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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 review under applicable information quality 

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

be construed to represent any Agency determination or policy. It is being circulated for review 

of its technical accuracy and science policy implications. 

U.S. Environmental Protection Agency 

Washington, DC 
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DISCLAIMER 

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

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

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

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

names or commercial products does not constitute endorsement or recommendation for use. 
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1 CONTENTS TOXICOLOGICAL REVIEW OF BENZO[a]PYRENE (CAS No. 50-32-8) 

2 

3 

4 LIST OF TABLES ....................................................................................................................... VII 

5 LIST OF FIGURES ....................................................................................................................... X 

6 LIST OF ABBREVIATIONS AND ACRONYMS .................................................................... XII 

7 FOREWORD ............................................................................................................................. XVI 

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

9 

10 1. INTRODUCTION ..................................................................................................................... 1

11 

12 2. CHEMICAL AND PHYSICAL INFORMATION ................................................................... 3

13 

14 3. TOXICOKINETICS .................................................................................................................. 9 

15 3.1. ABSORPTION .................................................................................................................... 9 

16 3.1.1. Inhalation Exposure ...................................................................................................... 9 

17 3.1.1.1. Inhalation Exposure in Humans ........................................................................... 9 

18 3.1.1.2. Inhalation Exposure in Animals.......................................................................... 11 

19 3.1.2. Oral Exposure ............................................................................................................. 17 

20 3.1.2.1. Oral Exposure in Humans .................................................................................. 18 

21 3.1.2.2. Oral Exposure in Animals ................................................................................... 18 

22 3.1.3. Dermal Exposure ........................................................................................................ 20 

23 3.1.3.1. Dermal Exposure in Humans .............................................................................. 20 

24 3.1.3.2. Dermal Exposure in Animals .............................................................................. 23 

25 3.1.4. Other Types of Exposure ............................................................................................ 25 

26 3.2. DISTRIBUTION ............................................................................................................... 26 

27 3.2.1. Inhalation Exposure .................................................................................................... 26 

28 3.2.2. Oral Exposure ............................................................................................................. 28 

29 3.2.3. Dermal and Other Exposures...................................................................................... 29 

30 3.3. METABOLISM ................................................................................................................ 32 

31 3.3.1. Phase I Metabolism .................................................................................................... 34 

32 3.3.1.1. CYP450-dependent Reactions ............................................................................. 34 

33 3.3.1.2. Non-CYP-related metabolic pathways ................................................................ 38 

34 3.3.2. Phase II Metabolism ................................................................................................... 40 

35 3.3.3. Tissue-specific Metabolism ........................................................................................ 43 

36 3.3.3.1. Respiratory Tract Tissues ................................................................................... 43 

37 3.3.3.2. GI Tract and Liver Tissues ................................................................................. 47 

38 3.3.3.3. Skin ...................................................................................................................... 48 

39 3.3.3.4. Reproductive Tissues and Fetal Metabolism ...................................................... 50 

40 3.3.3.5. Other Tissues ...................................................................................................... 53 

41 3.4. ELIMINATION ................................................................................................................ 53 

42 3.4.1. Inhalation Exposure .................................................................................................... 53 

43 3.4.2. Oral Exposure ............................................................................................................. 56 

44 3.4.3. Dermal Exposure and Other Exposure Routes ........................................................... 57 

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

46 

47 4. HAZARD IDENTIFICATION ................................................................................................ 64 

48 4.1. HUMANS STUDIES ........................................................................................................ 64 

49 4.1.1. Sources of Human Exposure ...................................................................................... 64 

50 4.1.2. Biomonitoring of benzo[a]pyrene Exposure and Effects In Humans ........................ 64 
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1 4.1.2.1. Urinary Excretion of benzo[a]pyrene ................................................................. 65 

2 4.1.2.2. Adduct Formation with DNA and Protein .......................................................... 66 

3 4.1.2.2.1. DNA Adduct measures in blood ........................................................................ 69 

4 4.1.2.2.2. Adduct measures in reproductive tissues .......................................................... 72 

5 4.1.2.3. Benzo[a]pyrene-Induced Cytogenetic Damage .................................................. 73 

6 4.1.3. Epidemiologic Findings in Humans ........................................................................... 75 

7 4.1.3.1. Molecular Epidemiology and Case-Control Cancer Studies .............................. 76 

8 4.1.3.2. Cohort Cancer Studies ........................................................................................ 77 

9 4.1.3.3. Noncancer Disease Caused by benzo[a]pyrene ................................................. 81 

10 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

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

12 4.2.1. Oral ............................................................................................................................. 85 

13 4.2.1.1. Subchronic Studies .............................................................................................. 85 

14 4.2.1.2. Chronic Studies and Cancer Bioassays .............................................................. 93 

15 4.2.2. Inhalation .................................................................................................................. 106 

16 4.2.2.1. Short-term and Subchronic Studies .................................................................. 106 

17 4.2.2.2. Chronic Studies and Cancer Bioassays ............................................................ 106 

18 4.2.3. Dermal Exposure ...................................................................................................... 108 

19 4.2.3.1. Skin-Tumor Initiation-Promotion Assays ......................................................... 108 

20 4.2.3.2. Carcinogenicity Dermal Bioassays................................................................... 109 

21 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL, INHALATION, AND 

22 DERMAL ........................................................................................................................ 118 

23 4.3.1. Oral ........................................................................................................................... 118 

24 4.3.2. Inhalation .................................................................................................................. 124 

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

26 4.4.1. Acute Neurological Studies ...................................................................................... 129 

27 4.4.2. Immunological Studies ............................................................................................. 133 

28 4.4.2.1. Oral Exposure Immunological Studies ............................................................. 133 

29 4.4.2.2. Inhalation Exposure Immunological Studies .................................................... 134 

30 4.4.2.3. Other Exposure Route Immunological Studies ................................................. 134 

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

32 4.4.3. Cancer Bioassays (Other Routes of Exposure) ........................................................ 135 

33 4.4.4. Atherogenesis Studies .............................................................................................. 136 

34 4.4.5. Reproductive Studies (Other Routes of Exposure) ................................................... 138 

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

36 4.5.1. Genotoxicity ............................................................................................................ 140 

37 4.5.2. Metabolic pathways .................................................................................................. 161 

38 4.5.3. Mechanistic Studies- Mutagenesis and Tumor Initiation .......................................... 164 

39 4.5.3. Tumor Promotion and Progression ........................................................................... 166 

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

41 4.6.1. Oral ........................................................................................................................... 171 

42 4.6.2. Inhalation ................................................................................................................. 177 

43 4.6.3. Dermal ...................................................................................................................... 180 

44 4.6.4. Mode-of-Action Information .................................................................................... 180 

45 4.6.4.1. Forestomach Lesions from Oral Exposure ....................................................... 180 

46 4.6.4.2. Immune System Effects ...................................................................................... 180 

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

48 4.7. EVALUATION OF CARCINOGENICITY ................................................................... 185 

49 4.7.1. Summary of Overall Weight-of-Evidence ................................................................ 185 
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1 4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence ............................... 185 

2 4.7.3. Mode-of-Action Information .................................................................................... 187 

3 4.7.3.1. Hypothesized MOA ........................................................................................... 187 

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

5 4.7.3.2. Other Possible MOAs ....................................................................................... 190 

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

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

8 4.8.1. Possible Childhood Susceptibility ............................................................................ 193 

9 4.8.2. Possible Gender Differences .................................................................................... 196 

10 4.8.3. Genetic Polymorphisms ............................................................................................ 198

11 

12 5. DOSE-RESPONSE ASSESSMENTS ................................................................................... 201 

13 5.1. ORAL REFERENCE DOSE (RfD) ................................................................................ 201 

14 5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification ... 201 

15 5.1.2. METHODS OF ANALYSIS ........................................................................................... 205 

16 5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)................... 209 

17 5.1.4. Previous RfD Assessment ........................................................................................ 210 

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

19 5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification ... 210 

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

21 5.2.3. RfC Derivation- Including Application of Uncertainty Factors (UFs) .................... 214 

22 5.2.4. Previous RfC Assessment......................................................................................... 215 

23 5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION 

24 REFERENCE CONCENTRATION ............................................................................... 216 

25 5.4. CANCER ASSESSMENT .............................................................................................. 217 

26 5.4.1. Oral Exposure—Oral Slope Factor .......................................................................... 217 

27 5.4.1.1. Choice of Study/Data—with Rationale and Justification—Oral Exposure ...... 217 

28 5.4.1.2. Dose-response Data—Oral Exposure............................................................... 218 

29 5.4.1.3. Dose Adjustments and Extrapolation Method(s)—Oral Exposure ................... 220 

30 5.4.1.4. Oral Slope Factor Derivation ........................................................................... 223 

31 5.4.2. Inhalation Exposure—Inhalation Unit Risk ............................................................. 225 

32 5.4.2.1. Choice of Study/Data—with Rationale and Justification—Inhalation Exposure

33 ......................................................................................................................................... 225 

34 5.4.2.2. Dose-response Data—Inhalation Exposure ..................................................... 226 

35 5.4.2.3. Dose Adjustments and Extrapolation Method(s)—Inhalation Exposure .......... 227 

36 5.4.2.4. Inhalation Unit Risk Derivation........................................................................ 227 

37 5.4.3. Dermal Exposure—Dermal Slope Factor ................................................................. 227 

38 5.4.3.1. Choice of Study/Data—with Rationale and Justification—Dermal Exposure . 227 

39 5.4.3.2. Dose-response Data—Dermal Exposure .......................................................... 228 

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

41 5.4.3.4. Dermal Slope Factor Derivation ...................................................................... 235 

42 5.4.3.5. Dermal Slope Factor Cross Species Scaling ..................................................... 236 

43 5.4.4. Application of Age-Dependent Adjustment Factors ................................................ 238 

44 5.4.5. Uncertainties in Cancer Risk Values ........................................................................ 240 

45 5.4.5.1. Oral Slope Factor ............................................................................................. 240 

46 5.4.5.2. Inhalation Unit Risk .......................................................................................... 240 

47 5.4.5.3 Dermal Slope Factor.......................................................................................... 241 

48 5.5.5. Previous Cancer Assessment .................................................................................... 242

49 
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1 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE 

2 RESPONSE ........................................................................................................................... 244 

3 6.1. HUMAN HAZARD POTENTIAL ................................................................................. 244 

4 6.2. DOSE RESPONSE ......................................................................................................... 244 

5 6.2.1. RfD ........................................................................................................................... 244 

6 6.2.2. RfC ........................................................................................................................... 246 

7 6.2.3. Cancer ....................................................................................................................... 247 

8 6.2.3.1. Cancer—Oral ......................................................................................................... 247 

9 6.2.3.2. Cancer—Inhalation................................................................................................ 248 

10 6.2.3.3. Cancer—Dermal .................................................................................................... 248

11 

12 7. REFERENCES ...................................................................................................................... 250

13 

14 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS 

15 AND DISPOSITION ............................................................................................................. 287 

16 APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR NONCANCER .......... 288 

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

18 APPENDIX D. TIME-TO-TUMOR MODELING FOR THE ORAL SLOPE FACTOR ........ 330 

19 APPENDIX E. TIME-TO-TUMOR MODELING FOR THE INHALATION UNIT RISK .... 360 

20 APPENDIX F. BENCHMARK MODELING FOR THE DERMAL SLOPE FACTOR ......... 367 

21 APPENDIX G. ADDITIONAL INFORMATION IN SUPPORT OF THE DERMAL SLOPE 

22 FACTOR ................................................................................................................................ 395 

23 APPENDIX H. ALTERNATIVE APPROACHES FOR CROSS-SPECIES SCALING OF THE 

24 DERMAL SLOPE FACTOR ................................................................................................. 398 

25 
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LIST OF TABLES 

Table 2-1. Physical properties and chemical identity 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 selected 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 metabolites in several species .......................... 56 

Table 4-1. Studies of PAH-DNA adducts in human populations 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 ± SDa

 for liver and thymus weights in Wistar rats exposed to benzo[a]pyrene 

by gavage 5 days/week for 90 days ........................................................................................ 92 

Table 4-5. Incidences of exposure-related neoplasms 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 in 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 neoplastic lesions in female B6C3F1 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 digestive tract tumors in male hamsters treated for 

life with benzo[a]pyrene by inhalation................................................................................. 107 

Table 4-10. Numbers of animals with pharynx and larynx tumors in male hamsters exposed by 

inhalation to benzo[a]pyrene for life .................................................................................... 108 

Table 4-11. Skin tumor incidence and time of appearance 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 appearance in male SWR, C3HeB, and A/He mice 

dermally exposed to benzo[a]pyrene for life or until a skin tumor was detected ................ 110 

Table 4-13. Tumor incidence in female Swiss mice dermally exposed to benzo[a]pyrene for up 

to 93 weeks ........................................................................................................................... 111 

Table 4-14. Skin tumor incidence in female NMRI and Swiss mice dermally exposed to 

benzo[a]pyrene ..................................................................................................................... 112 

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

 .............................................................................................................................................. 114 

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

 .............................................................................................................................................. 114 

Table 4-17. Skin tumor incidence and time of appearance in female CFLP mice dermally 

exposed to benzo[a]pyrene for 104 weeks ........................................................................... 115 

Table 4-18. Skin tumor incidence in female NMRI mice 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 female 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 benzo[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 effects in animals repeatedly exposed to 

benzo[a]pyrene by the oral route .......................................................................................... 172 

Table 4-28. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to 

benzo[a]pyrene by the inhalation route ................................................................................ 179 

Table 5-1. Means ± SD for ovary weight in female SD-rats ..................................................... 204 

Table 5-2. Summary of BMDs and BMDLs for modeled noncancer 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 rats exposed to benzo[a]pyrene by gavage, 5 

days/week for 104 weeks ..................................................................................................... 220 

Table 5-5. Incidence data for tumors in female B6C3F1 mice exposed to benzo[a]pyrene in the 

diet for 104 weeks ................................................................................................................ 220 

Table 5-6. Human equivalent PODs and oral slope factors derived from multistage-Weibull 

modeling of tumor incidence data at multiple tissue sites in Wistar rats and B6C3F1 mice 

exposed to benzo[a]pyrene orally for 2 years ...................................................................... 223 

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

life......................................................................................................................................... 226 

Table 5-8. Skin tumor incidence, benign or malignant, in C57L male mice dermally exposed to 

benzo[a]pyrene ..................................................................................................................... 232 

Table 5-9. Skin tumor incidence, benign or malignant in female Swiss or NMRI mice dermally 

exposed to benzo[a]pyrene ................................................................................................... 233 

Table 5-10. Skin tumor incidence, benign or malignant, in female CFLP mice dermally exposed 

to benzo[a]pyrene ................................................................................................................. 234 

Table 5-11. Skin tumor incidence, benign or malignant, 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 gavage for 

90 days.................................................................................................................................. 288 

Table B-2. BMD modeling results for increased liver weight in male rats, with BMR=10%... 288 

Table B-3. Means ± SDa

 for thymus weight in male Wistar rats exposed to benzo[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 ± SDa

 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 Wistar 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 hyperplasia in Wistar rats and B6C3F1 rats 

orally exposed to benzo[a]pyrene for 2 years ...................................................................... 312 

Table B-12. Summary of BMDs and BMDLs from the best fitting model forestomach 

hyperplasia—oral exposure .................................................................................................. 313 

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

 .............................................................................................................................................. 313 

Table B-14. Model predictions for forestomach hyperplasia in female Wistar rats in a 2-year 

study ..................................................................................................................................... 316 

Table B-15. Model predictions for forestomach hyperplasia 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 modeling 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) ............................ 337 

Table F-1. Summary of model selection and modeling results for best-fitting multistage models, 

for multiple data sets of skin tumors in mice following 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 
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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 noncancer effects from repeated oral exposure 

to benzo[a]pyrene. ................................................................................................................ 175 

Figure 4-3. Proposed principal pathways and key events in the benzo[a]pyrene carcinogenic 

MOA..................................................................................................................................... 188 

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

days....................................................................................................................................... 289 

Figure B-2. Fit of linear model (nonconstant variance) 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 decreased spleen B-cells in male Wistar rats—35 

days....................................................................................................................................... 308 

Figure B-6. Fit of log logistic model to data on forestomach hyperplasia in male Wistar rats in a 

2-year study. ......................................................................................................................... 314 

Figure B-7. Fit of log logistic model to data on forestomach hyperplasia in female Wistar rats in 

a 2-year study. ...................................................................................................................... 317 

Figure B-8. Fit of log logistic model to data on forestomach hyperplasia in female B6C3F1 mice 

in a 2-year study. .................................................................................................................. 320 

Figure B-9. Fit of linear/polynomial (1°) model to data on decreased ovary weight ................ 324 

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 model 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 model 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 model 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 model 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 model output. ....................................... 385 

Figure F-9. Fit of log-logistic model to skin tumors in female CFLP mice exposed dermally to 

benzo[a]pyrene (Grimmer et al., 1984); graph and model output. ....................................... 388 
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Figure F-11. Fit of multistage model to skin tumors 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 tumors in female CFLP mice exposed dermally to 

benzo[a]pyrene (Sivak et al., 1997); graph and model output. ........................................... 393
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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 
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CYP450 cytochrome P450 

dG deoxyguanosine 

dG-N2-BPDE 10β-(deoxyguanosin-N2-yl)-7β,8α,9α-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 

Fe2O3 ferrous oxide 

FEL frank effect level 

Ga2O3 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 

IC50 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 
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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 
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FOREWORD 

The purpose of this Toxicological Review is to provide 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 intended 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, where applicable, and to characterize the overall 

confidence in the quantitative and qualitative aspects 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 assessor 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 (202) 566-1676 (phone), (202) 566-1749 (fax), or 

hotline.iris@epa.gov (email address). 
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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 
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Toxicology Excellence for Risk Assessment 

Cincinnati, OH 

Glenn Talaska 

University of Cincinnati 

Cincinnati, OH 

REVIEWERS 

This document has been provided for review to EPA scientists. 

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 
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1 1. INTRODUCTION 

2 

3 This document presents background information and justification for the Integrated Risk 

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

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

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

7 carcinogenicity assessment. 

8 The RfD and RfC, if derived, provide quantitative information for use in risk assessments 

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

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

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

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

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m313 ) is 

14 analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The 

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

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

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

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

19 lifetime) exposure durations, all of which are derived based on an assumption of continuous 

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

21 derived for chronic exposure duration. 

22 The carcinogenicity assessment provides information on the carcinogenic hazard 

23 potential of the substance in question and quantitative estimates of risk from oral and inhalation 

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

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

26 effects may be expressed. Quantitative risk estimates may be derived from the application of a 

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

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

an upper bound on the estimate of risk per μg/m329 air breathed. 

30 There is evidence in humans and animal studies, demonstrating an increased incidence of 

31 skin tumors with increasing dermal exposure to polycyclic aromatic hydrocarbons (PAHs) 

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

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

34 upper bound on the estimate of risk per μg/day of dermal exposure. 

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

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

37 National Research Council (NRC, 1983). EPA Guidelines and Risk Assessment Forum 

38 Technical Panel Reports that may have been used in the development of this assessment include 
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1 the following: Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 

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

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

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

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

6 for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry

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

8 1995), Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for 

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

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

11 EPA, 2000b), Supplementary Guidance for Conducting Health Risk Assessment of Chemical 

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

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

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

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

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

17 2006b). 

18 The literature search strategy employed for this compound was based on the Chemical 

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

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

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

22 2010. 

23 
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Benzo[a]pyrene

1 2. CHEMICAL AND PHYSICAL INFORMATION 

2 

3 

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

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

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

7 sources and removed from the atmosphere by photochemical oxidation; reaction with nitrogen 

8 oxides, hydroxy and hydroperoxy radicals, ozone, sulfur oxides, and peroxyacetyl nitrate; and 

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

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

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

12 Table 2-1. 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

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

25 

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

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

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

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

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

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

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

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

34 Occupational exposure to PAHs occurs primarily through inhalation and skin contact during the 

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

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

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

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

39 carbide. 

40 
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Table 2-1. Physical properties and chemical identity 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/cm3 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 Kow 6.04 Verschueren (2001) 

Odor threshold No data 

Molecular weight 252.32 O’Neil et al. (2001) 

Conversion factorsa 1 ppm = 10.32 mg/m3 Verschueren (2001) 

Empirical formula C20H12 http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CHEM

aCalculated based on the ideal gas law, PV= nRT at 25°C: ppm = mg/m3

 × 24.45 ÷ molecular weight. 

1 

2 

3 Inhalation 

4 

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

ng/m3 for smokers and 0.27 to 0.58 ng/m36 for non-smokers. Naumova et al. (2002) measured 

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

indoor benzo[a]pyrene levels ranged from 0.02 to 0.078 ng/m38 . They also reported outdoor 

benzo[a]pyrene levels ranging from 0.025 to 0.14 ng/m39 . They concluded that indoor levels of 

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

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

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

smokers. They measured an average outdoor air concentration of 1.38 ng/m313 and indoor 

concentrations of 0.07 ng/m3 for homes with electrical utilities, 0.91 ng/m314 for homes with gas 

utilities, 0.80 ng/m3 for homes with gas utilities and a fireplace, 2.75 ng/m315 for homes with gas 

utilities and smokers, and 1.82 ng/m316 for homes with gas utilities, smokers, and a fireplace. 

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

an average of 0.77 ng/m3 inside homes and 0.23 ng/m318 outdoors. Park et al. (2001) measured 

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

measurements over this period, they found an average of 0.05 ng/m320 (vapor plus particulate). 

Parke et al. (2001) also reports average ambient air levels in ng/m321 from other studies conducted 

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

23 Christie, TX. Petry et al. (1996) conducted personal air sampling during 1992 at 5 workplaces in 
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1 Switzerland: carbon anode production, graphite production, silicon carbide production, bitumen 

2 paving work, and metal recycling. These data are summarized in Table 2-2. 

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

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

1990’s: rural areas: 0.1 – 1 ng/m3 and urban areas: 0.5 – 3 ng/m35 . They estimated the mean 

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

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

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

0.14 ng/m3 which would result in 2 ng/day assuming a 13 m39 /day inhalation rate (adult average 

10 based on USEPA, 1997). 

11 

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

Setting Year n Concentration 

(ng/m3 ) 

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., 2002 

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., 2002 

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 

13 NA = Not Available 

14 

15 Airborne intake of benzo[a]pyrene in the environment predominantly occurs via 

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

17 compounds, such as PAHs, are adsorbed. Reliable, quantitative measurements of the percent 
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1 absorption of benzo[a]pyrene from insoluble particles are not available; however, studies in 

2 experimental animals indicate that benzo[a]pyrene is readily absorbed from carbonaceous 

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

4 

5 

6 Oral 

7 The processing and cooking of foods is viewed as the dominant pathway of PAH 

8 contamination in foods (as reviewed by Bostrom, 2002). Among the cooking methods that lead 

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

10 and eggs will normally not contain high levels of PAH due to rapid metabolism of these 

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

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

13 example by oil spills. Vegetables and cereal grains can become contaminated primarily through 

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

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

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

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

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

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

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

21 reported as bread cereal and grain (29%) and grilled/barbecued meats (21%). 

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

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

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

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

26 intake of benzo[a]pyrene for an adult person was estimated in the range 0.05 to 0.29 μg/day 

(European Commission[EC], 2002). In the UK, average intakes on a ng kg-1 day-1 27 basis were 

28 estimated for the following age groups: adults – 1.6, 15 to 18 years – 1.4, 11 to 14 years – 1.8, 7 

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

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

31 grilled foods appeared less important in Europe than the U.S. because grilled foods are consumed 

32 less often (EC, 2002). 

33 

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

 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 
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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 

1 Source: Kazerouni et al., 2001 

2 

3 Estimates of oral bioavailability from animal studies range from about 30-70% (Ramesh 

4 et al, 2001b; Cavret et al., 2003; Hecht et al., 1979). Direct information regarding absorption of 

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

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

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

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

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

10 (Kawamura et al., 1988; O’Neill et al., 1991; Mirvish et al., 1981). 

11 

12 Dermal 

13 

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

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

16 use of dermally applied pharmaceutical products which contain coal tars, including formulations 

17 used to treat conditions such as eczema and psoriasis (IARC, 2010). 

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

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

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

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

22 are affected by proximity to roads/combustion sources, use of sewage sludge derived 

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

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

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

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

27 

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

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 
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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 276 

Nadal 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 

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

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

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

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

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

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

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

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

11 receptor fluid was quantified. 

12 These studies of benzo[a]pyrene absorption from soil suggest that reduced absorption of 

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

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

15 between soil and chemical). 

16 
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1 3. TOXICOKINETICS 

2 

3 

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

5 primary reports and secondary reviews are present in the scientific literature that describe the 

6 toxicokinetics of benzo[a]pyrene following oral, inhalation, and dermal exposures. 

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

8 and extent of absorption are dependent upon the exposure medium. For example, bioavailability 

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

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

11 benzo[a]pyrene metabolites in serum and excreta indicate wide tissue distribution. 

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

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

14 [GI] tract) and in reproductive tissues. Stable metabolic products identified in body tissues and 

15 excreta are very diverse and include phenols, quinones, and dihydrodiols. These classes of 

16 metabolites are typically isolated as glucuronide or sulfate ester conjugates in the excreta but can 

17 also include glutathione conjugates formed from quinones or intermediary epoxides. The 

18 primary route of metabolite elimination is in the feces, particularly following exposure by the 

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

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

21 

22 3.1. ABSORPTION 

23 3.1.1. Inhalation Exposure 

24 3.1.1.1. Inhalation Exposure in Humans

25 The absorption of benzo[a]pyrene is frequently assessed by identification of 

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

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

28 accompany exposure through the inhalation route, rendering estimates of inhalation-only 

29 exposure rather imprecise. For these reasons, quantitative estimates of absorption via the 

30 respiratory tract cannot be derived from these studies. Nevertheless, the observation of 

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

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

33 be via the respiratory tract. 

34 Becher and Bjørseth (1983) studied urinary excretion of 11 PAHs in highly exposed 

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

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

37 of hydroxy-metabolites (determined as parent compound following a chemical reduction 

38 procedure) and found that, in the case of benzo[a]pyrene, 92% of the total excreted were 
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1 metabolites. These values differed widely for the other 10 PAHs, several of which could be 

2 detected only following the reduction procedure. Exposed nonsmoking workers excreted 0.104 

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

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

5 excreted 0.025 ± 0.016 μg/mmol creatinine (range: 0.01–0.063); the difference was not 

6 significant. Total PAH excretion in the urine of aluminum plant workers was also higher in 

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

concentrations of PAHs in the aluminum reduction plant were typically 100 μg/m38 . The authors 

9 concluded that neither high occupational exposure to PAHs nor smoking status provided accurate 

10 determinants for PAH body burdens and that interindividual differences in absorption or 

11 metabolism played a major role. 

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

13 workers whose exposure to PAHs had been monitored with personal air samplers on 

14 4 consecutive workdays. They observed a correlation between benzo[a]pyrene amounts 

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

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

17 5 among the four workers, which the authors attributed to differences in genetically determined 

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

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

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

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

22 fivefold higher. 

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

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

25 provided concentrations in the air to which the workers were exposed. In the case of 

benzo[a]pyrene, the median for personal air samplers was 1.07 μg/m326 (range: 0.043–2.96), and 

the median for stationary air sampling was 1.31 μg/m327 (range: 0.63–5.41). Other PAH air 

concentrations ranged from 0.11 μg/m3 (dibenz[a,h]anthracene) to 4.85 μg/m328 (chrysene). The 

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

30 evaluated) was 1.58 ng/mmol creatinine (range: 0.34–22.6). This was by far the lowest level of 

31 PAH metabolites found in the urine of exposed workers; for comparison, phenanthrene, which 

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

(1.08 μg/m333 ), produced a total of 679 ng/mmol creatinine in the form of metabolites (range: 

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

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

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

37 detect a correlation between the levels of individual PAH exposures and urinary excretion of 

38 related metabolites. 
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1 Wu et al. (2002) found a statistically significant correlation between trans-anti2 benzo[a]pyrene-tetrol in the urine of coke oven workers and PAH concentrations in benzene 

3 extracts obtained from personal air monitoring devices. These workers were exposed to a variety 

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

5 consumption habits. However, genetic factors had some influence on urinary trans-anti6 benzo[a]pyrene-tetrol levels (e.g., workers homozygous for the cytochrome (CYP)1A1 MspI

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

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

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

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

11 PAH exposure. 

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

13 exposure by measurement of urinary metabolites of phenanthrene. They compared levels of 

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

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

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

17 nonsmoking unexposed control persons. They demonstrated statistically significant correlations 

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

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

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

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

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

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

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

25 unexposed controls but not in exposed coke oven workers, who displayed significantly elevated 

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

27 The available data from human exposure studies provide qualitative evidence of 

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

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

30 assume that, in occupational settings, exposure occurs predominantly by the inhalation route. 

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

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

33 These qualitative observations in humans are supported by inhalation and instillation 

34 toxicokinetic studies in animals. 

35 

36 3.1.1.2. Inhalation Exposure in Animals

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

38 the respiratory tract of dogs. Seven-year-old female beagle dogs (n = 3) were exposed to a bolus 
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1 of 77 mg aerosolized benzo[a]pyrene crystals (particle size <1 μm) injected directly into the 

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

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

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

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

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

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

8 phenanthrene, which is cleared more quickly, the authors concluded that the clearance rate was 

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

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

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

12 portion of the respiratory tract of animals (as opposed to the tracheal, bronchial, and bronchiolar 

13 portions) is nearly complete and very rapid. 

14 In a second study in this series, Gerde et al. (1993b) determined the disposition of 

15 benzo[a]pyrene in the conducting airways by instilling benzo[a]pyrene (dissolved in 20 μL saline 

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

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

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

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

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

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

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

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

24 Absorption into the airway walls was evaluated by instilling benzo[a]pyrene solutions into the 

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

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

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

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

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

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

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

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

33 dose. About 8% of the administered dose was covalently bound to tissues. The authors 

34 concluded that the significantly higher retention time in the bronchi, as compared to pulmonary 

35 epithelium, made the conducting portion of the respiratory tract a possible target of 

36 benzo[a]pyrene toxicity. 

37 In the third paper of the series, Gerde et al. (1993c) used the results from the previous 

38 two studies to evaluate benzo[a]pyrene dosimetry in the respiratory tract. The authors concluded 
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1 that benzo[a]pyrene uptake occurs via diffusion-limited transport through the lung epithelium. 

2 Uptake by the alveolar epithelium took only a few minutes while uptake by the airway 

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

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

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

6 benzo[a]pyrene. 

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

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

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

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

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

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

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

14 benzo[a]pyrene-coated diesel soot (mass median aerodynamic diameter [MMAD] 1.3 ± 0.2 μm), 

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

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

17 tests, the amount of benzo[a]pyrene deposited was determined to be 36 ± 20 μg (n = 6). The 

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

19 absorption was approximately 4 minutes. Although one dog received approximately seven times 

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

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

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

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

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

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

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

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

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

29 from particles transported to the lymph nodes to approximately 10% of a monolayer coating, 

30 which may reflect the more reactive chemical environment provided by alveolar macrophages. 

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

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

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

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

35 rate. 

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

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

concentrations of 0.1, 1.0, or 2.5 mg/m338 of benzo[a]pyrene adsorbed to carbon black (CB) 
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1 particles. The particle size distribution was monodisperse and largely in the respirable range (the 

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

3 that the results reflect absorption from the entire lung, since particles of the size distribution used 

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

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

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

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

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

9 difference in the time course of plasma levels between male and female rats. Female plasma 

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

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

12 administered aerosol concentration was reported instead of deposited dose, plasma samples were 

13 not collected during the 4-hour exposure period, and the study could not distinguish between 

14 absorption from the respiratory tract and mucociliary clearance followed by absorption from the 

15 gut. 

16 Rapid absorption through the lungs was also shown following intratracheal 

administration of 1 μg/kg body weight [317 H]-benzo[a]pyrene dissolved in triethylene glycol in 

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

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

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

21 suggesting rapid absorption from the upper respiratory tract. The authors noted that it was 

22 unlikely that the appearance of radiolabel in organs was due to GI tract absorption after 

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

24 and levels of radioactivity in the stomach increased only slowly. Based on a comparison of 

25 benzo[a]pyrene concentrations in the blood following intratracheal administration versus 

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

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

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

29 systemic circulation is limited by first-pass metabolism in the lung. 

Petridou-Fischer et al. (1988) applied 10 μL aliquots of [14 30 C]-benzo[a]pyrene in a 

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

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

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

34 total radioactivity administered (93 μCi per animal) and the specific activity of the radiolabeled 

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

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

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

38 in monkeys), and very little radioactivity was identified in excreta. Urinary excretion reached a 
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1 maximum of 0.69% of the administered dose in dogs and 0.07% in monkeys, while in feces a 

2 maximum of 6.42% of the administered dose was recovered in dogs and 1.17% in monkeys over 

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

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

5 to more traditional inhalation or intratracheal instillation experiments, which have demonstrated 

6 significant absorption via other portions of the respiratory tract following inhalation or 

7 intratracheal instillation. Little radioactivity was recovered from the mucus, blood, or excreta, 

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

9 Several studies demonstrated rapid desorption of benzo[a]pyrene bound to particles. 

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

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

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

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

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

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

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

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

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

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

20 diesel soot. Furthermore, Gerde and Scholander (1989) found in an in vitro study that the release 

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

22 bronchial epithelium. 

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

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

25 following intratracheal administration of benzo[a]pyrene crystals (0.5–1.0 μm in size) or 

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

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

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

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

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

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

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

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

adsorbed onto gallium oxide (67 34 Ga2O3) particles was investigated by Sun et al. (1982). Male and 

female F344 rats were exposed nose-only for 30 minutes to atmospheres containing 0.6 mg/m335 

[

3H]-benzo[a]pyrene absorbed onto 67Ga2O3 or to 1.0 μg/L neat (i.e., the pure chemical) [3

36 H]-

37 benzo[a]pyrene (MMADs were reported as approximately 0.1 μm in both cases). Radiolabel was 

38 detected in the esophagus, stomach, small and large intestines, cecum, liver, kidney and blood; 
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1 however, the time to reach peak tissue concentrations differed considerably between the two 

2 exposure regimens. Based on the total amount excreted, 22% of the inhaled dose of 

3 benzo[a]pyrene on Ga2O3 was released over 16 days, but only 10% of the inhaled dose of pure 

4 benzo[a]pyrene was released. Since the amount excreted can reflect differences in absorption, 

5 i.e., uptake via pulmonary epithelium vs. ingestion of cleared particles, and hence alternative 

6 tissue distribution and metabolism, this result cannot be used quantitatively to estimate 

7 bioavailability of inhaled benzo[a]pyrene. The study established that benzo[a]pyrene adsorbed 

8 to particles had a longer respiratory tract retention period. For benzo[a]pyrene coated on Ga2O3, 

1 day was required to clear 90% of the [39 H]-benzo[a]pyrene lung and trachea burdens that were 

10 present 30 minutes after exposure. In contrast, only 1.5 and 4 hours were required to clear 90% 

11 of pure benzo[a]pyrene burdens from the lung and trachea, respectively. A different effect was 

12 observed in the nose, where clearance of 90% benzo[a]pyrene coated on Ga2O3 required 7 hours 

13 as compared to 20 hours for pure benzo[a]pyrene aerosol. Thereafter, clearance curves for 

14 particle-bound and neat benzo[a]pyrene were similar; the authors attributed this to the absence of 

15 a mucociliary escalator in the nose. Inhalation of benzo[a]pyrene on Ga2O3 also increased the 

16 dose of the compound and its metabolites to the stomach, liver, and kidneys, which the authors 

17 suggest may have resulted from mucociliary clearance with subsequent ingestion. 

18 benzo[a]pyrene absorption from the respiratory tract may also be affected by the 

19 characteristics of the vehicle. Following intratracheal administration in hydrophilic triethylene 

20 glycol, approximately 70% of the benzo[a]pyrene administered was excreted within 6 hours by 

21 male Sprague-Dawley rats (Bevan and Ulman, 1991). In contrast, 58.4% and 56.2% of 

22 administered benzo[a]pyrene were excreted within a 6-hour period when the lipophilic solvents 

23 ethyl laurate and tricaprylin, respectively, were the vehicles. 

24 Pregnant Wistar rats were exposed head-only for 95 minutes on gestational day (GD) 17 

to 200, 350, 500, 650, or 800 mg/m3 of a [325 H]-benzo[a]pyrene microcondensate generated from 

26 heated pure material (Withey et al., 1993). Particle sizes ranged from 0.61 to 0.88 μm MMAD. 

27 Immediately following exposure (no time estimate was provided), blood radiolabel 

28 concentrations varied >eightfold over the fourfold dose range (2.66 ± 0.51 vs. 21.96 ± 1.37 μg/g 

29 at lowest and highest dose, respectively). Six hours after exposure, blood radiolabel 

30 concentrations had decreased two- to fourfold from the earlier observation but retained a >10-

31 fold difference over the dose range (0.74 ± 0.12 vs. 9.56 ± 2.1 μg/g). Therefore, the difference in 

32 the ratio of blood levels to dose range was not likely due to an initial rapid phase of 

33 benzo[a]pyrene absorption only at high exposures. However, the study authors did not provide 

34 an explanation for this finding. Radiolabel was detected at 0 and 6 hours in all maternal tissues 

35 and in fetuses examined, indicating that systemic absorption had occurred. 

36 Hood et al. (2000) exposed male and timed pregnant female Sprague-Dawley rats to 

100 μg/m337 benzo[a]pyrene-CB aerosol (nose only for 4 hours on GD 15), and collected blood 

38 was analyzed at 30, 60, 120, 180, and 240 minutes for concentrations of benzo[a]pyrene. The 
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1 benzo[a]pyrene aerosol particle distribution was trimodal with a significant portion of particles 

2 <1 μm in size. The particle size distribution was expected to result in deposition across all 

3 regions of the respiratory tract, including the pulmonary region. Following exposure, blood 

4 benzo[a]pyrene levels peaked at 30 minutes (the first time point reported), with females 

5 exhibiting approximately a 1.7-fold higher peak concentration than males. At later time points, 

6 the female benzo[a]pyrene blood concentrations were similar to those observed in males. By 

7 240 minutes postexposure benzo[a]pyrene in blood had diminished to <5% of the peak level. 

8 The authors did not report a mass balance to allow for the determination of the percentage of 

9 dose that was absorbed. Furthermore, benzo[a]pyrene metabolites were not measured. 

10 Nevertheless, the appearance of benzo[a]pyrene in the blood at the earliest time point measured 

11 is consistent with the conclusion that benzo[a]pyrene is rapidly absorbed from the respiratory 

12 tract. Although the peak came earlier than in the Ramesh et al. (2001a) study, the same trend of 

13 gender differences was observed, with females displaying higher peak blood levels than males. 

14 In summary, although quantitative estimates of human lung absorption are not available, 

15 existing toxicity and biological monitoring studies suggest that benzo[a]pyrene is absorbed in the 

16 respiratory tract, albeit rather poorly, following inhalation exposure of humans. The evidence 

17 suggests, however, that in humans it is difficult to establish a relationship between 

18 benzo[a]pyrene exposure and urinary excretion of its metabolites due to large interindividual 

19 variation, most likely the result of different genetic makeups and varying background exposures. 

20 Numerous controlled studies indicate that benzo[a]pyrene is well absorbed in animals following 

21 inhalation or intratracheal instillation. In general, the animal studies show that benzo[a]pyrene is 

22 absorbed rapidly (within minutes) and extensively. The rate of absorption varies across regions 

23 of the respiratory tract, with more rapid absorption in the pulmonary regions and slower 

24 absorption in the conducting airways and nose. In some studies in rats, blood benzo[a]pyrene 

25 peak levels at early exposure time points differed between females and males, but leveled out at 

26 later time points. Quantitative estimates of benzo[a]pyrene absorption from the respiratory tract 

27 are difficult to derive because the contribution of absorption from the GI tract following 

28 mucociliary clearance and metabolism of benzo[a]pyrene in the respiratory tract itself are often 

29 difficult to determine. Another complication in interpreting these studies is that benzo[a]pyrene 

30 absorption from the lung depends on the characteristics of the exposure vehicle or the nature of 

31 the particle to which benzo[a]pyrene is adsorbed. In general the data indicate that 

32 benzo[a]pyrene is released to a greater extent from hydrophilic vehicles than from lipophilic 

33 solvents; particle-bound benzo[a]pyrene is released more slowly than the neat compound; and 

34 desorption from large particles is slower than from smaller particles. Because much of the 

35 environmental benzo[a]pyrene is adsorbed onto particles of other materials, the effect of the 

36 carrier particle is highly relevant to environmental exposures. 

37 

38 3.1.2. Oral Exposure 
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1 3.1.2.1. Oral Exposure in Humans

2 In a study with eight volunteers who ingested broiled meat containing approximately 

3 8.6 μg of benzo[a]pyrene, the concentration of benzo[a]pyrene in feces was below detection 

4 limits (<0.1 μg/person) (Hecht et al., 1979). Although the analytical method used in this study 

5 assessed only parent compound, not fecal metabolites, the result can be interpreted as indicating 

6 that the ingested benzo[a]pyrene was absorbed completely from the GI tract. In addition, studies 

7 were conducted to assess DNA adduct levels or benzo[a]pyrene metabolites in humans exposed 

8 to PAHs by the oral route. In general, these human dietary studies are not adequate to develop 

9 quantitative estimates of oral bioavailability; in one case no measurable relationship between 

10 benzo[a]pyrene intake and internal dose measure was found (Scherer et al., 2000). 

11 

12 3.1.2.2. Oral Exposure in Animals

13 The bioavailability of benzo[a]pyrene was evaluated in F344 rats dosed by gavage with 

14 100 mg/kg benzo[a]pyrene dissolved in peanut oil and sacrificed 0, 0.5, 1.0, 2.0, 4.0, 8.0, 24, 48, 

15 or 72 hours after dosing (Ramesh et al., 2001b). Blood, liver, reproductive tissues, urine and 

16 feces were analyzed for benzo[a]pyrene and metabolites. Plasma benzo[a]pyrene levels peaked 

17 at 8 hours and fecal levels at 2.4 hours postexposure. Lipophilic metabolites of benzo[a]pyrene 

18 peaked at 2 hours in liver, 8 hours in feces, 24 hours in blood and lung, and 48 hours 

19 postexposure in urine. Water-soluble metabolites reached their maxima at 4 hours in liver and 

20 lung, 8 hours in blood and feces, and 48 hours in urine. Some of the disposition patterns 

21 displayed minor peaks at earlier time points. Based on comparison with plasma levels following 

22 i.v. injection, the oral bioavailability was estimated by the authors as approximately 40%; 

23 however, the details of this determination were not presented. 

24 Foth et al. (1988) conducted several experiments to determine the oral bioavailability of 

25 benzo[a]pyrene. In male Sprague Dawley rats, the areas under the blood concentration-time 

26 curve (AUCs) following oral versus i.v. bolus doses of benzo[a]pyrene (dissolved in Krebs 

27 Ringer buffer with 4% bovine serum albumin) were compared. The oral bioavailability was 

estimated as 7.8 and 11.5% for doses of 3.2 and 4.0 nmol [328 H]-benzo[a]pyrene per rat 

29 (approximately 1.8–2.7 and 2.2–3.4 μg/kg, respectively, calculated as 3.2–4 nmol/rat × 

30 0.252 μg/nmol ÷ (0.30–0.46) kg body weight as stated by the authors). This result may reflect 

31 limited systemic absorption of benzo[a]pyrene at low doses. In contrast to this result, analysis of 

32 benzo[a]pyrene concentrations in arterial blood and bile after continuous intraduodenal infusion 

33 of radiolabeled benzo[a]pyrene (in the same vehicle as above) showed that approximately 40% 

34 of the administered dose was absorbed by the duodenum over a 240-minute period (Foth et al., 

35 1988). In a similar experimental design, bile- and duodenum-cannulated male Sprague-Dawley 

rats were given [336 H]-benzo[a]pyrene in corn oil with and without exogenous bile (Rahman et al., 

37 1986). The absorption of benzo[a]pyrene was estimated from the cumulative recovery of 

38 radioactivity in the bile and urine over 24 hours. This study showed that absorption of 
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1 benzo[a]pyrene was enhanced by bile as absorption in the presence of endogenous bile only was 

2 22.9% of that when exogenous bile was administered.

3 Cavret et al. (2003) reported that absorption of benzo[a]pyrene (as measured by the 

4 percentage of orally administered radioactivity appearing in the portal blood in 24 hours) was 

30.5% in pigs fed 1 L of 4% fat milk containing 235 μg/L [14 5 C]-benzo[a]pyrene (this 

6 corresponds to an oral dose of approximately 6 μg/kg based on the reported body weight of 40 

7 kg). The level of radioactivity increased rapidly between 1 and 6 hours, with maximum uptake 

8 between 3 and 6 hours after dosing. 

In male F344 rats administered [14 9 C]-benzo[a]pyrene in peanut oil via gavage at doses

10 from 0.04 to 4.0 μmol/rat (approximately 0.03–0.04 mg/kg and 3.4–4 mg/kg, respectively, 

11 calculated as 0.04–4 μmol/rat × 0.252 mg/μmol ÷ 0.25–0.30 kg body weight as stated by the 

12 authors) approximately 85% of the radiolabel was recovered in the feces and 1–3% in the urine 

13 after 168 hours (Hecht et al., 1979). Because radiolabel in feces may represent unabsorbed as 

14 well as absorbed parent compound that is subsequently eliminated via biliary excretion, 

15 bioavailability cannot be estimated from this study. However, the percent of radioactivity 

16 recovered as parent benzo[a]pyrene was small (ranging from 6 to 13% of the administered 

17 radioactivity), suggesting that a minimum of 73% of the administered dose was absorbed (i.e., 

18 total in urine + total in feces – feces as benzo[a]pyrene). 

19 Dietary matrices may have an impact on the absorption of benzo[a]pyrene from the GI 

20 tract. For example, intestinal absorption of benzo[a]pyrene was enhanced in rats when the 

21 compound was solubilized in lipophilic compounds such as triolein, soybean oil, and high-fat 

22 diets, as compared with fiber- or protein-rich diets (O’Neill et al., 1991; Kawamura et al., 1988). 

23 This may be relevant for the absorption of benzo[a]pyrene from charbroiled meats and other fatty 

24 foods. 

O’Neill et al. (1990) assessed the intestinal absorption of [14 25 C]-benzo[a]pyrene given to 

26 rats by gavage in olive oil with regular rat chow or with low-fat diets high or low in either fiber 

27 or beef protein (used to represent human diets). Benzo[a]pyrene and its metabolites were 

28 recovered from feces, where they had been trapped by microcapsules given by gavage 2 hours 

29 prior to benzo[a]pyrene. Benzo[a]pyrene was absorbed from rat chow differently than from 

30 representative human diets. Dietary fiber decreased the availability of benzo[a]pyrene, as 

31 evidenced by the appearance of lower metabolite amounts in the GI tract, while the beef32 enriched diet affected absorption to result in increased formation of 1,6- and 3,6-benzo[a]pyrene 

33 diones. Urinary excretion of benzo[a]pyrene was decreased in rats given the high fiber diet but 

34 not the beef-enriched diet. The total amount of benzo[a]pyrene excreted in feces and the 

35 feces/urine ratio were increased by the high-fiber diet but not by the beef-enriched diet. These 

36 results indicated that bioavailability of benzo[a]pyrene from the GI tract is affected by the type 

37 of diet and that bioavailability studies in animals, using typical laboratory animal chow, may not 

38 appropriately model the situation with varied human diets. 
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1 The potential influence of diet on PAH bioavailability was investigated also by Wu et al. 

2 (1994). Female mice were fed either gel or powder diets containing coal tar with detectable 

3 levels of benzo[a]pyrene, Py, and other PAHs. Urine samples were collected on the first, 

4 seventh, and fourteenth day of treatment. Measurement of the amount of the Py metabolite 1-

5 OH-Py in the urine showed that the diet matrix did not influence the bioavailability of the PAHs. 

6 Stavric and Klassen (1994) administered radiolabeled benzo[a]pyrene dissolved in various 

7 vehicles (water, corn oil, liquid paraffin, or 50% ethanol) by gavage and monitored intestinal 

8 absorption in bile-cannulated rats. The animals were fed diets with or without added carbon 

9 particles and typical food components, such as quercetin or chlorogenic acid. They observed that 

10 aqueous vehicles, quercetin, chlorogenic acid, or carbon particles reduced biliary excretion of 

11 benzo[a]pyrene, while lipid media such as corn oil increased it strongly. The authors postulated 

12 that absorption of benzo[a]pyrene from food was affected by its solubility in the vehicle, by 

13 physical adsorption, and/or by adduction of benzo[a]pyrene to certain food ingredients. On the 

14 other hand, Mirvish et al. (1981) observed that varying the corn oil content of a synthetic diet 

15 containing 100 μg/g benzo[a]pyrene had little influence on fecal excretion of unmetabolized 

16 benzo[a]pyrene. However, addition of 5% wheat bran to the synthetic diet, or using standard lab 

17 chow, increased the fecal excretion of parent compound 13-fold. They suggested that the 

18 insoluble dietary fiber sequestered benzo[a]pyrene in the GI tract. 

19 In summary, absorption of ingested benzo[a]pyrene was demonstrated qualitatively in 

20 exposed humans by the excretion of metabolites or the presence of DNA adducts. However, 

21 these studies are not sufficient to determine the rate and extent of absorption from the GI tract in 

22 humans. Animal studies have produced variable results, in part due to different study designs. 

23 Standard approaches in animal studies suggest that the oral bioavailability ranges from 10 to 

24 40%. However, some studies have indicated that the standard diets of laboratory animals may 

25 not model the human oral exposure to benzo[a]pyrene appropriately. No data on species or 

26 gender-based differences in absorption were identified. 

27 

28 3.1.3. Dermal Exposure 

29 Several studies in humans and experimental animals have investigated the dermal 

30 absorption of benzo[a]pyrene. Benzo[a]pyrene metabolites or DNA adducts were measured in 

31 humans exposed dermally to benzo[a]pyrene-containing mixtures in biological monitoring 

32 studies. These studies provide only qualitative support for assessing the rate and degree of 

33 dermal absorption of benzo[a]pyrene through human skin. However, some studies provide 

34 quantitative information on the degree of benzo[a]pyrene absorption through the skin in 

35 volunteers or in explanted viable skin samples from tissue donors. 

36 

37 3.1.3.1. Dermal Exposure in Humans
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1 Van Rooij et al. (1993) demonstrated differences in absorption rates of PAHs in coal tar 

ointment at various skin sites in volunteers. A dose of 2.5 mg/cm22 of coal tar ointment, which 

consisted of 10% coal tar in a vehicle of zinc oxide paste, was applied to 24 cm23 of skin at the 

4 forehead, shoulder, volar forearm, palmar side of the hand, groin, or ankle and allowed to stand 

5 for 45 minutes before removing the residue. Dermal absorption of PAH was determined through 

6 measuring the disappearance of PAH fluorescence from the skin surface. Absorption rate 

7 constants ranged from 0.036 to 0.135/hour, across application sites, suggesting that 20–56% of 

8 the dose would be absorbed within 6 hours. A 69% difference in dermal absorption rates 

9 between anatomical sites was reported, while only a 7% difference between individual 

10 volunteers was observed. The skin on the shoulder absorbed the greatest dose of PAH, followed 

11 by the forehead, forearm, and groin, with the ankles and palms absorbing the least amount. 

12 There was, however, no correlation between anatomical site of exposure and excretion of 1-OH13 Py in urine. The authors estimated that 0.3–1.4% of the PAH dose (assessed as 1-OH-Py) 

14 became systemically available, although systemic measurement of benzo[a]pyrene or its 

15 metabolites was not reported. 

16 Quantitative comparisons of dermal penetration of human skin and other mammalian 

17 species have been conducted. Kao et al. (1985) compared benzo[a]pyrene permeation through 

18 skin in short-term organ cultures using skin harvested from mice, rats, rabbits, guinea pigs, 

19 marmosets, and human donors. Two test systems were used, a dynamic one where skin samples 

20 were held on top of chambers flushed with fresh organ culture medium at a constant rate and a 

21 static one where skin samples were incubated on a filter disk in a culture dish over culture 

22 medium at 36°C. The culture medium was a modified minimal essential medium with Earle’s 

23 salts and 10% fetal calf serum. In addition, all experiments were conducted with fresh, 

24 metabolically viable skin and with metabolically nonviable skin (previously frozen or poisoned 

with cyanide). Using the static system, [325 H]-benzo[a]pyrene dissolved in acetone was applied to 

full-thickness skin samples (2.5 μg/cm226 ) and medium collected after 24 hours to be assayed for 

[

3

27 H]-benzo[a]pyrene and metabolites. The overall penetration rate of benzo[a]pyrene was about 

28 2.6% of the dose in 24 hours from viable human skin and only about 0.5% from nonviable skin. 

29 After 24 hours penetration through viable skin, 52% of the radioactivity in medium consisted of 

30 water-soluble benzo[a]pyrene metabolites and 18% was parent compound. By contrast, after 

31 penetrating through nonviable skin, 90% of the recovered radioactivity was parent compound, 

32 and the ratio of water- to lipid-soluble metabolites was much lower. Results in marmoset, rat, 

33 and rabbit were similar. Skin from the mouse allowed more than 10% of the dose to penetrate, 

34 while that of guinea pig let only a negligible percentage of the dose penetrate. In all species, 

35 metabolism was an important determinant of permeation, with very low rates observed in 

36 nonviable skin. 

37 Using the dynamic test system, Kao et al. (1985) studied the influence of dermal 

38 metabolism on the rate of penetration of benzo[a]pyrene in more detail. Comparing rat to mouse 
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1 skin they again found a 10-fold difference in the amount penetrating in 16 hours through viable 

2 skin, approximately 0.7% of the dose in rat skin vs. approximately 7% in murine skin, but most 

3 of this species difference disappeared when previously frozen skin was used or potassium 

4 cyanide was added to the medium to destroy metabolic capability. An additional factor was 

5 responsiveness to aryl hydrocarbon (Ah)-receptor agonists: dermal penetration in responsive 

6 mice given an inducing dose of 3-methylcholanthrene (3-MC) was more than twice that in 

7 noninduced Ah-responsive mice, and the latter was similar to nonresponsive mice (see Section 

8 3.3 for further discussion of Ah-responsiveness). These results show that functional enzyme 

9 systems facilitate penetration of benzo[a]pyrene or its metabolites. 

Wester et al. (1990) compared skin penetration of [14 10 C]-benzo[a]pyrene dissolved in 

11 acetone versus benzo[a]pyrene adsorbed onto soil using skin from human cadavers. Skin was 

12 dermatomed to 500 μm and stored in medium at 4°C to preserve viability. The authors used a 

13 dynamic system with undiluted human serum as the receptor fluid and 24-hour exposure. 

14 Radiolabeled benzo[a]pyrene was applied to the skin, and concentrations of radioactivity in the 

15 receptor fluid, skin, and surface skin wash were determined. For benzo[a]pyrene applied in 

16 acetone, 23.7% of the applied dose was recovered in the skin, 0.09% was recovered in the 

17 receptor fluid, and 53.0% was recovered in the surface wash. The amount of radiolabel 

18 recovered from exposure to benzo[a]pyrene in soil was 1.4% of the dose in skin and 0.01% in 

19 receptor fluid, indicating that the exposure matrix greatly impacted dermal absorption. The 

20 considerable difference in penetration through human skin observed in this study (only 0.09% of 

21 the dose per 24 hours) and that of Kao et al. (1985) (2.6% of the dose) is at least in part a result 

22 of differences in experimental design, such as full thickness (approximately 1.5 mm) vs. 

23 dermatomed (0.5 mm) skin and synthetic receptor fluid with 10% serum vs. pure human serum. 

24 Together, these studies suggest that benzo[a]pyrene is absorbed into human skin but does not 

25 penetrate through the skin rapidly. 

26 In a second part of the same study, Wester et al. (1990) also evaluated the dermal 

27 penetration of benzo[a]pyrene in female rhesus monkeys in vivo. Radiolabeled benzo[a]pyrene 

was applied to a 12 cm228 area of the abdominal skin that was protected by a nonocclusive cover. 

29 The material was maintained on the skin for 24 hours, after which time the skin area was 

30 washed. Urine was collected from the animals during the initial 24-hour exposure period and 6 

31 days after. Skin penetration was determined as the ratio of urinary radiolabel for topically 

32 exposed animals compared to monkeys administered the same dose by i.v. injection. When 

33 benzo[a]pyrene was dissolved in acetone, 51% of the applied dose was absorbed as compared to 

34 13.2% when benzo[a]pyrene was applied in soil. These results further stress the fact that the 

35 dermal absorption of benzo[a]pyrene depends on the vehicle of administration (pure substance 

36 vs. contaminated environmental material). 

37 Also using human tissues, van der Bijl and van Eyk (1999) compared the mean flux of 

38 benzo[a]pyrene across vaginal and buccal mucosa samples from human donors. No significant 
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1 difference between the two tissue types was observed, and steady-state flux was very low, 

approximately 0.01% of the dose per cm2/minute (400 cpm/cm22 /minute in a figure in the paper, 

or 4,000 dpm [assuming 10% [33 H]-liquid scintillation counting efficiency] out of a 17.33 μCi 

dose, or 3.8 × 107 dpm, equals about 0.01% of the dose/cm24 /minute). These results indicate that 

5 benzo[a]pyrene can be absorbed from and metabolized by the female reproductive tract. 

6 However, the results cannot be compared to those obtained with human skin. First, there was the 

7 difference in absorbing surface, namely, the quasi-dry, mainly lipophilic stratum corneum vs. 

8 fresh epithelium. Second, there were also fundamental differences in experimental design: 

9 although van der Bijl and van Eyk (1999) used a dynamic system, the tissues had been frozen 

10 previously, benzo[a]pyrene was administered in aqueous solution in a large compartment on top 

11 of the tissue sample, receptor fluid was a simple buffer, and incubation was performed at 20°C. 

12 Potter et al. (1999) also demonstrated that the exposure matrix can affect the dermal 

13 absorption of benzo[a]pyrene in mice in vivo as well as in human skin in vitro. In particular, the 

14 uptake of benzo[a]pyrene decreased as the viscosity of the oil product used as vehicle increased. 

15 Although mouse skin absorbed more radiolabel than human skin, the trend of decreasing 

16 absorption with increasing viscosity found in the mouse was also present in human skin. 

17 Vehicles tested included mineral oils (32–198 centi-Stoke [cSt], measure of viscosity), residual 

aromatic extracts (5,160–5,400 cSt), and bitumens (0.65–69 × 10618 cSt). Most likely, differences 

19 in diffusion coefficients for benzo[a]pyrene in the various vehicles would adequately describe 

20 the differences in dermal absorption. 

21 

22 3.1.3.2. Dermal Exposure in Animals 

23 Yang et al. (1989) compared dermal absorption of benzo[a]pyrene in crude oil adsorbed 

24 to soil (particle size of 150 μm or less) versus benzo[a]pyrene in the crude oil alone. In the in 

vivo portion of the study, test materials spiked with 100 ppm [325 H]-benzo[a]pyrene were placed 

over a 7-cm226 portion of shaved dorsal skin of female Sprague-Dawley rats. Urine and feces were 

27 collected daily over 4 days, and at the end of the experiment animals were sacrificed and 

28 radioactivity in tissues was determined. The total percent of the absorbed dose recovered in 

29 excreta was greater for benzo[a]pyrene in crude oil (35.3%) than oil adsorbed to soil 

30 (approximately 9.2%). While the latter number refers to soil applied to the skin as a monolayer, 

31 when six times the amount of soil, and thus a sixfold dose, was applied, the absolute amount of 

32 benzo[a]pyrene absorbed remained virtually the same. The authors also found that using viable 

33 skin samples in vitro gave almost identical results to those obtained in vivo. 

34 Ng et al. (1992) examined the percutaneous absorption of radiolabeled benzo[a]pyrene in 

35 the hairless guinea pig. A single dose of 28 μg benzo[a]pyrene dissolved in 50 μL acetone was 

applied to 4 cm236 of dorsal skin and covered with a protective pad for 24 hours. Urine and feces 

37 were collected at 6 and 12 hours and then daily for the next 7 days postexposure. Approximately 

38 34% of the radiolabel was absorbed and eliminated in 24 hours. Most excretion had occurred by 
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1 day 3 and then continued at a lower rate to reach about 73% by day 7. Benzo[a]pyrene in the 

2 skin wash accounted for 11% of the applied dose. Comparison with the amount excreted 

3 following intramuscular injection (which reached about 85% in 7 days) suggested that the dermal 

4 bioavailability of benzo[a]pyrene was high (about 85%). Ng et al. (1992) noted that, in an in 

5 vitro study, only metabolites were present in the receptor solution, indicating that metabolism in 

6 the skin preceded systemic absorption. This is consistent with the findings of Kao et al. (1985) 

7 that dermal absorption may be aided by a high metabolic capacity of the skin but leaves open the 

8 question how much of the reactive metabolites escape from the dermal compartment. 

9 In an earlier study, Kao et al. (1984) used viable mouse skin to study the dose 

10 dependence of dermal absorption of benzo[a]pyrene. They administered 1, 2, 4, and 6 μg 

benzo[a]pyrene per cm211 full-thickness, shaved skin incubated at 36°C for 24 hours. The 

12 percentage of absorbed benzo[a]pyrene dose decreased with increasing dose: 24, 17, 12, and 7% 

with 1, 2, 4, and 6 μg/cm213 , respectively. The authors also found that, with doses of 4 and 

6 μg/cm214 , the total amount of penetrated benzo[a]pyrene-derived radioactivity remained similar, 

15 indicating saturation of the skin’s absorption and metabolic capacity. When the donor mice were 

16 pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce cytochrome P450 

17 (CYP450) enzymes in the skin, amounts of penetrated benzo[a]pyrene increased to 38, 33, 30, 

18 and 19% of the dose, respectively. In addition to the increased rate of absorption, a shift in 

19 metabolic pattern was observed among the benzo[a]pyrene metabolites in the medium. CYP450 

20 induction caused a large increase in metabolite conjugates at the expense of polar metabolites 

21 and benzo[a]pyrene diols, and a strong increase in covalent binding of benzo[a]pyrene to skin 

22 DNA. 

23 Kao et al. (1988) also investigated the influence of skin hair on the rate of benzo[a]pyrene 

24 penetration. Using the same dynamic system, six strains of haired and two strains of hairless 

mice were administered a dose of 1 μg/cm225 benzo[a]pyrene. After 16 hours of incubation, 

26 between 4.4 and 9.4% of the dose had penetrated through viable skin of haired mice but only 2–

27 2.9% of the dose through skin of hairless mice. Since the authors found that permeation of 

28 testosterone, which was used as a comparison compound with similar physiochemical properties 

29 as benzo[a]pyrene, was the same in haired and hairless mice, 70% of the dose, they concluded 

30 that skin appendages have considerable metabolic capacity for benzo[a]pyrene. Extending these 

31 findings to humans implies that in the assessment of dermal absorption of benzo[a]pyrene, 

32 presence of dense hair may play an additional role.

33 Morse and Carlson (1985) compared in vivo dermal absorption of benzo[a]pyrene in 

34 BALB/c and SENCAR mice to find an explanation for the higher sensitivity of the latter strain 

35 toward benzo[a]pyrene-induced skin tumors. Animals were treated with 50 mg/kg 

36 benzo[a]pyrene dissolved in acetone. Levels of benzo[a]pyrene in skin of BALB/c mice did not 

37 reach higher peak levels, but stayed up to three times higher than in SENCAR mice between 6 

38 and 24 hours postexposure. The levels of benzo[a]pyrene-derived radioactivity in liver, lung, 
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1 and stomach also peaked at comparable levels, but stayed at up to twice the levels in SENCAR 

2 mice between 6 and 48 hours posttreatment, compared with BALB/c mice. There were no strain 

3 differences in these tissues when benzo[a]pyrene was administered orally. The metabolic 

4 capacity of the skin was assessed by measuring DNA binding of benzo[a]pyrene in skin. 

5 Six hours after exposure DNA-binding of benzo[a]pyrene was higher in SENCAR than in 

6 BALB/c mice, but by 24 hours after topical administrations, DNA adducts were higher in 

7 BALB/c than in SENCAR mice, possibly indicating differences in metabolism. Kao et al. 

8 (1988) had included the same two strains of mice in one of their studies and found comparable 

9 results, with 9.2% of the dose penetrating over 16 hours in BALB/c and 4.4% in SENCAR mice. 

10 However, the results of Morse and Carlson (1985) did not provide the expected toxicokinetic 

11 explanation for the difference in skin tumor sensitivity between the two mouse strains. 

12 In summary, due to the use of coal tar products for medicinal purposes, quantitative data 

13 on dermal absorption of benzo[a]pyrene in humans are more abundant than for other routes of 

14 exposure. The animal-to-human differences are significant. Generally, mice have greater 

15 absorption than humans, followed by rats and rabbits. The 24-hour penetration values range 

16 from 1 to 3% in viable human skin; much greater amounts of material are absorbed into the skin 

17 but do not readily permeate through it. Dermal absorption of PAH is strongly dependent on 

18 anatomical site (69% difference across six sites), while inter-individual variation is much smaller 

19 (7% difference across nine volunteers). Furthermore, dermal absorption is highly dependent on 

20 underlying metabolic activity of the skin. The vehicle of exposure also impacts dermal 

21 absorption, with vehicles that absorb benzo[a]pyrene, such as soil, or vehicles with low diffusion 

22 coefficients (high viscosity) decreasing the rate and degree of absorption. 

23 

24 3.1.4. Other Types of Exposure 

25 Ewing et al. (2006) used isolated, perfused rat lungs and delivered benzo[a]pyrene coated 

26 onto silica carrier particles (average size: 3.5 μm) at mean doses of 2.2, 36, and 8,400 ng to each 

27 lung within <1 minute. Perfusate was collected for 77 minutes thereafter. Lungs and perfusates 

28 were analyzed for benzo[a]pyrene and metabolites. Absorption was strongly dose-dependent: at 

29 the low and mid exposure levels benzo[a]pyrene concentration increased rapidly in the perfusate 

30 to reach a maximum within <5 minutes, then decreased over the remaining observation period. 

