1: Do responses and/or variances differ amon g Dose levels? 74 (A2 vs. R) 75 Test 2: Are Variances Homogeneous? (A1 vs A2) 76 Test 3: Are variances adequately modeled? (A2 vs. A3) 77 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 78 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 79 80 Tests of Interest 81 82 Test -2*log(Likelihood Ratio) Test df p-value 83 84

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Test 1 21.4456 6 0. 001525 1

Test 2 2.11227 3 0.5494 2 Test 3 2.11227 3 0.5494 3

Test 4 1.08718 2 0.5807 4 5 The p-value for Test 1 is less than .05. There app ears to be a 6 difference between response and/or variances among the dose levels 7 It seems appropriate to model the data 8 9 The p-value for Test 2 is greater than .1. A homog eneous variance 10 model appears to be appropriate here 11 12 13 The p-value for Test 3 is greater than .1. The mod eled variance appears 14 to be appropriate here 15 16 The p-value for Test 4 is greater than .1. The mod el chosen seems 17 to adequately describe the data 18 19 20 Benchmark Dose Computation 21 22 Specified effect = 0.1 23 24 Risk Type = Relative risk 25 26 Confidence level = 0.95 27 28

BMD = 8.10857 29 30 31

BMDL = 5.80436 32 33 34 35 36 37

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1 

### Decreased thymus weight (males) Kroese et al., 2001 2

3 4 

### Table B-3. Means ± SD a 

### for thymus weight in male Wistar rats exposed to

benzo[a]pyrene by gavage 5 days/week for 90 days

Organ Dose (mg/kg-d)

0 3 10 30 Thymus weight (mg) Males 380 ± 60 380 ± 110 330 ± 60 270 ± 40 b

a Reported as SE, but judged to be SD (and confirmed by study authors).

b Significantly (

p < 0.05) different from control mean; student t-tes t (unpaired, two-tailed); n = 10/sex/group.

Source: Kroese et al. (2001).



### Table B-4. Model predictions for decreased thymus weight in male Wistar rats—90 days

Model Variance

p -value a Goodness-of- fit

p -value AIC BMD 1SD (mg/kg-d) BMDL 1SD

(mg/kg-d)

Constant variance

Linear 0.01 0.74 384.84 12.97 8.97

Nonconstant variance

Hill c NA Linear, Polynomial (2-degree), Power c 0.30 0.23 380.71 16.40 11.30

NA = not applicable, model failed;

5

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1 2 BMDs and BMDLs indicated are associated with a chan ge of 1 SD from the control, and are in 3 units of mg/kg-day. 4 5 Source: Kroese et al. (2001). 6 7 

### Figure B-2. Fit of linear model (nonconstant varia nce) to data on decreased 8 

### thymus weight in male Wistar rats—90 days. 9 10 Linear (nonconstant variance) 11 12 13 14

================================================== ================== 15 Polynomial Model. (Version: 2.13; Date: 04/0 8/2008) 16 Input Data File: 17 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\t hymusweight\male\durationadjusted\2 18 Linkrolin.(d) 19 Gnuplot Plotting File: 20 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\t hymusweight\male\durationadjusted\2 21 Linkrolin.plt 22

Thu Oct 15 15:33:37 2009 23

================================================== ================== 24 25

BMDS Model Run 26

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 27 28 The form of the response function is: 29 30 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 31 250

300 350 400 450

0 5 10 15 20Mean Response

dose Linear Model with 0.95 Confidence Level

15:33 10/15 2009 BMD BMDL Linear

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1 2 Dependent variable = mean 3 Independent variable = dose 4 The polynomial coefficients are restricted to be negative 5 The variance is to be modeled as Var(i) = exp(la lpha + log(mean(i)) * rho) 6 7 Total number of dose groups = 4 8 Total number of records with missing values = 0 9 Maximum number of iterations = 250 10 Relative Function Convergence has been set to: 1 e-008 11 Parameter Convergence has been set to: 1e-008 12 13 14 15 Default Initial Parameter Values 16 lalpha = 8.56121 17

rho = 0 18 beta_0 = 380.763 19 beta_1 = -5.3285 20 21 22 Asymptotic Correlation Matrix of Paramet er Estimates 23 24 lalpha rho beta_0 beta_1 25 26 lalpha 1 -1 0.048 -0.061 27 28

rho -1 1 -0.048 0.061 29 30 beta_0 0.048 -0.048 1 -0.84 31 32 beta_1 -0.061 0.061 -0.84 1 33 34 35 36 Parameter Estimate s 37 38 95.0% Wald Confidence 39

Interval 40 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 41 Limit 42 lalpha -18.8293 9.75429 -37.9473 43

0.288754 44

rho 4.66515 1.67581 1.38062 45

7.94967 46 beta_0 378.954 16.5291 346.558 47

411.351 48 beta_1 -5.14219 1.00497 -7.11189 - 49

3.17249 50 51 52 53 Table of Data and Estimated Values of Interest 54 55

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled Res. 56

------ --- -------- -------- ---------- - ----------- ---------- 57 58

0 10 380 379 60 84.3 0.0392 59

2.1 10 380 368 110 78.8 0.475 60

7.1 10 330 342 60 66.6 -0.591 61

21.4 10 270 269 40 37.9 0.0908 62 63 64 65 Model Descriptions for likelihoods calculated 66

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1 2 Model A1: Yij = Mu(i) + e(ij) 3 Var{e(ij)} = Sigma^2 4 5 Model A2: Yij = Mu(i) + e(ij) 6 Var{e(ij)} = Sigma(i)^2 7 8 Model A3: Yij = Mu(i) + e(ij) 9 Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 10 Model A3 uses any fixed variance parameters th at 11 were specified by the user 12 13 Model R: Yi = Mu + e(i) 14 Var{e(i)} = Sigma^2 15 16 17 Likelihoods of Interest 18 19 Model Log(likelihood) # Param's AIC 20

A1 -189.116991 5 388.233982 21

A2 -183.673279 8 383.346558 22

A3 -184.883626 6 381.767253 23 fitted -186.353541 4 380.707081 24

R -196.353362 2 396.706723 25 26 27 Explanation of Tests 28 29 Test 1: Do responses and/or variances differ amon g Dose levels? 30 (A2 vs. R) 31 Test 2: Are Variances Homogeneous? (A1 vs A2) 32 Test 3: Are variances adequately modeled? (A2 vs. A3) 33 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 34 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 35 36

Tests of Interest 37 38 Test -2*log(Likelihood Ratio) Test df p-value 39 40

Test 1 25.3602 6 0.0 002928 41

Test 2 10.8874 3 0 .01235 42

Test 3 2.42069 2 0.2981 43

Test 4 2.93983 2 0.2299 44 45 The p-value for Test 1 is less than .05. There app ears to be a 46 difference between response and/or variances among the dose levels 47 It seems appropriate to model the data 48 49 The p-value for Test 2 is less than .1. A non-homo geneous variance 50 model appears to be appropriate 51 52 The p-value for Test 3 is greater than .1. The mod eled variance appears 53 to be appropriate here 54 55 The p-value for Test 4 is greater than .1. The mod el chosen seems 56 to adequately describe the data 57 58 59 Benchmark Dose Computation 60 61 Specified effect = 1 62 63 Risk Type = Estimated standard deviation s from the control mean 64 65 Confidence level = 0.95 66

### 296 DRAFT - DO NOT CITE OR QUOTE

1

BMD = 16.4008 2 3 4

BMDL = 11.2965 5 6 7 8 9 10 11 12

### 297 DRAFT - DO NOT CITE OR QUOTE

Decreased thymus weight females (Kroese et al., 200 1) 1 2 3 

### Table B-5. Means ± SD a 

### for thymus weight in female Wistar rats exposed to

benzo[a]pyrene by gavage 5 days/week for 90 days

Organ Dose (mg/kg-d)

0 3 10 30 Thymus weight (mg) Females 320 ± 60 310 ± 50 300 ± 40 230 ± 30 b

a Reported as SE, but judged to be SD (and confirmed by study authors).

b Significantly (

p < 0.05) different from control mean; student t-tes t (unpaired, two-tailed); n = 10/sex/group.

Source: Kroese et al. (2001).

4 5 

### Table B-6. Model predictions for decreased thymus weight in female Wistar

rats—90 days

Model (constant variance) Variance

p -value a Means

p -value a AIC BMD 1SD

(mg/kg-d) BMDL 1SD

(mg/kg-d)

Hill b NA

Linear c 0.17 0.81 349.12 10.52 7.64 Polynomial (2-degree) c,d 0.17 0.77 350.80 13.29 7.77

Power b NA

a Values <0.10 fail to meet conventional goodness-of- fit criteria.

b Power restricted to ≥ 1.

c Coefficients restricted to be negative.

d Lowest degree polynomial with an adequate fit is re ported.

BMD/BMC = maximum likelihood estimate of the dose/c oncentration associated with the selected BMR; NA = not applicable; model failed to generate

Source: Kroese et al. (2001).

6

### 298 DRAFT - DO NOT CITE OR QUOTE

1 BMDs and BMDLs indicated are associated with a chan ge of 1 SD from the control, and are in 2 units of mg/kg-day. 3 4 Source: Kroese et al. (2001). 5 6 

### Figure B-3. Fit of linear model (constant variance ) to data on decreased 7 

### thymus weight in female Wistar rats—90 days. 8 9 10 11

================================================== ================== 12 Polynomial Model. (Version: 2.13; Date: 04/0 8/2008) 13 Input Data File: 14 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\t hymusweight\female\durationadjusted 15 \2Linkrolin.(d) 16 Gnuplot Plotting File: 17 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\t hymusweight\female\durationadjusted 18 \2Linkrolin.plt 19

Thu Oct 15 16:27:44 2009 20

================================================== ================== 21 22

BMDS Model Run 23

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 24 25 The form of the response function is: 26 27 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 28 29 30 Dependent variable = mean 31 200

220 240 260 280 300 320 340 360

0 5 10 15 20Mean Response

dose Linear Model with 0.95 Confidence Level

16:27 10/15 2009 BMD BMDL Linear

### 299 DRAFT - DO NOT CITE OR QUOTE

Independent variable = dose 1 rho is set to 0 2 The polynomial coefficients are restricted to be negative 3 A constant variance model is fit 4 5 Total number of dose groups = 4 6 Total number of records with missing values = 0 7 Maximum number of iterations = 250 8 Relative Function Convergence has been set to: 1 e-008 9 Parameter Convergence has been set to: 1e-008 10 11 12 13 Default Initial Parameter Values 14 alpha = 1 15 rho = 0 Sp ecified 16 beta_0 = 322.144 17 beta_1 = -4.2018 18 19 20 Asymptotic Correlation Matrix of Paramet er Estimates 21 22 ( *** The model parameter(s) -rho 23 have been estimated at a boundary point, or have been specified by 24 the user, 25 and do not appear in the correlati on matrix ) 26 27 alpha beta_0 beta_1 28 29 alpha 1 2.4e-008 -2.3e-008 30 31 beta_0 2.4e-008 1 -0.68 32 33 beta_1 -2.3e-008 -0.68 1 34 35 36 37 Parameter Estimate s 38 39 95.0% Wald Confidence 40

Interval 41 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 42 Limit 43 alpha 1954.92 437.134 1098.16 44

2811.69 45 beta_0 322.144 9.48287 303.558 46

340.73 47 beta_1 -4.2018 0.837537 -5.84334 - 48

2.56026 49 50 51 52 Table of Data and Estimated Values of Interest 53 54

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled Res. 55

------ --- -------- -------- ---------- - ----------- ---------- 56 57

0 10 320 322 60 44.2 -0.153 58

2.1 10 310 313 50 44.2 -0.237 59

7.1 10 300 292 40 44.2 0.55 60

21.4 10 230 232 30 44.2 -0.159 61 62 63 64 Model Descriptions for likelihoods calculated 65 66

### 300 DRAFT - DO NOT CITE OR QUOTE

1 Model A1: Yij = Mu(i) + e(ij) 2 Var{e(ij)} = Sigma^2 3 4 Model A2: Yij = Mu(i) + e(ij) 5 Var{e(ij)} = Sigma(i)^2 6 7 Model A3: Yij = Mu(i) + e(ij) 8 Var{e(ij)} = Sigma^2 9 Model A3 uses any fixed variance parameters th at 10 were specified by the user 11 12 Model R: Yi = Mu + e(i) 13 Var{e(i)} = Sigma^2 14 15 16 Likelihoods of Interest 17 18 Model Log(likelihood) # Param's AIC 19

A1 -171.357252 5 352.714504 20

A2 -168.857234 8 353.714467 21

A3 -171.357252 5 352.714504 22 fitted -171.562118 3 349.124237 23

R -181.324151 2 366.648303 24 25 26 Explanation of Tests 27 28 Test 1: Do responses and/or variances differ amon g Dose levels? 29 (A2 vs. R) 30 Test 2: Are Variances Homogeneous? (A1 vs A2) 31 Test 3: Are variances adequately modeled? (A2 vs. A3) 32 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 33 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 34 35

Tests of Interest 36 37 Test -2*log(Likelihood Ratio) Test df p-value 38 39

Test 1 24.9338 6 0.0 003512 40

Test 2 5.00004 3 0.1718 41

Test 3 5.00004 3 0.1718 42

Test 4 0.409733 2 0.8148 43 44 The p-value for Test 1 is less than .05. There app ears to be a 45 difference between response and/or variances among the dose levels 46 It seems appropriate to model the data 47 48 The p-value for Test 2 is greater than .1. A homog eneous variance 49 model appears to be appropriate here 50 51 52 The p-value for Test 3 is greater than .1. The mod eled variance appears 53 to be appropriate here 54 55 The p-value for Test 4 is greater than .1. The mod el chosen seems 56 to adequately describe the data 57 58 59 Benchmark Dose Computation 60 61 Specified effect = 1 62 63 Risk Type = Estimated standard deviation s from the control mean 64 65 Confidence level = 0.95 66

### 301 DRAFT - DO NOT CITE OR QUOTE

1

BMD = 10.5228 2 3 4

BMDL = 7.64037 5 6 7 8

### 302 DRAFT - DO NOT CITE OR QUOTE

Decreased thymus weight males (DeJong et al., 1999) 1 2 3 

### Table B-7. Means ± SD for thymus weight in male Wi star rats exposed to

benzo[a]pyrene by gavage 5 days/week for 35 days

4

Organ Dose (mg/kg-d)

0 3 10 30 90 Thymus weight (mg) (means; n = 7–8) 517 ± 47 472 ± 90 438 ± 64 a 388 ± 71 a 198 ± 65 a

5 6 

### Table B-8. Model predictions for decreased thymus weight in male Wistar

rats—35 days

Model (constant variance) Variance

p -value b Means

p -value b AIC BMD 1SD (mg/kg-d) BMDL 1SD

(mg/kg-d)

Hill c 0.50 0.42 382.91 11.15 6.19

Linear d , Polynomial (2-degree) d,e 0.50 0.52 381.41 14.41 11.58

Power d NA

a Number of animals was reported to be 7–8 per dose g roup, and was not specified for each group; forBMD modeling purposes, n = 8 was used.

b Values <0.10 fail to meet conventional goodness-of- fit criteria.

c Power restricted to ≥ 1.

d Coefficients restricted to be negative.

e Lowest degree polynomial with an adequate fit is re ported.

BMD/BMC = maximum likelihood estimate of the dose/c oncentration associated with the selected BMR; NA = not applicable; model failed to generate

Source: De Jong et al. (1999).

7 8

### 303 DRAFT - DO NOT CITE OR QUOTE

1 2 BMDs and BMDLs indicated are associated with a chan ge of 1 SD from the control, and are in 3 units of mg/kg-day. 4 5 Source: De Jong et al. (1999). 6

7 

### Figure B-4. Fit of linear model (constant variance ) to data on decreased 8 

### thymus weight in male Wistar rats—35 days. 9

10 Model Output: 11 12

=================================================== ================= 13 Polynomial Model. (Version: 2.13; Date: 04/0 8/2008) 14 Input Data File: 15 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\t hymusweightmale\durationadjusted\2L 16 indejlin.(d) 17 Gnuplot Plotting File: 18 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\t hymusweightmale\durationadjusted\2L 19 indejlin.plt 20

Mon Oct 19 04:31:24 2009 21

================================================== ================== 22 23

BMDS Model Run 24

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 25 26 The form of the response function is: 27 28 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 29 30 150

200 250 300 350 400 450 500 550

0 10 20 30 40 50 60Mean Response

dose Linear Model with 0.95 Confidence Level

04:31 10/19 2009 BMD BMDL Linear

### 304 DRAFT - DO NOT CITE OR QUOTE

1 Dependent variable = mean 2 Independent variable = dose 3 rho is set to 0 4 The polynomial coefficients are restricted to be negative 5 A constant variance model is fit 6 7 Total number of dose groups = 5 8 Total number of records with missing values = 0 9 Maximum number of iterations = 250 10 Relative Function Convergence has been set to: 1 e-008 11 Parameter Convergence has been set to: 1e-008 12 13 14 15 Default Initial Parameter Values 16 alpha = 1 17 rho = 0 Sp ecified 18 beta_0 = 489.769 19 beta_1 = -4.5927 20 21 22 Asymptotic Correlation Matrix of Paramet er Estimates 23 24 ( *** The model parameter(s) -rho 25 have been estimated at a boundary point, or have been specified by 26 the user, 27 and do not appear in the correlati on matrix ) 28 29 alpha beta_0 beta_1 30 31 alpha 1 -3.1e-009 -3.2e-009 32 33 beta_0 -3.1e-009 1 -0.62 34 35 beta_1 -3.2e-009 -0.62 1 36 37 38 39 Parameter Estimate s 40 41 95.0% Wald Confidence 42

Interval 43 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 44 Limit 45 alpha 4382.3 979.911 2461.71 46

6302.89 47 beta_0 489.769 13.3751 463.555 48

515.984 49 beta_1 -4.5927 0.438716 -5.45257 - 50

3.73283 51 52 53 54 Table of Data and Estimated Values of Interest 55 56

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled Res. 57

------ --- -------- -------- ---------- - ----------- ---------- 58 59

0 8 517 490 47 66.2 1.16 60

2.1 8 472 480 90 66.2 -0.347 61

7.1 8 438 457 64 66.2 -0.819 62

21.4 8 388 391 71 66.2 -0.149 63

64.3 8 198 194 65 66.2 0.151 64 65 66

### 305 DRAFT - DO NOT CITE OR QUOTE

1 Model Descriptions for likelihoods calculated 2 3 4 Model A1: Yij = Mu(i) + e(ij) 5 Var{e(ij)} = Sigma^2 6 7 Model A2: Yij = Mu(i) + e(ij) 8 Var{e(ij)} = Sigma(i)^2 9 10 Model A3: Yij = Mu(i) + e(ij) 11 Var{e(ij)} = Sigma^2 12 Model A3 uses any fixed variance parameters th at 13 were specified by the user 14 15 Model R: Yi = Mu + e(i) 16 Var{e(i)} = Sigma^2 17 18 19 Likelihoods of Interest 20 21 Model Log(likelihood) # Param's AIC 22

A1 -186.580733 6 385.161466 23

A2 -184.896632 10 389.793264 24

A3 -186.580733 6 385.161466 25 fitted -187.706565 3 381.413130 26

R -214.086904 2 432.173809 27 28 29 Explanation of Tests 30 31 Test 1: Do responses and/or variances differ amon g Dose levels? 32 (A2 vs. R) 33 Test 2: Are Variances Homogeneous? (A1 vs A2) 34 Test 3: Are variances adequately modeled? (A2 vs. A3) 35 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 36 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 37 38

Tests of Interest 39 40 Test -2*log(Likelihood Ratio) Test df p-value 41 42

Test 1 58.3805 8 <.0001 43

Test 2 3.3682 4 0.4982 44

Test 3 3.3682 4 0.4982 45

Test 4 2.25166 3 0.5218 46 47 The p-value for Test 1 is less than .05. There app ears to be a 48 difference between response and/or variances among the dose levels 49 It seems appropriate to model the data 50 51 The p-value for Test 2 is greater than .1. A homog eneous variance 52 model appears to be appropriate here 53 54 55 The p-value for Test 3 is greater than .1. The mod eled variance appears 56 to be appropriate here 57 58 The p-value for Test 4 is greater than .1. The mod el chosen seems 59 to adequately describe the data 60 61 62 Benchmark Dose Computation 63 64 Specified effect = 1 65 66

### 306 DRAFT - DO NOT CITE OR QUOTE

Risk Type = Estimated standard deviation s from the control mean 1 2 Confidence level = 0.95 3 4

BMD = 14.4139 5 6 7

BMDL = 11.577 8 9

### 307 DRAFT - DO NOT CITE OR QUOTE

Decreased Splenic B cells 1

2

3 

### Table B-9. Exposure-related effects in male Wistar rats exposed to

benzo[a]pyrene by gavage 5 days/week for 5 weeks

Effect Dose (mg/kg-d)

0 3 10 30 90 Spleen cell distribution (%) B cells T cells Th cells Ts cells 39± 4 40 ± 9 23 ± 7 24 ± 5 36 ± 2

48 ± 12 26 ± 7 26 ± 6 34 ± 3 a

40 ± 9 24 ± 5 24 ± 7 32 ± 4 a

36 ± 2 22 ± 4 19 ± 2 23 ± 4 a

44 ± 6 26 ± 4 27 ± 5

a Significantly (

p < 0.05) different from control mean. 4 5 Source: De Jong et al. (1999). 6

7

8 9 

### Table B-10. Model predictions for decreased spleen B-cells in male Wistar

rats—35 days

Model Variance

p -value a Means

p -value a AIC BMD 1SD

(mg/kg-d) BMDL 1SD

(mg/kg-d)

Constant variance

Hill b 0.30 0.18 146.18 10.24 5.31

Linear c , Polynomial (2-degree) c,d

, Power b 0.30 0.21 145.28 15.58 12.43

a Values <0.10 fail to meet conventional goodness-of- fit criteria.

b Power restricted to ≥ 1.

c Coefficients restricted to be negative.

d Lowest degree polynomial with an adequate fit is re ported.