31 At the high dose, benzo[a]pyrene in the perfusate reached the maximum at about 30 minutes 

32 after exposure and stayed at a constant level from there on, i.e., the absorption of benzo[a]pyrene 

33 proceeded at zero order until all deposited solid benzo[a]pyrene was dissolved. The mass 

34 balances for benzo[a]pyrene equivalents in lung vs. perfusate were lung ca. one-third perfusate at 

35 the low dose, lung = perfusate at the mid dose, and lung about twice that of perfusate at the high 

36 dose. At the low exposure level metabolism was apparently able to convert most of the parent 

37 compound, while at the highest exposure level most of the absorbed benzo[a]pyrene remained 

38 unmetabolized even at the end of the experiment. The results suggest that, at low doses (2.2 
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1 ng/lung) benzo[a]pyrene is absorbed very efficiently in rat lung (two-thirds absorbed in 

2 77 minutes), while at higher doses the rate of absorption decreases markedly, either because of 

3 diffusion limitation, or by saturation of local metabolism. 

4 

5 3.2. DISTRIBUTION 

6 No adequate quantitative studies of benzo[a]pyrene tissue distribution in exposed humans 

7 were identified. Obana et al. (1981) observed low levels of benzo[a]pyrene in liver and fat 

8 tissues from autopsy samples. However, prior exposure histories were not available for the 

9 donors. Nevertheless, the identification of benzo[a]pyrene metabolites or DNA adducts in 

10 tissues and excreta of PAH-exposed populations suggest that benzo[a]pyrene is widely 

11 distributed. 

12 

13 3.2.1. Inhalation Exposure 

14 Numerous studies evaluated the disposition of benzo[a]pyrene or its metabolites 

15 following inhalation or instillation in the respiratory tract in animals. The distribution of 

16 benzo[a]pyrene following inhalation exposure was shown to be similar in various species. Male 

17 and female F344 rats were exposed nose-only for 30 minutes to atmospheres containing 

0.6 mg/m3 [3H]-benzo[a]pyrene adsorbed onto 67Ga2O3 or to 1.0 mg/m3 neat [318 H]-

19 benzo[a]pyrene (Sun et al., 1982). There were qualitative differences in time and amount of 

20 absorption between the exposure regimens. With either exposure regimen, high tissue levels of 

21 radiolabel were found in the small and large intestines and cecum, followed by liver, kidney, and 

22 blood. Levels in the upper GI tract (esophagus and stomach) differed, with significant levels of 

radioactivity in the stomach following exposure to [

3H]-benzo[a]pyrene adsorbed onto 67 23 Ga2O3 

24 but only minimal levels in the stomach following exposure to neat benzo[a]pyrene aerosol. 

25 Twelve hours after exposure, the highest tissue concentrations of radiolabel were found in the 

26 lower GI tract for both regimes. The differences in distribution of radiolabel likely reflect the 

27 relative contribution of two mechanisms for the delivery of benzo[a]pyrene-related radioactivity: 

28 for particle-adsorbed benzo[a]pyrene, mucociliary clearance followed by ingestion may be a 

29 significant factor, while aerosolized benzo[a]pyrene is likely to be more readily absorbed in the 

30 respiratory tract only. 

31 Other inhalation studies measured levels of administered radioactivity as benzo[a]pyrene 

32 parent compound or metabolites in whole blood, plasma or lung, but not in any other tissues 

33 (Ramesh et al., 2001a; Gerde et al., 1993a, b). The rapid appearance of benzo[a]pyrene and 

34 metabolites in the blood is consistent with the conclusion that benzo[a]pyrene is readily 

35 bioavailable following exposure by the inhalation route. The degree to which absorbed 

36 benzo[a]pyrene or metabolites was delivered to target tissues was not determined in these 

37 studies. 
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1 Weyand and Bevan (1986) examined benzo[a]pyrene disposition and metabolism in male 

Sprague-Dawley rats following intratracheal instillation of 1 μg/kg [32 H]-benzo[a]pyrene 

3 dissolved in triethylene glycol. The amount of radioactivity in various organs was determined at 

4 timed intervals between 5 and 360 minutes after dosing. Peak levels of radioactivity as the 

5 percentage of the administered dose per organ/tissue as well as time profiles were as follows: 

6 

7 • Early peak levels: lungs (59.5% at 5 minutes, declining 10-fold over 360 minutes); blood 

8 (3–4% between 5 and 15 minutes, declining only twofold by 360 minutes); spleen (0.5% 

9 at 5 minutes, declining very slowly); liver (20.8% at 10 minutes, gradually declining); 

10 heart/thymus (1.6% at 10 minutes, barely declining from 0.5% after 30 minutes). 

11 

12 • Medium term peak levels: carcass (27.1% at 60 minutes, mostly stable at around 

13 approximately 25% between 10 and 90 minutes and at approximately around 22% 

14 thereafter); kidney (2.4% at 90 minutes but rather stable at approximately around 2% 

15 between 10 and 360 minutes); testes (1.3% at 90 minutes but rather stable at 

16 approximately around 1% between 15 and 360 minutes). 

17 

18 • Late peak levels: stomach (6.9% at 120 minutes, decreasing slowly); intestinal contents 

19 (44.7% at 360 minutes, increasing over time period); intestines (14.9% at 360 minutes, 

20 continually increasing). 

21 

22 These profiles are consistent with rapid uptake and delivery of benzo[a]pyrene to well23 perfused tissues, followed by clearance from the tissues via metabolism and excretion 

24 (particularly in the feces). The profile of radioactivity appearing in the GI tract suggests partial 

25 removal of benzo[a]pyrene from the lungs via mucociliary escalator. Total recovery of 

26 radiolabel at 5 minutes was 96% of the dose and 104% at 360 minutes, indicating complete 

27 recovery from the tissues. 

28 Weyand and Bevan (1986) also investigated benzo[a]pyrene metabolites in tissues. 

29 Quinones were at highest concentrations in both lung and liver 5 minutes after instillation, 

30 accounting for 12 and 7% of radiolabeled extractable material, respectively. Benzo[a]pyrene 

31 disposition was also investigated in male rats with and without biliary cannulas. Distribution 

32 patterns among organs were similar, though the amount excreted in bile and intestinal contents 

33 was 77% of the administered dose in cannulated rats and 53% in animals that were not 

34 cannulated. The intestinal contents carried lower fractions of the administered dose as thioether 

35 and glucuronic acid conjugates than the bile, indicating enterohepatic recirculation of 

36 benzo[a]pyrene metabolites. 

37 A comparative intratracheal instillation study with Sprague-Dawley rats, Gunn rats, 

38 guinea pigs, and hamsters gave results (Weyand and Bevan, 1987) qualitatively similar to those 

reported by Weyand and Bevan (1986). Doses of 0.16 or 350 μg [339 H]-benzo[a]pyrene per animal 

40 were administered intratracheally, and the distribution of benzo[a]pyrene-derived radioactivity 

41 was determined in various tissues. Relative amounts of radiolabel recovered per gram of tissue 
28 DRAFT - DO NOT CITE OR QUOTE 

1 at 6 hours were: lung (1.7%) > kidney (0.76%) ≈ liver (0.67%) > testes (0.21%) = spleen 

2 (0.21%) = heart (0.21%) ≈ GI tract (0.19%) > stomach (0.13%) > carcass (0.071%). This pattern 

3 of distribution was qualitatively similar among all species tested at both doses, but the relative 

4 values differed between species. For example, in Sprague-Dawley rats the liver burden 

5 represented 0.67% of the recovered radiolabel/gram of tissue, while this value was 2.16% for 

6 hamsters, 0.35% for guinea pigs, and 1.02% for Gunn rats after the 0.16 μg/animal dose. The 

7 study did not include the intestine or its contents, where likely the majority of radioactivity was. 

8 When benzo[a]pyrene was instilled intratracheally into mice, Schnizlein et al. (1987) found that 

9 radioactivity in the lung declined steadily throughout the 144-hour investigation period, while 

10 levels in liver, spleen, and intestine peaked at 8 hours after dosing, decreased slowly until 

11 24 hours, and declined rapidly thereafter. It was also noted that as the lung burden of the 

12 radiolabel decreased, radioactivity increased in lung-associated lymph nodes (LALN) over the 6-

13 day study, suggesting distribution of benzo[a]pyrene or its metabolites via the lymph. 

Bevan and Ulman (1991) administered 1 μg/kg [314 H]-benzo[a]pyrene intratracheally to 

15 male Sprague-Dawley rats in three different liquid vehicles. At 6 hours after dosing, 56.2, 58.4, 

16 and 70.5% of the dose delivered from tricaprylin, ethyl laurate, and triethylene glycol, 

17 respectively, were recovered from bile. Recovery from whole lung after 6 hours was 13.0% of 

18 the administered dose for tricaprylin and 15.6% for ethyl laurate but only 2.6% for triethylene 

19 glycol, indicating that pulmonary absorption of benzo[a]pyrene was more efficient from a less 

20 hydrophobic vehicle than from a highly hydrophobic one. Among the other organs, kidney and 

21 liver retained rather high levels of radioactivity (around 2 and 5%, respectively, for the whole 

22 organs). Two percent or less of the administered dose was recovered from intestine and its 

23 contents in this study. 

24 Pregnant Wistar rats were exposed for 95 minutes on GDs 17 to 200, 350, 500, 650, or 

800 mg/m3 [325 H]-benzo[a]pyrene generated as a microcondensate from heated pure material 

26 (Withey et al., 1993). Immediately (time not specified) following exposure, the ranking of 

27 benzo[a]pyrene concentrations was maternal lung > blood > liver > kidney > fat > fetus. When 

28 total metabolites (as measured by detection of radiolabel) were measured immediately following 

29 dosing, the ranking was maternal lung > blood > liver > kidney > fetus > fat. Six hours after 

30 exposure, benzo[a]pyrene concentrations were fat > lung > kidney > liver > blood > fetus, while 

31 total metabolite concentrations were lung = fat > kidney > liver = blood > fetus. Concentrations 

32 of benzo[a]pyrene and metabolites in the GI tract were not reported. This study is consistent 

33 with other studies in showing wide tissue distribution of benzo[a]pyrene. In addition, the results 

34 also demonstrated placental transfer of benzo[a]pyrene and its metabolites. 

35 

36 3.2.2. Oral Exposure 

37 Saunders et al. (2002) evaluated the neurotoxicity of benzo[a]pyrene in male F344 rats 

38 following a single gavage dose in peanut oil at doses of 0, 25, 50, 100, or 200 mg/kg body 
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1 weight (10/sex/dose). Benzo[a]pyrene and metabolite concentrations were monitored for up to 

2 96 hours after administration in plasma, cerebellum, and cerebral cortex. Unmetabolized 

3 benzo[a]pyrene was observed in brain tissue only at the two highest doses, peaking at 2 to 

4 4 hours and then gradually decreasing. Total metabolite concentrations in plasma as well as 

5 brain tissue peaked at 2 hours, remained at similar levels until 6 hours and then gradually 

6 decreased. By 96 hours after dosing benzo[a]pyrene or its metabolites had dropped to trace 

7 levels. The distribution of metabolites shifted over the observation period, with diol metabolites 

8 (4,5-, 7,8-, and 9,10-benzo[a]pyrene diols) predominating for the first 12 hours and hydroxy 

9 metabolites (3-OH, 9-OH-benzo[a]pyrene) predominating at later time points. The distribution 

10 of metabolites was similar in plasma and brain. 

Neubert and Tapken (1988) administered a single 12 mg/kg oral dose of [14 11 C]-

12 benzo[a]pyrene to groups of five pregnant NMRI:Han-mice on GDs 11, 12, and 13 to determine 

13 whether placental transfer occurred. Six, 24, and 48 hours after treatment radiolabel was found 

14 in the maternal lung, liver, and kidney (between 5 and 17% of the dose were recovered per gram 

15 tissue at 6 hours post dosing, decreasing to 0.5–1.3% by 48 hours). Radiolabel was also found in 

16 the placenta and embryonic liver at one to two orders of magnitude less than that found in 

17 maternal tissues. Similar results were found with five pregnant albino rats that received a single 

18 oral dose of 200 mg/kg benzo[a]pyrene in sunflower oil (Shendrikova and Aleksandrov, 1974). 

19 Three hours after treatment, fetal levels of radiolabel were approximately 10% of the maternal 

20 concentration. 

21 Taken together it is apparent that, in rats, benzo[a]pyrene can penetrate the blood barrier, 

22 and in pregnant mice or rats it crosses the placental barrier and reaches the fetus. Otherwise, 

23 distribution data on benzo[a]pyrene following oral administration are insufficient to establish a 

24 cogent picture of organ or tissue distribution. 

25 

26 3.2.3. Dermal and Other Exposures 

27 Some studies have evaluated the distribution of benzo[a]pyrene and its metabolites 

28 following dermal or other dose routes. Morse and Carlson (1985) sought to determine whether 

29 differences in toxicokinetic parameters could explain the difference in tumor response between 

30 SENCAR (high susceptibility) and BALB/c mice (low susceptibility). The mice were 

31 administered radiolabeled benzo[a]pyrene via dermal application or orally, and the time course 

32 of radioactivity levels was assessed in several organs. Following topical application of 

33 benzo[a]pyrene, organ levels of radioactivity were generally 1.5–4 times higher in SENCAR 

34 than in BALB/c mice. Radioactivity levels were liver > lung ≈ stomach, with an approximately 

35 twofold difference. DNA adduct levels were liver > lung > stomach, with a threefold difference. 

36 Following oral dosing, tissue radioactivity levels differed little between the two strains and 

37 essentially followed the same pattern of distribution. However, DNA adduct levels following 

38 oral dosing were much higher than after topical administration, approximately 6 times higher in 
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1 stomach and liver and 10 times higher in lung. RNA and protein binding showed patterns 

2 similar to DNA binding. This study seems to indicate that the route of administration exerts little 

3 influence on the tissue distribution of benzo[a]pyrene. 

4 Moir et al. (1998) also measured the toxicokinetics of benzo[a]pyrene in male Wistar rats 

dosed i.v. with 2, 6, or 15 mg/kg of [14 5 C]-benzo[a]pyrene dissolved in an emulphor/saline 

6 emulsion. The concentrations of both benzo[a]pyrene-derived radioactivity and parent 

7 compound were determined in blood, adipose tissue, lung, liver, kidney, heart, spleen, brain, and 

8 testes as well as in urine and feces. The concentrations in all tissues examined except lung 

9 appeared to follow a similar pattern of smoothly increasing and decreasing curves, while the lung 

10 data were rather erratic (this pattern may reflect temporary trapping of lipid vesicles from the 

11 benzo[a]pyrene emulsion in the fine lung vessels). The authors also extracted selected tissues 

12 and determined parent compound levels by high-performance liquid chromatography (HPLC). 

Peak concentrations of [14 13 C]-benzo[a]pyrene equivalents and of parent compound and time to 

14 peak for the 2 mg/kg dose group are given in Table 3-1. Similar patterns were observed at 

15 higher doses, with notable exceptions noted below. 

16 

Table 3-1. Distribution of benzo[a]pyrene in selected tissues of male rats 

following i.v. dosing with 2 mg/kg 

Tissue/organa Blood Adiposeb Kidneyc Liver Lungd

Total tissue radioactivity 

Peak level (μg/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 (μg/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 

aMean ± SD, n = 3–4. 

bAdipose tissue levels showed broad maxima between 20 and 480 min; the highest values are shown. 

cTotal radioactivity levels in kidney changed little between 30 and 480 min. 

d

Parent compound lung had another maximum of 14.65 ± 1.67 μg/g at 120 min; a second maximum at this time 

point was observed at the higher doses, too, but those did not exceed the value of the first maximum at 5 min. 

Source: Moir et al. (1998). 

17 

18 At all exposure levels blood maintained the lowest initial concentrations (with some 

19 exceptions in white adipose tissue discussed below). Half-lives for benzo[a]pyrene parent 

20 compound in blood after the 2 and 6 μg/kg doses were 36 and 25 minutes, respectively, while no 

21 parent compound could be detected at time points >480 minutes. Following the 15 μg/kg dose, 

22 blood showed an initial benzo[a]pyrene decline similar to the lower doses, but benzo[a]pyrene 

23 could be recovered up to the end of the study at 32 hours post dosing, and with these additional 

24 data a second elimination phase half-life for benzo[a]pyrene was estimated at 408 minutes. The 
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1 final half-lives for elimination of benzo[a]pyrene “metabolites” (total benzo[a]pyrene-derived 

2 radioactivity minus parent compound) from blood were 666, 848, and 179 minutes for the 2, 6, 

3 and 15 mg/kg doses, respectively. It is fair to speculate that the second phase of elimination is 

4 the result of redistribution of parent compound (and subsequent metabolism) from a large, deep 

5 compartment such as adipose tissue and/or enterohepatic circulation. 

6 Similar to the results in the lung, erratic patterns of radioactivity occurred in adipose 

7 tissue. Peaks and dips in radioactivity levels in lung occurred roughly with a pattern opposite to 

8 that in adipose tissue. This might suggest occasional redistribution of benzo[a]pyrene parent 

9 compound between a pool of vesicles trapped in the lung and adipose tissue. Data derived from 

10 these tissues should therefore be viewed with caution. Moir et al. (1998) noted that, as a general 

rule, increasing tissue radioactivity levels following administration of [14 11 C]-benzo[a]pyrene 

12 reflected metabolite accumulation. Kinetics models were fit to the data to derive rate constants 

13 for clearance of benzo[a]pyrene. Many organs displayed rapid uptake phases for 

14 benzo[a]pyrene: liver (uptake complete at the first time point, 5 minutes) kidney, brain, testis, 

15 and adipose tissue, which was followed by a bi-exponential decline (except in adipose tissue). 

16 Half-lives for benzo[a]pyrene parent compound elimination changed with the dose administered 

17 (half-life in minutes at doses of 2, 6, and 15 μg/kg, respectively): liver (28, 163, and 281), 

18 kidney (498, 456, and 389), and adipose tissue (239, 945, and 781). The following organs were 

19 evaluated for the 2 μg/kg dose only: brain (2,326 minutes), heart (25 minutes), and spleen (38 

20 minutes); the latter, most likely due to nondetectable radioactivity levels at later time points, 

21 reflect the initial elimination phase only. Elimination half-lives of benzo[a]pyrene metabolites in 

22 liver and several other organs were in the range of 10–15 hours and independent of dose, 

23 suggesting first-order elimination. Overall, the results of Moir et al. (1998) suggest a pattern 

24 consistent with initial wide distribution determined by blood flow, with lipophilicity and rates of 

25 metabolism contributing to temporal patterns of organ levels after the initial distribution period. 

26 Some studies showed that reactive metabolites of benzo[a]pyrene are transported in the 

27 blood and may be distributed to tissues incapable of benzo[a]pyrene metabolism. Ginsberg and 

28 Atherholt (1989) evaluated DNA adduct formation after intraperitoneal (i.p.) administration of 

29 benzo[a]pyrene in mice. Serum of benzo[a]pyrene-treated mice incubated with splenocytes or 

30 salmon sperm DNA resulted in adduct formation suggesting that reactive benzo[a]pyrene 

31 metabolites were systemically distributed and available for interaction with target tissues. DNA 

32 adduct levels formed in vivo were highest in liver, lung, and spleen, with levels in kidney and 

33 stomach significantly lower. 

34 Taken together, the limited human data and few toxicokinetic studies in animals 

35 demonstrate that benzo[a]pyrene and its metabolites are widely distributed throughout the body. 

36 Inhalation of particle-bound or pure benzo[a]pyrene results in significant levels of 

37 benzo[a]pyrene and metabolites in numerous tissues. Distribution of inhaled benzo[a]pyrene and 

38 its metabolites to the GI tract is a result of both mucociliary clearance of particulates from the 
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1 lung and of biliary excretion following metabolism. Following absorption, benzo[a]pyrene and 

2 metabolites are initially found predominantly in the highly perfused tissues, such as the lung, 

3 liver, and kidneys. Lower amounts are distributed to other tissues, including the male 

4 reproductive organs, central nervous system, and adipose tissue. Despite its high lipophilicity 

5 benzo[a]pyrene has no specific affinity for lipid-rich tissues, most likely because of its rapid 

6 metabolism to more hydrophilic compounds. There is some indication that the distribution of 

7 benzo[a]pyrene is not much influenced by the route of administration. Reactive benzo[a]pyrene 

8 metabolites are also distributed via blood where they may form protein adducts or reach tissues 

9 themselves unable to form reactive benzo[a]pyrene metabolites. Benzo[a]pyrene or metabolites 

10 are also transferred to the fetus at concentrations one to two orders of magnitude lower than 

11 those found in maternal tissues (Withey et al., 1993; Neubert and Tapkin, 1988; Shendrikov and 

12 Aleksandrov, 1974). 

13 

14 3.3. METABOLISM 

15 The metabolism of benzo[a]pyrene is a critical aspect of the assessment of its potential 

16 toxicity because for many endpoints reactive metabolites are likely to contribute to the toxic 

17 response. Numerous reviews on the metabolism of benzo[a]pyrene are available (Miller and 

18 Ramos, 2001; WHO, 1998; ATSDR, 1995; Conney et al., 1994; Grover, 1986; Levin et al., 

19 1982; Gelboin, 1980). Key concepts have been adapted largely from these reviews and 

20 supplemented with recent findings. 

21 benzo[a]pyrene is metabolized extensively by Phase I reactions to form numerous 

22 oxidative or reactive metabolites that are targets for further metabolism through diverse phase II 

23 reactions. Many of the enzymes involved in benzo[a]pyrene metabolism are isoenzymes within 

24 gene families, the members of which have varying metabolic specificities. Many of these 

25 enzymes are encoded by genes that show functional polymorphism. Many of the critical 

26 enzymes are inducible by a variety of agents, including benzo[a]pyrene itself, and, therefore, 

27 studies that evaluate benzo[a]pyrene kinetics following single short-term exposures may not be 

28 representative of the kinetics of benzo[a]pyrene following longer-term exposure conditions or 

29 exposure to mixtures. Metabolism of benzo[a]pyrene is species-, strain-, and organ-system30 specific. There are age- and gender-related differences in the expression of many of the key 

31 enzymes that must be considered. 

32 The metabolism of benzo[a]pyrene has been extensively studied using both in vivo and in 

33 vitro models, and a schematic representation of metabolic pathways is provided in Figure 3-1. 

34 Only Phase I reaction products are shown. Phase II reactions include glutathione conjugation of 

35 diol epoxides, sulfation and glucuronidation of phenols, and reduction of quinones by 

36 NADPH:quinone oxidoreductase (NQO)with subsequent conjugation. 
1 

2 

3 Source: Miller and Ramos (2001).

4 

5 Figure 3-1. Metabolic pathways for 

6 

7 Some enzymes involved in the metabolism of 

8 (although benzo[a]pyrene itself is a relatively weak inducer compared to other environmental 

9 pollutants, such as certain dioxins and polychlorinated biphenyls). The cellular mechanisms 

10 underlying the inducibility of these enzymes, as well as the relative p

11 has been reviewed in detail (Miller and Ramos, 2001; Whitlock, 1999; Nebert et al., 1993). 

12 Inducibility of genes that metabolize 

13 genetic locus that imparts inducibility is designated the Ah locus, so named for the enzyme 

14 activity aryl hydrocarbon hydroxylase (AHH) (now known to be catalyzed by enzyme activities 

15 from the CYP1 family. The Ah gene encodes a cytosolic receptor that regulates the inducible 

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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 other environmental 

pollutants, such as certain dioxins and polychlorinated biphenyls). The cellular mechanisms 

underlying the inducibility of these enzymes, as well as the relative potency 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 designated the Ah locus, so named for the enzyme 

activity aryl hydrocarbon hydroxylase (AHH) (now known to be catalyzed by enzyme activities 

gene encodes a cytosolic receptor that regulates the inducible 

DO NOT CITE OR QUOTE

are highly inducible 

itself is a relatively weak inducer compared to other environmental 

pollutants, such as certain dioxins and polychlorinated 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 designated the Ah locus, so named for the enzyme 

activity aryl hydrocarbon hydroxylase (AHH) (now known to be catalyzed by enzyme activities 

gene encodes a cytosolic receptor that regulates the inducible 
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1 expression of genes that encode Phase I (CYP1A and CYP1B isoforms) enzymes and may 

2 interact coordinately in regulating genes that encode isoforms of the Phase II enzymes uridine 

3 diphosphate (UDP)-dependent glucuronosyltransferases [UGTs], GSTs, NQO1) that metabolize 

4 the products of Phase I metabolism. The role of these enzyme systems in benzo[a]pyrene 

5 metabolism is discussed below. Due to the inducibility of benzo[a]pyrene metabolism, 

6 interpretation of toxicity studies should consider whether the studies were conducted in species 

7 and strains that have inducible metabolism, whether duration of exposure was sufficient to 

8 induce benzo[a]pyrene metabolism, and whether or not known inducers were administered. 

9 

10 3.3.1. Phase I Metabolism 

11 3.3.1.1. CYP450-dependent Reactions

12 Phase I reactions are catalyzed primarily by the mixed function oxidase system of 

13 enzymes associated with CYP450 to form arene oxides. This initial phase I metabolic reaction 

14 of benzo[a]pyrene is carried out primarily by the inducible activities of CYP1A1 or CYP1B1 and 

15 the constitutively expressed and somewhat inducible CYP1A2, depending on the tissue. Other 

16 CYP isoforms may also catalyze the initial oxidation reactions. NAPDH:CYP reductase is an 

17 important cofactor for this reaction as a supplier of redox equivalents (Byczkowski and Gessner, 

18 1989). In addition to the CYP450 enzymes there are also a number of oxidoreductases that are 

19 not typically considered Phase I enzymes, yet can play an important role in the oxidative 

20 metabolism of benzo[a]pyrene (see below). 

21 The isomerization of the arene oxides to their respective phenol metabolites is thought to 

22 be a nonenzymatic process; however, physical/chemical studies have shown that rearrangement 

23 is susceptible to catalysis by amines (Johnson and Bruice, 1975). This would suggest that 

24 rearrangements in vivo could be catalyzed by the amino or sulfhydryl groups of proteins. 

25 Typically, a single phenolic isomer tends to be produced and the direction of regioselective ring 

26 opening is predictable based on the relative stability of the two possible cationic intermediates 

27 (Fu et al., 1978). In accordance with these predictions, exclusively 3-OH-benzo[a]pyrene but not 

28 the other possible phenol metabolite, 2-OH-benzo[a]pyrene, is formed from the 2,3-oxide of 

29 benzo[a]pyrene (Yang et al., 1977). The other monophenol metabolites of benzo[a]pyrene 

30 include 1-, 6-, 7-, and 9-OH-benzo[a]pyrene. Benzo[a]pyrene epoxide formation may yield both 

31 phenols and dihydrodiols. Arene oxides that are poor substrates for epoxide hydrolase (EH) or 

32 the less stable ones that rearrange rapidly, such as benzo[a]pyrene 2,3-oxide, are less likely to 

33 yield dihydrodiols. 

34 The arene oxides can be hydrolyzed by EH to form dihydrodiols (Oesch, 1980). The 

35 dihydrodiols may be further metabolized by CYPs to form diol epoxides, which are the DNA36 reactive metabolites that have been the subject of most studies. In particular, much of the study 

37 of oxidative products of benzo[a]pyrene metabolism has been done for the 7,8-oxide, since it is a 

38 precursor to the potent DNA-binding metabolites. The metabolism of benzo[a]pyrene, as well as 
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1 of PAHs in general, proceeds with a high degree of stereoselectivity. Since most aromatic bonds 

2 are prochiral, their epoxidation catalyzed by CYPs often results in optically active products. 

3 Liver microsomes from rats stereospecifically oxidize the 7,8-bond of benzo[a]pyrene to yield 

4 almost exclusively the (+)-benzo[a]pyrene-(7,8)-oxide (see Figure 3-2). Each enantiomer of the 

5 7,8-oxide is stereospecifically converted by EH to a different dihydrodiol via attack of water at 

6 the 8-position. The (+)-benzo[a]pyrene-7,8-oxide gives rise to the (-)-benzo[a]pyrene-7,8-

7 dihydrodiol, while the (-)-benzo[a]pyrene-7,8-oxide yields the (+)-benzo[a]pyrene-7,8-di8 hydrodiol. 

9 

10 Source: Grover (1986). 

11 

12 Figure 3-2. The stereospecific activation of benzo[a]pyrene. 

13 

14 Further metabolism of the (-)-benzo[a]pyrene-7,8-dihydrodiol enantiomer by rat CYP 

15 enzymes preferentially yields (+)-benzo[a]pyrene-7R,8S-diol-9S,10R-epoxide [(+)-anti-BPDE], 

16 which is believed to be the most potent carcinogen among the four stereoisomers (Figure 3-2). 

17 Formation of these stereoisomers does not occur at equimolar ratios, and the ratios differ 

18 between biological systems. For example, in a study with rabbit livers, purified microsomes 

19 oxidized the (-)-benzo[a]pyrene-7,8-dihydrodiol to isomeric diol epoxides in a ratio ranging from 

20 1.8:1 to 11:1 in favor of the (+)-anti-BPDE isomer (Deutsch et al., 1979). 

21 Another important factor in evaluating variability in the metabolic activation of 

22 benzo[a]pyrene is the degree to which functional polymorphisms plays a role. Schwarz et al. 

23 (2001) used recombinant CYP1A1 allelic variants to determine catalytic activity in vitro. 

24 Catalytic activity of the variant allele 1A1.4 was 70%, and that of variant allele 1A1.2 was only 

1

2

3

4

7 6 5

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 

1 50% of the WT allele, CYP1A1.1. Km values were generally lower for variants than for the 

2 WT. Each variant produced BPDE, with the activity of CYP1A1.1 > 1.2 > 1.4. The formation 

3 of diol epoxides was stereospecific, with the allelic variants producing about three times the 

4 amount of (±)-anti-BPDE isomers, the suspected ultimate carcinogens, as compared to the 

5 noncarcinogenic stereoisomers (±)-syn-BPDE. Wu et al. (2002) found no relationship between 

6 benzo[a]pyrene metabolite formation and the CYP1A1 MspI polymorphism. The identification 

7 and characterization of CYP polymorphisms has been the subject of numerous reviews (e.g., 

8 Wormhoudt et al., 1999). 

9 Several studies have attempted to clarify the question of which CYP isozyme is 

10 predominantly responsible for the metabolism of benzo[a]pyrene. The studies used knock-out 

11 (ko) animals in which either one of the isozymes in question, CYP1A1 1A2, or 1B1, or the Ah 

12 receptor (AhR) had been removed or inactivated (CYP 1A1 and 1B1 levels respond to AhR 

13 induction, while 1A2 is expressed constitutively). Kleiner et al. (2004) measured DNA adduct 

formation in the epidermis of 1B1–/–, 1A2–/–, AhR–/–, and WT mice. Six [314 H]-PAHs were 

15 administered topically in one dose of 10–2000 nmol and animals were sacrificed after 24 hours. 

Absence of CYP1A2 had very little effect on benzo[a]pyrene adduct formation; in 1B1–/– 16 mice 

adducts were about 150% of WT (not significant), while in AhR–/– 17 they were only 27% of WT. 

18 These findings differed considerably with other PAHs. The benzo[a]pyrene-DNA adduct was 

19 identified as being derived from (+)-anti-BPDE. The authors concluded that 1A1 was the 

20 primary CYP to metabolize benzo[a]pyrene, and that 1B1 rather serves detoxification. 

21 Sagredo et al. (2006) conducted a similar experiment, but with various types of AhR 

knock out mice. AhR+/+, 

+/–, and –/– 22 mice were treated once with 100 mg/kg benzo[a]pyrene by 

23 gavage. Twenty-four hours after treatment gene expression for CYP1A1, 1B1, and AhR was 

24 measured in lung, liver, spleen, kidney, heart, and blood by real-time or reverse transcriptase 

PCR (RT-PCR). CYP1A1 expression was increased following benzo[a]pyrene treatment in +/+ 25 

and +/– mice (generally higher in heterozygotes), but –/– 26 mice expressed no 1A1. There was a low 

27 level of basic 1B1 expression in all three genotypes that was inducible by benzo[a]pyrene in 

lung, but not in liver in AhR+/+ and +/–, but not at all in either tissue of AhR–/– 28 mice. Expression 

29 of 1A1 was 25–40 times that of 1B1. There was an AhR gene-dose-response relationship for the 

30 basal CYP1A1 expression, i.e., +/+ > +/– > –/–, but no such dependence was seen for 1B1. 

31 Protein adduct levels were spleen > liver > lung > heart > plasma > kidney. The tissue levels of 

32 adducts showed an inverse relationship with AhR gene dose, i.e., –/– > +/– > +/+. Similarly, the 

33 levels of unmetabolized benzo[a]pyrene and of free benzo[a]pyrene-tetrol metabolites were 

higher in all organs of AhR– mice, as compared with AhR+ mice. Also, in the livers of AhR–34 

35 mice the levels of the less carcinogenic tetrol II were much higher than those of the tetrol I, 

opposite to the situation in AhR+36 mice. The authors suggested that the high levels of free 

benzo[a]pyrene metabolites in AhR–/– 37 mice were the result of delayed bioactivation, and that a 

38 powerful AhR-independent pathway for benzo[a]pyrene metabolism must exist. The authors 
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1 explained the very high levels of benzo[a]pyrene adducts in organs outside the liver as the result 

of slow detoxification of the agent in the liver of AhR–2 mice, allowing high concentrations of the 

3 parent compound to reach distant tissues. These findings establish important roles in 

4 benzo[a]pyrene metabolism for both CYP1A1 and 1B1, but they do not clarify which enzyme is 

5 responsible for biological activation, and which for detoxification. 

Uno et al. (2006) investigated the finding that CYP1A1 (1A1–/– 6 ) knock out mice are more 

7 sensitive than WT animals to the toxic effects of orally administered benzo[a]pyrene. They 

produced a series of C57BL/6-based single- or double knock out mice, 1A2–/–, 1B1–/–, 1A1/1B1–8 

/– and 1A2/1B1–/– 9 . Benzo[a]pyrene was administered in the feed at 1.25, 12.5, or 125 mg/kg for 

10 18 days (this dose is well tolerated by WT C57BL/6 mice for 1 year, but lethal within 30 days to 

the 1A1–/– 11 type). Steady-state blood levels of benzo[a]pyrene, reached within 5 days of 

treatment, were ~25 times higher in 1A1–/– and ~75 times higher in 1A1/1B1–/– 12 than in WT mice, 

13 while in the other knock out types clearance differed little from that in WT animals. 

14 Pretreatment of the animals with TCDD to induce the CYP1 enzyme family resulted in decreased 

15 benzo[a]pyrene peak blood levels and AUCs in WT, but doubled peak blood levels and vastly 

increased AUCs in the two 1A1–/– 16 types. A lower-than-WT benzo[a]pyrene clearance was 

observed only in the two 1A1–/– types. DNA adduct levels, measured by [32 17 P]-postlabeling in 

liver, spleen, and bone marrow, were highest in the 1A1–/– 18 mice at the higher doses, and in the 

1A1/1B1–/– mice at the mid dose only. Only 1A1–/– 19 mice, but not the other genotypes, showed 

20 signs of severe toxicity. In conclusion, the authors of the study painted a rather complex picture 

21 of how the three CYP1 family enzymes affect the toxicokinetics of benzo[a]pyrene. 

22 Detoxification of benzo[a]pyrene is mostly achieved by 1A1, probably not only in liver, but also 

23 in the intestine. Second, in spleen and bone marrow 1B1 brings about metabolic activation of 

24 benzo[a]pyrene, which, in the absence of 1A1, results in damage to the immune system. The 

25 authors suggested that tissue-specific expression of 1A1 and 1B1, respectively, determine an 

26 organism’s susceptibility to benzo[a]pyrene toxicity and, possibly, carcinogenicity. 

27 Van Lipzig et al. (2005) conducted experiments concerning potential estrogenic activity 

28 of mono- and dihydroxylated metabolites of benzo[a]pyrene. Estrogen receptor (ER) affinity 

29 and estrogenic activity were tested in T47D human breast adenocarcinoma cells (for 

30 experimental details see Section 4.5.2). Benzo[a]pyrene metabolite mixtures were generated 

31 using β-naphthoflavone (β-NF)-induced rat liver microsomes. Several hydroxylated 

32 benzo[a]pyrene metabolites had measurable estrogenic activity. In an attempt to identify 

33 enzymes involved in benzo[a]pyrene hydroxylation, the researchers added a specific CYP1A2 

34 inhibitor to the metabolic activation mixture and found a ~15-fold increased estrogenic activity 

35 of the benzo[a]pyrene metabolite mix. They suggested that 1A2 inhibition drove the metabolism 

36 of benzo[a]pyrene towards formation of more estrogenic metabolites. These findings impart a 

37 role to CYP1A2 in the metabolism of benzo[a]pyrene.
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1 To summarize, there is experimental evidence to suggest that three members of the CYP1 

2 family, 1A1, 1A2, and 1B1, contribute to the metabolism of benzo[a]pyrene. However, the 

3 available data do not attribute precise roles to either of these enzymes. There is evidence that 

4 their expressions, and thus activities, are organ- or tissue-specific and that local isozyme 

5 activities determine not only the ratio of toxification vs. detoxification, but also the pattern of 

6 highly toxic vs. less toxic metabolites. 

7 

8 3.3.1.2. Non-CYP-related metabolic pathways

9 Other bioactivation processes independent of CYP-mediated diolepoxide formation have 

10 been demonstrated for benzo[a]pyrene. One-electron oxidation (via CYPs or peroxidases) can 

11 generate radical cations, which, in turn, can generate benzo[a]pyrene quinones (see bottom 

12 portion of Figure 3-1). These metabolites may generate DNA damage through redox cycling or 

13 the formation of depurinating adducts (McCoull et al., 1999; Cavalieri et al., 1990). Kim et al. 

14 (2000) treated male Sprague-Dawley rats with 20 mg/rat benzo[a]pyrene by i.p. injection and 

15 reported that the pattern of lipid peroxidation and increase in antioxidant enzymes correlated 

16 with formation of quinone metabolites of benzo[a]pyrene. Direct i.v. injection of 

17 benzo[a]pyrene and a series of metabolites confirmed the quinone metabolites indeed to be 

18 associated with the observed increase in lipid peroxidation. Secondary oxidation of 6-OH19 benzo[a]pyrene can generate hydroquinone and quinone metabolites at the 1,6-, 3,6-, or 6,12-

20 positions. Quinone formation may be catalyzed by dihydrodiol dehydrogenases (DHHs), such as 

21 aldo-keto reductase (AKR) (Penning et al., 1999). Tsuruda et al. (2001) showed in vitro that the 

22 expression of rat DHH in human breast cancer MCF-7 cells generated 7,8-benzo[a]pyrene-diones 

23 from benzo[a]pyrene-7,8-diol. 

24 Mallet et al. (1991) treated tetradecanoylphorbol acetate-stimulated human 

25 polymorphonuclear leukocytes (PMNs) with (±)-trans-7,8-dihydroxy-7,8-dihydro26 benzo[a]pyrene. They found that the cells were able to transform the benzo[a]pyrene-diol into 

27 the diolepoxide and tetrols with a stereochemical anti/syn ratio of six. The kinetics of the 

28 reaction suggested that hydrogen peroxide or a ferryl-oxygen-transfer were involved. Because 

29 myeloperoxidase (MPO) uses hydrogen peroxide in its reaction, the authors inhibited this 

30 enzyme specifically with azide and found that the formation of tetrols from benzo[a]pyrene-diol 

31 was reduced. Thus, MPO is able to execute the metabolic activation of benzo[a]pyrene. 

32 Byczkowski and Kulkarni (1990) observed that benzo[a]pyrene diol can be cooxygenated 

33 during lipid peroxidation to form the diolepoxide. To reduce interference from CYP-catalyzed 

34 reactions they used term human placental microsomes, which are low in CYP450. Peroxidative 

35 conditions were created by a redox cycling system comprised of partially chelated ferrous ions 

36 and NADPH:CYP450 oxidase; this system quadrupled the formation of malonic dialdehyde (a 

37 measure of lipid peroxidation) compared with placental microsomes alone. The peroxidative 

38 system increased overall metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by 27–
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1 28%, compared with placental microsomes alone. The amounts of individual metabolites were 

2 changed to various extents in the presence of peroxidative conditions; the most striking 

3 observation was the more than doubled formation of trans-anti-benzo[a]pyrene-tetrol (the 

4 proximate carcinogen) and an almost tripled binding to protein. There was a highly significant 

5 correlation (p < 0.0005) between malondialdehyde and trans-anti-benzo[a]pyrene-tetrol 

6 formation. The authors pointed out that by means of this metabolic pathway the human fetus 

7 could be exposed to BPDE despite the absence of pronounced CYP450 activity in term placenta. 

8 Similarly, a redox cycling system based on vanadate-IV ions was able to increase formation of 

9 trans-anti-benzo[a]pyrene-tetrol five- to sixfold in the presence of term human placental 

10 microsomes (Byczkowski and Kulkarni, 1992). 

11 Flowers et al. (1996) investigated the role of radical and reactive oxygen species (ROS) 

12 formation in benzo[a]pyrene-quinone-induced redox cycling, a reaction involving DHH, which 

13 can oxidize BP-diol to benzo[a]pyrene-7,8-dione (BPQ). BPQ is mutagenic and genotoxic. In 

isolated rat hepatocytes BPQ was incorporated covalently into DNA (30 ± 17 adducts/10614 base 

15 pairs) while extensive DNA fragmentation took place. DNA fragmentation was also observed in 

16 hepatocytes treated with BP-diol; the effect was partially abolished when an inhibitor of DHH 

17 was added to the reaction. Hepatocytes treated with either BP-diol or BPQ produced superoxide 

18 anion radical, formation of which could be blocked by DHH inhibitors. In an in vitro experiment 

19 it was shown that BPQ at 0.05–10 μM caused DNA strand scission in the presence of NADPH 

20 and CuCl2, suggesting that redox-cycling took place. DNA strand scission was prevented by 

21 catalase and hydroxyl radical scavengers but not by superoxide dismutase. The authors 

22 concluded that DHH metabolizes (+/-)-anti-BPDE to BPQ, which in turn causes extensive DNA 

23 fragmentation via the generation of ROS. 

24 In a subsequent study, Flowers et al. (1997) provided more detail on the redox cycling 

25 reaction of BPQ. They showed that the reaction required the presence of NADPH (1 mM) and 

Cu2+ 26 (10 μM). During the reaction superoxide anion radicals, benzo[a]pyrene semiquinone 

27 radicals, hydroxyl radicals, and H2O2 were formed. Hydroxyl radical scavengers, such as

28 mannitol, sodium benzoate, or formic acid prevented the redox cycling (as assessed by DNA 

strand scission), as did the Cu+29 chelators bathocuproine or neocuproine. The results were 

interpreted as indicating that redox cycling of benzo[a]pyrene quinone involves a Cu+30 -catalyzed 

31 Fenton reaction. 

32 Other peroxidases such as prostaglandin H synthase (PHS) may also generate radical 

33 cations from benzo[a]pyrene (Parman and Wells, 2002). Marnett (1990) reviewed the role of 

34 PHS in benzo[a]pyrene metabolism. Peroxy radicals epoxidize the procarcinogen 

35 benzo[a]pyrene-7,8-diol via PHS to the epoxide BPDE. Stereochemical experiments have 

36 allowed the distinction between peroxide-mediated and CYP450-mediated epoxide formation 

37 from benzo[a]pyrene. Thus, peroxy radical-dependent epoxidation of benzo[a]pyrene-7,8-diol 

38 occurs in rat liver microsomes, mouse skin homogenates, cultured fibroblasts, cultured hamster 
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1 trachea, and freshly isolated mouse epidermal cells. Peroxy radical-generated metabolites are 

2 predominant in uninduced animals, while in ß-NF-induced animals the CYP450-produced 

3 metabolites prevail. There are other pathways by which peroxides can oxidize benzo[a]pyrene4 7,8-diol because inhibition of PGH synthase with non-steroidal anti-inflammatory drug 

5 (NSAIDs) does not prevent BPDE formation. 

6 Redox-active quinones are formed through the oxidative metabolism of benzo[a]pyrene, 

7 particularly at the 6-position (see Figure 3-1). NQO1 is an important enzyme for the 

8 detoxification of reactive quinones. Joseph and Jaiswal (1998) reported that NQO1 expression 

9 inhibited the formation of benzo[a]pyrene-quinone adducts with DNA and mutagenicity in vitro. 

10 There are also potential ring opening mechanisms for benzo[a]pyrene. Stansbury et al. (2000) 

11 used activated polymorphonuclear monocytes or a reconstituted in vitro system to generate a 

12 ring-opening dialdehyde metabolite from the benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) that 

13 can react with DNA to generate unique DNA adducts that are different from the ones formed by 

14 BPDE. 

15 Nordling et al. (2002) identified a novel benzo[a]pyrene metabolite (7-oxo16 benz[d]anthracene-3,4-dicarboxylic acid anhydride) in the urine of benzo[a]pyrene-treated rats. 

17 Follow-up in vitro experiments with this compound found it to be weakly mutagenic in 

18 salmonella strain TA102, able to induce DNA stand breaks in HT-29 cells, capable of inducing 

19 cytotoxicity via an apoptotic mechanism, and increasing gene expression through a 

20 cyclooxygenase (COX)-2 promoter in HCT 116 cells. These results demonstrate that novel 

21 benzo[a]pyrene metabolites have toxicological properties distinct from those of the better studied 

22 BPDE. 

23 The roles of PHS-2 (now mostly called COX-2), MPO, NQO1, and other enzymes in the 

24 oxidative metabolism of benzo[a]pyrene may be crucial, but not enough data are as yet available 

25 to attempt a quantitative comparison with the CYP-mediated pathways. This may be of some 

26 importance in the assessment of cancer risks because not only CYP450 isozymes, but also MPO 

27 and NQO1 exhibit gene polymorphism. This is an area where much more research is necessary. 

28 

29 3.3.2. Phase II Metabolism 

30 The reactive products of phase I metabolism are subject to the action of several phase II 

31 conjugation and detoxification enzyme systems that display preferential activity for specific 

32 oxidation products of benzo[a]pyrene. These phase II reactions play a critical role in protecting 

33 cellular macromolecules from binding with reactive benzo[a]pyrene diolepoxides, radical 

34 cations, or ROS. Therefore, the balance between Phase I activation of benzo[a]pyrene and its 

35 metabolites and detoxification by Phase II processes is an important determinant of toxicity. 

36 The diol epoxides formed from benzo[a]pyrene metabolism are not usually found as 

37 urinary metabolites, rather they are detected as adducts of nucleic acids or proteins if not further 

38 metabolized. Detoxification of the diol epoxide metabolites of benzo[a]pyrene is through 
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1 rearrangement to form tetrols or via conjugation with glutathione. Early work by Gozukara et al. 

2 (1981) demonstrated that exogenous EH added to benzo[a]pyrene-treated human monocytes 

3 reduced DNA binding, suggesting a role for this enzyme in the inactivation of diol epoxides. 

4 Furthermore, nonenzymatic hydration of diol epoxides proceeds rapidly in aqueous media in the 

5 absence of EH to yield tetrol products via cis or trans addition. A second mechanism for the 

6 detoxification of reactive diol epoxides formed from benzo[a]pyrene is through glutathione 

7 (GSH) conjugation. This Phase II reaction is catalyzed by GSTs. GSTs are a family of enzymes 

8 with varying substrate specificity and distribution among tissues (reviewed in Hayes and 

9 Strange, 2000; Eaton and Bammler, 1999; Hayes and Pulford, 1995). Primary isoforms of 

10 relevance for conjugation of BPDE include the α, μ, π, and θ isoforms (GSTA, GSTM, GSTP, 

11 GSTT, respectively). 

12 Numerous studies using human GSTs expressed in mammalian cell lines have 

13 demonstrated the ability of GST to metabolize benzo[a]pyrene diol epoxides. For example, Dreij 

14 et al. (2002) demonstrated that GST isozymes, including alpha class GSTA1-1, GSTM1-1, and 

15 GSTP1-1 isoforms, had significant catalytic activity toward benzo[a]pyrene-derived diol 

16 epoxides. Robertson et al. (1986) incubated isolated human GST isoforms with GSH and 

17 (±)anti-BPDE to assess differences in their catalytic properties. Maximum substrate turnover 

velocity (Vmax) values for α, μ, and π were 38, 570, and 825 nmol mg-1 minute-1 18 , and Km values 

19 were 28, 27, and 54 μM, respectively. Rojas et al. (1998) reported that no BPDE adducts were 

20 formed in GSTM1-positive cells, but adducts were present in GSTM1-negative cells. This body 

21 of in vitro studies suggests that GST is an important detoxification mechanism for 

22 benzo[a]pyrene-derived epoxides. This compelling evidence for a role of GSTs in protecting 

23 against reactive benzo[a]pyrene metabolites has triggered several molecular epidemiology 

24 studies. However, recent studies on the impact of polymorphism on adduct levels in PAH25 exposed human populations did not succeed in showing clear relationships between CYP1A1, 

26 EH, or GSTM1 polymorphisms and DNA (Hemminki et al., 1997) or blood protein adduct 

27 formation (Pastorelli et al., 1998). 

28 Conjugation with glucuronide is another important detoxification mechanism for 

29 oxidative benzo[a]pyrene metabolites. Most of the phenolic metabolites of benzo[a]pyrene are 

30 further metabolized by glucuronidation or sulfation, and significant portions of total metabolites 

31 in excreta or tissues can be recovered in this form. Bevan and Sadler (1992) administered a 

32 single 2 μg/kg benzo[a]pyrene dose by intratracheal instillation to male Sprague-Dawley rats and 

33 assessed the benzo[a]pyrene metabolite profiles in bile after 6 hours. Identified metabolites were 

34 31.2% quinol diglucuronides, 30.4% thioether conjugates, 17.8% monoglucuronide, 6.2% sulfate 

35 conjugates, and 14.4% unconjugated metabolites. 

36 The UGTs are a family of enzymes that catalyze the conjugation of UDP-glucuronide 

37 with endogenous substrates (e.g., bilirubin) as well as xenobiotics (reviewed in Guillemette, 

38 2003). UGT isoforms as well as their allelic variants show different patterns of tissue 
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1 distribution and catalytic activity toward benzo[a]pyrene-derived phenols and diols. For 

2 example, Fang and Lazarus (2004) assessed the ability of the allelic variants UGT1A1 and 1A9 

3 in human liver to catalyze glucuronidation of benzo[a]pyrene-7,8-diol. Microsomes from 

4 subjects homozygous for the UGT1A1*28 variant (present in approximately 12% of the 

5 Caucasian population) had approximately twofold lower UGT1A1 protein levels than liver 

6 microsomes from individuals with the WT allele. Addition of a UGT1A9 inhibitor to the 

7 incubation decreased benzo[a]pyrene-glucuronide formation three- to sixfold, suggesting that 

8 UGT1A9 also has significant catalytic activity toward benzo[a]pyrene-7,8-diols. With UGT1A9 

9 activity blocked, glucuronide formation in UGT1A1*28 homozygotes was significantly lower 

10 than in WT and heterozygous individuals. The apparent Km for this reaction did not differ 

11 among microsomes from allelic variants. 

12 UGT activity also shows significant interindividual variability. Hu and Wells (2004) 

13 evaluated glucuronidation of benzo[a]pyrene metabolites in human lymphocytes (HL) in vitro. 

14 The degree to which glucuronide conjugates were formed varied over 200-fold (percent of 

15 metabolites as glucuronide conjugates ranged from 0.01 to 5% of total benzo[a]pyrene 

16 metabolites). Incubation of lymphocytes with benzo[a]pyrene, benzo[a]pyrene-7,8-diol, or 

17 benzo[a]pyrene-4,5-diol resulted in covalent binding to protein with, in the case of 

18 benzo[a]pyrene, a 220-fold inter-individual variability. Addition of the UGT substrate, UDP 

19 glucuronic acid (UDPGA), lowered the inter-individual variability to 143-fold. For 

20 benzo[a]pyrene or its diols there was a statistically highly significant relationship between 

21 increase in glucuronidation and decrease in protein binding. Cytotoxicity also was inversely 

22 correlated to conjugation of diols and diones, suggesting that glucuronidation is an important 

23 pathway for protection from chemically reactive benzo[a]pyrene metabolites. 

24 Sulfation, normally a defoliation process, can produce a DNA-damaging intermediate in 

25 the case of benzo[a]pyrene. It was shown that in rat or mouse liver cytosolic sulfotransferase (in 

26 the presence of 3'-phosphoadenosine 5'-phosphosulfate) catalyzes formation of sulfates of 

27 benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol, benzo[a]pyrene-7,8-dihydrodiol, and benzo[a]pyrene28 7,8,9,10-tetrol. All three sulfates were tested for their ability to bind to DNA, but only the 

29 benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol-sulfate formed DNA adducts (Surh and Tannenbaum, 

30 1995). 

31 Although not specific for benzo[a]pyrene, there is now considerable evidence that genetic 

32 polymorphisms of the GST, UGT, and EH genes impart an added risk to humans for developing 

33 cancer. Of some significance to the assessment of benzo[a]pyrene may be that smoking, in 

34 combination with genetic polymorphism at several gene loci (for detail, see Section 4.8.3), 

35 increases the risk for bladder cancer (Moore et al., 2004; Choi et al., 2003; Park et al., 2003) and 

36 lung cancer (Alexandrie et al., 2004; Lin et al., 2003). Also, Leng et al. (2004), according to the 

37 English abstract of a paper in Chinese, showed that coke oven workers (who are exposed to 

38 PAHs, including benzo[a]pyrene) homozygous at the P187S site of the NQO1 gene or carrying 
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1 the null variant of the GSTM1 gene had a significantly increased risk of chromosomal damage in 

2 peripheral blood lymphocytes, while the risk was much lower than controls in subjects with a 

3 variant allele at the H113Y site of the EH gene. 

4 

5 3.3.3. Tissue-specific Metabolism 

6 3.3.3.1. Respiratory Tract Tissues

7 benzo[a]pyrene treatment has been associated with the induction of respiratory tract 

8 tumors. This finding is consistent with the ability of the lung to metabolize benzo[a]pyrene, 

9 which has been demonstrated in numerous studies. Ewing et al. (2006) investigated the 

10 hypothesis that lung cancer following PAH induction may be a result of slow absorption and 

11 extensive metabolism in the thick respiratory epithelia. The researchers used isolated, perfused 

12 rat lung to investigate these processes. Benzo[a]pyrene was coated onto 3.5 μm silica carrier 

13 particles at concentrations to deliver an average of 2.2, 36, and 8,400 ng to each lung within <1 

14 minute. Perfusate was collected for 77 minutes thereafter. Both perfusates and lungs were 

15 analyzed for benzo[a]pyrene and metabolites. Absorption and metabolism were both strongly 

16 dose-dependent: at the low and mid exposure levels benzo[a]pyrene concentration increased 

17 rapidly in the perfusate to a maximum after <5 minutes, then decreased over the remaining 

18 observation period. At the high dose, benzo[a]pyrene in perfusate reached the maximum at 

19 about 30 minutes after exposure and stayed at a constant level from there on, i.e., the absorption 

20 of benzo[a]pyrene proceeded at zero order until all deposited solid benzo[a]pyrene was 

21 dissolved. The mass balances for benzo[a]pyrene equivalents in lung vs. perfusate were lung ca. 

22 one-third perfusate at the low dose, lung = perfusate at the mid dose, and lung about twice that of 

23 perfusate at the high dose. At the low exposure level metabolism was apparently able to convert 

24 most of the parent compound, while at the highest exposure level most of the absorbed 

25 benzo[a]pyrene remained unmetabolized even at the end of the experiment. The authors pointed 

26 out that these findings may explain why many attempts have failed to inducer lung cancer in 

27 animals using high-dose particle inhalation protocols. The results further confirm that 

28 benzo[a]pyrene metabolism is organ specific. 

29 Autrup et al. (1980) compared the metabolic capacity of tracheobronchial tissues in 

30 culture among several species, including humans, mice, rats, hamsters and bovines. Results from 

31 this study are summarized in Table 3-2. Benzo[a]pyrene was metabolized extensively in tissues 

32 from all species tested, with lower amounts of metabolites identified in rats and nonresponsive 

33 mice. Patterns of metabolism differed among the species but showed formation of a complex 

34 array of metabolites, including phenols, diols, tetrols, and quinones. Data summarized from the 

35 study suggest that under the conditions tested: (1) upper respiratory tract tissues for all species 

36 were able to metabolize benzo[a]pyrene, (2) the degree of phase II conjugation products was 

37 greatest in humans, followed by hamsters, Ah-responsive mice, bovines, rats, and Ah38 nonresponsive mice, (3) multiple phase II conjugation pathways were operative in tissues of all 
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1 species, although the relative proportions of conjugate formation varied, and (4) conducting 

2 airway tissues from all species were able to metabolize benzo[a]pyrene into DNA-reactive 

3 metabolites, with DNA binding greatest in hamster trachea followed by human and bovine 

4 bronchus. 

5 DNA binding in human, rat, and mouse tissue was similar but considerably higher in 

6 hamster (Table 3-2). The results were quite variable among individuals, a 33-fold difference in 

7 human bronchus, a fivefold variation in human trachea, and a threefold difference in bovine 

8 bronchus, but minimal variation among individuals of the laboratory animal species. Overall, 

9 these results show that human lung tissue metabolizes benzo[a]pyrene in a manner that is 

10 qualitatively similar to that of species that are susceptible to lung tumors, although some 

11 quantitative differences in specific metabolic pathways are observed (Autrup et al., 1980). 

12 

Table 3-2. Species differences in tracheobronchial benzo[a]pyrene 

metabolism 

Species 

Total 

metabolitesa

Ratio organic/water 

soluble metabolites

Percent water soluble metabolites as 

sulfate esters, glucuronides, and 

glutathione conjugates 

DNA 

bindingb

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 

aMean ± SD in pmol/μg DNA. 

bMean ± SD in pmol/mg DNA; results are from trachea in mouse, rat, and hamster. 

Source: Autrup et al. (1980). 

13 

14 In vitro studies with human bronchial epithelial and lung tissue showed that 

15 benzo[a]pyrene is metabolized to the 7,8- and 9,10-diols and, to a lesser extent, to the 4,5-diol 

16 and 3-OH metabolites (Autrup et al., 1978; Cohen et al., 1976). The metabolites were identified 

17 as glutathione and sulfate conjugates; no glucuronide metabolites were found. The ability of 

18 human tissues to metabolize benzo[a]pyrene has also been demonstrated in lung-derived cell 

19 lines. Kiefer et al. (1988) demonstrated that benzo[a]pyrene was metabolized in vitro in the 

20 human lung cancer cell line NCI-H322. These cells were also able to form benzo[a]pyrene-7,8-

21 diol, suggesting that human lung cells are able to generate carcinogenic metabolites of 

22 benzo[a]pyrene. Approximately 30% of the detected metabolites were water-soluble, about 30% 
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1 of which were glutathione conjugates. Sulfates, but not glucuronide conjugates, were also 

2 detected. 

3 A complement of enzymes for the oxidative metabolism of benzo[a]pyrene in the lungs 

4 has been identified in both humans and animal tissues, and these activities are inducible with 

5 prior exposure. Wei et al. (2001) evaluated CYP1A1 levels in fresh lung tissue from nine human 

6 donors. CYP1A1 and CYP1A2 were present at variable levels in lung tissues based on mRNA, 

7 protein levels, enzyme activity, and ability of S9 fractions to induce mutagenicity in an Ames 

8 assay. CYP1B1 was not identified. The authors emphasized that, in contrast to some previous 

9 studies, they were able to identify CYP1A2 in human lung (Wei et al., 2001). In a subsequent 

10 study, Wei et al. (2002) lung tissue from 27 human donors was evaluated for CYP1A status. 

11 CYP1A1 and CYP1A2 transcripts were present at variable levels in nearly all samples and were 

12 inducible by benzo[a]pyrene treatment. Mean inducible CYP1A2 levels were roughly four times 

13 lower than CYP1A1. Microsomes prepared from these tissues pretreated with benzo[a]pyrene 

14 resulted in a threefold increase in DNA adduct formation, while pretreatment with the potent 

15 AhR ligand TCDD increased benzo[a]pyrene-DNA adduct formation to 24-fold over controls. 

16 This result shows that that CYP1A activity was highly inducible via the AhR pathway in human 

17 lung tissues. 

18 These results using human donor tissues are consistent with the body of literature 

19 demonstrating the induction of benzo[a]pyrene metabolism in lungs of rodents. For example, 

20 Vainio et al. (1976) compared the metabolism of intratracheally-instilled benzo[a]pyrene in the 

21 isolated perfused rat lung of both control and rats induced with 3-MC. Pretreated rat lungs had 

increased covalent binding of [322 H]-benzo[a]pyrene to lung tissue, decreased appearance of 

23 unmetabolized benzo[a]pyrene in perfusion liquid, and increased formation of water soluble 

24 metabolites. Bompart and Clamens (1990) assessed AHH activity in male Sprague-Dawley rats 

25 given 2 mg/kg benzo[a]pyrene by i.p. injection weekly for 30 weeks. Every third week, five 

26 animals were sacrificed and lung and liver microsomes were prepared for determination of AHH 

27 activity (as measured by formation of the 3-OH metabolite of benzo[a]pyrene). Control levels of 

28 AHH in lung were much lower than in liver. AHH activity in the lung increased with repeated 

29 benzo[a]pyrene doses until approximately week 15, when it reached levels approximately 

30 eightfold over controls. Even after this induction, lung AHH activity was still approximately 30-

31 fold lower than in liver. Benzo[a]pyrene treatments had no effect on AHH activity in the liver in 

32 this study. 

33 Petridou-Fischer et al. (1988) instilled radiolabeled benzo[a]pyrene into the nasal 

34 turbinates (ethmoid and maxillary) of monkeys and dogs. Metabolic activity in these tissues was 

35 demonstrated by the formation of diverse metabolic products (phenols, diols, tetrols, and 

36 quinones). No region-specific metabolism was identified. Even though ethmoid versus 

37 maxillary turbinates contain different CYP activities, the pattern of metabolites was qualitatively 
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1 similar. Differences were not compared quantitatively due to the small number of animals used 

2 (four dogs and two monkeys). 

3 Bond et al. (1988) showed differential levels of Phase I and Phase II activities in 

4 respiratory tract regions of dogs. Benzo[a]pyrene metabolism was greater in nasal tissue than in 

5 lung tissue. Metabolic activities towards benzo[a]pyrene in various areas of the upper 

6 respiratory tract ranged from 5 to 15 pmol/mg protein/minute; however, in the ethmoid turbinates 

7 they reached approximately 45 pmol/mg-minute. The was no difference in the regional patterns 

8 of metabolite formation. Although CYP isozyme activity was similar in liver and lung, 

9 benzo[a]pyrene metabolism in respiratory tract tissues was about one-tenth that in liver (one10 third for the ethmoid turbinates). EH activity was highest in the lower generations of the 

11 conducting airways, followed by liver then nasal tissues. GST activity was highest in liver, 

12 followed by nasal tissues. UGT activity was more evenly distributed among lung regions, and 

13 was similar to levels in the liver. These data show that in dogs the nasal region and lungs have 

14 greater metabolic capability for benzo[a]pyrene and its metabolites than the conducting airways, 

15 and levels of metabolism are generally similar to those observed in the liver. 

16 Dahl et al. (1985) evaluated benzo[a]pyrene metabolism in the respiratory tract tissues 

17 from Syrian hamsters. All regions of the respiratory tract had metabolic activity as assessed by 

18 the formation of benzo[a]pyrene metabolites. Activity was highest in the nasal tissues on a per 

19 gram tissue basis, with similar activities observed in esophagus, forestomach, trachea, larynx, 

20 and lungs. Total metabolism on a per organ basis was highest for the lung and trachea. Similar 

21 results were obtained for lung and nasal tissue of rats that had inhaled benzo[a]pyrene (Wolff et 

22 al., 1989). 

Persson et al. (2002) showed that [323 H]-benzo[a]pyrene instilled nasally in female 

24 Sprague- Dawley rats was taken up in nasal structures (sustenacular cells and Bowman’s glands). 

25 Transport of benzo[a]pyrene or metabolites (as determined by radiography) via axons of 

26 olfactory neurons to the olfactory bulb was identified, indicating uptake into nasal structures and 

27 transport to central nervous system (CNS) structures via neurons. 

28 Weyand and Bevan (1986) examined benzo[a]pyrene disposition and metabolism in male 

Sprague-Dawley rats following intratracheal instillation of 1 μg/kg body weight [329 H]-

30 benzo[a]pyrene. The overall concentration of benzo[a]pyrene metabolites in lung and liver 

31 decreased over the 360-minute period, with a shift from predominately lipid-soluble metabolites 

32 to an increasing component of water-soluble metabolites at later times. In the lung, 

33 benzo[a]pyrene metabolites represented 47.7% of the administered dose in the organic and 

34 11.9% in the aqueous fraction, respectively, at 5 minutes, while at 360 minutes the corresponding 

35 fractions were 2.16 and 1.83%, respectively. The metabolite profile determined in the organic 

36 phase at 360 minutes was as follows: conjugates or polyhydroxylated compounds, 16.3%; 9,10-

37 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-

38 quinone, 2.23%; 9-OH, 2.26%; 3-OH, 4.59%; and benzo[a]pyrene, 20.0%. Over the 360-minute 
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1 period, notable shifts in the relative proportion of benzo[a]pyrene and metabolite levels in the 

2 lung indicated a net increase in the contribution of conjugates and tetrols, stable to decreasing 

3 levels of quinones, increasing levels of diols and phenols, and decreasing levels of parent 

4 benzo[a]pyrene. A similar pattern was observed for concentrations of benzo[a]pyrene 

5 metabolites in the liver, except that quinone levels also increased in this tissue over the 6-hour 

6 period. This identification of significant levels of quinone metabolites early in the lungs is 

7 consistent with in vitro studies, such as that of Autrup et al. (1980) in which rat lung had a high 

8 capacity to form quinones originating from the oxidation of benzo[a]pyrene at the 6 position. 

9 Most of the metabolism studies have focused on the role of the CYP1 isoforms on 

10 benzo[a]pyrene metabolism in the lung. However, other CYPs may also be important for 

11 benzo[a]pyrene metabolism among species. For example, Shultz et al. (2001) reported that 

12 benzo[a]pyrene was metabolized using recombinant CYP2F2 from mouse lung in a cell-free in 

13 vitro assay, although metabolic capability was less than that of other isoforms. CYP2F2 

14 expression in mouse lung airways was greater than that in tracheal parenchyma, showing region15 specific metabolic differences. 