BMD/BMC = maximum likelihood estimate of the dose/c oncentration associated with the selected BMR; NA = not applicable; model failed to generate these values

Source: De Jong et al. (1999).

10

### 308 DRAFT - DO NOT CITE OR QUOTE

1 BMDs and BMDLs indicated are associated with a chan ge of 1 SD from the control, and are in 2 units of mg/kg-day. 3 4 Source: De Jong et al. (1999). 5 6 

### Figure B-5. Fit of linear model to data on decreas ed spleen B-cells in male 7 

### Wistar rats—35 days. 8

9 Model Output: 10 11

================================================== ================== 12 Polynomial Model. (Version: 2.13; Date: 04/0 8/2008) 13 Input Data File: 14 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\s pleenBcell\durationadjusted\2Lindej 15 lin.(d) 16 Gnuplot Plotting File: 17 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\s pleenBcell\durationadjusted\2Lindej 18 lin.plt 19

Mon Oct 19 05:06:33 2009 20

================================================== ================== 21 22

BMDS Model Run 23

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 24 25 The form of the response function is: 26 27 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 28 29 30 20

25 30 35 40

0 10 20 30 40 50 60Mean Response

dose Linear Model with 0.95 Confidence Level

05:06 10/19 2009 BMD BMDL Linear

### 309 DRAFT - DO NOT CITE OR QUOTE

Dependent variable = mean 1 Independent variable = dose 2 rho is set to 0 3 The polynomial coefficients are restricted to be negative 4 A constant variance model is fit 5 6 Total number of dose groups = 5 7 Total number of records with missing values = 0 8 Maximum number of iterations = 250 9 Relative Function Convergence has been set to: 1 e-008 10 Parameter Convergence has been set to: 1e-008 11 12 13 14 Default Initial Parameter Values 15 alpha = 12.2 16 rho = 0 Sp ecified 17 beta_0 = 37.0148 18 beta_1 = -0.222068 19 20 21 Asymptotic Correlation Matrix of Paramet er Estimates 22 23 ( *** The model parameter(s) -rho 24 have been estimated at a boundary point, or have been specified by 25 the user, 26 and do not appear in the correlati on matrix ) 27 28 alpha beta_0 beta_1 29 30 alpha 1 -2.4e-009 3.8e-009 31 32 beta_0 -2.4e-009 1 -0.62 33 34 beta_1 3.8e-009 -0.62 1 35 36 37 38 Parameter Estimate s 39 40 95.0% Wald Confidence 41

Interval 42 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 43 Limit 44 alpha 11.9647 2.6754 6.72106 45

17.2084 46 beta_0 37.0148 0.698873 35.6451 47

38.3846 48 beta_1 -0.222068 0.0229237 -0.266997 - 49

0.177138 50 51 52 53 Table of Data and Estimated Values of Interest 54 55

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled Res. 56

------ --- -------- -------- ---------- - ----------- ---------- 57 58

0 8 39 37 4 3.46 1.62 59

2.1 8 36 36.5 2 3.46 -0.449 60

7.1 8 34 35.4 3 3.46 -1.18 61

21.4 8 32 32.3 4 3.46 -0.215 62

64.3 8 23 22.7 4 3.46 0.216 63 64 65 66

### 310 DRAFT - DO NOT CITE OR QUOTE

Model Descriptions for likelihoods calculated 1 2 3 Model A1: Yij = Mu(i) + e(ij) 4 Var{e(ij)} = Sigma^2 5 6 Model A2: Yij = Mu(i) + e(ij) 7 Var{e(ij)} = Sigma(i)^2 8 9 Model A3: Yij = Mu(i) + e(ij) 10 Var{e(ij)} = Sigma^2 11 Model A3 uses any fixed variance parameters th at 12 were specified by the user 13 14 Model R: Yi = Mu + e(i) 15 Var{e(i)} = Sigma^2 16 17 18 Likelihoods of Interest 19 20 Model Log(likelihood) # Param's AIC 21

A1 -67.358091 6 146.716182 22

A2 -64.934513 10 149.869025 23

A3 -67.358091 6 146.716182 24 fitted -69.639287 3 145.278575 25

R -93.795081 2 191.590163 26 27 28 Explanation of Tests 29 30 Test 1: Do responses and/or variances differ amon g Dose levels? 31 (A2 vs. R) 32 Test 2: Are Variances Homogeneous? (A1 vs A2) 33 Test 3: Are variances adequately modeled? (A2 vs. A3) 34 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 35 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 36 37

Tests of Interest 38 39 Test -2*log(Likelihood Ratio) Test df p-value 40 41

Test 1 57.7211 8 <.0001 42

Test 2 4.84716 4 0.3033 43

Test 3 4.84716 4 0.3033 44

Test 4 4.56239 3 0.2068 45 46 The p-value for Test 1 is less than .05. There app ears to be a 47 difference between response and/or variances among the dose levels 48 It seems appropriate to model the data 49 50 The p-value for Test 2 is greater than .1. A homog eneous variance 51 model appears to be appropriate here 52 53 54 The p-value for Test 3 is greater than .1. The mod eled variance appears 55 to be appropriate here 56 57 The p-value for Test 4 is greater than .1. The mod el chosen seems 58 to adequately describe the data 59 60 61 Benchmark Dose Computation 62 63 Specified effect = 1 64 65 Risk Type = Estimated standard deviation s from the control mean 66

### 311 DRAFT - DO NOT CITE OR QUOTE

1 Confidence level = 0.95 2 3

BMD = 15.5764 4 5 6

BMDL = 12.4286 7

8

9

### 312 DRAFT - DO NOT CITE OR QUOTE

Forestomach hyperplasia 1

2 

### All available dichotomous models in the EPA BMDS (v ersion 2.1) were fit to the 3



### incidence data shown in Table B-9 for forestomach h yperplasia in rats and mice orally exposed 4 

### to benzo[a]pyrene for 2 years (Kroese et al., 2001; Beland and Culp, 1998). In accordance with 5 

### U.S. EPA (2000) guidance, BMDs and BMDLs associated with an extra risk of 10% are 6



### calculated for all models. 7 

### Adequate model fit was judged by three criteria: g oodness-of-fit p -value ( p > 0.1), visual 8 

### inspection of the dose-response curve, and scaled r esidual at the data point (except the control) 9 

### closest to the predefined benchmark response (BMR). Among all of the models providing 10 

### adequate fit to the data, the lowest BMDL is select ed as the POD when the difference between 11



### the BMDLs estimated from these models are more than threefold; otherwise, the BMDL from 12



### the model with the lowest Akaike’s Information Crit erion (AIC) is chosen. If an adequate fit to 13 

### the data was not achieved using the protocol above for the full dataset, doses were dropped 14 

### (starting with the highest dose) until an adequate fit was achieved. 15

16 

### Table B-11. Dose-response data for forestomach hyp erplasia in Wistar rats

and B6C3F 1 

### rats orally exposed to benzo[a]pyrene for 2 years

Species, sex (reference) Administered dose (mg/kg-day)

0 3 10 30 Duration-adjusted dose (× 5/7)

0 2.1 7.14 21.4 Wistar rat, female (Kroese et al., 2001) 1/52 8/51 13/51 2/52 Wistar rat, male (Kroese et al., 2001) 2/50 8/52 8/ 52 0/52 Administered dose (mg/kg-day)

0 0.7 3.3 16.5

B6C3F 1 mice, female (Beland and Culp, 1998) 13/48 23/47 3 3/46 46/47

17 18 

### All data sets provided adequate descriptions of the dose-response relationship for 19 

### forestomach hyperplasia from chronic oral exposure to benzo[a]pyrene, but at the highest dose 20 

### level in rats, the incidence of hyperplasia was not increased. It is possible that the forestomach 21 

### hyperplasia observed following benzo[a]pyrene expos ure may be a precursor to the development 22 

### of forestomach tumors, but specific data supporting this conclusion are unavailable. Regardless, 23 

### the male and female data sets in rats (Kroese et al ., 2001) were modeled without the data from 24 

### the highest dose group due to the nonmontonic incre ase in response to increasing dose (Kroese et 25 

### al., 2001). 26

27

28 29

### 313 DRAFT - DO NOT CITE OR QUOTE

Table B-12. Summary of BMDs and BMDLs from the bes t fitting model forestomach hyperplasia—oral exposure

Endpoint/data Strain/species Exposure duration Best fitting

model BMD

(mg/kg-d) BMDL

(mg/kg-d) Reference

Forestomach hyperplasia (highest dose excluded) a Wistar rat (male) 2 yrs Log-logistic 5.31 2.39 Kro ese et al., 2001

Forestomach hyperplasia (highest dose excluded) a Wistar rat

(female) 2 yrs Log-logistic 2.15 1.35 Kroese et al., 2001

Forestomach hyperplasia B6C3F 1 mouse

(female) 2 yrs Log-logistic 0.33 0.12 Beland and Culp, 1998

a Data for the high-dose group were excluded in the m odeled datset due to the absence of an increase in incidence at 1 the high dose. 2 3 4 5 6 

### Forestomach hyperplasia- Male Wistar Rats, 2 yrs (K roese et al., 2001) 7 8 9 

### Table B-13. Model predictions for forestomach hype rplasia in male Wistar

rats in a 2-year study

Model Degrees of

freedom χ

2 χ

2 Goodnessof-fit p -value a AIC BMD 10 (mg/kg-d) BMDL 10

(mg/kg-d) Highest dose excluded b

Gamma c , Multistage d

, Weibull c 1 2.39 0.12 112.37 5.63 2.67

Logistic 1 2.83 0.09 112.93 7.25 4.35 LogLogistic 1 2.28 0.13 112.27 5.31 2.39 LogProbit 1 3.64 0.06 113.88 8.36 4.52

Probit 1 2.78 0.10 112.87 7.09 4.13

a Values <0.10 fail to meet conventional goodness-of- fit criteria.

b Data for the high-dose group were excluded in the m odeled datset due to the absence of an increase in incidence at the high dose; likely related to the statistically significantly increased incidence of forestomach tu mors in these animals.

c Power restricted to ≥ 1.

d Betas restricted to ≥ 0; lowest degree polynomial with an adequate fit is reported (1-degree polynomial).

Source: Kroese et al. (2001).

10 11

12

### 314 DRAFT - DO NOT CITE OR QUOTE

1 2 3 

### Figure B-6. Fit of log logistic model to data on f orestomach hyperplasia in 4 

### male Wistar rats in a 2-year study. 5 6

7

8 Model output: 9

10

================================================== ================== 11 Logistic Model. (Version: 2.12; Date: 05/16/2 008) 12 Input Data File: 13 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic \forestomachhyperplasia\male\durati 14 onadjusted\3Logkrolog.(d) 15 Gnuplot Plotting File: 16 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic \forestomachhyperplasia\male\durati 17 onadjusted\3Logkrolog.plt 18

Tue Oct 13 21:42:15 2009 19

================================================== ================== 20 21

BMDS Model Run 22

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 23 24 The form of the probability function is: 25 26 P[response] = background+(1-background)/[1+EXP(- intercept-slope*Log(dose))] 27 28 29 Dependent variable = incidence 30 Independent variable = dose 31 Slope parameter is restricted as slope >= 1 32 33 0 0.05 0.1 0.15 0.2 0.25 0.3

0 1 2 3 4 5 6 7Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

08:57 10/14 2009 BMDL BMD Log-Logistic

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Total number of observations = 3 1 Total number of records with missing values = 0 2 Maximum number of iterations = 250 3 Relative Function Convergence has been set to: 1 e-008 4 Parameter Convergence has been set to: 1e-008 5 6 7 8 User has chosen the log transformed model 9 10 11 Default Initial Parameter Values 12 background = 0.04 13 intercept = -3.27842 14 slope = 1 15 16 17 Asymptotic Correlation Matrix of Paramet er Estimates 18 19 ( *** The model parameter(s) -slope 20 have been estimated at a boundary point, or have been specified by 21 the user, 22 and do not appear in the correlati on matrix ) 23 24 background intercept 25 26 background 1 -0.7 27 28 intercept -0.7 1 29 30 31 32 Parameter Estimate s 33 34 95.0% Wald Confidence 35

Interval 36 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 37 Limit 38 background 0.0623861 * * * 39 intercept -3.86644 * * * 40 slope 1 * * * 41 42 * - Indicates that this value is not calculated. 43 44 45 46 Analysis of Deviance Table 47 48 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 49

Full model -53.0468 3 50 Fitted model -54.1335 2 2.1 7337 1 0.1404 51

Reduced model -55.5429 1 4.9 9229 2 0.0824 52 53

AIC: 112.267 54 55 56 Goodness of Fit 57

Scaled 58 Dose Est._Prob. Expected Observed Size Residual 59

------------------------------------------------- ----------------------- 60

0.0000 0.0624 3.119 2.000 50 -0.654 61

2.1000 0.1019 5.297 8.000 52 1.239 62

7.1400 0.1843 9.584 8.000 52 -0.566 63 64 Chi^2 = 2.28 d.f. = 1 P-value = 0.1306 65 66

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1 Benchmark Dose Computation 2 3 Specified effect = 0.1 4 5 Risk Type = Extra risk 6 7 Confidence level = 0.95 8 9

BMD = 5.308 10 11

BMDL = 2.38692 12 13

14

15 

### Forestomach hyperplasia- Female Wistar Rats, 2 yrs (Kroese et al., 2001) 16

17 

### Table B-14. Model predictions for forestomach hype rplasia in female

Wistar rats in a 2-year study

Model Degrees of

freedom χ

2 χ

2 Goodnessof-fit p -value a AIC BMD 10 (mg/kg-d) BMDL 10

(mg/kg-d) Highest dose excluded b Logistic 1 3.68 0.06 120.02 4.23 3.28 LogLogistic 1 0.98 0.32 117.04 2.15 1.35 LogProbit 1 5.09 0.02 121.13 3.91 2.57

Gamma c , Multistage d

, Weibull c 1 1.40 0.24 117.42 2.40 1.59

Probit 1 3.47 0.06 119.74 3.99 3.06

a Values <0.10 fail to meet conventional goodness-of- fit criteria.

b Data for the high-dose group were excluded in the m odeled datset due to the absence of an increase in incidence at the high dose; likely related to the statistically significantly increased incidence of forestomach tu mors in these animals.

c Power restricted to ≥ 1.

d Betas restricted to ≥ 0; lowest degree polynomial with an adequate fit is reported (1-degree polynomial).

BMD = maximum likelihood estimate of the dose/conce ntration associated with the selected BMR

Source: Kroese et al. (2001).

18

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1

2 

### Figure B-7. Fit of log logistic model to data on f orestomach hyperplasia in 3 

### female Wistar rats in a 2-year study. 4 5

6 Model output: 7

=================================================== ================= 8 Logistic Model. (Version: 2.12; Date: 05/16/2 008) 9 Input Data File: 10 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic \forestomachhyperplasia\female\dura 11 tionadjusted\3Logkrolog.(d) 12 Gnuplot Plotting File: 13 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic \forestomachhyperplasia\female\dura 14 tionadjusted\3Logkrolog.plt 15

Wed Oct 14 08:59:23 2009 16

================================================== ================== 17 18

BMDS Model Run 19

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 20 21 The form of the probability function is: 22 23 P[response] = background+(1-background)/[1+EXP(- intercept-slope*Log(dose))] 24 25 26 Dependent variable = incidence 27 Independent variable = dose 28 Slope parameter is restricted as slope >= 1 29 30 Total number of observations = 3 31 Total number of records with missing values = 0 32 Maximum number of iterations = 250 33 Relative Function Convergence has been set to: 1 e-008 34 Parameter Convergence has been set to: 1e-008 35 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

0 1 2 3 4 5 6 7Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

08:59 10/14 2009 BMDL BMD Log-Logistic

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1 2 3 User has chosen the log transformed model 4 5 6 Default Initial Parameter Values 7 background = 0.0192308 8 intercept = -2.7983 9 slope = 1 10 11 12 Asymptotic Correlation Matrix of Paramet er Estimates 13 14 ( *** The model parameter(s) -slope 15 have been estimated at a boundary point, or have been specified by 16 the user, 17 and do not appear in the correlati on matrix ) 18 19 background intercept 20 21 background 1 -0.46 22 23 intercept -0.46 1 24 25 26 27 Parameter Estimate s 28 29 95.0% Wald Confidence 30

Interval 31 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 32 Limit 33 background 0.0238694 * * * 34 intercept -2.96044 * * * 35 slope 1 * * * 36 37 * - Indicates that this value is not calculated. 38 39 40 41 Analysis of Deviance Table 42 43 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 44

Full model -56.048 3 45 Fitted model -56.5181 2 0.94 0072 1 0.3323 46

Reduced model -63.1579 1 14. 2198 2 0.000817 47 48

AIC: 117.036 49 50 51 Goodness of Fit 52

Scaled 53 Dose Est._Prob. Expected Observed Size Residual 54

------------------------------------------------- ----------------------- 55

0.0000 0.0239 1.241 1.000 52 -0.219 56

2.1000 0.1196 6.101 8.000 51 0.819 57

7.1400 0.2874 14.658 13.000 51 -0.513 58 59 Chi^2 = 0.98 d.f. = 1 P-value = 0.3216 60 61 62 Benchmark Dose Computation 63 64 Specified effect = 0.1 65 66

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Risk Type = Extra risk 1 2 Confidence level = 0.95 3 4

BMD = 2.14515 5 6

BMDL = 1.34776 7

8

9 

### Forestomach hyperplasia- female mice, 2 yrs (Beland and Culp, 1998) 10 

### Table B-15. Model predictions for forestomach hype rplasia in female

B6C3F1 mice in a 2-year study

Model Degrees of

freedom χ 2 χ

2 Goodnessof-fit p -value a AIC BMD 10 (mg/kg-d) BMDL 10

(mg/kg-d) Logistic 2 5.71 0.06 194.7 0.757 0.545 LogLogistic 2 1.55 0.21 193.3 0.329 0.115 LogProbit 2 2.49 0.29 192.1 0.670 0.448 Multistage c , Weibull b

, Gamma b 2 1.74 0.42 191.3 0.421 0.295

Probit 2 7.04 0.03 196.6 0.946 0.711

a Values <0.10 fail to meet conventional goodness-of- fit criteria.

b Power restricted to ≥ 1.

c Betas restricted to ≥ 0; lowest degree polynomial with an adequate fit is reported (1-degree polynomial).

BMD = maximum likelihood estimate of the dose/conce ntration associated with the selected BMR Source: Beland and Culp (1998).