16 

17 3.3.3.2. GI Tract and Liver Tissues

18 Fontana et al. (1999) reported that in healthy volunteers fed diets enriched with char19 grilled meat, CYP1A1 and 1A2 activities were induced in the liver and CYP1A1 protein levels 

20 were increased in small intestine biopsies. No change in CYP3A4 or 3A5 levels was observed. 

21 DNA adducts in peripheral blood cells were inversely correlated to CYP1A levels in enterocytes 

22 and CYP1A2 levels in liver. These findings point to the presence of AhR ligands, such as 

23 benzo[a]pyrene, in char-grilled meat and lend further support to the complexity of 

24 benzo[a]pyrene metabolism by CYP isozymes. 

In a human hepatoma cell line (HepG2) incubated with [325 H]-benzo[a]pyrene, 

26 radiolabeled metabolites were recovered primarily in the medium (88.4% of the radioactive 

27 material) (Diamond et al., 1980). Sixty-four percent of the metabolites were unidentified water28 soluble metabolites. Chloroform extractable metabolites (36% of the radiolabel) included 7,8-

29 diol, 9,10-diol, quinones, 3-OH (16% combined) metabolites, and unmetabolized benzo[a]pyrene 

30 (20%). The cell lysate contained the same metabolites, but the proportions of the 3-OH 

31 metabolite and parent compound were relatively higher. Enzymatic treatment for conjugate 

32 formation did not change recovery of radioactivity, suggesting that at least this tumor cell line 

33 did not extensively form phenol products. The authors noted that the HepG2 cell line did not 

34 utilize the major phenol detoxification pathway of rodent cell cultures. 

35 Similar results were obtained with human hepatocytes in culture (Monteith et al., 1987). 

Following incubation for 24 hours, the primary metabolites of [336 H]-benzo[a]pyrene were 

37 unidentified highly polar, water-soluble conjugates. The next four most prevalent metabolites 

38 consisted of 3-OH-benzo[a]pyrene and the 4,5-, 9,10- and 7,8-diols. As the dose of 
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1 benzo[a]pyrene was increased from 10 to 100 μmol, the amount of metabolites increased in a 

2 linear fashion, suggesting that the capacity of human hepatocytes to metabolize benzo[a]pyrene 

3 was not saturated at benzo[a]pyrene concentrations up to 100 μmol. 3-MC induced rat 

4 microsomes convert benzo[a]pyrene to the BPDE approximately 10 times faster than 

5 microsomes from uninduced rats, likely through the induction of CYP1A1 (Keller et al., 1987). 

6 The rate-limiting step appeared to be competition for CYP1A1 binding sites between parent 

7 benzo[a]pyrene and the benzo[a]pyrene-7,8-diol. 

8 The metabolism of benzo[a]pyrene in the GI tract and liver has been studied. Zheng et al. 

9 (2002) determined the expression of UGT isozyme levels and activity toward benzo[a]pyrene 

10 from human tissue samples of the liver, lung, and regions of the aerodigestive tract (tongue, 

11 tonsil, floor of the mouth, larynx, esophagus). Glucuronidation of phenolic benzo[a]pyrene 

12 metabolites was detected in all aerodigestive tract tissues examined and, of the isoforms tested, 

13 activity was identified for UGT1A7, 1A8, and 1A10. No UGT expression or glucuronidation 

14 activity was detected in lung tissue. 

15 Bentsen-Farmen et al. (1999a) measured CYP1A1 induction and DNA adduct levels in 

16 Wistar rats given i.p. doses of 3-MC and followed by a single dose of benzo[a]pyrene, or a single 

17 benzo[a]pyrene dose without pretreatment. 3-MC pretreatment increased CYP1A1 activity in 

18 the liver and DNA adduct levels in both liver and lung, with significant correlation to CYP1A1 

19 activity in lung but not in liver. The results indicate the difficulty in using DNA adducts as a 

20 biomarker in short-term exposures. The authors further reported that the study results were 

21 highly dependent on the analytical technique used. 

22 Ramesh et al. (2001b) exposed F344 rats for up to 90 days to benzo[a]pyrene in the diet 

23 at doses of 0, 5, 50, or 100 mg/kg-day. AHH activity (as measured by formation of the 3-OH24 benzo[a]pyrene) in the liver was increased in both males and females (approximately twofold 

25 higher in females at the end of the study) in a dose- and duration-dependent manner. 

Granberg et al. (2000) treated female NMRI mice with [326 H]-benzo[a]pyrene by i.v. 

27 injection and identified tissue-bound radiolabel in the lung, liver, and cardiovascular endothelial 

28 cells. The levels of tissue-bound radioactivity were correlated to CYP1A1, as determined by 

29 7-ethoxyresorufin-O-deethylase (EROD) activity. Pretreatment with the AhR ligand β-NF 

30 increased tissue binding in lung and heart cells. 

31 

32 3.3.3.3. Skin

33 Indirect evidence for metabolism of benzo[a]pyrene in skin is plentiful and includes 

34 numerous studies of dermally exposed humans or animals with subsequent detection of 

35 benzo[a]pyrene metabolites in tissues or excreta. These types of studies provide qualitative 

36 evidence for dermal metabolism but are confounded by potential contribution of metabolism in 

37 other tissues following systemic circulation. However, direct measurements of benzo[a]pyrene 

38 metabolism have been made in in vitro models using human skin or skin cells. 
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1 Hall and Grover (1988) also showed that human skin samples can metabolize 

2 benzo[a]pyrene. In skin samples from 11 subjects, the majority of benzo[a]pyrene-7,8-diol 

3 formed from benzo[a]pyrene was recovered as the (-) enantiomer. However, the stereospecific 

4 formation of tetrol metabolites was highly variable, suggesting significant inter-individual 

5 variability in the relative formation of the DNA reactive epoxide metabolites from 

6 benzo[a]pyrene. Bowman et al. (1997) assessed levels of the 7,8,9,10-tetrol metabolite in the 

7 urine of 43 psoriasis patients treated with coal tar medication. Urinary benzo[a]pyrene 

8 metabolites were detected in 40% of patients vs. 10% of matched controls. Amounts of 

9 metabolites detected were highly variable, but precise measures of applied dose were not 

10 available. These studies show that human skin can metabolize benzo[a]pyrene to diverse 

11 products, the metabolism of benzo[a]pyrene is inducible and variable with regard to 

12 stereospecific formation of downstream metabolites, and there may be significant interindividual 

13 variation. 

14 Merk et al. (1987) used hair follicles from volunteers to study benzo[a]pyrene 

15 metabolism and the effect of coal-tar-containing shampoos. Hair follicles were able to form 

16 numerous metabolites of benzo[a]pyrene. The results with coal-tar-exposed individuals showed 

17 that AHH activity was inducible in these cells and inhibition of CYP activity decreased the 

18 metabolism of benzo[a]pyrene and the formation of DNA binding activity. Alexandrov et al. 

19 (1990) demonstrated that hair follicles from human subjects (10 healthy female smokers and 10 

20 healthy female nonsmokers) were able to generate benzo[a]pyrene tetrols from diol epoxide 

21 precursors. There was stereospecific metabolism with most of the tetrol formed from (-)-

22 benzo[a]pyrene-7,8-diol consistent with extensive formation of (±)-anti-BPDE rather than the 

23 (±)-syn-BPDE metabolite. Agarwal et al. (1991) reported that human melanocytes treated in 

24 culture formed diverse benzo[a]pyrene metabolites, including dihydrodiols, hydroxyl 

25 compounds, quinones, and their glucuronide and sulfate conjugates. 

26 Kao et al. (1985) investigated the metabolism of benzo[a]pyrene in skin from several 

27 species, including humans. In the case of human skin, TCDD induction could not be studied, but 

28 the authors investigated the influence of metabolic viability of the skin on metabolism. Skin 

samples were treated with 2 μg/cm2 [14 29 C]-benzo[a]pyrene in acetone and were incubated for

30 24 hours. Medium under the skin was then extracted with ethyl acetate but was not subjected to 

31 hydrolysis to identify conjugate formation. Fifty-two percent of the radioactivity in culture 

32 medium below viable human skin was composed of water-soluble metabolites, 8% were lipid33 soluble polar metabolites, 17% were diols, 1.2% were monophenols, 2.5% were quinones, and 

34 18% were parent compound. By contrast, previously frozen, nonviable skin allowed mostly 

35 parent compound to pass (50% of radioactivity in the medium) and considerably smaller portions 

36 of water-soluble and polar metabolites and diols to pass, but, surprisingly, relatively increased 

37 portions of monophenols and quinines to pass. Results for skin from other species—marmoset, 

38 rabbit, rat, mouse—were similar: water-soluble metabolites varied between 55 and 77% of the 
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1 radioactivity in the medium; polar metabolites, 5.8–13.2%; diols, 7.9–15.2%; monophenols, 0.7–

2 1.8%; quinones, 0.6–2.1%; and parent compound, 0% (mouse), to 6.7% (rabbit). Metabolites 

3 found below nonviable skin from animal species were not reported. 

4 Animal studies are consistent with the metabolism data in human skin. Ng et al. (1992) 

5 used an in vitro culture system with skin from female hairless guinea pigs and concluded that the 

6 degree of penetration of benzo[a]pyrene was dependent on metabolism, since the collection of 

7 the administered radioactivity was much greater for viable than nonviable skin. At the lowest 

dose tested (32.1 nmol/cm28 ), 37% of the applied radioactivity was recovered in the receptor fluid 

9 within 24 hours, 84% of which were metabolites, including OH derivatives, dihydrodiol diones, 

10 and conjugates. The tetrol metabolite of the DNA-reactive epoxide accounted for 2.56% of the 

administered dose. MacNicoll et al. (1980) assessed metabolism of [311 H]-benzo[a]pyrene in 

12 Swiss mouse skin maintained in short-term organ culture. After incubation for 24 hours, 

13 radioactivity derived from benzo[a]pyrene consisted of 3 benzo[a]pyrene-equivalents of lipid14 soluble metabolites, 147 benzo[a]pyrene-equivalents water-soluble metabolites, and 5 

15 benzo[a]pyrene-equivalents bound to skin. The high proportion of water-soluble metabolites 

16 indicates that benzo[a]pyrene was readily metabolized. 

17 Kao et al. (1984) evaluated metabolism of benzo[a]pyrene as a factor in the dermal 

18 penetration of benzo[a]pyrene in mouse skin. Skin samples formed predominantly polar 

19 metabolites and diols (approximately 20% of the radioactivity each), with small shares 

20 (approximately 1% of the penetrated material) of conjugates, monophenols, and quinones. 

21 Parent compound was recovered at about 1.5%. TCDD induction changed the portion of 

22 conjugates to almost 8%, at the expense of polar metabolites, diols, and parent compound, but 

23 did not affect monophenol or quinone formation. Investigation of the radioactivity left in the 

24 skin revealed that it was mostly parent compound (almost 50% of the dose in uninduced and a 

25 little more than 30% in induced skin), with about 12–16% water-soluble metabolites and polar 

26 metabolites, diols, monophenols, and quinones decreasing in that order from ~5% of the dose to 

27 <2%. 

28 

29 3.3.3.4. Reproductive Tissues and Fetal Metabolism

30 Several toxicity studies have demonstrated the ability of benzo[a]pyrene to impair 

31 reproductive function in male and female rodents as well as induce developmental toxicity. 

32 Therefore, the ability of benzo[a]pyrene to be metabolized in tissues that affect reproduction or 

33 development is of interest. Williams et al. (2000) reported the presence of CYP1A1, CYP1A2, 

34 and CYP1B1 transcripts in prostate tissue from human donors exposed in short-term organ 

35 culture and the activation of benzo[a]pyrene as indicated by DNA adduct formation. Primary 

36 human prostate cells have also been demonstrated to metabolize benzo[a]pyrene in vitro (Martin 

37 et al., 2002). 
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1 Bao et al. (2002) exposed a human endometrium epithelial cell line, which expresses low 

2 constitutional levels of EROD activity (used as a measure of CYP1 activity), to benzo[a]pyrene 

3 (1 mM) in vitro and observed a 12-fold induction of CYP1A1. Specific inhibitors of CYP1B1 

4 and CYP1A2 had no effect on EROD activity, suggesting little metabolic contribution from these 

5 two isoforms in endometrial tissues, while the CYP1A1 inhibitor α-NF (100 nM) inhibited 

6 EROD activity more than 60%. Melikian et al. (1999) measured DNA adducts in cervical 

7 epithelial and stromal tissues in smokers and nonsmokers. Increased levels of adducts in the 

8 tissues of smokers suggested that delivery of benzo[a]pyrene to the cervical tissues with 

9 subsequent metabolism may occur, although transport of reactive metabolites generated in other 

10 tissues was not specifically ruled out. 

11 Ramesh et al. (2003, available only as abstract) investigated benzo[a]pyrene metabolism 

in multiple organ systems in rats exposed by nose-only inhalation to 75 μg/m312 benzo[a]pyrene 

13 adsorbed on CB 4 hours/day for 60 days. AHH activity and benzo[a]pyrene metabolism were 

14 reported for various tissues. A diverse spectrum of metabolites was identified, including 

15 dihydrodiols and 3- and 9-monophenols. The concentrations of BPDE were highest in testes, 

16 providing mechanistic support for previously observed effects of benzo[a]pyrene on male 

17 reproductive parameters in animal toxicity studies.

18 Exposure to reactive benzo[a]pyrene metabolites may be a concern both in utero and 

19 during lactation. Wu et al. (2003) measured the generation of benzo[a]pyrene metabolites in F1 

20 generation pups and determined the mRNA development profile for the AhR in the absence and 

21 presence of subacute exposure concentrations of benzo[a]pyrene in preweaning rats. Pregnant 

F344 rats were exposed to 25, 75, or 100 μg/m322 benzo[a]pyrene aerosols via nose-only 

23 inhalation, 4 hours/day for 10 days (GDs 11–21). Benzo[a]pyrene metabolites and mRNA and 

24 protein expression profiles of AhR and CYP1A1 were analyzed in the cerebral cortex, 

25 hippocampus, liver, and plasma. Plasma and cerebral cortex benzo[a]pyrene levels on postnatal 

26 day (PND) 0 changed with the dose. Benzo[a]pyrene decreased steadily with time in these 

27 tissues, always reflecting the administered dose, reaching nondetectable levels by PND 30. In 

28 plasma, diols represented approximately 60% of the total metabolites over the period of 

29 20 PNDs. In the cerebral cortex, diols represented close to 80% of metabolites soon after 

30 parturition, decreasing to approximately 40% by PND 20. Benzo[a]pyrene-7,8-diol represented 

31 a maximum of approximately 25% of the recovered metabolites in plasma and approximately 

32 30% of recovered metabolites in the cerebral cortex. 

33 There was a statistically significant (p < 0.05) up-regulation of AhR mRNA, a subsequent 

34 induction of CYP1A1 mRNA, and a significant increase in CYP1A1 protein levels in pup livers 

at 100 μg/m335 compared to unexposed controls (data were presented only for PND 60 and the 

100 μg/m336 concentration). The AhR mRNA expression profile in the developing cerebral cortex 

37 and hippocampus indicated up-regulation of AhR during the first 3 postnatal weeks at all 

38 concentrations, although these differences were not statistically significant due to large 
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1 individual variation. At the high concentration, AhR mRNA abundance was more than twice 

2 that of controls by PND 30. However, this up-regulation of AhR mRNA was not accompanied 

3 by a concomitant up-regulation of CYP1A1 mRNA in the CNS. In fact, expression of CYP1A1 

4 in these tissues was very low or absent during the 1-month postnatal period after exposure to 

100 μg/m35 of benzo[a]pyrene (the only dose tested). Based on these findings, the authors 

6 suggested that the up-regulation of the AhR may increase the potential for benzo[a]pyrene 

7 neurotoxicity via the activation of CYP450 in liver and the subsequent deposition of toxic 

8 metabolites in the developing CNS. The results do not suggest that benzo[a]pyrene metabolism 

9 by CYP1A1 is induced in the developing CNS. 

10 Pregnant Swiss Webster mice were administered radiolabeled benzo[a]pyrene by i.v. 

11 injection, and maternal and fetal levels of benzo[a]pyrene and radiolabel were determined. Fetal 

12 tissue levels of radiolabel increased while maternal tissues decreased. The level of 

13 benzo[a]pyrene decreased in fetal tissue during this period, suggesting the accumulation of 

14 benzo[a]pyrene metabolites. Increasing the capacity of maternal plasma to bind benzo[a]pyrene 

15 by administering a benzo[a]pyrene antiserum decreased fetal accumulation of radiolabel, 

16 suggesting that bioavailability had decreased (McCabe and Flynn, 1990). Other investigators 

17 have also demonstrated the ability of benzo[a]pyrene to be transported to the fetus (Neubert and 

18 Tapken, 1988; Shendrikova and Aleksandrov, 1974). Martin et al. (2000) exposed exfoliated 

19 cells from breast milk of human donors to benzo[a]pyrene in vitro. Treated cells showed 

20 increased DNA single strand breaks (SSB) relative to controls, indicating that these cells can 

21 activate benzo[a]pyrene. Taken together, the results from these various experiments suggest that 

22 placental and lactational transfer of benzo[a]pyrene and active metabolites may be of concern. 

23 The developmental expression patterns of metabolizing enzymes can be an important 

24 determinant of childhood susceptibility. Numerous in vivo mechanistic tumor screening assays 

25 have been conducted in newborn mice (see Section 4.4.1 on mechanistic cancer studies), and the 

26 observed increases in tumors provide evidence for the ability of young animals to metabolize 

benzo[a]pyrene to DNA reactive metabolites. Melikian et al. (1989) administered [327 H]-

benzo[a]pyrene or [328 H]-BPDE via i.p. injection to CD-1 mice on PND 1, 8, or 15 and formation 

29 of benzo[a]pyrene metabolites was determined in lung and liver over time periods ranging from 

30 2 minutes to 24 hours after the last dose. In the lung, metabolites included diones, quinones, and 

31 phenols, and metabolite levels in the lung were higher on day 1 than on days 8 or 15. In liver, a 

32 different spectrum of metabolites was observed, dominated by unidentified polar metabolites. 

33 The percentage of radiolabel as metabolites was increased on days 8 and 15, perhaps reflecting 

34 greater inducibility of benzo[a]pyrene metabolism in liver versus the lung. Formation of 

35 glucuronide or sulfate conjugates was greater than glutathione conjugates in both tissues. The 

36 total amount of benzo[a]pyrene metabolized by Phase II enzymes was greater in liver than in 

37 lung on day 1 but was similar on days 8 and 15. Age-dependent expression of genes that encode 
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1 metabolizing enzymes has been the subject of reviews (e.g., Cresteil, 1998) that are addressed in 

2 Section 4.4. 

3 

4 3.3.3.5. Other Tissues

Moore et al. (1982) exposed organ cultures of human and rat bladder tissues to [35 H]-

6 benzo[a]pyrene for 24 hours and evaluated metabolite profiles. Benzo[a]pyrene was metabolized 

7 by bladder tissues of both species. Total mean amount of metabolites formed was higher in 

8 human than rat bladders (twofold). A similar spectrum of diverse metabolites was generated, 

9 although relative proportions varied. Formation of benzo[a]pyrene-7,8-diol was similar in both 

10 species and similar levels of DNA binding was observed. These results indicate that bladder 

11 tissues have metabolic capability for benzo[a]pyrene and that the metabolic capacity is similar 

12 between humans and rats. 

13 Several other target tissues for benzo[a]pyrene toxicity have been found to have the 

14 capacity to metabolize benzo[a]pyrene. For example, Moorthy et al. (2003) treated mouse aortic 

15 smooth muscle cells with benzo[a]pyrene, 3-OH-benzo[a]pyrene, or benzo[a]pyrene-3,6-quinone 

16 in vitro. Several DNA adducts were identified that were attributed to the 3-OH and BPQ 

17 metabolites. Benzo[a]pyrene treatment increased CYP1B1 but not CYP1A1 in these cells. The 

18 authors suggest that CYP1B1 activates benzo[a]pyrene to 3-OH and BPQ metabolites, which 

19 induce the DNA damage responsible for changes that are important precursors for 

20 atherosclerosis. 

21 

22 3.4. ELIMINATION 

23 3.4.1. Inhalation Exposure 

24 Human studies of benzo[a]pyrene exposure have generally been limited to individuals 

25 exposed to coke oven emissions, coal tars, or other products containing a mixture of PAHs and 

26 are of limited value in assessing urinary benzo[a]pyrene exposure biomarkers. Numerous 

27 studies have evaluated 1-OH pyrene in urine as a general marker for PAH exposure, but, because 

28 1-OH pyrene is not a metabolite of benzo[a]pyrene, these data are not directly useful in 

29 evaluating the toxicokinetics of benzo[a]pyrene. Some studies have unsuccessfully attempted to 

30 quantify exposure to benzo[a]pyrene via measurement of parent compound or its metabolites. 

31 Bentsen-Farmen et al. (1999b) compared air concentrations of PAHs to PAH metabolites in the 

32 urine of 17 electrode paste plant workers and detected 1-OH-Py but no benzo[a]pyrene 

33 metabolites despite the fact that benzo[a]pyrene in personal air samples showed mean exposure 

levels of 0.3 μg/m334 . Waidyanatha et al. (2003) also attempted to measure PAH exposure via 

35 urinary metabolite levels of coke oven workers; several PAHs but no benzo[a]pyrene were 

36 detected. In other cases, exposure and urinary metabolites were positively correlated (Hecht et 

37 al., 2003; Wu et al., 2002; Gündel et al., 2000; Grimmer et al., 1993; Becher and Bjørseth, 1983). 
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1 Wu et al. (2002) reported a correlation between benzo[a]pyrene-tetrols and total PAH exposure 

2 in coke oven workers. 

3 Several studies evaluated the elimination of benzo[a]pyrene in animals following 

exposure via the respiratory tract. Petridou-Fischer et al. (1988) applied 10 μL aliquots of [14 4 C]-

5 benzo[a]pyrene in a gelatin:saline solution over a 2-hour period to the ethmoid and maxillary 

6 nasal turbinates of monkeys and dogs to assess differences in benzo[a]pyrene disposition in 

7 portions of the nose. Urine levels reached a maximum of 0.69% of the administered dose in 

8 dogs and 0.07% in monkeys, while fecal levels reached a maximum of 6.42% of the dose in dogs 

9 and 1.17% in monkeys over 48 hours. The pattern of metabolites in the excreta was consistent 

10 with results from other respiratory tract deposition studies, showing that benzo[a]pyrene is 

11 excreted preferentially in the feces. Wolff et al. (1989) investigated the effects of nose-only 

12 exposure of male and female F344 rats to unlabeled benzo[a]pyrene for 4 weeks, followed by a 

single exposure to [14 13 C]-benzo[a]pyrene. Four weeks after treatment with the radiolabel, the rats 

14 had eliminated approximately 96% of the total dose in the feces. The mean half-life was 

15 calculated as 22 hours for feces and 28 hours for urine. Sun et al. (1982) exposed male and 

16 female F344 rats via nose-only inhalation for 30 minutes to atmospheres containing 0.6 μg/L 

[

3H]-benzo[a]pyrene absorbed onto 67Ga2O3 or to 1.0 μg/L neat [3

17 H]-benzo[a]pyrene and 

18 measured levels of radioactivity in tissues and excreta. With either exposure, benzo[a]pyrene 

19 excretion declined rapidly over the first 3 days after exposure, with little additional excretion 

20 occurring by 5 days postexposure. Excretion in feces was much greater than in urine for either 

21 exposure regimen. Following exposure of rats to neat benzo[a]pyrene, feces accounted for 86% 

22 of total excreted radioactivity and urine for 14%. Metabolite profiles were not evaluated. Wang 

23 et al. (2003) exposed B6C3F1 mice for 10 days to asphalt fumes in an inhalation chamber at a 

concentration of approximately 180 mg/m324 ; the benzo[a]pyrene content of the fumes was not 

25 reported. Benzo[a]pyrene metabolites measured in the urine of exposed mice were in the ng/100 

26 mL range and were identified at the following mass ratios (with benzo[a]pyrene arbitrarily set as 

27 1): 7,8,9,10-benzo[a]pyrene-tetrol, 14; benzo[a]pyrene-7,8-diol epoxides, 17; benzo[a]pyrene28 7,8-diol, 3.6; 3-OH-benzo[a]pyrene, 10. 

In a study investigating the disposition of benzo[a]pyrene, 1 μg/kg of the [329 H]-labeled 

30 compound dissolved in triethylene glycol was instilled into the trachea of male Sprague-Dawley 

31 rats (Weyand and Bevan, 1986). Approximately 60% of the administered dose was associated 

32 with the intestine and intestinal contents, and 2.2% of the radiolabel was recovered in the urine 

33 6 hours after dosing. The relative contribution of water- and lipid-soluble metabolites in the 

34 intestinal contents was 18.2 and 26.5% of the administered dose, respectively. The amounts of 

35 metabolites determined in the organic phase 6 hours after the end of exposure were as follows 

36 (percent of total in the organic phase): conjugates or polyhydroxylated compounds (24.1%), 

37 9,10-diol (7.75%), 4,5-diol (8.43%), 7,8-diol (5.73%), 1,6-quinone (7.13%), 3,6-quinone 

38 (7.85%), 6,12-quinone (7.10%), 9-OH (2.24%), 3-OH (4.66%), benzo[a]pyrene (1.55%). The 
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1 total amounts of metabolites in feces and urine were not reported in this study, and, due to 

2 enterohepatic circulation, the intestinal content concentrations may not reflect the final amounts 

3 of each metabolite in the excreta. Bevan and Sadler (1992) administered a single intratracheal 

4 instillation of benzo[a]pyrene (2 μg/kg) to male Sprague-Dawley rats and assessed metabolite 

5 profiles in bile after 6 hours. Relative metabolite levels were 31.2% diglucuronides, 30.4% 

6 thioether conjugates, 17.8% monoglucuronides, 6.2% sulfate conjugates, and 14.4% 

7 unconjugated metabolites. 

8 Weyand and Bevan (1987) studied the differences in benzo[a]pyrene elimination among 

9 rats, hamsters, and guinea pigs. Male Sprague-Dawley and Gunn rats (the latter a strain 

10 genetically deficient in bilirubin glucuronidation), male Syrian golden hamsters, and male 

Dunkin-Hartley guinea pigs were given single intratracheal doses of 0.16 μg or 350 μg [311 H]-

12 benzo[a]pyrene per animal. Tissue levels and the amount of radiolabel excreted into the urine 

13 and bile 6 hours after treatment are given in Table 3-3. Urinary excretion of benzo[a]pyrene 

14 metabolites amounted only to a small fraction of biliary excretion in all tested species. The 

15 results suggested that the metabolic capacity for benzo[a]pyrene became saturated in guinea pigs 

16 and Sprague-Dawley and Gunn rats but not in hamsters at the 350 μg/animal dose (note: rats 

17 weighed 200–250 grams, average high dose 1.56 mg/kg; hamsters 100–140 grams, 2.92 mg/kg; 

18 and guinea pigs 600–850 grams, 0.48 mg/kg), indicating that hamsters, who received about 6 

19 times the dose of guinea pigs, command a metabolic system that handles benzo[a]pyrene very 

20 well. Moir et al. (1998) administered benzo[a]pyrene i.v. to Wistar rats and recovered, at 8 hours 

21 after a 2 mg/kg dose, 4.27% of the dose in urine but only 0.06% in feces, confirming the fact of 

22 enterohepatic circulation of benzo[a]pyrene metabolite conjugates (Hirom et al., 1983; Weyand 

23 and Bevan, 1986). 

24 

Table 3-3. Excretion of benzo[a]pyrene metabolites in several animal species 

Dose 

Route of 

excretion Gunn rata

Sprague-Dawley 

rata

Syrian golden 

hamstera

Dunkin-Hartley 

guinea piga

0.16 μg/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 μg/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 

aValues are percent of the applied dose at 6 hours after intratracheal instillation. 

Source: Weyand and Bevan (1987). 

25 

26 The pattern of metabolites in bile was also reported; results are compiled in Table 3-4. 

27 At each dose thioether conjugates predominated but to a varying extent in each species. Guinea 

28 pigs evidently used preferentially thioether conjugation for biliary benzo[a]pyrene elimination, 

29 with the other conjugates (or nonconjugated metabolites) making up a small portion of biliary 
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1 excretion. At the high dose a shift in the relative proportion of metabolites was observed, with 

2 thioethers decreasing, glucuronides and sulfates increasing, and total nonconjugated remaining 

3 unchanged in both rat strains and hamster. The data also indicate that glucuronidation and 

4 sulfation became saturated at the high dose only in guinea pigs, while thioether formation 

5 capacity became overwhelmed by the high dose in all species. Glucuronide levels in the bile of 

6 Gunn rats were about one-half those of Sprague-Dawley rats; this strain also excreted fewer 

7 nonconjugated metabolites and appeared to have a generally lower metabolic capacity for 

8 benzo[a]pyrene. 

9 

Table 3-4. Biliary excretion of benzo[a]pyrene metabolites in several 

species 

Metabolite group 

Dose 

(μg/animal) Gunn rata

SpragueDawley rataSyrian golden 

hamstera

Dunkin-Hartley 

guinea piga

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 

aValues are percent of the applied dose at 6 hours after intratracheal instillation. 

Source: Weyand and Bevan (1987). 

10 

Following intratracheal administration of 1 μg/kg body weight [311 H]-benzo[a]pyrene to 

12 male Sprague-Dawley rats in hydrophilic triethylene glycol, 70.5% of the administered 

13 benzo[a]pyrene was excreted into bile within 6 hours (Bevan and Ulman, 1991). In contrast, 

14 benzo[a]pyrene excretion was 58.4 and 56.2% in the same time period when the lipophilic 

15 solvents ethyl laurate and tricaprylin, respectively, were the vehicles. Benzo[a]pyrene (in 

16 triethylene glycol) excretion in bile was described as biphasic with half-lives of 31 and 100 

17 minutes, respectively. Individual metabolite concentrations were not monitored. 

18 

19 3.4.2. Oral Exposure 

20 Only limited data were identified on the elimination of benzo[a]pyrene following 

21 exposure by the oral route. Although the dietary/oral route is likely to be the predominant route 

22 of exposure for the general population not occupationally exposed, it has received very little 

23 attention (Stavric and Klassen, 1994). In the only study investigating this issue in humans, the 

24 concentration of benzo[a]pyrene was below detection limits (<0.1 μg/person) in the feces of 
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1 eight volunteers who had ingested broiled meat containing approximately 8.6 μg of 

2 benzo[a]pyrene (Hecht et al., 1979). 

3 Ramesh et al. (2001b) evaluated benzo[a]pyrene disposition in F344 rats dosed via 

4 gavage with 100 mg/kg benzo[a]pyrene dissolved in peanut oil. Recovery of unmetabolized 

5 benzo[a]pyrene from feces reached a maximum of 35% of the dose at 24 hours but was very low 

6 at 48 hours and thereafter. Lipid-soluble metabolites in feces reached approximately 28% of all 

7 metabolites by 8 hours after dosing then declined sharply. Lipid-soluble metabolites in urine 

8 reached 35–40% of total by 48 hours then declined to approximately 10% by 72 hours. Diol 

9 metabolites were most numerous in feces, while phenols predominated in urine. 

10 Hecht et al. (1979) conducted a study of fecal excretion of benzo[a]pyrene and its 

metabolites in rats. In male F344 rats administered [14 11 C]-benzo[a]pyrene via gavage (0.04, 0.4, 

12 or 4 μmol/animal), approximately 85% of radiolabel was recovered in feces, and 1–3% in urine 

13 after 168 hours. The portion of benzo[a]pyrene recovered from feces as parent compound 

14 ranged from 13 to 6% of the administered dose within 48 hours. In rats fed charcoal-broiled 

15 hamburger containing 52.7 μg benzo[a]pyrene/kg meat, 11% of the benzo[a]pyrene was excreted 

16 unchanged in feces. 

17 

18 3.4.3. Dermal Exposure and Other Exposure Routes 

19 Bowman et al. (1997) detected benzo[a]pyrene-tetrols in 40% of the urine samples from 

20 psoriasis patients treated with coal tar medication, as compared to only 10% of those of controls. 

21 No specific measures of applied dose were available. 

In a dermal absorption study, Yang et al. (1989) evaluated the recovery of [322 H]-

23 benzo[a]pyrene, 100 ppm in crude oil applied topically, in urine and feces of female Sprague24 Dawley rats. Total recovery of applied radioactivity over 96 hours was 5.3% in urine and 27.5% 

25 in feces. Individual metabolite concentrations were not measured. Ng et al. (1992) examined the 

26 percutaneous absorption of radiolabeled benzo[a]pyrene in the hairless guinea pig. Following a 

single application of 28 μg benzo[a]pyrene (dissolved in 50 μL acetone applied to 4 cm227 of 

28 dorsal skin), approximately 34% of the administered radiolabel was eliminated within 24 hours. 

29 Most excretion had occurred by day 3 and continued slowly to reach 73% by day 7. Relative 

30 amounts of benzo[a]pyrene or metabolites in urine versus feces were not reported. 

Moir et al. (1998) dosed male Wistar rats i.v. with 2, 6, or 15 mg/kg of [14 31 C]-

32 benzo[a]pyrene and examined excretion in urine and feces over 32 hours. At 8 hours after 

33 injection, urinary excretion was 4.3, 2.6, and 3.2% of the dose, while fecal excretion was only 

34 0.06, 5.6, and 0.43% of the 2, 6, and 15 mg/kg doses, an indication of enterohepatic circulation. 

35 The amount of radioactivity excreted in the urine after 32 hours was similar in each dose group 

36 (6–7% of the administered dose). However, the proportion of the administered dose in the feces 

37 was dose-dependent. At the low dose (2 mg/kg) fecal excretion accounted for 26% of the dose, 
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1 while at the mid- and high-doses, fecal excretion accounted for 56 and 50% of the dose, 

2 respectively. No measurement of specific metabolites or whole body clearance was conducted. 

Following i.v. administration of 3 μmol [14 3 C]-benzo[a]pyrene to male New Zealand white 

4 rabbits, approximately 30% of the dose was recovered in the bile and 12% in the urine within 

5 6 hours after treatment (Chipman et al., 1982). Excretion in the bile was biphasic, with estimated 

6 half-lives of 0.27 and 4.62 hours. Further analysis of metabolite profiles in bile and urine were 

7 conducted. Treatment of bile or urine with β-glucuronidase or aryl sulfatase increased the 

8 amount of radioactivity recovered in ethyl acetate extracts, suggesting that sulfate and 

9 glucuronide conjugation are important contributors to benzo[a]pyrene metabolites in excreta. 

10 Analysis of these extracts revealed the primary metabolite as 9,10-diol, with lower amounts of 

11 numerous other metabolites identified (diols, quinones, and monophenols). 

12 Likhachev et al. (1992) measured the excretion of benzo[a]pyrene-7,8-diol and 3-OH13 benzo[a]pyrene in L10 rats given a single i.p. dose of 200 mg/kg benzo[a]pyrene. Urine and 

14 feces were collected over a 15-day period and then again at 30 days after exposure for another 5 

15 days. Both metabolites were excreted in feces at two to three times the amount excreted in urine. 

16 Metabolites in urine and feces decreased steadily over the 15-day postexposure period, but no 

17 more metabolites were detected at 30 days. A comparative study of metabolism in male Macaca 

18 fascicularis monkeys (five animals) and male L10 rats (four animals) was also performed. Both 

19 species were given a single i.p. dose of 100 mg/kg benzo[a]pyrene, and levels of benzo[a]pyrene 

20 metabolites in feces were evaluated over 8 days. Total benzo[a]pyrene metabolites were 

21 significantly lower in monkeys than in rats, but the results were hampered by infrequent feces 

22 collection in monkeys. Monkeys had an approximately fourfold higher benzo[a]pyrene-7,8-diol 

23 to 3-OH-benzo[a]pyrene ratio in feces than rats. Together these data suggest that monkeys have 

24 lower excretion of benzo[a]pyrene (and perhaps lower metabolism), and formation of the 

25 proximate carcinogenic metabolite is higher than in rats. 

26 In another part of this study (Likhachev et al., 1992), benzo[a]pyrene metabolism and 

27 excretion were assessed following multiple exposures. L10 rats were given i.p. injections of 

28 1 mg/kg benzo[a]pyrene every 11th day for 10 treatments. Benzo[a]pyrene metabolites were 

29 measured in excreta for 8 days after 1, 5, and 10 treatments. After the single dose, excretion in 

30 feces was favored over urine, and benzo[a]pyrene-7,8-diol was the predominant metabolite. The 

31 rate of metabolite excretion was decreased considerably after 5 and 10 doses, respectively. The 

32 authors attributed this finding to age-related changes in metabolic capacity or “exhaustion of 

33 enzymes.” They also compared metabolite profiles for individual rats with their tumor responses 

34 and based on the results suggested a link between tumor latency and elimination of 

35 benzo[a]pyrene metabolites (although small sample size and variability limited the power of the 

36 analysis). A direct correlation between benzo[a]pyrene-7,8-diol excreted in urine and tumor 

37 latency was observed. It was postulated that higher excretion of reactive benzo[a]pyrene 
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1 metabolites caused fewer of these metabolites to bind to DNA targets and thus increased tumor 

2 latency. 

3 In addition to benzo[a]pyrene and its metabolites, adducts of benzo[a]pyrene with 

4 nucleotides have also been identified in feces and urine of animals but only as a small fraction of 

5 the administered dose. Autrup and Seremet (1986) administered i.p. doses of 0, 10, 50, or 

6 100 μg/kg tritiated benzo[a]pyrene to male Wistar rats and collected urine over 24-hour periods 

7 for 72 hours. The level of BPDE adducts with guanine detected in urine was dose-dependent. 

8 The authors reported that, at the high dose, 0.15% of the administered benzo[a]pyrene dose was 

9 excreted in the urine as this adduct within 48 hours. Rogan et al. (1990, only study abstract was 

10 available for review) reported that 0.02% of a benzo[a]pyrene dose was excreted as an adduct 

11 with guanine in urine and feces over 5 days. 

12 Overall, the data on benzo[a]pyrene elimination in humans are too limited to estimate 

13 quantitative rates of elimination. The situation is further complicated by the existence of 

14 multiple pathways in which many of the key enzymes exhibit polymorphisms. In the context of 

15 biomonitoring studies, benzo[a]pyrene metabolites have been detected in the urine of exposed 

16 humans, but the fecal excretion has not been investigated in any detail. The animal data are 

17 consistent in showing that feces are the primary route of elimination of benzo[a]pyrene, while 

18 urinary excretion plays a lesser role. A diverse array of metabolites, as well as parent 

19 benzo[a]pyrene, is found in the excreta. Enterohepatic circulation of benzo[a]pyrene metabolite 

20 conjugates has been demonstrated in animals and may exist in humans as well, but its impact on 

21 the toxicokinetics of benzo[a]pyrene is not understood. 

22 Similar considerations apply to the toxicokinetics of benzo[a]pyrene as a whole. A few 

23 animal studies that have attempted comprehensive assessments of benzo[a]pyrene metabolism. 

24 Although a lot is known from ex vivo studies about which humans tissue can metabolize 

25 benzo[a]pyrene to what extent, comprehensive studies to estimate overall metabolic capacities or 

26 the balance between Phase I and Phase II metabolism in humans have not been conducted. 

27 There are also no comprehensive data on the tissue distribution of benzo[a]pyrene in humans, not 

28 counting a number of studies that have reported post mortem levels in a few tissues in the 

29 absence of any exposure assessments. Thus, despite the fact the humans are universally exposed 

30 to benzo[a]pyrene, there is a great need for more knowledge concerning its toxicokinetics. 

31 

32 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS 

33 Several toxicokinetic or pharmacokinetic models of benzo[a]pyrene have been developed 

34 for rodents (rat and hamster), but none has been calibrated in humans. Bevan and Weyand 

35 (1988) performed compartmental pharmacokinetic analysis of distribution of radioactivity in 

36 male Sprague-Dawley rats, following the intratracheal instillation of benzo[a]pyrene to normal 

37 and bile duct-cannulated animals (Weyand and Bevan, 1987, 1986). The authors used the 

38 Simulation, Analysis and Modeling (SAAM) and Conversational SAAM (CONSAAM) 
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1 computer programs to model the disposition of labeled benzo[a]pyrene and its metabolites. A 

2 good fit to the experimental data was achieved with 10 assumed compartments corresponding to 

3 lung, blood, liver, other tissues (reflecting the sum of radioactivity in kidney, stomach, testis, 

4 spleen, heart, and thymus), carcass (reflecting the sum of radioactivity in skin, fat, bones, 

5 muscle, and blood in those organs), and several additional hypothetical compartments that linked 

6 the central compartment with intestines (including their contents) and urine. Enterohepatic 

7 circulation and intestinal secretion were both included in the model. The model allowed 

calculation of linear rate constants for moving radioactivity among compartments (in minute-1 8 ), 

9 representing the probability per unit time that radioactivity from one compartment would be 

10 transferred to another. The model adequately simulated disposition of benzo[a]pyrene and its 

11 metabolites, measured as total radioactivity in blood, organs and excreta, under the assumption 

12 that the kinetics of benzo[a]pyrene and its metabolites are the same. The authors emphasized 

13 differences between their approach in building the implicit SAAM model and the approach used 

14 to build explicit PBPK models. Roth and Vinegar (1990) reviewed the capacity of the lung to 

15 impact the disposition of chemicals and used benzo[a]pyrene as a case study. A PBPK model 

16 was presented based on data from Wiersma and Roth (1983a, b) and was evaluated against tissue 

17 concentration data from Schlede et al. (1970). The model was structured with compartments for 

18 arterial blood, venous blood, lung, liver, fat, and slowly as well as rapidly perfused tissues. 

19 Metabolism in liver and lung was estimated using kinetic data from control rats and rats 

20 pretreated with 3-MC to induce benzo[a]pyrene metabolism. Benzo[a]pyrene binding was 

21 accounted for in blood, liver, and lung, but only one binding component was used, which, as the 

22 authors suggested, resulted in a rather poor fit for liver. The model was built based on blood and 

23 tissue concentrations measured over 5 hours in rats given a dose of 117 nmol/kg by i.v. injection 

24 into the arterial circulation or the venous blood supply of the liver. The model was tested against 

25 the data of Schlede et al. (1970). The number of data points was limited (six time points for the 

26 venous circulation, liver, and fat and only a single time point for the lung). The model predicted 

27 the data of Schlede et al. (1970) reasonably well, although deviations were apparent for several 

28 compartments. Most notably, the model overpredicted concentrations in the induced lung, 

29 uninduced and induced liver, and induced fat compartments. The results of PBPK simulations 

30 showed that induction of metabolizing enzymes increased the amount of benzo[a]pyrene cleared 

31 by the lungs relative to the liver. 

32 An interesting result of the Roth and Vinegar (1990) modeling study was that even 

33 though the metabolic clearance of benzo[a]pyrene in the lungs was low in comparison to the 

34 liver, under simulated pathological conditions of reduced hepatic blood flow, a substantial 

35 metabolic clearance was carried on by well perfused lungs. This illustrates that changes in tissue 

36 perfusion can shift the organ pattern of benzo[a]pyrene metabolic clearance in vivo, and 

37 consequently, might also shift the organ pattern of benzo[a]pyrene-induced disease, such as 

38 cancer. The authors emphasized that their PBPK model with the results of simulations should 
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1 not be taken as a definite model for benzo[a]pyrene but that it could be used for designing further 

2 studies of benzo[a]pyrene metabolic clearance. They also suggested that it would be appropriate 

3 to incorporate into the PBPK model a description of the appearance of various metabolites of 

4 benzo[a]pyrene. 

5 Moir et al. (1998) conducted a pharmacokinetic study on benzo[a]pyrene to obtain data 

for model development. Rats were injected with [14 6 C]-benzo[a]pyrene at doses of 2, 6, or 

7 15 mg/kg and blood, liver, fat, and richly perfused tissue were sampled at 15 time points from 5 

8 minutes to 32 hours after dosing. Moir (1999) then described a model for lung, liver, fat, richly 

9 and slowly perfused tissues, and venous blood, with saturable metabolism occurring in the liver. 

10 The fat and richly perfused tissues were modeled as diffusion-limited, while the other tissues 

11 were flow-limited. The model was developed using the 15 mg/kg dose group data from the Moir 

12 et al. (1998) study and validated using the 2 and 6 mg/kg data. The model predicted the blood 

13 benzo[a]pyrene concentrations well, although it overestimated the 6 mg/kg results at longer 

14 times (>100 minutes). The fat and richly perfused tissue benzo[a]pyrene levels were reproduced 

15 by the model fairly well at the two highest dose levels but underestimated at the 2 mg/kg dose. 

16 The model produced a poor fit to the liver data, underestimating benzo[a]pyrene tissue levels at 

17 the two lowest doses. At the high dose the model overestimated the liver benzo[a]pyrene 

18 concentration at times <200 minutes but afterwards underestimated them increasingly more. It 

19 appeared that the model could not accommodate a slow elimination phase at times beyond 200 

20 minutes for the mid and high dose levels. The model simulations were also compared to data of 

21 Schlede et al. (1970) who had injected rats with 0.056 mg/kg body weight of benzo[a]pyrene. 

22 Again the model predicted blood and fat benzo[a]pyrene concentrations quite well but 

23 underestimated liver benzo[a]pyrene concentrations.

24 Moir (1999) suggested that the poor prediction of fat benzo[a]pyrene concentrations after 

25 injection of 2 mg/kg benzo[a]pyrene in their own study (Moir et al., 1998) was due to analytical 

26 error, causing the model to fail. This explanation is credible because the measured fat 

27 benzo[a]pyrene concentrations following injection of 2 mg/kg benzo[a]pyrene were 

28 indistinguishable from those obtained following injection of 6 mg/kg benzo[a]pyrene (Moir et 

29 al., 1998). The author also speculated that the poor fit to liver benzo[a]pyrene concentration data 

30 was due to binding of benzo[a]pyrene to receptors in the liver. The model predicted blood and 

31 fat benzo[a]pyrene concentrations fairly accurately over a wide range of doses (0.056–

32 15 mg/kg), but it was limited by the failure to accurately predict liver benzo[a]pyrene 

33 concentrations. The model included only one saturable metabolic pathway, and only parent 

34 chemical concentrations were used to establish the model. Metabolites were not modeled.An 

35 attempt to scale the rodent PBPK model to humans, relevant to risk assessment of oral exposures 

36 to benzo[a]pyrene, was presented by Zeilmaker et al. (1999a, b). The PBPK model for 

37 benzo[a]pyrene was derived from an earlier model for TCDD in rats (Zeilmaker and van 

38 Eijkeren, 1997). The structure of the mainly perfusion-limited PBPK model included 
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1 compartments for blood, adipose tissue (with diffusion limitation), slowly and richly perfused 

2 tissues, and the liver (Figure 3-3). However, there was no separate compartment for the lung. 

3 The liver compartment featured the AhR-dependent CYP450 induction mechanism and DNA 

4 adduct formation as a marker for formation of genotoxic benzo[a]pyrene metabolites. It was 

5 assumed that DNA adduct formation and the bulk benzo[a]pyrene metabolism were mediated by 

6 two different metabolic pathways. The model was experimentally calibrated in rats with the data 

7 for EROD and formation of DNA adducts in the liver after i.v. administration of a single dose 

8 and per oral (p.o.) administration of a single or repeated doses of benzo[a]pyrene (Zeilmaker et 

9 al., 1999a). 

10 In order to scale this rat PBPK model to human, Zeilmaker et al. (1999b) assumed 

11 identical values for several parameters in rats and humans, respectively: benzo[a]pyrene tissue 

12 partition coefficients, AhR concentration in liver, rate constant for the decay of the 

13 benzo[a]pyrene-CYP450 complex, half-life of the CYP450 protein, fraction and rate of GI 

14 absorption of benzo[a]pyrene, and rates of formation and repair of DNA adducts in liver. The 

15 basal CYP450 activity in humans was assumed to be lower than that in rat liver (ranging from 

16 almost absent up to equal to that in the rat). The mechanism of AhR-dependent induction of 

17 CYP450 dominated the simulated benzo[a]pyrene-DNA adduct formation in the liver. The 

18 results of PBPK model simulations indicated that the same dose of benzo[a]pyrene administered 

19 to rats or humans might produce one order of magnitude higher accumulation of DNA adducts in 

20 human liver when compared with the rat (Zeilmaker et al., 1999b). 

21 Even though the model of Zeilmaker et al. (1999b) represents a major improvement in 

22 predictive modeling of benzo[a]pyrene toxicokinetics, the results of modeling, which included 

23 interspecies extrapolation, bear significant uncertainties. As emphasized by the authors, the 

24 conversion of benzo[a]pyrene to its mutagenic and carcinogenic metabolites could not be 

25 explicitly modeled in human liver because no suitable experimental data were available. 

26 According to the authors, to improve the model would require direct measurements of basal 

27 activities of CYP1A1 and CYP1A2 and formation of benzo[a]pyrene-DNA adducts in human 

28 liver. Moreover, despite the prior results of the study by Roth and Vinegar (1990), the metabolic 

29 clearance of benzo[a]pyrene in the lungs was not addressed. Also, the toxicokinetic modeling by 

30 Zeilmaker et al. (1999b) addressed only one pathway of benzo[a]pyrene metabolic activation, a 

31 single target organ (the liver), and one route of administration (oral). 

32 For modeling and predicting of the health outcomes of exposures to benzo[a]pyrene, a 

33 mechanistically accurate PBPK model needs to follow, over time and in several target organs, 

34 the rate of accumulation of benzo[a]pyrene-DNA adducts and/or the distribution and fate of 

35 benzo[a]pyrene metabolites (e.g., BPDE) that bind to DNA and other macromolecules. 

36 Alternatively, a stable derivative of the "ultimately carcinogenic" metabolite (e.g., 

37 benzo[a]pyrene trans-anti-tetrol) may be used as an internal dose surrogate. Calibration of such 

38 a model requires quantitation of these benzo[a]pyrene metabolites in biological samples, which, 
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1 in turn, would require refined and sufficiently sensitive analytical methods. Therefore, while the 

2 metabolic pattern of benzo[a]pyrene has been relatively well characterized qualitatively in 

3 animals, both in vitro and in vivo, the quantitative kinetic relationships between overlapping and 

4 sometimes competing metabolic reactions in potential target organs, essential for meaningful 

5 PBPK modeling, are yet not well defined. 

6 Furthermore, the potential for scaling this model to humans without complete 

7 reparameterization is questionable. According to Liao (2004), the recombinant human CYP1A1 

8 has a higher activity for the formation of benzo[a]pyrene-hydroxy metabolites and a lower 

9 activity for the formation of benzo[a]pyrene-diones, when compared with recombinant rat 

10 CYP1A1. The RN-PBPK model overestimated the benzo[a]pyrene concentration in the liver, 

11 but after 4 hours, it underestimated benzo[a]pyrene concentration in fat. In contrast to the 

12 findings of Zeilmaker et al. (1999a), the adipose tissue compartment was modeled only as 

13 perfusion-limited. Tissue-blood partition coefficients for benzo[a]pyrene and its metabolites 

14 used in the RN-PBPK model were estimated but not validated experimentally or otherwise. 

15 Also, no adjustment was made for using in vitro enzyme activities, especially when measured in 

16 recombinant proteins as was done in the calibration of this model, although they typically differ 

17 from those in vivo. 

18 The published PBPK models for benzo[a]pyrene were evaluated to determine whether the 

19 existing models could be used to extrapolate from rats to humans or for a route to route 

20 extrapolation from oral exposure to the inhalation route. The focus was concentrated on models 

21 for the inhalation route since several well conducted studies by the oral route exist from which to 

22 derive toxicity values. No full PBPK model for the inhalation route was identified. It was 

23 concluded that at present, none of the published models allow for computation of the resulting 

24 internal doses used in the only cancer bioassay available for the inhalation route (Thyssen et al. 

25 1981), nor is there a model for humans that simulates the typical inhalation exposure to 

26 benzo[a]pyrene on poorly soluble carbonaceous particles. 
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1 4. HAZARD IDENTIFICATION 

2 

3 

4 4.1. HUMANS STUDIES 

5 4.1.1. Sources of Human Exposure 

6 Although it has no commercial production or significant uses, benzo[a]pyrene is 

7 nevertheless a ubiquitous environmental contaminant resulting from the incomplete combustion 

8 of organic matter. Essentially all humans experience repeated exposure to benzo[a]pyrene, 

9 which can begin in utero and continue throughout life. The magnitude of benzo[a]pyrene 

10 exposure depends on several factors related to lifestyle (e.g., diet, tobacco smoking), occupation, 

11 and living conditions (e.g., urban versus rural setting, domestic heating and cooking methods). 

12 A distinguishing feature concerning benzo[a]pyrene exposure is that environmental sources 

13 always occur as complex mixtures, which may consist of numerous PAHs, including 

14 heterocyclic and nonheterocyclic forms as well as aza arenes and nitro-substituted PAHs 

15 (reviewed in Bostrom et al., 2002; WHO, 1998; ATSDR, 1995; IARC, 1973). Many of these 

16 complex mixture components are carcinogens, and some can exceed the carcinogenic potency of 

17 benzo[a]pyrene, as observed in animal bioassays. 

18 With the exception of certain occupational exposure sources such as aluminum 

19 production and the conversion of coal to coke and coal tar, the major contributor to total PAH 

20 (and thus benzo[a]pyrene) exposure in nonsmokers is the diet (Cogliano et al., 2008; Straif et al., 

21 2005; Bostrom et al., 2002; Cenni et al., 1993; Andersson et al., 1983; Bjorseth et al., 1978a, b). 

22 In particular, charbroiled and grilled meats, and certain grain products, are important 

23 determinants of PAH exposure in most populations. In very limited populations receiving 

24 clinical coal tar treatment of the skin for conditions such as psoriasis, acute dermal exposure to 

25 benzo[a]pyrene may greatly exceed that received from both occupational and nonoccupational 

26 sources (Godschalk et al., 2001; Pavanello et al., 1999). 

27 

28 4.1.2. Biomonitoring of Benzo[a]pyrene Exposure and Effects In Humans 

29 Quantitative exposure assessment becomes very challenging for complex mixture 

30 components such as benzo[a]pyrene, where individual exposures vary depending on background 

31 concentrations in the environment, lifestyle factors, and occupation. An alternative to measuring 

32 concentrations in various environmental media, human biomonitoring focuses on biomarkers of 

33 internal dose which may also serve as mechanistic indicators of early biological response 

34 following exposure to a genotoxic agent. Carcinogen exposure biomarkers are generated by 

35 uptake and metabolic processes which may result either in detoxification and excretion or in 

36 bioactivation to reactive forms that can bind covalently with DNA and other macromolecules. 

37 Following exposure to complex PAH mixtures, biomonitoring of benzo[a]pyrene uptake can 

38 involve evaluation of urinary metabolites of benzo[a]pyrene or surrogates, DNA adducts in 
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1 peripheral blood cells such as leukocytes and lymphocytes, protein adducts in hemoglobin and 

2 serum albumin, and cytogenetic damage in lymphocytes (Gyorffy et al., 2008; Godschalk et al., 

3 2003; Bostrom et al., 2002; Scherer et al., 2000). Biomarkers offer advantages over traditional 

4 benzo[a]pyrene exposure concentration monitoring because they can be quantitative indicators of 

5 an individual’s environmental exposure and internal carcinogen dose irrespective of time, route 

6 of exposure, and inter-individual metabolic differences. Associations between increased cancer 

7 risk and levels of specific biomarkers, such as benzo[a]pyrene-DNA adducts in target or 

8 surrogate tissues, also provide important mechanistic information in studies of human disease 

9 etiology. 

10 Considerable progress has been achieved in the application of biomarker methods for 

11 human biomonitoring of carcinogens (Vineis and Perera, 2007; Perera and Weinstein, 2000; 

12 Poirier et al., 2000). However, further validation is needed before using biomarkers to routinely 

13 predict disease risk. Successful application of biomarkers in benzo[a]pyrene exposure 

14 assessment currently depends on several assumptions: 1) urinary metabolites, whether derived 

15 from benzo[a]pyrene or surrogate PAH compounds such as Py or phenanthrene, provide 

16 information about recent benzo[a]pyrene exposures; 2) DNA and protein adduct measurements 

17 in easily accessible tissues serve as surrogate biomarkers of long-term benzo[a]pyrene exposure 

18 and internal dose for less accessible target tissues (e.g., lung); 3) and cytogenetic biomarkers 

19 reflect early biological effects that correlate with relevant preclinical events occurring at target 

20 sites, and with biomarkers of biologically effective benzo[a]pyrene dose. Evidence is 

21 accumulating to support these assumptions for carcinogenic PAHs, in general, and for 

22 benzo[a]pyrene, in particular, although conflicting and variable results among some studies limit 

23 their application for dose-response assessment. Important issues to address include differences 

24 in biomarker assay sensitivity and specificity, acquired and inherited variations in PAH 

25 bioactivation, detoxification, and DNA repair, and uncertain carcinogen intake(Gyorffy et al., 

26 2008; Divi et al., 2002; Poirier et al., 2000; Scherer et al., 2000; Santella et al., 1994). 

27 

28 4.1.2.1. Urinary Excretion of Benzo[a]pyrene

29 Based on animal studies, urinary excretion of benzo[a]pyrene is a minor pathway of 

30 elimination compared with elimination in the feces. The hydroxylated metabolites of 

31 benzo[a]pyrene, 3-hydroxy-benzo[a]pyrene (3-OH-benzo[a]pyrene) and 9-hydroxy32 benzo[a]pyrene, have been identified in the urine of humans exposed to PAH, although they are 

33 often not detectable in human urine despite known exposure to benzo[a]pyrene (Rossella et al., 

34 2009; Hecht, 2002; Bentsen-Farmen et al., 1999b). In numerous human studies, the 

35 hydroxylated metabolite of Py, 1-OH-Py, is typically used as a surrogate indictor of internal 

36 exposure to carcinogenic PAH mixtures based on the assumption that levels of 1-OH-Py in urine 

37 correlate with exposure to individual PAH compounds of higher molecular weight, including 

38 benzo[a]pyrene. Although urinary 1-OH-Py appears suitable as a sensitive biomarker for total 
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1 PAH exposure, results are variable for the correlation of 1-OH-Py with the benzo[a]pyrene 

2 metabolite, 3-OH-benzo[a]pyrene, in urine (Forster et al., 2008; Godschalk et al., 1998a). In a 

3 study of urinary PAHs and their hydroxylated metabolites in 55 coke oven workers, 1-OH-Py 

4 was found in 100% of the urine samples, whereas 3-OH-benzo[a]pyrene was always below the 

5 quantification limit (Rossella et al., 2009). In addition, reported correlations between urinary 1-

6 OH-Py levels and PAH-DNA adducts in white blood cells (WBCs) are conflicting, although in 

7 one study, 3-OH-benzo[a]pyrene levels in urine were significantly correlated with specific 

8 benzo[a]pyrene diol epoxide adducts in skin DNA following dermal application of coal tar 

9 ointments (Gyorffy et al., 2008; Godschalk et al., 1998a). 

10 Buckley et al. (1995) found that excretion of benzo[a]pyrene metabolites in residents 

11 (non-smokers not employed in high PAH occupational environments) of Phillipsburg, New 

12 Jersey, correlated better with ingestion of benzo[a]pyrene from food rather than with 

13 environmental inhalation exposures. Benzo[a]pyrene metabolites in urine were measured 

14 following “reverse metabolism,” a procedure that involves enzymatic hydrolysis of 

15 benzo[a]pyrene conjugates followed by chemical conversion of all hydroxylated benzo[a]pyrene 

16 metabolites back to the parent compound for subsequent thin-layer chromatography and 

17 scanning spectrofluorometry. Buckley et al. (1995) estimated pulmonary uptake at 11 and 2.3 ng 

18 benzo[a]pyrene/person/day for winter and summer, respectively, based on 24-hour personal air 

19 measurements. The median intake of benzo[a]pyrene from the diet was estimated at 176 ng/day. 

20 The median urinary excretion of benzo[a]pyrene and metabolites was 121 and 129 ng/day in 

21 winter and summer, respectively. Based on multiple regression analyses of estimated inhaled 

22 and ingested doses, change in urinary excretion of benzo[a]pyrene was only marginally 

23 predictive of benzo[a]pyrene exposure. Most of the variation in urinary benzo[a]pyrene 

24 excretion was explained by the ingested dose. These results confirm an earlier study of 

25 occupationally exposed (aluminum plant) workers using the same reverse metabolism procedure 

26 to measure urinary PAH elimination (Becher and Bjørseth, 1983). Although a significant 

27 difference in urinary PAH excretion was seen in nonoccupationallly exposed smokers versus 

28 nonsmokers, PAH metabolite levels in urine of aluminum plant workers did not reflect the large 

29 difference in inhalation exposure relative to controls (Becher and Bjørseth, 1983). 

30 Gündel et al. (2000) investigated PAH exposure and urinary metabolite excretion in 

31 19 workers from a fireproof stone factory. Along with other PAH metabolites in urine, they 

32 measured 3-OH-benzo[a]pyrene levels following airborne exposures to benzo[a]pyrene ranging 

from 0.043 to 5.41 μg/m333 based on personal and stationary air sampling. The study authors 

34 failed to identify a correlation between benzo[a]pyrene inhalation exposure concentration and 

35 urinary 3-OH-benzo[a]pyrene excretion, nor between 1-OH-Py and 3-OH-benzo[a]pyrene levels 

36 in urine. 

37 

38 4.1.2.2. Adduct Formation with DNA and Protein 
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1 A large body of literature supports DNA and protein adducts as biomarkers of a 

2 biologically effective dose (ED) for exposures to some DNA reactive human carcinogens 

3 (Gyorffy et al., 2008; Vineis and Perera, 2007; Hecht, 2004; Godschalk et al., 2003; Bostrom et 

4 al., 2002; Perera and Weinstein, 2000; Poirier et al., 2000). DNA adduct formation is considered 

5 to be a necessary early event in tumor formation for many such compounds, particularly large, 

6 bulky carcinogens such as benzo[a]pyrene and PAHs in general. In PAH exposed populations, 

7 there is evidence demonstrating the formation of DNA adducts with tobacco-derived PAH 

8 including benzo[a]pyrene, not only sites directly exposed, but also in distant organs and the 

9 peripheral blood (see Table 4-1). 

10 

11 Table 4-1. Studies of PAH-DNA adducts in human populations or tissues 

12 exposed to PAHs 

13 

Reference Study description 

Arnould et al. (1998) DNA adducts from leukocytes from heavy smokers 

Arnould et al. (1999) DNA adducts in leukocytes from 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 blood 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 smokers, tobacco chewers, coke oven workers 

Bhattacharya et al. (2003) benzo[a]pyrene-DNA adducts in human urine 

Casale et al. (2001) benzo[a]pyrene adducts in urine 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 cigarette or coal smoke 

Godschalk et al. (1998a) DNA adducts in alveolar macrophages 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 treated with coal tar 

Hemminki et al. (1997 DNA adduct formation in exposed humans 

Izzotti et al. (1991, 1992) DNA adducts in pulmonary alveolar macrophages following exposure to 

cigarette smoke 

Li et al. (2001) DNA adduct levels in human peripheral blood lymphocytes of SCC patients 

and controls 

Li et al. (2002) BPDE-DNA adducts in human breast tissues 

Lodovici et al. (1998) benzo[a]pyrene levels in autoptic lungs of smokers and nonsmokers 

Lodovici et al. (1999) DNA adduct formation in human 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 and stromal cervical tissue samples from women 

smokers and self-reported nonsmokers after hysterectomy for nonmalignant 

conditions 

Paleologo et al. (1992) BPDE-DNA adducts in the white 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 parenchyma of coke oven workers and 

smokers 

Scherer et al. (2000) benzo[a]pyrene adducts of hemoglobin 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, damage, cell cycle, and apoptosis) in smokers and 

nonsmokers 

Shinozaki et al. (1999) DNA adduct formation in aging 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 and SCEs in human lymphocytes 

Zenzes et al. (1999a) DNA adducts in sperm cells from smokers and nonsmokers 

Zenzes et al. (1999b) DNA adducts in embryos from smoking couples 

Zhang et al. (1995) DNA adduct levels in oral mucosa cells from nonsmokers and smokers 
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1 

2 Measurement of DNA adducts in target tissues (e.g., lung) can be difficult and invasive; 

3 however, readily accessible nucleated tissue (e.g., WBCs, sperm cells, cervical cells) and 

4 proteins (e.g., hemoglobin and albumin in blood) can serve as surrogate biomarkers of exposure. 

5 DNA adducts in WBCs reflect exposure over a relatively long period, and are indicative of the 

6 individual’s metabolic and DNA repair capability, both of which are genetically influenced. 

7 Although protein adducts are not thought to be mechanistic intermediates in PAH-initiated 

8 carcinogenesis, they offer several advantages as biomarkers. For example, hemoglobin and 

9 albumin are much more abundant than DNA; their adducts are not subject to removal by 

10 enzymatic repair, and they can integrate exposures over the protein lifespan of days to weeks. 

11 In humans, PAH albumin and hemoglobin adducts have often been used to investigate 

12 internal dosimetry of direct tobacco and environmental tobacco smoke (ETS) exposures. 

13 Elevated benzo[a]pyrene adducts are often reported in smokers versus nonsmokers and in 

14 children exposed to ETS from their smoking mothers (Hecht, 2004; Philips, 2002). 

15 Benzo[a]pyrene-protein adduct levels in smokers are typically increased twofold compared with 

16 nonsmokers (Scherer et al., 2000; Sherson et al., 1990). 

17 

18 4.1.2.2.1. DNA adduct measures in blood

19 Major advances in methodology have greatly increased the sensitivity and specificity 

20 (Himmelstein et al., 2009; Jarabek et al., 2009) of DNA adducts measurements. The various 

techniques utilized include immunoassays and immunohistochemistry, [32P]- and [33 21 P]-

22 postlabelling of modified nucleotides paired with thin layer or high performance liquid 

23 chromatography (TLC and HPLC, respectively), fluorescence and phosphorescence 

24 spectroscopy, electrochemical detection, and mass spectrometry (Arlt et al., 2007; Poirier et al., 

2000). [32 25 P]-postlabelling assays and immunological methods are the most commonly used. The 

26 immunoassays employ an antiserum generated against benzo[a]pyrene-DNA adducts. In 

27 addition to DNA adducts formed by benzo[a]pyrene, the antibody cross reacts with DNA adducts 

28 formed by other carcinogenic polycyclic aromatic hydrocarbons (PAHs) (Weston, et al, 1989) 

29 but has no affinity for DNA or PAHs alone. DNA adducts detected in non-laboratory samples 

using this technique are therefore referred to as “PAH-DNA” adducts. The [32 30 P]-postlabelling 

31 method is highly sensitive in detecting bulky DNA adducts, including but not limited to 

benzo[a]pyrene- and PAH-DNA adducts, enabling quantitation down to 1 DNA adduct/10932 

33 normal nucleotides, and achieving high resolution of individual adducts particularly when 

34 combined with HPLC. Unless specific chromatographic standards are available to assist in 

35 identification, adducts detected using this technique are referred to in general as “bulky DNA 

36 adducts”. Accelerator mass spectrometry is a relatively new method for adduct determination 

37 that has demonstrated even greater sensitivity. Most studies do not identify the specific DNA 

38 adduct structures, but instead report total “PAH-DNA,” “bulky-DNA,” “hydrophobic-DNA,” or 
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1 “aromatic-DNA” adducts (Alexandrov et al., 2002). Further investigations are needed to 

2 examine correlations between DNA adduct levels determined by different methods, and 

3 correlations between DNA adducts and urinary PAH metabolites. 

4 benzo[a]pyrene-DNA and PAH-DNA adducts have been measured in WBCs in several 

5 PAH-exposed human groups and found to be associated with, and predictive of, elevated cancer 

6 risks (Vineis and Perera, 2007; Pavanello et al., 1999). Coke oven workers experience especially 

7 high exposures to PAHs and demonstrate increased lung cancer rates. Pavanello and coworkers 

8 (2005) studied a group of 67 highly exposed coke oven workers for genetic factors that can 

9 modulate individual responses to carcinogenic PAHs. Levels of BPDE-DNA adducts in 

10 mononuclear WBCs (lymphocyte plus monocyte fraction) were associated with workplace PAH 

11 exposure as indicated by urinary 1-OH-Py excretion. The authors concluded that the elevated 

12 levels of BPDE-DNA adducts reflected both exposure and individual variation in expression of 

13 genes involved with glutathione conjugation activity and DNA excision repair capacity. 

14 In a similar study, Pavanello et al. (2006) screened 585 Caucasian municipal workers 

15 (52% males, 20–62 years old) from northeast Italy for BPDE-DNA adduct formation in 

16 peripheral lymphocytes. Forty-two percent of the participants had elevated anti-BPDE-DNA 

adduct levels, defined as >0.5 adducts/108 nucleotides (mean, 1.28 ± 2.80 adducts/10817 

18 nucleotides). Comparison of adduct levels with questionnaire responses indicated that smoking, 

19 frequent consumption of PAH-rich meals (>52 times/year vs. <52 times/year), and long time 

20 periods spent outdoors (>4 hours/day vs. <4 hours/day) were risk factors as all increased BPDE21 DNA adduct levels significantly. Exposure to indoor combustion sources (use of fireplace, coal22 or wood stove >5 times/year) significantly increased the frequency of subjects positive for 

23 BPDE-DNA adducts. Exposure to heavy traffic did not alter lymphocyte BPDE-DNA adduct 

24 levels. Smoking and high-PAH diets were associated with increased BPDE-DNA adduct levels. 

25 In nonsmokers, high-PAH diets and extended time spent indoors associated with increased 

26 BPDE-DNA adduct formation, while in smokers, personal cigarette smoking was the only factor 

27 that was positively correlated with adduct levels. These results demonstrate the potential utility 

28 of BPDE-DNA adduct measurement as a biomarker of PAH exposure not only in heavily PAH29 exposed occupations and tobacco smokers, but also in the general population, including 

30 nonsmokers. 

31 Several studies investigated the level of DNA adducts in the WBCs of foundry workers 

32 (Perera et al., 1988) or coke oven workers (Arnould et al., 2000; Assennato et al., 1993a, b) using 

33 an immunoassay. The antibody’s cross-reactivity with adducts originating from PAHs other than 

benzo[a]pyrene generally results in higher reported adduct levels than the [32 34 P]-postlabelling 

35 method which can isolate benzo[a]pyrene-DNA adducts. All three studies involved several 

36 levels of potential benzo[a]pyrene exposure as assessed by environmental sampling: 0.02, 5, 25, 

and 45 μg/m3 (Arnould et al., 2000); <0.05, 0.05–0.2, and >0.2 μg/m337 (Perera et al., 1988); and 

0.03–12.6 μg/m338 (Assennato et al., 1993a, b). Adduct levels ranged from 0.05 to 7 fmol PAH-
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1 DNA per μg DNA, and variation within exposure groups was high; however, all three studies 

2 reported statistically significant correlations between exposure and adduct levels. However, 

3 there are a large number of studies, many of them conducted by the same laboratories cited here, 

4 that did not report positive correlations between benzo[a]pyrene exposure and DNA adduct 

5 formation in people exposed occupationally or to cigarette smoke (e.g., van Delft et al., 2001; 

6 Arnould et al., 1999; Pan et al., 1998; Peluso et al., 1998; Lewtas et al., 1997; Assennato et al., 

7 1993a, b; Kriek et al., 1993; Paleologo et al., 1992; Herbert et al., 1990). These studies illustrate

8 that attempting to correlate DNA adducts with benzo[a]pyrene exposure by a single route of 

9 exposure (e.g., inhalation), or by occupation alone, may produce highly variable or misleading 

10 results. 

11 Perera et al. (2005a) measured BPDE-DNA adduct levels in maternal and umbilical cord 

12 blood obtained following normal delivery from 329 nonsmoking pregnant women exposed to 

13 emissions from fires during the 4 weeks following the collapse of the World Trade Center 

14 (WTC) building in New York City on 09/11/2001. BPDE-DNA adduct levels were highest in 

15 study participants who lived within 1 mile of the WTC, with inverse correlation between cord 

16 blood levels and distance from WTC. For the group of participants that resided within 1 mile of 

the WTC, maternal levels were 0.30 ± 0.16 adducts/10

8

17 nucleotides, umbilical cord levels were 

18 0.28 ± 0.08; for the group employed within 1 mile of WTC, the corresponding values were 0.25 

19 ± 0.11 and 0.24 ± 0.12, and for the unexposed referent group, the values were 0.22 ± 0.10 and 

20 0.23 ± 0.10. Differences between referent and exposed subjects were marginally significant (0.1 

21 > p > 0.05); however, the trend for adducts in maternal blood decreasing with increasing distance 

22 from the WTC was statistically significant (p = 0.02) as was the finding that the percentage of 

23 participants with detectable BPDE-DNA adducts increased significantly (trend p = 0.05) with 

24 increasing time and proximity to WTC–from 52.6% (reference group) to 66.7% (employed 

25 group) to 80.0% (resident group). 

26 Tuntawiroon et al. (2007) evaluated airborne PAH concentrations and internal biomarker 

27 levels in 115 Thai school boys (8–12 years old) attending schools adjacent to high-density traffic 

28 areas in Bangkok (high exposure group), compared with 69 boys (9–13 years old) attending 

29 schools located in a provincial area (low exposure group). Ambient air concentrations (roadside 

30 and school areas) and personal breathing zone air concentrations of 10 particulate PAHs were 

31 measured in Bangkok and in the provincial location. Peripheral blood samples were collected 

from the school boys for [32 32 P]-postlabeling determination of bulky DNA adducts in lymphocytes. 

33 Based on toxic equivalency factors (TEFs, Nisbet and LaGoy, 1992) for PAH, benzo[a]pyrene 

34 equivalent exposures from personal breathing zone measurements in Bangkok children were 

35 about 3.5-fold greater than in rural provincial children (p < 0.001). Interestingly, although bulky 

36 DNA adducts were increased fivefold in Bangkok children compared with provincial children (p

37 < 0.001), adduct levels were negatively correlated with total PAH and benzo[a]pyrene equivalent 

38 exposures. 
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1 

2 4.1.2.2.2. Adduct measures in reproductive tissues

3 

4 Zenzes et al. (1998) used immunostaining to detect PAH-DNA adducts in ovarian 

5 granulosa-lutein cells from women undergoing procedures for reproductive assistance. The 32 

6 women in the sample were separated by smoking status and consisted of 14 active smokers (1-20 

7 cigarettes per day), 7 passive smokers (nonsmoker but husband smoked), and 11 non-smokers 

8 with a non-smoking partner. The collected cells were fixed and stained with the anti-BP-DNA 

9 antibody which is known to cross-react with several carcinogenic PAH-DNA adducts. In this 

10 assay, darker nuclear staining results from a greater concentration of PAH-DNA adducts and is 

11 assigned a higher score. The proportion of nuclei exhibiting respective intensities of staining 

12 were combined to obtain an overall intensity score for the sample. The observed staining 

13 intensity was related to smoking status, with a mean ± SE of 1.91 ± 0.10 among active smokers, 

14 1.21 ± 0.20 among passive smokers, and 0.62 ± 0.17 among nonsmokers (p < 0.0001). The 

15 authors concluded that smoking-related seminal DNA adducts could be a potential source of 

16 transmissible zygotic DNA damage. 

17 In another study using the same immunostaining assay, Zenzes et al. (1999a) investigated 

18 the occurrence of PAH-DNA adducts in semen in relation to tobacco use. The study included 23 

19 men (11 smokers and 12 nonsmokers), mean age 37 years, recruited through couples attending 

20 an in vitro fertilization – embryo transfer clinic in Toronto. Among smokers, the mean amount 

21 smoked was 20.6 cigarettes per day. The PAH-DNA adduct staining intensity was higher in the 

22 sperm samples from smokers (mean ± SE, 1.73 ± 0.09) when compared with nonsmokers (0.93 ± 

23 0.10) (p < 0.0001); 5.7% and 30.9% of the sperm from smokers and nonsmokers, respectively, 

24 exhibited negative staining and 21.2% and 3.8%, respectively, exhibited strong staining. The 

25 authors concluded that smoking-related DNA adducts in semen could potentially be a source of 

26 zygotic DNA damage. 

27 A related study examined the presence of PAH-DNA adducts in 112 blastomere cells 

28 from 22 pre-implantation embryos available through an in vitro fertilization – embryo transfer 

29 clinic (Zenzes et al., 1999b). The donated embryos were grouped with respect to the maternal 

30 and paternal smoking status (n = 8 both parents smoked, n = 12 father smoked but mother did not 

31 smoke, and n=7 neither parent smoked). Five of the embryos (4 from the father-only smoking 

32 and 1 from a non-smoking couple) were very fragmented and were not used in the analysis of 

33 PAH-DNA staining intensity. Among the smoking parents, women smoked less per day than 

34 men (mean ± SE, 13.0 ± 3.0 and 16.8 ± 1.5 cigarettes per day for women and men, respectively). 

35 The intensity score was higher for embryos with at least one parent who smoked (1.40 ± 0.28) 

36 compared with embryos from nonsmoking parents (0.38 ± 0.14) (p = 0.015), but there was little 

37 difference between embryos with two compared with one smoking parent (1.34 ± 0.30 and 1.48 

38 ± 0.55 for both smokers and one smoker, respectively). Similar results were seen using the 
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1 proportion of embryos exhibiting any staining rather than a mean staining measure, and with the 

2 proportion of blastomere cells exhibiting staining. Intensity score was correlated with the 

3 amount smoked by the father (p = 0.020) but not by the mother. Analysis of the presence of 

4 PAH-DNA adducts in sperm cells also revealed an increased proportion and staining intensity in 

5 samples from smokers compared with nonsmokers (p < 0.0001 for categorical analysis of 

6 negative, weak, moderate and strong staining intensity). The authors noted that the demonstrated 

7 presence of these adducts in the embryos, reflecting most strongly a paternal origin, may affect 

8 the viability of the pregnancy. 

9 

10 4.1.2.3. Benzo[a]pyrene-Induced Cytogenetic Damage

11 Many studies measure cytogenetic damage as biomarkers of early biological effects 

12 which also reflect exposure to genotoxic chemicals. Standard cytogenetic end points include 

13 chromosomal aberration (CA), sister chromatid exchange (SCE), micronucleus (MN) formation, 

14 hypoxanthine guanine phosphoribosyl transferase (hprt) mutation frequency, and GPA mutation 

15 frequency (Gyorffy et al., 2008). These biomarkers are often incorporated in multi-endpoint 

16 studies with other biomarkers of exposure. Because they indicate related but different endpoints, 

17 there is often a lack of correlation between the different categories of biomarkers. 

Merlo et al. (1997) evaluated DNA adduct formation (measured by [32 18 P]-postlabelling) 

19 and micronuclei in WBCs of 94 traffic policemen vs. 52 residents from the metropolitan area of 

20 Genoa, Italy. All study subjects wore personal air samplers for 5 hours of one work shift, and 

21 levels of benzo[a]pyrene and other PAHs. Policemen were exposed to 4.55 ng 

benzo[a]pyrene/m3 air, compared with urban residents who were exposed to 0.15 ng/m322 . DNA 

23 adduct levels in policemen were 35% higher than in urban residents (p = 0.007), but micronuclei 

24 in urban residents were 20% higher than in policemen (p = 0.02). Linear regressions of DNA 

25 adducts and MN incidence, respectively, vs. benzo[a]pyrene exposure levels did not reveal 

26 significant correlations. 

27 Perera and coworkers assessed DNA damage in Finnish iron foundry workers in two 

28 separate studies and using three methodologies. Based on results from personal sampling and 

29 stationary monitoring in both studies, three levels of benzo[a]pyrene air concentrations were 

defined: low (<5 ng/m3 benzo[a]pyrene), medium (5–12 ng/m3), and high (>12 ng/m330 ). (Perera 

31 et al., 1994, 1993). In the first study, involving 48 workers, several biomarkers were analyzed 

32 for dose-response and interindividual variability (Perera et al., 1993). PAH-DNA adducts were 

33 determined in WBCs using an immunoassay as described in Section 4.1.2.2.1 and enzyme-linked 

34 immunosorbent assay (ELISA) with fluorescence detection. Mutations at the hprt locus were 

35 also measured in WBC DNA. The latter assay is based on the fact that each cell contains only 

36 one copy of the hprt gene, which is located on the X-chromosome. While male cells have only 

37 one X-chromosome, female cells inactivate one of the two X-chromosomes at random. The gene 

38 is highly sensitive to mutations such that in the event of a crucial mutation in the gene, enzyme 
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1 activity disappears completely from the cell. In addition, mutations at the GPA gene locus were 

2 measured in red blood cells (RBCs). The GPA mutation frequency was not correlated with 

3 either benzo[a]pyrene exposure or PAH-DNA adduct formation. However, both PAH-DNA 

4 adduct levels and hprt mutation frequency increased with increasing benzo[a]pyrene exposure. 

5 In addition, there was a highly significant correlation between incidence of hprt mutations and 

6 PAH-DNA adduct levels (p = 0.004). 

7 In a second study, Perera et al. (1994) surveyed 64 iron foundry workers with 

8 assessments conducted in 2 successive years; 24 of the workers provided blood samples in both 

9 years. Exposure to benzo[a]pyrene, collected by personal and area sampling in the 1st year of 

the study, ranged from <5 to 60 ng/m3 10 and was estimated to have decreased by 40% in the 2nd 

11 year. The levels of PAH-DNA adducts were roughly 50% lower in the 2nd year, presumably 

12 reflecting decreased exposure. The longer-lived hprt mutations were not as strongly influenced 

13 by the decreasing exposure to benzo[a]pyrene. Study subjects who did not have detectable levels 

14 of DNA adducts were excluded from the study. As in the previous study, a strong correlation 

15 between DNA adduct levels and incidence of hprt mutations was observed (Perera et al., 1993). 

16 Kalina et al. (1998) studied several cytogenetic markers in 64 coke oven workers and 

17 34 controls employed at other locations within the same plant. Airborne benzo[a]pyrene and 

18 seven other carcinogenic PAHs were collected by personal air samplers, which showed ambient 

benzo[a]pyrene concentrations ranging widely from 0.002 to 50 μg/m319 in coke oven workers and 

from 0.002 to 0.063 μg/m320 in controls. CAs, SCEs, high-frequency cells (HFCs), and SCE 

21 heterogeneity index (SCE-H) were all significantly increased with benzo[a]pyrene exposure. 

22 Except for increases in HFCs, no effect of smoking was observed. Consistent with studies of 

23 PAH-DNA adduct formation, reduced cytogenetic response at high exposure levels produced a 

24 nonlinear dose-response relationship. The authors also evaluated the potential influence of 

25 polymorphisms in enzymes involved in the metabolism of benzo[a]pyrene. Glutathione S26 transferase M1 (GSTM-1) and N-acetyl transferase (NAT)-2 polymorphisms were studied and 

27 no evidence of the two gene polymorphisms having any influence on the incidence of 

28 cytogenetic damage was found. 

29 Motykiewicz et al. (1998) conducted a similar study of genotoxicity associated with 

30 benzo[a]pyrene exposure in 67 female residents of a highly polluted industrial urban area of 

31 Upper Silesia, Poland, and compared the results to those obtained from 72 female residents of 

32 another urban but less polluted area in the same province of Poland. Urinary mutagenicity and 

33 1-OH-Py levels, PAH-DNA adducts in oral mucosa cells (detected by immunoperoxidase 

34 staining), SCE, HFC, CA, bleomycin sensitivity, and GSTM-1 and CYP1A1 polymorphisms in 

35 blood lymphocytes were investigated. High volume air samplers and gas chromatography were 

used to quantify ambient benzo[a]pyrene levels which during the summer were 3.7 ng/m336 in the 

polluted area and 0.6 ng/m3 in the control area. During winter, levels rose to 43.4 and 7.2 ng/m3 37 

38 in the two areas, respectively. The cytogenetic biomarkers (CA and SCE/HFC), urinary 
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1 mutagenicity, and urinary 1-OH-Py excretion were significantly increased in females from the 

2 polluted area, and differences appeared to be more pronounced during winter time. PAH-DNA 

3 adduct levels were significantly increased in the study population, when compared to the 

4 controls, only in the winter season. No difference in sensitivity to bleomycin-induced 

5 lymphocyte chromatid breaks was seen between the two populations. As with the study by 

6 Kalina et al. (1998), genetic polymorphisms assumed to affect the metabolic transformation of 

7 benzo[a]pyrene were not associated with any difference in the incidence of DNA damage. 

8 In a study of Thai school boys in urban (Bangkok) and rural areas, bulky (including but 

9 not limited to BPDE-type) DNA adduct levels were measured in lymphocytes along with DNA 

10 single strand breaks (SSBs), using the comet assay, and DNA repair capacity (Tuntawiroon et al., 

11 2007). Ambient air and personal breathing zone measurements indicated that Bangkok school 

12 children experienced significantly higher exposures to benzo[a]pyrene and total PAHs. A 

13 significantly higher level of SSBs (tail length 1.93 ± 0.09 vs. 1.28 ± 0.12 μm, +51%; p < 0.001) 

14 was observed in Bangkok school children when compared with rural children, and this parameter 

15 was significantly associated with DNA adduct levels. A significantly reduced DNA repair 

16 capacity (0.45 ± 0.01 vs. 0.26 ± 0.01 γ-radiation-induced deletions per metaphase, -42%; 

17 p < 0.001) was also observed in the city school children, again significantly associated with 

18 DNA adduct levels. It was not evident why higher environmental PAH exposure would be 

19 associated with lowered DNA repair capacity. However, because the personal breathing zone 

20 PAH levels and DNA adduct levels were not associated with each other, it is conceivable that the 

21 city school children had a priori lower DNA repair capacities that contributed significantly to the 

22 high adduct levels. The authors considered genetic differences between the two study 

23 populations as a possible reason for this observation. 

24 

25 4.1.3. Epidemiologic Findings in Humans 

26 The association between human cancer and contact with PAH-containing substances, 

27 such as soot, coal tar, and pitch, has been widely recognized since the early 1900s (Bostrom et 

28 al., 2002). Although numerous epidemiology studies establish an unequivocal association 

29 between PAH exposure and human cancer, defining the causative role for benzo[a]pyrene and 

30 other specific PAHs remains a challenge. In essentially all reported studies, either the 

31 benzo[a]pyrene exposure and/or internal dose are not known, or the benzo[a]pyrene carcinogenic 

32 effect cannot be distinguished from the effects of other PAH and non-PAH carcinogens. 

33 Nevertheless, three types of investigations provide support for the involvement of 

34 benzo[a]pyrene in some human cancers: molecular epidemiology studies; population- and 

35 hospital-based case-control studies; and occupational cohort studies. In some cohort studies, 

36 benzo[a]pyrene exposure concentrations were measured and thus provide a means to link 

37 exposure intensity with observed cancer rates. In case-control studies, by their nature, 

38 benzo[a]pyrene and total PAH doses can only be estimated. 
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1 

2 4.1.3.1. Molecular Epidemiology and Case-Control Cancer Studies

3 Defective DNA repair capacity leading to genomic instability and, ultimately, increased 

4 cancer risk is well documented (Wu et al., 2007, 2005). Moreover, sensitivity to mutagen5 induced DNA damage is highly heritable and thus represents an important factor that determines 

6 individual cancer susceptibility. Based on studies comparing monozygotic and dizygotic twins, 

7 the genetic contribution to BPDE mutagenic sensitivity was estimated to be 48.0% (Wu et al., 

8 2007). BPDE has been used as an etiologically relevant mutagen in case-control studies to 

9 examine the association between elevated lung and bladder cancer risk and individual sensitivity 

10 to BPDE-induced DNA damage. Mutagen sensitivity is determined by quantifying chromatid 

11 breaks or DNA adducts in phytohemagglutinin-stimulated peripheral blood lymphocytes as an 

12 indirect measure of DNA repair capacity. 

13 In a hospital-based case-control study involving 221 lung cancer cases and 229 healthy 

14 controls, DNA adducts were measured in stimulated peripheral blood lymphocytes after 

15 incubation with BPDE in vitro (Li et al., 2001). Lung cells from cancer cases showed consistent 

16 statistically significant elevations in induced BPDE-DNA adducts, compared with controls, 

17 regardless of subgroup by age, sex, ethnicity, smoking history, weight loss, or family history of 

18 cancer. The BPDE-induced DNA adduct levels, when grouped by quartile using the levels in 

19 controls as cutoff points, were significantly dose-related with lung cancer risk (odds ratios [ORs] 

20 1.11, 1.62, and 3.23; trend test, p < 0.001). In a related hospital-based case-control study 

21 involving 155 lung cancer patients and 153 healthy controls, stimulated peripheral blood 

22 lymphocytes were exposed to BPDE in vitro (Wu et al., 2005). DNA damage/repair was 

23 evaluated using the comet assay, and impacts on cell cycle checkpoints measured using a 

24 fluorescence-activated cell-sorting method. The lung cancer cases exhibited significantly higher 

25 levels of BPDE-induced DNA damage than the controls (p < 0.001), with lung cancer risk 

26 positively associated with increasing levels of DNA damage when grouped in quartiles (trend 

27 test, p < 0.001). In addition, lung cancer patients demonstrated significantly shorter cell cycle 

28 delays in response to BPDE exposure, which correlated with increased DNA damage. 

29 Sensitivity to BPDE-induced DNA damage in bladder cancer patients supports the results 

30 observed in lung cancer cases. In a hospital-based case-control study involving 203 bladder 

31 cancer patients and 198 healthy controls, BPDE-induced DNA damage was specifically 

32 evaluated at the chromosome 9p21 locus in stimulated peripheral blood lymphocytes (Gu et al., 

33 2008). Deletions of 9p21, which includes critical components of cell cycle control pathways, are 

34 associated with a variety of cancers. After adjusting for age, sex, ethnicity, and smoking status, 

35 individuals with high BPDE-induced damage at 9p21 were significantly associated with 

36 increased bladder cancer risk (OR 5.28; 95% confidence interval [CI] 3.26–8.59). 

37 Categorization of patients into tertiles for BPDE sensitivity relative to controls demonstrated a 

38 dose-related association between BPDE-induced 9p21 damage and bladder cancer risk. 
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1 Collectively, the results of molecular epidemiology studies with lung and bladder cancer patients 

2 indicate that individuals with a defective ability to repair BPDE-DNA adducts are at increased 

3 risk for cancer and, moreover, that specific genes linked to tumorigenesis pathways may be 

4 molecular targets for benzo[a]pyrene and other carcinogens. 

5 Due to the importance of the diet as a benzo[a]pyrene exposure source, several 

6 population- and hospital-based case-control studies have investigated the implied association 

7 between dietary intake of benzo[a]pyrene and risk for several tumor types. In a study involving 

8 193 pancreatic cancer cases and 674 controls (Anderson et al., 2005), another involving 

9 626 pancreatic cancer cases and 530 controls (Li et al., 2007), and a third involving 

10 146 colorectal adenoma cases and 228 controls (Sinha et al., 2005), dietary intake of 

11 benzo[a]pyrene was estimated using food frequency questionnaires. In all studies, the primary 

12 focus was on estimated intake of benzo[a]pyrene (and other carcinogens) derived from cooked 

13 meat. Overall, cases when compared with controls had higher intakes of benzo[a]pyrene and 

14 other food carcinogens, leading to the conclusion that benzo[a]pyrene plays a role in the etiology 

15 of these tumors in humans. In a supportive follow-up case-control study of colorectal adenomas, 

16 increased leukocyte PAH-DNA adducts were measured in cases when compared with controls, 

17 using a method that recognizes BPDE and several other PAHs bound to DNA (Gunter et al., 

18 2007). 

19 

20 4.1.3.2. Cohort Cancer Studies

21 Epidemiologic studies of workers in PAH-related occupations indicate increased human 

22 cancer risks associated with iron and steel production, roofing, carbon black production, and 

23 exposure to diesel exhaust (Bosetti et al., 2007). Exposure to benzo[a]pyrene is only one of 

24 numerous contributors to the cancer risk from complex PAH-containing mixtures that occur in 

25 the workplace. Although some occupational cohort studies report measured or estimated 

26 inhalation exposure concentrations for benzo[a]pyrene, none report biomarkers of internal 

27 benzo[a]pyrene dose in study subjects (reviewed in Bosetti et al., 2007; Armstrong et al., 2004). 

28 Several of these cohort studies (summarized below) demonstrate a positive exposure-response 

29 relationship with cumulative PAH exposure using benzo[a]pyrene—or a proxy such as benzene30 soluble matter (BSM) that can be converted to benzo[a]pyrene—as an indicator substance. 

31 These studies provide insight and support for the causative role of benzo[a]pyrene in human 

32 cancer. 

33 
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1 4.1.3.2.1. Cancer incidence in aluminum and electrode production plants. 

2 Exposure to benzo[a]pyrene and BSM in aluminum smelter workers is strongly 

3 associated with bladder cancer and weakly associated with lung cancer (Boffetta et al., 1997; 

4 Tremblay et al., 1995; Armstrong et al., 1994; Gibbs, 1985; Theriault et al., 1984). In an 

5 analysis of pooled data from nine cohorts of aluminum production workers, 688 respiratory tract 

6 cancer cases were observed versus 674.1 expected (pooled RR 1.03; CI 0.96–1.11) (Bosetti et al., 

7 2007). A total of 196 bladder cancer cases were observed in eight of the cohorts, compared with 

8 155.7 expected (pooled RR 1.29; CI 1.12–1.49). Based on estimated airborne benzo[a]pyrene 

9 exposures from a meta-analysis of eight cohort studies, the predicted lung cancer relative risk 

(RR) per 100 μg/m310 -years of cumulative benzo[a]pyrene exposure was 1.16 (95% CI 1.05–1.28) 

11 (Armstrong et al., 2004). 

12 Spinelli et al. (2006) reported a 14-year update to a previously published historical cohort 

13 study (Spinelli et al., 1991) of Canadian aluminum reduction plant workers. The results 

14 confirmed and extended the findings from the earlier epidemiology study. The study surveyed a 

15 total of 6,423 workers with ≥3 years of employment at an aluminum reduction plant in British 

16 Columbia, Canada, between the years 1954 and 1997, and evaluated all types of cancers. The 

17 focus was on cumulative exposure to coal tar pitch volatiles, measured as BSM and as 

18 benzo[a]pyrene. Benzo[a]pyrene exposure categories were determined from the range of 

19 predicted exposures over time from statistical exposure models. There were 662 cancer cases, of 

20 which approximately 98% had confirmed diagnoses. The overall cancer mortality rate 

21 (standardized mortality ratio [SMR] 0.97; CI 0.87–1.08) and cancer incidence rate (standardized 

22 incidence ratio [SIR] 1.00; CI 0.92–1.08) were not different from that of the British Columbia 

23 general population. However, this study identified significantly increased incidence rates for 

24 cancers of the bladder (SIR 1.80; CI 1.45–2.21) and the stomach (SIR 1.46; CI 1.01–2.04). The 

25 lung cancer incidence rate was only slightly higher than expected (SIR = 1.10; CI 0.93–1.30). 

26 Significant dose-response associations with cumulative benzo[a]pyrene exposure were seen for 

27 bladder cancer (p trend <0.001), stomach cancer (p trend <0.05), lung cancer (p trend <0.001), 

28 non-Hodgkin lymphoma (p trend <0.001), and kidney cancer (p trend <0.01), although the 

29 overall incidence rates for the latter three cancer types were not significantly elevated versus the 

30 general population. Similar cancer risk results were obtained using BSM as the exposure 

31 measure; the cumulative benzo[a]pyrene and BSM exposures were highly correlated (r = 0.94). 

32 In several occupational cohort studies of workers in Norwegian aluminum production 

33 plants, personal and stationary airborne PAH measurements were performed. 

34 In a study covering 11,103 workers and 272,554 person × years of PAH exposure, cancer 

35 incidence was evaluated in six Norwegian aluminum smelters (Romundstad et al., 2000a, b). 

Reported estimates of PAH exposure concentrations reached a maximum of 3,400 μg/m336 PAH 

(680 μg/m337 benzo[a]pyrene). The overall number of cancers observed in this study did not differ 

38 significantly from control values (SIR 1.03; CI 1.0–1.1). The data from this study showed 
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1 significantly increased incidences for cancer of the bladder (SIR 1.3; CI 1.1–1.5), and elevated, 

2 but not significant, SIRs for larynx (SIR 1.3; CI 0.8–1.9), thyroid (SIR 1.4; CI 0.7–2.5), and 

3 multiple myeloma (SIR 1.4; CI 0.9–1.9). Incidence rates for bladder, lung, pancreas, and kidney 

4 cancer (the latter three with SIRs close to unity) were subjected to a cumulative exposure5 response analysis. The incidence rate for bladder cancer showed a trend with increasing 

6 cumulative exposure and with increasing lag times (up to 30 years) at the highest exposure level. 

7 The incidence of both lung and bladder cancers was greatly increased in smokers. The authors 

8 reported that using local county rates rather than national cancer incidence rates as controls 

9 increased the SIR for lung cancer (SIR 1.4; CI 1.2–1.6) to a statistically significant level. 

10 

11 4.1.3.2.2. Cancer incidence in coke oven, coal gasification, and iron and steel foundry 

12 workers. 

13 An increased risk of death from lung and bladder cancer is reported in some studies 

14 involving coke oven, coal gasification, and iron and steel foundry workers (Bostrom et al., 2002; 

15 Boffetta et al., 1997). An especially consistent risk of lung lung cancer across occupations is 

16 noted when cumulative exposure is taken into consideration (e.g. RR of 1.16 per 100 unity-yrs 

17 for aluminum smeleter workers, 1.17 for coke oven workers, and 1.15 for coal gasificiation 

18 workers). In an analysis of pooled data from 10 cohorts of coke production workers, 762 lung 

19 cancer cases were observed versus 512.1 expected (pooled RR 1.58; CI 1.47–1.69) (Bosetti et al., 

20 2007). Significant variations in risk estimates among the studies were reported, particularly in 

21 the large cohorts (RRs of 1.1, 1.2, 2.0, and 2.6). There was no evidence for increased bladder 

22 cancer risk in the coke production workers. Based on estimated airborne benzo[a]pyrene 

23 exposures from a meta-analysis of 10 cohort studies, the predicted lung cancer RR per 

100 μg/m324 -years of cumulative benzo[a]pyrene exposure was 1.17 (95% CI 1.12–1.22) 

25 (Armstrong et al., 2004). 

26 A meta-analysis of data from five cohorts of gasification workers reported 251 deaths 

27 from respiratory tract cancer, compared with 104.7 expected (pooled RR 2.58; 95% CI 2.28–

28 2.92) (Bosetti et al., 2007). Pooled data from three of the cohorts indicated 18 deaths from 

29 urinary tract cancers, versus 6.0 expected (pooled RR 3.27; 95% CI 2.06–5.19). Based on 

30 estimated airborne benzo[a]pyrene exposures from a meta-analysis of four gas worker cohort 

studies, the predicted lung cancer RR per 100 μg/m331 -years of cumulative benzo[a]pyrene 

32 exposure was 1.15 (95% CI 1.11–1.20) (Armstrong et al., 2004). 

33 Increased risks were reported in iron and steel foundry workers for cancers of the 

34 respiratory tract, bladder and kidney. In an analysis of pooled data from 10 cohorts, 

35 1,004 respiratory tract cancer cases were observed versus 726.0 expected (pooled RR 1.40; CI 

36 1.31–1.49) (Bosetti et al., 2007). A total of 99 bladder cancer cases were observed in seven of 

37 the cohorts, compared with 83.0 expected (pooled RR 1.29; CI 1.06–1.57). For kidney cancer, 
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1 40 cases were observed compared with 31.0 expected based on four studies (pooled RR 1.30; 

2 95% CI 0.95–1.77). 

3 Xu et al. (1996) conducted a nested case-control study, surveying the cancer incidence 

4 among 196,993 active or retired workers from the Anshan Chinese iron and steel production 

5 complex. A large number of historical benzo[a]pyrene measurements (1956–1995) were 

6 available. The study included 610 cases of lung cancer and 292 cases of stomach cancer, with 

7 959 matched controls from the workforce. After adjusting for nonoccupational risk factors such 

8 as smoking and diet, significantly elevated risks for lung cancer and stomach cancer were 

9 identified for subjects employed for 15 or more years, with ORs varying among job categories. 

10 For either type of cancer, highest risks were seen among coke oven workers: lung cancer, OR = 

11 3.4 (CI 1.4–8.5); stomach cancer, OR = 5.4 (CI 1.8–16.0). 

12 There were significant trends for long-term cumulative benzo[a]pyrene exposure vs. lung 

13 cancer (p = 0.004) or stomach cancer (p = 0.016) incidence. For cumulative total 

14 benzo[a]pyrene exposures of < 0.84, 0.85-1.96, 1.97-3.2 and ≥ 3.2 the ORs for lung cancer were 

15 1.1 (CI 0.8-1.7), 1.6 (CI 1.2-2.3), 1.6 (1.1-2.3) and 1.8 (CI 1.2-2.5). For cumulative total 

16 benzo[a]pyrene exposures of < 0.84, 0.85-1.96, 1.97-3.2 and ≥ 3.2 the ORs for stomach cancer 

17 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 

18 investigators noted that additional workplace air contaminants were measured, which might have 

19 influenced the outcome. Of these, asbestos, silica, quartz, and iron oxide-containing dusts may 

20 have been confounders. For lung cancers, cumulative exposures to total dust and silica dust both 

21 showed significant dose-response trends (p = 0.001 and 0.007, respectively), while for stomach 

22 cancer, only cumulative total dust exposure showed a marginally significant trend (p = 0.061). 

For cumulative total dust exposures of < 69, 69-279, 280-882 and ≥ 883 mg/m323 the ORs for lung 

24 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), 

25 respectively. For cumulative silica dust exposures of < 3.7, 3.7-10.39, 10.4-27.71 and ≥ 27.72 

mg/m326 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 

27 (CI 1.2-2.5), respectively. For cumulative total dust exposures of < 69, 69-279, 280-882 and ≥

883 mg/m328 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 

29 1.6 (CI 1.1-2.5), respectively. 

30 Exposure-response data from studies of coke oven workers in the United States have 

31 often been used to derive quantitative risk estimates for PAH mixtures, and for benzo[a]pyrene 

32 as an indicator substance (Bostrom et al., 2002). However, there are numerous studies of coke 

33 oven worker cohorts that do not provide estimates of benzo[a]pyrene exposure. An overview of 

34 the results of these and other studies can be obtained from the review of Boffetta et al. (1997). 

35 

36 4.1.3.2.3. Cancer incidence in asphalt workers and roofers. 

37 These groups encompass different types of work (asphalt paving vs. roofing) and also 

38 different types of historical exposure that have changed from using PAH-rich coal tar pitch to the 
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1 use of bitumen or asphalt, both of which are rather low in PAHs due to their source (crude oil 

2 refinery) and a special purification process. Increased risks for lung cancer were reported in 

3 large cohorts of asphalt workers and roofers; evidence for increased bladder cancer risk is weak 

4 (Burstyn et al., 2007; Partanen and Boffetta, 1994; Chiazze et al., 1991; Hansen, 1991, 1989; 

5 Hammond et al., 1976). In an analysis of pooled data from two cohorts of asphalt workers, 822 

6 lung cancer cases were observed versus 730.7 expected (pooled RR 1.14; CI 1.07–1.22) (Bosetti 

7 et al., 2007). In two cohorts of roofers, analysis of pooled data indicated that 138 lung cancer 

8 cases were observed, compared with 91.9 expected (pooled RR 1.51; CI 1.28–1.78) (Bosetti et 

9 al., 2007). 

10 

11 4.1.3.3. Noncancer Disease Caused by Benzo[a]pyrene

12 Because accumulating evidence indicates that PAH exposure is a risk factor for ischemic 

13 heart disease (IHD), Burstyn et al. (2005) investigated 418 cases of fatal IHD in a cohort of 

14 12,367 asphalt paving workers exposed to low-level PAH from bitumen and coal tar. The 

15 follow-up started in 1953 and ended in 2000, with an average exposure of 17 ± 9 years 

16 (minimum: one work season), resulting in 193,889 person-years of observation. Previous 

17 analyses of this cohort indicated no association between PAH exposure and excess mortality 

18 from cancer or by all causes. Quantitative estimates of exposure to benzo[a]pyrene were 

19 obtained for paving operations on the basis of previously available personal exposure 

20 measurements from workers in the asphalt industry (but not necessarily from cohort members). 

21 Exposures were calculated as average (0–68 [reference], 68–105, 106–146, 147–272, and 273+ 

ng/m3) and cumulative (0–189 [reference], 189–501, 502–931, 932–2,012, and 2,013+ ng/m322 -

23 years), respectively. 

24 Cumulative and average exposure indices for benzo[a]pyrene were positively associated 

25 with mortality from IHD; the highest RR coincided with an average exposure to benzo[a]pyrene 

of 273 ng/m326 or higher (RR = 1.64; CI = 1.13–2.28). A similar risk was observed for the highest 

cumulative benzo[a]pyrene exposure group (>2,013 ng/m327 -years) (RR = 1.58; CI = 0.98–2.55). 

28 Length of employment had no influence on this result. A dose-response was evident for IHD, 

29 but not for other types of cardiovascular disease. The RR remained elevated even with 

30 adjustment for smoking as a confounder; the RR was 1.24 under the extreme assumption of 0% 

31 never smokers, 30% former smokers, and 70% current smokers in the highest-exposed group. 

32 The authors discussed the possibility of bias because some of their study subjects might have 

33 been exposed to other IHD-causing factors that were not controlled for in their study, or might 

34 have been misclassified. 

35 An occupational study of Canadian aluminum smelter workers investigated the effect of 

36 benzo[a]pyrene exposure on cardiopulmonary mortality (Friesen et al., 2010). Adjusted internal 

37 comparisons for smoking were conducted using Cox regression for male subjects (n = 6,423). 

38 Ischemic heart disease (IHD) was associated with cumulative benzo[a]pyrene exposure with a 
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1 hazard ratio of 1.62 (95% CI 1.06-2.46) in the highest benzo[a]pyrene exposure category. For 

2 active employment, the hazard ratio for IHD was 2.39 (95% CI 0.95-6.05) in the highest 

3 cumulative benzo[a]pyrene exposure category. 

4 Other studies have reported potential prenatal effects and associated birth outcomes 

5 induced by inhalation exposure to PAHs, including benzo[a]pyrene. Perera et al. (2005a) studied 

6 329 nonsmoking pregnant women (30 ± 5 years old) possibly exposed to PAHs from fires during 

7 the 4 weeks after 09/11/2001. Maternal and umbilical cord blood levels of benzo[a]pyrene 

8 (BPDE)-DNA adducts were highest in study participants who lived within 1 mile of the WTC, 

9 with an inverse correlation between cord blood levels and distance from the WTC. Neither cord 

10 blood adduct level nor ETS alone was positively correlated with adverse birth outcomes. 

11 However, the interaction between ETS exposure and cord blood adducts was significantly 

12 associated with reduced birth weight and head circumference. Among babies exposed to ETS in 

13 utero, a doubling of cord blood benzo[a]pyrene-DNA adducts was associated with an 8% 

14 decrease in birth weight (p = 0.03) and a 3% decrease in head circumference (p = 0.04). 

15 Perera et al. (2005b) compared various exposures—ETS, nutrition, pesticides, material 

16 hardship—with birth outcomes (length, head circumference, cognitive development). ETS 

17 exposure and intake of PAH-rich foods by pregnant women were determined by questionnaire. 

18 Levels of benzo[a]pyrene(BPDE)-DNA adducts were determined in umbilical cord blood 

19 collected at delivery. The study population consisted of Dominican or African-American 

20 nonsmoking pregnant women (n = 529; 24 ± 5 years old) free of diabetes, hypertension, HIV, 

21 and drug or alcohol abuse. Benzo[a]pyrene adducts, ETS, and dietary PAHs were not 

22 significantly correlated with each other. However, the interaction between benzo[a]pyrene-DNA 

23 adducts and ETS exposure was significantly associated with reduced birth weights (-6.8%; p = 

24 0.03) and reduced head circumference (-2.9%; p = 0.04). 

25 Tang et al. (2006) measured benzo[a]pyrene(BPDE)-DNA adducts in maternal and 

26 umbilical cord blood obtained at delivery from a cohort of 150 nonsmoking women and their 

27 newborns in China. Exposure assessment was related to the seasonal operation of a local, coal28 fired power plant; however, airborne PAH concentrations were not measured. Dietary PAH 

29 intake was not included as a covariate because it did not significantly contribute to the final 

30 models, but ETS, sex, and maternal height and weight were considered as covariates. DNA 

31 adduct levels were compared to several birth outcomes and physical development parameters, 

32 such as gestational age at birth; infant sex, birth weight, length, head circumference, and 

33 malformations; maternal height and pregnancy weight total weight gain; complications of 

34 pregnancy and delivery; and medications used during pregnancy. 

35 High cord blood adduct levels were significantly associated with reduced infant/child 

36 weight at 18 months (β = -0.048, p = 0.03), 24 months (β = -0.041, p = 0.027), and 30 months of 

37 age (β = -0.040, p = 0.049); decreased birth head circumference was marginally associated with 

38 DNA adduct levels (β = -0.011, p = 0.057). Maternal adduct levels were correlated neither with 
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1 cord blood adduct levels nor with fetal and child growth. Among female infants, cord blood 

2 adduct levels were significantly associated with smaller birth head circumference (p = 0.022) and 

3 with lower weight at 18 months (p = 0.014), 24 months (p = 0.012), and 30 months of age (p = 

4 0.033), and with decreased body length at 18 months of age (p = 0.033). Among male infants, 

5 the corresponding associations were also inverse but statistically not significant. 

6 Considerable evidence of a deleterious effect of smoking on male and female fertility has 

7 accumulated from epidemiological studies of time to pregnancy, ovulatory disorders, semen 

8 quality, and spontaneous abortion (reviewed in Waylen et al., 2009; Cooper and Moley, 2008; 

9 Soares and Melo, 2008). In addition, the effect of smoking, particularly during the time of the 

10 perimenopausal transition, on acceleration of ovarian senescence (menopause) has also been 

11 established (Midgette and Baron, 1990). More limited data is available pertaining specifically to 

12 measures of benzo[a]pyrene and reproductive outcomes. 

13 Neal et al. (2008, 2007) examined levels of benzo[a]pyrene and other PAHs in follicular 

14 fluid and serum sample from 36 women undergoing in vitro fertilization at a clinic in Toronto, 

15 and compared the successful conception rate in relation to benzo[a]pyrene levels. The women 

16 were classified by smoking status, with 19 who were current cigarette smokers, 7 with passive or 

17 sidestream smoke exposure (i.e., non-smoker with a partner who smoked) and 10 non-smoked 

18 exposed. An early follicular phase blood sample and follicular fluid sample from the follicle at 

19 the time of ovum retrieval were collected and analyzed for the presence of benzo[a]pyrene, 

20 acenapthelene, phenanthrene, pyrene and chrysene using GC/MS (detection limit 5 pg/ml). The 

21 frequency of non-dectable levels of serum benzo[a]pyrene was highest in the non-smoking group 

22 (60.0%, 14.3%, and 21.0% below detection limit in non-smoking, sidestream smoke, and active 

23 smoking groups, respectively). A similar pattern was seen with follicular fluid benzo[a]pyrene 

24 (30.0%, 14.3%, and 10.5% below detection limit limit in non-smoking, sidestream smoke, and 

25 active smoking groups, respectively). In the analyses comparing mean values across groups, an 

26 assigned value of 0 was used for non-detectable samples. Follicular fluid benzo[a]pyrene levels 

27 were higher in the active smoking group (mean ± SE, 1.32 ± 0.68 ng/ml) than in the sidestream 

28 (0.05 ± 0.01 ng/ml) or non-smoking (0.03 ± 0.01 ng/ml) groups (p = 0.04). The between-group 

29 differences in serum benzo[a]pyrene levels were not statistically significant (0.22 ± 0.15, 0.98 ± 

30 0.56, and 0.40 ± 0.13 ng/ml in non-smoking, sidestream smoke, and active smoking groups, 

31 respectively), and there were no differences in relation to smoking status. Among active 

32 smokers, the number of cigarettes smoked per day was strongly correlated with follicular fluid 

33 benzo[a]pyrene levels (r= 0.7, p < 0.01). Follicular fluid benzo[a]pyrene levels were 

34 significantly higher among the women who did not conceive (1.79 ng/ml ± 0.86) compared with 

35 women who did get pregnant (mean approximately, 0.10 ng/ml, as estimated from graph) (p < 

36 0.001), but serum levels of benzo[a]pyrene were not associated with successful conception. 

37 A small case-control study conducted between August 2005 and February 2006 in 

38 Lucknow city (Uttar Pradesh), India examined PAH concentrations in placental tissues (Singh et 
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1 al., 2008) in relation to risk of preterm birth. The study included 29 cases (delivery between 28 

2 and < 36 weeks gestation) and 31 term delivery controls. Demographic data smoking history, 

3 reproductive history and other information were collected by interview, and a 10 g sample of 

4 placental tissue was collected form all participants. Concentration of specific PAHs in placental 

5 tissue was determined using HPLC. In addition to benzo[a]pyrene, the PAHs assayed were 

6 naphthalene, acenapththylene, phenanthrene, fluorene, anthracene, benzo(a)anthracene, 

7 fluoranthene, pyrene, beno(k)fluoranthene, benzo(b)fluoranthene, benzo(g,h,i)perylene, and 

8 dibenzo(a,h)anthracene. PAH exposure in this population was from environmental sources and 

9 from cooking. The age of study participants ranged from 20 to 35 years. There was little 

10 difference in birthweight between cases and controls (mean 2.77 and 2.75 in the case and control 

11 groups, respectively). Placental benzo[a]pyrene levels were lower than the levels of the other 

12 PAHs detected (mean 8.83 ppb in controls for benzo[a]pyrene compared with 25-30 ppb for 

13 anthracene, beno(k)fluoranthene, benzo(b)fluoranthene, and dibenzo(a,h)anthracene, 59 ppb for 

14 acenaphthylene, and 200 – 380 naphthalene, phenanthrene, fluoranthene, and pyrene; non15 detectable levels of fluorine, benzo(a)anthracene, and benzo(g,h,i)perylene were found). There 

16 was little difference in benzo[a]pyrene levels between cases (mean ± SE 13.85 ± 7.06 ppb) and 

17 controls (8.83 ± 5.84 ppb), but elevated levels of fluoranthene (325.91 ± 45.14 and 208.6 ± 

18 21.93 ppb in cases and controls, p < 0.05) and benzo(b)fluoranthene (61.91 ± 12.43 and 23.84 ± 

19 7.01 ppb in cases and controls, p < 0.05) were seen. 

20 Wu et al. (2010) conducted a study of benzo[a]pyrene-DNA adduct levels in relation to 

21 risk of fetal death in Tianjin, China. This case-control study included women who experienced a 

22 missed abortion before 14 weeks gestational age, that is a fetal death that remained in utero and 

23 so required surgical intervention. Cases were matched by age and gravidity to controls (women 

24 undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded 

25 women who smoked, women with chronic disease and pregnancy complications, and women 

26 with occupational exposures to PAHs. Residency within Tianjin for at least one year was also an 

27 eligibility criterion. The participation rate was high: 81 of 84 eligible cases participanted and 81 

28 of 89 eligible controls participated. Data pertaining to demographic characteristics, reproductive 

29 history, and factors relating to potential PAH exposure were collected using a structured 

30 interview, and samples from the aborted tissue were obtained. In two of the four hospitals used 

31 in the study, blood samples from the women (n=51 cases and 51 controls) were also collected. 

32 The presence of benzo[a]pyrene-BPDE adducts was assessed in the blood and tissue samples 

33 using HPLC. There was no correlation between blood and aborted tissue levels of 

34 benzo[a]pyrene adducts (r = –0.12 for the 102 blood-tissue pairs, r = –0.02 for the 51 case paris 

35 and r = –0.21 for the 51 control pairs). (The authors noted that there was little difference 

36 between women with and without blood samples in terms of the interview- based measures 

37 collected or in terms of the DNA-adduct levels in aborted tissue.) benzo[a]pyrene-adduct levels 

38 were similar but slightly lower in the aborted tissue of cases compared with controls (mean ± SD 
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1 4.8 ± 6.0 in cases and 6.0 ± 7.4 in controls, p = 0.29). In the blood samples, however, 

2 benzo[a]pyrene-adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ± 2.2 in cases and controls, 

3 respectively, p < 0.001). In logistic regression analyses using a continuous adduct measure, the 

OR was 1.35 (95% CI 1.11 – 1.64) per adduct/1084 nucleotide. These results adjusted for 

5 education and household income, but were very similar to the unadjusted results. Categorizing 

6 exposure at the median value resulted in an adjusted OR of 4.27 (95% CI 1.41 – 12.99) in the 

7 high compared with low benzo[a]pyrene-adduct group. There was no relation between 

8 benzo[a]pyrene-adduct levels in the aborted tissue and missed abortion in the logistic regression 

9 analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93 – 1.02) or dichotomous 

10 exposure measure (adjusted OR 0.76, 95% CI 0.37 – 1.54). Associations between missed 

11 abortion and several interview-based measures of potential PAH exposure were also seen: 

12 adjusted OR 3.07 (95% CI 1.31 – 7.16) for traffic congestion near residence, 3.52 (95% CI 1.44 

13 – 8.57) for commuting by walking, 3.78 (95% CI 1.11 – 12.87) for routinely cooked during 

14 pregnancy, and 3.21 (95% CI 0.98 – 10.48) for industrial site or stack near residence, but there 

15 was no association with other types of commuting (e.g., by bike, car, or bus). 

16 

17 

18 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

19 ANIMALS—ORAL, INHALATION, AND DERMAL 

20 4.2.1. Oral 

21 4.2.1.1. Subchronic Studies 

22 

23 De Jong et al. (1999) 35-day rat study

24 De Jong et al. (1999) treated male Wistar rats (eight/dose group) with benzo[a]pyrene 

25 (98.6% purity) dissolved in soybean oil by gavage 5 days/week for 35 days at doses of 0, 3, 10, 

26 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 

27 exposure period, rats were necropsied, organ weights were determined, and major organs and 

28 tissues were prepared for histological examination (adrenals, brain, bone marrow, colon, caecum, 

29 jejunum, heart, kidney, liver, lung, lymph nodes, esophagus, pituitary, spleen, stomach, testis, 

30 and thymus). Blood was collected for examination of hematological endpoints, but there was no 

31 indication that serum biochemical parameters were analyzed. Immune parameters included 

32 determinations of serum immunoglobulin levels (IgG, IgM, IgE, and IgA), relative spleen cell 

33 distribution, and spontaneous cytotoxicity of spleen cell populations determined in a natural34 killer (NK) cell assay. 

35 Body weight gain was decreased beginning at week 2 at the high dose of 90 mg/kg-day; 

36 there was no effect at lower doses (De Jong et al., 1999). Hematology revealed a dose-related 

37 decrease in RBC count, hemoglobin, and hematocrit at ≥10 mg/kg-day (Table 4-2). A minimal 

38 but significant increase in mean cell volume and a decrease in mean cell hemoglobin 
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1 concentration were noted at 90 mg/kg-day, and may indicate dose-related toxicity for the RBCs 

2 and/or RBC precursors in the bone marrow. A decrease in WBCs, attributed to a decrease in the 

3 number of lymphocytes (approximately 50%) and eosinophils (approximately 90%), was 

4 observed at 90 mg/kg-day; however, there was no effect on the number of neutrophils or 

5 monocytes. A decrease in the cell number in the bone marrow observed in the 90 mg/kg-day 

6 dose group was consistent with the observed decrease in the RBC and WBC counts at this dose 

7 level. In the 90 mg/kg-day dose group, brain, heart, kidney, and lymph node weights were 

8 decreased and liver weight was increased (Table 4-2). Decreases in heart weight at 3 mg/kg-day 

9 and in kidney weight at 3 and 30 mg/kg-day were also observed, but these changes did not show 

10 dose-dependent responses. Dose-related decreases in thymus weight were statistically 

11 significant at ≥10 mg/kg-day (Table 4-2). 

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 (109

/L) 

Red blood cells (109

/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 ± 0.2a

0.47 ± 0.01a

13.58 ± 2.9 a

7.8 ± 0.4 

9.5 ± 0.4a

0.46 ± 0.02a

8.53 ± 1.1a 

7.1 ± 0.4a

8.6 ± 0.6a

0.43 ± 0.02a

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 ± 16a

106 ± 19 

72 ± 22 

145 ± 176 

81 ± 26 

99 ± 29 

39 ± 19a

75 ± 55 

Cellularity (mean ± SD; n = 7–8)

Spleen (cell number ×107

) 

Bone marrow (G/L) 

59 ± 15 

31 ± 7 

71 ± 14 

36 ± 5 

59 ± 13 

31 ± 8 

63 ± 10 

27 ± 8 

41 ± 10a

19 ± 4a

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 ± 3a

40 ± 9 

24 ± 5 

24 ± 7 

32 ± 4a

36 ± 2 

22 ± 4 

19 ± 2 

23 ± 4a

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 

282a

1,864 

934a

1,761a

9,567 

472 ± 90 

590 

123 

148 

18 

300 

1,859 

1,000 

1,899 

11,250 

438 ± 64a

538 

160 

130a

19 

293 

1,784 

967 

1,790a

11,118 

388 ± 71a

596 

141 

158 

17 

250a

1,743a

863a

1,626a

12,107a

198 ± 65a

505 

89a 

107a

10a

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 ± 5.2a

a

Significantly (p < 0.05) different from control mean. For body weight and organ weight means, SDs were only 

reported for thymus weights. 

Source: De Jong et al. (1999). 

1 

2 Statistically significant reductions were also observed in the relative cortex surface area 

3 of the thymus and thymic medullar weight at 90 mg/kg-day, but there was no difference in cell 

4 proliferation between treated and control animals using the proliferating cell nuclear antigen 

5 (PCNA) technique. Changes in the following immune parameters were noted: dose-related and 

6 statistically significant decrease in the relative number of B cells in the spleen at 10 (13%), 30 

7 (18%), and 90 mg/kg-day (41%); significant decreases in absolute number of cells harvested in 
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1 the spleen (31%), in the number of B cells in the spleen (61%), and NK cell activity in the spleen 

2 (E:T ratio was 40.9 ± 28.4% that of the controls) at 90 mg/kg-day; and a decrease in serum IgM 

3 (33%) and IgA (61%) in rats treated with 30 and 90 mg/kg-day, respectively. The decrease in 

4 the spleen cell count was attributed by the study authors to the decreased B cells and suggested a 

5 possible selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The study 

6 authors considered the decrease in IgA and IgM to be due to impaired production of antibodies, 

7 suggesting a role of thymus toxicity in the decreased (T-cell dependent) antibody production. In 

8 addition to the effects on the thymus and spleen, histopathologic examination revealed treatment9 related lesions only in the liver and forestomach at the two highest dose levels, but the incidence 

10 data for these lesions were not reported by De Jong et al. (1999). Increased incidence for 

11 forestomach basal cell hyperplasia (p < 0.05 by Fisher’s exact test) was reported at 30 and 

12 90 mg/kg-day, and increased incidence for oval cell hyperplasia in the liver was reported at 

13 90 mg/kg-day (p < 0.01, Fisher’s exact test). The results indicate that 3 mg/kg-day was a no14 observed-adverse-effect level (NOAEL) for effects on hematological parameters (decrease in 

15 RBC count, hemoglobin, and hematocrit) and immune parameters (decreased thymus weight and 

16 percent of B cells in the spleen) noted in Wistar rats at 10 mg/kg-day (the lowest-observed17 adverse-effect level [LOAEL]) and above. Lesions of the liver (oval cell hyperplasia) and 

18 forestomach (basal cell hyperplasia) occurred at doses ≥30 mg/kg-day. 

19 

20 Knuckles et al. (2001) 90-day rat study 

21 Knuckles et al. (2001) exposed male and female F344 rats (20/sex/dose group) to 

22 benzo[a]pyrene (98% purity) at doses of 0, 5, 50, or 100 mg/kg-day in the diet for 90 days. Food 

23 consumption and body weight were monitored, and the concentration of benzo[a]pyrene in the 

24 food was adjusted every 3–4 days to maintain the target dose. The authors indicated that actual 

25 intake of benzo[a]pyrene by the rats was within 10% of the calculated intake, and the nominal 

26 doses were not corrected to actual doses. Hematology and serum chemistry parameters were 

27 evaluated. Urinalysis was also performed. Animals were examined for gross pathology, and 

28 histopathology was performed on selected organs (stomach, liver, kidney, testes, and ovaries). 

29 Statistically significant decreases in RBC counts and hematocrit level (decreases as much as 10 

30 and 12%, respectively) were observed in males at doses ≥50 mg/kg-day and in females at 

31 100 mg/kg-day. A maximum 12% decrease (statistically significant) in hemoglobin level was 

32 noted in both sexes at 100 mg/kg-day. Blood chemistry analysis showed a significant increase in 

33 blood urea nitrogen (BUN) only in high-dose (100 mg/kg-day) males. Histopathology 

34 examination revealed an apparent increase in the incidence of abnormal tubular casts in the 

35 kidney in males at 5 mg/kg-day (40%), 50 mg/kg-day (80%) and 100 mg/kg-day (100%), 

36 compared to 10% in the controls. Only 10% of the females showed significant kidney tubular 

37 changes at the two high dose levels compared to zero animals in the female control group. The 

38 casts were described as molds of distal nephron lumen and were considered by the study authors 
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1 to be indicative of renal dysfunction. From this study, male F344 rats appeared to be affected 

2 more severely by benzo[a]pyrene treatment than the female rats. However the statistical 

3 significance of the kidney lesions are unclear. Several reporting gaps and inconsistencies 

4 regarding the reporting of kidney abnormalities in Knuckles et al (2001) make interpretation of 

5 the results difficult. Results of histopathological kidney abnormalities (characterized primarily 

6 as kidney casts) were presented graphically and the data were not presented numerically in this 

7 report. No indication was given in the graph that any groups are statistically different than 

8 controls, though visual examination of the magnitude of response and error bars appears to 

9 indicate a four fold increase in kidney casts in males compared to the control group (40 

10 compared to 10%). The figure legend reports the data as “percentage incidence of abnormal 

11 kidney tissues” and reports values are mean plus or minus standard deviation. However, text 

12 under the materials and methods section states that for histopathological data, Fisher’s Exact Test 

13 was used. This would involve the pairwise comparison of incidence and not means. There are 

14 additional internal inconsistencies in the data presented. Data appear to indicate that incidences 

15 for males are as follows: control: 10%, 5mg/kg-day: 40%, 50 mg/kg-day: 80% and 100 mg/kg16 day: 100%, however, these incidences are inconsistent with the size of the study groups which 

17 were reported as 6-8 animals per group. The study authors were contacted but did not respond to 

18 EPA’s request for clarification of study design and/or results. Due to issues of data reporting, a 

19 LOAEL could not be established for the increased incidence of kidney lesions. Based on the 

20 statistically significant hematological effects including decreases in RBC counts, hematocrit, and 

21 BUN, the NOAEL in males was 5 mg/kg-day and the LOAEL was 50 mg/kg-day, based on in 

22 F344 rats. No exposure-related histological lesions were identified in the stomach, liver, testes, 

23 or ovaries in this study. 

24 

25 Kroese et al. (2001) 5-week rat study 

26 In a range-finding study, Wistar (specific pathogen-free [SPF] Riv:TOX) rats 

27 (10/sex/dose group) were administered benzo[a]pyrene (97.7% purity) dissolved in soybean oil 

28 by gavage at dose levels of 0, 1.5, 5, 15, or 50 mg/kg body weight, 5 days/week for 5 weeks 

29 (Kroese et al., 2001). Behavior, clinical symptoms, body weight, and food and water 

30 consumption were monitored. None of the animals died during the treatment period. Animals 

31 were sacrificed 24 hours after the last dose. Urine and blood were collected for standard 

32 urinalysis and hematology and clinical chemistry evaluation. Liver enzyme induction was 

33 monitored based on EROD activity in plasma. Animals were subjected to macroscopic 

34 examination, and organ weights were recorded. The esophagus, stomach, duodenum, liver, 

35 kidneys, spleen, thymus, lung, and mammary gland (females only) from the highest-dose and 

36 control animals were evaluated for histopathology. Intermediate dose-groups were examined if 

37 abnormalities were observed in the higher-dose groups. 
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1 A significant, but not dose-dependent, increase in food consumption in males at 

2 ≥1.5 mg/kg-day and a decrease in females at ≥5 mg/kg-day was observed (Kroese et al., 2001). 

3 Water consumption was statistically significantly altered in males only, a decrease at 1.5, 5, and 

4 15 mg/kg-day and an increase at 50 mg/kg-day. Organ weights of lung, spleen, kidneys, 

5 adrenals, and ovaries were not affected by treatment. There was a dose-related, statistically 

6 significant decrease in thymus weight in males at 15 and 20 mg/kg-day (decreased by 28 and 

7 33%, respectively) and a significant decrease in females at 50 mg/kg-day (decreased by 17%) 

8 (Table 4-3). In both sexes, liver weight was statistically significantly increased only at 

9 50 mg/kg-day by about 18% (Table 4-3). 

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 ± 0.18a

5.03 ± 0.15a

Thymus weight (mg; mean ± SD) 

Males 

Females 

471 ± 19 

326 ± 12 

434 ± 20 

367 ± 23 

418 ± 26 

351 ± 25 

342 ± 20a

317 ± 30 

317 ± 21a

271 ± 16a

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 

3/10a

7/10 

7/10a

a

Significantly (p < 0.05) different from control mean; n = 10/sex/group. 

Source: Kroese et al. (2001). 

11 

12 Hematological evaluation revealed only statistically nonsignificant, small dose-related 

13 decreases in hemoglobin in both sexes, and RBC counts in males. Clinical chemistry analysis 

14 showed a small, but statistically significant, increase in creatinine levels in males only at 

15 1.5 mg/kg-day, but this effect was not dose-dependent. A dose-dependent induction of liver 

16 microsomal EROD activity was observed, with a five-fold induction at 1.5 mg/kg-day compared 

17 to controls, reaching 36-fold in males at 50 mg/kg-day; the fold induction in females at the top 

18 dose was less than in males. At necropsy, significant, dose-dependent macroscopic findings 

19 were not observed. 

20 Histopathology examination revealed a statistically significant increase in basal cell 

21 hyperplasia in the forestomach of females at doses ≥15 mg/kg-day (Kroese et al., 2001). The 

22 induction of liver microsomal EROD was not accompanied by any adverse histopathologic 

23 findings in the liver at the highest dose, 50 mg/kg-day, so the livers from intermediate-dose 
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1 groups were, therefore, not examined. An increased incidence of brown pigmentation of red 

2 pulp (hemosiderin) in the thymus was observed in treated animals of both sexes. However, this 

3 tissue was not examined in intermediate-dose groups. This range-finding 5-week study 

4 identified a NOAEL of 5 mg/kg-day and a LOAEL of 15 mg/kg-day, based on decreased thymus 

5 weight and forestomach hyperplasia in Wistar rats. 

6 

7 Kroese et al. (2001) 90-day rat study

8 Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (10/sex/dose group) to 

9 benzo[a]pyrene (98.6% purity, dissolved in soybean oil) by gavage at 0, 3, 10, or 30 mg/kg body 

10 weight, 5 days/week for 90 days. The rats were examined daily for behavior and clinical 

11 symptoms and by palpation. Food and water consumption, body weights, morbidity, and 

12 mortality were monitored. At the end of the exposure period, rats were subjected to macroscopic 

13 examination and organ weights were recorded. Blood was collected for hematology and serum 

14 chemistry evaluation and urine was collected for urinalysis. All gross abnormalities, particularly 

15 masses and lesions suspected of being tumors were also evaluated. The liver, stomach, 

16 esophagus, thymus, lung, spleen, and mesenteric lymph node were examined histopathologically. 

17 In addition, cell proliferation in forestomach epithelium was measured as the prevalence of S18 phase epithelial cells displaying bromodeoxyuridine (BrdU) incorporation. 

19 There were no obvious effects on behavior of the animals, and no difference was 

20 observed in survival or food consumption between exposed animals and controls (Kroese et al. 

21 2001). Higher water consumption and slightly lower body weights than the controls were 

22 observed in males but not females at the high dose of 30 mg/kg-day. Hematological 

23 investigations showed only nonsignificant, small dose-related decreases in RBC count and 

24 hemoglobin level in both sexes. Clinical chemistry evaluation did not show any treatment25 related group differences or dose-response relationships for alanine aminotransferase (ALT), 

26 serum aspartate transaminase (AST), lactate dehydrogenase (LDH), or creatinine, but a small 

27 dose-related decrease in γ-glutamyl transferase (GGT) activity was observed in males only. 