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1 

### Figure B-8. Fit of log logistic model to data on f orestomach hyperplasia in 2 

### female B6C3F 1 

### mice in a 2-year study. 3 4 5 Model output: 6

7

================================================== ================== 8 Logistic Model. (Version: 2.12; Date: 05/16/2 008) 9 Input Data File: 10 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_BelandCulp_ mice_4s_hyperplasia_generic_dich_10 11 .(d) 12 Gnuplot Plotting File: 13 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_BelandCulp_ mice_4s_hyperplasia_generic_dich_10 14 .plt 15 16

================================================== ================== 17 18

BMDS Model Run 19

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 20 21 The form of the probability function is: 22 23 P[response] = background+(1-background)/[1+EXP(- intercept-slope*Log(dose))] 24 25 26 Dependent variable = NumAff 27 Independent variable = dose 28 Slope parameter is restricted as slope >= 1 29 30 Total number of observations = 4 31 Total number of records with missing values = 0 32 Maximum number of iterations = 250 33 Relative Function Convergence has been set to: 1 e-008 34 Parameter Convergence has been set to: 1e-008 35 36 37 38 User has chosen the log transformed model 39 0.2

0.4 0.6 0.8 1

0 2 4 6 8 10 12 14 16Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

BMDL BMD Log-Logistic

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1 2 Default Initial Parameter Values 3 background = 0.270833 4 intercept = -0.637972 5 slope = 1.38091 6 7 8 Asymptotic Correlation Matrix of Paramet er Estimates 9 10 background intercept slope 11 12 background 1 -0.66 0.46 13 14 intercept -0.66 1 -0.8 15 16 slope 0.46 -0.8 1 17 18 19 20 Parameter Estimate s 21 22 95.0% Wald Confidence 23

Interval 24 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 25 Limit 26 background 0.286381 * * * 27 intercept -0.789676 * * * 28 slope 1.26641 * * * 29 30 * - Indicates that this value is not calculated. 31 32 33 34 Analysis of Deviance Table 35 36 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 37

Full model -92.8312 4 38 Fitted model -93.6497 3 1.6 3686 1 0.2008 39

Reduced model -125.58 1 65. 4982 3 <.0001 40 41

AIC: 193.299 42 43 44 Goodness of Fit 45

Scaled 46 Dose Est._Prob. Expected Observed Size Residual 47

------------------------------------------------- ----------------------- 48

0.0000 0.2864 13.746 13.000 48 -0.238 49

0.7000 0.4464 20.979 23.000 47 0.593 50

3.3000 0.7667 35.270 33.000 46 -0.791 51

16.5000 0.9575 45.005 46.000 47 0.720 52 53 Chi^2 = 1.55 d.f. = 1 P-value = 0.2127 54 55 56 Benchmark Dose Computation 57 58 Specified effect = 0.1 59 60 Risk Type = Extra risk 61 62 Confidence level = 0.95 63 64

BMD = 0.329083 65 66

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BMDL = 0.115446 1

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Decreased ovary weight- female rats, 60 days (Xu e t al., 2010) 1

2 3 

### Table B-16. Means ± SDa for ovary weight in female SD-rats

Organ Dose (mg/kg-d) a

0 2.5 5

Ovary weight (mg) 0.160 ± 0.0146 0.143 ± 0.0098 b 0.136 ± 0.0098 b

a TWA doses over the 60 day study period

b Statistically different (p < 0.05) from controls u sing one-way ANOVA



### Table B-17. Model predictions for decreased ovary weight in female SDrats—60 days

Model Goodness-of-fit

p -value AIC BMD 1SD (mg/kg-d) BMDL 1SD

(mg/kg-d)

Power N/A

Linear, Polynomial (1°) 0.39 -138.67 2.27 1.49

NA = not applicable, model failed; 4 5

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1 2 

### Figure B-9. Fit of linear/polynomial (1°) model to data on decreased ovary 3 

### weight 4 5 6 7 Model Output: 8 9

=================================================== ================= 10 Polynomial Model. (Version: 2.16; Date: 05/2 6/2010) 11 Input Data File: 12 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWe ight/Xu2010_AbsOvaryWeight_Linear_1 13 SD.(d) 14 Gnuplot Plotting File: 15 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWe ight/Xu2010_AbsOvaryWeight_Linear_1 16 SD.plt 17

Tue Dec 14 13:51:32 2010 18

================================================== ================== 19

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 20 21 The form of the response function is: 22 23 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 24 25 26 Dependent variable = Mean 27 Independent variable = Dose 28 rho is set to 0 29 Signs of the polynomial coefficients are not res tricted 30 A constant variance model is fit 31 32 Total number of dose groups = 3 33 0.13

0.14 0.15 0.16 0.17 0.18

0 1 2 3 4 5Mean Response

dose Linear Model with 0.95 Confidence Level

16:03 12/14 2010 BMD BMDL Linear

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Total number of records with missing values = 0 1 Maximum number of iterations = 250 2 Relative Function Convergence has been set to: 1 e-008 3 Parameter Convergence has been set to: 1e-008 4 5 6 7 Default Initial Parameter Values 8 alpha = 0.000136 9 rho = 0 Sp ecified 10 beta_0 = 0.158333 11 beta_1 = -0.0048 12 13 14 Asymptotic Correlation Matrix of Paramet er Estimates 15 16 ( *** The model parameter(s) -rho 17 have been estimated at a boundary point, or have been specified by 18 the user, 19 and do not appear in the correlati on matrix ) 20 21 alpha beta_0 beta_1 22 23 alpha 1 4e-010 -4.5e-010 24 25 beta_0 4e-010 1 -0.77 26 27 beta_1 -4.5e-010 -0.77 1 28 29 30 31

Parameter Estimate s 32 33

95.0% Wald Confidence Interval 34

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 35 alpha 0.000118889 3.96296e-005 4.12162e-005 0.000196562 36 beta_0 0.158333 0.00406354 0.150369 0.166298 37 beta_1 -0.0048 0.00125904 -0.00726768 -0.00233232 38 39 40 41 Table of Data and Estimated Values of Interest 42 43

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled Res. 44

------ --- -------- -------- ---------- - ----------- ---------- 45 46

0 6 0.16 0.158 0.0147 0.0109 0.374 47

2.5 6 0.143 0.146 0.0098 0.0109 -0.749 48

5 6 0.136 0.134 0.0098 0.0109 0.374 49 50 51 52 Model Descriptions for likelihoods calculated 53 54 55 Model A1: Yij = Mu(i) + e(ij) 56 Var{e(ij)} = Sigma^2 57 58 Model A2: Yij = Mu(i) + e(ij) 59 Var{e(ij)} = Sigma(i)^2 60 61 Model A3: Yij = Mu(i) + e(ij) 62 Var{e(ij)} = Sigma^2 63 Model A3 uses any fixed variance parameters th at 64 were specified by the user 65 66 Model R: Yi = Mu + e(i) 67 Var{e(i)} = Sigma^2 68

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1 2 Likelihoods of Interest 3 4 Model Log(likelihood) # Param's AIC 5

A1 72.766595 4 -137.533190 6

A2 73.468565 6 -134.937129 7

A3 72.766595 4 -137.533190 8 fitted 72.335891 3 -138.671782 9

R 67.008505 2 -130.017010 10 11 12 Explanation of Tests 13 14 Test 1: Do responses and/or variances differ amon g Dose levels? 15 (A2 vs. R) 16 Test 2: Are Variances Homogeneous? (A1 vs A2) 17 Test 3: Are variances adequately modeled? (A2 vs. A3) 18 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 19 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 20 21

Tests of Interest 22 23 Test -2*log(Likelihood Ratio) Test df p-value 24 25

Test 1 12.9201 4 0 .01167 26

Test 2 1.40394 2 0.4956 27

Test 3 1.40394 2 0.4956 28

Test 4 0.861408 1 0.3533 29 30 The p-value for Test 1 is less than .05. There app ears to be a 31 difference between response and/or variances among the dose levels 32 It seems appropriate to model the data 33 34 The p-value for Test 2 is greater than .1. A homog eneous variance 35 model appears to be appropriate here 36 37 38 The p-value for Test 3 is greater than .1. The mod eled variance appears 39 to be appropriate here 40 41 The p-value for Test 4 is greater than .1. The mod el chosen seems 42 to adequately describe the data 43 44 45 Benchmark Dose Computation 46 47 Specified effect = 1 48 49 Risk Type = Estimated standard deviation s from the control mean 50 51 Confidence level = 0.95 52 53

BMD = 2.27159 54 55 56

BMDL = 1.49968 57 58

59

### APPENDIX C. ADDITIONAL CALCULATIONS FOR THE RfC

1

2

3

4 5 6 7 

### Figure C- 1. Human Fractional Deposition 8 9 10 11 Species = humanlimited 12 FRC = 3300.0 13 Head volume = 50.0 14 Density = 1.0 15 Number of particles calculated = single 16 Diameter = 1.7000000000000 002 μm MMAD 17 Inhalability = yes 18 GSD = 1.0 19 Breathing interval: One single breath 20 Concentration = 4.2 21 Breathing Frequency = 16.0 22 Tidal Volume = 860.0 23 Inspiratory Fraction = 0.5 24 Pause Fraction = 0.0 25 Breathing Route = nasal 26 27 Region: Entire Lung 28 Region: Entire Lung 29 Region Deposition Fraction 30 

### 327 DRAFT - DO NOT CITE OR QUOTE APPENDIX C. ADDITIONAL CALCULATIONS FOR THE RfC

1. Human Fractional Deposition

Number of particles calculated = single 002 μm MMAD

Breathing interval: One single breath



### DO NOT CITE OR QUOTE

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-- -- 1 Head 0.449 2

TB 0.045 3

P 0.127 4 Total 0.621 5

61

2 

### Figure C- 2. Rat Fractional Deposition 3 4 5 Species = rat 6 FRC = 4.0 7 Head volume = 0.42 8 Density = 1.0 9 Number of particles calculated = single 10 Diameter = 1.700000000000 0002 μm MMAD 11 Inhalability = yes 12 GSD = 1.0 13 Breathing interval: One single breath 14 Concentration = 4.2 15 Breathing Frequency = 102.0 16 Tidal Volume = 1.8 17 Inspiratory Fraction = 0.5 18 Pause Fraction = 0.0 19 Breathing Route = nasal 20 21 Region: Entire Lung 22 Region: Entire Lung 23 Region Deposition Fraction 24

-- -- 25 Head 0.072 26

TB 0.041 27

P 0.068 28 Total 0.181 29



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2. Rat Fractional Deposition

Number of particles calculated = single 0002 μm MMAD

Breathing interval: One single breath

Breathing Frequency = 102.0

Deposition Fraction



### DO NOT CITE OR QUOTE

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APPENDIX D. TIME-TO-TUMOR MODELING FOR THE ORAL SL OPE FACTOR

Table D-1. Tumor incidence data, with time to death with tumor; male rats exposed by gavage to Benzo[a]pyrene (Kroese et al., 2001)

Dose Group (mg/kg-day) Week of

Death Total

Examined Numbers of Animals with

Oral Cavity or Forestomach Tumors Liver Tumors Duodenum or Jejunum Tumors Skin or Mammary

Gland Kidney

Urothelial Carcinoma Basal Cell Tumors Squamous

Cell Tumors

Incid. a Fatal a Incid. Fatal Incid. Incid. Incid. Incid.

0 44

80 82 84 89 90 91 92 93 94 95 96 97 98 100 104 105 108 109 1

1 1 1 1 3 1 1 1 1 2 2 1 1 3 1 1 7 22 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3 29

40 74 76 79 82 92 93 94 95 98 107 108 109 1

1 1 1 1 1 2 1 1 2 1 10 15 14 0

1 0 0 0 0 0 0 0 0 0 4 2 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 1 3 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 1 0 0

0 0 0 0 0 0 0 0 0 0 0 1 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

10 39

47 63 68 69 77 80 81 84 86 90 95 97 100 102 103 104 107 108 109 1

2 1 2 1 1 1 1 1 1 1 3 1 1 1 1 3 12 11 6 0

0 1 2 1 0 0 1 1 0 1 3 1 1 1 1 3 12 11 5 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 1 1 0 0 1 0 2 0 1 1 1 3 11 11 3 0

0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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Table D-1. Tumor incidence data, with time to death with tumor; male rats exposed by gavage to Benzo[a]pyrene (Kroese et al., 2001)

Dose Group (mg/kg-day) Week of

Death Total

Examined Numbers of Animals with

Oral Cavity or Forestomach Tumors Liver Tumors Duodenum or Jejunum Tumors Skin or Mammary

Gland Kidney

Urothelial Carcinoma Basal Cell Tumors Squamous

Cell Tumors

Incid. a Fatal a Incid. Fatal Incid. Incid. Incid. Incid.

30 32

35 37 44 45 47 48 49 50 51 52 53 56 58 59 60 61 62 63 64 65 66 67 68 70 71 73 76 1

1 1 1 2 1 1 1 1 1 4 1 2 2 2 2 3 5 5 2 3 1 3 1 2 1 1 1 1

1 1 0 2 1 1 1 1 1 3 1 1 2 2 1 2 5 5 2 2 1 1 1 2 1 0 1 0

0 0 1 0 0 0 0 0 0 1 0 1 0 0 1 1 0 0 0 1 0 2 0 0 0 1 0 0

1 0 1 2 1 1 1 1 1 3 1 1 2 2 1 1 0 4 1 1 0 2 1 1 1 1 0 0

0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 2 4 1 1 2 1 1 0 1 0 0 1 0

0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 3 1 0 0 0 1 0 1 0 0 0 0

0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 2 0 3 0 1 0 1 1 1 1 0

0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 2 0 1 0 0 1 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 0 0 0 0 0 0 0 0

a Incidental, denotes presence of tumors not known t o have caused death of particular animals. “Fatal” denotes incidence of tumors reported by the study investigators to have caused death of par ticular animals.

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Table D-2. Tumor incidence data, with time to death with tumor; female rats exposed by gavage to Benzo[a]pyrene (Kroese et al., 2001)

Dose Group (mg/kg-day) Week

of Death Total Examined Numbers of Animals with

Oral Cavity or Forestomach

Tumors Liver Tumors Duodenum or Jejunum Tumors

Incidental a Fatal a Incidental Fatal Incidental

0 64

69 75 104 106 107 108 109 1

1 1 1 4 7 7 30 0

0 0 0 0 0 0 1 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0

3 8

47 52 60 65 76 77 83 85 86 88 93 94 97 107 108 109 1

1 1 1 1 1 1 2 1 1 1 2 1 1 6 9 21 0

0 0 0 0 0 0 0 0 0 0 0 0 1 2 2 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

10 42

43 44 45 48 55 59 75 76 77 80 81 82 83 85 86 87 88 89 91 95 96 98 99 102 104 105 106 107 108 109 1

1 1 1 1 1 1 1 2 2 1 1 1 1 2 1 1 2 1 1 1 1 2 3 1 1 2 1 5 7 4 0

0 0 0 0 0 0 0 0 0 1 1 1 0 1 1 0 1 1 0 0 0 2 3 1 1 1 1 5 7 2 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 1 0 1 1 0 1 0 1 1 1 0 1 1 0 0 0 0 1 1 0 1 1 0 5 7 2 0

0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 0 1 1 1 0 0 1 2 1 0 1 1 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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Table D-2. Tumor incidence data, with time to death with tumor; female rats exposed by gavage to Benzo[a]pyrene (Kroese et al., 2001)

Dose Group (mg/kg-day) Week

of Death Total Examined Numbers of Animals with

Oral Cavity or Forestomach

Tumors Liver Tumors Duodenum or Jejunum Tumors

Incidental a Fatal a Incidental Fatal Incidental

30 26

44 47 48 54 55 56 57 58 59 60 61 62 63 64 66 67 68 69 71 72 1

4 3 1 1 3 2 2 4 2 1 2 2 3 5 3 2 1 4 4 2 0

4 3 1 0 3 2 2 3 1 0 2 2 3 5 3 1 1 3 3 1 0

0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 1 0 1 1 1 0

3 2 0 1 1 0 2 0 0 1 0 1 0 0 0 0 0 1 1 0 0

1 1 1 0 2 2 0 4 2 0 2 1 3 5 3 2 1 3 3 2 0

0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 1 0 0

a “Incidental” denotes presence of tumors not known to have caused death of particular animals. “Fatal” denotes incidence of tumors indicated by the study investigators to have caused death of par ticular animals.

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Table D-3. Tumor incidence data, with time to death with tumor; female mice exposed to Benzo[a]pyrene via diet (Beland and Culp, 1998)

Dose Group (ppm in diet) Week

of Death Total

Examined Number of Animals With Alimentary

Tract squamous cell tumors

Fatal a Incidental

0 31

74 89 91 93 94 97 98 99 100 101 104 105 1

1 2 1 2 2 2 2 1 2 2 1 29 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

5 25

55 83 86 87 88 90 94 95 96 97 98 101 102 105 1

1 1 1 2 2 1 1 2 1 2 2 2 2 27 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

25 44

47 64 70 77 80 81 84 85 86 88 89 90 93 94 96 97 98 99 100 101 102 104 105 1

1 1 1 1 1 1 2 1 1 1 1 4 3 2 3 1 1 2 1 1 2 1 13 1

0 0 1 1 0 1 1 1 1 1 0 4 2 2 0 1 1 1 1 0 2 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 2 0 0 1 0 0 0 0 10

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Table D-3. Tumor incidence data, with time to death with tumor; female mice exposed to Benzo[a]pyrene via diet (Beland and Culp, 1998)

Dose Group (ppm in diet) Week

of Death Total

Examined Number of Animals With Alimentary

Tract squamous cell tumors

Fatal a Incidental

100 39

40 42 47 49 50 53 55 56 57 58 59 60 61 62 63 64 65 66 68 69 70 71 72 73 74 79 1

1 1 2 1 1 1 3 1 1 1 3 1 3 5 4 3 2 3 1 2 2 1 1 1 1 1 1

1 1 2 0 1 0 3 1 1 1 3 1 3 5 4 3 2 3 1 2 2 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

a “Incidental” denotes presence of tumors not known to have caused death of particular animals. “Fatal” denotes incidence of tumors indicated by the study investigators to have caused death of par ticular animals.

### 336 DRAFT - DO NOT CITE OR QUOTE Table D-4. Derivation of HEDs to use for BMD modeling of Wistar rat tumor incidence data from Kroese et al. (2001)

benzo[a]pyrene dose (mg/kg-d) TWA body weight (kg) Interspecies

Scaling factor a HED b

(mg/kg-d)

Male

3 0.349 0.27 0.54

10 0.349 0.27 1.81

30 0.288 0.25 5.17

Female

3 0.222 0.24 0.49

10 0.222 0.24 1.62

30 0.222 0.24 4.85

a Scaling factors were calculated using U.S. EPA (19 88) reference body weights for humans (70 kg), and the TWA body weights for each d ose group: rat-to-human = (TWA body weight/70) 0.25 = scaling factor.

b HED = administered dose × scaling factor.



### Table D-5. Derivation of HEDs for BMD modeling of B6C3F1 female mouse tumor incidence data from Beland and Culp (1998)

benzo[a]pyrene dose in diet (ppm) Intake ( μ g/d) TWA body weight average (kg) Administered

Dose a (mg/kg-d) Scaling factor b HED c (mg/kg-d)

5 21 0.032 0.7 0.15 0.10

25 104 0.032 3.3 0.15 0.48

100 430 0.027 16.5 0.14 2.32

a Administered doses in mg/kg/day were calculated fro m dietary concentrations of benzo[a]pyrene using th e TWA body weight and reported food intakes for mice.

b Scaling factors were calculated using U.S. EPA (19 88) reference body weights for humans (70 kg), and the TWA body weights for each dose group: mouse-to-human = (TWA body weight/70) 0.25 = scaling factor. HED = administered dose × scaling factor.

c HED = administered dose × scaling factor337 DRAFT - DO NOT CITE OR QUOTETable D-6.Summary of Model Selection and Modeling Results forbest-fitting multistage-Weibull models, usingtime-to-tumor data for rats (Kroese et al., 2001)SexEndPointsModelstages LLaχ2bNumberofparam.AIC BMD10Responses at mg/kg-d levelscSelected modelparameterestimatesModel Selection Rationale0 0.54 1.8 5.2 c t0MalesOral Cavity andForestomach:Squam. CellTumors0 8 45 521 -284.891NR 4 577.80.104 0.021.139.838.72.2442 -198.795172.25 407.60.678 0.03.325.448.01.3443 -108.512180.66 229.00.453 0.06.841.750.03.741Lowest AIC, best fit to low dose dataHepatocellularTumors0 4 38 491 -179.664NR 4 367.30.181 013.632.1421522 -145.74967.85 301.50.472 0.06.537.2492.348.43 -138.54414.46 289.10.651 0.03.436.849.63.540.2Lowest AIC, best fit to low dose dataDuodenum andJejunumTumors0 0 1 91 -31.781NR 3 69.62.64 0.01.13.45.71NR2 -28.9415.74 65.93.04 0.00.21.88.21NR3 -28.4391.05 66.93.03 0.00.01.09.01.8NR Best fit to dataKidney:UroethelialCarcinoma0 0 0 31 -12.956NR 3 31.99.16 0.0 0.311.71NR2 -11.8372.24 31.75.71 0.0 0.10.52.51NR3 -11.3980.95 32.84.65 0.0 0.00.32.71.7NR Best fit to dataSkin andMammaryGland: BasalCell Tumors2 1 1 131 -52.314NR 3 110.61.88 1.0 2.55.58.31NR2 -48.5707.54 105.12.58 1.1 1.33.411.41NR3 -47.3622.45 104.72.86 1.2 1.22.312.51.4NR Lowest AIC, best fit to low dose dataSkin andMammaryGland: Squam.Cell Tumors0 1 1 61 -28.745NR 3 63.53.36 0.0 0.92.74.51NR Lowest AIC, best fit to low dose data2 -28.1451.24 64.32.75 0.0 0.42.45.21.9NR3 -27.6521.05 65.32.64 0.0 0.42.15.53.0NRFemalesOral Cavity andForestomach:Squam. CellTumors1 6 30 501 -134.532NR 4 277.10.245 1.0 10.123.735.11582 -100.80967.45 211.60.428 0.8 6.833.247.92.5523-94.51212.66 201.00.539 1.1 4.931.849.43.547Lowest AIC, best fit to low dose dataHepatocellularTumors0 1 39 511 -293.771NR 4 595.50.146 0.0 14.632.643.81442 -382.470177.45 774.90.370 0.0 8.138.550.12.2443-228.170308.66 468.30.575 0.0 3.038.451.43.139Lowest AIC, best fit to low dose dataDuodenum andJejunumTumors0 0 0 41 -15.948NR 3 37.96.00 0.00.41.32.41NR2 -14.5182.94 37.04.33 0.00.00.73.31.1NR3 -13.8781.35 37.83.43 0.00.00.43.62.3NR Best fit to low dose dataaLL=log-likelihood.bχ2= chi-squared statistic for testing the differencebetween 2 model fits, from 2 × |(LLi– LLj)| evaluated for i-j degrees of freedom. In all cases the difference was evaluated for consecutive numbers ofstages; i-j = 1, andχ2atα= 0.05 is 3.84.c“Responses” describes the number of animals with each tumor type; observed responses are in italics, and expected responses (predicted by each model fit)are given to one decimal place for comparisonwith the observed data.338 DRAFT - DO NOT CITE OR QUOTETable D-6.Summary of Model Selection and Modeling Results forbest-fitting multistage-Weibull models, usingtime-to-tumor data for rats (Kroese et al., 2001)SexEndPointsModelstages LLaχ2bNumberofparam.AIC BMD10Responses at mg/kg-d levelscSelected modelparameterestimatesModel Selection Rationale0 0.54 1.8 5.2 c t0MalesOral Cavity andForestomach:Squam. CellTumors0 8 45 521 -284.891NR 4 577.80.104 0.021.139.838.72.2442 -198.795172.25 407.60.678 0.03.325.448.01.3443 -108.512180.66 229.00.453 0.06.841.750.03.741Lowest AIC, best fit to low dose dataHepatocellularTumors0 4 38 491 -179.664NR 4 367.30.181 013.632.1421522 -145.74967.85 301.50.472 0.06.537.2492.348.43 -138.54414.46 289.10.651 0.03.436.849.63.540.2Lowest AIC, best fit to low dose dataDuodenum andJejunumTumors0 0 1 91 -31.781NR 3 69.62.64 0.01.13.45.71NR2 -28.9415.74 65.93.04 0.00.21.88.21NR3 -28.4391.05 66.93.03 0.00.01.09.01.8NR Best fit to dataKidney:UroethelialCarcinoma0 0 0 31 -12.956NR 3 31.99.16 0.0 0.311.71NR2 -11.8372.24 31.75.71 0.0 0.10.52.51NR3 -11.3980.95 32.84.65 0.0 0.00.32.71.7NR Best fit to dataSkin andMammaryGland: BasalCell Tumors2 1 1 131 -52.314NR 3 110.61.88 1.0 2.55.58.31NR2 -48.5707.54 105.12.58 1.1 1.33.411.41NR3 -47.3622.45 104.72.86 1.2 1.22.312.51.4NR Lowest AIC, best fit to low dose dataSkin andMammaryGland: Squam.Cell Tumors0 1 1 61 -28.745NR 3 63.53.36 0.0 0.92.74.51NR Lowest AIC, best fit to low dose data2 -28.1451.24 64.32.75 0.0 0.42.45.21.9NR3 -27.6521.05 65.32.64 0.0 0.42.15.53.0NRFemalesOral Cavity andForestomach:Squam. CellTumors1 6 30 501 -134.532NR 4 277.10.245 1.0 10.123.735.11582 -100.80967.45 211.60.428 0.8 6.833.247.92.5523-94.51212.66 201.00.539 1.1 4.931.849.43.547Lowest AIC, best fit to low dose dataHepatocellularTumors0 1 39 511 -293.771NR 4 595.50.146 0.0 14.632.643.81442 -382.470177.45 774.90.370 0.0 8.138.550.12.2443-228.170308.66 468.30.575 0.0 3.038.451.43.139Lowest AIC, best fit to low dose dataDuodenum andJejunumTumors0 0 0 41 -15.948NR 3 37.96.00 0.00.41.32.41NR2 -14.5182.94 37.04.33 0.00.00.73.31.1NR3 -13.8781.35 37.83.43 0.00.00.43.62.3NR Best fit to low dose dataNR = not relevant.

### 339 DRAFT - DO NOT CITE OR QUOTE

Male Rat (Kroese et al., 2001): Squamous Cell Papi lloma or Carcinoma in Oral Cavity or 1 

### Forestomach 2 3

================================================== ===================== 4 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 6 Input Data File: OralForstKroeseM3.(d) 7

================================================== ===================== 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = CONTEXT 15 Independent variables = DOSE, TIME 16 17 Total number of observations = 208 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 0 21 Degree of polynomial = 3 22 23 Maximum number of iterations = 64 24 Relative Function Convergence has been set to: 2.2 2045e-016 25 Parameter Convergence has been set to: 1.49012e-00 8 26 27 28 Default Initial Parameter Values 29

c = 3.6 30 t_0 = 39.1111 31 beta_0 = 0 32 beta_1 = 8.8911e-009 33 beta_2 = 1.60475e-031 34 beta_3 = 1.95818e-008 35 36 37 Asymptotic Correlation Matrix of Paramet er Estimates 38 ( *** The model parameter(s) -beta_0 -beta_2 39 have been estimated at a boundary point, or have been specified by the user, 40 and do not appear in the correlati on matrix ) 41 42 c t_0 beta_1 beta_3 43 44

c 1 -0.53 -0.93 -0.99 45 46 t_0 -0.53 1 0.47 0.57 47 48 beta_1 -0.93 0.47 1 0.9 49 50 beta_3 -0.99 0.57 0.9 1 51 52 53 Parameter Estimate s 54 95.0% Wald Confidence Interval 55 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 56

c 3.74559 0.447309 2.86888 4.6223 57 t_0 41.4581 2.14975 37.2447 45.6716 58 beta_0 0 NA 59 beta_1 4.37816e-009 1.07528e-008 -1.6697e-008 2.54533e-008 60 beta_2 0 NA 61 beta_3 1.01904e-008 1.94164e-008 -2.78651e-008 4.82458e-008 62 63 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 64 and thus has no standard error. 65 66 67 Log(likelihood) # Param AIC 68 Fitted Model -108.512 6 2 29.024 69 70 71 Data Summary 72

CONTEXT 73 C F I U Total Expec ted Response 74

DOSE 75

0 52 0 0 0 52 0. 00 76 0.54 44 0 8 0 52 6. 77 77 1.8 7 0 45 0 52 41. 69 78

5.2 0 9 43 0 52 49. 97 79 80 Minimum observation time for F tumor context = 44 81 82 83

### 340 DRAFT - DO NOT CITE OR QUOTE

Benchmark Dose Computation 1 Risk Response = Incidental 2 Risk Type = Extra 3 Confidence level = 0.9 4 Time = 104 5 6 7

Specified effect =

BMD = BMDL = BMDU = 0.1 0.453471 0.281044 0.612462 0.01

0.0633681 0.0286649 0.248377 0.001

0.00636659 0.00285563 > 0.0509326

8

9 10 11

12 13

### 341 DRAFT - DO NOT CITE OR QUOTE

Male Rat (Kroese et al., 2001): Hepatocellular Ad enoma or Carcinoma 1 2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: LiverKroeseM3.(d) 6

=================================================== ==================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = CONTEXT 15 Independent variables = DOSE, TIME 16 17 Total number of observations = 208 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 0 21 Degree of polynomial = 3 22 23 24 Maximum number of iterations = 64 25 Relative Function Convergence has been set to: 2.2 2045e-016 26 Parameter Convergence has been set to: 1.49012e-00 8 27 28 29 Default Initial Parameter Values 30 c = 3.6 31 t_0 = 34.6667 32 beta_0 = 0 33 beta_1 = 2.73535e-009 34 beta_2 = 8.116e-028 35 beta_3 = 1.43532e-008 36 37 38 Asymptotic Correlation Matrix of Paramet er Estimates 39 ( *** The model parameter(s) -beta_0 -beta_2 40 have been estimated at a boundary point, or have been specified by the user, 41 and do not appear in the correlati on matrix ) 42 43 c t_0 beta_1 beta_3 44 45

c 1 -0.84 -0.88 -1 46 47 t_0 -0.84 1 0.71 0.86 48 49 beta_1 -0.88 0.71 1 0.86 50 51 beta_3 -1 0.86 0.86 1 52 53 54 Parameter Estimate s 55 95.0% Wald Confidence Interval 56 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 57

c 3.49582 0.629257 2.26249 4.72914 58 t_0 40.2211 5.65421 29.1391 51.3032 59 beta_0 0 NA 60 beta_1 4.43906e-009 1.76051e-008 -3.00664e-008 3.89445e-008 61 beta_2 0 NA 62 beta_3 2.35065e-008 6.47999e-008 -1.03499e-007 1.50512e-007 63 64 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 65 and thus has no standard error. 66 67 68 Log(likelihood) # Param AIC 69 Fitted Model -138.544 6 2 89.088 70 71 72 Data Summary 73

CONTEXT 74 C F I U Total Expec ted Response 75

DOSE 76 0 52 0 0 0 52 0. 00 77 0.54 48 0 4 0 52 3. 38 78 1.8 14 2 36 0 52 36. 81 79

5.2 3 17 32 0 52 49. 55 80 81 Minimum observation time for F tumor context = 52 82 83 84

### 342 DRAFT - DO NOT CITE OR QUOTE

Benchmark Dose Computation 1 Risk Response = Incidental 2 Risk Type = Extra 3 Confidence level = 0.9 4 Time = 104 5 6 7

Specified effect =

BMD = BMDL = BMDU = 0.1 0.6507 0.44868 0.772467 0.01

0.173556 0.0530469 0.352684 0.001

0.0199908 0.00530386 > 0.159927

8 9 10 11 12 13

14 15 

### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.00



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.54



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 1.81



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 5.17



### TimeProbability Incidental Risk: Hepatocellular_Kroese_M3 points show nonparam. est. for Incidental (unfilled ) and Fatal (filled)

### 343 DRAFT - DO NOT CITE OR QUOTE

Male Rat (Kroese et al., 2001): Duodenum or Jejunu m Adenocarcinoma 1

2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: DuoJejKroeseM3.(d) 6

=================================================== ==================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = CONTEXT 15 Independent variables = DOSE, TIME 16 17 Total number of observations = 208 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 1 21 Degree of polynomial = 3 22 23 24 25 User specifies the following parameters: 26 t_0 = 0 27 28 Maximum number of iterations = 64 29 Relative Function Convergence has been set to: 2.2 2045e-016 30 Parameter Convergence has been set to: 1.49012e-00 8 31 32 33 Default Initial Parameter Values 34 c = 1.63636 35 t_0 = 0 Sp ecified 36 beta_0 = 4.31119e-027 37 beta_1 = 2.96347e-025 38 beta_2 = 0 39 beta_3 = 1.76198e-006 40 41 42 Asymptotic Correlation Matrix of Paramet er Estimates 43 ( *** The model parameter(s) -t_0 -beta_0 -beta_1 -beta_2 44 have been estimated at a boundary point, or have been specified by the user, 45 and do not appear in the correlati on matrix ) 46 47 c beta_3 48 49 c 1 -1 50 51 beta_3 -1 1 52 53 54 Parameter Estimate s 55 95.0% Wald Confidence Interval 56 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 57

c 1.77722 2.03042 -2.20233 5.75677 58 beta_0 0 NA 59 beta_1 0 NA 60 beta_2 0 NA 61 beta_3 9.82635e-007 8.29355e-006 -1.52724e-005 1.72377e-005 62 63 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 64 and thus has no standard error. 65 66 67 Log(likelihood) # Param AIC 68 Fitted Model -28.4387 5 6 6.8773 69 70 71 Data Summary 72

CONTEXT 73 C F I U Total Expec ted Response 74

DOSE 75

0 52 0 0 0 52 0. 00 76 0.54 52 0 0 0 52 0. 03 77 1.8 51 0 1 0 52 1. 04 78

5.2 43 0 9 0 52 8. 96 79 80 81 Benchmark Dose Computation 82 Risk Response = Incidental 83

### 344 DRAFT - DO NOT CITE OR QUOTE Risk Type = Extra 1 Specified effect = 0.1 2 Confidence level = 0.9 3 Time = 104 4 5 6

Specified effect =

BMD = BMDL = BMDU = 0.1 3.03291 2.37782 3.87183 0.01

1.38578 0.418285 1.76166 0.001

0.642252 0.0420835 0.811476

7 8 9 10 11

12 13

14 0 20 40 60 80 1000.00 0.15 Dose = 0.00

TimeProbability

0 20 40 60 80 1000.00 0.15 Dose = 0.54

TimeProbability

0 20 40 60 80 1000.00 0.15 Dose = 1.81

TimeProbability

0 20 40 60 80 1000.00 0.15 Dose = 5.17

TimeProbability Incidental Risk: DuoJej_Kroese_M3

### 345 DRAFT - DO NOT CITE OR QUOTE

Male Rat (Kroese et al., 2001): Skin or Mammary Gl and Basal Cell Tumors 1

2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: SKinMamBasalKroeseM3.(d) 6

=================================================== ==================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = CONTEXT 15 Independent variables = DOSE, TIME 16 17 Total number of observations = 208 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 1 21 Degree of polynomial = 3 22 23 24 25 User specifies the following parameters: 26 t_0 = 0 27 28 Maximum number of iterations = 64 29 Relative Function Convergence has been set to: 2.2 2045e-016 30 Parameter Convergence has been set to: 1.49012e-00 8 31 32 33 Default Initial Parameter Values 34 c = 1.38462 35 t_0 = 0 Sp ecified 36 beta_0 = 3.84298e-005 37 beta_1 = 1.06194e-028 38 beta_2 = 0 39 beta_3 = 6.84718e-006 40 41 42 Asymptotic Correlation Matrix of Paramet er Estimates 43 ( *** The model parameter(s) -t_0 -beta_1 -beta_2 44 have been estimated at a boundary point, or have been specified by the user, 45 and do not appear in the correlati on matrix ) 46 47 c beta_0 beta_3 48 49 c 1 -1 -1 50 51 beta_0 -1 1 0.99 52 53 beta_3 -1 0.99 1 54 55 56 Parameter Estimate s 57 95.0% Wald Confidence Interval 58 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 59

c 1.47227 1.76686 -1.9907 4.93525 60 beta_0 2.54786e-005 0.000211261 -0.000388585 0.000439542 61 beta_1 0 NA 62 beta_2 0 NA 63 beta_3 4.81611e-006 3.49e-005 -6.35866e-005 7.32188e-005 64 65 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 66 and thus has no standard error. 67 68 69 Log(likelihood) # Param AIC 70 Fitted Model -47.3623 5 1 04.725 71 72 73 Data Summary 74

CONTEXT 75 C F I U Total Expec ted Response 76

DOSE 77 0 50 0 2 0 52 1. 18 78

0.54 51 0 1 0 52 1. 22 79 1.8 51 0 1 0 52 2. 32 80

5.2 39 0 13 0 52 12. 54 81 82 83

### 346 DRAFT - DO NOT CITE OR QUOTE Benchmark Dose Computation 1 Risk Response = Incidental 2 Risk Type = Extra 3 Confidence level = 0.9 4 Time = 104 5 6 7 8

Specified effect =

BMD = BMDL = BMDU = 0.1 2.86276 2.35118 3.62258 0.01

1.30804 0.415897 1.69571 0.001

0.606222 0.0424277 0.761447

9 10 11 12 13

14 15 

### 0 20 40 60 800.0 0.4 0.8 Dose = 0.54



### TimeProbability



### 0 20 40 60 800.0 0.4 0.8 Dose = 1.81



### TimeProbability



### 0 20 40 60 800.0 0.4 0.8 Dose = 5.17



### TimeProbability Incidental Risk: Skin_Mam_Basal_Kroese_M3

### 347 DRAFT - DO NOT CITE OR QUOTE

Male Rat (Kroese et al., 2001): Skin or Mammary G land Squamous Cell Tumors 1 2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: SKinMamSCCKroeseM3.(d) 6

================================================== ===================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = CONTEXT 15 Independent variables = DOSE, TIME 16 17 Total number of observations = 208 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 1 21 Degree of polynomial = 3 22 23 24 25 User specifies the following parameters: 26 t_0 = 0 27 28 Maximum number of iterations = 64 29 Relative Function Convergence has been set to: 2.2 2045e-016 30 Parameter Convergence has been set to: 1.49012e-00 8 31 32 33 Default Initial Parameter Values 34

c = 3 35 t_0 = 0 Sp ecified 36 beta_0 = 0 37 beta_1 = 1.25256e-008 38 beta_2 = 1.25627e-030 39 beta_3 = 3.34696e-009 40 41 42 Asymptotic Correlation Matrix of Paramet er Estimates 43 ( *** The model parameter(s) -t_0 -beta_0 -beta_2 44 have been estimated at a boundary point, or have been specified by the user, 45 and do not appear in the correlati on matrix ) 46 47 c beta_1 beta_3 48 49

c 1 -0.99 -1 50 51 beta_1 -0.99 1 0.99 52 beta_3 -1 0.99 1 53 54 55 Parameter Estimate s 56 95.0% Wald Confidence Interval 57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 58

c 2.96213 2.591 -2.11613 8.04039 59 beta_0 0 NA 60 beta_1 1.50104e-008 1.86972e-007 -3.51447e-007 3.81468e-007 61 beta_2 0 NA 62 beta_3 3.9084e-009 4.15374e-008 -7.75033e-008 8.53201e-008 63 64 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 65 and thus has no standard error. 66 67 68 Log(likelihood) # Param AIC 69 Fitted Model -27.652 5 65.304 70 71 72 Data Summary 73

CONTEXT 74 C F I U Total Expec ted Response 75

DOSE 76 0 52 0 0 0 52 0. 00 77 0.54 51 0 1 0 52 0. 42 78 1.8 51 0 1 0 52 2. 12 79

5.2 46 0 6 0 52 5. 51 80 81 82 Benchmark Dose Computation 83 Risk Response = Incidental 84

### 348 DRAFT - DO NOT CITE OR QUOTE

Risk Type = Extra 1 Confidence level = 0.9 2 Time = 104 3 4 5

Specified effect =

BMD = BMDL = BMDU = 0.1 2.6414 1.76931 4.42145 0.01

0.64109 0.211043 2.03605 0.001

0.070558 0.0210552 > 0.564463

6 7

8 

### Dose Response plot 9 10

11

### 349 DRAFT - DO NOT CITE OR QUOTE

Male Rat (Kroese et al., 2001): Kidney Urothelial Carcinomas 1

2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: KidneyUrothelialCarKroeseM3.( d) 6

=================================================== ==================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = CONTEXT 15 Independent variables = DOSE, TIME 16 17 Total number of observations = 208 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 1 21 Degree of polynomial = 3 22 23 24 25 User specifies the following parameters: 26 t_0 = 0 27 28 Maximum number of iterations = 64 29 Relative Function Convergence has been set to: 2.2 2045e-016 30 Parameter Convergence has been set to: 1.49012e-00 8 31 32 33 Default Initial Parameter Values 34 c = 1.63636 35 t_0 = 0 Sp ecified 36 beta_0 = 3.78734e-027 37 beta_1 = 1.59278e-027 38 beta_2 = 2.718e-024 39 beta_3 = 4.96063e-007 40 41 42 Asymptotic Correlation Matrix of Paramet er Estimates 43 ( *** The model parameter(s) -t_0 -beta_0 -beta_1 -beta_2 44 have been estimated at a boundary point, or have been specified by the user, 45 and do not appear in the correlati on matrix ) 46 47 c beta_3 48 49 c 1 -1 50 51 beta_3 -1 1 52 53 54 Parameter Estimate s 55 95.0% Wald Confidence Interval 56 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 57

c 1.74897 3.79403 -5.68719 9.18512 58 beta_0 0 NA 59 beta_1 0 NA 60 beta_2 0 NA 61 beta_3 3.11107e-007 4.90313e-006 -9.29885e-006 9.92107e-006 62 63 NA - Indicates that this parameter has hit a 64 bound implied by some inequality constraint 65 and thus has no standard error. 66 67 68 Log(likelihood) # Param AIC 69 Fitted Model -11.3978 5 3 2.7956 70 71 72 Data Summary 73

CONTEXT 74 C F I U Total Expec ted Response 75

DOSE 76 0 52 0 0 0 52 0. 00 77 0.54 52 0 0 0 52 0. 01 78

1.8 52 0 0 0 52 0. 29 79 5.2 49 0 3 0 52 2. 71 80 81 82 Benchmark Dose Computation 83

### 350 DRAFT - DO NOT CITE OR QUOTE

Risk Response = Incidental 1 Risk Type = Extra 2 Confidence level = 0.9 3 Time = 104 4 5 6

Specified effect =

BMD = BMDL = BMDU = 0.1 4.64886 2.49972 9.01023 0.01

2.12413 0.734665 3.49311 0.001

0.984449 0.0748097 1.61892

7 8

9 10 0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 0.00

TimeProbability

0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 0.54

TimeProbability

0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 1.81

TimeProbability

0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 5.17

TimeProbability Incidental Risk: Kidney_Kroese_M3

### 351 DRAFT - DO NOT CITE OR QUOTE

Female Rat (Kroese et al., 2001): Oral Cavity or Forestomach, Squamous Cell Papilloma 1 