28 Urinalysis revealed an increase in urine volume in males at 30 mg/kg-day, which was not dose 

29 related. At the highest dose, both sexes showed increased levels of urinary creatinine and a dose30 related increase in urinary protein. However, no further investigation was conducted to 

31 determine the underlying mechanisms for these changes. At necropsy, reddish to brown/gray 

32 discoloration of the mandibular lymph nodes was consistently noted in most rats; occasional 

33 discoloration was also observed in other regional lymph nodes (axillary). Statistically significant 

34 increases in liver weight were observed at 10 and 30 mg/kg-day in males only (15 and 29% ) and 

35 a decrease in thymus weight in both sexes only at 30 mg/kg-day (17 and 33% decrease in 

36 females and males, respectively, compared with controls) (Table 4-4). At 10 mg/kg-day, thymus 

37 weight in males was decreased by 15%, but the decrease did not reach statistical significance. 

38 
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Table 4-4. Means ± SDa for liver and thymus weights in Wistar rats exposed 

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 ± 1.30b

5.76 ± 0.71 

9.67 ± 1.17b

6.48 ± 0.78b

Thymus weight (mg) 

Males 

Females 

380 ± 60 

320 ± 60 

380 ± 110 

310 ± 50 

330 ± 60 

300 ± 40 

270 ± 40b

230 ± 30b

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-test (unpaired, two-tailed); n = 10/sex/group. 

Source: Kroese et al. (2001). 

1 

2 Histopathologic examination revealed what was characterized by Kroese et al. (2001) as 

3 basal cell disturbance in the epithelium of the forestomach in males (p < 0.05) and females 

4 (p < 0.01) at 30 mg/kg-day. The basal cell disturbance was characterized by increased number of 

5 basal cells, mitotic figures, and remnants of necrotic cells; occasionally early nodule 

6 development; infiltration by inflammatory cells (mainly histiocytes); and capillary hyperemia, 

7 often in combination with the previous changes (Kroese et al., 2001). Incidences for these 

8 lesions (also described as “slight basal cell hyperplasia”) in the 0, 3, 10, and 30-mg/kg-day 

9 groups were 0/10, 2/10, 3/10, and 7/10 in female rats and 2/10, 0/10, 6/10, and 7/10 for male rats. 

10 Nodular hyperplasia was noted in one animal of either sex at 30 mg/kg-day. A significant 

11 (p < 0.05) increase in proliferation of forestomach epithelial cells was detected at doses 

12 ≥10 mg/kg-day by morphometric of analysis of nuclei with BrdU incorporation. The mean 

13 numbers of BrdU-staining nuclei per unit surface area of the underlying lamina muscularis 

14 mucosa were increased by about two- and three- to four-fold at 10 and 30 mg/kg-day, 

15 respectively, compared with controls. A reduction of thymus weight and increase in the 

16 incidence of thymus atrophy (the report described the atrophy as slight, but did not specify the 

17 full severity scale used in the pathology examination) was observed in males only at 30 mg/kg18 day (p < 0.01 compared with controls). Incidences for thymus atrophy for the control through 

19 high-dose groups were 0/10, 0/10, 0/10, and 3/10 for females and 0/10, 2/10, 1/10, and 6/10 for 

20 males. No significant differences were observed in the lungs of control and treated animals. In 

21 the esophagus, degeneration and regeneration of muscle fibers and focal inflammation of the 

22 muscular wall were judged to be a result of the gavage dosing rather than of benzo[a]pyrene 

23 treatment. 

24 The target organs of benzo[a]pyrene toxicity in this 90-day dietary study of Wistar rats 

25 were the forestomach, thymus, and liver. The LOAEL for forestomach hyperplasia, decreased 
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1 thymus weight, thymus atrophy, and increased liver weight was 30 mg/kg-day and the NOAEL 

2 was 10 mg/kg-day. 

3 

4 4.2.1.2. Chronic Studies and Cancer Bioassays

5 

6 Kroese et al. (2001) 2-year rat study 

7 Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (52/sex/dose group) to 

8 benzo[a]pyrene (98.6% purity) in soybean oil by gavage at nominal doses of 0, 3, 10, or 

9 30 mg/kg-day, 5 days/week, for 104 weeks. Mean achieved dose levels were 0, 2.9, 9.6, and 

10 29 mg/kg-day. Additional rats (6/sex/group) were sacrificed after 4 and 5 months of exposure 

11 for analysis of DNA adduct formation in blood and major organs and tissues. The rats were 

12 6 weeks old at the start of exposure. The rats were examined daily for behavior and clinical 

13 symptoms and by palpation. Food and water consumption, body weights, morbidity, and 

14 mortality were monitored during the study. Complete necropsy was performed on all animals 

15 that died during the course of the study, were found moribund, or at terminal sacrifice (organ 

16 weight measurement was not mentioned in the report by Kroese et al., 2001). The organs and 

17 tissues collected and prepared for microscopic examination included: brain, pituitary, heart, 

18 thyroid, salivary glands, lungs, stomach, oesophagus, duodenum, jejunum, ileum, caecum, colon, 

19 rectum, thymus, kidneys, urinary bladder, spleen, lymph nodes, liver pancreas, adrenals, sciatic 

20 nerve, nasal cavity, femur, skin including mammary tissue, ovaries/uterus, and testis/accessory 

21 sex glands. Some of these tissues were examined only when gross abnormalities were detected. 

22 All gross abnormalities, particularly masses and lesions that appeared to be tumors, were also 

23 examined. 

24 At 104 weeks, survival in the control group was 65% (males) and 50% (females), 

25 whereas mortality in the 30 mg/kg-day dose group was 100% after about week 70. At 80 weeks, 

26 survival percentages were about 90, 85 and 75% in female rats in the 0, 3, and 10 mg/kg-day 

27 groups, respectively; in males, respective survival percentages were about 95, 90, and 85% at 

28 80 weeks. Survival of 50% of animals occurred at 104, 104, about 90, and 60 weeks for control 

29 through high-dose females; for males, the respective times associated with 65% survival were 

30 104, 104, 104, and about 60 weeks. The high mortality rate in high-dose rats was attributed to 

31 liver or forestomach tumor development, not to noncancer systemic effects. After 20 weeks, 

32 body weight was decreased (compared with controls by >10%) in 30-mg/kg-day males, but not 

33 in females. This decrease was accompanied by a decrease in food consumption. Body weights 

34 and food consumption were not adversely affected in the other dose groups compared to 

35 controls. In males, there was a dose-dependent increase in water consumption starting at week 

36 13, but benzo[a]pyrene treatment had no significant effects on water consumption in females. 

37 Tumors were detected at significantly elevated incidences at several tissue sites in female 

38 and male rats at doses ≥10 and ≥3 mg/kg-day, respectively (Table 4-4; Kroese et al., 2001). The 
94 DRAFT - DO NOT CITE OR QUOTE 

1 tissue sites with the highest incidences of tumors were the liver (hepatocellular adenoma and 

2 carcinoma) and forestomach (squamous cell papilloma and carcinoma) in both sexes (Table 4-4). 

3 The first liver tumors were detected in week 35 in high-dose male rats. Liver tumors were 

4 described as complex, with a considerable proportion (59/150 tumors) metastasizing to the lungs. 

5 At the highest dose level, 95% of rats with liver tumors had malignant carcinomas (95/100; 

6 Table 4-4). Forestomach tumors were associated with the basal cell proliferation observed 

7 (without diffuse hyperplasia) in the forestomach of rats in the preliminary range-finding and 

8 90-day exposure studies described previously in Section 4.2.1. At the highest dose level, 59% of 

9 rats with forestomach tumors had malignant carcinomas (60/102; Table 4-4). Other tissue sites 

10 with distinctly elevated incidences of tumors in the 30 mg/kg-day dose group included: the oral 

11 cavity (papilloma and squamous cell carcinoma [SCC]) in both sexes, and the jejunum 

12 (adenocarcinoma), kidney (cortical adenoma) and skin (basal cell adenoma and carcinoma) in 

13 male rats (Table 4-4). In addition, auditory canal tumors (carcinoma or squamous cell papilloma 

14 originating from pilo-sebaceous units including the Zymbal’s gland) were also detected in both 

15 sexes at 30 mg/kg-day, but auditory canal tissue was not histologically examined in the lower 

16 dose groups and the controls (Table 4-4). Gross examination revealed auditory canal tumors 

17 only in the high-dose group. 

18 

19 
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Table 4-5. Incidences of exposure-related neoplasms in Wistar rats treated 

by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks 

Dose (mg/kg-d) 

0 3 10 30b

Site Femalesa

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 

9/31c

9/31c

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 

20/51c

10/51c

25/52c

25/52c

Liver 

 Hepatocellular adenoma 

 Hepatocellular carcinoma 

 Cholangiocarcinoma 

 Anaplastic carcinoma 

0/52 

0/52 

0/52 

0/52 

2/52 

0/52 

0/52 

0/52 

7/52c

32/52c

1/52 

1/52 

1/52 

50/52c

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 

13/20c

Malesa

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 

10/38c

11/38c

2/38 

2/38 

Forestomach 

 Squamous cell papilloma 

 SCC 

0/52 

0/52 

7/52c

1/52 

18/52c

25/52c

17/52c

35/52c

Jejunum 

 Adenocarcinoma 0/51 0/50 1/51 8/49c

Liver 

 Hepatocellular adenoma 

 Hepatocellular carcinoma 

 Cholangiocarcinoma 

0/52 

0/52 

0/52 

3/52 

1/52 

0/52 

15/52c

23/52c 

0/52 

4/52 

45/52c 

1/52 

Kidney 

 Cortical adenoma 

 Adenocarcinoma 

 Urothelial carcinoma 

0/52 

0/52 

0/52 

0/52 

0/52 

0/52 

7/52c

2/52 

0/52 

8/52c

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 

19/33c

1/33 
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Table 4-5. Incidences of exposure-related neoplasms in Wistar rats treated 

by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks 

Dose (mg/kg-d) 

0 3 10 30b

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 

10/51c

4/51 

5/51 

4/51 

8/51c

0/51 

1/52 

a

Incidences are for number of rats with tumors compared with number of tissues examined histologically. Auditory 

canal and oral cavity tissues were only examined histologically when abnormalities were observed upon 

macroscopic examination. 

bThis group had significantly decreased survival. 

c

Statistically significant difference (p ≤ 0.01), Fisher’s exact test; analysis of auditory canal tumor incidence was 

based on assumption of n = 52 and no tumors in the controls. 

Source: Kroese et al. (2001). 

1 

2 Kroese et al. (2001) did not systematically investigate nonneoplastic lesions detected in 

3 rats sacrificed during the 2-year study, because the focus was to identify and quantitate tumor 

4 occurrence. However, incidences were reported for nonneoplastic lesions in tissues or organs in 

5 which tumors were detected (i.e., oral cavity, oesophagus, forestomach, jejunum, liver, kidney, 

6 skin, mammary, and auditory canal). The reported nonneoplastic lesions associated with 

7 exposure were the forestomach basal cell hyperplasia and clear cell foci of cellular alteration in 

8 the liver. Incidences for forestomach basal cell hyperplasia in the control through high-dose 

9 groups were: 1/52, 8/51, 13/51, and 2/52 for females and 2/50, 8/52, 8/52, and 0/52 in males. 

10 Incidences for hepatic clear cell foci of cellular alteration were 22/52, 33/52, 4/52, and 2/52 for 

11 females and 8/52, 22/52, 1/52, and 1/52 for males. These results indicate that the lowest dose 

12 group, 3 mg/kg-day, was a LOAEL for increased incidence of forestomach hyperplasia and 

13 hepatic histological changes in male and female Wistar rats exposed by gavage to 

14 benzo[a]pyrene for up to 104 weeks. The lack of an increase in incidence of these nonneoplastic 

15 lesions in the forestomach and liver at the intermediate and high doses (compared with controls) 

16 may be associated with increased incidences of forestomach and liver tumors at these dose levels 

17 (see Table 4-4). 

18 As an adjunct study to the 2-year gavage study with Wistar rats, Kroese et al. (2001) 

19 sacrificed additional rats (6/sex/group) after 4 and 5 months of exposure (0, 1, 3, 10, or 

20 30 mg/kg-day) for analysis of DNA adduct formation in WBCs and major organs and tissues. 

21 Additional rats (6/sex/time period) were exposed to 0.1 mg/kg-day benzo[a]pyrene for 4 and 

5 months for analysis of DNA adduct formation. Using the [32 22 P]-postlabeling technique, five 
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1 benzo[a]pyrene-DNA adducts were identified in all of the examined tissues at 4 months (WBCs, 

2 liver, kidney, heart, lung, skin, forestomach, glandular stomach, brain). Only one of these 

3 adducts (adduct 2) was identified based on co-chromatography with a standard. This adduct, 

identified as dG-N24 -BPDE, was the predominant adduct in all organs of female rats exposed to 

5 10 mg/kg-day, except the liver and kidney, in which another adduct (unidentified adduct 4) was 

predominant. Levels of total adducts (number of benzo[a]pyrene-DNA adducts per 1010 6 

7 nucleotides) in examined tissues (from the single 10 mg/kg-day female rat) showed the following 

8 order: liver > heart > kidney > lung > skin > forestomach ≈ WBCs > brain. Mean values for 

female levels of total benzo[a]pyrene-DNA adducts (number per 1010 9 nucleotides) in four organs 

10 showed the same order, regardless of exposure group: liver > lung > forestomach ≈ WBCs; 

11 comparable data for males were not reported). Mean total benzo[a]pyrene-DNA adduct levels in 

livers increased in both sexes from about 100 adducts per 1010 12 nucleotides at 0.1 mg/kg-day to 

about 70,000 adducts per 1010 13 nucleotides at 30 mg/kg-day. In summary, these results suggest 

14 that total benzo[a]pyrene-DNA adduct levels in tissues at 4 months were not independently 

15 associated with the carcinogenic responses noted after 2 years of exposure to benzo[a]pyrene. 

16 The liver showed the highest total DNA adduct levels and a carcinogenic response, but total 

17 DNA adduct levels in heart, kidney, and lung (in which no carcinogenic responses were 

18 detected) were higher than levels in forestomach and skin (in which carcinogenic responses were 

19 detected). 

20 

21 Brune et al. (1981) 2-year rat study 

22 Groups of Sprague-Dawley rats (32/sex/dose) were fed diets delivering a daily dose of 

0.15 mg benzo[a]pyrene/kg body weight every 9th 23 day or 5 times/week (Brune et al., 1981). 

24 Other groups (32/ sex/dose) were given gavage doses of 0.15 mg benzo[a]pyrene (in aqueous 

1.5% caffeine solution)/kg every 9th day, every 3rd 25 day, or 5 times/week. The study included an 

26 untreated control group (to compare with the dietary exposed groups) and a gavage vehicle 

27 control group (each with 32 rats/sex). Rats were treated until moribundity or death occurred, 

28 with average annual doses are reported in Table 4-2 (mg/kg-year, calculated by Brune et al. 

29 [1981]). The following tissues were prepared for histopathological examination: tongue, larynx, 

30 lung, heart, trachea, esophagus, stomach, small intestine, colon, rectum, spleen, liver, urinary 

31 bladder, kidney, adrenal gland, and any tissues showing tumors or other gross changes. Survival 

32 was similar among the groups, with the exception that the highest gavage-exposure group 

33 showed a decreased median time of survival (Table 4-5). Increased incidences of portal-of-entry 

34 tumors (forestomach, esophagus, and larynx) were observed in all of the gavage-exposed groups 

35 and in the highest dietary exposure group (Table 4-5). Following dietary administration, all 

36 observed tumors were papillomas. Following gavage administration, two malignant forestomach 

37 tumors were found (one each in the mid- and high-dose groups) and the remaining tumors were 

38 benign. The data in Table 4-5 show that the carcinogenic response to benzo[a]pyrene was 
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1 stronger with the gavage protocol compared with dietary exposure, and that no distinct difference 

2 in response was apparent between the sexes. Tumors at distant sites (mammary gland, kidney, 

3 pancreas, lung, urinary bladder, testes, hematopoietic, and soft tissue) were not considered 

4 treatment-related as they were also observed at similar rates in the control group (data not 

5 provided). The study report did not address noncancer systemic effects. 

6 

Table 4-6. Incidences of alimentary tract tumors in Sprague-Dawley rats 

chronically exposed to benzo[a]pyrene in the diet or by gavage in caffeine 

solution 

Average annual 

dose (mg/kg-yr) 

Estimated average 

daily dosea

(mg/kg-d) Forestomach tumorsb

Total alimentary tract 

tumorsc

 (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 

aAverage annual dose divided by 365 d. 

bNo sex-specific forestomach tumor incidence data were 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 

8 Beland and Culp (1998; Culp et al., 1998) 2-year mouse study 

9 In the other modern cancer bioassay with benzo[a]pyrene, female B6C3F1 mice (48/dose 

10 group) were administered benzo[a]pyrene (98.5% purity) at concentrations of 0 (acetone 

11 vehicle), 5, 25, or 100 ppm in the diet for 2 years (Beland and Culp, 1998; Culp et al., 1998). 

12 This study was designed to compare the carcinogenicity of coal tar mixtures with that of 

13 benzo[a]pyrene and included groups of mice fed diets containing one of several concentrations 

14 of two coal tar mixtures. Benzo[a]pyrene was dissolved in acetone before mixing with the feed. 

15 Control mice received only acetone-treated feed. Female mice were chosen because they have a 

16 lower background incidence of lung tumors than male B6C3F1 mice. Culp et al. (1998) reported 
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1 that the average daily intakes of benzo[a]pyrene in the 25- and 100-ppm groups were 104 and 

2 430 μg/day, but did not report intakes for the 5-ppm group. Based on the assumption that daily 

3 benzo[a]pyrene intake at 5 ppm was one-fifth of the 25-ppm intake (about 21 μg/day), average 

4 daily doses for the three benzo[a]pyrene groups are estimated at 0.7, 3.3, and 16.5 mg/kg-day. 

5 Estimated doses were calculated using time-weighted average (TWA) body weights of 0.032 kg 

6 for the control, 5- and 25-ppm groups and 0.026 kg for the 100-ppm group (estimated from 

7 graphically presented data). Food consumption, body weights, morbidity, and mortality were 

8 monitored at intervals, and lung, kidneys, and liver were weighed at sacrifice. Necropsy was 

9 performed on all mice that died during the experiment or survived to the end of the study period. 

10 Limited histopathologic examinations (liver, lung, small intestine, stomach, tongue, esophagus) 

11 were performed on all control and high-dose mice and on all mice that died during the 

12 experimental period, regardless of treatment group. In addition, all gross lesions found in mice 

13 of the low- and mid- dose groups were examined histopathologically. 

14 None of the mice administered 100 ppm benzo[a]pyrene survived to the end of the study, 

15 and morbidity/mortality was 100% by week 78. Decreased survival was also observed at 25 ppm 

16 with only 27% survival at 104 weeks, compared with 56 and 60%, in the 5-ppm and control 

17 groups, respectively. In the mid- and high-dose group, 60% of mice were alive at about 90 and 

18 60 weeks, respectively. Early deaths in exposed mice were attributed to tumor formation rather 

19 than other causes of systemic toxicity. Food consumption was not statistically different in 

20 benzo[a]pyrene-exposed and control mice. Body weights of mice fed 100 ppm were similar to 

21 those of the other treated and control groups up to week 46, and after approximately 52 weeks, 

22 body weights were reduced in 100-ppm mice compared with controls. Body weights for the 5- 

23 and 25-ppm groups were similar to controls throughout the treatment period. Compared with the 

24 control group, no differences in liver, kidney, or lung weights were evident in any of the treated 

25 groups (other organ weights were not measured). 

26 Papillomas and/or carcinomas of the forestomach, esophagus, tongue, and larynx at 

27 elevated incidences occurred in groups of mice exposed to 25 or 100 ppm, but no exposure28 related tumors occurred in the liver or lung (Table 4-6; Beland and Culp [1998]; Culp et al. 

29 [1998]). The forestomach was the most sensitive tissue, and demonstrated the highest tumor 

30 incidence among the examined tissues and was the only tissue with an elevated incidence of 

31 tumors at 25 ppm (Table 4-6). In addition, most of the forestomach tumors in the exposed 

32 groups were carcinomas, as 1, 31, and 45 mice had forestomach carcinomas in the 5-, 25-, and 

33 100-ppm groups respectively. Nonneoplastic lesions were also found in the forestomach at 

34 significantly (p < 0.05) elevated incidences: hyperplasia at ≥5 ppm and hyperkeratosis at ≥25 

35 ppm (Table 4-6). The esophagus was the only other examined tissue showing elevated incidence 

36 of a nonneoplastic lesion (basal cell hyperplasia, see Table 4-6). Tumors (papillomas and 

37 carcinonas) were also significantly elevated in the esophagus and tongue at 100 ppm (Table 4-6). 

38 Esophogeal carcinomas were detected in 1 mouse at 25 ppm and in 11 mice at 100 ppm. Tongue 
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1 carcinomas were detected in seven 100-ppm mice; the remaining tongue tumors were 

2 papillomas. Although incidences of tumors of the larynx were not significantly elevated in any 

3 of the exposed groups, a significant dose-related trend was apparent (Table 4-6). 

4 

Table 4-7. Incidence of nonneoplastic and neoplastic lesions in female 

B6C3F1 mice fed benzo[a]pyrene in the diet for up to 2 years 

Tissue and lesion 

Incidence (%) 

benzo[a]pyrene concentration (ppm) in dieta

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)b1/48 

(2) 

3/47 

(6) 

36/46b

(78) 

46/47b

(98) 

Forestomach (hyperplasia)b13/48 

(27) 

23/47a

(49) 

33/46b

(72) 

37/47b

(79) 

Forestomach (hyperkeratosis)b 13/48 

(27) 

22/47 

(47) 

33/46b

(72) 

38/47b

(81) 

Esophagus (papilloma and/or carcinoma)b 0/48 

(0) 

0/48 

(0) 

2/45 

(0) 

27/46b

(59) 

Esophagus (basal cell hyperplasia)b 1/48 

(2) 

0/48 

(0) 

5/45 

(11) 

30/46b

(65) 

Tongue (papilloma and/or carcinoma)b 0/49 

(0) 

0/48 

(0) 

2/46 

(4) 

23/48b

(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 incidences 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 

6 Neal and Rigdon (1967; Rigdon and Neal 1969, 1966) mouse study 

7 Neal and Rigdon (1967) fed BAP (purity not reported) at concentrations of 0, 1, 10, 20, 

8 30, 40, 45, 50, 100 and 250 ppm in the diets of male and female CFW-Swiss mice. 

Corresponding doses (in mg/kg-day) were calculated19 as 0, 0.2, 1.8, 3.6, 5.3, 7.1, 8, 8.9, 17.8, 

10 44.4 mg/kg-day. The age of the mice ranged from 17-180 days old and the treatment time from 

11 1-197 days; the size of the treated groups ranged from 9 to 73. There were 289 mice (number of 

 

1

 Calculation: mg/kg-day = (ppm in feed x kg food/day)/kg body weight. Reference food consumption rates of 

0.0062 kg/day (males) and 0.0056 kg/day (females) and 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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1 mice/sex not stated) in the control group. No forestomach tumors were reported in the 0-, 0.2- 

2 and 1.8 mg/kg-day dose groups. The incidence of forestomach tumors in the 20-, 30-, 40-, 45-, 

3 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, 

4 1/40, 4/40, 23/34, 19/23 and 66/73, respectively. 

5 

6 Other oral exposure cancer bioassays in mice 

7 Numerous other oral exposure cancer bioassays in mice have limitations which restrict 

8 their usefulness for characterizing dose-response relationships between chronic-duration oral 

9 exposure to benzo[a]pyrene and noncancer effects or cancer, but collectively, they provide strong 

10 evidence that oral exposure to benzo[a]pyrene can cause portal-of-entry site tumors (see Table 4-

11 8 for references). 

12 
102 DRAFT - DO 

1 

Table 4-8. Tumor incidence in oral exposure rodent cancer bioassays with limitations for describing d

relationships for lifetime exposure to benzo[a]pyrene 

Species/strain Exposure Results Comments 

Mouse/HaICR Groups of nine mice (9 wks old) were fed 

benzo[a]pyrene in the diet (0, 0.2, or 0.3 mg/g diet) for 12 wks and sacrificed. Estimated doses were 0, 27.3, or 41 mg/kg-d. 

Incidence with forestomach tumors: Control 0/9 Low 6/9 High 9/9 

Less than lifetime exposure durationglandular stomach, lung, and livers control and exposed mice showed ntumors. 

Mouse/HaICR Groups of 12–20 mice (10 wks old) were 

fed 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, or 192 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 durationstomachs were examined for tumorstumors found only in forestomach. 

Mouse/CD-1 20 female mice (9 wks old) were given 

1 mg benzo[a]pyrene by gavage 2 times/wk for 4 wks and observed for 19 wks. Estimated dose is 33 mg/kg-d, using an average body weight of 0.030 kg 

from reported data. 

Incidence with forestomach tumors: Exposed 17/20 (85%) Controls 0/24 

Less than lifetime exposure durationstomach were examined for tumors; found only in forestomach. 

Mouse/BALB 25 mice (8 wks old) were given 0.5 mg 

benzo[a]pyrene 2 times/wk for 15 wks. 

5/25 mice had squamous carcinomas of the forestomach; tumors were detected 28–65 wks after treatment. 

Less than lifetime exposure durationfollowing details were not reported: inclusion of controls, methods for dtumors, and body weight data. 

Mouse/C3H 19 mice (about 3 mo old) were given 

0.3 mL of 0.5% benzo[a]pyrene in polyethylene glycol-400 by gavage, once/d for 3 d. 

By 30 wks, 7/10 mice had papillomas; no carcinomas were evident 

Less than lifetime exposure duration

Mouse/albino Groups of 17–18 mice were given single

doses of benzo[a]pyrene and allowed to survive until terminal sacrifice at 569 d. 

Incidence of mice (that survived at least to 60 d) with forestomach papillomas: Dose (

μg) Incidence 

 Experiment 1 Experiment 2 Control 0/17 0/18 12.5 3/17 2/18 50 0/17 1/17200 8/17 ------

a

Less than lifetime exposure durationtract examined for tumors with hand body weight data not reported. 
103 DRAFT - DO 

Table 4-8. Tumor incidence in oral exposure rodent cancer bioassays with limitations for describing d

relationships for lifetime exposure to benzo[a]pyrene 

Species/strain Exposure Results Comments 

Mouse/HaICR 20 mice (9 wks old) were given 

benzo[a]pyrene in the diet (0.3 mg benzo[a]pyrene/g diet) for 6 wks and sacrificed after 20 wks in the study. 

8/20 exposed mice had forestomach tumors. 

Less than lifetime exposure durationstomachs were examined for tumorstumors found only in forestomach; nnonexposed controls were mentione

Mouse/A/HeJ 12 female mice (9 wks old) were given 

standard diet for 25 d, and 3 mg benzo[a]pyrene by gastric intubation on d 

7 and 21 of the study. Mice were killed at 31 wks of age and examined for lung tumors. 

12/12 exposed mice had lung tumors Less than lifetime exposure duration

lungs examined for tumors; no nonecontrols were mentioned. 

Mouse/white Groups of 16–30 mice were given 

benzo[a]pyrene in triethylene glycol (0.001–10 mg) wkly for 10 wks and observed until 19 mo. 

Tumors in stomach antrum Dose (mg) – Carcinoma - Papilloma 0.001 0/16 0/16 0.01– 0/26 2/26 0.1 0/24 5/24 1.0 11/30 12/30 10 16/27 7/27 

Less than lifetime exposure duration

Mouse/albino Groups of about 160 female mice (70 d 

of age; strain unknown) were given 0 or 8 mg benzo[a]pyrene mixed in the diet over a period of 14 mo. 

Gastric tumors were observed at the following incidence: Control 0/158 8 mg benzo[a]pyrene total 13/160 

Close to lifetime exposure duration; dose levels and methods of detectintumors were not clearly reported. 

Mouse/A/J Groups 40 female mice (8 wks old) were 

given 0 or 0.25 mg benzo[a]pyrene (in 2% emulphor) by gavage 3 times/wk for 8 wks. Mice were killed at 9 mo of age and examined for lung or forestomach tumors. 

Incidence for mice surviving at 9 mo of age: Lung tumors  Control 11/38  Exposed 22/36 Forestomach tumors  Control 0/38  Exposed 33/36 

Less than lifetime duration of exposonly lungs and GI tract were examintumors. 

Mouse/Swiss albino 

Groups of mice (9–14 wks old) were given single doses of 0 or 0.05 mg benzo[a]pyrene in polyethylene glycol400 by gavage. Surviving mice were killed at 18 mo of age and examined for macroscopic tumors. 

Forestomach tumor incidence: Dose (

μg) – Carcinoma – Papilloma 

0 0/65 2/65 50 1/61 20/61 

Less than lifetime duration of exposexposure-related tumors only found forestomach. 
104 DRAFT - DO 

Table 4-8. Tumor incidence in oral exposure rodent cancer bioassays with limitations for describing d

relationships for lifetime exposure to benzo[a]pyrene 

Species/strain Exposure Results Comments 

Mouse/ICR Groups of 20 or 24 mice (71 d old) were 

given 1.5 mg benzo[a]pyrene by gavage 2 times/wk for 4 wks; terminal sacrifice was at 211 d of age. Estimated dose was about 50 mg benzo[a]pyrene/kg, using an average body weight of 0.03 kg during exposure from reported data. 

Incidence of mice with forestomach neoplasms. Experiment 1 23/24 Experiment 2 19/20 

Less than lifetime duration of exposonly stomachs were examined for tutumors found only in forestomach; nonexposed controls were not menti

Mouse/CFW Groups of mice (mixed sex) were fed 

benzo[a]pyrene in the diet (dissolved in benzene and mixed with diet) at 0, 1, 10, 20, 30, 40, 45, 50, 100, or 250 ppm in the diet. 

ppm Exposure Forestomach tumor  (d) incidence 1 110 0/25 10 110 0/24 20 110 1/23 30 110 0/37 40 110 1/40 45 110 4/40 50 152 24/34 100 110 19/23 250 118 66/73 

Less than lifetime exposure durationvehicle control group; animals rangethree wks to 6 mo old at the start of only alimentary tract was examined tumors; (see also Rigdon and Neal, 1967, 1966). 

Rat/ SpragueDawley Groups of Sprague-Dawley rats (32/sex/dose) were fed diets delivering a 

daily dose of 0.15 mg benzo[a]pyrene/kg body weight every 9th day or 

5 times/week (Brune et al., 1981). Other groups (32/ sex/dose) were given gavage doses of 0.15 mg benzo[a]pyrene (in aqueous 1.5% caffeine solution)/kg every 9th day, every 3rd day, or 5 times/week. 

Dose larynx, esopogus, and forestomach (gavage) tumors 0 6/64 0.016 13/64 0.049 26/64 0.107 14/64 (diet) 0 3/64 0.016 3/64 0.107 10/64 

Doses are annual averages. Non-statreatment protocol involved animals treated for 5 days a week or fewer; relatively high control incidence comto other gavage studies; 

Mouse/A/J Groups of female mice were fed 

benzo[a]pyrene in the diet at 0, 16, or 98 ppm for 260 d. Average intakes of benzo[a]pyrene were 0, 40.6, and 256.6 

μg/mouse/d. Estimated doses are 

0, 1.6, and 9.9 mg/kg-d using a chronic reference body weight value of 0.026 kg (U.S. EPA, 1988). 

Incidence of mice surviving to 260 d: Lung tumors  Control 4/21  16 ppm 9/25  98 ppm 14/27 Forestomach tumors  Control 0/21  16 ppm 5/25  98 ppm 27/27 

Close to lifetime exposure duration; strain of mice particularly sensitive chemically induced cancer; only lunstomachs were examined for tumors
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a 1 ---- = not evaluated
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1 4.2.2. Inhalation 

2 4.2.2.1. Short-term and Subchronic Studies

3 Wolff et al. (1989) exposed groups of 40 male and 40 female F344/Crl rats, via nose 

only, to 7.5 mg benzo[a]pyrene/m34 for 2 hours/day, 5 days/week for 4 weeks (corresponding to a 

TWA of 0.45 mg/m35 ). Rats were 10–11 weeks old at the beginning of the experiment. 

6 Benzo[a]pyrene (>98% pure) aerosols were formed by heating and then condensing the 

7 vaporized benzo[a]pyrene. The particle MMAD was 0.21 μm. Subgroups of these animals 

8 (six/sex/dose) were exposed for 4 days or 6 months after the end of the 4-week exposure, 

9 respectively, to radiolabeled aluminosilicate particles. Lung injury was assessed by analyzing 

10 clearance of radiolabeled aluminosilicate particles and via histopathologic evaluations. Body 

11 and lung weights, measured in subgroups from 1 day to 12 months after the exposure did not 

12 differ between controls and treated animals. Radiolabeled particle clearance did not differ 

13 between the control and treated groups, and there were no significant lung lesions. This study 

identified a NOAEL for lung effects of 0.45 mg/m314 -day for a short-term exposure. 

15 

16 4.2.2.2. Chronic Studies and Cancer Bioassays

17 Thyssen et al. (1981) conducted an inhalation study in which male Syrian golden 

18 hamsters were exposed to benzo[a]pyrene for their natural lifetime. Groups of 20–30 male 

19 Syrian golden hamsters (8 weeks old) were exposed by nose-only inhalation to NaCl aerosols 

(controls; 240 μg NaCl/m320 ) or benzo[a]pyrene condensed onto NaCl aerosols at three nominal 

concentrations of 2, 10, or 50 mg benzo[a]pyrene/m321 for 3–4.5 hours/day, 5 days/week for 1–

22 41 weeks, followed by 3 hours/day, 7 days/week for the remainder of study (until hamsters died 

23 or became moribund). Thyssen et al. (1981) reported average measured benzo[a]pyrene 

concentrations to be 0, 2.2, 9.5, or 46.5 mg/m324 . More than 99% of the particles were between 0.2 

25 and 0.5 μm in diameter, and over 80% had diameters between 0.2 and 0.3 μm. The particle 

26 analysis of the aerosols was not reported to modern standards (MMAD and geometric SD were 

27 not reported). Each group initially consisted of 24 hamsters; final group sizes were larger as 

28 animals dying during the first 12 months of the study were replaced. 

29 Survival was similar in the control, low-dose, and mid-dose groups, but was significantly 

30 decreased in the high-dose group. Average survival times in the control, low-, mid-, and high31 dose groups were 96.4 ± 27.6, 95.2 ± 29.1, 96.4 ± 27.8, and 59.5 ± 15.2 weeks, respectively. 

After the 60th 32 week, body weights decreased and mortality increased steeply in the highest dose 

33 group. Histologic examination of organs (a complete list of organs examined histologically was 

34 not reported by Thyssen et al. [1981]) revealed a dose-related increase in tumors in the upper 

35 respiratory tract, including the nasal cavity, pharynx, larynx, and trachea and in the digestive 

36 tract in the mid- and high-dose groups (Table 4-8). A statistical analysis was not included in the 

37 Thyssen et al. (1981) report. No lung tumors were observed. Squamous cell tumors in the 

38 esophagus and forestomach were also observed in the high-dose group, presumably as a 
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1 consequence of mucociliary particle clearance. Tumors were detected in other sites, but none of 

2 these appeared to be related to exposure. The results indicated that the pharynx and larynx, 

3 including the epiglottis, were the main cancer targets (Table 4-8). 

4 

Table 4-9. Incidence of respiratory and upper digestive tract tumors in male 

hamsters treated for life with benzo[a]pyrene by inhalation 

Tumor site 

Reported benzo[a]pyrene concentration (mg/m3

) 

0

a

 2b 10 50 

Tumor incidence (latency in wksc)

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) 

aEffective number of animals in control group: n = 27. 

bEffective number of animals in 2 mg/m3

 dose group: n = 27. 

cMean ± SD. 

Source: Thyssen et al. (1981). 

5 

6 Under contract to the U.S. EPA, Clement Associates (1990) obtained the individual 

7 animal data (including individual animal pathology reports, time-to-death data, and exposure 

8 chamber monitoring data) collected by Thyssen et al. (1981). Re-analysis of the original data 

9 revealed several errors and omissions in the published report. The actual exposure protocol was 

10 as follows: 4.5 hours/day 5 days/week on weeks 1–12, 3 hours/day 5 days/week on weeks 13–

11 29, 3.7 hours/day 5 days/week on week 30, 3 hours/day 5 days/week on weeks 31–41, and 

12 3 hours/day 7 days/week for the reminder of the experiment. In addition, actual exposure 

13 concentrations varied widely from week to week. Because different animals were started at 

14 different times, each individual animal had an exposure history somewhat different than others in 

15 the same exposure group. In order to deal with this problem, Clement Associates (1990) used 

16 the orignial individual animal data to calculate average continuous lifetime exposures for each 

17 individual hamster. Group averages of individual average continuous lifetime exposure 

concentrations were 0, 0.25, 1.01, and 4.29 mg/m318 for the control through high-exposure groups. 

19 For this assessment, the individual animal pathology reports prepared by Thyssen et al. 

20 (1981) and obtained by Clement Associates (1990) were examined to independently assess the 

21 numbers of hamsters with tumors in the larynx, pharynx, and nose in each group. Table 4-9 

22 presents the number of animals with tumors in the larynx and pharynx and the numbers of 

23 animals in each exposure group. Numbers of animals with either laryngeal or pharyngeal tumors 
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1 are also noted in Table 4-10, since these two types of tumors arise in close anatomical proximity 

2 from similar cell types. Examination of the individual animal pathology reports also showed that 

3 all of the nasal, forestomach, esophageal, and tracheal tumors occurred in animals that also had 

4 either laryngeal or pharyngeal tumors, except for two animals in the mid-dose group that 

5 displayed nasal tumors (one malignant and one benign) without displaying tumors in the pharynx 

6 or larynx. 

7 

Table 4-10. Number of animals with pharynx and larynx tumors in male 

hamsters exposed by inhalation to benzo[a]pyrene for life 

Average 

continuous 

benzo[a]pyrene 

concentrationa 

(mg/m3) 

Number of 

hamsters in 

groupb

Larynxb Pharynxb

Larynx or pharynx, 

combinedc

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 

aAs calculated by Clement Associates (1990) from air monitoring data collected by Thyssen and colleagues. 

bAs counted from information in Table D-1 in Appendix D, which was obtained from examination of individual 

animal pathology reports prepared by Thyssen and colleagues and obtained by Clement Associates. 

cAs counted from information in Table D-1 in Appendix D. Nasal, forestomach, esophageal, and tracheal tumors 

occurred in hamsters that also had tumors in the larynx 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 

9 Several studies have investigated the carcinogenicity of benzo[a]pyrene in hamsters 

10 exposed by intratracheal instillation. Single-dose studies verified that benzo[a]pyrene is 

11 tumorigenic, but do not provide data useful characterizing dose-response relationships because of 

12 their design (Kobayashi, 1975; Reznik-Schuller and Mohr, 1974; Henry et al., 1973; Mohr, 1971; 

13 Saffiotti et al., 1968; Gross et al., 1965; Herrold and Dunham, 1962). One multiple-dose study, 

14 which utilized very low doses (0.005, 0.02, and 0.04 mg, once every 2 weeks), failed to find any 

15 tumorigenic response (Kunstler, 1983). Tumorigenic responses (mostly in the respiratory tract) 

16 were found at higher dosage levels (0.25–2 mg benzo[a]pyrene once per week for 30–52 weeks) 

17 in four multiple-dose studies (Feron and Kruysse, 1978; Ketkar et al., 1978; Feron et al., 1973; 

18 Saffiotti et al., 1972). These studies identify the respiratory tract as a cancer target with exposure 

19 to benzo[a]pyrene by intratracheal instillation and provide supporting evidence for the 

20 carcinogenicity of benzo[a]pyrene at portal-of-entry sites. 

21 

22 4.2.3. Dermal Exposure 

23 4.2.3.1. Skin-Tumor Initiation-Promotion Assays
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1 Results from numerous studies indicate that acute dermal exposure to benzo[a]pyrene 

2 induces skin tumors in mice when followed by repeated exposure to a potent tumor promoter 

3 (Weyand et al., 1992; Cavalieri et al., 1991, 1981; Rice et al., 1985; El-Bayoumy et al., 1982; 

4 LaVoie et al., 1982; Raveh et al., 1982; Slaga et al., 1980, 1978; Wood et al., 1980; Hoffmann et 

5 al., 1972). The typical exposure protocol in these studies involved the application of a single 

6 dose of benzo[a]pyrene (typically ≥20 nmol per mouse) to dorsal skin of mice followed by 

7 repeated exposure to a potent tumor promoter, such as 12-O-tetradecanoylphorbol-13-acetate 

8 (TPA). 

9 

10 4.2.3.2. Carcinogenicity Dermal Bioassays

11 

12 Poel (1959) 

13 Poel (1959) applied benzo[a]pyrene in toluene to shaved interscapular skin of groups of 

14 13–56 male C57L mice at doses of 0, 0.15, 0.38, 0.75, 3.8, 19, 94, 188, 376, or 752 μg, 

15 3 times/week for up to 103 weeks or until the appearance of a tumor by gross examination 

16 (3 times weekly). Some organs (not further specified) and interscapular skin in sacrificed mice 

17 were examined histologically. With increasing dose level, the incidence of mice with skin 

18 tumors increased and the time of tumor appearance decreased (see Table 4-10). Doses >3.8 μg 

19 were associated with 100% mortality after increasingly shorter exposure periods, none greater 

20 than 44 weeks. Poel (1959) did not mention the appearance of exposure-related tumors in tissues 

21 other than interscapular skin. 

22 

Table 4-11. Skin tumor incidence and time of appearance in male C57L 

mice dermally exposed to benzo[a]pyrene for up to 103 weeks 

Dose (μg)a

Incidence of mice with 

gross skin tumors 

Time of first tumor 

appearance (wks) 

Incidence of mice 

with epidermoid 

carcinomab

Length of exposure 

period(wks) 

0 (Toluene) 0/33 (0%) − 0/33 (0%) 92 

0.15 5/55 (9%) 42–44c0/55 (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%) 25-44c

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 appearance of a 

grossly detected skin tumor. 

bCarcinomas were histologically confirmed. 
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cRanges reflect differing information in Tables 4 and 6 of Poel (1959). 

Source: Poel (1959). 

1 

2 Poel (1960) applied benzo[a]pyrene in a toluene vehicle to shaved interscapular skin of 

3 groups of 14–25 male SWR, C3HeB, or A/He mice 3 times/week at doses of 0, 0.15, 0.38, 0.75, 

4 3.8, 19.0, 94.0, or 470 μg benzo[a]pyrene per application, until mice died or a skin tumor was 

5 observed. Time ranges for tumor observations were provided, but not times of death for mice 

6 without tumors, so it was not possible to evaluate differential mortality among all dose groups or 

7 the length of exposure for mice without tumors. With increasing dose level, the incidence of 

8 mice with skin tumors increased and the time of tumor appearance decreased (Table 4-11). The 

9 lowest dose level did not induce an increased incidence of mice with skin tumors in any strain, 

10 but strain differences in susceptibility were evident at higher dose levels. SWR and C3HeB mice 

11 showed skin tumors at doses ≥0.38 μg benzo[a]pyrene, whereas AH/e mice showed tumors at 

12 doses ≥19 μg benzo[a]pyrene (Table 4-11). Except for metastases of the skin tumors to lymph 

13 nodes and lung, Poel (1960) did not mention the appearance of exposure-related tumors in 

14 tissues other than interscapular skin. 

15 

Table 4-12. Skin tumor incidence and time of appearance in male SWR, 

C3HeB, and A/He mice dermally exposed to benzo[a]pyrene for life or until 

a skin tumor was detected 

Dose (μg)a

SWR mice C3HeB mice A/He mice 

Tumor 

incidenceb

Time of first 

tumor 

appearance 

(weeks) 

Tumor 

incidenceb

Time of first 

tumor 

appearance 

(weeks) 

Tumor 

incidenceb

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 (91%) 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 (100%) 4–21 

a

Indicated doses were applied 3times/week for life or 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 skin tumor. 

Source: Poel (1960). 

16 

17 
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1 Roe et al. (1970) treated groups of 50 female Swiss mice with 0 (acetone vehicle), 0.1, 

2 0.3, 1, 3, or 9 μg benzo[a]pyrene applied to the shaved dorsal skin 3 times/week for up to 

3 93 weeks; all surviving mice were killed and examined for tumors during the following 3 weeks. 

4 The dorsal skin of an additional control group was shaved periodically but was not treated with 

5 the vehicle. Mice were examined every 2 weeks for the development of skin tumors at the site of 

6 application. Histologic examinations included: (1) all skin tumors thought to be possibly 

7 malignant; (2) lesions of other tissues thought to be neoplastic; and (3) limited nonneoplastic 

8 lesions in other tissues. As shown in Table 4-13, markedly elevated incidences of mice with skin 

9 tumors were only found in the two highest dose groups (3 or 9 μg ), compared with no skin 

10 tumors in the control groups. Malignant skin tumors (defined as tumors with invasion or 

11 penetration of the panniculus carnosus muscle) were detected in 4/41 and 31/40 mice in the 

12 3- and 9-μg groups, respectively, surviving to at least 300 days. Malignant lymphomas were 

13 detected in all groups, but the numbers of cases were not elevated compared with expected 

14 numbers after adjustment for survival differences. Lung tumors were likewise detected in 

15 control and exposed groups at incidences that were not statistically different. 

16 

Table 4-13. Tumor incidence in female Swiss mice dermally exposed to 

benzo[a]pyrene for up to 93 weeks 

Dose (μg)a

Cumulative number of mice with skin 

tumor/survivors Skin tumor 

incidenceb

Malignant 

Lymphoma 

incidencec 

Lung 

Tumor 

incidencec 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%) 12/47 (26%) 10/46 (22%) 

0.1 0/45 1/42 1/35 1/31 1/22 1/0 1/42 (2%) 11/43 (26%) 10/40 (25%) 

0.3 0/46 0/42 0/37 0/30 0/19 0/0 0/42 (0%) 10/43 (23%) 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%) 

aDoses were applied 3 times/wk for up to 93 wks to shaved dorsal skin. 

bNumerator: number of mice detected with a skin tumor. Denominator: number of mice surviving to 300 d for all 

groups except the highest dose group. For the highest dose group (in which skin tumors were first detected 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 300 d 

unless a tumor was detected earlier, in which case the number dying before 300 d without a tumor was subtracted 

from the number of animals reported to have been examined. 

Source: Roe et al. (1970). 

17 

18 Schmidt et al. (1973) dermally administered benzo[a]pyrene in acetone to female NMRI 

19 mice (100/group) and female Swiss mice. Benzo[a]pyrene was applied to the shaved dorsal skin 

20 twice weekly with doses of 0, 0.05, 0.2, 0.8, or 2 μg until spontaneous death occurred or until an 

21 advanced carcinoma was observed. Skin carcinomas were identified by the presence of crater-
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1 shaped ulcerations, infiltrative growth and the beginning of physical wasting (i.e., cachexia). 

2 Necropsy was performed for all animals, and histopathological examination of the dermal site of 

3 application and any other tissues with gross abnormalities was conducted. Skin tumors were 

4 observed at the two highest doses in both strains of female mice (see Table 4-14), with induction 

5 periods of 53.0 and 75.8 weeks for the 0.8 and 2.0 μg NMRI mice and 57.8 and 60.7 weeks for 

6 the Swiss mice, respectively. The authors indicated that the latency period for tumor formation 

7 was highly variable and significant differences among exposure groups could not be identified, 

8 but no further timing information was available, including overall survival. Carcinoma was the 

9 primary tumor type seen after lifetime application of benzo[a]pyrene to mouse skin. 

10 

Table 4-14. Skin tumor incidence in female NMRI and Swiss mice dermally 

exposed to benzo[a]pyrene 

Dose (μg)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%) 

aMice 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 skin of the back. 

Source: Schmidt et al. (1973). 

11 

12 Schmähl et al. (1977) applied benzo[a]pyrene 2 times/week to the shaved dorsal skin of 

13 female NMRI mice (100/group) at doses of 0, 1, 1.7, or 3 μg in 20 μL acetone. The authors 

14 reported that animals were observed until natural death or until they developed a carcinoma at 

15 the site of application. The effective numbers of animals at risk was about 80% of the nominal 

16 group sizes, which the authors attributed to autolyis; no information was provided concerning 

17 when tumors appeared in the relevant groups, how long treatment lasted in each group, or any 

18 times of death. Necropsy was performed on all mice and the skin of the back, as well as any 

19 organs that exhibited macroscopic changes, were examined histopathologically. The incidence 

20 of all types of skin tumors was increased in a dose related manner compared to controls (see 

21 Table 4-15). Carcinoma was the primary tumor type observed following chronic dermal 
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1 exposure to benzo[a]pyrene, and skin papillomas occurred infrequently. Dermal sarcoma was not 

2 observed. 

3 
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Table 4-15. Skin tumor incidence in female NMRI mice dermally exposed to 

benzo[a]pyrene 

Dose (μg)a,b

Skin tumor incidence 

(all types) Incidence of papilloma Incidence of carcinoma 

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%) 

aMice 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 skin of the back. 

Source: Schmähl et al. (1977). 

1 

2 Habs et al. (1980) applied benzo[a]pyrene to the shaved interscapular skin of female 

3 NMRI mice (40/group) at doses of 0, 1.7, 2.8 or 4.6 μg in 20 μL acetone twice weekly, from 

4 10 weeks of age until natural death or gross observation of infiltrative tumor growth. Latency of 

5 tumors, either as time of first appearance or average time of appearance of tumors, was not 

6 reported. Necropsy was performed on all animals, and the dorsal skin, as well as any organs 

7 showing gross alterations at autopsy, was prepared for histopathological examination. Age8 standardized mortality rates, using the total population of the experiment as the standard 

9 population, were used to adjust tumor incidence findings in the study. Benzo[a]pyrene 

10 application was associated with a statistically significant increase in the incidence of skin tumors 

11 at each dose level (see Table 4-17). 

12 

Table 4-16. Skin tumor incidence in female NMRI mice dermally exposed to 

benzo[a]pyrene 

Dose (μg)a,b Skin tumor incidence Age-standardized tumor incidencec

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% 

aMice 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 skin of the back. 

cMortality data of the total study population were used to derive the age-standardized tumor incidence.

Source: Habs et al. (1980). 

13 

14 Grimmer et al. (1983) and Grimmer et al. (1984) applied benzo[a]pyrene (in 0.1 mL of a 

15 1:3 solution of acetone:dimethyl sulfoxide [DMSO]) to the interscapular skin of female CFLP 

16 mice (65–80/group) 2 times/week for 104 weeks. Doses were 0, 3.9, 7.7 and 15.4 μg in the 1983 
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1 experiment, and 0, 3.4, 6.7, and 13.5 μg in the 1984 experiment. Mice were observed until

2 spontaneous death, unless an advanced tumor was observed or if animals were found moribund. 

3 Survival information was not provided; incidences reflect the number of animals placed on 

4 study. Necropsy was performed on all mice. Histopathological examination of the skin and any 

5 other organ showing gross abnormalities was performed. Chronic dermal exposure to 

6 benzo[a]pyrene produced a dose-related increase in skin tumor incidence and a decrease in tumor 

7 latency (see Table 4-17). Carcinoma was the primary tumor type observed and a dose-response 

8 relationship was evident for carcinoma formation and incidence of all types of skin tumors. 

9 

Table 4-17. Skin tumor incidence and time of appearance in female CFLP 

mice dermally exposed to benzo[a]pyrene for 104 weeks 

Dose (μg)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 ± 16.78b

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 skin of the back. 

b

 Mean ± SD. 

c

 Median with 95% confidence interval. 

10 Habs et al. (1984) applied benzo[a]pyrene (in 0.01 mL acetone) to the shaved 

11 interscapular skin of female NMRI mice at doses of 0, 2 or 4 μg, 2 times/week for life. Animals 

12 were observed twice daily until spontaneous death, unless an invasive tumor was observed. All 

13 animals were necropsied and histopathological examination was performed on the dorsal skin 

14 and any other organ with gross abnormalities. Chronic dermal exposure to benzo[a]pyrene did 

15 not affect body weight gain, but appeared to reduce survival at the highest dose with mean 

16 survival times of 691, 648, and 528 days for the 0, 2, and 4 μg/day groups, respectively. The 

17 total length of exposure for each group was not reported, but can be inferred from the survival 

18 data. Latency also was not reported. Benzo[a]pyrene application resulted in a dose-related 

19 increase the incidence of total skin tumors and skin carcinomas (see Table 4-18). Hematopoietic 

20 tumors (at 6/20, 3/20, and 3/20) and lung adenomas (at 2/20, 1/20, and 0/20) were observed in 
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1 the controls and in the benzo[a]pyrene treatment groups, but did not appear to be treatment 

2 related according to the study authors. 

3 

Table 4-18. Skin tumor incidence in female NMRI mice dermally exposed to 

benzo[a]pyrene for life 

Dose (μg)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) 

aMice were exposed until natural death or until they developed an invasive tumor at the site of application. 

b

Indicated doses were applied 2 times/wk to shaved interscapular skin. 

Source: Habs et al. (1984). 

4 

5 Higginbotham et al. (1993) 

6 Groups of 23–27 female Ah-receptor-responsive Swiss mice were treated on a shaved 

7 area of dorsal skin with 0, 1, 4, or 8 nmol (0, 0.25, 1, or 2 μg/treatment) benzo[a]pyrene (>99% 

8 pure) in acetone 2 times weekly for 40 weeks (Higginbotham et al., 1993). Surviving animals 

9 were sacrificed 8 weeks later. Complete necropsies were performed, and tissues from the treated 

10 area, lung, liver, kidney, spleen, urinary bladder, ovary, and uterus were harvested for 

11 histopathologic examination. Histopathologic examination was performed on tissues from the 

12 treated area, lungs, liver, kidneys, spleen, urinary bladder, uterus, and ovaries, as well as any 

13 other grossly abnormal tissue. Lung adenomas occurred in each group (1/27, 2/24, 1/23, 1/23), 

14 and other tumors were noted in isolated mice (i.e., malignant lymphoma (spleen) in one low-dose 

15 and one mid-dose mouse; malignant lymphoma with middle organ involvement in one high-dose 

16 mouse, and hemangioma (liver) in one mid-dose mouse) and were not considered dose related. 

17 In addition, benzo[a]pyrene showed no skin tumors under the conditions of this bioassay. 

18 

19 Sivak et al. (1997)

20 Sivak et al. (1997) designed a study to compare the carcinogenicity of condensed asphalt 

21 fumes (including benzo[a]pyrene and other PAHs) with several doses of benzo[a]pyrene alone. 

22 For the purposes of this assessment, the exposure groups exposed to PAH mixtures are not 

23 discussed. Groups of 30 male C3H /HeJ mice were treated dermally twice/week to 0, 0.0001, 

24 0.001, or 0.01% (0, 0.05, 0.5, or 5 μg) benzo[a]pyrene in a 50 μl volume of 

25 cyclohexanone/acetone (1:1) for 104 weeks beginning at 8 weeks of age. All mice were 

26 necropsied, and skin samples from all as well as any grossly observed lesions were subjected to 

27 histopathological examination. The incidence of skin tumors and mean survival times for each 
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1 group are shown in Table 4-19. All high dose mice died before the final sacrifice. The extent of 

2 deaths prior to one year in each group was not provided, so that the reported incidence may 

3 underestimate the tumor rate of animals exposed long enough to develop tumors. However, the 

4 crude skin tumor rates show an increasing trend in incidence. 

5 

Table 4-19. Skin tumor incidence in male C3H /HeJ mice dermally exposed 

to benzo[a]pyrene for 24 months 

Dose (μg)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 dorsal skin. 

Source: Sivak et al. (1997). 

6 

7 

8 Albert et al. (1991) 

9 To examine dose-response relationships and the time course of benzo[a]pyrene-induced 

10 skin damage, DNA adduct formation, and tumor formation, groups of 43–85 female Harlan mice 

11 were treated dermally with 0, 16, 32, or 64 μg of benzo[a]pyrene in 50 μL of acetone once per 

12 week for 29 weeks (Albert et al., 1991). Interscapular skin of each mouse was clipped 3 days 

13 before the first application and every 2 weeks thereafter. Additional groups of mice were treated 

14 for 9 weeks with 0, 8, 16, 32, or 64 μg radiolabeled benzo[a]pyrene to determine benzo[a]pyrene 

15 diolepoxide-DNA (BPDE-DNA) adduct formation in the epidermis at several time points (1, 2, 

16 4, and 9 weeks). Tumor formation was monitored only in the skin. 

17 No tumors were present in vehicle-treated or untreated control mice. In exposed groups, 

18 incidences of mice with skin tumors were not reported, but time-course data for cumulative 

19 number of tumors per mouse, corrected for deaths from nontumor causes, were reported. 

20 Tumors began appearing after 12–14 weeks of exposure for the mid- and high-dose groups and 

21 at 18 weeks for the low-dose group. At study termination (35 weeks after start of exposure), the 

22 mean number of tumors per mouse was approximately one per mouse in the low- and mid-dose 

23 groups and eight per mouse in the high-dose group; indicating that most, if not all, mice in each 

24 exposure group developed skin tumors and that the tumorigenic response was greatest in the 

25 highest dose group. The majority of tumors were initially benign, with an average time of 

26 8 weeks for progression from benign papillomas to malignant carcinomas. Epidermal damage 

27 occurred in a dose-related manner (more severe in the high-dose group than in the low- and middose groups) and included statistically significant increases (compared with controls) in: [3

28 H]-

29 thymidine labeling and mitotic indices; incidence of pyknotic and dark cells (signs of apoptosis); 
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1 and epidermal thickness. Only a minor expansion of the epidermal cell population was observed. 

2 In the high-dose group, indices of epidermal damage increased to a plateau by 2 weeks of 

3 exposure. The early time course of epidermal damage indices was not described in the low- and 

4 mid-dose groups, since data for these endpoints were only collected at 20, 24, and 30 weeks of 

5 exposure. An increased level of BPDE-DNA adducts, compared with controls, was apparent in 

6 all exposed groups after 4 weeks of exposure in the following order: 64>32>16>8 μg/week. The 

7 time-course data indicate that benzo[a]pyrene-induced increases in epidermal damage indices 

8 and BPDE-DNA adducts preceded the appearance of skin tumors. 

9 

10 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL, INHALATION, AND 

11 DERMAL 

12 As discussed in Section 4.1.4.3, several studies of human cohorts have examined possible 

13 associations between lower body weights or head circumference in newborns or infants with 

14 benzo[a]pyrene-DNA adducts levels in cord blood and exposure to ETS (Tang et al., 2006; 

15 Perera et al., 2005a, b). Available studies of reproductive or developmental endpoints in 

16 animals exposed orally or by inhalation to benzo[a]pyrene are reviewed as follows in this 

17 section. No studies that evaluated reproductive or developmental effects following dermal 

18 exposure were identified. 

19 

20 4.3.1. Oral 

21 Mohamed et al. (2010) investigated multi-generational effects in male mice following 

22 exposure of six-week old C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day 

23 benzo[a]pyrene for 6 weeks by daily gavage. Following final treatment, male mice were allowed 

24 to stabilize for one week prior to being mated with two untreated female mice to produce an F1 

25 generation. Male mice were sacrificed one week after mating. F1 males were also mated with 

26 untreated female mice as were F2 males. The mice of the F1, F2, and F3 generations were not 

27 exposed to benzo[a]pyrene. The F0, F1, F2 and F3 mice were all sacrificed at the same age (14 

28 weeks) and endpoints including testis histology, sperm count, sperm motility, and in vitro sperm 

29 penetration (of hamster oocytes) were evaluated. These endpoints were analyzed statistically 

30 using ANOVA and Tukey’s honest significance test and results were reported graphically as 

31 means +/- SD. 

32 Testicular atrophy was observed in the benzo[a]pyrene treatment groups, but was not 

33 statistically different than controls. Statistically significant reductions were observed in 

34 epididymal sperm counts of F0 and F1 generations treated with the high or low dose of 

35 benzo[a]pyrene. For F0 and F1 generations, epididymal sperm counts were reduced 

36 approximately 50% and 70%, respectively, in the low and high dose groups. Additionally, sperm 

37 motility was statistically significantly decreased in the high dose in the F0 and F1 generations. 

38 Sperm parameters of the F3 generation were not statistically different from controls. An in vitro 
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1 sperm penetration assay revealed statistically significantly reduced fertilization in F0 and F1 

2 generations of the low and high dose groups. However, the value of this in vitro test is limited as 

3 it bypasses essential components of the intact animal system (US EPA, 1996). Based on 

4 decreased epididymal sperm counts of F0 and F1 generations, a LOAEL of 1 mg/kg-day was 

5 established from this study (no NOAEL was identified). 

6 Xu et al., (2010) treated female Sprague-Dawley rats (6/group) to 0 (corn oil only), 5, or 

7 10 mg/kg-day benzo[a]pyrene by gavage every other day for a duration of 60 days. This resulted 

8 in time weighted average doses of 0, 2.5, and 5 mg/kg-day over the study period of 60 days. 

9 Endpoints examined included ovary weight, estrous cycle, 17B-estradiol blood level, and ovarian 

10 follicle populations (including primordial, primary, secondary, atretic, and corpora leutea). 

11 Animals were observed daily for any clinical signs of toxicity and following sacrifice, gross 

12 pathological examinations were made and any findings were recorded. All animals survived to 

13 necropsy. A difference in clinical signs was not observed for the treated groups and body 

14 weights were not statistically different in treated animals (though they appear to be depressed 6% 

15 at the high dose). Absolute ovary weight was statistically significantly reduced in the both the 

16 low and high dose groups, 11 and 15% respectively (see Table 4-20). Animals treated with the 

17 high dose were noted to have a statistically significantly prolonged duration of the estrous cycle 

18 and non-estrus phase compared to controls. Animals in the high dose group also had statistically 

19 significantly depressed levels of estradiol (by approximately 25%) and decreased numbers of 

20 primordial follicles (by approximately 20%). This study also indicated a strong apoptotic 

21 response of ovarian granulosa cells as visualized through TUNEL labeling, however, the 

22 strongest response was seen at the low dose; decreased apoptosis was also observed at the high 

23 dose. Based on decreased ovary weight following 60 day oral exposure to benzo[a]pyrene, a 

24 LOAEL of 2.5 mg/kg-day was established from this study (no NOAEL was identified). 

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 ± 0.0098b0.136 ± 0.0098b

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) using one-way ANOVA 

Source: Xu et al. (2010). 

26 
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1 Zheng et al., (2010) treated male Sprague-Dawley rats to 0 (corn oil only), 1, or 5 mg/kg2 day benzo[a]pyrene by daily gavage for a duration of 30 (8/group) or 90 days (8/group). At 

3 necropsy, the left testis of each animal was collected and weighed. Testes testosterone 

4 concentrations were determined by radioimmunassay and results were expressed as ng/g testis 

5 and reported graphically. Testicular testosterone was statistically significantly decreased in the 

6 high dose group approximately 15% following 90 days of exposure. The low dose group also 

7 appeared to have a similar average depression of testosterone levels; however, the change did not 

8 reach statistical significance. Testosterone levels measured in animals sacrificed following 30 

9 days of benzo[a]pyrene exposure were not statistically different than controls. Based on 

10 decreased testicular testosterone levels following 90 day oral exposure to benzo[a]pyrene, a 

11 LOAEL of 5 mg/kg-day and a NOAEL of 1 mg/kg-day were identified. 

12 McCallister et al., (2008) administered 0 or 300 ug/kg benzo[a]pyrene by oral gavage in 

13 peanut oil to pregnant Long Evans rats (n= 5 or 6) on GDs 14 to 17. At this exposure level, no 

14 significant changes were see in number of pups per litter, pup growth, or liver to body weight 

15 ratios in control compared to benzo[a]pyrene exposed offspring. Treatment related differences 

16 in brain to body weight ratios were observed only on PND 15 and PND 30. Decreases in 

17 cerebrocortical mRNA expression of the glutamatergic NMDA receptor subunit was 

18 significantly reduced (50%) in treated offspring compared to controls. In addition, in utero 

19 exposed offpring exhibited decreased evoked cortical neuronal activity in the barrel field cortex 

20 when tested at PN 90-120. 

21 Rigdon and Neal (1965) administered diets containing 1,000 ppm benzo[a]pyrene to 

22 pregnant mice (nine/group) on GDs 10–21 or 5–21. The pups were reported as appearing 

23 generally normal at birth, but cannibalism was elevated in the exposed groups. These results 

24 contrast with an earlier study (Rigdon and Rennels, 1964) in which rats (strain not specified) 

25 were fed diets containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to 

26 mating and during gestation. In the earlier study, five of eight treated females mated with 

27 untreated males became pregnant, but only one delivered live young. The treated dam that 

28 delivered had two live and two stillborn pups; one dead pup was grossly malformed. In the 

29 remaining treated females, vaginal bleeding was observed on GDs 23 or 24. In the inverse 

30 experimental design, three of six controls mated to benzo[a]pyrene-treated males became 

31 pregnant and delivered live young. Visceral and skeletal examinations of the pups were not 

32 conducted. These studies were limited by the small numbers of animals, minimal evaluation of 

33 the pups, lack of details on days of treatment (food consumption, weight gain), and the 

34 occurrence of cannibalism. 

35 

36 Reproductive effects of in utero exposure via oral route 

37 MacKenzie and Angevine (1981) conducted a two-generation reproductive and 

38 developmental toxicity study for benzo[a]pyrene in CD-1 mice. Benzo[a]pyrene was 
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1 administered by gavage in 0.2 mL of corn oil to groups of 30 or 60 pregnant (the F0 generation) 

2 mice at doses of 0, 10, 40, or 160 mg/kg-day on GDs 7–16 only. Therefore, unlike the standard 

3 two-generation study, F1 animals were exposed only in utero. F1 offspring were evaluated for 

4 postnatal development and reproductive function as follows. F1 pups (four/sex when possible) 

5 were allowed to remain with their mothers until weaning on PND 20. Crossover mating studies 

6 were then conducted. Beginning at 7 weeks of age, each F1 male mouse (n = 20–45/group) was 

7 allowed to mate with two untreated virgin females for 5-day periods for 25 days (for a total 

8 exposure of 10 untreated females/F1 male), after which time the males were separated from the 

9 females. Fourteen days after separation from the males, (i.e., on days 14–19 of gestation), the 

10 females were sacrificed and the numbers of implants, fetuses, and resorptions were recorded. 