### or Carcinoma 2

3

=================================================== ==================== 4 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 6 Input Data File: OralForstKroeseF3.(d) 7

=================================================== ==================== 8 9 The form of the probability function is: 10 P[response] = 1-EXP{-(t - t_0)^c * 11 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 12 13 The parameter betas are restricted to be positiv e 14 15 Dependent variable = CONTEXT 16 Independent variables = DOSE, TIME 17 18 Total number of observations = 208 19 Total number of records with missing values = 0 20 Total number of parameters in model = 6 21 Total number of specified parameters = 0 22 Degree of polynomial = 3 23 24 25 Maximum number of iterations = 64 26 Relative Function Convergence has been set to: 2.2 2045e-016 27 Parameter Convergence has been set to: 1.49012e-00 8 28 29 30 Default Initial Parameter Values 31

c = 3.6 32 t_0 = 45.1111 33 beta_0 = 1.11645e-009 34 beta_1 = 4.85388e-009 35 beta_2 = 0 36 beta_3 = 1.95655e-008 37 38 Asymptotic Correlation Matrix of Paramet er Estimates 39 ( *** The model parameter(s) -beta_2 40 have been estimated at a boundary point, or have been specified by the user, 41 and do not appear in the correlati on matrix ) 42 43 c t_0 beta_0 beta_1 beta_3 44 45

c 1 -0.79 -0.92 -0.93 -1 46 47 t_0 -0.79 1 0.73 0.72 0.8 48 49 beta_0 -0.92 0.73 1 0.79 0.92 50 51 beta_1 -0.93 0.72 0.79 1 0.91 52 53 beta_3 -1 0.8 0.92 0.91 1 54 55 56 Parameter Estimate s 57 95.0% Wald Confidence Interval 58 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 59

c 3.52871 0.701117 2.15454 4.90287 60 t_0 46.553 5.93306 34.9244 58.1816 61 beta_0 1.53589e-009 5.40523e-009 -9.05817e-009 1.21299e-008 62 beta_1 7.57004e-009 2.9647e-008 -5.05369e-008 6.5677e-008 63 beta_2 0 NA 64 beta_3 2.53126e-008 7.66404e-008 -1.249e-007 1.75525e-007 65 66 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 67 and thus has no standard error. 68 69 70 Log(likelihood) # Param AIC 71 Fitted Model -94.5119 6 2 01.024 72 73 74 Data Summary 75

CONTEXT 76 C F I U Total Expec ted Response 77

DOSE 78

0 51 0 1 0 52 1. 14 79

0.49 46 0 6 0 52 4. 90 80 1.6 22 0 30 0 52 31. 81 81

4.6 2 7 43 0 52 49. 43 82

### 352 DRAFT - DO NOT CITE OR QUOTE

1 Minimum observation time for F tumor context = 58 2 3 Benchmark Dose Computation 4 Risk Response = Incidental 5 Risk Type = Extra 6 Confidence level = 0.9 7 Time = 104 8 9

Specified effect =

BMD = BMDL = BMDU = 0.1 0.538801 0.328135 0.717127 0.01

0.0981283 0.0345104 0.325909 0.001

0.0100797 0.00344714 > 0.0806373 10 11 12

13 14 

### 0 20 40 60 80 1000.0 0.4 0.8 

### Dose = 0.00



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 

### Dose = 0.49



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 

### Dose = 1.62



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 

### Dose = 4.58



### TimeProbability 

### Incidental Risk: OralForstKroeseF3 points show nonparam. est. for Incidental (unfilled ) and Fatal (filled)

### 353 DRAFT - DO NOT CITE OR QUOTE

Female Rat (Kroese et al., 2001): Hepatocellular Adenoma or Carcinoma 1

2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: LiverKroeseF3.(d) 6 Fri Apr 16 09:08:03 2010 7

================================================== ===================== 8 9 Timer to Tumor Model, Liver Hepatocellular Tumors, Kroese et al, Female 10 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14 P[response] = 1-EXP{-(t - t_0)^c * 15 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 16 17 The parameter betas are restricted to be positiv e 18 19 Dependent variable = CONTEXT 20 Independent variables = DOSE, TIME 21 22 Total number of observations = 208 23 Total number of records with missing values = 0 24 Total number of parameters in model = 6 25 Total number of specified parameters = 0 26 Degree of polynomial = 3 27 28 29 Maximum number of iterations = 64 30 Relative Function Convergence has been set to: 2.2 2045e-016 31 Parameter Convergence has been set to: 1.49012e-00 8 32 33 34 Default Initial Parameter Values 35

c = 3.6 36 t_0 = 31.7778 37 beta_0 = 0 38 beta_1 = 4.9104e-031 39 beta_2 = 5.45766e-030 40 beta_3 = 3.44704e-008 41 42 43 Asymptotic Correlation Matrix of Paramet er Estimates 44 ( *** The model parameter(s) -beta_0 -beta_1 -beta_2 45 have been estimated at a boundary point, or have been specified by the user, 46 and do not appear in the correlati on matrix ) 47 48 c t_0 beta_3 49 50

c 1 -0.9 -1 51 52 t_0 -0.9 1 0.92 53 54 beta_3 -1 0.92 1 55 56 57 Parameter Estimate s 58 95.0% Wald Confidence Interval 59 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 60

c 3.11076 0.549208 2.03434 4.18719 61 t_0 38.6965 5.21028 28.4846 48.9085 62 beta_0 0 NA 63 beta_1 0 NA 64 beta_2 0 NA 65 beta_3 2.94354e-007 7.19418e-007 -1.11568e-006 1.70439e-006 66 67 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 68 and thus has no standard error. 69 70 71 Log(likelihood) # Param AIC 72 Fitted Model -228.17 6 468.34 73 74 75 Data Summary 76

CONTEXT 77 C F I U Total Expec ted Response 78

DOSE 79 0 52 0 0 0 52 0. 00 80

0.49 51 0 1 0 52 3. 02 81

1.6 13 12 27 0 52 38. 36 82 4.6 1 38 13 0 52 51. 36 83

### 354 DRAFT - DO NOT CITE OR QUOTE

1 Minimum observation time for F tumor context = 44 2 3 4 Benchmark Dose Computation 5 Risk Response = Incidental 6 Risk Type = Extra 7 Confidence level = 0.9 8 Time = 104 9 10

Specified effect =

BMD = BMDL = BMDU = 0.1 0.575127 0.506633 0.629806 0.01

0.262783 0.134213 0.287232 0.001

0.12179 0.0152934 0.133064

11 12

13 14 

### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.00



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.49



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 1.62



### TimeProbability



### 0 20 40 60 80 1000.0 0.4 0.8 Dose = 4.58



### TimeProbability Incidental Risk: Hepatocellular_Kroese_F3 points show nonparam. est. for Incidental (unfilled ) and Fatal (filled)

### 355 DRAFT - DO NOT CITE OR QUOTE

Female Rat (Kroese et al., 2001): Duodenum or Jeju num Adenocarcinoma 1

2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: DuoJejKroeseF3.(d) 6

================================================== ===================== 7 The form of the probability function is: 8 P[response] = 1-EXP{-(t - t_0)^c * 9 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 10 11 The parameter betas are restricted to be positiv e 12 13 Dependent variable = CONTEXT 14 Independent variables = DOSE, TIME 15 16 Total number of observations = 208 17 Total number of records with missing values = 0 18 Total number of parameters in model = 6 19 Total number of specified parameters = 1 20 Degree of polynomial = 3 21 22 23 User specifies the following parameters: 24 t_0 = 0 25 26 Maximum number of iterations = 64 27 Relative Function Convergence has been set to: 2.2 2045e-016 28 Parameter Convergence has been set to: 1.49012e-00 8 29 30 31 Default Initial Parameter Values 32

c = 2.25 33 t_0 = 0 Sp ecified 34 beta_0 = 0 35 beta_1 = 0 36 beta_2 = 0 37 beta_3 = 7.289e-008 38 39 40 Asymptotic Correlation Matrix of Paramet er Estimates 41 ( *** The model parameter(s) -t_0 -beta_0 -beta_1 -beta_2 42 have been estimated at a boundary point, or have been specified by the user, 43 and do not appear in the correlati on matrix ) 44 45 c beta_3 46 47

c 1 -1 48 49 beta_3 -1 1 50 51 52 Parameter Estimate s 53 95.0% Wald Confidence Interval 54 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 55

c 2.32531 3.58729 -4.70565 9.35626 56 beta_0 0 NA 57 beta_1 0 NA 58 beta_2 0 NA 59 beta_3 5.32209e-008 7.98487e-007 -1.51178e-006 1.61823e-006 60 61 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 62 and thus has no standard error. 63 64 65 Log(likelihood) # Param AIC 66 Fitted Model -13.8784 5 3 7.7569 67 68 69 Data Summary 70

CONTEXT 71 C F I U Total Expec ted Response 72

DOSE 73 0 52 0 0 0 52 0. 00 74

0.49 52 0 0 0 52 0. 01 75

1.6 52 0 0 0 52 0. 44 76 4.6 48 0 4 0 52 3. 57 77 78 79 Benchmark Dose Computation 80 Risk Response = Incidental 81 Risk Type = Extra 82 Confidence level = 0.9 83

### 356 DRAFT - DO NOT CITE OR QUOTE

Time = 104 1 2

Specified effect =

BMD = BMDL = BMDU = 0.1 3.43129 1.94745 5.70108 0.01

1.56781 0.560867 2.61447 0.001

0.726615 0.0584891 1.21046

3 4

5 6 0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 0.00

TimeProbability

0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 0.49

TimeProbability

0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 1.62

TimeProbability

0 20 40 60 80 1000.00 0.05 0.10 0.15 Dose = 4.58

TimeProbability Incidental Risk: DuoJej_Kroese_F3

### 357 DRAFT - DO NOT CITE OR QUOTE

Table D-7. Summary of human equivalent overall can cer risk values, based on male and female rat tumor incidence (Kroese et al., 2001)

Data set Tumor Site BMD 001 BMDL 001 Risk value a at

SD SD 2 Prop. of total variance BMD 001 BMDL 001 Males Oral cavity/forestomach 6.37E-03 2.86E-03 1.57E-01 3.50E-01 1.17E-01 1.38E-02 0.64

Liver 2.00E-02 5.30E-03 5.00E-02 1.89E-01 8.42E-02 7.09E-03 0.33

Duodenum/ jejunum 6.42E-01 4.21E-02 1.56E-03 2.38E- 02 1.35E-02 1.82E-04 0.01

Skin/mammary gland: basal cell 6.06E-01 4.24E-02 1.65E-03 2.36E-02 1.33E-02 1.78E- 04 0.01

Skin/mammary gland: squam. cell 7.06E-02 2.11E-02 1.42E-02 4.75E-02 2.03E-02 4.10E- 04 0.02

Kidney 9.84E-01 7.48E-02 1.02E-03 1.34E-02 7.51E-03 5.64E-05 0.00

Sum, risk values at BMD 001 : 2.25E-01 Sum, SD 2 : 2.17E-02

Overall SD b : 1.47E-01

Upper bound on sum of risk estimates c : 4.68E-01

Females Oral cavity/forestomach 3.45E-03 1.01E-02 2.90E-01 9.92E-02 1.16E-01 1.35E-02 0.91

Liver 1.53E-02 1.22E-01 6.54E-02 8.21E-03 3.48E-02 1.21E-03 0.08

Duodenum/ jejunum 5.85E-02 7.27E-01 1.71E-02 1.38E- 03 9.56E-03 9.13E-05 0.01

Sum, risk values at BMD 001 : 1.09E-01 Sum, SD 2 : 1.48E-02

Overall SD: 1.22E-01

Upper bound on sum of risk estimates c : 3.09E-01

a Risk value=0.001/BMDL 001 b Overall SD = (Sum, SD 2 ) 0.5

c Upper bound on the overall risk estimate = Sum of BMD 001 risk values + 1.645 × Overall SD.

1

2 3 4 

### Table D-8. Summary of model selection among multistage-Weibull models fit to alimentary tract tumor data for female mice (Beland and Culp, 1998)

Model stages LL a χ 2 b Number of

param. AIC BMD 10 Responses @ mg/kg-d levels c Selected model parameter estimates Model Selection

Rationale 0 0.1 0.48 2.3 c t0 1 3 38 46 1 -340.271 NR 4 688.5 0.104 0.6 14.6 34.1 36.3 3.4 18 2 -309.620 61.3 5 629.2 0.102 0.7 4.5 33.2 41.8 5.5 16

3

- 306.265 6.7 6 624.5 0.127 0.9 3.2 30.8 41.9 6.9 14 Lowest AIC, best fit to low dose data

a LL=log-likelihood.

b χ 2 = chi-squared statistic for testing the difference between 2 model fits, from 2 × |(LL i – LL j )| evaluated for i-j degrees of freedom. In all cases the difference was evaluated for consecutive numbers of stages; i-j = 1, and χ 2 at α = 0.05 is 3.84.

c “ Responses” describes the number of animals with eac h tumor type; observed responses are in italics, an d expected responses (predicted by each model fit) are given to one decimal place for comparison with the observed data. NR = not relevant. 5

### 358 DRAFT - DO NOT CITE OR QUOTE

Female Mice (Beland and Culp, 1998): Alimentary Tr act Squamous Cell Tumors 1 2

================================================== ===================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: C:\msw10-09\benzo[a]pyrene_Fe maleSquamF3i.(d) 6

=================================================== ==================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = Class 15 Independent variables = Dose, time 16 17 Total number of observations = 191 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 0 21 Degree of polynomial = 3 22 23 24 Maximum number of iterations = 64 25 Relative Function Convergence has been set to: 2.2 2045e-016 26 Parameter Convergence has been set to: 1.49012e-00 8 27 28 29 30 User Inputs Initial Parameter Valu es 31

c = 2 32 t_0 = 15 33 beta_0 = 1.6e-014 34 beta_1 = 0 35 beta_2 = 5.5e-012 36 beta_3 = 4.4e-012 37 38 39 Asymptotic Correlation Matrix of Paramet er Estimates 40 c t_0 beta_0 beta_1 beta_2 beta_3 41 42 c 1 -0.78 -0.97 -0.42 -0.99 -0.99 43 44 t_0 -0.78 1 0.76 0.39 0.74 0.84 45 46 beta_0 -0.97 0.76 1 0.33 0.97 0.96 47 48 beta_1 -0.42 0.39 0.33 1 0.31 0.46 49 50 beta_2 -0.99 0.74 0.97 0.31 1 0.97 51 52 beta_3 -0.99 0.84 0.96 0.46 0.97 1 53 54 55 Parameter Estimate s 56 95.0% Wald Confidence Interval 57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 58

c 6.92317 1.33874 4.29929 9.54705 59 t_0 13.9429 4.96646 4.20881 23.677 60 beta_0 2.46916e-016 1.47619e-015 -2.64636e-015 3.14019e-015 61 beta_1 0 1.30525e-014 -2.55825e-014 2.55825e-014 62 beta_2 5.85452e-014 3.75144e-013 -6.76723e-013 7.93813e-013 63 beta_3 9.76542e-014 5.62017e-013 -1.00388e-012 1.19919e-012 64 65 66 Log(likelihood) # Param AIC 67 Fitted Model -306.265 6 624.53 68 69 70 Data Summary 71 Class 72 C F I U Total Expec ted Response 73

Dose 74

0 47 0 1 0 48 0. 93 75

0.1 45 0 3 0 48 3. 21 76 0.48 8 23 15 1 47 30. 82 77 2.3 1 46 0 1 48 41. 91 78 79 Minimum observation time for F tumor context = 39 80 81 82 Benchmark Dose Computation 83 Risk Response = Incidental 84

### 359 DRAFT - DO NOT CITE OR QUOTE

Risk Type = Extra 1 Specified effect = 0.1 2 Confidence level = 0.9 3 4 Time = 104 5 6 BMD = 0.126983 7 BMDL = 0.0706103 8 BMDU = 0.179419 9 10 11 12 13

14

15

### 360 DRAFT - DO NOT CITE OR QUOTE

APPENDIX E. TIME-TO-TUMOR MODELING FOR THE INHALAT ION UNIT RISK 1

2 3 

### Table E-1. Individual pathology and tumor occurren ce data for male Syrian hamsters exposed to benzo[a]pyrene via inhalation f or lifetime (Thyssen et al., 1981)

Admin. Exposure Conc. (mg/m 3 ) Time

on Study Number

Examined Larynx Pharynx Trachea Esophagus Forestoma ch Nasal Cavity

0 17

39 45 79 83 85 86 88 89 90 101 102 103 106 108 109 112 115 116 122 123 124 125 127 132 1

1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a 0 0 0 0 a

0 0 0 0 0 a 0 0 0 0 0 0 0 0 0 0 0 0 0 a 0 0 0 0 0 a 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 14

35 53 59 71 78 80 85 87 88 93 98 99 102 103 108 111 113 114 115 116 117 120 122 133 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 0 a

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a 0 0 a

0 0 0 0 0 0 0 0 0 0 0 a 0 0 0 0 0 0 0 0 0 0 0 0 a 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

### 361 DRAFT - DO NOT CITE OR QUOTE

Table E-1. Individual pathology and tumor occurren ce data for male Syrian hamsters exposed to benzo[a]pyrene via inhalation f or lifetime (Thyssen et al., 1981)

Admin. Exposure Conc. (mg/m 3 ) Time

on Study Number

Examined Larynx Pharynx Trachea Esophagus Forestoma ch Nasal Cavity

10 31

32 52 67 73 76 80 85 94 100 102 105 111 113 114 115 116 117 118 122 124 125 1

1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 0

0 0 0 0 0 1 0 1 0 0 1 0 0 1 1 0 1 3 1 1 0 0

0 0 0 0 2 0 0 0 0 1 1 1 1 1 0 a 0 0 1 b

0 1 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1

50 20

21 25 29 30 34 36 37 40 41 43 47 48 51 56 57 60 63 64 66 68 70 71 72 73 79 1

1 2 1 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 4 0 a

0 a 0 a 0 a 0 a 0 a 0 a 0 a 1 a 0 0 1 0 0 1 0 0 0 0 1 0 1 1 1 2 3 0 a

0 a 0 a 0 a 0 a 0 a 0 a 0 a 1 a 0 0 1 1 0 a 1 1 1 0 1 1 1 1 1 1 2 4 0 a

0 a 0 a 0 a 0 a 0 a 0 a 0 a 1 a 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1

a Tissue was not examined for one animal of total ex amined. 2

b Tissue was not examined for two animals of total ex amined. 3

### 362 DRAFT - DO NOT CITE OR QUOTE

Table E-2. Summary of model selection among multistage-Weibull models fit to tumor data for male hamsters a 

### (Thyssen et al., 1981)

Model stages LL b χ 2 c Number of

param. AIC BMD 10 Responses @ mg/kg-d levels d Selected model parameter estimates Model Selection

Rationale 0 0.25 1.01 4.29 c t 0 Oral tract tumors: all tumors considered incidenta l to cause of death 0 0 18 18

1 -26 NR 3 58 0.090 0.0 5.6 15.9 17.2 2.2 NR

2 -19.967 12.1 4 47.9 0.285 0.0 1.9 16.0 18.2 4.2 N R Lowest AIC, best fit to data; maximum number of stages that could be fit Oral tract tumors: all tumors considered to be cau se of death 0 0 18 18

1 -160.646 NR 3 327.292 0.136 Not available 4.9 NR

2 -147.428 26.4 4 302.857 0.421 Not available 6.7 NR

3 -144.522 5 299.043 0.648 Not available 9.0 NR Lowest AIC; best fit to data (see graphs)

a All animals with missing tissues were omitted.

b LL=log-likelihood.

c χ 2 = chi-squared statistic for testing the difference between 2 model fits: χ 2 = 2 × |(LL i – LL j )| evaluated for |i-j| degrees of freedom (df). In all cases the difference was evaluated for consecut ive numbers of stages; i-j = 1, and χ 2 for 1 df at α = 0.05 is 3.84.

d “ Responses” describes the number of animals with eac h tumor type; observed responses are in italics, an d expected responses (predicted by each model fit) are given to one decimal place for comparison with the observed data. NR = not relevant. 1

2

### 363 DRAFT - DO NOT CITE OR QUOTE

Output for Oral tract tumors: all tumors considere d incidental to cause of death 1 2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: C:\msw\benzo[a]pyrene-Thyssen _inc2st.(d) 6

=================================================== ==================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = Class 15 Independent variables = Conc, Time 16 17 Total number of observations = 96 18 Total number of records with missing values = 0 19 Total number of parameters in model = 5 20 Total number of specified parameters = 1 21 Degree of polynomial = 2 22 23 24 25 User specifies the following parameters: 26 t_0 = 0 27 28 Maximum number of iterations = 32 29 Relative Function Convergence has been set to: 1e- 008 30 Parameter Convergence has been set to: 1e-008 31 32 33 Default Initial Parameter Values 34

c = 3.6 35 t_0 = 0 Sp ecified 36 beta_0 = 1.18657e-031 37 beta_1 = 1.49e-030 38 beta_2 = 6.10362e-008 39 40 41 Asymptotic Correlation Matrix of Paramet er Estimates 42 ( *** The model parameter(s) -t_0 -beta_0 -beta_1 43 have been estimated at a boundary point, or have been specified by the user, 44 and do not appear in the correlati on matrix ) 45 46 c beta_2 47 48

c 1 -1 49 50 beta_2 -1 1 51 52 53 Parameter Estimate s 54 95.0% Wald Confidence Interval 55 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 56

c 4.21938 0.840997 2.57105 5.8677 57 beta_0 0 NA 58 beta_1 0 NA 59 beta_2 4.00402e-009 1.495e-008 -2.52974e-008 3.33054e-008 60 61 NA - Indicates that this parameter has hit a 62 bound implied by some inequality constraint 63 and thus has no standard error. 64 65 66 Log(likelihood) # Param AIC 67 Fitted Model -19.967 4 4 7.9339 68 69 70 Data Summary 71 Class 72 C F I U Total Expec ted Response 73

Conc 74 0 23 0 0 0 23 0. 00 75

0.25 24 0 0 0 24 1. 92 76 1 8 0 18 0 26 16. 04 77 4.3 5 0 18 0 23 18. 22 78 79 80 Benchmark Dose Computation 81 Risk Response = Incidental 82 Risk Type = Extra 83

### 364 DRAFT - DO NOT CITE OR QUOTE Specified effect = 0.1 1 Confidence level = 0.9 2 3 Time = 104 4 5

BMD = 0.284958 6 BMDL = 0.197807 7 BMDU = 0.350247 8 9 10 11

12 

### 0 20 60 1000.0 0.4 0.8 Dose = 0.00



### TimeProbability



### 0 20 60 1000.0 0.4 0.8 Dose = 0.25



### TimeProbability



### 0 20 60 1000.0 0.4 0.8 Dose = 1.00



### TimeProbability



### 0 20 60 1000.0 0.4 0.8 Dose = 4.29



### TimeProbability Incidental Risk: BaP-Thyssen_inc2st

### 365 DRAFT - DO NOT CITE OR QUOTE

Output for Oral tract tumors: all tumors considere d to be cause of death 1 2

=================================================== ==================== 3 Multistage Weibull Model. (Version: 1.6.1; D ate: 11/24/2009) 4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 5 Input Data File: C:\msw\benzo[a]pyrene-Thyssen _allfatal_noU_3st.(d) 6

=================================================== ==================== 7 8 The form of the probability function is: 9 P[response] = 1-EXP{-(t - t_0)^c * 10 (beta_0+beta_1*dose^1+beta_2*dose^ 2+beta_3*dose^3)} 11 12 The parameter betas are restricted to be positiv e 13 14 Dependent variable = Class 15 Independent variables = Conc, Time 16 17 Total number of observations = 96 18 Total number of records with missing values = 0 19 Total number of parameters in model = 6 20 Total number of specified parameters = 1 21 Degree of polynomial = 3 22 23 24 25 User specifies the following parameters: 26 t_0 = 0 27 28 Maximum number of iterations = 32 29 Relative Function Convergence has been set to: 1e- 008 30 Parameter Convergence has been set to: 1e-008 31 32 33 Default Initial Parameter Values 34

c = 4.5 35 t_0 = 0 Sp ecified 36 beta_0 = 0 37 beta_1 = 1.37501e-010 38 beta_2 = 2.84027e-010 39 beta_3 = 1.44668e-037 40 41 42 Asymptotic Correlation Matrix of Paramet er Estimates 43 ( *** The model parameter(s) -t_0 -beta_0 -beta_1 -beta_2 44 have been estimated at a boundary point, or have been specified by the user, 45 and do not appear in the correlati on matrix ) 46 47 c beta_3 48 49

c 1 -1 50 51 beta_3 -1 1 52 53 54 Parameter Estimate s 55 95.0% Wald Confidence Interval 56 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 57

c 8.95016 0.896607 7.19284 10.7075 58 beta_0 0 NA 59 beta_1 0 NA 60 beta_2 0 NA 61 beta_3 3.43452e-019 1.39727e-018 -2.39515e-018 3.08205e-018 62 63 NA - Indicates that this parameter has hit a 64 bound implied by some inequality constraint 65 and thus has no standard error. 66 67 68 Log(likelihood) # Param AIC 69 Fitted Model -144.522 5 2 99.043 70 71 72 Data Summary 73 Class 74 C F I U Total 75

Conc 76 0 23 0 0 0 23 77 0.25 24 0 0 0 24 78

1 8 18 0 0 26 79

4.3 5 18 0 0 23 80 81 Minimum observation time for F tumor context = 40 82 83

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1 Benchmark Dose Computation 2 Risk Response = Fatal 3 Risk Type = Extra 4 Specified effect = 0.1 5 Confidence level = 0.9 6 7 Time = 104 8 9

BMD = 0.647659 10

BMDL = 0.461415 11 BMDU = 0.719325 12 13

14

15 16 17

18

19

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APPENDIX F. BENCHMARK MODELING FOR THE DERMAL SLOP E FACTOR 1

2 

### Table F-1. Summary of model selection and modeling results for bestfitting multistage models, for multiple data sets o f skin tumors in mice following dermal benzo[a]pyrene exposure

Data Set (See Section 5.4.3.2., Tables 5-7 to 5-10) Degree of Model df Goodnessof-fit

p -value LL b χ 2 c BMD 10 (μg/d) BMDL 10

(μg/d) Model selection rationale, with best

fitting model in boldface a Figure number Poel (1959) male C57L 2 3 4 2 2 2 0.027 0.053 0.068 -91.28

-90.43 -90.12 NR

1.7 0.58 NA 0.127 0.122 NA

0.078 0.077 (Inadequate fit for 1-2 stage models) Most parsimonious fit Improvement in fit not statistically significant over 2-stage fit F-1

Roe et al. (1970) female Swiss 1 2-5 5 3 0.110 0.463 -64.56

-58.81 NR

11.5 0.299 0.689 0.233

0.394 Most parsimonious fit ; significant improvement over 1-stage fit F-2

Schmidt et al. (1973) female NMRI 1 2 3 4 4 4 0.008 0.609 0.999 -80.34

-72.68 -70.95 NR

15.3 3.5 0.256 0.329 0.381 0.194

0.287 0.326 Most parsimonious fit Improvement in fit not statistically significant over 2-stage fit F-3

Schmidt et al. (1973) female Swiss 1 2 3 -4 4 4 3 <0.01 0.514 0.983 -87.99

-75.66 -73.66 NR

24.7 4.0 0.116 0.216 0.282 0.093

0.192 0.223 Most parsimonious fit ; significant improvement over 2-stage fit F-4

Schmähl et al. (1977)

female NMRI 1 2 2 1 0.136 0.939 -147.20

-145.13 NR

4.14 0.140 0.233 0.117

0.149 Most parsimonious fit ; significant improvement over 1-stage fit F-5

Habs et al. (1980) female NMRI 2 3 3 3 0.009 0.207 -41.18

-37.34 NR

7.7 NA 0.294 NA

0.215 (Inadequate fit for 1-2 stage models) Most parsimonious fit F-6 Habs et al. (1984) female NMRI 1 2 2 1 0.577 1.000 -22.78

-22.22 NR

1.1 0.078 0.171 0.056

0.060 Most parsimonious fit F-7

Grimmer et al. (1983) female CFLP 1 2-3 3 2 0.850 0.972 -108.94

-108.56 NR

0.76 0.245 0.292 0.208

0.213 Most parsimonious fit Improvement in fit not statistically significant over 1-stage fit F-8

Grimmer et al. (1984), female CFLP Multistage 1-3 3 0.003 205.3 b NR NA NA Inadequate fit F-9

Other: LogLogistic Dich.-Hill LogProbit Gamma, Weibull Logistic Probit 2

1 3

— 2 2 0.919 1.000 0.047

— <0.01 <0.01 195.8

197.7 200.2

— 250.5 255.4 NR

NR NR

— NR NR 1.07 0.902 NA

— NA NA 0.479 0.533 NA

— NA NA Best fit ; slope parameter unrestricted Slope parameter unrestricted Inadequate fit Same model as multistage (above) Inadequate fit Inadequate fit F-10

Multistage, high dose dropped d 1 -2 2 0.499 NR NR 0.106 0.088 Best fit from multistage model F-11

Sivak et al. (1997) male CeH/HeJ 1 2 -3 3 3 0.059 0.998 -27.92

-23.30 9.2 0.036

0.109 0.026

0.058 Most parsimonious fit F-12

a Adequate fit: goodness-of-fit p>0.05, scaled resi duals <2.0, good fit near BMR, lack of extreme curv ature not reflected in the observed data.

b LL=Log-likelihood; values for Grimmer et al. (1984 ) are AICs, in order to compare across models.

c χ 2 = 2 × |(LL

i – LL j )|, where i and j are consecutive numbers of stages . The test was evaluated for 1 degree of freedom ( df). χ 2 for 1 df at

α = 0.05 is 3.84.

d The preferred multistage model did not adequately fit the data for Grimmer et al. (1984), thus, the r emaining suite of models were fit to the data. The POD for Grimmer et al. (1984) was b ased on the LogLogistic model. For comparison purp oses, the multistage model was it fit to these data for Grimmer et al. (1984) with th e highest dose dropped.

### 368 DRAFT - DO NOT CITE OR QUOTE

Figure F-1. Fit of multistage model to skin tumors in C57L mice exposed 1 

### dermally to benzo[a]pyrene (Poel, 1959); graph and model output. 2 3

4

================================================== ================== 5 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 6 Input Data File: C:\Usepa\BMDS21\Data\msc_benz o[a]pyrene_Poel_1959_MultiCanc3_0.1.(d) 7 Gnuplot Plotting File: 8 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_1959_M ultiCanc3_0.1.plt 9 10

================================================== ================== 11 12 [add notes here] 13

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 14 15 The form of the probability function is: 16 17 P[response] = background + (1-background)*[1-EXP ( 18 -beta1*dose^1-beta2*dose^2-beta3*d ose^3)] 19 20 The parameter betas are restricted to be positiv e 21 22 23 Dependent variable = NumAff 24 Independent variable = LADD 25 26 Total number of observations = 5 27 Total number of records with missing values = 0 28 Total number of parameters in model = 4 29 Total number of specified parameters = 0 30 Degree of polynomial = 3 31 32 33 Maximum number of iterations = 250 34 Relative Function Convergence has been set to: 1e- 008 35 Parameter Convergence has been set to: 1e-008 36 37 38 39 Default Initial Parameter Values 40 Background = 0.0449589 41 Beta(1) = 0.490451 42 Beta(2) = 0 43 Beta(3) = 2.68146 44 45 46 Asymptotic Correlation Matrix of Paramet er Estimates 47 48 0

0.2 0.4 0.6 0.8 1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 369 DRAFT - DO NOT CITE OR QUOTE

( *** The model parameter(s) -Beta(2) 1 have been estimated at a boundary point, or have been specified by the user, 2 and do not appear in the correlati on matrix ) 3 4 Background Beta(1) Beta(3) 5 6 Background 1 -0.87 0.74 7 8 Beta(1) -0.87 1 -0.92 9 10 Beta(3) 0.74 -0.92 1 11 12 13 14 Parameter Estimate s 15 16 95.0% Wald Confidence Interval 17 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 18 Background 0.0176699 * * * 19 Beta(1) 0.79766 * * * 20 Beta(2) 0 * * * 21 Beta(3) 2.17146 * * * 22 23 * - Indicates that this value is not calculated. 24 25 26 27 Analysis of Deviance Table 28 29 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 30 Full model -87.1835 5 31 Fitted model -90.4265 3 6.4 8606 2 0.03905 32 Reduced model -141.614 1 10 8.86 4 <.0001 33 34

AIC: 186.853 35 36 37 Goodness of Fit 38 Scaled 39 Dose Est._Prob. Expected Observed Size Residual 40

------------------------------------------------- ----------------------- 41 0.0000 0.0177 0.583 0.000 33 -0.770 42

0.0500 0.0563 3.098 5.000 55 1.112 43

0.1600 0.1430 7.866 11.000 55 1.207 44 0.2400 0.2128 11.917 7.000 56 -1.605 45

0.8000 0.8293 40.635 41.000 49 0.139 46 47 Chi^2 = 5.88 d.f. = 2 P-value = 0.0528 48 49 50 Benchmark Dose Computation 51 52 Specified effect = 0.1 53 54 Risk Type = Extra risk 55 56 Confidence level = 0.95 57 58

BMD = 0.126567 59 60

BMDL = 0.0777875 61 62 BMDU = 0.272961 63 64 Taken together, (0.0777875, 0.272961) is a 90 % two-sided confidence 65 interval for the BMD 66 67 Multistage Cancer Slope Factor = 1.28555 68 69 70

71

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1 

### Figure F-2. Fit of multistage model to skin tumors in female Swiss mice 2 

### exposed dermally to benzo[a]pyrene (Roe et al., 197 0); graph and model 3 

### output. 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 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 30 Input Data File: C:\Usepa\BMDS21\Data\msc_benz o[a]pyrene_Roe_1970_Setting.(d) 31 Gnuplot Plotting File: C:\Usepa\BMDS21\Data\m sc_benzo[a]pyrene_Roe_1970_Setting.plt 32 33

================================================== ================== 34 35 BMDS Model Run 36

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 37 38 The form of the probability function is: 39 40 P[response] = background + (1-background)*[1-EXP ( 41 -beta1*dose^1-beta2*dose^2-beta3*d ose^3-beta4*dose^4-beta5*dose^5)] 42 43 The parameter betas are restricted to be positiv e 44 45 46 Dependent variable = tumors 47 Independent variable = LADD 48 49 Total number of observations = 6 50 Total number of records with missing values = 0 51 Total number of parameters in model = 6 52 Total number of specified parameters = 0 53 Degree of polynomial = 5 54 55 56 Maximum number of iterations = 250 57 Relative Function Convergence has been set to: 1e- 008 58 Parameter Convergence has been set to: 1e-008 59 60 61 62 Default Initial Parameter Values 63 Background = 0 64 0

0.2 0.4 0.6 0.8

0 0.5 1 1.5 2 2.5Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer

Linear extrapolation

0 0.2 0.4 0.6 0.8

0 0.5 1 1.5 0 0.2 0.4 0.6 0.8

0 0.5 1 1.5 2 2.5Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer

Linear extrapolation

### 371 DRAFT - DO NOT CITE OR QUOTE

Beta(1) = 0.0962491 1 Beta(2) = 0.141689 2 Beta(3) = 0 3 Beta(4) = 0 4 Beta(5) = 0 5 6 7 Asymptotic Correlation Matrix of Paramet er Estimates 8 9 ( *** The model parameter(s) -Beta(3) -Beta(4) -Beta(5) 10 have been estimated at a boundary point, or have been specified by the user, 11 and do not appear in the correlati on matrix ) 12 13 Background Beta(1) Beta(2) 14 15 Background 1 -0.57 0.45 16 17 Beta(1) -0.57 1 -0.94 18 19 Beta(2) 0.45 -0.94 1 20 21 22 23 Parameter Estimate s 24 25 95.0% Wald Confidence Interval 26 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 27 Background 0.00584893 * * * 28 Beta(1) 0.0379152 * * * 29 Beta(2) 0.166839 * * * 30 Beta(3) 0 * * * 31 Beta(4) 0 * * * 32 Beta(5) 0 * * * 33 34 * - Indicates that this value is not calculated. 35 36 37 38 Analysis of Deviance Table 39 40 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 41 Full model -56.1835 6 42 Fitted model -57.5694 3 2.7 7176 3 0.4282 43 Reduced model -118.948 1 125 .529 5 <.0001 44 45

AIC: 121.139 46 47 48 Goodness of Fit 49 Scaled 50 Dose Est._Prob. Expected Observed Size Residual 51

------------------------------------------------- ----------------------- 52

0.0000 0.0058 0.275 0.000 47 -0.526 53

0.0300 0.0071 0.321 1.000 45 1.204 54

0.0900 0.0106 0.444 0.000 42 -0.670 55

0.3100 0.0331 1.423 1.000 43 -0.361 56

0.9200 0.1664 6.821 8.000 41 0.494 57

2.7600 0.7488 34.444 34.000 46 -0.151 58 59 Chi^2 = 2.57 d.f. = 3 P-value = 0.4626 60 61 62 Benchmark Dose Computation 63 64 Specified effect = 0.1 65 66 Risk Type = Extra risk 67 68 Confidence level = 0.95 69 70

BMD = 0.689131 71 72

BMDL = 0.393806 73 74 BMDU = 0.952365 75 76 Taken together, (0.393806, 0.952365) is a 90 % two-sided confidence 77 interval for the BMD 78 79 Multistage Cancer Slope Factor = 0.253932 80 81

82

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Figure F-3. Fit of multistage model to skin tumors in female NMRI mice 1 

### exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph and model 2 

### output. 3 4 5

=================================================== ================= 6 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 7 Input Data File: 8 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schm idt1973femaleNMRI\2MulSchMS_.(d) 9 Gnuplot Plotting File: 10 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schm idt1973femaleNMRI\2MulSchMS_.plt 11 12

================================================== ================== 13 14 BMDS Model Run 15

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 16 17 The form of the probability function is: 18 19 P[response] = background + (1-background)*[1-EXP ( 20 -beta1*dose^1-beta2*dose^2)] 21 22 The parameter betas are restricted to be positiv e 23 24 25 Dependent variable = incidence 26 Independent variable = dose 27 28 Total number of observations = 5 29 Total number of records with missing values = 0 30 Total number of parameters in model = 3 31 Total number of specified parameters = 0 32 Degree of polynomial = 2 33 34 35 Maximum number of iterations = 250 36 Relative Function Convergence has been set to: 2.2 2045e-016 37 Parameter Convergence has been set to: 1.49012e-00 8 38 39 0.4

0 0.1 0.2 0.3 0.4 0.5Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 373 DRAFT - DO NOT CITE OR QUOTE

**** We are sorry but Relative Function and Parame ter Convergence   1 ** ** are currently unavailable in this model. Ple ase keep checking   2 ** ** the web sight for model updates which will ev entually   3 ** ** incorporate these convergence criterion. Def ault values used.   4 5 6 7 Default Initial Parameter Values 8 Background = 0 9 Beta(1) = 0 10 Beta(2) = 1.11271 11 12 13 Asymptotic Correlation Matrix of Paramet er Estimates 14 15 ( ** * The model parameter(s) -Backgroun d -Beta(1) 16 have been estimated at a boundary point, or have been specified by the user, 17 and do not appear in the correlati on matrix ) 18 19 Beta(2) 20 21 Beta(2) 1 22 23 24 25 Parameter Estimate s 26 27 95.0% Wald Confidence Interval 28 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 29 Background 0 * * * 30 Beta(1) 0 * * * 31 Beta(2) 0.970648 * * * 32 33 * - Indicates that this value is not calculated. 34 35 36 37 Analysis of Deviance Table 38 39 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 40 Full model -70.8903 5 41 Fitted model -72.6831 1 3.5 8562 4 0.465 42 Reduced model -118.917 1 96 .054 4 <.0001 43 44 AIC: 147.366 45 46 47 Goodness of Fit 48 Scaled 49 Dose Est._Prob. Expected Observed Size Residual 50

------------------------------------------------- ----------------------- 51

0.0000 0.0000 0.000 0.000 100 0.000 52

0.0100 0.0001 0.010 0.000 100 -0.099 53

0.0600 0.0035 0.349 0.000 100 -0.592 54

0.2300 0.0501 5.005 2.000 100 -1.378 55

0.5700 0.2705 27.048 30.000 100 0.665 56 57 Chi^2 = 2.70 d.f. = 4 P-value = 0.6091 58 59 60 Benchmark Dose Computation 61 62 Specified effect = 0.1 63 64 Risk Type = Extra risk 65 66 Confidence level = 0.95 67 68 BMD = 0.329464 69 70

BMDL = 0.286624 71 72

BMDU = 0.384046 73 74 Taken together, (0.286624, 0.384046) is a 90 % two-sided confidence 75 interval for the BMD 76 77 Multistage Cancer Slope Factor = 0.348889 78 79

80

### 374 DRAFT - DO NOT CITE OR QUOTE

Figure F-4. Fit of multistage model to skin tumors in female Swiss mice 1 

### exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph and model 2 

### output. 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 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 35 Input Data File: 36 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schm idt1973swissmice\3MulSchMS_.(d) 37 Gnuplot Plotting File: 38 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schm idt1973swissmice\3MulSchMS_.plt 39

=================================================== ================= 40 41 BMDS Model Run 42

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 43 44 The form of the probability function is: 45 46 P[response] = background + (1-background)*[1-EXP ( 47 -beta1*dose^1-beta2*dose^2-beta3*d ose^3)] 48 49 The parameter betas are restricted to be positiv e 50 51 52 Dependent variable = incidence 53 Independent variable = dose 54 55 Total number of observations = 5 56 Total number of records with missing values = 0 57 Total number of parameters in model = 4 58 Total number of specified parameters = 0 59 Degree of polynomial = 3 60 61 62 Maximum number of iterations = 250 63 Relative Function Convergence has been set to: 2.2 2045e-016 64 Parameter Convergence has been set to: 1.49012e-00 8 65 66 **** We are sorry but Relative Function and Parame ter Convergence   67 ** ** are currently unavailable in this model. Ple ase keep checking   68 ** ** the web sight for model updates which will ev entually   69 0