11 The F2 fetuses were then examined for gross abnormalities. Similarly, each F1 female mouse (n 

12 = 20–55/group), beginning at 6 weeks of age, was paired with an untreated male for a period of 6 

13 months. Males were replaced if the females failed to produce a litter during the first 30-day 

14 period. All F2 young were examined for gross abnormalities on day 1 of life and their weights 

15 were recorded on day 4 of age. This F2 group was sacrificed on day 20 postpartum, while the 

16 F1 female was left with a male until the conclusion of the study. At 6 weeks of age, gonads of 

17 groups of 10 male and 10 female F1 mice exposed to 0, 10, or 40 mg/kg-day benzo[a]pyrene in 

18 utero were subjected to gross pathology and histologic examinations. 

19 No maternal toxicity was observed. The number of F0 females with viable litters at 

20 parturition at the highest dose was statistically significantly reduced by about 35% (Table 4-21), 

21 but progeny were normal by gross observation. Parturition rates of the low- and mid-dose 

22 groups were unaffected by treatment, and litter sizes of all treated groups were similar to the 

23 control group throughout lactation. However, body weights of the F1 pups in the mid-dose and 

24 high-dose groups were statistically significantly decreased on PND 20, by 7 and 13%, 

25 respectively, and in all treated pups on PND 42, 6, 6, and 10% for the low, mid, and high dose, 

26 respectively (Table 4-21). The number of F1 pups surviving to PNDs 20 and 42 was 

27 significantly reduced at the high dose (p < 0.01), by 8 and 16%, respectively. When F1 males 

28 were bred to untreated females and F1 females were mated with untreated males, a marked dose29 related decrease in fertility of > 30% was observed in both sexes, starting at the lowest exposure. 

30 There were no treatment-associated gross abnormalities or differences in body weights in the F2 

31 offspring. 

32 

Table 4-21. Reproductive effects in male and female 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 indexc

F1 female fertility indexd

46/60 (77%) 

11.2 ± 0.1 

29.9 ± 0.2 

80.4 

100.0 

21/30 (70%) 

11.6 ± 0.1 

28.2 ± 0.3b 

52.0b

65.7b

44/60 (73%) 

10.4 ± 0.1b

28.0 ± 0.2b 

4.7b

0.0b

13/30 (43%)b

9.7 ± 0.2b

26.8 ± 0.4b 

0.0b

0.0b

a

Pregnant F0 mice were administered daily doses of benzo[a]pyrene in corn oil on GDs 7–16. 

b

Significantly (p < 0.05) different from control by unspecified tests. 

cBeginning at 7 wks of age, each F1 male mouse (20–45/group) was exposed to 10 untreated females over a period 

of 25 d. Index = (females pregnant/females exposed to males) × 100. 

dBeginning at 6 wks of age, each F1 female mouse (20–55/group) was cohabitated with an untreated male for a 

period of 6 mo. 

Source: MacKenzie and Angevine (1981). 

1 

2 Exposure to benzo[a]pyrene caused a marked dose-related decrease in the size of the 

3 gonads. In F1 males, testes weights were statistically significantly reduced. Testes from animals 

4 exposed in utero to 10 and 40 mg/kg-day weighed approximately 60 and 18%, respectively, of 

5 the weight of testes from the control animals (no F2 offspring were produced in the high dose 

6 group). This was confirmed by histopathologic observation of atrophic seminiferous tubules in 

7 the 40 mg/kg-day group that were smaller than those of controls and were empty except for a 

8 basal layer of cells. The number of interstitial cells in the testes was also increased in this group. 

9 Males from the 10 mg/kg-day group showed limited testicular damage; although all exhibited 

10 evidence of tubular injury, each animal had some seminiferous tubules that displayed active 

11 spermatogenesis. Ovarian tissue was absent or reduced in F1 females such that organ weights 

12 were not possible to obtain. Examination of available tissue in these females revealed 

13 hypoplastic ovaries with few follicles and corpora lutea (10 mg/kg-day) or with no evidence of 

14 folliculogenesis (40 mg/kg-day). Ovarian tissue was not examined in highest-dose females. 

15 The LOAEL in this study was 10 mg/kg-day, based on decreases in mean pup weight 

16 (<5%) at PND 42 of F1 offspring of dams treated with 10, 40, or 160 mg/kg-day benzo[a]pyrene, 

17 marked decreases in the reproductive capacity (as measured by fertility index) of both male and 

18 female F1 offspring exposed at all three treatment levels of benzo[a]pyrene (by approximately 

19 30% in males and females), decreased litter size (by about 20%) in offspring of F1 dams, and 

20 also the dramatic decrease in size and alteration in anatomy of the gonads of both male and 

21 female F1 mice exposed to 10 and 40 mg/kg-day benzo[a]pyrene in utero. A NOAEL was not 

22 identified. 

23 In another reproductive and developmental toxicity study, benzo[a]pyrene was 

24 administered by gavage in corn oil to nine female NMRI mice at a dose of 10 mg/kg-day on GDs 

25 7–16; a group of nine controls received corn oil (Kristensen et al., 1995). Body weights were 

26 monitored. F0 females were kept with their offspring until after weaning (21 days after 

27 delivery). At 6 weeks of age, one F1 female from each litter (n = 9) was caged with an untreated 

28 male. The F2 offspring were inspected for gross deformities at birth, weight and sex were 

29 recorded 2 days after birth, and the pups were sacrificed. The F1 females were sacrificed after 6 
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1 months of continuous breeding. The effects of benzo[a]pyrene treatment on fertility, ovary 

2 weights, follicles, and corpora lutea were evaluated. F0 females showed no signs of general 

3 toxicity, and there was no effect on their fertility. F1 females had statistically significantly lower 

4 median numbers of offspring, number of litters, and litter sizes and a statistically significantly 

5 greater median number of days between litters as compared with the controls (Table 4-22). At 

6 necropsy, the F1 females from treated F0 females had statistically significantly reduced ovary 

7 weights; histologic examination of the ovaries revealed decreased numbers of small, medium, or 

8 large follicles and corpora lutea (Table 4-22). Only one dose group was used in this study, with 

9 decreased F1 female fertility observed following in utero exposure at the LOAEL of 10 mg/kg10 day; no NOAEL was identified. 

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) Controla benzo[a]pyrene exposeda(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) 

22b (0–86) 

3

b

 (0–8) 

8

b

 (3–11) 

21b (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) 

aGroups of nine female NMRI F0 mice were administered 0 or 10 mg benzo[a]pyrene/kg by gavage in corn oil on 

GDs 7–16. One F1 female from each litter was continuously bred with an untreated male for 6 mo. 

b

Significantly (p < 0.05) different from control group by Wilcoxon rank sum test or Kruskall-Wallis two-tailed test.

Source: Kristensen et al. (1995). 

12 

13 Reproductive effects in adults and repeated oral exposure 

14 Rigdon and Neal (1965) conducted a series of experiments to assess the reproductive 

15 effects of orally administered benzo[a]pyrene to Ah-responsive white Swiss mice. Female 

16 animals (number not stated) were administered benzo[a]pyrene at 250, 500, or 1,000 ppm in the 

17 feed before or during a 5-day mating period. Based on the initial body weight, the doses can be 

18 estimated as 32, 56, and 122 mg/kg-day, respectively. No effect on fertility was observed at any 

19 treatment dose, even when animals were fed 1,000 ppm benzo[a]pyrene for 20 days prior to 

20 mating, but interpretation of this finding was marred by large variability in numbers of pregnant 

21 females and litter sizes for both treated and control mice. In separate experiments, the fertility of

22 five male mice/group was not affected by exposure to 1,000 ppm in food for up to 30 days prior 

23 to mating with untreated females. Histologic examinations showed that male mice fed 500 ppm 

24 benzo[a]pyrene for 30 days had spermatozoa present in their testes; further details were not 
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1 provided. The only treatment-related effect was a lack of weight gain related to feed 

2 unpalatabilty. While this study suggests that premating exposure of male or female mice to 

3 doses up to 122 mg/kg-day for 20 days may not affect fertility, the sample sizes were too small 

4 and study designs were too inconsistent to provide reliable NOAELs and LOAELs for 

5 reproductive/developmental toxicity. 

6 In an earlier study (Rigdon and Rennels, 1964) rats (strain not specified) were fed diets 

7 containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and during 

8 gestation. In this study, five of eight treated females mated with untreated males became 

9 pregnant, but only one delivered live young. The treated dam that delivered had two live and 

10 two stillborn pups; one dead pup was grossly malformed. In the remaining treated females, 

11 vaginal bleeding was observed on GDs 23 or 24. In the inverse experimental design, three of six 

12 controls mated to benzo[a]pyrene-treated males became pregnant and delivered live young. 

13 Visceral and skeletal examinations of the pups were not conducted. These studies are 

14 insufficiently reported and of insufficient design (e.g., inadequate numbers of animals for 

15 statistical analysis) to provide reliable NOAELs or LOAELs for reproductive effects from 

16 repeated oral exposure to benzo[a]pyrene. 

17 

18 Immunosuppression effects and in utero exposure via oral route 

19 No studies were found that examined immune system endpoints following in utero 

20 exposure via the oral route. The abstract of a report by Holladay and Smith (1994) referred to 

21 gavage dosing in a study of immune endpoints in fetuses of pregnant mice exposed to 

22 benzo[a]pyrene on GDs 13–17, but the methods section of the report described an i.p. injection 

23 procedure in detail (see Section 4.4.2). 

24 

25 4.3.2. Inhalation 

26 
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1 Reproductive toxicity and in utero exposure via inhalation 

2 Archibong et al. (2002) evaluated the effect of exposure to inhaled benzo[a]pyrene on 

3 fetal survival and luteal maintenance in timed-pregnant F344 rats. Prior to exposure on GD 8, 

4 laparotomy was performed to determine the number of implantation sites, and confirmed 

5 pregnant rats were divided into three groups, consisting of rats that had four to six, seven to nine, 

6 or more than nine conceptuses in utero. Rats in these groups were then assigned randomly to the 

7 treatment groups or control groups to ensure a similar distribution of litter sizes. Animals 

8 (10/group) were exposed to benzo[a]pyrene:CB aerosols at concentrations of 25, 75, or 

100 μg/m39 via nose-only inhalation, 4 hours/day on GDs 11–20. Control animals were either 

10 sham-exposed to CB or remained entirely unexposed. Results of particle size analysis of 

11 generated aerosols were reported by several other reports from this laboratory (Inyang et al., 

12 2003; Ramesh et al., 2001a; Hood et al., 2000). Aerosols showed a trimodal distribution with 

13 averages of 95% cumulative mass with diameters <15.85 μm; 89% <10 μm; 55% <2.5 μm; and 

14 38% <1 μm (Inyang et al., 2003). Ramesh et al. (2001a) reported that the (MMAD ± GSD) for 

15 the 55% mass fraction with diameters <2.5 μm was 1.7 ± 0.085. Progesterone, estradiol-17β, 

16 and prolactin concentrations were determined in plasma collected on GDs 15 and 17. Fetal 

17 survival was calculated as the total number of pups divided by the number of all implantation 

18 sites determined on GD 8. Individual pup weights and crown-rump length per litter per 

19 treatment were determined on PND 4 (PND 0 = day of parturition). 

20 Archibong et al. (2002) reported that exposure of rats to benzo[a]pyrene caused 

21 biologically and statistically significant (p ≤ 0.05) reductions in fetal survival compared with the 

22 two control groups; fetal survival rates were 78.3, 38.0, and 33.8% per litter at 25, 75, and 

100 μg/m323 , respectively, and 96.7% with CB or 98.8% per litter in untreated controls (see Table 

24 4-23). Consequently, the number of pups per litter was also decreased in a concentrationdependent manner. The decrease was ~50% at 75 μg/m3

 and ~65% at 100 μg/m325 , compared with 

26 sham-exposed and unexposed control groups. No effects on hormone levels were observed on 

27 GDs 15 or 17 at the low-dose. Biologically significant decreases in mean pup weights 

(expressed as g per litter) of >5% were observed at doses ≥75 μg/m3 28 (14 and 16% decreases at 75 

and 100 μg/m329 , respectively, p < 0.05). Exposure to benzo[a]pyrene did not affect crown-rump 

30 length (see Table 4-22). 

31 

Table 4-23. Pregnancy outcomes in female F344 rats treated with 

benzo[a]pyrene on GDs 11–21 by inhalation 

Parametera

Administered concentration of benzo[a]pyrene (μg/m3

) 

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 ± 0.5b 4.2 ± 0.1b 3.0 ± 0.2b
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Survival (litter %) 98.9 ± 1.1 96.7 ± 1.7 78.3 ± 4.1

b

 38.0 ± 2.1b 33.8 ± 1.3b

Pup weight (g/litter) 10.6 ± 0.1 8.8 ± 0.1 10.5 ± 0.2 9.1 ± 0.2b 8.9 ± 0.1b

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 μg/m32 caused a statistically significant decrease in plasma 

3 progesterone, estradiol, and prolactin on GD 17; these levels were not determined in the rats 

exposed to 100 μg/m34 (Archibong et al., 2002). Plasma prolactin is an indirect measure of the 

5 activity of decidual luteotropin, a prolactin-like hormone whose activity is necessary for luteal 

6 maintenance during pregnancy in rats. Control levels of prolactin increased from GDs 15 to 17, 

but this increase did not occur in the rats exposed to 75 μg/m37 . Although the progesterone 

concentration at 75 μg/m38 was significantly lower than in controls on GD 17, the authors thought 

9 that the circulating levels should have been sufficient to maintain pregnancy; thus, the increased 

10 loss of fetuses was thought to be caused by the lower prolactin levels rather than progesterone 

11 deficiency. The reduced circulating levels of progesterone and estradiol-17β among 

12 benzo[a]pyrene-treated rats were thought to be a result of limited decidual luteotropic support for 

13 the corpora lutea. The authors proposed the following mechanism for the effects of 

14 benzo[a]pyrene on fertility: benzo[a]pyrene or its metabolites decreased prolactin and decidual 

15 luteotropin levels, compromising the luteotropic support for the corpora lutea and thereby 

16 decreasing the plasma levels of progesterone and estradiol-17β. The low estradiol-17β may 

17 decrease uterine levels of progesterone receptors, thereby resulting in fetal mortality. Based on 

18 biologically and statistically significant decreases in pups/litter and percent fetal survival/per 

litter, the LOAEL was 25 μg/m319 ; no NOAEL was identified. 

20 

21 Neurotoxicity and in utero exposure via inhalation 

22 To evaluate the effects of benzo[a]pyrene on the developing central nervous system, 

23 Wormley et al. (2004) exposed timed-pregnant F344 rats (10/group) to benzo[a]pyrene:CB 

aerosols by nose-only inhalation on GDs 11–21 for 4 hours/day at a concentration of 100 μg/m324 . 

25 Results of particle size analysis of genenerated aerosols were reported by other reports from this 

laboratory (Ramesh et al., 2001a; Hood et al., 2000). Particle size analysis of a 100-μg/m3 26 

27 aerosol showed a trimodal distribution with averages of 95% cumulative mass with diameters 

28 <15.85 μm; 90% <10 μm; 67.5% <2.5 μm; and 66.2% <1 μm; the MMAD ± GSD for the latter 

29 fraction was 0.4 ± 0.02 μm (Hood et al., 2000). Dams were maintained to term and pups were 

30 weaned on PND 30. Benzo[a]pyrene reduced the number of live pups to one-third of control 

31 values without affecting the number of implantation sites. During PNDs 60–70 electrical 

32 stimulation and evoked field potential responses were recorded via electrodes implanted into the 
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1 brains of the animals. Direct stimulation of perforant paths in the entorhinal region revealed a 

2 diminution in long-term potentiation of population spikes across the perforant path-granular cell 

3 synapses in the dentate gyrus of the hippocampus of F1 generation benzo[a]pyrene-exposed 

4 animals; responses in exposed offspring were about 25% weaker than in control offspring. 

5 Additionally, NMDA receptor subunit 1 protein (important for synaptic functioning) was down6 regulated in the hippocampus of benzo[a]pyrene exposed F1 pups. The authors interpreted their 

7 results as suggesting that gestational exposure to benzo[a]pyrene inhalation attenuates the 

8 capacity for long-term potentiation (a cellular correlate of learning and memory) in the F1 

9 generation. 

10 In a later study by this same group of investigators, Wu et al. (2003) evaluated the 

11 generation of benzo[a]pyrene metabolites in F1 generation pups, as well as the developmental 

12 profile for AhR and mRNA. In this study, confirmed pregnant F344 rats were exposed to 

benzo[a]pyrene:CB aerosols at 25, 75, or 100 μg/m313 via nose-only inhalation, 4 hours/day, for 

14 10 days (GDs 11–21). Control animals were exposed to CB (sham) to control for inert carrier 

15 effects or they remained untreated. Each benzo[a]pyrene concentration had its own set of 

16 controls (CB and untreated). Two randomly selected pups were sacrificed on each of PND 0, 3, 

17 5, 10, 15, 20, and 30. Body, brain, and liver weights were recorded. Benzo[a]pyrene metabolites 

18 were analyzed in the cerebral cortex, hippocampus, liver, and plasma. A dose-related increase in 

19 plasma and cortex benzo[a]pyrene metabolite concentrations in pups was observed. 

20 Dihydrodiols (4,5-; 7,8-; 9,10-) dominated the metabolite distribution profile up to PND 15 and 

21 the hydroxy (3-OH-benzo[a]pyrene; 9-OH-benzo[a]pyrene) metabolites after PND 15 at 

100 μg/m322 (the only exposure concentration reported). Results indicated a dose-related decrease 

23 in the ratio of the total number of pups born per litter to the total number of implantation sites per

litter. The number of resorptions at 75 and 100 μg/m3, but not at 25 μg/m324 , was statistically 

25 significantly increased compared with controls. 

26 

27 Adult male reproductive effects and repeated inhalation exposure 

28 Inyang et al. (2003) evaluated the effect of sub-acute exposure to inhaled benzo[a]pyrene 

29 on testicular steroidogenesis and epididymal function in rats. Male F344 rats (10/group), 

13 weeks of age, were exposed to benzo[a]pyrene:CB aerosols at 25, 75, or 100 μg/m330 via nose31 only inhalation, 4 hours/day for 10 days. Control animals were either exposed to CB (sham) to 

32 control for exposure to the inert carrier, or they remained untreated. Each benzo[a]pyrene 

33 concentration had its own set of controls (CB and untreated). Aerosols showed a trimodal 

34 distribution with averages of 95% cumulative mass <15.85 μm; 89% <10 μm; 55% <2.5 μm; and 

35 38% <1 μm (Inyang et al., 2003); an earlier report from this laboratory indicated that the 55% 

36 mass fraction had a MMAD ± GSD of 1.7 ± 0.085 (Ramesh et al., 2001a). Blood samples were 

37 collected at 0, 24, 48, and 72 hours after cessation of exposure to assess the effect of 

38 benzo[a]pyrene on systemic concentrations of testosterone and luteinizing hormone (LH), 
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1 hormones that regulate testosterone synthesis. Reproductive endpoints such as testis weight and 

2 motility and density of stored (epididymal) sperm were evaluated. 

3 Regardless of the exposure concentration, inhaled benzo[a]pyrene did not affect testis 

4 weight or the density of stored sperm compared with controls. However, inhaled benzo[a]pyrene 

5 caused a concentration-dependent reduction in the progressive motility of stored sperm. 

Progressive motility was similar at 75 and 100 μg/m36 , but these values were significantly lower 

7 (p < 0.05) than in any other group. The reduction in sperm motility post-cessation of exposure 

8 was thought to be the result of benzo[a]pyrene limiting epididymal function. Benzo[a]pyrene 

exposure to 75 μg/m39 caused a decrease in circulating concentrations of testosterone compared 

10 with controls from the time of cessation of exposure (time 0) to 48 hours post-termination of 

11 exposure (p < 0.05). However, the decrease was followed by a compensatory increase in 

testosterone concentration at 72 hours post-cessation of exposure. Exposure to 75 μg/m312 caused 

13 a nonsignificant increase in plasma LH concentrations at the end of exposure compared with 

14 controls, which increased further and turned significant (p < 0.05) for the remaining time of the 

15 study period. The decreased plasma concentration of testosterone, accompanied by an increased 

16 plasma LH level, was thought to indicate that benzo[a]pyrene did not have a direct effect on LH. 

17 The authors also noted that the decreased circulating testosterone may have been secondary to 

18 induction of liver CYP450 enzymes by benzo[a]pyrene. The authors concluded that subacute 

19 exposure to benzo[a]pyrene contributed to impaired testicular endocrine function that ultimately 

impaired epididymal function. Based on this study, the NOAEL was 25 μg/m320 and the LOAEL 

was 75 μg/m321 , based on a statistically significant reduction in the progressive motility of stored 

sperm and impairment of testicular function with 10 days of exposure at 75 μg/m322 . 

23 In a follow up study with longer exposure duration, adult male F344 rats (10 per group) 

were exposed to benzo[a]pyrene:CB aerosols at 75 μg/m3 24 via nose-only inhalation, 4 hours/day 

25 for 60 days (Archibong et al., 2008; Ramesh et al., 2008). Rats in the control group were 

26 subjected to the nose-only restraint, but were not exposed to the CB carrier. Blood samples were 

27 collected at 0, 24, 48, and 72 hours after exposure terminated, and the animals sacrificed for 

28 tissue analyses following the last blood sampling. Data were analyzed statistically for 

29 benzo[a]pyrene effects on weekly body weights, total plasma testosterone and LH 

30 concentrations, testis weights, density of stored spermatozoa, sperm morphological forms and 

31 motility, benzo[a]pyrene metabolite concentrations and AHH activity, and morphometric 

32 assessments of testicular histologies. Relative to controls, the results indicated 34% reduced 

33 testis weight (p < 0.025), reduced daily sperm production (p < 0.025) and reduced intratesticular 

34 testosterone concentrations (p < 0.025). Plasma testosterone concentrations were reduced to 

35 about one-third of the level in controls on the last day of exposure (day 60) and at 24, 48, and 72 

36 hours later (p < 0.05). However, plasma LH concentrations in benzo[a]pyrene exposed rats were 

37 elevated throughout the blood sampling time periods compared with controls (p < 0.05). In 

38 testis, lung, and liver, AHH activity, and benzo[a]pyrene-7,8-dihydrodiol (precursor to the DNA-
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1 reactive BPDE) and benzo[a]pyrene-3,6-dione metabolites were significantly (p < 0.05) elevated 

2 relative to controls. Progressive motility and mean density of stored spermatozoa were 

3 significantly reduced (p < 0.05). Weekly body weight gains were not affected by benzo[a]pyrene 

4 exposure. These results indicate that 60-day exposure of adult male rats to benzo[a]pyrene:CB 

aerosols at 75 μg/m35 produced decreased testis weight; decreased intratesticular and plasma 

6 testosterone concentrations; and decreased sperm production, motility, and density. 

7 

8 4.4. OTHER DURATION OR ENDPOINT-SPECIFIC STUDIES 

9 4.4.1. Acute Neurological Studies 

10 Saunders et al. (2001) administered benzo[a]pyrene (>97% pure in peanut oil) to F344 

11 rats (10/sex/dose group) via a single gavage dose of 0, 12.5, 25, 50, 100, or 200 mg/kg. Separate 

12 groups of animals were used for motor activity assessment and functional observational battery. 

13 Motor activity (horizontal, vertical, total distance, and stereotypic activity) was measured over 

14 2-hour intervals during the nocturnal phase (12 hours) for 5 consecutive days after treatment 

15 (day 1). The functional observational battery, consisting of 29 tests assessing autonomic, 

16 neuromuscular, CNS excitability, CNS activity, sensorimotor, and physiological activity, was 

17 administered before dosing and at 2, 4, 6, 12, 24, 48, 72, and 96 hours after dosing. Body weight 

18 was measured after the functional observational battery. 

19 In both sexes of rat, body weight gain was significantly reduced on day 4 and/or 5 at 

20 doses ≥25 mg/kg (Saunders et al., 2001). Body weight gains were comparable to controls in the 

21 25 and 50 mg/kg groups by day 6. Higher doses of benzo[a]pyrene resulted in prolonged 

22 reductions in body weight gain, with reductions of 21–26% in both sexes on day 9 after 

23 treatment. Weight gain had returned to control levels after 2 weeks postdosing. At doses of 

24 50 mg/kg and higher, all measures of motor activity were significantly depressed in both sexes 

25 beginning 2 hours after dosing and persisting through the 12-hour post-dosing measurement. At 

26 the 25 mg/kg dose, significant changes in motor activity were not observed until 4–6 hours after 

27 treatment. When motor activity was measured over 24-hour intervals, significant depression of 

28 motor activity (all measures) was observed at all doses on day 1 post-treatment and at ≥50 mg/kg 

29 on day 2. The results of the functional observational battery showed significant effects on 

30 neuromuscular endpoints (decreased mobility and grip strength, abnormal gait, loss of righting 

31 reflex), autonomic endpoints (increased defecation and urination), and sensorimotor endpoints 

32 (decreased response to sound, touch, and pain) at all doses and in both sexes. Effects on most 

33 parameters peaked at 6 hours post-dosing, with return to control levels by 72 hours post-dosing. 

34 The severity of effects on the FOB tests was greater in males than in females. This study 

35 identified a LOAEL of 25 mg/kg benzo[a]pyrene for acute neurotoxicity; the NOAEL is 

36 12.5 mg/kg. 

37 In a study with nearly identical design, Saunders et al. (2002) treated male F344 rats 

38 (10/dose) with single gavage doses of 0, 25, 50, 100, or 200 mg/kg benzo[a]pyrene (>97% pure, 
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1 in peanut oil). As in the study by Saunders et al. (2001), separate groups of animals were used 

2 for motor activity assessment and functional observational battery (using the same endpoints 

3 measured at the same time intervals); in addition, a third group of animals was treated at the 

4 same doses and used for measurement of benzo[a]pyrene and its metabolites in plasma and brain 

5 tissue. At 2, 4, 6, 12, 24, 48, 72, and 96 hours after dosing, rats in the metabolism groups were 

6 sacrificed for analysis of benzo[a]pyrene and its metabolites in plasma and brain tissue. Dose7 and time-dependent effects on locomotor activity were observed in all treated groups. In 

8 measurements conducted over the 12 hours on day 1 after treatment, significant (p < 0.05 relative 

9 to vehicle controls) reductions in total distance traveled (considered by the authors to represent 

10 the most accurate measure of locomotor activity) were observed at doses ≥50 mg/kg beginning at 

11 2–4 hours post dosing and persisting up to the 12-hour time point. When assessed on a daily 

12 basis over the 5 posttreatment days, total distance traveled was significantly lower than controls 

13 in all treated groups on day 1 and in groups exposed to ≥50 mg/kg on day 2. Significant (p < 

14 0.05) dose-dependent effects on neuromuscular, sensorimotor, and autonomic parameters were 

15 observed with benzo[a]pyrene treatment. Significant increases in the severity of abnormal gait 

16 and impaired sound and tail pinch responses, as well as decreased forelimb grip strength, 

17 occurred in all dose groups; the severity of effects peaked at 4 or 6 hours after treatment. 

18 Increased severity of landing foot splay was observed at doses ≥50 mg/kg, also peaking in 

19 severity at 6 hours after treatment. Effects on this endpoint persisted from 2 to 24 hours post 

20 treatment in the two high dose groups. Autonomic effects, consisting of increased frequency of 

21 urination and defecation, occurred at doses ≥50 mg/kg; in the two high dose groups, these effects 

22 began 4 hours after dosing and persisted through 24 hours. The onset and duration of the effects 

23 observed in this study corresponded well with the plasma and brain tissue concentrations of 

24 benzo[a]pyrene and its metabolites. In particular, levels of benzo[a]pyrene metabolites in brain 

25 tissue peaked between 2 and 6 hours after dosing, and plasma levels peaked at 6 hours after 

26 dosing, in all treated groups. By 72 hours after dosing, metabolite levels had returned to 

27 baseline. Unmetabolized benzo[a]pyrene was detected in brain tissue only in the two highest 

28 dose groups; levels in both brain tissue and plasma peaked between 2 and 6 hours after dosing. 

29 Analysis of specific metabolite levels over time showed that the diol metabolites comprised a 

30 larger percentage of the total metabolites at earlier time points (up to 12 hours after dosing), 

31 while the hydroxyl metabolites predominated at later times. The authors postulated that the 

32 observed toxic effects were associated with the production of benzo[a]pyrene diol metabolites 

33 and/or related generation of ROS rather than an effect of the parent compound or hydroxyl 

34 metabolites. The LOAEL in this study was 25 mg/kg based on suppression of locomotor activity 

35 and evidence of impairment in the functional observational battery. 

36 In a follow-up study, Saunders et al. (2006) attempted to correlate neurobehavioral 

37 changes with levels of benzo[a]pyrene metabolites, antioxidant enzyme levels, and measures of 

38 lipid peroxidation in selected brain regions. Single oral doses of 0, 25, 50, 100, or 200 mg/kg 
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1 benzo[a]pyrene (97% pure, in peanut oil) were administered by gavage to groups of 10 male 

2 F344 rats. Motor activity, measured as total distance traveled over a 2-hour interval during the 

3 nocturnal phase, was assessed at 0, 2, 4, 6, 12, 24, 48, 72, and 96 hours post treatment. 

4 Additional groups of animals were exposed to the same doses and sacrificed for collection of 

5 blood and specific brain tissues (hippocampus, striatum) at the same time points; benzo[a]pyrene 

6 and its metabolites were measured by reverse phase HPLC in blood and brain tissues. In 

7 addition, the activities of superoxide dismutase, catalase, glutathione peroxidase, and levels of 

8 malondialdehyde in striatum and hippocampus were determined at 6 and 96 hours after 

9 treatment. The authors reported that motor activity was significantly (p < 0.01) suppressed as 

10 early as 2 hours after treatment and remained suppressed through the 48-hour time point in the 

11 groups exposed to 50 and 200 mg/kg benzo[a]pyrene; however, the data were not reported, and it 

12 was not clear whether the 100 mg/kg dose group was affected. The authors indicated that the 

13 maximum suppression occurred at 12 hours, when motor activity was 72% lower than controls; 

14 however, the affected dose group(s) was not reported. Data were reported graphically for the 

15 25 mg/kg group only; based on the graph, a significant (p < 0.05) suppression of motor activity 

16 occurred by 4 hours after treatment and persisted through the 12-hour measurement. Activity 

17 had returned to control levels by 72 or 96 hours in all dose groups. As in the study reported by 

18 Saunders et al. (2002), the levels of benzo[a]pyrene metabolites in the plasma and brain 

19 correlated with the onset and duration of behavioral effects; metabolite concentrations peaked 

20 between 2 and 6 hours after treatment, when suppression of motor activity occurred. In addition, 

21 the toxification/detoxification ratio of benzo[a]pyrene metabolites (measured as the ratio of 7,8-

22 dihydrodiol 9,10-epoxide to 3[OH]benzo[a]pyrene) in plasma, cortex, cerebellum, hippocampus, 

23 and striatum was higher (between 2 and 7) for the first 6 hours, indicating higher levels of the 

24 more toxic epoxide metabolite. From 24 to 96 hours after treatment, the ratio was <1, indicating 

25 that the hydroxyl form predominated. Measurement of antioxidant enzyme levels in the striatum 

26 and hippocampus at 6 and 96 hours after dosing showed significant dose-related decreases in the 

27 activities of superoxide dismutase and glutathione peroxidase, but enhanced catalase activity and 

28 increased lipid peroxidation products in the striatum and hippocampus. The authors suggested 

29 that benzo[a]pyrene-induced acute neurobehavioral effects may be associated with oxidative 

30 stress resulting from generation of ROS and inhibition of brain antioxidants. 

31 Evidence of neurotoxicity has also been reported in studies of acute exposure to 

32 benzo[a]pyrene administered via parenteral routes. As part of a study of the neurotoxicity of 

33 motorcycle exhaust, Liu et al. (2002) administered benzo[a]pyrene dissolved in corn oil via i.p. 

34 injection to ICR mice (sex not specified; 4–6/group) at doses of 0, 50, or 100 mg/kg-day for 

35 3 consecutive days. One day after the last treatment, motor nerve conduction velocity was 

36 measured in the tails. The mice were then sacrificed for removal of the sciatic nerve, which was 

assayed for Na+/K+37 -ATPase activity. Although the methods section indicated that 

38 benzo[a]pyrene-treated animals were tested for rotarod performance, results of this evaluation 
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were not reported. Data on motor nerve conduction velocity and Na+/K+1 -ATPase activity were 

2 presented graphically. Exposure to benzo[a]pyrene at resulted in significant (p < 0.05) 

3 depression of motor nerve conduction velocity (about 25 and 40% decrease from control for 50 

4 and 100 mg/kg-day doses, respectively, based on visual inspection data presented graphically), 

and decreased the Na+/K+5 -ATPase activities of sciatic nerves (about 20 and 30% decreases from 

6 control, respectively). 

7 Grova et al. (2007) evaluated the effects on short-term exposure to benzo[a]pyrene on 

8 learning, memory, locomotor activity, and motor coordination. Groups of 10 female Balb/c mice 

9 were treated with i.p. injections of benzo[a]pyrene (>97% pure, in vegetable oil) at doses of 0, 

10 0.02, 0.2, 2, 20, or 200 mg/kg-day for 10 consecutive days. At the end of exposure, locomotor 

11 activity was measured in open field activity, and motor coordination was assessed. Memory and 

12 learning were evaluated using the Y maze (measures spontaneous alternation behavior) and the 

13 Morris water maze (measures learning as escape latency in repeated trials). Finally, the animals 

14 were sacrificed for removal of the brain; expression of the NMDA R1 receptor (involved in 

15 cognitive function) subunit gene was measured in eight brain regions (cerebral trunk, cerebellum, 

16 mesencephalum, hippocampus, hypothalamus, thalamus, frontal cortex, and temporal cortex) by 

17 quantitative real-time reverse transcription PCR assay. In contrast to oral studies of 

18 benzo[a]pyrene exposure (Saunders et al., 2006, 2002, 2001), injection of benzo[a]pyrene did not 

19 affect locomotor activity at any dose in this study. At the lowest doses (0.02 and 0.2 mg/kg20 day), benzo[a]pyrene exposure resulted in reductions in the percentage of spontaneous 

21 alternation in the Y maze. However, at the higher doses (≥2 mg/kg-day), there was no difference 

22 from controls. The authors attributed this finding to increased activity and arousal at the higher 

doses, postulated to result from an anxiolytic effect of benzo[a]pyrene. In the 5th 23 trial of the 

24 water maze, all benzo[a]pyrene groups showed impairment; the escape latency was significantly 

25 higher than controls. In contrast, the higher doses of benzo[a]pyrene resulted in significantly 

reduced latency during the first trial. No differences from control were observed in the 2nd, 3rd 26 , 

and 4th 27 trials, or 1 day after the last dose. Benzo[a]pyrene exposure resulted in modulation of 

28 NMDA-R1 subunit gene expression; expression was significantly (p < 0.05) increased in the 

29 cerebellum, mesencephalus, and hippocampus, but decreased in the frontal cortex and cerebral 

30 trunk. Effects on gene expression in the cerebellum, frontal cortex, and hippocampus occurred at 

31 all doses, while gene expression in the cerebral trunk was affected only at doses of ≥0.2 mg/kg32 day, and expression in the mesencephalus occurred only at doses of ≥2.0 mg/kg-day. 

33 In a follow-up study, Grova et al. (2008) evaluated the effects of exposure to 

34 benzo[a]pyrene on anxiety-related behaviors (performance in elevated-plus maze and hole-board 

35 apparatus). The animals, group sizes, and doses were the same as in Grova et al. (2007), but 

36 exposure occurred over 11 days. Behavioral tests were administered 30 minutes after the final 

37 dose, and the animals were sacrificed 1 hour after the tests. Benzo[a]pyrene exposure at 20 and 

38 200 mg/kg-day resulted in reduction in anxiety-related behavior as measured by the increased 
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1 number of head dippings in the hole-board apparatus. In the elevated-plus maze test, only the 

2 highest dose resulted in a significant reduction in anxiety (as measured by higher percentage of 

3 open arm entries and time spent in open arms). 

4 

5 4.4.2. Immunological Studies 

6 Immunological effects (e.g., decreased thymus weight, decreased number of B cells in 

7 spleen) have been reported in Wistar rats repeatedly exposed to benzo[a]pyrene doses ≥10–

8 15 mg/kg-day in standard oral toxicity studies (Kroese et al., 2001; De Jong et al., 1999). 

9 Diminished immune responses elicited by the dermal sensitizer, 2,4-dinitrochlorobenzene 

10 (DNCB) have been observed in C56BL/6 mice orally exposed to 13 mg/kg-day benzo[a]pyrene, 

11 3 times/week for 4 weeks (van den Berg et al., 2005). No studies were located examining 

12 immune system endpoints following inhalation exposure of animals to benzo[a]pyrene. Results 

13 from studies of immune system endpoints in mice following i.p., subcutaneous (s.c.), or 

14 intratracheal instillation exposure are consistent with immune suppression at dose levels 

15 generally ≥40 mg/kg-day. The available animal studies identify immune suppression as a 

16 potential hazard of repeated oral exposure to benzo[a]pyrene at doses ≥10–15 mg/kg-day. 

17 

18 4.4.2.1. Oral Exposure Immunological Studies 

19 As discussed in Section 4.2.1.1, dose-related decreases in thymus weight and relative 

20 number of B cells in the spleen were observed in male Wistar rats administered gavage doses 

21 ≥10 mg/kg-day for 35 days (De Jong et al., 1999). At higher doses (≥30 mg/kg-day), serum IgM 

22 and IgA levels were decreased. At the highest dose tested (90 mg/kg-day), the relative cortex 

23 surface area of thymus and thymic medullar weight were significantly reduced; NK cell activity 

24 in the spleen was also reduced at this dose. No effects on the immune system were observed at 

25 3 mg/kg-day (De Jong et al., 1999). In two additional subchronic gavage studies, thymus weight 

26 was decreased in a dose-related manner in male Wistar rats exposed to doses ≥15 mg/kg-day 

27 (5 days/week) for at least 5 weeks and in females exposed to doses of ≥30 mg/kg-day 

28 (5 days/week) for 90 days (Kroese et al., 2001). No other immune system parameters were 

29 assessed in these studies. In an adaptation of the sensitization-specific murine local lymph node 

30 assay for use in testing immune function, van den Berg et al. (2005) tested several 

31 immunomodulating compounds, including benzo[a]pyrene, for effects on the T-cell-dependent 

32 immune response induced by the contact sensitizer, DNCB. Groups of eight male and eight 

33 female C56BL/6 mice were given gavage doses of 13 mg/kg-day benzo[a]pyrene (purity not 

34 reported) 3 times/week for 4 weeks, followed by sensitization with DNCB (0, 0.33, 0.66, or 1% 

35 solutions in acetone:corn oil) applied topically to the backs of both ears for 3 consecutive days. 

36 Three days after the last DNCB treatment, the lymph nodes under the application area were 

37 excised, weighed, and homogenized for preparation of cell suspensions. The lymph node cell 

suspensions were tested for cell proliferation capacity via measurement of [338 H]-thymidine 
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1 incorporation. Releases of the cytokines interferon (IFN)-γ and interleukin (IL)-4 following 

2 concanavalin A stimulation were assayed by ELISA. At the highest concentration of the 

sensitizer, benzo[a]pyrene treatment reduced [33 H]-thymidine incorporation into lymphocytes by 

4 approximately 30% (based on visual inspection of data presented graphically; p = 0.008) 

5 compared with untreated controls. In this treatment group, benzo[a]pyrene also reduced the 

6 release of IFN-γ (approximately 75% less than controls based on graphical data, not significant) 

7 and IL-4 (approximately 60% less than controls based on graphical data, p < 0.001). The results 

8 indicated that benzo[a]pyrene modulates the immune response elicited by the sensitizer DNCB. 

9 

10 4.4.2.2. Inhalation Exposure Immunological Studies 

11 No studies were located that examined immune system endpoints following inhalation 

12 exposure of animals to benzo[a]pyrene. 

13 

14 4.4.2.3. Other Exposure Route Immunological Studies 

15 A number of studies have shown suppression of both humoral and cell-mediated immune 

16 responses in mice following i.p., s.c., or intratracheal administration. Dose-related decreases in 

17 spleen and serum IgM levels after challenge by sheep red blood cells (SRBC) were reported in 

18 rats (10, 40 mg/kg-day) and mice (5, 20, 40 mg/kg-day) following s.c. injection of 

19 benzo[a]pyrene for 14 days (Temple et al. 1993). Reduced spleen cell response to SRBC and 

20 lipopolysaccharides were observed in B6C3F1 mice exposed to doses ≥40 mg/kg-day 

21 benzo[a]pyrene by i.p. or s.c. injection for 4–14 days (Lyte and Bick, 1985; Dean et al., 1983; 

22 Munson and White, 1983) or by intratracheal instillation for 7 days (Schnizlein et al., 1987). 

23 B6C3F1 mice exhibited dose-dependent decreased resistance to Streptococcus pneumonia or 

24 Herpes simplex type 2 following s.c. injection of 5, 20, or 40 mg/kg benzo[a]pyrene for 14 days 

25 (Munson et al., 1985). Galvan et al. (2006) reported that single i.p. injections of mice with 

26 50 mg/kg benzo[a]pyrene caused decreased pro/pre B-lymphocytes and neutrophils in bone 

27 marrow, without affecting numbers of immature and mature B-lymphocytes or GR-1+ myeloid 

28 cells. Several i.p. injection studies reported immune suppression effects in mice exposed to 

29 benzo[a]pyrene in utero at doses ranging from 50 to 150 mg/kg; effects included decreased 

30 spleen or thymus weights, suppression of antibody forming cells in response to sheep red blood 

31 cells, decreased spleen, thymic, or bone marrow cellularity, and disrupted T-cell development 

32 (Rodriguez et al. 1999; Holladay and Smith, 1995; Lummus and Henningsen, 1995; Holladay 

33 and Smith, 1994; Urso and Johnson 1988; Urso et al., 1988; Urso and Gengozian, 1984, 1982, 

34 1980). 

35 In contrast to the studies that have shown decrements in immune response, 

36 benzo[a]pyrene may also induce sensitization responses. Epicutaneous application of 

37 benzo[a]pyrene (100 μg benzo[a]pyrene to C3H/HeN mice followed by ear challenge with 20 μg 
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1 benzo[a]pyrene 5 days later) produced a contact hypersensitivity (a significant ear swelling) 

2 response (Klemme et al., 1987). 

3 

4 4.4.2.4. Other Exposure Route Developmental Immunotoxicy 

5 While there are no oral or inhalation studies of benzo[a]pyrene on the developing 

6 immune system, several i.p. injection studies indicate that this is an area of concern for both cell7 mediated and humoral immune ontogeny. In terms of cell-mediated effects, Urso and Gengozian 

8 (1984) reported severe suppression of the mixed lymphocyte response and moderate suppression 

9 of the graft-versus-host response in mice exposed in utero to 150 mg/kg from GD 11 to 17. Both 

10 effects persisted until 18 mo of age. Holladay and Smith (1994) found that mice exposed to 0, 

11 50, 100, or 150 mg/kg from GD 13 to 17 exhibited severe fetal thymic atrophy when examined 

12 on GD 18. In the same study, expression data of cell surface markers (e.g. CD4, CD8) indicate 

13 that benzo[a]pyrene may inhibit and/or delay thymocyte maturation, possibly contributing to the 

14 observed thymic atrophy. Several other studies also show decreased thymocyte numbers and 

15 disrupted T cell maturation after in utero exposure to benzo[a]pyrene (Rodriguez et al., 1999; 

16 Holladay and Smith, 1995; Lummus and Henningsen, 1995; Urso et al., 1992; Urso and Johnson, 

17 1987). 

18 In addition to direct thymus effects, Holladay and Smith (1994) reported a large reduction 

19 in total cellularity in the fetal liver, which is the primary hematopoietic organ during gestation 

20 and a major source of thymocyte precursors beginning around GD 10-11 in mice (Pennit and 

21 Vaddeur, 1989; Landreth and Dodson, 2005). This was accompanied by decreased expression of 

22 terminal deoxynucleotidyl transferase (TdT), an intracellular marker known to be present in 

23 cortical thymocyte progenitors in the fetal liver (Fine et al., 1990; Silverstone et al., 1976). This 

24 data suggests that benzo[a]pyrene also disrupts liver hematopoiesis during gestation and may 

25 interfere with prolymphoid seeding of the thymus, possibly contributing to thymic atrophy and 

26 cell-mediated immunosuppression. Rodriguez et al. (1999) assessed downstream affects of T cell 

development by showing that CD4+27 T-cells were reduced in the spleen of 1-week old mice 

28 following in utero benzo[a]pyrene exposure. 

29 There is also some evidence of humoral immune disruption by benzo[a]pyrene during 

30 fetal life. In a series of related studies, mice exposed to benzo[a]pyrene during mid (GD 11 to 

31 13) or late (GD 16-18) gestation or both (GD 11 to 17) exhibited severe suppression of the 

32 plaque-forming cell response to sheep red blood cells from 1 wk up to 18 mo after birth (Urso 

33 and Gengozian, 1984, 1982, 1980). In their analysis of fetal liver cells, Holladay and Smith 

34 (1994) reported large decreases in expression of TdT and CD45R cellular markers, both of which 

35 are present on pre-B lymphocytes. 

36 

37 4.4.3. Cancer Bioassays (Other Routes of Exposure)
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1 Cancer bioassays following i.p. injection of mice with benzo[a]pyrene have consistently 

2 found cancer responses. Newborn mouse bioassays involving postnatal injections of 

3 benzo[a]pyrene (generally in the dose range of 0.5–1 μmol/mouse on PNDs 1, 8, and 15) 

4 consistently found increases in liver or lung tumors, either increases in incidence of animals with 

5 tumors or increased numbers of tumors per animal (LaVoie et al., 1994, 1987; Busby et al., 1989, 

6 1984; Weyand and LaVoie, 1988; Wislocki et al., 1986; Buening et al., 1978; Kapitulnik et al., 

7 1978). Likewise, i.p. injection of pregnant mice with benzo[a]pyrene (100–150 mg/kg) during 

8 gestation induced increased incidences of offspring with liver or lung tumors, compared with 

9 controls (Urso and Gengozian, 1984; Bulay and Wattenberg, 1971). A/J adult mice given single 

10 i.p. injections of benzo[a]pyrene showed a dose-related increase in the number of lung tumors 

11 per mouse with doses ranging from about 5 to 200 mg/kg (Mass et al., 1993). 

12 Tumorigenic responses to s.c. administered benzo[a]pyrene have been observed mostly at 

13 the site of injection in studies with mice (Nikonova, 1977; Pfeiffer, 1977; Homburger et al., 

14 1972; Roe and Walters, 1967; Grant and Roe, 1963; Steiner, 1955; Rask-Nielson, 1950; Pfeiffer 

15 and Allen, 1948; Bryan and Shimkin, 1943; Barry et al., 1935). 

16 Positive cancer responses from other routes of exposure have included: (1) mammary 

17 tumors in rats with intramammilary administration (Cavalieri et al., 1991, 1988a, b, c); 

18 (2) cervical tumors in mice with intravaginal application (Naslund et al., 1987); (3) injection site 

19 sarcomas with intramuscular injection (Sugiyama, 1973); (4) respiratory tract tumors in hamsters 

20 with intratracheal instillation (Henry et al., 1973); and (5) tracheal epithelial tumors in rats with 

21 intratracheal implantation (Topping et al., 1981, Nettesheim et al., 1977). 

22 

23 4.4.4. Atherogenesis Studies 

24 Cigarette smoking (see Ramos and Moorthy, 2005; Miller and Ramos, 2001; Thirman et 

25 al., 1994, for review) and, to a more limited degree, occupational exposure to PAH mixtures 

26 (Burstyn et al., 2005) have been identified as risk factors associated with the development of 

27 atherosclerotic vascular disease and increased risk for cardiovascular mortality. Based on results 

28 from in vivo and in vitro animal studies, reactive metabolites of PAHs, including 

29 benzo[a]pyrene, are thought to play a role in the progression of atherosclerosis leading to 

30 hardening and thickening of the arteries (see Ramos and Moorthey, 2005; Miller and Ramos, 

31 2001 for review). For example, in vivo exposure of Sprague-Dawley rats to 10 mg/kg 

32 benzo[a]pyrene i.p. injections (once/week for 8 weeks) induced aortic wall lesions related to 

33 atherosclerosis including loss of endothelial integrity and increase of smooth muscle cell mass 

34 (Zhang and Ramos, 1997). The molecular mechanisms responsible for PAH-induced vascular 

35 injury and the development of atherosclerosis are not well established, but current hypotheses 

36 include roles for cell proliferative responses to injury of endothelial cells from reactive 

37 metabolites (including ROS) and genomic alterations in smooth muscle cells from reactive 
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1 metabolites leading to transformed vasculature cells and eventual plaque formation (Ramos and 

2 Moorthy, 2005). 

3 Although many studies have been conducted in animal systems to study the mechanisms 

4 by which PAHs may participate in the initiation and promotion of atherosclerosis, no studies 

5 were located that examined relationships between levels of exposure to benzo[a]pyrene (via 

6 environmentally relevant routes) and the development of aortic wall lesions related to 

7 atherosclerosis, with the exception of a series of experiments involving repeated exposure of 

8 Apolipoprotein E knock out (ApoE-/-) mice to oral doses of 5 mg/kg benzo[a]pyrene (Knaapen 

9 et al., 2007; Curfs et al., 2005, 2004; Godschalk et al., 2003). ApoE-/- mice develop 

10 spontaneous atherosclerosis, which is thought to be due to enhanced oxidative stress from the 

11 lack of ApoE (Godschalk et al., 2003). 

12 Treatment of male ApoE-/- mice with gavage doses of 5 mg/kg-day benzo[a]pyrene for 

13 4 days produced increased levels of lipid peroxidation-derived DNA modifications (etheno-DNA 

14 adducts) and BPDE-DNA adducts in aorta, compared with unexposed ApoE-/- controls 

15 (Godschalk et al., 2003). Repeated exposure of male ApoE-/- mice to 5 mg/kg once a week for 

16 12 or 24 weeks did not cause enhancement of the initiation of plaques in the aortic arch 

17 (compared with unexposed ApoE-/- controls), but caused larger plaques with increased plaque 

18 layering and number of lipid cores, and increased plaque content of T-lymphocytes, compared 

19 with unexposed ApoE-/- controls (Curfs et al., 2004). In another study, gavage exposure of male 

20 ApoE-/- mice with 5 mg/kg benzo[a]pyrene or 5 mg/kg benzo[e]pyrene (BeP) once per week for 

21 24 weeks similarly increased plaque size and T-lymphocyte content (Curfs et al., 2005). In 

22 addition, exposure to benzo[a]pyrene, and to a lesser extent BeP, was associated with increased 

23 transforming growth factor beta (TGFβ1) protein levels in plaque macrophages; TGFβ1 is 

24 thought to play a role in the migration of T-lymphocytes. No exposure-related differences were 

25 noted in the location or number of plaques, oxidative DNA damage (assessed by immunostaining 

26 for 8-hydroxydeoxyguanosine, 8-OHdG), or apoptosis in the plaques (Curfs et al., 2005). As 

27 expected, the lungs of benzo[a]pyrene-exposed mice showed several benzo[a]pyrene-DNA 

28 adducts, which were not detectable in the lungs of BeP-exposed or control ApoE-/- mice (Curfs 

29 et al., 2005). In another study, increased expression of monocyte-chemoattractant protein-1 

30 (MCP-1) was found in aortic tissue from male ApoE-/- mice exposed to 5 mg/kg benzo[a]pyrene 

31 once per week by gavage for 2 weeks; this protein is thought to recruit monocytes into 

32 atherosclerotic lesions (Knaapen et al., 2007). 

33 In summary, the results of the studies with ApoE-/- mice indicate that repeated oral 

34 exposure to 5 mg/kg gavage doses of benzo[a]pyrene enhance the progression of (but do not 

35 initiate) atherosclerosis through a general local inflammatory process. The involvement of PAH36 DNA adducts was not evident in these studies, as indicated by observations that BeP, which does 

37 not cause DNA adducts, elicited similar plaque responses in ApoE-/- mice as benzo[a]pyrene. 

38 Although these results demonstrate that repeated oral exposure to 5 mg/kg benzo[a]pyrene can 
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1 enhance atherosclerosis in animals, the altered genetic disposition of ApoE-/- mice limits their 

2 usefulness in describing human-relevant dose-response relationships for oral exposure to 

3 benzo[a]pyrene and atherosclerosis. 

4 

5 4.4.5. Reproductive Studies (Other Routes of Exposure) 

6 Mattison et al. (1980) examined the response to i.p. exposure to a single dose of 

7 benzo[a]pyrene in female DBa/2N mice (n=15 per dose group); effects on fertility, primordial 

8 oocyte destruction, and response to pregnant mare’s serum gonadotropins were evaluated. In the 

9 10 week breeding study, dose groups included the vehicle control (corn oil) and 10, 100, 200 and 

10 500 mg benzo[a]pyrene/kg. Complete infertility was seen in the 200 and 500 mg/day groups, 

11 with decreased fertility seen in the 10 and 100 mg/kg dose groups, too. The total number of pups 

12 born was 137, 91, 28, 0 and 0 and the mean number of pups per mouse per week was 0.91, 0.61, 

13 0.20, 0.0, 0.0 in the 0, 10, 100, 200 and 500 mg/kg dose groups, respectively (p < 0.05 for 

14 comparison of 0 to 10 mg/kg groups and 20 to 100 mg/kg groups). In a parallel study using a 

15 single i.p. dose administered 21 days before sacrifice, the percent of primordial oocytes 

16 destroyed (compared with controls) was 0, 18, 19, 56, 88, 100% for doses of 0, 5, 10, 50, 100, 

17 and 200 mg benzo[a]pyrene/kg. The differences at doses ≥ 50 mg/kg were statistically 

18 significant (p < 0.05) compared with controls. The results from these studies were used to 

19 calculated an ED50 (i.e., dose producing a reduction in fertility or number of oocytes) of 25.5 

20 mg/kg for fertility reduction and 24.5 mg/kg for primordial oocyte destruction. There was no 

21 effect of benzo[a]pyrene exposure on ovary weight or response to pregnant mare’s serum 

22 gonadotropin, indicating that the effect of exposure did not involve ovulation inhibition. 

23 Another acute exposure study examined the effect of benzo[a]pyrene exposure on 

24 ovulatory response (as determined by number of corpora lutea) in female C57BL/6N mice 

25 (Swartz and Mattison, 1985). Benzo[a]pyrene was given as a control dose (corn oil vehicle), and 

26 1, 5, 50, 100, 500 mg/kg i.p., 20 animlals were included per dose group and 5 were sacrificed at 

27 weekly intervals. Ovaries were removed and serial sections were examined for histological 

28 changes and counts of corpora lutea. There was a 35% mortality rate in the 500 mg/kg group, 

29 but no evidence of treatment-related mortality in the other groups. Mean number of corpora 

30 lutera in controls varied between 5.5 and 10.0 for the samples taken at 1, 2, 3, and 4 weeks post31 dose administration, with no time-related trend of increasing or decreasing number. The 1 mg/kg 

32 dose group exhibited no decrease in number of corpora lutea compared with controls at any time 

33 period (mean count varying between 6.2 and 7.2). The number of corpora lutea was decreased (p 

34 < 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 

35 5.0, 10, 50, 100 and 500 mg/kg compared with 6.8 and 10.0 in the control and 1 mg/kg groups, 

36 respectively); at 2 weeks decreases were seen at ≥ 50 mg/kg and at weeks 3 and 4 a decrease was 

37 seen only at doses ≥ 100 mg/kg. Thus in addition to the destruction of primordial follicles seen 
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1 in Mattison et al. (1980), this study also demonstrated an inhibition of ovulation by 

2 benzo[a]pyrene that was time- and dose-related. 

3 Miller et al. (1992) used the protocol described by Swartz and Mattison (1985) to further 

4 examine the ovarian effects of acute benzo[a]pyrene exposure. As in the previous experiment, 

5 doses of 1, 5, 10, 50, 100 and 500 mg/kg i.p. (with corn oil vehicle control) were administered to 

6 female C57BL/6N mice (20 per group; 5 sacrificed at 1, 2, 3, and 4 weeks post-dose 

7 administration). In addition to counts of corpora lutea, total ovarian volume and total and 

8 individual corpora lutea volume were measured. The results pertaining to dose- and time9 dependent descreases in corpora lutea matched the results seen in Swartz and Mattison, 1985), 

10 and similar trends were seen in the total ovarian volume and total corpora luteal volume 

11 measures. However, volume of individual corpora lutea was increased in the treated animals 

12 compared with controls at weeks 2, 3 and 4 post-treatment. The authors note that the recovery 

13 seen in the effect on corpora lutea number by 2 weeks post-treatment at the lower doses may 

14 reflect an effect specifically on antral follicles, whereas the longer recover period at higher doses 

15 indicates an additional effect on growing follicles. 

16 Borman et al. (2000) compared the ovarian effects of benzo[a]pyrene, two other PAHs 

17 (9,10-dimethylbenzanthracene and 3-methylcholanthrene in female B6C3F1 mice and Fischer 

18 344 rats); the ovotoxic 4-vinylcyclohexene (VHC)and its diepoxide metabolite (4-

19 vinylcyclohexene diexpoxide, VHD) were also included to allow calculation of an “ovotoxic 

20 index.” as positive controls. Doses of 0.0, 0.0075, 0.015, 0.075, 0.15, 0.75, 3.5, 7.5 and 15 

21 mg/kg in the mouse, and an additional dose of 60 mg/kg for the rat were administered i.p. 

22 (sesame oil vehicle) daily for 15 days (6-7 animals per treatment group). The size of the ovaries 

23 and the number of primordial, primary and secondary (containing an oocyte) follicles was 

24 determined was determined after sacrifice (4 hours after the last dose administration). The 

25 ovotoxic index was defined as the lowest dose that resulted in a 50% loss (ED50) of primordial 

26 follicles. For benzo[a]pyrene in mice, the ED50 was 3 mg/kg (0.012 mmol/kg) for primordial 

27 follicles, a 50% loss in primary follicles was seen at 7.5 mg/kg (0.03 mmol/kg), but this level of 

28 loss of secondary follicles was not seen even at the highest dose used. The ED50 for primordial 

29 follicle loss was 0.02 mg/kg for dimethylbenzanthracene and 0.045 mg/kg for 3-

30 methylcholanthrene. In rats, the 60 mg/kg (0.24 mmol/kg) dose of benzo[a]pyrene resulted in a 

31 50% loss of primary and secondary follicles, but a much smaller decrease in primordial follicles 

32 was seen (approximately 75% of control counts in the 15 and 60 mg/kg groups). 

33 

34 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MOA 

35 benzo[a]pyrene is a complete carcinogen in that it both initiates and promotes tumor 

36 formation. Several mechanistic processes have been associated with benzo[a]pyrene 

37 carcinogenicity, including oxidative metabolism, which gives rise to reactive intermediates (see 

38 Section 3.3), and formation of DNA adducts, both of which can lead to genotoxicity and 
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1 mutations in specific cancer-related genes. The ability of benzo[a]pyrene to function as a tumor 

2 promoter may be related to cytotoxicity, AhR affinity, and upregulation of genes related to 

3 biotransformation, growth, and differentiation. 

4 The following sections discuss mechanistic evidence for possible key events in the MOA 

5 for cancer (a topic that is further discussed in Section 4.7.3). Information regarding MOAs for 

6 noncancer effects noted above is discussed in Section 4.6.3. 

7 

8 4.5.1. Genotoxicity 

9 The ability of benzo[a]pyrene to cause mutations and other forms of DNA damage in 

10 both in vivo and in vitro studies is well documented (see Tables 4-24, 4-25 and 4-26). With 

11 metabolic activation (inclusion of S9) benzo[a]pyrene is consistently mutagenic in the 

12 prokaryotic Salmonella/Ames and E. coli assays (Table 4-24). A rare exception was observed, in 

13 which benzo[a]pyrene did not induce mitotic recombination in eukaryotic S. cerevisiae

14 regardless of the presence of S9. In mammalian in vitro studies, benzo[a]pyrene is consistently 

15 mutagenic, clastogenic and induces cell transformation both with and without metabolic 

16 activation (Table 4-25). Cytogenetic damage in the form of chromosomal aberrations, 

17 micronuclei, sister chromatid exchanges and aneuploidy are commonplace following 

18 benzo[a]pyrene exposure as are DNA adduct formation, single strand breaks, and induction of 

19 DNA repair and unscheduled DNA synthesis. The in vitro mammalian cell assays were 

20 conducted in various test systems, including human cell lines. 

21 In in vivo studies, benzo[a]pyrene consistently tested positive in multiple species and 

22 strains and under various test conditions in the following assays: cell transformation, 

23 chromosomal aberrations, DNA adducts, DNA strand breaks, micronuclei formation, gene 

24 mutations (H-ras, K-ras, p53, lacZ, Hprt), sister chromatid exchanges, sperm abnormality, and 

25 unscheduled synthesis. Negative results were nominally interspersed throughout the in vivo 

26 mammalian assays, except for consistently negative results observed for unscheduled DNA 

27 synthesis. 

28 In human in vivo studies, exposures were to mixed PAHs through cigarette smoke or 

29 occupational exposure. In a subset of these studies, benzo[a]pyrene-specific DNA adducts have 

30 been detected, and it has been demonstrated qualitatively that benzo[a]pyrene metabolites 

31 damage DNA in exposed humans (see Table 4-26). 