0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 375 DRAFT - DO NOT CITE OR QUOTE

** ** incorporate these convergence criterion. Def ault values used.   1 2 3 4 Default Initial Parameter Values 5 Background = 0 6 Beta(1) = 0 7 Beta(2) = 0.338951 8 Beta(3) = 3.8728 9 10 11 Asymptotic Correlation Matrix of Paramet er Estimates 12 13 ( ** * The model parameter(s) -Backgroun d -Beta(1) 14 have been estimated at a boundary point, or have been specified by the user, 15 and do not appear in the correlati on matrix ) 16 17 Beta(2) Beta(3) 18 19 Beta(2) 1 -0.99 20 21 Beta(3) -0.99 1 22 23 24 25 Parameter Estimate s 26 27 95.0% Wald Confidence Interval 28 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 29 Background 0 * * * 30 Beta(1) 0 * * * 31 Beta(2) 0.108125 * * * 32 Beta(3) 4.31441 * * * 33 34 * - Indicates that this value is not calculated. 35 36 37 38 Analysis of Deviance Table 39 40 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 41 Full model -73.5285 5 42 Fitted model -73.6628 2 0.26 8637 3 0.9658 43 Reduced model -150.708 1 154 .359 4 <.0001 44 45

AIC: 151.326 46 47 48 Goodness of Fit 49 Scaled 50 Dose Est._Prob. Expected Observed Size Residual 51

------------------------------------------------- ----------------------- 52

0.0000 0.0000 0.000 0.000 80 0.000 53

0.0100 0.0000 0.001 0.000 80 -0.035 54

0.0600 0.0013 0.106 0.000 80 -0.325 55

0.2300 0.0566 4.524 5.000 80 0.230 56

0.5700 0.5657 45.260 45.000 80 -0.059 57 58 Chi^2 = 0.16 d.f. = 3 P-value = 0.9833 59 60 61 Benchmark Dose Computation 62 63 Specified effect = 0.1 64 65 Risk Type = Extra risk 66 67 Confidence level = 0.95 68 69

BMD = 0.282007 70 71 BMDL = 0.223401 72 73

BMDU = 0.309888 74 75 Taken together, (0.223401, 0.309888) is a 90 % two-sided confidence 76 interval for the BMD 77 78 Multistage Cancer Slope Factor = 0.447626 79 80 81 82

83

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Figure F-5. Fit of multistage model to skin tumors in female NMRI mice 1 

### exposed dermally to benzo[a]pyrene (Schmähl et al., 1977); graph and model 2 

### output. 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 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 36 Input Data File: 37 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schm ahl1977femaleNMRI\2MulschMS_.(d) 38 Gnuplot Plotting File: 39 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schm ahl1977femaleNMRI\2MulschMS_.plt 40

=================================================== ================= 41 42 BMDS Model Run 43

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 44 45 The form of the probability function is: 46 47 P[response] = background + (1-background)*[1-EXP ( 48 -beta1*dose^1-beta2*dose^2)] 49 50 The parameter betas are restricted to be positiv e 51 52 53 Dependent variable = incidence 54 Independent variable = dose 55 56 Total number of observations = 4 57 Total number of records with missing values = 0 58 Total number of parameters in model = 3 59 Total number of specified parameters = 0 60 Degree of polynomial = 2 61 62 63 Maximum number of iterations = 250 64 Relative Function Convergence has been set to: 2.2 2045e-016 65 Parameter Convergence has been set to: 1.49012e-00 8 66 67 **** We are sorry but Relative Function and Parame ter Convergence   68 ** ** are currently unavailable in this model. Ple ase keep checking   69 ** ** the web sight for model updates which will ev entually   70 0

0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer

Linear extrapolation

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer

Linear extrapolation

### 377 DRAFT - DO NOT CITE OR QUOTE

** ** incorporate these convergence criterion. Def ault values used. **** 1 2 3 4 Default Initial Parameter Values 5 Background = 0.0115034 6 Beta(1) = 0.284955 7 Beta(2) = 0.750235 8 9 10 Asymptotic Correlation Matrix of Paramet er Estimates 11 12 Background Beta(1) Beta(2) 13 14 Background 1 -0.67 0.47 15 16 Beta(1) -0.67 1 -0.94 17 18 Beta(2) 0.47 -0.94 1 19 20 21 22 Parameter Estimate s 23 24 95.0% Wald Confidence Interval 25 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 26 Background 0.0123066 * * * 27 Beta(1) 0.274413 * * * 28 Beta(2) 0.764244 * * * 29 30 * - Indicates that this value is not calculated. 31 32 33 34 Analysis of Deviance Table 35 36 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 37 Full model -145.127 4 38 Fitted model -145.13 3 0.0057 9898 1 0.9393 39 Reduced model -184.158 1 78. 0608 3 <.0001 40 41 AIC: 296.261 42 43 44 Goodness of Fit 45 Scaled 46 Dose Est._Prob. Expected Observed Size Residual 47

------------------------------------------------- ----------------------- 48

0.0000 0.0123 0.997 1.000 81 0.003 49

0.2900 0.1446 11.137 11.000 77 -0.045 50

0.4900 0.2813 24.756 25.000 88 0.058 51

0.8600 0.5567 45.096 45.000 81 -0.022 52 53 Chi^2 = 0.01 d.f. = 1 P-value = 0.9393 54 55 56 Benchmark Dose Computation 57 58 Specified effect = 0.1 59 60 Risk Type = Extra risk 61 62 Confidence level = 0.95 63 64

BMD = 0.232893 65 66 BMDL = 0.148895 67 68 BMDU = 0.320396 69 70 Taken together, (0.148895, 0.320396) is a 90 % two-sided confidence 71 interval for the BMD 72 73 Multistage Cancer Slope Factor = 0.671616 74 75 76 77

78

### 378 DRAFT - DO NOT CITE OR QUOTE

Figure F-6. Fit of multistage model to skin tumors in female NMRI mice 1 

### exposed dermally to benzo[a]pyrene (Habs et al., 19 80); graph and model 2 

### output. 3

4 0 0.2 0.4 0.6 0.8 1

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dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer

Linear extrapolation

### 379 DRAFT - DO NOT CITE OR QUOTE

1 2 3 4 5

=================================================== ================= 6 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 7 Input Data File: M:\_BMDS\msc_BAP_HABS1980_Mul tiCanc3_0.1.(d) 8 Gnuplot Plotting File: M:\_BMDS\msc_BAP_HABS1 980_MultiCanc3_0.1.plt 9 10

================================================== ================== 11 12 BMDS Model Run 13

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 14 15 The form of the probability function is: 16 17 P[response] = background + (1-background)*[1-EXP ( 18 -beta1*dose^1-beta2*dose^2-beta3*d ose^3)] 19 20 The parameter betas are restricted to be positiv e 21 22 23 Dependent variable = NumAff 24 Independent variable = LADD 25 26 Total number of observations = 4 27 Total number of records with missing values = 0 28 Total number of parameters in model = 4 29 Total number of specified parameters = 0 30 Degree of polynomial = 3 31 32 33 Maximum number of iterations = 250 34 Relative Function Convergence has been set to: 1e- 008 35 Parameter Convergence has been set to: 1e-008 36 37 38 39 Default Initial Parameter Values 40 Background = 0 41 Beta(1) = 0 42 Beta(2) = 4.23649 43 Beta(3) = 0 44 45 46 Asymptotic Correlation Matrix of Paramet er Estimates 47 48 ( *** The model parameter(s) -Backgroun d -Beta(1) -Beta(2) 49 have been estimated at a boundary point, or have been specified 50 by the user, 51 and do not appear in the correlati on matrix ) 52 53 Beta(3) 54 55 Beta(3) 1 56 57 58 59 Parameter Estimate s 60 61 95.0% Wald Confidence 62 Interval 63 Variable Estimate Std. Err. Lower Conf. Limit Upper 64 Conf. Limit 65 Background 0 * * * 66 Beta(1) 0 * * * 67 Beta(2) 0 * * * 68 Beta(3) 4.1289 * * * 69 70 * - Indicates that this value is not calculated. 71 72 73 74 Analysis of Deviance Table 75 76 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 77 Full model -34.8527 4 78 Fitted model -37.3373 1 4.9 6903 3 0.1741 79 Reduced model -82.5767 1 95. 4478 3 <.0001 80 81

AIC: 76.6745 82 83 84

### 380 DRAFT - DO NOT CITE OR QUOTE Goodness of Fit 1 Scaled 2 Dose Est._Prob. Expected Observed Size Residual 3

------------------------------------------------- ----------------------- 4

0.0000 0.0000 0.000 0.000 35 0.000 5

0.4900 0.3848 13.082 8.000 34 -1.791 6 0.7400 0.8123 21.933 24.000 27 1.019 7 0.8000 0.8792 21.102 22.000 24 0.563 8 9 Chi^2 = 4.56 d.f. = 3 P-value = 0.2067 10 11 12 Benchmark Dose Computation 13 14 Specified effect = 0.1 15 16 Risk Type = Extra risk 17 18 Confidence level = 0.95 19 20 BMD = 0.294407 21 22

BMDL = 0.215151 23 24

BMDU = 0.320955 25 26 Taken together, (0.215151, 0.320955) is a 90 % two-sided confidence 27 interval for the BMD 28 29 Multistage Cancer Slope Factor = 0.46479 30 31 32

### 381 DRAFT - DO NOT CITE OR QUOTE

Figure F-7. Fit of multistage model to skin tumors in female NMRI mice 1 

### exposed dermally to benzo[a]pyrene (Habs et al., 19 84); graph and model 2 

### output. 3

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=================================================== ================= 31 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 32 Input Data File: C:\Usepa\BMDS21\mscDax_Settin g.(d) 33 Gnuplot Plotting File: C:\Usepa\BMDS21\mscDax _Setting.plt 34 35

================================================== ================== 36 37 BMDS Model Run 38

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 39 40 The form of the probability function is: 41 42 P[response] = background + (1-background)*[1-EXP ( 43 -beta1*dose^1)] 44 45 The parameter betas are restricted to be positiv e 46 47 48 Dependent variable = tumors 49 Independent variable = LADD 50 51 Total number of observations = 3 52 Total number of records with missing values = 0 53 Total number of parameters in model = 2 54 Total number of specified parameters = 0 55 Degree of polynomial = 1 56 57 58 Maximum number of iterations = 250 59 Relative Function Convergence has been set to: 1e- 008 60 Parameter Convergence has been set to: 1e-008 61 62 63 0

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dose

Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

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0 0.2 0.4 0 0.2 0.4 0.6 0.8 1

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dose

Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 382 DRAFT - DO NOT CITE OR QUOTE

1 Default Initial Parameter Values 2 Background = 0 3 Beta(1) = 1.66414 4 5 6 Asymptotic Correlation Matrix of Paramet er Estimates 7 8 ( *** The model parameter(s) -Backgroun d 9 have been estimated at a boundary point, or have been specified by the user, 10 and do not appear in the correlati on matrix ) 11 12 Beta(1) 13 14 Beta(1) 1 15 16 17 18 Parameter Estimate s 19 20 95.0% Wald Confidence Interval 21 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 22 Background 0 * * * 23 Beta(1) 1.35264 * * * 24 25 * - Indicates that this value is not calculated. 26 27 28 29 Analysis of Deviance Table 30 31 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 32 Full model -22.217 3 33 Fitted model -22.7878 1 1.1 4175 2 0.565 34 Reduced model -41.0539 1 37. 6739 2 <.0001 35 36

AIC: 47.5757 37 38 39 Goodness of Fit 40 Scaled 41 Dose Est._Prob. Expected Observed Size Residual 42

------------------------------------------------- ----------------------- 43

0.0000 0.0000 0.000 0.000 20 0.000 44 0.5700 0.5375 10.749 9.000 20 -0.784 45

1.1400 0.7860 15.721 17.000 20 0.697 46 47 Chi^2 = 1.10 d.f. = 2 P-value = 0.5765 48 49 50 Benchmark Dose Computation 51 52 Specified effect = 0.1 53 54 Risk Type = Extra risk 55 56 Confidence level = 0.95 57 58

BMD = 0.0778926 59 60

BMDL = 0.0558466 61 62 BMDU = 0.111853 63 64 Taken together, (0.0558466, 0.111853) is a 90 % two-sided confidence 65 interval for the BMD 66 67 Multistage Cancer Slope Factor = 1.79062 68 69

### 383 DRAFT - DO NOT CITE OR QUOTE

Figure F-8. Fit of multistage model to skin tumors in female CFLP mice 1 

### exposed dermally to benzo[a]pyrene (Grimmer et al, 1983); graph and model 2 

### output. 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 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 36 Input Data File: 37 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grim mer1983CFLPmice\1MulGriMS_.(d) 38 Gnuplot Plotting File: 39 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grim mer1983CFLPmice\1MulGriMS_.plt 40 41

================================================== ================== 42 43 BMDS Model Run 44

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 45 46 The form of the probability function is: 47 48 P[response] = background + (1-background)*[1-EXP ( 49 -beta1*dose^1)] 50 51 The parameter betas are restricted to be positiv e 52 53 54 Dependent variable = incidence 55 Independent variable = dose 56 57 Total number of observations = 4 58 Total number of records with missing values = 0 59 Total number of parameters in model = 2 60 Total number of specified parameters = 0 61 Degree of polynomial = 1 62 63 64 Maximum number of iterations = 250 65 Relative Function Convergence has been set to: 2.2 2045e-016 66 Parameter Convergence has been set to: 1.49012e-00 8 67 68 0

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BMD BMDL Multistage Cancer Linear extrapolation

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0 0.5 1 0 0.2 0.4 0.6 0.8 1

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dose

Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 384 DRAFT - DO NOT CITE OR QUOTE

**** We are sorry but Relative Function and Parame ter Convergence   1 ** ** are currently unavailable in this model. Ple ase keep checking   2 ** ** the web sight for model updates which will ev entually   3 ** ** incorporate these convergence criterion. Def ault values used.   4 5 6 7 Default Initial Parameter Values 8 Background = 0 9 Beta(1) = 0.478645 10 11 12 Asymptotic Correlation Matrix of Paramet er Estimates 13 14 ( ** * The model parameter(s) -Backgroun d 15 have been estimated at a boundary point, or have been specified by the user, 16 and do not appear in the correlati on matrix ) 17 18 Beta(1) 19 20 Beta(1) 1 21 22 23 24 Parameter Estimate s 25 26 95.0% Wald Confidence Interval 27 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 28 Background 0 * * * 29 Beta(1) 0.430366 * * * 30 31 * - Indicates that this value is not calculated. 32 33 34 35 Analysis of Deviance Table 36 37 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 38 Full model -108.532 4 39 Fitted model -108.943 1 0.82 3537 3 0.8438 40 Reduced model -186.434 1 155 .805 3 <.0001 41 42

AIC: 219.887 43 44 45 Goodness of Fit 46 Scaled 47 Dose Est._Prob. Expected Observed Size Residual 48

------------------------------------------------- ----------------------- 49

0.0000 0.0000 0.000 0.000 80 -0.000 50

1.1100 0.3798 24.687 22.000 65 -0.687 51

2.2000 0.6120 39.169 39.000 64 -0.043 52

4.4000 0.8495 54.366 56.000 64 0.571 53 54 Chi^2 = 0.80 d.f. = 3 P-value = 0.8496 55 56 57 Benchmark Dose Computation 58 59 Specified effect = 0.1 60 61 Risk Type = Extra risk 62 63 Confidence level = 0.95 64 65

BMD = 0.244816 66 67 BMDL = 0.208269 68 69 BMDU = 0.289606 70 71 Taken together, (0.208269, 0.289606) is a 90 % two-sided confidence 72 interval for the BMD 73 74 Multistage Cancer Slope Factor = 0.480148 75 76 77

78

### 385 DRAFT - DO NOT CITE OR QUOTE

Figure F-9. Fit of multistage model to skin tumors in female CFLP mice 1 

### exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and 2 

### model output. 3

4 0

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dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 386 DRAFT - DO NOT CITE OR QUOTE

1

2

================================================== ================== 3 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 4 Input Data File: 5 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984 _MultiCanc1_0.1.(d) 6 Gnuplot Plotting File: 7 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984 _MultiCanc1_0.1.plt 8 Wed Apr 27 17:11:28 2011 9 10 [add notes here] 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14 15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19 20 21 Dependent variable = NumAff 22 Independent variable = LADD 23 24 Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29 30 31 Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34 35 36 37 Default Initial Parameter Values 38 Background = 0.311241 39 Beta(1) = 0.502556 40 41 42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been specified 46 by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52 53 54 55 Parameter Estimate s 56 57 95.0% Wald Confidence 58 Interval 59 Variable Estimate Std. Err. Lower Conf. Limit Upper 60 Conf. Limit 61 Background 0 * * * 62 Beta(1) 0.796546 * * * 63 64 * - Indicates that this value is not calculated. 65 66 67 68 Analysis of Deviance Table 69 70 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 71 Full model -95.8385 4 72 Fitted model -101.643 1 1 1.61 3 0.008846 73 Reduced model -175.237 1 158 .797 3 <.0001 74 75

AIC: 205.287 76 77 78 Goodness of Fit 79 Scaled 80 Dose Est._Prob. Expected Observed Size Residual 81

------------------------------------------------- ----------------------- 82 0.0000 0.0000 0.000 0.000 65 0.000 83

### 387 DRAFT - DO NOT CITE OR QUOTE 0.9700 0.5382 34.446 43.000 64 2.145 1

1.9100 0.7816 50.804 53.000 65 0.659 2 3.9000 0.9552 62.091 57.000 65 -3.054 3 4 Chi^2 = 14.36 d.f. = 3 P-value = 0.0025 5 6 7 Benchmark Dose Computation 8 9 Specified effect = 0.1 10 11 Risk Type = Extra risk 12 13 Confidence level = 0.95 14 15

BMD = 0.132272 16 17 BMDL = 0.113427 18 19

BMDU = 0.154848 20 21 Taken together, (0.113427, 0.154848) is a 90 % two-sided confidence 22 interval for the BMD 23 24 Multistage Cancer Slope Factor = 0.881621 25 26

### 388 DRAFT - DO NOT CITE OR QUOTE

Figure F-9. Fit of log-logistic model to skin tumo rs in female CFLP mice 1 

### exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and 2 

### model output. 3

4

5

=================================================== ================= 6 Logistic Model. (Version: 2.12; Date: 05/16/2 008) 7 Input Data File: 8 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmer1984 _Grimmer1984_0.70u.(d) 9 Gnuplot Plotting File: 10 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmer1984 _Grimmer1984_0.70u.plt 11 12

================================================== ================== 13 14 BMDS Model Run 15

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 16 17 The form of the probability function is: 18 19 P[response] = background+(1-background)/[1+EXP(- intercept-slope*Log(dose))] 20 21 22 Dependent variable = NumAff 23 Independent variable = LADD 24 Slope parameter is not restricted 25 26 Total number of observations = 4 27 Total number of records with missing values = 0 28 Maximum number of iterations = 250 29 Relative Function Convergence has been set to: 1 e-008 30 Parameter Convergence has been set to: 1e-008 31 32 33 34 User has chosen the log transformed model 35 36 37 Default Initial Parameter Values 38 background = 0 39 intercept = 0.799142 40 slope = 0.894129 41 42 43 Asymptotic Correlation Matrix of Paramet er Estimates 44 45 ( *** The model parameter(s) -backgroun d 46 have been estimated at a boundary point, or have been specified 47 by the user, 48 0

0.2 0.4 0.6 0.8 1

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dose Log-Logistic Model with 0.95 Confidence Level

BMDL BMD

Log-Logistic

### 389 DRAFT - DO NOT CITE OR QUOTE and do not appear in the correlati on matrix ) 1 2 intercept slope 3 4 intercept 1 -0.68 5 6 slope -0.68 1 7 8 9 10 Parameter Estimate s 11 12 95.0% Wald Confidence 13 Interval 14 Variable Estimate Std. Err. Lower Conf. Limit Upper 15 Conf. Limit 16 background 0 * * * 17 intercept 0.783559 * * * 18 slope 0.922655 * * * 19 20 * - Indicates that this value is not calculated. 21 22 23 24 Analysis of Deviance Table 25 26 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 27 Full model -95.8385 4 28 Fitted model -95.9236 2 0.1 7031 2 0.9184 29 Reduced model -175.237 1 158 .797 3 <.0001 30 31

AIC: 195.847 32 33 34 Goodness of Fit 35 Scaled 36 Dose Est._Prob. Expected Observed Size Residual 37

------------------------------------------------- ----------------------- 38 0.0000 0.0000 0.000 0.000 65 0.000 39

0.9700 0.6804 43.543 43.000 64 -0.146 40

1.9100 0.7991 51.941 53.000 65 0.328 41 3.9000 0.8849 57.516 57.000 65 -0.200 42 43 Chi^2 = 0.17 d.f. = 2 P-value = 0.9190 44 45 46 Benchmark Dose Computation 47 48 Specified effect = 0.7 49 50 Risk Type = Extra risk 51 52 Confidence level = 0.95 53 54

BMD = 1.07152 55 56

BMDL = 0.478669 57 58

### 390 DRAFT - DO NOT CITE OR QUOTE

Figure F-11. Fit of multistage model to skin tumor s in female CFLP mice 1 

### exposed dermally to benzo[a]pyrene (Grimmer et al., 1984), highest dose 2 

### dropped; graph and model output. 3 4

0

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dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

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dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 391 DRAFT - DO NOT CITE OR QUOTE

1 2 3

=================================================== ================= 4 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 5 Input Data File: 6 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984 _drophidose_Setting.(d) 7 Gnuplot Plotting File: 8 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984 _drophidose_Setting.plt 9 10

================================================== ================== 11 12 BMDS Model Run 13

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 14 15 The form of the probability function is: 16 17 P[response] = background + (1-background)*[1-EXP ( 18 -beta1*dose^1-beta2*dose^2)] 19 20 The parameter betas are restricted to be positiv e 21 22 23 Dependent variable = tumors 24 Independent variable = LADD 25 26 Total number of observations = 3 27 Total number of records with missing values = 0 28 Total number of parameters in model = 3 29 Total number of specified parameters = 0 30 Degree of polynomial = 2 31 32 33 Maximum number of iterations = 250 34 Relative Function Convergence has been set to: 1e- 008 35 Parameter Convergence has been set to: 1e-008 36 37 38 39 Default Initial Parameter Values 40 Background = 0.0806622 41 Beta(1) = 0.88595 42 Beta(2) = 0 43 44 45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Backgroun d -Beta(2) 48 have been estimated at a boundary point, or have been specified 49 by the user, 50 and do not appear in the correlati on matrix ) 51 52 Beta(1) 53 54 Beta(1) 1 55 56 57 58 Parameter Estimate s 59 60 95.0% Wald Confidence 61 Interval 62 Variable Estimate Std. Err. Lower Conf. Limit Upper 63 Conf. Limit 64 Background 0 * * * 65 Beta(1) 0.997118 * * * 66 Beta(2) 0 * * * 67 68 * - Indicates that this value is not calculated. 69 70 71 72 Analysis of Deviance Table 73 74 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 75 Full model -71.5928 3 76 Fitted model -72.2756 1 1.3 6568 2 0.5052 77 Reduced model -134.46 1 125 .735 2 <.0001 78 79 AIC: 146.551 80 81 82 Goodness of Fit 83 Scaled 84

### 392 DRAFT - DO NOT CITE OR QUOTE Dose Est._Prob. Expected Observed Size Residual 1

------------------------------------------------- ----------------------- 2 0.0000 0.0000 0.000 0.000 65 0.000 3

0.9700 0.6199 39.671 43.000 64 0.857 4

1.9100 0.8511 55.322 53.000 65 -0.809 5 6 Chi^2 = 1.39 d.f. = 2 P-value = 0.4992 7 8 9 Benchmark Dose Computation 10 11 Specified effect = 0.1 12 13 Risk Type = Extra risk 14 15 Confidence level = 0.95 16 17 BMD = 0.105665 18 19

BMDL = 0.0881529 20 21

BMDU = 0.149328 22 23 Taken together, (0.0881529, 0.149328) is a 90 % two-sided confidence 24 interval for the BMD 25 26 Multistage Cancer Slope Factor = 1.13439 27 28 29

30 31 32 33

### 393 DRAFT - DO NOT CITE OR QUOTE

Figure F-12. Fit of multistage model to skin tumor s in female CFLP mice 1 

### exposed dermally to benzo[a]pyrene (Sivak et al., 1997); graph and model 2 

### output. 3 4 5

6 7

=================================================== ================= 8 Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) 9 Input Data File: C:\Usepa\BMDS21\Data\msc_benz o[a]pyrene_Sivak1993_MultiCanc2_0.1.(d) 10 Gnuplot Plotting File: 11 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivak1993_M ultiCanc2_0.1.plt 12 13

================================================== ================== 14 15 [add notes here] 16

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 17 18 The form of the probability function is: 19 20 P[response] = background + (1-background)*[1-EXP ( 21 -beta1*dose^1-beta2*dose^2)] 22 23 The parameter betas are restricted to be positiv e 24 25 26 Dependent variable = NumAff 27 Independent variable = LADD 28 29 Total number of observations = 4 30 Total number of records with missing values = 0 31 Total number of parameters in model = 3 32 Total number of specified parameters = 0 33 Degree of polynomial = 2 34 35 36 Maximum number of iterations = 250 37 Relative Function Convergence has been set to: 1e- 008 38 Parameter Convergence has been set to: 1e-008 39 40 41 42 Default Initial Parameter Values 43 Background = 0 44 Beta(1) = 0.0936505 45 Beta(2) = 8.67239 46 47 48 0

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dose Multistage Cancer Model with 0.95 Confidence Level

BMD BMDL Multistage Cancer Linear extrapolation

### 394 DRAFT - DO NOT CITE OR QUOTE

Asymptotic Correlation Matrix of Paramet er Estimates 1 2 ( *** The model parameter(s) -Backgroun d -Beta(1) 3 have been estimated at a boundary point, or have been specified by the user, 4 and do not appear in the correlati on matrix ) 5 6 Beta(2) 7 8 Beta(2) 1 9 10 11 12 Parameter Estimate s 13 14 95.0% Wald Confidence Interval 15 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 16 Background 0 * * * 17 Beta(1) 0 * * * 18 Beta(2) 8.9375 * * * 19 20 * - Indicates that this value is not calculated. 21 22 23 24 Analysis of Deviance Table 25 26 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 27 Full model -23.2693 4 28 Fitted model -23.3009 1 0.063 1003 3 0.9959 29 Reduced model -69.5898 1 92 .641 3 <.0001 30 31

AIC: 48.6018 32 33 34 Goodness of Fit 35 Scaled 36 Dose Est._Prob. Expected Observed Size Residual 37

------------------------------------------------- ----------------------- 38 0.0000 0.0000 0.000 0.000 30 0.000 39

0.0100 0.0009 0.027 0.000 30 -0.164 40

0.1400 0.1607 4.821 5.000 30 0.089 41 0.5100 0.9022 27.065 27.000 30 -0.040 42 43 Chi^2 = 0.04 d.f. = 3 P-value = 0.9982 44 45 46 Benchmark Dose Computation 47 48 Specified effect = 0.1 49 50 Risk Type = Extra risk 51 52 Confidence level = 0.95 53 54

BMD = 0.108575 55 56

BMDL = 0.058484 57 58

BMDU = 0.129641 59 60 Taken together, (0.058484, 0.129641) is a 90 % two-sided confidence 61 interval for the BMD 62 63 Multistage Cancer Slope Factor = 1.70987 64 65

### 395 DRAFT - DO NOT CITE OR QUOTE

APPENDIX G. Additional Information in Support of t he Dermal Slope factor 1

2 

### Mouse Dermal Carcinogenesis Exposure Methods 3 

### Studies which macroscopically examined systemic org ans for tumors include Roe et al., 4 

### 1970; Schmidt et al., 1973; Schmahl et al., 1977; H abs et al., 1980, 1984; Grimmer et al., 1983, 5 

### 1984. The studies by Roe et al. 1970 and Habs et a l. 1984 observed systemic tumors which the 6



### authors did not consider to be treatment related. The other studies which conducted post mortem 7 

### macroscopic examinations of abnormal tissues, did n ot report any treatment related systemic 8



### effects. 9396 DRAFT - DO NOT CITE OR QUOTETable G-1: Exposure methods for selected lifetimedermal exposure mouse cancer bioassays for benzo[a]pyreneinduced skin tumorsMousestrainsexnApplied dose ofbenzo[a]pyrene,times per week,vehicleApplication method, volume Application region Duration(weeks)Comments ReferenceCeH/HeJmale300, 0.05, 0.5, or 5μg2x/wkcyclohexane/acetone (1:1)not specified0.050 mlshaved dorsal skin 104 Sivak et al.,1997C57Lmale13-550, 0.15, 0.38, 0.75, 3.8, 19,94, 188, 376, or 752μg3x/wktoluenecalibrated needle dropper0.0075 ml(solvent let dry between drops tolimit spread)shaved interscapularskin103 mice were 18-20 weeks ofage at the start of study“principal organs”examinedPoel, 1959SWRmale14-250, 0.15, 0.38, 0.75, 3.8,19.0, 94.0, or 470μg3x/wktolueneCalibrated needle pipette0.0075 mlShaved interscapularskinlifetime1exposed until tumordevelopment or deathPoel, 1960C3HeBmale14-250, 0.15, 0.38, 0.75, 3.8,19.0, 94.0, or 470μgbenzo[a]pyrene3x/wktolueneCalibrated needle pipette0.0075mlShaved interscapularskin104lifetime1exposed until tumordevelopment or deathPoel, 1960A/Hemale14-250, 0.15, 0.38, 0.75, 3.8,19.0, 94.0, or 470μgbenzo[a]pyrene3x/wktolueneCalibrated needle pipette0.0075 mlShaved interscapularskin104lifetime1exposed until tumordevelopment or deathPoel, 1960Swiss SPFfemale500, 0.1, 0.3, 1, 3, or 9μg3x/wkacetoneCalibrated pipette0.25 mlEntire shaved dorsalarea93 systemic post-mortemexamRoe et al.,1970397 DRAFT - DO NOT CITE OR QUOTE1Treated until natural death or sacrifice followingtumor formation12NMRIfemale1000, 0.05, 0.2, 0.8, or 2μg2x/wkacetoneDrop burette (dose delivered insingle drop; volume not given)Shaved back 104lifetime1macroscopic examinationof internal organsSchmidt etal., 1973Swiss SPFfemale800, 0.05, 0.2, 0.8, or 2μg2x/wkacetoneDrop burette (dose delivered insingle drop; volume not given)Shaved back 104lifetime1macroscopic examinationof internal organsSchmidt etal., 1973NMRIfemale1000, 1, 1.7, or 3μg2x/wkacetoneDrop application with syringe0.02 mlShaved back 104lifetime1macroscopic examinationof internal organsSchmahl etal., 1977NMRIfemale650, 1.7, 2.8, and 4.6 ug2x/wkDrop application by calibratedsyringe,0.02 mldorsal skin ininterscapular area104lifetime1macroscopic examinationof internal organsHabs et al.,1980CFLPfemale650, 3.9, 7.7 and 15.4μgacetone/DMSO (1:3)2x/wkDrop0.1 mlDorsal skin,interscapular area104 macroscopic examinationof internal organsGrimmer etal., 1983CFLPfemale650, 3.4, 6.7, and 13.5μgacetone:DMSO (1:3)2x/wkDrop0.1 mlDorsal skin,interscapular area104 macroscopic examinationof internal organsGrimmer etal., 1984NMRIfemale200, 2 or 4μgacetone2x/wkdrop application by calibratedsyringe0.01 mldorsal skin ininterscapular arealifetime1macroscopic examinationof internal organsHabs et al.,1984

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APPENDIX H. Alternative Approaches for Cross-Speci es Scaling of the Dermal Slope 1



### Factor 2

3 

### Several publications which develop a dermal slope f actor for benzo[a]pyrene are 4 

### available in the peer reviewed literature (Knafla e t al., 2010; 2006; Hussain et al., 1998; LaGoy 5 

### and Quirk 1994; Sullivan et al., 1991). With the e xception of the 2010 Knafla et al. publication, 6 

### none of these approaches applied quantitative adjus tments to account for interspecies differences, 7 

### though the proposed slope factors were developed to account for human risk. Knafla et al. 8 

### (2010) qualitatively discuss processes which could affect the extrapolation between mice and 9 

### humans including skin metabolic activity adduct for mation, stratum corneum thickness, 10 

### epidermal thickness, etc. Ultimately, the authors apply an adjustment based on the increased 11 

### epidermal thickness of human skin on the arms and h ands compared to mouse interscapular 12 

### epidermal thickness. They hypothesize that the car cinogenic potential of benzo[a]pyrene may be 13 

### related to the thickness of the epidermal layer. 14 

### Because there is no established methodology for cro ss-species extrapolation of dermal 15 

### toxicity, several alternative approaches were evalu ated. Each approach begins with the POD of 16 

### 0.066 μg/day that was based on a 10% extra risk for skin tumors in male mice (see Section 17 

### 5.4.3). Based on the assumptions of each approach, a dermal slope factor for humans is 18 

### calculated. The discussion of these approaches use s the following abbreviations: 19 20 DSF = dermal slope factor 21 POD M = point of departure (for 10% extra risk) from mou se bioassay, in μg/day 22 BW M = mouse body weight = 0.035 kg (assumed) 23 BW H = human body weight = 70 kg (assumed) 24 SA H = total human surface area = 19,000 cm 2 (assumed) 25 SA M = total mouse surface area = 100 cm 2 (assumed) 26

27 

### Approach 1. No interspecies adjustment to daily ap plied dose (POD) in mouse model 28 

### Under this approach, a given mass of benzo[a]pyrene , applied daily, would pose the same 29 

### risk in an animal or in humans, regardless of wheth er it is applied to a small surface area or to a 30 

### larger surface area at a proportionately lower conc entration. 31

32 

### DSF = 0.1/ POD M 33

34 

### DSF= 0.1/0.066 μg/day = 2 (μg/day) -1 35

36 

### Assumptions: The same mass of benzo[a]pyrene, applied daily, wo uld have same potency in 37 

### mice as in human skin regardless of treatment area. 38

39

40 

### Approach 2. Cross-species adjustment based on whol e body surface-area scaling 41 

### Under this approach, animals and humans are assumed to have equal lifetime cancer risk 42 

### with equal average whole body exposures in loading units (μg/cm 2 

### -day). As long as doses are 43 

### low enough that risk is proportional to the mass of applied compound, the daily dermal dose of 44 

### benzo[a]pyrene can be normalized over the total sur face area. 45

46 

### POD (μg/cm 2 

### -day) = POD M/SA 

### (μg/cm 2 

### -day) = POD M 

### (μg/day) / SA M 

### (cm 2 

### ) 47

48 

### POD = (0.066 μg/day) / 100 cm 2 49

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= 0.00066 μg/cm 2 

### -day 1

2 

### DSF = 0.1/(0.00066 μg/cm 2 

### -day) ≈ 152 (μg/cm 2 

### -day) -1 3

4 

### Assumptions : Mouse and human slope factors are equipotent if t otal dermal dose is averaged 5 

### over equal fractions of the entire surface area. T umor potency of benzo[a]pyrene is assumed to 6 

### be related to overall dose and not dose per unit ar ea. For example, someone exposed to 0.01 7 

### μg/day on 10 cm 2 

### would be assumed to have the same potential to for m a skin tumor as someone 8 

### treated with 0.01 μg/day over 10,000 cm 2



### . 9

10

11 

### Approach 3. Cross-species adjustment based on body weight 12 

### Under this approach, a given mass of benzo[a]pyrene is normalized relative to the body 13 

### weight of the animal or human. This approach has b een used for oral doses for noncancer 14 

### effects. 15

16 

### POD M 

### / BW M 

### = 0.066 μg/0.035 kg-day = 1.9 μg/kg-day 17

18 

### DSF = 0.1/1.9 μg/kg-day = 0.05 (μg/kg-day) -1 19

20 

### Assumptions : The potency of point of contact skin tumors is re lated to bodyweight and humans 21 

### and mice would have an equal likelihood of developi ng skin tumors based on a dermal dose per 22 

### kg basis. 23

24 

### Issues : Skin cancer following benzo[a]pyrene exposure is a local effect and not likely dependent 25 

### on body weight. 26

27

28 

### Approach 4. Cross-species adjustment based on allo metric scaling using body weight to 29 

### the 3/4 power 30 

### Under this approach, rodents and humans exposed to the same daily dose of a carcinogen, 31 

### adjusted for BW 3/4 

### , would be expected to have equal lifetime risks of cancer. That is, a lifetime 32 

### dose expressed as μg/kg 3/4 

### -day would lead to an equal risk in rodents and hum ans. This scaling 33 

### reflects the empirically observed phenomena of more rapid distribution, metabolism, and 34 

### clearance in smaller animals. The metabolism of be nzo[a]pyrene to reactive intermediates is a 35 

### critical step in the carcinogenicity of benzo[a]pyr ene, and this metabolism occurs in the skin. 36

37 

### POD (μg/day) = POD M 

### (μg/day) × (BW H 

### / BW M 

### ) 3/4 38

39 

### POD (μg/day) = 0.066 μg/day × (70 kg / 0.035 kg) 3/4 40 

### = 19.7 μg/day 41

42 

### DSF = 0.1/(19.7 μg/day) ≈ 0.005 (μg/day) -1 43

44 

### Assumptions: Risk at low doses of benzo[a]pyrene is dependent on absolute dermal dose and not 45 

### dose per unit of skin, meaning a higher exposure co ncentration of benzo[a]pyrene contacting a 46 

### smaller area of exposed skin could carry the same r isk of skin tumors as a lower exposure 47 

### concentration of benzo[a]pyrene that contacts a lar ger area of skin. 48

49

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Issues: It is unclear if scaling of doses based on bodywe ight ratios will correspond to differences 1 

### in metabolic processes in the skin of mice and huma ns. 2

3

4 

### Synthesis of the alternative approaches to cross-sp ecies scaling 5 

### A comparison of the above approaches is provided i n Table H-1 below. The lifetime risk 6 

### from a nominal human dermal exposure to benzo[a]pyr ene over a 5% area of exposed skin 7 

### (approximately 950 cm 2 

### ), estimated at 1 x 10 -4 

### μg/day*, is calculated for each of the approaches 8 

### in order to judge whether the method yields risk es timates that are unrealistically high. 9

10 

### Other potential interspecies adjustments 11 

### The above discussion presents several mathematical approaches that result from varying 12 

### assumptions about what is the relevant dose metric for determining equivalence across species. 13 

### Biological information (that is not presently compr ehensive or detailed enough to develop robust 14 

### models) that could be used in future biologically b ased models for cross-species extrapolation 15 

### include: 16

17



### a. Quantitative information on interspecies difference s in partitioning from exposure 18 

### medium to the skin and absorption through the skin 19 

### b. Thickness of the stratum corneum between anatomical sites and between species 20



### c. Thickness of epidermal layer 21 

### d. Skin permeability 22



### e. Metabolic activity of skin 23

f. 

### Formation of DNA adducts in skin 24

25401 DRAFT - DO NOT CITE OR QUOTE1Table H-1. Alternative approaches to cross-speciesscaling2ApproachAssumptionsDose metric DSFRisk at nominalexposure(0.0001 μg/day)*1. Mass-perday scalingEqual mass per day (μg /d), if applied to equal areasof skin (cm2), will affect similarnumbers of cells across species. Cancer risk is proportional to the area (cm2) exposed ifthe loading rate (μg /cm2-d) is the same. This approach assumes that risk is proportionalto dose expressed as mass per day. This approach implies that any combination of loadingrate (μg /cm2-day) and skin area exposed (cm2) that have the same product whenmultiplied, will result in the same risk.μg/day 1 per μg/day2 x 10-42. Surfacearea scalingEqual mass per day (μg /d), if applied to equal fractionsof total skin surface (cm2) willhave similar cancer risks. That is, whole-body lifetime exposure [e.g., 5%-of-the-bodylifetime exposure] at the same loading rate (μg /cm2-d) gives similar cancer risks acrossspecies. This approach assumes that risk is proportional to dose expressed as mass perarea per day. This approach implies that risk doesnot increase with area exposed as longas dose per area remains constant.μg/cm2-day152 perμg/cm2-day8 x 10-73. BodyweightscalingThe skin is an organ with thickness and volume; benzo[a]pyrene is distributed within thisvolume of skin. Cancer risk is proportional to theconcentration of benzo[a]pyrene in theexposed volume of skin. Equal mass per day (μg /d), if distributed within equal fractionsof total body skin will have similar cancer risks.That is, whole-body lifetime exposure[e.g., 5%-of-the-body lifetime exposure] at the same loading rate (μg /cm2-d) gives similarcancer risks across species. This approach assumesthat risk is proportional to doseexpressed as mass per kg body weight per day. This approach implies that anycombination of dose (μg /day) and body weight (kg)that have the same result whendivided, will result in the same risk.μg/kg-day0.05 perμg/kg-day8 x 10-84. Allometricscaling(BW3/4)Same as for body-weight scaling, except that benzo[a]pyrene distribution and metabolismtakes place within this volume of skin. Allometricscaling is generally regarded asdescribing the relative rate of toxicokinetic processes across species. This approach alsois used by EPA to scale oral exposures.μg/day0.005 per μg/day5 x 10-7* Nominal exposure calculated as a geometric mean of average daily doses (μg/day) calculated from a range of benzo[a]pyrene soil concentrations (1- 1000ppb) reported3from non-contaminated rural/agricultural soils (ATSDR, 1995) and a range of standard exposure assumptions.4