32 
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Table 4-24. In vitro genotoxicity studies of benzo[a]pyrene in non-mammallian cells 

Result 

+S9 – S9 Reference 

Endpoint/Test System: prokaryotic cells Forward mutation S. typhimurium TM677 + – Rastetter et al. (1S. typhimurium TM677 + ND Babson et al. (19Reverse mutation S. typhimurium TA1537; TA1538 + – Ames et al. (1973S. typhimurium TA1535 – – Ames et al. (1973S. typhimurium TA98; TA1538 + ND Ames et al. (1975S. typhimurium TA1537; TA1538 + – Glatt et al. (1975S. typhimurium TA 1535 – – Glatt et al. (1975S. typhimurium TA98; TA100; TA1538 + ND McCann et al. (1S. typhimurium TA 1535 – ND McCann et al. (1S. typhimurium TA1538 + ND Egert and Greim S. typhimurium TA1537 + ND Oesch et al. (197S. typhimurium TA1538, TA98 + – Wood et al. (197S. typhimurium TA98; TA100; TA1537 + – Epler et al. (1977S. typhimurium TA1535 – – Epler et al. (1977S. typhimurium TA98; TA100 + – Obermeier and FS. typhimurium TA100 + ND Tang and FriedmS. typhimurium TA98 + – Pitts et al. (1978) S. typhimurium TA100 + ND Bruce and HeddlS. typhimurium TA98, TA100 + ND LaVoie et al. (19S. typhimurium TA1538 + – Rosenkranz and PS. typhimurium TA98, TA100 + – Simmon et al. (19S. typhimurium TA98 + ND Hermann (1981) S. typhimurium TA98, TA100 + ND Alfheim and RanS. typhimurium TA100 + ND Norpoth et al. (19S. typhimurium TA98, TA100, TA 1538 ND – Glatt et al. (1985S. typhimurium TA97, TA98, TA100 + – Sakai et al. (1985
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S. typhimurium TA100 + – Carver et al. (198S. typhimurium TA98 + – Alzieu et al. (198S. typhimurium TA97, TA98, TA100, TA1537 + – Glatt et al. (1987S. typhimurium TA97, TA98, TA100 + ND Marino (1987) S. typhimurium TA100 + ND Pahlman and PelS. typhimurium TA 98, TA100 + – Prasanna et al. (1S. typhimurium TA98 + ND Ampy et al. (198S. typhimurium TA98, TA100 + ND Bos et al. (1988) S. typhimurium TA98 + ND Lee and Lin (198S. typhimurium TA100 + ND Phillipson and IoS. typhimurium TA98 + ND Antignac et al. (1S. typhimurium TA98 – ND Gao et al. (1991) S. typhimurium TA98 + ND Balansky et al. (1S. typhimurium TA100 – ND Balansky et al. (1DNA damage E. coli/pol A + – Rosenkranz and PE. coli/differential killing test + – Tweats (1981) E. coli WP2-WP100/rec-assay + ND Mamber et al. (19E. coli/SOS chromotest Pq37 + – Mersch-SundermEndpoint/Test System: Non-mammalian eukaryotesMitotic recombination S. cerevisiae D4-RDII ND – Siebert et al. (198S. cerevisiae D3 – – Simmon (1979b) 
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1 2 

Table 4- 25. In vitro genotoxicity studies of benzo[a]pyrene in mammalian cells 

Assay/Test System

Result 

+S9 – S9 Reference 

Forward Mutation Human AHH-1 lymphoblastoid cells ND + Danheiser et al. (1989) Human lymphoblast (AHH-1) cells (Hprt) ND + Crespi et al. (1985) Human lymphoblastoid (AHH-1) cell line ND + Chen et al. (1996) Human fibroblast (MRC5CV1) cell line (Hprt) – ND Hanelt et al. (1997) Human lymphoblast (TK) cells ND + Barfknecht et al. (1982) Human lymphoblast (TK6) cells + ND Crespi et al. (1985) Human embryonic epithelial (EUE) cells ND + 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 + ND 

Amacher et al. (1980); Amacher and Turner (1

Mouse lymphoma (L5178Y/TK+/-) cells + – Amacher and Paillet (19Mouse lymphoma (L5178Y/TK+/-) cells + ND Arce et al. (1987) Human HSC172 lung fibroblasts + – Gupta and Goldstein (1Human Q3-wp normal lung keratinocytes + ND Allen-Hofmann and RhHuman SCC-13Y lung keratinocytes ND + Allen-Hofmann and RhChinese 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/OuaR ND + Mishra et al. (1978) DNA damage DNA adducts 
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Human fibroblast (MRC5CV1) cell line + ND Hanelt et al. (1997) Human peripheral blood lymphocytes ND + Wienke et al. (1990) Hamster tracheal cells ND + Roggeband et al. (1994Chinese 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 cells ND + Roggeband et al. (1994DNA damage/single strand breaks Human fibroblast (MRC5CV1) cell line + ND Hanelt et al. (1997) Human hepatoma (HepG2) cell line ND + Tarantini et al. (2009) Human prostrate carcinoma (DU145) cell line ND + Nwagbara (2007) Mouse embryo fibroblast (C3H/10T1/2 CL 8) cells ND + Lubet et al. (1983) Rat C18 trachea epithelial cells ND + 

Cosma and Marchok, 19Cosma et al. (1988) 

Rat lymphocytes ND + Gao et al. (1991) Unscheduled DNA synthesis HeLa cells + ND Martin et al. (1978) Human fibroblasts + ND Agrelo and Amos (1981Human fibroblasts + – Robinson and Mitchell Human HepG2 [+] Valentin-Severin et al. (Hamster Primary embryo cells ND + Casto et al. (1976) Hamster tracheal cells ND + Roggeband et al. (1994Rat Hepatocytes [+] Michalopoulos et al. (19Rat tracheal cells ND – Roggeband et al. (1994DNA repair Human mammary epithelial cells ND + Leadon et al. (1988) Human skin fibroblasts ND + Milo et al. (1978) Baby hamster kidney (BHK21/c13) cells [+] Feldman et al. (1978) 
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secondary mouse embryo fibroblasts (C57BL/6) and human lymphocytes [+] Shinohara and Cerutti (Rat/F344 hepatocytes ND + Williams et al. (1982) Cytogenetic damage Chromosomal aberrations Human blood cells ND + 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) Micronuclei Human AHH-1 lymphoblastoid cells ND + Crofton-Sleigh et al. (19Human HepG2 liver cells ND + Wu et al. (2003b) Human lymphoblastoid (TK) cells ND + Fowler et al. (2010) Human MCL-5 lymphoblastoid cells ND + Crofton-Sleigh et al. (19Human peripheral blood lymphocytes + ND Lo Jacono et al. (1992) Chinese hamster V79 cells ND + Whitwell et al. (2010) Chinese hamster V79-MZ cells ND + Matsuoka et al. (1999) Sister chromatid exchanges Human C-HC-4 and C-HC-20 hepatoma cells ND + Abe et al. (1983a,b) Human diploid fibroblast (TIG-II) cell line + + Huh et al. (1982) Human fibroblasts ND + Juhl et al. (1978) Human blood cells ND + Salama et al. (2001) Human peripheral blood lymphocytes ND + Rudiger et al. (1976) Human peripheral blood lymphocytes ND + Craig-Holmes and Shaw Human peripheral blood lymphocytes ND + Schoenwald et al. (1977Human peripheral blood lymphocytes ND + Wienke et al. (1990) Human peripheral blood lymphocytes ND + Wienke et al. (1990) 
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Human peripheral blood lymphocytes + – Tohda et al. (1980) Human peripheral blood lymphocytes + ND Lo Jacono et al. (1992) Chinese hamster Don-6 cells ND + 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. (1Chinese hamster V79 lung epithelial cells + ND Arce et al. (1987) Chinese hamster V79 lung epithelial cells ND + Kulka et al. (1993) Chinese hamster ovary (CHO) cells + – de Raat (1979) Chinese hamster ovary (CHO) cells + – Husgafvel-Pursiainen eChinese hamster ovary (CHO) cells ND + Wolff and Takehisa (19Chinese hamster ovary (CHO) cells ND + Pal et al. (1978) Hamster Chl cells ND + Shimizu et al. (1984) Rabbit peripheral blood lymphocytes ND + Takehisa and Wolff (19Rat ascites hepatoma AH66-B ND + Abe et al. (1983a,b) Rat esophageal tumor R1 ND + 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) cells ND + Dean et al. (1983) Rat liver cell line ARL18 ND + Tong et al. (1981) Rat pleural mesothelial cells ND + Achard et al. (1987) Aneuploidy Chinese hamster V79-MZ cells ND + Matsuoka et al. (1998) Cell transformation Human BEAS-2B lung cells + van Agen et al. (1997) Human breast epithelial (MCF-10F, MCF-7, T24) cell lines 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) cells ND + DiPaolo et al. (1969, 19Syrian hamster embryo cells ND + Dunkel et al. (1981) 
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Syrian hamster embryo cells + LeBoeuf et al. (1996) Syrian hamster embyro (SHE) cells/focus assay ND + Casto et al. (1977) Fetal Syrain hamster lung (FSHL) cells ND + Emura et al. (1980, 198Virus infected rat embryo RLV/RE and RAT cells; mouse embryo AKR/Me cells; Syrain hamster embryo cells + Heidelberger et al. (198Virus transformed Syrian hamster embryo and mouse C3H10T1/2 cells ND + Arce et al. (1987) Mouse C3H/10T1/2 embryo fibroblasts + Nesnow et al. (2002, 19Mouse embryo fibroblast (C3H/10T1/2 CL 8) cells ND + Peterson et al. (1981) Mouse embryo fibroblast (C3H/10T1/2 CL 8) cells ND + Lubet et al. (1983) Mouse SHE cells; BALB/c-3t3 cells; C3H/10T1/2 cells; prostate cells + Heidelberger et al. (198Mouse BALB/c-3T3 cells ND + Dunkel et al. (1981) Mouse BALB/c-3T3 cells + Matthews (1993) Mouse BALB/c-3T3 clone A31-1-1 ND + Little and Vetroys (198Rat embyro cells/SA7 virus transformation ND + DiPaolo and Casto (197Rat/Fischer, embryo cells (leukemia virus transformed) ND + Dunkel et al. (1981) Rat/Fischer, embryo cells/OuaR ND + Mishra et al. (1978) Key: [+] = S9 status not given; “+” = positive; “-“ = negative; ND = not determined. 

1 2 
148 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-26. In vivo genotoxicity studies of benzo[a]pyrene 

Endpoint Test System Test Conditions Results Dose Comment 

Mutations 

Human/Blood T 

lymphocytes (smokers 

and nonsmokers); HPRT

locus mutation assay 

T-cells of lung cancer patients 

(smokers and nonsmokers from 

lung cancer patients and population 

controls with known smoking 

status) analyzed for HPRT locus 

mutations. 

+ 

Smokers and 

nonsmokers 

Splicing mutations, bas

pair substitutions, frames

and deletion mutation

observed. Smokers an

nonsmokers had GC-->T

transversions (13% and 

respectively) and GC-->

transitions (24% and 35

respectively) in HPRT g

consistent with in vitr

mutagenicity of 

benzo[a]pyrene 

Mutations 

Mouse/strains:T-stock, 

(SEC×C57BL)F1, 

(C3H×101)F1, 

(C3H×C57BL)F1 for 

females; (101×C3H)F1

or (C3H×101)F1 for 

males; dominant-lethal 

mutation assay 

12-wk old males dosed with 

benzo[a]pyrene i.p. and mated 3.5-

6.5 days post-treatment with 12-wk 

old females from different stocks; 

sacrificed on days 12-15 after 

vaginal plug was observed; 

females kept in a 5 hr-dark phase 

to synchronize ovulation 5 wks 

before the start of the expt.; 

fertilized eggs collected from 9-11 

hrs after mating and first-cleavage 

metaphase chromosomes prepared 

20 hrs after mating 

+ 500 mg/kg b.w. 

The % of dominant leth

mutations were in the or

of T-stock = (C3H×101)

(SEC×C57BL)F1> 

(C3H×C57BL)F1

Mutations, GC 

Mice/strains: Male 

stocks: (101×C3H)F1; 

Female stocks (A): 

(101xC3H)F1, (B): (C3Hx101)F1, (C): 

(C3HxC57BL)F1, 

(D):(SECxC57BL)F1, 

(E):T-stock females; 

dominant lethal 

mutations 

In dominant lethal assay (DLA), 

12-wk-old males dosed i.p. with 

benzo[a]pyrene and mated with 10-

12 wk-old (#1) stock A females; or 

(#2) stock B females on the day of 

dosing; or with (#3a) with stocks 

B, C and D females 3.5-7.5 days 

post-dosing, or with (#3b) with 

stocks B, C, D and E females 3.5-

6.5 days post-dosing. Control 

group mated at time corresponding 

to 1.5-4.5 days post-treatment in 

positive 

for 

DLA; 

negative 

for HT 

500 mg/kg 

b.w. 

Dominant lethal effect

were observed in early 

middle (4.5-5.5 and 6.5-

days post-treatment, 

respectively) spermatoz

and in preleptotene 

spermatocytes (32.5-33

and 34.5-35.5 days pos

treatment). In the HTA, 

significant difference

observed between treat

and control progeny. 
149 DRAFT - DO NOT CITE OR QUOTE 

the test groups. 

Mutations, GC 

Mice/strains: Male 

stocks: (101×C3H)F1; 

Female stocks (A): 

(101xC3H)F1, (B): (C3Hx101)F1, (C): 

(C3HxC57BL)F1, 

(D):(SECxC57BL)F1, 

(E):T-stock females; 

heritable translocations 

For heritable translocation assay 

(HTA), males were mated with 

stocks B and D females 3.5-7.7 

days post-benzo[a]pyrene 

treatment and male progeny 

screened for translocation 

heterozygosity. 

– 

500 mg/kg 

b.w. 

No significant differenc

observed between treat

and control progeny. 

Mutations, GC/ 

spot test 

Mouse/C57BL female × 

T-strain male; somatic 

mutation assay 

Mice mated for a 5-day period; 

101/4 days post-appearance of 

vaginal plug, females injected i.p. 

with benzo[a]pyrene or vehicle; 

offspring (pups) scored for 

survival, morphology and presence 

of white near-midline ventral spots 

(WMVS) and recessive spots (RS). 

+ 

100 and 500 

mg/kg b.w. 

Induced coat color mosa

represent genetic chang

(e.g. point mutations) 

somatic cells. WMVS a

RS represent melanocy

cell killing and 

mutagenicity, respective

Benzo[a]pyrene caused h

incidence of RS but did 

correlated with WMVS

Mutations Mouse/lacZ transgenic (MutaTM Mouse) 

benzo[a]pyrene given orally in 

corn oil for 5 consecutive days; 

sacrificed 14 days after last dosing; 

Eleven organs analyzed for lacZ

MF 

+ 

125 

mg/kg/day 

Highest MF observed 

colon followed by ileum 

forestomach > bone mar

= spleen > glandular 

stomach > liver = 

lung>kidney = heart 

Mutations 

Mouse/C57BL/6J Dlb-1

congenic; Dlb-1locus 

assay 

Animals dosed i) i.p. with vehicle 

or benzo[a]pyrene 2, 4, or 6 doses 

at 96 hr intervals; or ii) single dose 

of benzo[a]pyrene given i.p. or p.o. 

alone or 96 hours following a 

single i.p. dosing with 10 μg/kg 

TCDD 

+ 40 mg/kg b.w. 

benzo[a]pyrene caused 

dose-dependent increase 

mutant frequency; i.p. ro

showed higher mutan

frequency than p.o. rou

induction of mutations w

associated with Ahresponsiveness. 
150 DRAFT - DO NOT CITE OR QUOTE 

Mutations (Hprt 

locus) 

Mice/C57BL/6 (lacz

negative and XPA+/+ and 

XPA-/-); T lymphocytes 

Gavage in corn oil 3 times/wk for 

0, 1, 5, 9, 13 wks; sacrificed 7 wks 

after last treatment 

+ 13 mg/kg Mutation sensitivity: XPA-/- > XPA+/+ 

Mutations 

Mouse/Cockayne 

syndrome-deficient (Csb- /-); heterozygous (Csb+/-) 

and wild type controls

(Csb+/+); Hprt mutation 

frequency assay 

Csb-/-/lacZ+/- and Csb+/-/lacZ+/- mice were dosed i.p. with 

benzo[a]pyrene thrice a wk for 5, 

9, or 13 wks; For Hprt MF analysis 

mice were sacrificed 3 wks after 

last treatment; spleenocytes 

collected; For lac Z MF analysis 

mice were sacrificed 3 days after 

last treatment and liver, lung and 

spleen collected.

+ 13 mg/kg 

lac Z MF detected in a

tissues but no differenc

between WT and Csb-

mice; Hprt mutations 

significantly higher in C

mice than control miceBPDE-dGuo adducts i

Hprt gene are preferenti

removed in WT mice th

Csb-/- mice.

Mutations 

Mouse/B6C3F1, 

forestomach H-ras, Kras & p53 mutations benzo[a]pyrene given in feed in a 2-year chronic feeding study; + 5, 25, 100 ppm 

68% K-ras (codons 12,1

10% H-ras (codon 13), 1

p53 mutations; all G-->

transversions 

Mutations 

Mouse/lacZ/galE

(MutaTM Mouse); Skin 

painting study 

Mice topically treated with a single 

dose or in five divided doses daily; 

sacrificed 7 or 21 days after the 

single or final treatment; DNA 

from skin, liver and lung analyzed 

for mutations. 

+Sk or –

Li,Lu

1.25 or 2.5 

mg/kg (25 or 

50 μg/mouse) 

Skin showed significan

dose- and time-depende

increase in mutation 

frequency; liver and lun

showed no mutations; M

for single or multiple-do

regimens were similar

Mutations/ spot 

test Mouse/T-strain 

benzo[a]pyrene given to pregnant mice by gavage in 0.5 ml corn oil 

on GDs 5-10 

+ 10 mg/mouse (5 x 2 mg) 

Mutations (Hprt 

locus) 

Mouse, 129/Ola (Wild 

type); splenic T 

lymphocytes 

Single i.p. injection followed by 

sacrifice 7 wks post-treatment + 

0, 50, 100, 

200, 400 

mg/kg 

dose-dependent increase 

Hprt MF 

Mutations Mouse, A/J, male Single i.p. injection followed by sacrifice 28 days post-treatment + 0, 0.05, 0.5, 5 50 mg/kg Dose-dependent increas

lung tissue K-ras codon 

GT mutation frequen

Mutations/ gene 

Mouse/CD-1; skin 

papillomas (Ha-ras

mutations) 

Female mice were initiated 

topically with a single dose of 

benzo[a]pyrene and 1 wk after 

+ 

600 

nmol/mouse 

About 90% of papillom

contained Ha-ras mutatio

all of them being 
151 DRAFT - DO NOT CITE OR QUOTE 

initiation promoted twice weekly 

with 5 nmol TPA for 14 wks. One 

month after stopping TPA 

application, papillomas collected 

and DNA from 10 individual 

papillomas were analyzed for Haras mutations by PCR and direct 

sequencing. 

transversions at codons 

(20% GGA-->GTA), 1

(50% GGC-->GTC), and 

(20% CAA--> CTA)

Mutation/In 

vivo-in vitro Rat, Wistar 

Single dose by gavage; urine and 

feces collected 0-24, 24-48 and 48-

72 hrs post-treatment; urine and 

extracts of feces tested in S. 

typhimurium TA100 strain with or 

without S9 mix and β- glucuronidase 

+ 0, 1, 5, 10, 100 mg/kg 

Fecal extracts and urin

showed mutagenicity at 

above 1 and 10 mg/kg b

Benzo[a]pyrene, 

respectively. Highes

mutagenic activity obser

for 0-24 hrs post-treatm

for feces and 24-48 hrs p

treatment for urine with 

glucuronidase ± S9 mi

Mutations, GC 

/gene 

D. melanogaster/ sexlinked recessive lethal 

test

Basc males exposed to 

benzo[a]pyrene were mated with 

virgin females of Berlin K or mei9L1strains; ± 10 mM 

Data inconclusive due 

low fertility rates of mei

females. 

Mutations, GC 

/gene 

D. melanogaster/ sexlinked recessive lethal 

test

Adult Berlin males treated orally 

with benzo[a]pyrene + 5 or 7.5 mM Low mutagenic activit

Mutations, GC 

/gene 

D. melanogaster/BerlinK and Oregon-K strains; 

sex-linked recessive 

lethal test

benzo[a]pyrene dissolved in 

special fat and injected into the 

abdomen of flies. 

– 2 and 5 mM Negative at both dose

Mutations, GC 

/gene 

D. melanogaster/ sexlinked recessive lethal 

test

Male Berlin K larvae treated with 

benzo[a]pyrene for 9-11 days + 0.1-4 mM 

Threefold enhancement 

lethals in treated versu

controls 
152 DRAFT - DO NOT CITE OR QUOTE 

Mutations, GC 

/gene 

D. melanogaster/CantonS (WT) males, FM6 

(homozygous for an X 

chromosome) females; 

sex-linked recessive 

lethal test

Adult male flies were fed on filters 

soaked in benzo[a]pyrene for 48 or 

72 hrs; Treated and control males 

mated with FM6 females, males 

transferred to new groups of 

females at intervals of 3, 2, 2, and 

3 days; four broods obtained; a 

group of 100 daughters of each 

male were mated again; scored for 

% lethal 

– 250, 500 ppm 

Authors report incompl

dissolution of 

benzo[a]pyrene in DMSO 

a possible cause of nega

result. 

Mutation/gene 

D. melanogaster; 

somatic mutation - eye 

color mosaicism

50 females and 20 females were 

mated in a culture bottle for 48 hrs 

allowing females to oviposit; 

adults then discarded and the eggs 

allowed to hatch; larvae fed on 

benzo[a]pyrene deposited on food 

surface and the emerging adult 

males scored for mosaicly colored 

eye sectors; 

+ 1, 2, or 3 mM 

benzo[a]pyrene was 

effective as a mutagen; 

dose-response observe

DNA adducts Human/white blood cells 

Workers were exposed for 6-8 

hrs/day for at least 4-6 months 

before blood collection; leukocyte 

DNA isolated, digested and 

benzo[a]pyrene tetrols analyzed by 

HPLC with fluorescent detection 

(HPLC-FD). Low, medium, and 

high exposure groups correspond 

to < 0.15, 0.15 to 4, > 4 mg/m3 of 

benzo[a]pyrene, respectively. 

+ 

< 0.15, 0.15 

to 4, > 4 

μg/m3 of 

benzo[a]pyre

ne 

PAH exposure, CYP1A

status and smoking 

significantly affected DN

adduct levels, i.e. 

CYP1A1(*1/*2 or *2A/*

> CYP1A1*1/*1; 

occupational > 

environmental exposur

smokers > nonsmoker

adducts increased with d

and duration of smokin

DNA adducts Human/white blood cells 

Coke oven workers were exposed 

to PAHs and benzo[a]pyrene-WBC 

DNA analyzed by HPLC-FD for 

BPDE-DNA adducts 

+ 0.14 μg/m3

BPDE-DNA adducts 

detectable; no significa

difference between smok

and nonsmokers; no 

correlation with air 

benzo[a]pyrene levels a

adduct levels 
153 DRAFT - DO NOT CITE OR QUOTE 

DNA adducts 

and Mutations 

Mouse/C57BL/6 lacZ

transgenic 

Mice dosed with single i.p. 

injection of benzo[a]pyrene in 

DMSO; sacrificed 1, 3, 5, 7, 14, 

21, and 28 days post-treatment; 

spleen, lung, liver, kidney and 

brain collected, DNA isolated and 

analyzed for mutations in lacz 

reporter gene in E. coli and adducts 

by 32P-postlabeling assay. 

+ 50 mg/kg b.w. 

BPDE-dG adduct levepeaked between 5 and 

days post-treatment, 

followed by gradual decl

rate of removal highest 

lung, liver and spleen a

lowest in kidney and bra

mutant frequencies peak

between 7 and 14 days 

lung, spleen, liver and 

kidney; brain was not 

significant at any time po

DNA adducts Mice/ (Ahr+/+, Ahr+/-,Ahr-/-) Gavage; sacrificed 24 hr post- treatment + 100 mg/kg b.w. No induction of CYP i

Ahr-/-, but all alleles posi

for adduct formation 

DNA adducts 

Mice/C57BL/6J 

Cyp1a1(+/-) and 

Cyp1a1(-/-) 

Single i.p. injection; sacrificed 24 

hrs post-treatment; liver DNA 

analyzed by 32P-postlabeling assay 

+ 500 mg/kg b.w. BPDE-DNA adduct lev

4-fold higher in Cyp1a1(

mice than Cyp1a1(+/-) m

DNA adducts Mouse/B6C3F1 

benzo[a]pyrene fed in diet for 4 

(100 ppm) or for 1, 2, 8, 16 and 32 

wks (5 ppm); sacrificed and liver, 

lungs, forestomach, small intestine 

collected; DNA analyzed by 32Ppostlabeling assay 

+ 

5 ppm (32 

wks) and 100 

ppm (4 wks) 

Linear dose-response in 

wk study; the 5 ppm gro

showed a plateau after 

wks of feeding 

DNA adducts Mouse/Balb/c; 

Single i.p. injection; sacrificed 12 

hrs post-injection; liver and 

forestomach collected; DNA 

binding of [3H]benzo[a]pyrene 

analyzed by scintillation counting. 

+ 

140 μCi/100 

g b.w. 

Liver DNA had 3-fold 

higher binding of 

benzo[a]pyrene than tha

forestomach 

DNA adducts 

Mice/BALB/cAnN 

(BALB), CBA/JN 

(CBA); 32P-postlabeling 

assay 

Animals dosed i.p. with or without 

24 hr pretreatment with TCDD + 

50 and 200 

mg/kg 

Adduct levels similar i

both strains dosed wit

benzo[a]pyrene alone

TCDD pre-treatment ha

greater suppressive effec
154 DRAFT - DO NOT CITE OR QUOTE 

adduct formation in BA

relative to CBA mice at 

dose but resulted in no 

significant difference i

adduct levels at high do

DNA adducts Mice/BALB/c, skin; 

Four doses of benzo[a]pyrene 

topically applied to the shaved 

backs of animals at 0, 6, 30 and 54 

hrs; sacrificed 1 day after last 

treatment; DNA analyzed by 32Ppostlabeling assay 

+ 

4 x 1.2 

μmol/animal Five adducts spots detec

DNA adducts Mice/Swiss, epidermal and dermal skin 

Single topical application on 

shaved backs; sacrificed 1, 3, and 7 

days post-treatment; epidermal and 

dermal cells separated; DNA 

isolated, digested with DNAseI 

and estimated DNA binding; 

adducts separated by HPLC. 

+ 

250 nmol in 

150 μl 

acetone 

Both cells positive fo

benzo[a]pyrene adduct

epidermis>dermis; addu

persisted up to 7 days wi

gradual decline in leve

DNA adducts Rats/CD, PBLs, lungs and liver 

Single i.p. injection; sacrificed 3 

days post-treatment; DNA 

analyzed by Nuclease P1-

endhanced 32P-postlabeling assay. 

+ 

2.5 

mg/animal 

BPDE-dG as major addu

and several minor addu

detected in all tissues 

DNA adducts Rats/Sprague-Dawley, liver 

Single i.p. injection followed by 

sacrifice at 4 hours post-treatment; 

liver DNA isolated and analyzed 

by 32P-postlabeling assay. 

+ 100 mg/kg b.w. Two adduct spots detec

DNA adducts Rat/Lewis; lung and liver 

Animals received a single oral 

dose of benzo[a]pyrene in 

tricaprylin; sacrificed 1, 2, 4, 11, 

and 21 days post-dosing; analyzed 

liver and lung DNA for BP-DNA 

adducts by 32P-postlabeling assay 

and urine for 8-oxodG adducts by 

HPLC-ECD. 

+ 10 mg/kg 

BPDE-dG levels peaked 

days after exposure in b

tissues, higher in lungs t

liver at all time points

decline faster in liver th

lung; Increased 8-oxod

levels in urine and 

decreased levels in liver 

lung. 
155 DRAFT - DO NOT CITE OR QUOTE 

DNA adducts Rats/F344; 32P- postlabeling assay 

benzo[a]pyrene given in the diet 

for 30, 60, or 90 days; animals 

sacrificed and liver and lung 

isolated and DNA extracted and 

analyzed for adducts. 

+ 0, 5, 50, 100 mg/kg Adduct levels linear at l

and intermediate dose

nonlinear at high dose

DNA adducts Rats/Wistar; liver and PBL adducts 

Single dose by gavage; sacrificed 

24 hrs post-dosing; PBL and liver 

DNA analyzed by 32P-postlabeling 

for BP-DNA adducts 

+ 0, 10, 100 mg/kg b.w. 

At 100 mg dose total add

levels in PBL were twof

higher than the levels i

liver; adduct profiles 

differed between PBL a

liver 

DNA strand 

breaks 

Rats/Sprague-Dawley; 

Comet assay 

instilled intratracheally with (i) 

single dose of benzo[a]pyrene in 

aqueous suspension; sacrificed at 

3, 24, 48 hrs post-treatment; 

alveolar macrophages, lung cells, 

lymphocytes, hepatocytes collected 

(ii) dose-response study and 

sacrificed at 24 hours posttreatment; lungs collected; 

Controls received normal saline 

instillation; All cells analyzed by 

comet assay. 

+ 

Expt#1: 3 mg 

of 

benzo[a]pyre

ne; Expt#2: 

dose-response 

study with 

0.75, 1.5, 3 

mg 

benzo[a]pyre

ne 

All time points showe

significant increase in S

(Expt#1); A dose-respon

in SSB observed (Expt#

DNA strand 

breaks 

Aquatic organisms:Carp 

(Cyprinus carpio), 

rainbow trout 

(Oncorhynchus mykiss), 

and clams (Spisula 

sachalinensis); Comet 

assay 

All organisms acclimatized in 

tanks for 2 days, water changed 

every 24 hrs; exposed to 

benzo[a]pyrene in DMSO in a 

tank; one third volume of tank 

contents changed every 12 hrs; 

organisms sacrificed at 24, 48, 72, 

and 96 hrs post-treatment; cell 

suspensions prepared from liver 

(carp and trout) or digestive gland 

(clam) for comet assay 

+ 

0.05, 0.25, 

0.5 and 1 

ppm 

Significant dose-respon

for strand breaks observ

carp and trout liver show

highest response at 48 h

and clam digestive glan

showed time-dependen

increase at highest con

DNA strand 

breaks Rat, Brown Norway UDS determined after 5 and 18 hrs of a single i.g. dosing – 62.5 mg/kg negative at both time po
156 DRAFT - DO NOT CITE OR QUOTE 

Unscheduled 

DNA synthesis Rats/F344; 

Single i.p. injection of 

benzo[a]pyrene or DMSO; 

sacrificed at 2 or 12 hrs postexposure; liver isolated, hepatocyte 

cultures were setup and incubated 

with 10 mCi/ml 3H-thymidine for 4 

hrs; washed and autoradiography 

performed 

– 

100 mg/kg 

b.w. 

benzo[a]pyrene was 

negative at both time po

Unscheduled 

DNA synthesis 

Mouse/HOS:HR-1 

hairless; skin 

Single topical application on two 

spots on the backs after stripping 

stratum corneum with adhesive 

tape to enhance penetration; 

sacrificed 24 hr post-treatment, 

skin isolated [3H]thymidine; cultured in ; epidermal UDS 

measured 

+ 

0, 0.25, 0.5 

and 1% (w/v) 

in acetone 

UDS index showed a do

dependent increase up 

0.5% benzo[a]pyrene do

and then plateaued 

Unscheduled 

DNA synthesis 

Rat/Brown Norway; 

liver 

Single intragastric injection; 

sacrificed at 5 and 18 hours postinjection – 62.5 mg/kg b.w. benzo[a]pyrene was negative at both time po

Unscheduled 

DNA synthesis 

Mouse/(C3Hf x 101)F1

hybrid, germ cells 

i.p. injection of benzo[a]pyrene; 

[3H]Thymidine injection later – 0.3 mL Concentration not specif

Unscheduled 

DNA synthesis Mouse, early spermatid i.p. injection – 250-500 mg/kg b.w. Reviewed by Sotomay

and Sega (2000) 

Chromosomal 

aberrations 

Hamster/Chinese bone 

marrow 

Single, i.p. injection of 

benzo[a]pyrene dissolved in 

tricapryline; animals sacrificed 24 

hours post-exposure 

+ 25, 50, 100 mg/kg b.w. 

benzo[a]pyrene induce

CAs at 50 mg/kg/bw on

with negative responses 

the low and high dose 

Chromosomal 

aberrations 

Mice/C57 (high AHH 

inducible) and DBA 

(low AHH inducible) 

strains; 11-day old 

embryos; adult bone 

marrows 

Study used 4 matings 

(female×male): C57×C57; 

DBA×DBA; C57×DBA; 

DBA×C57; Pregnant mice treated 

orally on GD11 with 

benzo[a]pyrene; sacrificed 15 hrs 

post-treatment; material liver, bone 

+ 150 mg/kg 

Levels of CAs: hybrid 

embryos > homozygou

DBA embryos > 

homozygous C57 embry

Tissue AHH activity: C

mothers and their embry

DBA females and thei
157 DRAFT - DO NOT CITE OR QUOTE 

marrow and placenta and embryos 

collected; male mice dosed 

similarly and bone marrows 

collected; individual embryo cell 

suspensions and bone marrow 

preparations scored for CAs. 

Tissue AHH activity measured. 

homozygous embryos. N

quantitative correlatio

between BP-induced C

and AHH inducibility. N

differences in bone marr

mitotic index of males 

different strains betwee

control and treatment 

groups. 

Chromosomal 

aberrations 

Mouse/1C3F1 hybrid 

(101/E1xC31×E1)F1; 

CAs in bone marrow 

Single dose by gavage; sacrificed 

30 hrs of post-dosing; bone 

marrow from femur isolated and 

analyzed for CAs 

+ 63 mg/kg 

Significant increase in C

in benzo[a]pyrene-treat

animals compared to 

controls. 

Chromosomal 

aberrations Rats/Wistar; PBLs 

Single dose by gavage; sacrificed 

6, 24 and 48 hrs post-treatment; 

blood from abdominal aorta 

collected, whole blood cultures set 

up, CAs scored in 100 firstdivision PBLs per animal 

– 

0, 10, 100, 

200 mg/kg 

b.w. 

No difference betwee

control and treatment 

groups at any dose or at 

sampling time observe

Micronuclei Hamster, Chinese, bone 

marrow 

Single, i.p. injection of 

benzo[a]pyrene dissolved in 

tricaprylin; animals sacrificed 30 

hours post-exposure 

– 

100, 300, 500 

mg/kg b.w. 

Micronuclei Mice/B6C3F1 (hybrid); I.p. injection; several doses given to calculate LD50 + 

232 mg/kg 

(LD50/7); 

259 mg/kg 

(LD50/4)

Study conducted to 

determine the toxicity 

benzo[a]pyrene (LD50

Micronuclei Mouse/CD-1 and BDF1; bone marrow Dosed orally once, twice or thrice at 24 hr intervals; sacrificed 24 hrs after last treatment + 250, 500, 1000, 2000 mg/kg b.w. significant increase at a

doses; no dose-respons

double dosing at 500 mg

dose gave best respons

Micronuclei 

Mouse/CD-1 & BDF1, 

peripheral blood 

reticulocytes 

Given single i.p injection; tail 

blood collected at 24 hr intervals 

from 0 to 72 hrs 

+ 

62.5, 125, 

250, 500 

mg/kg b.w. 

maximum response seen 

48 hrs post-treatment 

Micronuclei 

Rat/Sprague-Dawley, 

peripheral blood 

reticulocytes 

Given single i.p injection; tail 

blood collected at 24 hr intervals 

from 0 to 96 hrs 

+ 

62.5, 125, 

250, 500, 

1000 mg/kg 

b.w. 

maximum response seen 

72 hrs post-treatment 
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Micronuclei Mouse/ICR [Hsd: (ICR)Br] 

benzo[a]pyrene was heated in olive 

oil and given orally as a single 

dose; males, females and pregnant 

mothers used; pregnant mice dosed 

on GDs 16-17 and sacrificed on 

GDs 17-18; micronuclei evaluated 

in adult bone marrow and fetal 

liver 

+ 150 mg/kg b.w. 

All groups significantl

higher than controls fo

MN; Fetal liver more 

sensitive than any othe

group 

Micronuclei Mouse/Swiss albino; bone marrow Given orally in corn oil; sacrificed 24 hr post-exposure + 75 mg/kg b.w. Micronuclei Mouse/Swiss; bone marrow PCE Given by gavage and sacrificed 36 hrs post-treatment + 75 mg/kg b.w. Micronuclei Mice/CD-1 and MS/Ae strains i.p. and p.o. administration + 62.5, 125, 250, 500 mg/kg 

good dose response by b

routes, strains; i.p. bett

than p.o.; MS/Ae strai

more sensitive than CD

strain 

Micronuclei Mouse/BDF1, bone 

marrow 

Male and female mice aged 12-15 

wks given single i.p. injection of 

benzo[a]pyrene or corn oil; 

sacrificed 24, 48, and 72 hrs posttreatment; bone marrow smears 

prepared, stained with MayGrunwald-Giemsa technique and 

scored for MN PCEs. 

+ 0, 25, 50, 60 mg/kg b.w. 

Positive at all doses, tim

points and sexes tested

Dose-dependent increas

MN observed in both sex

males responded better t

females; highest positiv

response observed at 72 

post-injection 

Micronuclei Mouse, HRA/Skh hairless, keratinocytes Single topical application + 0.5, 5, 50, 100, 500 mg/mouse Micronuclei 

Mouse/HOS:HR-1, 

hairless; skin 

micronuclei 

Topical application once daily for 

3 days; sacrificed 24 hrs after last 

treatment 

+ 0.4, 1, 2, 4 

mg 

Micronuclei 

Mice/HR-1 hairless, skin 

(benzo[a]pyrene with 

slight radiation) 

 + 

Exposure to sunlight 

simulator to evaluate 

photogenotoxicity and 

chemical exposure 

Micronuclei 

Rat, Sprague-Dawley, 

pulmonary alveolar 

macrophages 

i.t. instillation, once/day for 3 days + 25 mg/kg b.w. 
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Micronuclei Rat, Sprague-Dawley, bone marrow cells i.t. instillation, once/day for 3 days – 25 mg/kg b.w. Micronuclei 

Fish (Carp, rainbow 

trout, clams); Blood and 

hemolymph 

 + 

0.05, 0.25, 

0.5 and 1 

ppm 

Sister 

chromatid 

exchanges 

Hamster/Chinese; SCEs 

in bone marrow 

8-12 wk-old animals dosed with 

two i.p. injections of 

benzo[a]pyrene given 24 hrs apart; 

animals sacrificed 24 hrs after last 

treatment, bone marrow from 

femur isolated and metaphases 

analyzed. 

+ 450 mg/kg b.w. 

Significant increase in 

metaphase SCEs in 

benzo[a]pyrene-treate

animals compared to 

vehicle-treated control

Sister 

chromatid 

exchanges 

Hamster/Chinese, 

Animals implanted s.c. with 

bromodeoxyuridine (BrdU) tablet; 

2 hrs later given phorone (125 or 

250 mg/kg) i.p.; another 2 hrs later 

dosed i.p. with benzo[a]pyrene; 24 

hrs post-BrdU dosing, animals 

injected with colchicine 10 mg/kg 

b.w., sacrificed 2 hrs later; bone 

marrow from femur prepared for 

SCE assay 

+ 

50 or 100 

mg/kg b.w. 

SCEs increased with lo

dose of phorone 

significantly. 

Sister 

chromatid 

exchanges 

Hamster/Syrian, fetal 

liver 

i.p. injection to pregnant animals 

on GDs 11, 13 or 15; fetal liver 

SCEs were analyzed 

+ 

50 and 125 

mg/kg b.w. 

Produced doubling of S

frequency 

Sister 

chromatid 

exchanges 

Hamster/Chinese, bone 

marrow 

NA + 

2.5, 25, 40, 50, 75, 100 

mg/kd b.w. 

Frequency of SCEs 

increased ≥40 mg/kg b.

Sister 

chromatid 

exchanges 

Mouse/DBA/2 & 

C57BL/6, bone marrow 

cells 

Two intragastric injections given; 

mice implanted with BrdU tablets, 

sacrificed on day 5, SCE estimated 

+ 

10 or 100 

mg/kg b.w. 

SCEs and BP-DNA addu

in the order of C57Bl/

(AHH-inducible) < DBA

(AHH-noninducible) 

Sister 

chromatid 

exchanges 

Mouse/DBA/2 & 

C57BL/6, spleenic 

lymphocytes 

Two intragastric injections given; 

mice killed on 5th day and cells 

cultured for 48 hrs with BrdU. 

+ 

10 or 100 

mg/kg b.w. 

SCEs and BP-DNA addu

in the order of C57Bl/

(AHH-inducible) < DBA

(AHH-noninducible) 
160 DRAFT - DO NOT CITE OR QUOTE 

Sister 

chromatid 

exchanges 

Rats/Wistar; PBLs 

Single dose by gavage; sacrificed 

6, 24 and 48 hrs post-treatement; 

blood from abdominal aorta 

collected, whole blood cultures set 

up, SCEs scored in 50 seconddivision metaphases in PBLs per 

animal 

+ 0, 10, 100, 200 mg/kg 

Linear dose-response at 

sampling time, howeve

significant at the highe

dose only; no interactio

between dose and sampl

time 

Cell 

transformation 

Hamsters/LVG:LAK 

strain (virus free); 

Transplacental hostmediated assay 

Pregnant animals dosed i.p. with 

benzo[a]pyrene on GD 10; 

sacrificed on GD 13, fetal cell 

cultures prepared, 10×106

cells/plate; 5 days post-culture 

trypsinized; subcultured every 4-6 

days thereafter and scored for 

plating efficiency and 

transformation. 

+ 3 mg/100 g b.w. 

8-oxodG, 8-oxodeoxyguanosine; AHH, aryl hydrocarbon hydroxylase; benzo[a]pyrene, benzo[a]pyrene; BPDE, benzo[a]pyrene diol epoxbromodeoxyuridine; CAs, chromosomal aberrations; CSB, Cockayne syndrome; CYP, cytochrome P450; DLA, dominant lethal assay; DM

dimethylsulfoxide; ECD, electrochemical detection; FD, fluorescence detection; FM6, First Multiple No. 6 is an X chromosome with a com

suppress cross-over) and visible markers such as yellow body, white eyes and narrow eyes. GC, germline cell; GD, gestational day; HPLC

liquid chromatography; HPRT, hypoxanthine-guanine phosphoribosyl transferase; HTA, heritable translocation assay; i.p., intraperitoneal

Liver; Lu, Lung; MF, mutation frequency; PBL, peripheral blood lympbocytes; PCE, polychromatic erythrocytes; SCEs, sister chromatid 

synchronous fluorescence spectrometry; Sk Skin TCDD, 2,3,7,8-tetrachlorodibenzodioxin; TPA, 12-tetradecanoyl-O-phorbol acetate; UD

synthesis; USERIA, ultrasensitive enzyme radioimmunoassay; WT, wild-type; XP, xeroderma pigmentosum; 
161 DRAFT - DO NOT CITE OR QUOTE 

1 

2 4.5.2. Metabolic pathways 

3 Diol epoxide pathway

4 benzo[a]pyrene diol epoxide metabolites interact preferentially with the exocyclic amino 

5 groups of deoxyguanine and deoxyadenine (Geacintov et al., 1997; Jerina et al., 1991). Adducts 

6 may give rise to mutations unless these adducts are removed by DNA repair processes prior to 

7 replication. The stereochemical nature of the diol epoxide metabolite (i.e., anti- vs. syn-diol 

8 epoxides) affects the number and type of adducts and mutation that occurs (Geacintov et al., 

9 1997). Transversion mutations (e.g., GC→TA or AT→TA) are the most common type of 

10 mutation found in mammalian cells following diol epoxide exposure (Bostrom et al., 2002). 

11 Strong evidence for the association between benzo[a]pyrene activation by the diol 

12 epoxide pathway and key DNA-reactive and mutational events associated with tumor initiation 

13 comes from the following observations: (1) (+)-anti-BPDE is very reactive with guanine 

14 residues in DNA (Koreeda et al., 1978; Jeffrey et al., 1976); (2) (+)-anti-BPDE is more potent 

15 than benzo[a]pyrene, benzo[a]pyrene phenols, and benzo[a]pyrene diols in mutagenicity assays 

16 in bacterial and mammalian cells (Malaveille et al., 1977; Newbold and Brookes, 1976); 

17 (3) When administered by ip injection to newborn mice, (+)-anti-BPDE is more potent than 

18 benzo[a]pyrene phenols and benzo[a]pyrene diols and much more potent than benzo[a]pyrene 

19 itself in lung tumorigenicity assays (Chang et al., 1987; Buening et al., 1978; Kapitulnik et al., 

20 1978); (4) (+)-anti-BPDE treatment resulted in ras gene codon 12 G→T point mutations, the 

21 activation of the H-ras-1 proto-oncogene and transformation of NIH/3T3 cells (Marshall et al., 

22 1984); (5) (+)-anti-BPDE forms DNA adducts at specific “hotspots” in the p53 tumor suppressor 

23 gene that are commonly mutated in lung and other cancer patients (Denissenko et al., 1996; 

24 Puisieux et al., 1991); (6) lung tumors from nonsmoking patients who were chronically exposed 

25 to smoky coal emissions contained mutated p53 and showed a spectrum of mutations consistent 

26 with (+)-anti-BPDE-associated mutations in the K-ras oncogene (DeMarini et al., 2001); (7) 

27 elevated blood BPDE-DNA adducts have been observed in coke oven workers and chimney 

28 sweeps, occupations associated with increased risks of cancer from PAH-containing complex 

29 mixtures (Pavanello et al., 1999); (8) the spectrum of mutation in the K-ras, H-ras, and p53 genes 

30 in forestomach tumors of mice fed benzo[a]pyrene in the diet for 2 years was consistent with (+)-

31 anti-BPDE DNA reactions (Culp et al., 2000); (9) K-ras mutations found in lung tumors from 

32 A/J mice given single i.p. injections of benzo[a]pyrene showed several guanine mutations at 

33 codon 12, which are indicative of (+)-anti-BPDE DNA adduct formation (Ross and Nesnow, 

34 1999; Nesnow et al., 1998a, b, 1996, 1995; Mass et al., 1993); and (10) the major DNA adduct 

35 formed in a murine embryonic fibroblast line transfected with human p53 DNA and exposed to 

36 1uM benzo[a]pyrene for 96 hours was (+)-anti-BPDE-DNA. The concomitant spectrum of p53 

37 mutations in the latter study had features similar to those found in human lung cancer: 
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1 predominance of G→T mutations, strand bias of transversions, and mutation hot spots at codons 

2 157 to 158 (Liu et al., 2005). 

3 As pointed out by Penning et al. (1999), the association between BPDE-DNA adducts 

4 and tumors from benzo[a]pyrene exposure is not entirely specific given that dihydrodiol and diol 

5 epoxides of benzo[a]pyrene are less potent tumorigenic agents in mouse skin than the parent 

6 material (Slaga et al., 1977; Chouroulinkov et al., 1976) and oxidative damage to DNA has been 

7 observed in rats treated with benzo[a]pyrene (Kim and Lee, 1997) and human mammary 

8 epithelial cells exposed to benzo[a]pyrene (Leadon et al., 1988). In addition, although BPDE9 DNA adduct levels in forestomach tissue were linearly related to the amount of benzo[a]pyrene 

10 consumed by mice in a 28-day study (Culp et al., 2000, 1998, 1996a; Culp and Beland, 1994), 

11 levels of BPDE-DNA adduct in lung and liver tissue (which did not develop tumors with 2 years 

12 of exposure to benzo[a]pyrene in the diet) were similar at 28 days to those in forestomach tumors 

13 (Goldstein et al., 1998). These observations suggest that BPDE-adduct levels alone are not the 

14 only path to benzo[a]pyrene-induced tumors and provide indirect evidence for the other 

15 mutagenic pathways to benzo[a]pyrene tumor initiation. 

16 

17 Radical cation pathway 

18 Radical cation formation involves a one-electron oxidation by CYP or peroxidase 

19 enzymes (i.e., horseradish peroxidase, prostaglandin H synthetase) that produces electrophilic 

20 radical cation intermediates (Cavalieri and Rogan, 1995, 1992). Radical cations can be further 

21 metabolized to phenols and quinones (Cavalieri et al., 1988d, e), or they can form unstable 

22 adducts with DNA that ultimately result in depurination (Cavalieri et al., 2005, 1993; Rogan et 

23 al., 1993). The predominant depurinating adducts occur at the N-3 and N-7 positions of adenine 

24 and the C-8 and N-7 positions of guanine (Cavalieri and Rogan, 1995). 

25 Abasic sites resulting from base depurination undergo error-prone excision repair and can 

26 induce mutations such as those found in the H-ras oncogene in mouse skin (Chakravarti et al., 

27 2000). One pathway to the formation of depurinating DNA adducts involves the formation of 

28 DNA-reactive radical cations from benzo[a]pyrene via CYP peroxidases (Cavalieri and Rogan, 

29 1995). In mouse skin exposed to 200 nmol benzo[a]pyrene for 4 hours, a mix of stable and 

30 depurinating DNA adducts was found: (+)-anti-BPDE DNA and depurinating adducts accounted 

31 for 22 and 74% of the identified adducts, respectively (Rogan et al., 1993). When mouse skin 

32 was exposed to either the benzo[a]pyrene-7,8-diol or BPDE, only stable BPDE-DNA adducts 

33 were found (Rogan et al., 1993). In mouse skin tumors induced by benzo[a]pyrene and 

34 promoted by TPA, 7/13 examined tumors had H-ras oncogene mutations attributed to apurinic 

35 sites generated by loss of N-7 and C-8 guanine adducts, and 2/13 tumors had H-ras mutations 

36 attributed to loss of N-7 adenine adducts (Chakravarti et al., 1995). Results from the Rogan et al. 

37 (1993) and Chakravarti (1995) studies provide strong in vivo evidence for the importance of both 
163 DRAFT - DO NOT CITE OR QUOTE 

1 the diol epoxide and the radical cation pathways in the activation of benzo[a]pyrene to initiate 

2 mouse skin tumors, possibly by inducing mutations in critical genes. 

3 

4 o-Quinone/ROS pathway 

5 The o-Quinone metabolites of PAHs are formed by enzymatic dehydrogenation of 

6 dihydrodiols (Bolton et al., 2000; Penning et al., 1999; Harvey, 1996; ATSDR, 1995). DHH 

7 enzymes are members of the α-keto reductase gene superfamily. o-Quinone metabolites are 

8 potent cytotoxins, are weakly mutagenic, and are capable of producing a broad spectrum of DNA 

9 damage. These metabolites can interact directly with DNA as well as result in the production of 

10 ROS (i.e., hydroxyl and superoxide radicals) that may produce further cytotoxicity and DNA 

11 damage. The DNA damage caused by o-quinones may include the formation of stable adducts 

12 (Balu et al., 2004), N-7 depurinating adducts (McCoull et al., 1999), oxidative base damage (i.e., 

13 8-oxo-2’-deoxyguanosine or 8-oxo-dG) (Park et al., 2006a), and strand scission (Flowers et al., 

14 1997). The ROS generated by the o-quinone metabolites of benzo[a]pyrene and other PAHs 

15 have been shown to induce mutation in the p53 tumor suppressor gene using an in vitro yeast 

16 reporter gene assay (Park et al., 2008; Shen et al., 2006; Yu et al., 2002). 

17 The o-quinone/ROS pathway also can produce depurinated DNA adducts from 

18 benzo[a]pyrene metabolites (Jiang et al., 2007; 2005). In this pathway, and in the presence of 

NAD(P)+19 , AKR oxidizes benzo[a]pyrene-7,8-diol to a ketol, which subsequently forms 

20 benzo[a]pyrene-7-8-dione. This and other PAH o-quinones react with DNA to form unstable, 

21 depurinating DNA adducts. In the presence of cellular reducing equivalents, o-quinones can also 

22 activate redox cycles which produce DNA-ROS (Penning et al., 1996). DNA damage in in vitro

23 systems following exposure to benzo[a]pyrene-7,8-dione or other o-quinone PAH derivatives 

24 occurs through the AKR pathway and can involve the formation of stable DNA adducts (Balu et 

25 al., 2004), N-7 depurinated DNA adducts (McCoull et al., 1999), DNA damage from ROS 

26 (8-oxo-dG) (Park et al., 2006a) and strand scission (Flowers et al., 1997, 1996). 

27 Benzo[a]pyrene-7,8-dione and other PAH o-quinones have been shown to induce mutations in 

28 the p53 tumor suppressor gene using an in vitro yeast reporter gene assay (Park et al., 2008; Shen 

29 et al., 2006; Yu et al., 2002). When the yeast were exposed to varying concentration of 

30 benzo[a]pyrene-7,8-dione or (+)-anti-BPDE, levels of 8-oxo-dG or (+)-anti-BPDE-DNA, 

31 adducts were linearly related to p53 mutagenic frequencies with similar slopes, suggesting that 

32 these two types of DNA lesions were equipotent in producing p53 mutations in this system (Park 

33 et al., 2008). When the p53 mutations were sorted into dominant and recessive mutants, the 

34 dominant mutations clustered to p53 mutation hotspots observed in human lung cancer tissue 

35 (Park, 2008). The combined results provide strong in vitro evidence for the potential for the o36 Quinone/ROS pathway to produce several DNA-damaging products from benzo[a]pyrene (e.g., 

37 benzo[a]pyrene-7,8-dione and ROS) that lead to p53 mutations associated with human lung 

38 cancer. In support of the operation of this pathway, and the other bioactivation pathways, in 
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1 humans, Jiang et al. (2007) used liquid chromatograpy-mass spectrometry (LC-MS) to provide 

2 evidence for the formation of radical cations, diol epoxides, and o-quinones in cultured human 

lung H358 cells following exposure to 4 μM [33 H]-benzo[a]pyrene. 

4 

5 

6 4.5.3. Mechanistic Studies- Mutagenesis and Tumor Initiation 

7 

8 Oncogene/tumor suppressor gene mutations (in vivo) 

9 DeMarini et al. (2001) demonstrated mutations in the p53 tumor suppressor gene and the 

10 K-ras oncogene in the lung tumors of nonsmokers, whose tumors were associated with exposure 

11 to smoky coal. Lung tumors were obtained from 24 nonsmoking women from China (age 30–

12 63, mean age 48.5 ± 8.8 years) who used smoky coal in their homes without chimneys. 

13 Bronchioloalveolar adenocarcinoma and acinar adenocarcinoma were observed in 54 and 46% of 

14 the women studied, respectively. The observed mutations in lung tumors were primarily G→T 

15 transversions at either K-ras or p53. Mutation hotspots in the lung tumors examined 

16 corresponded with hot spots for PAH adducts (codon 154), cigarette smoke associated mutations 

17 (codon 249), and both of these events together (codon 273). The mutation spectrum was 

18 described as unique and consistent with exposure to PAHs in the absence of cigarette smoke. 

19 Mutations in the K-ras, H-ras, and p53 genes were assessed in forestomach tumors 

20 (n = 31) of mice fed benzo[a]pyrene in the diet (0, 5, 25, or 100 ppm) for 2 years (Culp et al., 

21 2000). Forestomach tumors had K-ras mutations (68% of tumors) that were G→T or C 

22 transversions in codon 12 or 13. H-ras (codon 13) and p53 mutations characterized as G→T or 

23 C transversions were also each found in 10% of forestomach tumors. 

24 K-ras mutations were observed in A/J mouse lung tumors (Nesnow et al., 1998a, b, 1996, 

25 1995; Mass et al., 1993). Benzo[a]pyrene was administered to male A/J mice (20/group) as a 

26 single i.p. injection (0, 10, 50, 100, or 200 mg/kg in tricaprylin) and the presence of lung 

27 adenomas were evaluated 8 months following injection. The number of lung adenomas/mouse 

28 was significantly greater than control (p < 0.05) for benzo[a]pyrene doses ≥50 mg/kg. Lung 

29 tumor DNA was isolated and DNA sequence analysis of K-ras mutations was performed for 19 

30 separate lung tumors. The DNA sequence analysis demonstrated several guanine mutations at 

31 codon 12, including GGT→TGT (56% of tumors), GGT→GTT (25% of tumors), and 

32 GGT→GAT (19% of tumors). 

33 H-ras mutations were studied in skin papillomas of SENCAR mice resulting from dermal 

34 initiation by benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol (400 nmol) followed by TPA 

35 promotion (Chakravarti et al., 2000, 1995). PCR amplification of the H-ras gene and sequencing 

36 revealed that codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas 

37 corresponded to the relative levels of depurinating adducts of guanine and adenine, despite the 

38 formation of significant amounts of stable DNA adducts. 
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1 

2 DNA adducts in target tissues detected following chemical exposure 

3 DNA adducts of benzo[a]pyrene have been measured in target tissues of humans exposed 

4 to PAH mixtures and experimental animals exposed to benzo[a]pyrene. Phillips et al. (2002) 

5 provided a review of smoking-related DNA adducts detected in human respiratory tract tissues. 

6 BPDE-DNA adducts were detected in the lung, parenchyma of cigarette smokers with lung 

7 cancer (Godschalk et al., 2002; Bartsch et al., 1999; Alexandrov et al., 1992). DNA was isolated 

8 from the normal tissue (i.e., noncancerous) of the lung which was obtained during surgery. A 

9 study using lung samples obtained on autopsy revealed that the average level of BPDE-DNA 

adducts was higher in smokers (4.46 ± 5.76 per 10810 bases) compared to ex-smokers (4.04 ± 2.37 

per 108 bases) and nonsmokers (1.76 ± 1.69 per 10811 bases) (Lodovici et al., 1998). 

12 BPDE-DNA adducts were measured in skin biopsies of eczema patients treated with coal 

13 tar preparations (Godschalk et al., 2001). Godschalk et al. (1998a) performed a study examining 

14 the level of DNA adducts in biopsies of treated skin and in WBCs in psoriasis patients being 

15 treated with coal tar. Urinary 1-OH-Py levels were serially monitored in all subjects. Skin 

16 biopsies were taken before and after five treatments, at which point the average number of DNA 

adduct levels increased from 2.9 to 63.3 per 108 17 nucleotides. Total WBC DNA adducts increased 

from 0.33 to 0.89 per 108 after five treatments, and then doubled to 1.59 per 108 18 when sampled 

19 1 week later. There was an increase in 1-OH-Py levels from 0.75 to 186 μg/L after one treatment 

20 and to 266 μg/L after five treatments. One week later, mean 1-OH-Py levels were reduced to 

21 2.4 μg/L. Adduct levels in the skin increased over 20-fold with five treatments, while WBC 

22 adduct levels approximately doubled over the same period. 

23 DNA adduct levels were examined in the forestomach of groups of female B6C3F1 mice 

24 fed benzo[a]pyrene in the diet at concentrations of 5, 25, or 100 ppm for 28 days (Culp et al., 

2000, 1998, 1996a, b; Culp and Beland, 1994). [32 25 P]-postlabeling of forestomach DNA of 

benzo[a]pyrene-treated mice revealed one major adduct characterized as dG-N226 -BPDE. There 

27 was a linear relationship between the amount of benzo[a]pyrene consumed and the concentration 

of dG-N228 -BPDE in the forestomach of mice. For benzo[a]pyrene, forestomach tumor incidence 

29 increased sharply with adduct concentrations between 50 and 140 fmol/mg DNA and in coal-tar 

fed mice. Tumor incidence increased sharply with dG-N230 -BPDE adduct levels between 20 and 

31 60 fmol/mg DNA. The same levels of adduct were present in lung and liver of benzo[a]pyrene32 treated mice, although only the forestomach exhibited benzo[a]pyrene-induced tumors 

33 (Goldstein et al., 1998). The presence of adducts in tumor-free tissue suggests that DNA adduct 

34 levels alone are not necessarily predictors of tumor outcome. 

35 DNA adducts were identified and quantified in experiments using the A/J mouse lung 

36 model which results in lung adenomas in male A/J mice 8 months following a single i.p. 

37 injection (Nesnow et al., 1998a, b, 1996, 1995; Ross et al., 1995). Benzo[a]pyrene was 

38 administered to male A/J mice (20-25/group) as a single i.p. injection (0, 20, 50, or 100 mg/kg in 
166 DRAFT - DO NOT CITE OR QUOTE 

1 tricaprylin) and DNA was isolated from lung tissues at several time points between 1 and 21 

2 days following injection. The primary DNA adduct identified in mouse lung tumors was a 

3 benzo[a]pyrene bay region diol epoxide adduct of guanine (7R, 8S, 9S-trihydroxy-10R-[2N-2’-

4 deoxyguanosyl]-7, 8, 9, 10-tetrahydro-benzo[a]pyrene). Two minor adducts were also observed 

5 to result from the metabolism of 9-hydroxy-benzo[a]pyrene and trans-7,8-dihydroxy-7,8-

dihydro-benzo[a]pyrene. Quantitative analysis of DNA adducts by [32 6 P]-postlabeling illustrated 

7 the importance of measuring DNA adduct levels over time. Total DNA adducts accumulated 

8 rapidly between 3 and 9 days after exposure followed by a gradual decrease. A time-integrated 

9 DNA adduct level (TIDAL) was linearly related to the administered dose of benzo[a]pyrene 

10 (Ross et al., 1995). 

11 Following i.p injection, benzo[a]pyrene also induced DNA adducts in the lungs, liver, 

12 and peripheral blood lymphocytes of rats (Ross et al., 1991, 1990), the liver of Lewis rats 

13 (Godschalk et al., 1998b), and lungs of BALB/c mice (Van Schooten et al., 1991). Qian et al. 

14 (1998) treated male CD rats with intratracheal instillation of fume condensates of roofing 

15 asphalts and evaluated adducts in lung cells and WBCs. Adducts were seen in the lungs but not 

16 in WBCs, leading the authors to conclude that WBCs may not be a suitable surrogate for lung 

17 cells. The adducts were not characterized, so the benzo[a]pyrene-specificity of the results cannot 

18 be evaluated. Formation of DNA adducts from benzo[a]pyrene metabolites also has been 

19 observed in the lung and liver of male Sprague-Dawley rats after intratracheal administration of 

20 benzo[a]pyrene (De Flora et al., 1991; Weyand and Bevan, 1987). 

21 DNA adducts have been reported in the lung and skin of dermally treated SENCAR mice 

22 (Mukhtar et al., 1986), in the epidermis of Swiss mice (Oueslati et al., 1992), and in the skin of 

23 an unspecified strain of mice (Ingram et al., 2000). When Talaska et al. (1996) compared the 

24 dose-duration-response of benzo[a]pyrene-induced adducts in the skin, lung, and liver of Hsd 

25 (ICR) BR mice treated dermally with 10, 25, or 50 nmol benzo[a]pyrene, accumulation of 

26 adducts was found to be linear with dose in the skin and lung. In skin painting studies with 

27 female SENCAR mice and various PAHs, Melendez-Colon et al. (1999) found that carcinogenic 

28 potency correlated with DNA adduct levels in epidermal DNA rather than in the formation of 

29 apurinic sites. Alexandrov and Rojas-Moreno (1990) found DNA adducts in epidermal 

30 keratinocytes and dermal fibroblasts of Swiss mice treated dermally with benzo[a]pyrene but not 

31 in similarly treated Wistar rats. BPDE-DNA adducts were measured in the lung, stomach, and 

32 skin of male Lewis rats (15/group) following a single exposure to 10 mg/kg benzo[a]pyrene via 

33 the intratracheal, gavage, and dermal routes, respectively (Godschalk et al., 2000). 

34 

35 4.5.3. Tumor Promotion and Progression 

36 benzo[a]pyrene has been shown to promote the growth of previously initiated cells, 

37 resulting in the formation of tumors in the skin (see Section 4.2.3.1). The tumor promotion 
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1 properties of benzo[a]pyrene may be due to a compensatory response to cytotoxicity or via an 

2 AhR-mediated effect on cell growth and differentiation. 

3 

4 Cytotoxicity and inflammatory response 

5 The cytotoxicity of benzo[a]pyrene metabolites may contribute to tumor promotion via 

6 inflammatory responses leading to cell proliferation (Burdick et al., 2003). Benzo[a]pyrene is 

7 metabolized to o-quinones, which are cytotoxic, and can generate ROS (Bolton et al., 2000; 

8 Penning, 1999). Benzo[a]pyrene o-quinones reduce the viability and survival of rat and human 

9 hepatoma cells (Flowers-Geary et al., 1996, 1993). Cytotoxicity was also induced by 

10 benzo[a]pyrene and BPDE in a human prostate carcinoma cell line (Nwagbara et al., 2007). 

11 Inflammatory responses to cytotoxicity may contribute to the tumor promotion process. For 

12 example, benzo[a]pyrene quinones (1,6-, 3,6-, and 6,12-benzo[a]pyrene-quinone) generated ROS 

13 and increased cell proliferation by enhancing the epidermal growth factor receptor 

14 (EFGR)pathway in cultured breast epithelial cells (Burdick et al., 2003). 

15 Several studies have demonstrated that exposure to benzo[a]pyrene increases the 

16 production of inflammatory cytokines which may contribute to cancer progression. Garçon et al. 

17 (2001a, b) exposed Sprague-Dawley rats by inhalation to benzo[a]pyrene with or without ferrous 

18 oxide (Fe2O3) particles. They found that benzo[a]pyrene alone or in combination with Fe2O3

19 particles elicited mRNA and protein synthesis of the inflammatory cytokine, IL-1. Tamaki et al. 

20 (2004) also demonstrated a benzo[a]pyrene-induced increase in IL-1 expression in a human 

21 fibroblast-like synoviocyte cell line (MH7A). Benzo[a]pyrene increases the expression of the 

22 mRNA for CCL1, an inflammatory chemokine, in human macrophages (N’Diaye et al., 2006). 

23 The benzo[a]pyrene-induced increase in CCL1 mRNA was inhibited by the potent AhR 

24 antagonist 3′-methoxy-4′-nitroflavone. 

25 

26 AhR-mediated effects 

27 The promotional effects of benzo[a]pyrene may also be related to AhR affinity and the 

28 upregulation of genes related to biotransformation (i.e., induction of CYP1A1), growth, and 

29 differentiation (Bostrom et al., 2002). Figure 4-1 illustrates the function of the AhR and depicts 

30 the genes regulated by this receptor as belonging to two major functional groups (i.e., induction 

31 of metabolism or regulation cell differentiation and proliferation). PAHs bind to the cytosolic 

32 AhR in complex with heat shock protein 90 (Hsp90). The ligand-bound receptor is then 

33 transported to nucleus in complex with the ARNT. The AhR complex interacts with the Ah 

34 responsive elements (AHRE) of the DNA to increase the transcription of proteins associated with 

35 induction of metabolism and regulation of cell differentiation and proliferation. 
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AHR Hsp90 AHR

Hsp90

AHR Hsp90

Hsp90

ARNT

Hsp90 ARNT

Hsp90

AHR

ARNT

AHREDNA

PAH

nucleus

Enhanced

specific

mRNA

production

Increased

synthesis of 

PAH metabolizing 

enzymes

Increased 

synthesis of 

proteins that

regulate cell

differentiation and

proliferation

1 

2 AHREDNA = Ah-responsive elements of DNA; Hsp90 = heat shock protein 90 

3 

4 Source: Okey et al. (1994). 

5 

6 Figure 4-1. Interaction of PAHs with the AhR. 

7 

8 Binding to the AhR induces enzymes that increase the formation of reactive metabolites, 

9 resulting in DNA binding and, eventually, tumor initiation. In addition, with persistent exposure, 

10 the ligand-activated AhR triggers epithelial hyperplasia, which provides the second step leading 

11 from tumor initiation to promotion and progression (Nebert et al., 1993). Ma and Lu (2007) 

12 reviewed several studies of benzo[a]pyrene toxicity and tumorigenicity in mouse strains with 

13 high and low affinity AhRs. Disparities were observed in the tumor pattern and toxicity of Ah14 responsive (+/+ and +/-) and Ah-nonresponsive (-/-) mice. Ah-responsive mice were more 

15 susceptible to toxicity and tumorigenicity in proximal target tissues such as the liver, lung, and 

16 skin. For example, Shimizu et al. (2000) reported that AhR knock out mice (-/-), treated with 

17 benzo[a]pyrene by s.c. injection or dermal painting, did not develop skin cancers at the treatment 

18 site, while AhR-responsive (+/+) or heterozygous (+/-) mice developed tumors within 18–

19 25 weeks after treatment. Benzo[a]pyrene treatment increased CYP1A1 expression in the skin 

20 and liver of AhR-positive mice (+/- or +/+), but CYP1A1 expression was not altered by 

21 benzo[a]pyrene treatment in AhR knock out mice (-/-). Talaska et al. (2006) also showed that 

22 benzo[a]pyrene adduct levels in skin were reduced by 50% in CYP1A2 knock out mice and by 
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1 90% in AhR knock out mice compared with WT C57Bl6/J mice following a single dermal 

2 application of 33 mg/kg benzo[a]pyrene for 24 hours. Ma and Lu (2007) further noted that Ah3 nonresponsive mice were at greater risk of toxicity and tumorigenicity in remote organs, distant 

4 from the site of exposure (i.e., bone marrow). As an example, Uno et al. (2006) showed that 

5 benzo[a]pyrene (125 mg/kg/-day, p.o. for 18 days) caused marked wasting, immunosuppression, 

6 and bone marrow hypocellularity in CYP1A1 knock out mice, but not in WT mice. 

7 Some studies have demonstrated the formation of DNA adducts in the liver of AhR 

8 knock out mice following i.p. or oral exposure to benzo[a]pyrene (Sagredo et al., 2006; Uno et 

9 al., 2006; Kondraganti et al., 2003). These findings suggest that there may be alternative (i.e., 

10 non-AhR mediated) mechanisms of benzo[a]pyrene activation in the mouse liver. Sagredo et al. 

11 (2006) studied the relationship between the AhR genotype and CYP metabolism in different 

organs of the mouse. AhR+/+, 

+/–, and –/– 12 mice were treated once with 100 mg/kg benzo[a]pyrene 

13 by gavage. CYP1A1, CYP1B1, and AhR expression was evaluated in the lung, liver, spleen, 

14 kidney, heart, and blood, via RT-PCR, 24 hours after treatment. CYP1A1 RNA was increased in 

15 the lung and liver and CYP1B1 RNA was increased in the lung following benzo[a]pyrene 

treatment in AhR+/+ and +/– 16 mice (generally higher in heterozygotes). Benzo[a]pyrene treatment 

did not induce CYP1A1 or CYP1B1 enzymes in AhR–/– 17 mice. The expression of CYP1A1 RNA, 

18 as standardized to β-actin expression, was generally about 40 times that of CYP1B1. The 

19 concentration of benzo[a]pyrene metabolites and the levels of DNA and protein adducts were 

20 increased in mice lacking the AhR, suggesting that there may be an AhR-independent pathway 

21 for benzo[a]pyrene metabolism and activation. The high levels of benzo[a]pyrene DNA adducts 

in organs other than the liver of AhR–/– 22 mice may be the result of slow detoxification of 

23 benzo[a]pyrene in the liver, allowing high concentrations of the parent compound to reach 

24 distant tissues. 

25 Uno et al. (2006) also demonstrated a paradoxical increase in liver DNA adducts in AhR 

26 ko mice following oral exposure to benzo[a]pyrene. WT C57BL/6 mice and several knock out 

mouse strains (CYP1A2–/– and CYP1B1–/– single ko, CYP1A1/1B1–/– and CYP1A2/1B1–/– 27 

28 double ko) were studied. Benzo[a]pyrene was administered in the feed at 1.25, 12.5, or 125 

29 mg/kg for 18 days (this dose is well tolerated by WT C57BL/6 mice for 1 year, but lethal within 

30 days to the CYP1A1–/– 30 mice). Steady-state blood levels of benzo[a]pyrene, reached within 5 

days of treatment, were ~25 times higher in CYP1A1–/– and ~75 times higher in CYP1A1/1B1–/– 31 

32 than in WT mice, while clearance was similar to WT mice in the other knock out mouse strains. 

DNA adduct levels, measured by [32 33 P]-postlabeling in liver, spleen, and bone marrow, were 

highest in the CYP1A1–/– mice at the two higher doses, and in the CYP1A1/1B1

–/– 34 mice at the 

35 mid dose only. Adduct patterns, as revealed by 2-dimensional chromatography, differed 

36 substantially between organs in the various knock out types. 

37 Dertinger et al. (2001, 2000) demonstrated that AhR signaling may play a role in 

38 cytogenetic damage caused by benzo[a]pyrene. The in vivo formation of micronuclei in 
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1 peripheral blood reticulocytes of C57Bl/6J mice induced by a single i.p. injection of 

2 benzo[a]pyrene (150 mg/kg) was eliminated by prior treatment with the potent AhR antagonist 

3 3'-methoxy-4'-nitroflavone. This antagonist also protected AhR null allele mice from 

4 benzo[a]pyrene-induced increases in micronuclei formation, suggesting that 3'-methoxy-4'-

5 nitroflavone may also act through a mechanism independent of the AhR (Dertinger et al., 2000). 

6 Several in vitro studies have suggested that the AhR plays a role in the disruption of cell 

7 cycle control, possibly leading to cell proliferation and tumor promotion following exposure to 

8 benzo[a]pyrene (Andrysik et al., 2007; Chung et al., 2007; Chen et al., 2003). Chung et al. 

9 (2007) showed that benzo[a]pyrene-induced cytotoxicity and apoptosis in mouse hepatoma 

10 (Hepa1c1c7) cells occurred through a p53 and caspace-dependent process requiring the AhR. 

11 An accumulation of cells in the S-phase of the cell cycle (i.e., DNA synthesis and replication) 

12 was also observed, suggesting that this process may be related to cell proliferation. Chen et al. 

13 (2003) also demonstrated the importance of the AhR in benzo[a]pyrene-7,8-dihydrodiol- and 

14 BPDE-induced apoptosis in human HepG2 cells. Both the dihydrodiol and BPDE affected Bcl2 

15 (a member of a family of apoptosis suppressors) and activated caspase and p38 mitogen16 activated protein (MAP) kinases, both enzymes that promote apoptosis. When the experiments 

17 were conducted in a cell line that does not contain ARNT (see Figure 4-1), the dihydrodiol was 

18 not able to initiate apoptotic event sequences, indicating that activation to BPDE by CYP1A1 

19 was required. BPDE did not induce apoptosis-related events in a p38-defective cell line, 

20 illustrating the importance of MAP kinases in this process. In rat liver epithelial cells (WB-F344 

21 cells), in vitro exposure to benzo[a]pyrene resulted in apoptosis, a decrease in cell number, an 

22 increase in the percentage of cells in S-phase (comparable to a proliferating population of WB23 F334 cells), and increased expression of cell cycle proteins (e.g., cyclin A) (Andrysik et al., 

24 2007). Benzo[a]pyrene-induced apoptosis was attenuated in cells transfected with a dominant25 negative mutation of the AhR. 

26 

27 Inhibition of gap junctional intercellular communication 

28 Gap junctions are channels between cells that allow substances of a molecular weight up 

29 to roughly 1 kDa to pass from one cell to the other. This process of metabolic cooperation is 

30 crucial for differentiation, proliferation, apoptosis, and cell death and consequently for the two 

31 epigenetic steps of tumor formation, promotion, and progression. Chronic exposure to many 

32 toxicants results in down-regulation of gap junctions. For tumor promoters, such as TPA or 

33 TCDD, inhibition of intercellular communication is correlated with their promoting potency 

34 (Sharovskaya et al., 2006; Yamasaki, 1990). 

35 Blaha et al. (2002) surveyed the potency of 35 PAHs, including benzo[a]pyrene, to 

36 inhibit gap junctional intercellular communication (GJIC). The scrape loading/dye transfer assay 

37 was employed using a rat liver epithelial cell line that was incubated in vitro for 15, 30, or 60 

38 minutes with 50 μM benzo[a]pyrene. After incubation, cells were washed, and then a line was 
171 DRAFT - DO NOT CITE OR QUOTE 

1 scraped through the cells with a surgical blade. Cells were exposed to the fluorescent dye lucifer 

2 yellow for 4 minutes and then fixed with formalin. Spread of the dye from the scrape line into 

3 cells remote from the scrape was estimated under a fluorescence microscope. Benzo[a]pyrene 

4 reduced spread of the dye after 30 minutes of exposure (approximately 50% of control). 

5 Recovery of GJIC was observed 60 minutes after exposure. 

6 Sharovskaya et al. (2006) studied the effects of carcinogenic and noncarcinogenic PAHs 

7 on GJIC in HepG2 cells. Individual carcinogenic PAHs inhibited GJIC in a temporary fashion 

8 (70–100% within 24 hours), but removal of the PAH from culture reversed the effect. 

9 Noncarcinogenic PAHs had very little effect on GJIC. Benzo[a]pyrene at 20 μM inhibited GJIC 

10 completely within 24 hours, while its noncarcinogenic homolog, BeP, produced <20% inhibition. 

11 The effect was not AhR-dependent, because benzo[a]pyrene inhibited GJIC in HepG2 cells to 

12 the same extent as in hepatoma G27 cells, which express neither CYP1A1 nor AhR. The authors 

13 concluded that the effects of benzo[a]pyrene and BeP on GJIC were direct (i.e., not caused by 

14 metabolites). 

15 

16 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 

17 4.6.1. Oral 

18 Numerous epidemiological studies are available which investigate associations between 

19 PAH (including benzo[a]pyrene) dietary intake and cancer incidence; however, no studies were 

20 found which evaluate the contribution of benzo[a]pyrene through dietary exposure in humans 

21 and noncancer health effects. Several studies in animal models are available evaluating the 

22 sensitive noncancer effects following subchronic or chronic exposure to benzo[a]pyrene. The 

23 types of effects observed following oral exposure were predominantly effects in the reproductive 

24 and immune systems. Additionally, some minor hematological effects and kidney and 

25 forestomach effects were observed. Studies that identified NOAELs and LOAELS for 

26 noncancer effects in animals repeatedly exposed to benzo[a]pyrene by the oral route are 

27 summarized in Table 4-27. 
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Table 4-27. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]py

route 

Species/sex Dose Duration

NOAEL LOAEL 

mg/kg-d Response at LOAEL Commen

Wistar rat/ male and female

0, 3, 10, or 30 mg/kg-d, gavage, 5 d/wk 

2 yrs ND 3 ↑ Forestomach hyperplasia, ↑ liver clear 

cell foci of alteration 

Hematological and orgvariables were not meaterminal sacrifice. No exposure-related chnoncancer histology in esophagus, forestomacliver, kidney, skin, maor auditory canal (nonwere only detected in tdeveloped tumors). ↑ Forestomach tumors ≥3 mg/kg-d. ↑ Forestomach, liver, a(males only) tumors at 30 mg/kg-d. 

B6C3F1

mouse/female only 

Estimated doses: 0, 0.7, 3.3, or 16.5 mg/kg-d in diet 

2 yrs ND 

0.7 3.3 

0.7 3.3 

16.5 

↑ Forestomach hyperplasia ↑ Forestomach hyperkeratosis ↑ Esophagus (basal cell hyperplasia) 

No exposure-related chweight or histology of or lung weight (other omeasured). Hematologic variables examined. ↑ Forestomach tumors 16.5 mg/kg-d; ↑ Esophagus and tongu16.5 mg/kg-d. 

Wistar rat/ male and female

0, 3, 10, or 30 mg/kg-d, 5 d/wk 

90 d 10 

3 

30 10 

↓ Thymus weight, ↑ liver weight, 

↑ forestomach hyperplasia, ↑ slight thymic 

atrophy ↑ Forestomach epithelial cell proliferation index (BrdU incorporation) 

No exposure-related ch

hematological variableof lung, spleen, or lym

F344 rat/ male and female

0, 5, 50, or 100 mg/kg-d in diet 

90 d ND 

5 

50 

5 

50 

100 

↑ renal tubular casts in males ↓ RBCs and hematocrit in males ↓ RBCs and hematocrit in females, ↓ hemoglobin in both sexes, ↑liver:body 

weight ratio in males 

No exposure-related chother organ weights m(stomach, testes, ovarihistology of stomach, ovaries (other tissues wexamined). 
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Table 4-27. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]py

route 

Species/sex Dose Duration

NOAEL LOAEL 

mg/kg-d Response at LOAEL Commen

SD male rats 0, 1 or 5 mg/kgday by gavage 90 d 1 5 ↓ testicular testosterone Testosterone levels me

animals sacrificed aftenot statistically differecontrols 

SD female rats 0, 2.5 or 5 

mg/kg-day, gavage

1

60 d ND 

2.5 

2.5 

5 

↓ ovary weight ↓ estrogen and primordial follicles; altered estrous cyclicity 

 

C57BL/6 male mice 

0, 1, or 10 mg/kg-day exposure to F0 generation 

42 d ND 

1 

1 

10 

↓ epididymal sperm count in F0 and F1 

generations ↓ sperm motility in F0 mice 

 

Wistar rat/male only 

0, 3, 10, 30, or 90 mg/kg-d, gavage, 5 d/wk 

35 d 3 

10 30 

10 30 90 

↓ RBCs, hemoglobin, and hematocrit, ↓ thymus weight, ↓ percent B cells in 

spleen ↑ Forestomach hyperplasia, ↓serum IgM 

and IgA ↑ Liver oval cell hyperplasia 

No exposure-related hchanges in adrenals, brmarrow, colon, caecumheart, kidney, lung, lymesophagus, pituitary, sstomach, testis, or thym

Wistar rat/ male and female

0, 1.5, 5, 15, or 50 mg/kg-d, gavage, 5 d/wk 

35 d 5 

15 

15 

50 

↓ Thymus weight, ↑ forestomach 

hyperplasia ↑ Liver weight 

No exposure-related chhematological variablekidney, spleen, lung, aovaries, or histology okidney, spleen, thymumammary gland. 

CD-1 mouse/ F0 female; F1 male and female 

0,10, 40, or 160 mg/kg-d, gavage 

GDs 7–16 of F0 pregnancy

40 10 

ND ND 

160 

40 10 10 

↓ Number of F0 females with viable litters ↓ F1 body weight at PND 20 ↓ F1 body weight at PND 42 ↓ F1 male and F1female fertility index 

Beginning at 6–7 wks F1 male mouse (20–45exposed to 10 untreateover a period of 25 d. 

6 wks of age, each F1 (20–55/group) was cohan untreated male for a 6 mo. 
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Table 4-27. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]py

route 

Species/sex Dose Duration

NOAEL LOAEL 

mg/kg-d Response at LOAEL Commen

NMRI mouse/ F0 female; F1 female 

0 or 10 mg/kg-d GDs 7–16 

of F0 pregnancy

ND 10 ↓ F1 female fertility (↓ number of F2 litters 

and F2 litter size; ↓ ovary weight, and ↓

numbers of small, medium, or large follicles and corpora lutea) 

Exposed F0 females shgross signs of toxicity effects on fertility. Onfrom each litter was cobred with an untreated 6 mo. 

ND = not determined 1 Time weighted average dose ; animals treated by gavage, every other day to 0, 5, or 10 mg/kg-day 

1 2 
1 

2 

3 (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) Zhe4 2010; (7) van den Berg et al., 2005; (8) MacKenzie and Angevine, 1981; (9) Kristensen et al.5 hyperplasia; wt = weight. 6 7 Figure 4-2. NOAELs and LOAELs for selected noncancer effects from repeated oral exposure to 8 benzo[a]pyrene. 

05

101520253035

FS hyp; rat; 

(1)

FS hyp; 

mouse; (2)

↓ thymus 

wt; rat; (1)

↓ thymus 

wt; rat; (3)

2 years

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(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., 20102010; (7) van den Berg et al., 2005; (8) MacKenzie and Angevine, 1981; (9) Kristensen et al., 1995; ↑= increased; ↓= decreased; FS = 

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 NO

ng et al., 2010

= decreased; FS = 

2. NOAELs and LOAELs for selected noncancer effects from repeated oral exposure to 

↓ immune 

response; 

mouse; (7)

↓ 

w

m

G
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1 The two oral chronic-duration studies identify forestomach hyperplasia in rats (gavage 

2 exposure) and mice (dietary exposure) as a sensitive effect—a LOAEL of 3 mg/kg-day for 

3 forestomach hyperplasia in male and female Wistar rats (Kroese et al., 2001) and a LOAEL of 

4 0.7 mg/kg-day for forestomach hyperplasia in female B6C3F1 mice (Beland and Culp, 1998; 

5 Culp et al., 1998). In both rats and mice, an increasing incidence of animals with forestomach 

6 tumors with increasing dose was also observed. 

7 Several immune related effects have been observed in animals treated subchronicly with 

8 benzo[a]pyrene, including decreased thymus weight, decreased % of B cells in the spleen, 

9 decreased RBCs, and decreased serum immunoglobulins. LOAELs for decreased thymus 

10 weights were 10 and 15 mg/kg-day in two different studies of Wistar rats exposed by gavage for 

11 35 days (Kroese et al., 2001; De Jong et al., 1999) and 30 mg/kg-day for Wistar rats exposed to 

12 benzo[a]pyrene in the diet for 90 days (Kroese et al., 2001). Thymus weights were not measured 

13 in the the available chronic studies. Decreased thymus weight was accompanied by a decreased 

14 percentage of B cells in spleen and decreased serum IgM and IgA in one 35-day Wistar rat study 

15 (De Jong et al., 1999) and increased incidence of slight thymic atrophy in the 90-day Wistar rat 

16 study (Kroese et al., 2001). Thymus atrophy, but no histological thymus lesions, was noted in 

17 the other 35-day study with Wistar rats (Kroese et al., 2001). Other support for immune effects 

18 as a potential effect from repeated oral exposure to benzo[a]pyrene is shown by decreased 

19 immune responses in lymph nodes to the dermal sensitizer, DNCB, in C56BL/6 mice given 13 

20 mg/kg-day (LOAEL) 3 times/week for 4 weeks (van den Berg et al., 2005; see Section 4.4.2). 

21 Effects on RBC counts were also observed across the rat subchronic duration studies 

22 (Table 4-25). LOAELs for decreased RBCs were 10 mg/kg-day in Wistar rats exposed by 

23 gavage for 35 days (De Jong et al., 1999) and 50 mg/kg-day in male F344 rats exposed in the 

24 diet for 90 days (Knuckles et al., 2001), but no significant exposure-related changes in RBC 

25 counts were observed in Wistar rats in another 35 day study at doses up to 50 mg/kg-day (Kroese 

26 et al., 2001) or at 30 mg/kg-day in Wistar rats exposed in the diet for 90 days (Kroese et al., 

27 2001; see Table 4-26). When observed, the magnitudes of the decreases in RBC, hemoglobin, or 

28 hematocrit were generally small: about 18% at 90 mg/kg-day and <10% at lower doses in Wistar 

29 rats (De Jong et al., 1999) and about 10% in F344 rats (Knuckles et al., 2001). Hematologic 

30 variables were not measured at the terminal sacrifices in the chronic duration studies in rats 

31 (Kroese et al., 2001) or mice (Beland and Culp, 1998; Culp et al., 1998). 

32 Kidney effects characterized as increased incidence of renal tubular casts in male F344 

33 rats were observed in a study by Knuckles et al 2001). The most sensitive effect observed in this 

34 study was an increase in abnormal tubular casts in the kidney in males at 5 mg/kg-day (40%), 

35 50 mg/kg-day (80%) and 100 mg/kg-day (100%), compared to 10% in the controls. In females, 
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1 only 10% showed significant kidney tubular changes at the two high dose levels compared to 

2 zero incidence in controls. 

3 Reproductive and developmental effects following gestational exposure to 

4 benzo[a]pyrene have been observed in animal models. Decreased male reproductive endpoints 

5 including decreased testicular testosterone, decreased epididymal sperm count, and decreased 

6 sperm motility have been observed in rodents treated subchronically (Mohamed et al., 2010; 

7 Zheng et al., 2010). In addition, female reproductive endpoints including decreased ovary 

8 weight, decreased estrogen, decreased primordial follicles, and estrus cyclicity have been 

9 observed in female rats treated for 60 days (Xu et al., 2010). Impaired reproductive performance 

10 in F1 mouse offspring (male and female) has been observed following exposure of F0 mice to 

11 oral doses as low as 10 mg/kg-day during GDs 7–16 (Kristensen et al., 1995; MacKenzie and 

12 Angevine, 1981). Effects observed included decreased ovary weight in F1 females and reduced 

13 fertility as reflected by decreased mean number of F2 litters. F1 females had statistically 

14 significantly lower median numbers of offspring, number of litters, and litter sizes and a 

15 statistically significantly greater median number of days between litters as compared with the 

16 controls (Kristensen et al., 1995). Another study of gestationally treated dams (GD 7-16) 

17 identified statistically significant decrements in fertility, pup weight, and reproductive organ 

18 weights and histology (MacKenzie and Angevine, 1981). These mouse developmental/

19 reproductive toxicity studies observed effects at the lowest dose tested (10 mg/kg-day). 

20 Reductions in motor activity, decreased grip strength, and decreased response to sound, touch, 

21 and pain were observed in F344 rats following administration of single gavage doses of 

22 ≥25 mg/kg (Saunders et al., 2006, 2002, 2001; see Section 4.4.1), but similar evaluations of 

23 neurological endpoints following repeated oral exposure of animals to benzo[a]pyrene were not 

24 located. 

25 Studies with ApoE-/- mice, which spontaneously develop atherosclerosis, show that 

26 repeated oral exposure to 5 mg/kg gavage doses of benzo[a]pyrene enhances the progression of 

27 atherosclerosis through a general local inflammatory process (Knaapen et al., 2007; Curfs et al., 

28 2005, 2004; Godschalk et al., 2003; see Section 4.4.4); however, available data are inadequate to 

29 assess oral exposure dose-response relationships for benzo[a]pyrene-induced atherosclerosis in 

30 normal test animals. 

31 

32 4.6.2. Inhalation 

33 Several epidemiological studies have associated increased occupational exposure of 

34 benzo[a]pyrene with cardiac endpoints, specifically ischemic heart disease (Friesen et al., 2010; 

35 Burstyn et al., 2005). Other studies have reported potential prenatal effects, birth outcomes, and 

36 decreased fertility associated with increased exposure to benzo[a]pyrene. Decreased head 
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1 circumference, decreased birth weight, and decreased postnatal weight have been reported (Tang 

2 et al., 2006; Perera et al., 2005a,b;) in addition to increased risk of early fetal death (Wu et al., 

3 2010). Furthermore, elevated levels of benzo[a]pyrene in follicular fluid have been associated 

4 with reduced fertility (Neal et al., 2008). 

5 In addition to epidemiological studies, several repeated-exposure inhalation toxicity studies in 

6 animals exist for benzo[a]pyrene (Archibong et al., 2002, 2008; Ramesh et al., 2008; Inyang et 

7 al., 2003; Wormley et al., 2004). A lifetime-exposure carcinogenicity study of Syrian golden 

8 hamsters exposed to benzo[a]pyrene condensed onto NaCl aerosols at nominal concentrations of 

2, 10, or 50 mg/m39 is available (Thyssen et al., 1981); however, noncancer effects were not 

10 evaluated. 

11 Although no standard developmental toxicity studies are available for benzo[a]pyrene via 

12 the inhalation route, decreased fetal survival and number of pups per litter were observed 

13 following exposure of pregnant F344 rats to aerosols of benzo[a]pyrene and CB at 

concentrations ≥25 μg/m314 on GDs 11–20 (Archibong et al., 2002). Decreased levels of plasma 

progesterone, estradiol, and prolactin were observed on GD 17 in dams exposed to 75 μg/m315 , but 

not in those exposed to 25 μg/m316 (Archibong et al., 2002). Other rat studies from the same 

laboratory have associated inhalation exposures to 100 μg/m317 benzo[a]pyrene:CB aerosols 

18 during gestation with changes in electrophysiological variables in the hippocampus (Wormley et 

19 al., 2004). Duration-dependent effects on male reproductive endpoints including increased 

20 luteinizing hormone, decreased circulating and intratesticular concentrations of testosterone, 

21 decreased testis weight, and decreased sperm motility have also been observed following 

exposure of adult male F344 rats to benzo[a]pyrene:CB aerosols at 75 μg/m322 for 10 or 60 days 

23 (Archibong et al., 2008; Ramesh et al., 2008; Inyang et al., 2003). 

24 
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Table 4-28. NOAELS and LOAELs for noncancer effects in animals repeatedly exposed to benzo[a]py

inhalation route 

Species/sex Dose Duration

NOAEL LOAEL 

μg/m Response at LOAEL Commen

3

F344 rats 0, 25, 75, 100 

μg/m3 

4hr/d 

GD 11-20 ND 

25 

25 75 

↓ pups/litter, litter survival (%) ↑ resorptions ↓ plasma progesterone, estradiol, and prolactin 

 carbon black used as c

F344 rats 0, 75 μg/m3

4hr/d 

60 d ND 75 ↑ luteinizing hormone 

↓ testosterone ↓ decreased testis weight ↓ sperm motility 

carbon black used as cin treatment group; coexposed to carbon blac

F344 rats 0, 100 μg/m3

4hr/d 

GD ND 100 ↓ pups/litter 

electrophysiological changes in the hippocampus 

 

1 
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1 

2 4.6.3. Dermal 

3 

4 Though numerous chronic cancer bioassays exist for benzo[a]pyrene by the dermal route, 

5 noncancer effects were not reported in these studies, nor are studies available evaluating 

6 noncancer effects in humans exposed dermally to benzo[a]pyrene. 

7 

8 4.6.4. Mode-of-Action Information 

9 4.6.4.1. Forestomach Lesions from Oral Exposure 

10 The development of forestomach hyperplasia in mice and rats from subchronic or chronic 

11 oral exposures (by gavage and diet) to benzo[a]pyrene is reasonably expected to involve a cell 

12 proliferative response to cytotoxicity from reactive benzo[a]pyrene metabolic intermediates, 

13 based on the extensive findings from research on the bioactivation of benzo[a]pyrene and 

14 carcinogenicity (see reviews on the bioactivation of benzo[a]pyrene by Xu et al., 2009; Jiang et 

15 al., 2007, 2005; Xue and Warshawsky, 2005; Penning et al., 1999; Harvey 1996; Cavalieri and 

16 Rogan, 1995). Reactive intermediates that can react with cellular macromolecules and 

17 potentially lead to cytotoxicity include BPDE, benzo[a]pyrene radical cations, benzo[a]pyrene o18 quinones, and ROS. Reactive benzo[a]pyrene metabolites are also well known to reduce the 

19 viability and survival of cultured cells involving mechanisms related to stimulation of apoptosis 

20 (Andrysik et al., 2007; Chung et al., 2007; Nwagbara et al., 2007; Chen et al., 2003; Jyonouchi et 

21 al., 1999; Flowers-Geary et al., 1996, 1993). Molecular details of cell proliferative responses to 

22 cytotoxicity or apoptosis from benzo[a]pyrene metabolites are poorly understood, but Burdick et 

23 al. (2006, 2003) provided evidence that benzo[a]pyrene o-quinones could inhibit apoptosis and 

24 increase cell proliferation in a model human mammary epithelial cell system (MCF10A) via 

25 activation of the epidermal growth factor receptor (EGFR) by ROS. The relationship of these 

26 findings to development of benzo[a]pyrene-induced forestomach hyperplasia is unknown. 

27 

28 4.6.4.2. Immune System Effects 

29 Decreased thymus weight, decreased number of B cells in spleen, and immune 

30 suppression have been observed following oral, i.p., s.c., or intratracheal instillation exposure to 

31 benzo[a]pyrene. DeJong et al. (1999) and Kroese et al. (2001) found decreased thymus weight 

32 due to benzo[a]pyrene at oral doses ≥10 mg/kg body weight. In addition, several studies report 

33 thymus effects at higher doses and/or by other routes of exposure (e.g. Rodriguez et al., 1999; 

34 Holladay and Smith, 1994). Reduced thymus size or weight have been noted to be among the 

35 first indicators of immunotoxicity (Schuurman et al. 1992; Luster et al. 1988), and correlate well 

36 with adverse histopathologic effects and the presence of lesions in the thymic cortex (Germolec 

37 et al. 2004a, 2004b; Wachsmuth 1983). 
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1 Interpretation of decreased thymus weight as an adverse effect is supported by general 

2 immunology literature as well as chemical-specific data. The thymic cortex is known to be a 

3 major site of thymocyte proliferation and selection for maturation, and impairment can lead to 

4 cell-mediated immune suppression (Kuper 2002, 1992; De Waal et al. 1997). Reduced thymus 

5 weight is often attributed to decreased thymocyte proliferation or increased thymocyte apoptosis 

6 in the thymic cortex (Kamath et al., 1997; Vandebriel et al., 1999). DeJong et al. (1999) reported 

7 a decrease in estimated thymic cortex weight at 10, 30, and 90 mg/kg benzo[a]pyrene, and 

8 reduced medulla weight at 90 mg/kg, but did not calculate the ratio between the two. This 

9 suggests that both parts of the thymus were affected by benzo[a]pyrene, but that the cortex may 

10 be more sensitive. De Jong also used immunohistochemistry data to show that cell proliferation 

11 was not affected, suggesting that thymic atrophy may be due to increased rates of thymocyte 

12 apoptosis. However, a study using the murine LLNA showed that proliferation activity 

13 decreased after a single 13 mg/ml oral dose of benzo[a]pyrene (van den Berg et al., 2005). 

14 In addition to thymus effects, decreased B cell percentages in the spleen were observed at 

15 10, 30, and 90 mg/kg-day benzo[a]pyrene in a dose response pattern. The absolute number of B 

16 cells, however, was not significantly lower than control animals until 90 mg-kg/day. There is 

17 also supportive evidence of humoral immune suppression at higher doses, as well as evidence of 

18 overall toxicity of the bone marrow. This theory is supported by the decrease in IgA and IgM 

19 (incating T-cell dependant effects), and the dose-related toxicity of RBCs observed by Dejong et 

20 al. (1999). 

21 The MOA by which benzo[a]pyrene produces immune system effects is not understood, 

22 but several in vitro studies have been conducted to investigate potential contributing 

23 mechanisms. Benzo[a]pyrene induced myelotoxicity in human cord blood cells (Carfi et al., 

24 2007) and mouse bone marrow cultures (Legraverend et al., 1983), suppressed mouse B cell 

25 lymphopoiesis (Hardin et al., 1992), and inhibited mitogen-induced proliferative responses of 

26 mouse spleen cell cultures (Lee and Urso, 2007). Benzo[a]pyrene inhibition of the proliferative 

27 responses of spleen cells to a mitogen was diminished by the presence of the AhR antagonist and 

28 CYP inhibitor, α-NF, indicating the potential importance of benzo[a]pyrene metabolites in the 

29 immune suppression effect (Lee and Urso, 2007). Similarly, the CYP1A1 inhibitor 1-(1-

30 propynyl)pyrene blocked B-cell growth inhibition by benzo[a]pyrene, but not through the 

31 metabolite BPDE (Allan et al. 2006). 

32 Carfi et al. (2007) described a series of in vitro assays designed to assess cytotoxicity, 

33 myelotoxicity, cytokine release, and mitogen responsiveness in rat, mouse, and human cells (i.e., 

34 peripheral lymphocytes and cord blood cells, and spleen cells from rats and mice only). The 

35 cytotoxicity half maximal inhibitory concentration (IC50) value for benzo[a]pyrene was >200 μM 

36 in human, rat, and mouse cells. Benzo[a]pyrene produced myelotoxicity as evaluated by a dose37 related decrease in colony scoring for the colony forming unit-granulocyte macrophage (CFU38 GM) assay using human cord blood cells. Benzo[a]pyrene reduced the release of specific 
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1 cytokines from HL following phytohemagglutinin (PHA), gamma-interferon (γ-INF), and 

2 lipopolysaccharide (LPS) tumor necrosis factor (TNF-α) stimulation. Mitogen responsiveness in 

3 rat and mouse spleen cells following stimulation with LPS or PHA was decreased by exposure to 

4 benzo[a]pyrene. T-lymphocyte proliferation induced by anti-CD3 antibody was also inhibited by 

5 benzo[a]pyrene in HL, but was not affected in mouse spleen cells at the highest concentration 

6 (160 μM). 

7 Myelotoxicity was also observed in mouse bone marrow cultures exposed to 

8 benzo[a]pyrene as evidenced by decreased cell survival (Legraverend et al., 1983). The findings 

9 in bone marrow cultures from Ah-responsive (C57BL/6) and Ah-nonresponsive (DBA/2) mice 

10 suggest that AhR affinity may play a role in benzo[a]pyrene-induced myelotoxicity. 

11 Benzo[a]pyrene is more toxic to bone marrow cells from C57BL/6 mice in vitro compared to 

12 cells cultured from DBA/2 mice. 

13 Hardin et al. (1992) treated cultured bone marrow cells from DBA/2 and C57BL/6 mice 

with benzo[a]pyrene at concentrations between 10-4 and 10-8 14 M. Benzo[a]pyrene suppressed B 

15 cell lymphopoiesis in a dose-dependent manner even at the lowest concentration used. Bone 

16 marrow cells from Ah-nonresponsive DBA/2 mice were less sensitive to the immunosuppressive 

17 action of benzo[a]pyrene, compared with Ah-responsive C57BL/6 mice. The AhR antagonist 

18 and CYP450 inhibitor α-NF prevented benzo[a]pyrene-induced inhibition of B cell 

19 lymphopoiesis from C57BL/6 mice in a concentration-dependent fashion. Benzo[a]pyrene also 

20 induced apoptosis in cultured bone marrow cells. 

21 Spleen cell cultures derived from C3H/HeJ and CBY/D2 mice were exposed to benzo[a]pyrene 

22 and assessed for T-lymphocyte proliferation in response to mitogenic or antigenic stimulation 

23 (Lee and Urso, 2007). Benzo[a]pyrene (10 μM) produced an 80% decrease in the allogeneic 

24 mixed lymphocyte response (MLR) assay, which is a measure of the proliferative response to 

25 antigenic stimulation. Benzo[a]pyrene (0.1, 1, and 10 μM) also produced a dose-dependent 

26 inhibition of the proliferative response to the mitogen Concanavalin A (Con A). This inhibition 

27 did not occur in spleen cells treated with the AhR antagonist and CYP450 inhibitor, α-NF. 

28 BPDE-DNA adducts were detected in CH3 spleen cells cultured with 10 μM benzo[a]pyrene in 

29 the presence of Con A. 

30 

31 4.6.4.3. Developmental and Reproductive Toxicity Effects 

32 Developmental and reproductive toxicity effects have been associated with oral and 

33 inhalation exposure to benzo[a]pyrene. Impaired fertility, with associated lesions in ovarian 

34 (decreased follicles) and testicular (atrophic seminiferous tubules) tissues, has been observed in 

35 male and female F1 offspring following exposure of F0 female mice to 10 mg/kg-day 

36 benzo[a]pyrene on GDs 7–16; a decrease in the number of F0 females with viable litters was 

37 observed at a higher dose level of 160 mg/kg-day (Kristensen et al., 1995; MacKenzie and 

38 Angevine, 1981). Inhalation exposure of pregnant rats to benzo[a]pyrene:CB aerosols during 
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1 gestation has also been associated with decreased fetal survival and number of pups per litter 

2 (Archibong et al., 2002), decreased levels of plasma progesterone, estradiol, and prolactin 

3 (Archibong et al., 2002), changes in electrophysiological variables in the hippocampus 

4 (Wormley et al., 2004) and decreased cortical neuron activity (McCallister et al., 2008). 

Inhalation exposure of adult male rats to benzo[a]pyrene:CB aerosols (75 μg/m35 ) for 10 or 60 

6 days caused decreased circulating and intratesticular concentrations of testosterone, decreased 

7 testis weight, and decreased sperm motility (Archibong et al., 2008; Ramesh et al., 2008; Inyang 

8 et al., 2003). ). Acute i.p. exposure studies in adult female DBa/2N or C57BL/6N mice 

9 demonstrated a 50% destruction of primordial follicles with a single dose of 25 mg/kg (Mattison 

10 et al., 1980) and a 15-day exposure of 3 mg/kg-day (Boorman et al., 2000). Other reproductive11 related effects in these studies include decreased fertility (number of pups) (Mattison et al., 1980) 

12 and ovulatory inhibition, as indicated by decreased number of corpora lutea, one week after a 

13 dose of 5 mg/kg and 3-4 weeks after a dose of 100 mg/kg (Miller et al., 1992; Swartz and 

14 Mattison, 1985). 

15 In vivo studies have suggested that the mechanism of decreased fertility in females and 

16 decreased fetal survival may result from changes in circulating hormones (i.e., decreased 

17 progesterone, estradiol-17β, and prolactin levels) responsible for maintaining the uterine 

18 environment in a state that can support embryonic and fetal development (Archibong et al., 2002, 

19 see Section 4.3.2). Several in vitro studies have demonstrated low affinity binding of 

20 benzo[a]pyrene to the estrogen receptor and alteration of estrogen-dependent gene expression 

21 (Liu et al., 2006; Van Lipzig et al., 2005; Vondracek et al., 2002; Fertuck et al., 2001; Charles et 

22 al., 2000); however, the role of these changes in benzo[a]pyrene-induced reproductive toxicity is 

23 unknown. Fertuck et al. (2001) showed in vitro effects of benzo[a]pyrene on estrogen-receptor24 mediated gene expression, but did not demonstrate estrogen-mediated uterotrophic effects 

25 (increased uterine weight or lactoferrin mRNA expression) in ovarectomized C57BL/J6 or 

26 DBA/2 mice following in vivo administration of benzo[a]pyrene (0.1–10 mg/kg-day, p.o., for 3 

27 consecutive days). 

28 The mechanism(s) by which benzo[a]pyrene or its metabolites impair the development of 

29 follicles in the ovary have been the focus of study for more than 30 years (Mattison and 

30 Thorgeirsson, 1977). AHH is found in the ovary, and inhibition of AHH activity reduces the 

31 level of oocyte destruction seen with benzo[a]pyrene exposures in mice (Mattison and 

32 Thorgeirsson, 1979). AHH activation is not sufficient to explain the variation across strains in 

33 oocyte destruction, however (Mattison and Nightingale, 1980). Studies using intraovarian 

34 injection of metabolites of benzo[a]pyrene indicate that the epoxide metabolite (+)-(7R,8S)-diol35 (9S,10R)-epoxide-2 is most strongly correlated with the oocyte counts in exposed mice 

36 (Takizawa et al.,1984). 

37 Most of the loss of oocytes that occurs in utero and throughout the reproductive lifespan 

38 in mice and in women occurs through programmed cell death (apoptosis), which is regulated by 
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1 the protein Bax. Activation of Bax leads to increased oocyte death, and PAHs (including 

2 benzo[a]pyrene) have been demonstrated to activate Bax gene transcription in mice (Matikainen 

3 et al., 2002; 2001). However, an in vitro study of mouse ovarian cells obtained from 4-day old 

4 pups did not show any evidence of increased markers of apoptosis with treatments of 

5 benzo[a]pyrene concentrations of up to 1000 ng/ml for 6 and for 24 hours (Tuttle et al., 2009). 

6 Other mechanisms may be more relevant for the ovulatory inhibition effects seen with 

7 benzo[a]pyrene exposures. In an intro experiment, Neal et al. (2007) demonstrated a dose8 dependent decrease in FSH-stimulated rat follicle growth, with 158, 99, 75, 38, 30 and 38% 

9 change in follicle area for benzo[a]pyrene exposure concentrations of 0, 1.5, 5.0, 15, 45 and 135 

10 ng /ml (p < 0.05 for all differences compared with controls). The authors noted that the lowest 

11 dose at which this effect was seen, 1.5 ng/ml, was similar to the mean concentration of 

12 benzo[a]pyrene seen in follicular fluid samples from women who smoked. 

13 Several in vitro studies have investigated the possible mechanisms for impaired 

14 spermatogenesis by benzo[a]pyrene including enhancement of apoptosis of spermatogonia 

15 (Revel et al., 2001), inhibition of spermatid meiosis (Georgellis et al., 1990), Sertoli cell 

16 cytotoxicity (Raychoudhury and Kubinski, 2003), and altered androgen hormone regulation 

17 (Inyang et al., 2003; Vinggaard et al., 2000). 

18 Revel et al. (2001) reported dose-related increases in apoptosis of spermatogonia 

19 harvested from the vas deferens of male BALB/c mice administered benzo[a]pyrene doses 

20 ranging from 0.5 to 50 mg/kg via s.c. injection for 5 weeks. The competitive AhR inhibitor, 

21 resveratrol (50 mg/kg, s.c.) given simultaneously with 5 mg/kg benzo[a]pyrene s.c. for 5 weeks, 

22 suppressed both BPDE DNA adduct formation and apoptosis, suggesting a role for the AhR in 

23 this benzo[a]pyrene-induced male reproductive toxicity (Revel et al., 2001). 

24 Georgellis et al. (1990) reported that concentrations of 0.1 μM benzo[a]pyrene incubated 

25 in vitro with seminiferous tubule segments from Sprague-Dawley rats with microsome 

26 preparations from the whole rat testes inhibited meiotic division of the spermatids and was 

27 highly cytotoxic. 

28 Raychoudhury and Kubinski (2003) isolated Sertoli cells from CD rats and incubated the 

29 cells in culture with benzo[a]pyrene. Benzo[a]pyrene was cytotoxic to these cells at 50 and 

30 100 μg/mL. Treatment of the cells for 24 hours with 10 μg/mL benzo[a]pyrene induced cell 

31 killing through an apoptotic response (as measured by fluorescence labeling of apoptotic DNA 

32 fragments). 

33 Inyang et al. (2003) demonstrated that benzo[a]pyrene inhalation altered circulating 

34 levels or cellular responsiveness to androgenic hormones such as testosterone (see Section 4.3.2). 

35 Vinggaard et al., 2000) showed that benzo[a]pyrene antagonized the human androgen receptor 

36 (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a 

37 hAR vector and an MMTV-LUC vector (antiandrogen IC50 of 3.9 μM). 

38 
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1 4.7. EVALUATION OF CARCINOGENICITY 

2 4.7.1. Summary of Overall Weight-of-Evidence 

3 Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 

4 benzo[a]pyrene is “carcinogenic to humans.” This conclusion is based on evidence of 

5 carcinogenicity in humans following exposure to different PAH mixtures containing 

6 benzo[a]pyrene, extensive and consistent evidence of carcinogenicity in laboratory animals 

7 exposed to benzo[a]pyrene via all routes of administration, and strong evidence that the 

8 biological processes leading to benzo[a]pyrene carcinogenesis in laboratory animals are also 

9 present in humans. Bioactivation of benzo[a]pyrene leads to the formation of DNA-reactive 

10 metabolites which can produce mutations in key genes, such as the p53 tumor suppressor gene 

11 and the ras oncogene, leading to tumor formation (see Section 4.7.3.). 

12 

13 4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 

14 There is a large body of evidence for human carcinogenicity for several PAH mixtures 

15 containing benzo[a]pyrene, such as soot, coal tars, coal-tar pitch, mineral oils, and shale oils 

16 (IARC, 2010; Baan et al., 2009; Straif et al., 2005). There is also evidence of carcinogenicity in 

17 occupations involving exposure to PAH mixtures containing benzo[a]pyrene, such as aluminum 

18 production, chimney sweeping, coal gasification, coal-tar distillation, coke production, iron and 

19 steel founding, and paving and roofing with coal tar pitch (IARC, 2010; Baan et al., 2009; Straif 

20 et al., 2005). Increased cancer risks have been reported among other occupations involving 

21 exposure to PAH mixtures such as carbon black and diesel exhaust (Bosetti et al., 2007; Straif et 

22 al., 2005). Benzo[a]pyrene is also a notable constituent of tobacco smoke (IARC 2004). An 

23 increasing number of studies report exposure biomarkers such as benzo[a]pyrene- or PAH-DNA 

24 adducts in white blood cells, and several cohort studies (summarized in Section 4.1) demonstrate 

25 a positive exposure-response relationship with cumulative PAH exposure using 

26 benzo[a]pyrene—or a proxy such as BSM that can be converted to benzo[a]pyrene—as an 

27 indicator substance. Because benzo[a]pyrene is only one of many PAHs that could contribute to 

28 these observed increases in cancer, the epidemiologic studies provide credible but limited 

29 support for a causative role of benzo[a]pyrene in human cancer. 

30 In laboratory animals (i.e., rats, mice, and hamsters), exposure to benzo[a]pyrene via the 

31 oral, inhalation, and dermal routes have been associated with carcinogenic responses both 

32 systemically and at the site of administration. Chronic oral exposure to benzo[a]pyrene was 

33 associated with forestomach and liver tumors in male and female Wistar rats (Kroese et al., 

34 2001), forestomach tumors in male and female Sprague-Dawley rats (Brune et al., 1981), and 

35 forestomach, esophagus, tongue, and larynx tumors in female B6C3F1 mice (Beland and Culp, 

36 1998; Culp et al., 1998). Auditory canal tumors were also observed in male and female Wistar 

37 rats (Kroese et al., 2001). Repeated or short-term oral exposure to benzo[a]pyrene was 

38 associated with forestomach tumors in more than 10 additional bioassays with several strains of 
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1 mice (see Table 4-4 in Section 4.2.1.2). Chronic inhalation exposure to benzo[a]pyrene was 

2 associated with tumors in the larynx and pharynx of male Syrian golden hamsters exposed to 

3 benzo[a]pyrene:NaCl aerosols (Thyssen et al., 1981). Intratracheal instillation of benzo[a]pyrene 

4 was associated with respiratory tract tumors in more than 10 additional studies with hamsters 

5 (see Section 4.2.2.2 for references). Chronic dermal application of benzo[a]pyrene (2–3 

6 times/week) has been associated with mouse skin tumors in 12 bioassays (see Section 4.2.3.2 for 

7 references). Skin tumors in rats, rabbits, and guinea pigs have also been associated with repeated 

8 application of benzo[a]pyrene to skin in the absence of exogenous promoters (WHO, 1998; 

9 ATSDR, 1995; IARC, 1983, 1973). When followed by repeated exposure to a potent tumor 

10 promoter, acute dermal exposure to benzo[a]pyrene induced skin tumors in numerous studies of 

11 mice, indicating that benzo[a]pyrene is a strong tumor-initiating agent in the mouse skin model 

12 (see Section 4.2.3.1 for references). 

13 Carcinogenic responses in animals exposed to benzo[a]pyrene by other routes of 

14 administration include: (1) liver or lung tumors in newborn mice given acute postnatal i.p. 

15 injections; (2) increased lung tumor multiplicity in A/J adult mice given single i.p. injections; (3) 

16 injection site tumors in mice following s.c. injection; (4) injection site sarcomas in mice 

17 following intramuscular injection; (5) mammary tumors in rats with intramammilary 

18 administration; (6) cervical tumors in mice with intravaginal application; and (7) tracheal tumors 

19 in rats with intratracheal implantation (see Section 4.4.3 for references). 

20 Benzo[a]pyrene is classified as an alternant PAH, or a compound composed solely of 

21 fused benzene rings. Nonalternant PAHs contain both benzene and five carbon rings. Among 

22 alternant PAHs, important structural features related to enhanced mutagenicity and 

23 carcinogenicity include the presence of at least four rings (Bostrom et al., 2002). The 

24 carcinogenic activity of PAH compounds is influenced by specific structural features. Recently, 

25 this knowledge has been exploited in an effort to derive quantitative structure activity 

26 relationship (QSAR) methods to evaluate the relationship between specific PAH structural 

27 features and mechanistic events related to carcinogenesis (Bruce et al., 2008; Vijayalakshmi et 

28 al., 2008). Alternant PAHs having four or more benzene rings exhibit greater carcinogenic 

29 potency than PAHs with two or three benzene rings (Bostrom et al., 2002). The carcinogenic 

30 activity of PAHs is also related to the specific arrangement of the benzene rings. As a general 

31 rule, PAHs with at least four rings and a classic bay- or fjord-region (formed entirely by benzene 

32 rings) may be characterized as containing structural alerts for carcinogenesis. However, this 

33 structural characterization is likely to be overly simplistic and other features may be important to 

34 carcinogenesis. 

35 As discussed in Section 4.5.1.1, several lines of evidence related to tumor initiation 

36 following mutagenicity are available for benzo[a]pyrene including: (1) in vivo detection of 

37 cancer-relevant oncogene/tumor suppressor gene mutations in target tissue; (2) in vivo detection 

38 of DNA adducts in target tissue; (3) in vivo DNA adducts, gene mutations, cytogenetic damage, 
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1 and other measures of primary DNA damage in non-target tissues; and (4) in vitro DNA adduct 

2 formation, mutations, cytogenetic damage, and primary DNA damage in cells from target and 

3 nontarget tissues. 

4 

5 4.7.3. Mode-of-Action Information 

6 4.7.3.1. Hypothesized MOA

7 The carcinogenicity of benzo[a]pyrene, the most studied and best characterized PAH, is 

8 well documented (Xu et al., 2009; Jiang et al., 2007, 2005; Xue and Warshawsky, 2005; Ramesh 

9 et al., 2004; Bostrom et al., 2002; Penning et al., 1999; WHO, 1998; Harvey, 1996; ATSDR, 

10 1995; Cavalieri and Rogan, 1995; U.S. EPA, 1991b). EPA has concluded that benzo[a]pyrene 

11 induces carcinogenicity via a mutagenic mode of action. Mutagenicity is a well-established 

12 cause of carcinogenicity. This hypothesized mode of action is presumed to apply to all tumor 

13 types and is relevant for all routes of exposure. The principal key events associated with the 

14 mode of action for benzo[a]pyrene include: (1) bioactivation of benzo[a]pyrene to reactive 

15 metabolites (2) direct DNA damage by the reactive metabolites, including the formation of DNA 

16 adducts (3) formation and fixation of DNA mutations, particularly in tumor suppressor genes or 

17 oncogenes and (4) clonal expansion of mutated cells. These events are depicted in Figure 4-3. 

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 Carcinogenicity

Binding to AhR 

Upregulation of 

genes related to 

biotransformation, 

growth, and 

differentiation
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1 Figure 4-3. Proposed principal pathways and key events in the 

2 benzo[a]pyrene carcinogenic MOA. 

3 

4 4.7.3.2. Experimental Support for the Hypothesized MOA

5 

6 Strength, consistency, specificity of association. There is an extensive database of in vitro and in 

7 vivo studies demonstrating the genotoxicity and mutagenicity of benzo[a]pyrene following 

8 metabolic activation (see Tables 4-24, 4-25 and 4-26). In vitro studies overwhelmingly support 

9 the formation of DNA adducts, mutagenesis in bacteria, yeast and mammalian cells, several 

10 measures of cytogenetic damage (CA, SCE, MN), and DNA damage. In vivo systems in animal 

11 models are predominantly positive for somatic mutations following benzo[a]pyrene exposure. 

12 Additionally, some evidence exists that benzo[a]pyrene can induce mutations in germ cells. 

13 Benzo[a]pyrene is thought to be converted into reactive intermediates via three principal 

14 metabolic pathways: (1) activation to a reactive diol epoxide via CYP1A1/1B1 and epoxide 

15 hydrolase; (2) activation to a reactive radical cation via CYP peroxidases and (3) activation to a 

16 reactive and redox active o-quinone metabolite via AKR1A1 and AKR1C1-1C4 (Xu et al., 2009; 

17 Jiang et al., 2007, 2005; Xue and Warshawsky, 2005; Penning et al., 1999; Harvey 1996; 

18 Cavalieri and Rogan, 1995). All three of these pathways (discussed in detail in Sections 3.3. and 

19 4.5.2) lead to DNA damage including DNA adducts, depurination, and/or oxidative damage to 

20 DNA. DNA damage, if not correctly repaired prior to replication, can subsequently give rise to 

21 mutations. 

22 Benzo[a]pyrene-DNA adducts, biomarkers of exposure and of effect, have been 

23 extensively demonstrated with in vitro cell systems, in vivo animals studies, and in human target 

24 tissues, including skin and lung (see Section 4.1.2.). Specifically, elevated BPDE-DNA adducts 

25 have been observed in coke oven workers and chimney sweepers, occupations associated with 

26 increased risks of cancer from complex PAH-containing mixtures (Pavanello et al., 1999). 

27 BPDE-DNA adducts were also found to be elevated in the lungs of cigarette smokers with lung 

28 cancer (Godschalk et al., 2002; Phillips et al., 2002; Bartsch et al., 1999; Alexandrov et al., 

29 1992). Multiple epidemiological studies have indicated that PAH exposed individuals who are 

30 homozygous for a CYP1A1 polymorphism which increases the inducability of this enzyme (thus 

31 increasing the production of reactive diol epoxide metabolites) have increased levels of 

32 benzo[a]pyrene-DNA adducts (Bartsch et al., 2006; Aklillu et al., 2005; Alexandrov et al., 2002; 

33 Perera and Weinstein, 2000). In addition, this population of individuals also has a greater risk of 

34 certain tumors, including those of the lung. 

35 Mutations in the K-ras, H-ras, and p53 genes were assessed in forestomach tumors of 

36 mice fed benzo[a]pyrene in the diet for 2 years (Culp et al., 2000). Forestomach tumors had K37 ras mutations (68% of tumors) that were G→T or C transversions in codon 12 or 13. H-ras 

38 (codon 13) and p53 mutations characterized as G→T or C transversions were also found. 
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1 K-ras mutations were observed in the A/J mouse lung tumor model following IP 

2 treatment with benzo[a]pyrene, and were observed to be qualitatively different than K-ras 

3 mutations in spontaneous lung tumors from control animals (Nesnow et al., 1996). Lung tumor 

4 DNA was isolated and DNA sequence analysis of K-ras mutations was performed for 19 separate 

5 lung tumors in the benzo[a]pyrene treated group and the control group. The DNA sequence 

6 analysis demonstrated several guanine mutations at codon 12, which were different than the 

7 spectrum of mutations found in untreated animals. Specifically, the frequency of GGT→TGT 

8 transversion mutations were significantly higher in the benzo[a]pyrene treated animals compared 

9 to controls (56% vs. 0%) whereas GGT→GAT transition mutations were the predominant 

10 mutation in lung tumors from control animals (58% vs. 19% in benzo[a]pyrene group). 

11 Some human data exist which correlate the frequency of PAH-DNA adducts with gene 

12 mutations in highly PAH-exposed populations. In a study of iron foundry workers (a high PAH 

13 exposure population which has been demonstrated to have an increased risk of lung cancer 

14 [Bosetti et al., 2007]), biological samples from workers were analyzed for DNA adducts and 

15 somatic gene mutations at the hprt locus (Perera et al.1994, 1993). A strong correlation between 

16 PAH-DNA adduct levels and incidence of hprt mutations was observed in individuals with 

17 detectable levels of adducts, indicating that somatic mutations were increased in parallel with 

18 PAH-DNA adducts in workers exposed to PAHs. 

19 Data in humans from a study by Marini et al. (2001) indicate that the types of mutations 

20 commonly found in response to benzo[a]pyrene exposure in in vitro and animal models are 

21 similar to the spectrum of mutations in critical tumor suppressor genes and/or oncogenes in 

22 populations highly exposed to PAHs. DeMarini et al. (2001) demonstrated mutations in the p53 

23 tumor suppressor gene and the K-ras oncogene in lung tumors obtained from 24 nonsmoking 

24 women from China, whose tumors were associated with exposure in their homes to smoky coal 

25 from the use of stoves with no chimneys. The observed mutations in lung tumors were primarily 

26 G→T transversions at either K-ras or p53. Mutation hotspots in the lung tumors corresponded 

27 with hot spots for PAH mutations (codon 154, codon 249, and codon 273). 

28 

29 Dose-response concordance and temporal relationship. The metabolism of benzo[a]pyrene to 

30 reactive metabolites is a necessary event which precedes mutagenesis. Mutation assays of 

31 benzo[a]pyrene in salmonella typhimurium are overwhelmingly positive with the inclusion of S9 

32 metabolic liver fractions, but are negative without the addition of the S9 metabolic enzymes (see 

33 Table 4-24). 

34 In mice, a dose-response and temporal relationship has been demonstrated between the 

35 formation of BPDE-DNA adducts and skin and forestomach tumors. In a study using mice 

36 treated dermally with benzo[a]pyrene once or twice per week for 15 weeks, a linear dose37 response of benzo[a]pyrene-induced adducts in the skin, lung, and liver was observed (Talaska et 

38 al. 1996). Another study examined the dose-response relationship and the time course of 
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1 benzo[a]pyrene-induced skin damage, DNA adduct formation, and tumor formation in female 

2 mice. Mice were treated dermally with 0, 16, 32, or 64 μg of benzo[a]pyrene once per week for 

3 29 weeks (Albert et al., 1991). Indices of skin damage and levels of BPDE-DNA adducts in skin 

4 reached plateau levels in exposed groups by 2–4 weeks of exposure. With increasing dose level, 

5 levels of BPDE-DNA adducts (fmol/μg DNA) initially increased in a linear manner and began to 

6 plateau at doses ≥32 μg/week. Tumors began appearing after 12–14 weeks of exposure for the 

7 mid- and high-dose groups and at 18 weeks for the low-dose group. At study termination 

8 (35 weeks after start of exposure), the mean number of tumors per mouse was approximately one 

9 per mouse in the low- and mid-dose groups and eight per mouse in the high-dose group. The 

10 time-course data indicate that benzo[a]pyrene-induced increases in BPDE-DNA adducts 

11 preceded the appearance of skin tumors, consistent with the formation of DNA adducts as a 

12 precursor event in benzo[a]pyrene induced skin tumors. 

13 Culp et al. (1996a) compared dose-response relationships for BPDE-DNA adducts and 

14 tumors in female B6C3F1 mice exposed to benzo[a]pyrene in the diet at 0, 18.5, 90, or 350 

15 μg/day for 28 days (to examine adducts) or 2 years (to examine tumors). The benzo[a]pyrene 

16 dose-tumor response data showed a sharp increase in forestomach tumor incidence between the 

17 18.5 μg/day group (6% incidence) and the 90 μg/day group (78% incidence). The BPDE-DNA 

18 adduct levels in forestomach showed a relatively linear dose-response throughout the 

19 benzo[a]pyrene dose range tested. The appearance of increased levels of BPDE-DNA adducts in 

20 the target tissue at 28 days is temporally consistent with the contribution of these adducts to the 

21 initiation of forestomach tumors. Furthermore, about 60% of the examined tumors had 

22 mutations in the K-ras oncogene at codons 12 and 13, which were G→T or G→C transversions 

23 indicative of BPDE reactions with DNA (Culp et al., 1996a). 

24 

25 Biological plausibility and coherence. A mutagenic MOA for benzo[a]pyrene is supported by a 

26 large body of research over time with consistent evidence of benzo[a]pyrene activation to 

27 reactive metabolites leading to DNA-damage and mutational events associated with tumor 

28 initiation. Mutagenicity as a mode of action for carcinogenicity in humans is generally accepted 

29 and is a biologically plausible mechanism for tumor induction. The formation of DNA adducts 

30 and oncogene/tumor suppressor mutations in organs that also displayed an increase in tumor 

31 incidence in rats and mice indicates coherence of these effects. Benzo[a]pyrene has been shown 

32 to be mutagenic in vivo and in vitro, across species and tissue types. 

33 

34 4.7.3.2. Other Possible MOAs 

35 In addition to mutagenicity, other MOAs which contribute to the carcinogenicity of 

36 benzo[a]pyrene are possible, but are not as well studied. The tumor promotion properties of 

37 benzo[a]pyrene may involve cell proliferative responses to cytotoxicity or apoptosis from 

38 benzo[a]pyrene metabolites, AhR-mediated effects on cell growth and differentiation, or anti-
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1 apoptotic signals elicited by metabolites (Burdick et al., 2006, 2003; Chen et al., 2003; Nebert et 

2 al., 1993). Results from some studies indicate that exposure to benzo[a]pyrene or its metabolites 

3 increases the production of inflammatory cytokines, such as IL-1, which may contribute to tumor 

4 promotion (N’Diaye et al., 2006; Tamaki et al., 2004; Garçon et al., 2001a, b). Benzo[a]pyrene 

5 also has been shown to inhibit GJIC, a characteristic associated with well-known tumor 

6 promoters such as TPA (Sharovskaya et al., 2006; Blaha et al., 2002). In summary, though there 

7 are limited data which support other processes that may contribute to the carcinogenicity of 

8 benzo[a]pyrene (inflammation, cytotoxicity, anti-apoptotic signaling, etc.); the available 

9 evidence indicates that the primary mode of action for benzo[a]pyrene involves DNA reactivity 

10 and mutagenicity leading to carcinogenesis. 

11 

12 4.7.3.4. Conclusions About the Hypothesized Mode of Action 

13 The MOA of mutagenicity of benzo[a]pyrene through reactive metabolites is extensively 

14 supported by a large body of research. Mutations from DNA reactive benzo[a]pyrene 

15 metabolites occur as early events in the carcinogenic process and are not believed to be acquired 

16 following cytoxicity or regenerative proliferation. Several lines of evidence relating to 

17 mutagenicity and tumor initiation are available for benzo[a]pyrene including: in vitro evidence 

18 of DNA adducts, mutations, cytogenetic damage, and primary DNA damage; in vivo DNA 

19 adducts, gene mutations, cytogenetic damage, and other measures of primary DNA damage; 

20 detection of DNA adducts in target tissue in vivo; and detection of cancer-relevant 

21 oncogene/tumor suppressor gene mutations in target tissue in vivo. Taken together, these data 

22 provide support for a mutagenic MOA for benzo[a]pyrene-induced cancer. 

23 

24 Support for the hypothesized MOA in test animals 

25 Benzo[a]pyrene induces gene mutations in a variety of in vivo and in vitro systems and 

26 produces tumors in all animal species tested and all routes of exposure. Strong, consistent 

27 evidence indicates that the postulated key events: the metabolism benzo[a]pyrene to a DNA28 reactive intermediates, the formation of DNA adducts, and the occurrence of subsequent 

29 mutations in oncogenes and tumor suppressor genes occur in animal models. 

30 

31 Relevance of the hypothesized MOA to humans 

32 Mutagenicity is a well-established cause of carcinogenicity. A substantial database of 

33 information on benzo[a]pyrene indicates that the postulated key events: the metabolism of 

34 benzo[a]pyrene to a DNA-reactive intermediates, the formation of DNA adducts, and the 

35 formation of subsequent mutations in oncogenes and tumor suppressor genes all occur in human 

36 tissues. The following lines of evidence from human studies provide support that the 

37 hypothesized mutagenic MOA is relevant to humans: the activation of benzo[a]pyrene to DNA 

38 reactive metabolites occurs in humans in qualitatively and quantitatively similar manner 
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1 compared to animals; DNA adducts specific to benzo[a]pyrene have been found in a wide variety 

2 of human tissues and are elevated in populations exposed to high levels PAHs; increased DNA 

3 mutations have been strongly associated with increasing levels of PAH-DNA adducts in workers 

4 occupationally exposed to PAHs; and the spectra of benzo[a]pyrene induced mutations in p53 

5 tumor suppressor genes and ras oncogenes observed in controlled in vitro cell systems and in 

6 vivo animal studies are similar to the spectra of mutations in tumors from PAH-exposed humans. 

7 

8 Populations or lifestages particularly susceptible to the hypothesized MOA

9 The mutagenic mode of action is considered relevant to all populations and lifestages. 

10 The current understanding of biology of cancer indicates that mutagenic chemicals, such as 

11 benzo[a]pyrene, are expected to exhibit a greater effect in early life versus later life exposure 

12 (U.S. EPA, 2005b; Vesselinovitch et al., 1979). Although the developing fetus and infants may 

13 have lower levels of some bioactivating enzymes than adults (e.g., CYP1A1/1B1), infants or 

14 children are expected to be more susceptible to benzo[a]pyrene-induced cancer at certain tissue 

15 sites. Newborn or infant mice developed liver and lung tumors more readily than young adult 

16 mice following acute i.p. exposures to benzo[a]pyrene (Vesselinovitch et al., 1975; see Section 

17 4.8.1). These results indicate that exposure to benzo[a]pyrene during early life stages presents 

18 additional risk for cancer, compared with exposure during adulthood. The Supplemental 

19 Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 

20 2005b) recommends the application of age-dependent adjustment factors (ADAFs) for 

21 carcinogens that act through a mutagenic mode of action. Given the weight of the available 

22 evidence, benzo[a]pyrene acts through a mutagenic mode of carcinogenic action and the ADAFs 

23 should be applied. 

24 Population variability in metabolism and detoxification of benzo[a]pyrene, in addition to 

25 DNA repair capability, may affect cancer risk. Polymorphic variations in the human population 

26 in CYP1A1, CYP1B1, and other CYPs have been implicated as determinants of increased 

27 individual lung cancer risk in some studies (Aklillu et al., 2005; Alexandrov et al., 2002; Perera 

28 and Weinstein, 2000). The Phase II cytosolic GST, mediated by variants of the GSTM1 and 

29 GSTT1 genes, prevents the formation of BPDE-DNA adducts. Some evidence suggests that 

30 humans lacking a functional GST gene have higher BPDE-DNA adduct levels and thus are at 

31 greater risk for cancer (Vineis et al., 2007a; Pavanello et al., 2004; Perera and Weinstein, 2000; 

32 Alexandrov et al., 2002). In addition, acquired deficiencies or inherited gene polymorphisms 

33 that affect the efficiency or fidelity of DNA repair may also influence individual susceptibility to 

34 cancer from environmental mutagens (Matullo et al., 2003; Shen et al., 2003; Cheng et al., 2000; 

35 Perera and Weinstein, 2000; Wei et al., 2000; Amos et al., 1999). In general, however, available 

36 support for the role of single polymorphisms in significantly modulating human PAH cancer risk 

37 is relatively weak or inconsistent. Combinations of metabolic polymorphisms, on the other hand, 
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1 may be critical determinants of cumulative DNA-damaging dose, and thus susceptibility to 

2 cancer from benzo[a]pyrene exposure. 

3 

4 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 

5 4.8.1. Possible Childhood Susceptibility 

6 Increased childhood susceptibility to benzo[a]pyrene is supported by several lines of 

7 evidence including epidemiological studies reporting associations between adverse birth 

8 outcomes and developmental effects and internal biomarkers of exposure to benzo[a]pyrene, 

9 presumably via exposure to complex PAH mixtures (Tang et al. 2008, 2006; Perera et al., 

10 2005a,b). The occurrence of BPDE-DNA in maternal and umbilical cord blood in conjunction 

11 with exposure to ETS was associated with reduced birth weight and head circumference in 

12 pregnant women living in the vicinity of fires from the 09/11/2001 disaster site in New York 

13 City (Perera et al., 2005a). In other studies, elevated levels of BPDE-DNA adducts in umbilical 

14 cord blood were associated with: (1) reduced birth weights or reduced head circumference in the 

15 offspring of 529 Dominican or African-American nonsmoking women (Perera et al., 2005b); and 

16 (2) decreased body weight at 18, 24, and 30 months and deficits in several areas of development 

17 as assessed by the Gesell Developmental Schedules at 24 months in the offspring of nonsmoking 

18 Chinese women living in the vicinity of a coal-fired power plant (Tang et al., 2008, 2006). 

19 

20 Developmental neurotoxicity 

21 Studies in humans and experimental animals indicate that exposure to PAHs in general, 

22 and benzo[a]pyrene in particular, may impact neurological development at relatively low 

23 exposure levels. Observational studies in humans have suggested associations between 

24 gestational exposure to PAHs and later measures of neurodevelopment (Perera et al., 2009; Tang 

25 et al., 2008). An observational study of a Chinese population living in close proximity to a coal 

26 fired power plant found increased levels of benzo[a]pyrene-DNA adducts in cord blood were 

27 associated with decreased developmental quotients in offspring (Tang et al., 2008). In addition, 

28 a study of pregnant women living or working near the World Trade Center site in NYC found 

29 high PAH exposure during pregnancy was associated with a reduction in verbal and full scale IQ 

30 of offspring at 5 years of age (Perera et al., 2009). 

31 A study in pregnant rats exposed by inhalation showed a dose related increase in 

32 benzo[a]pyrene metabolites in the cerebral cortex and hippocampus of pups, indicating the fetal 

33 brain is exposed to benzo[a]pyrene and/or its metabolites following maternal inhalation exposure 

34 (Wu et al., 2003). Another study which treated pregnant rat dams to benzo[a]pyrene by 

35 inhalation found a decrease in long term potentiation (LTP) in the hippocampus of gestationally 

36 treated pups compared with controls, indicating a possible effect on learning and memory in 

37 benzo[a]pyrene exposed animals, though functional tests were not conducted (Wormley et al., 

38 2004). Another study by the same group treated rat dams by gavage with low levels of 
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1 benzo[a]pyrene (300 μg/kg) on GD 14-17 and observed benzo[a]pyrene metabolites in the brains 

2 of pups and diminished cortical neuronal activity to sensory input in exposed offspring compared 

3 with controls (McCallister et al., 2008). In addition, a study of mice exposed to benzo[a]pyrene 

4 lactationally observed statistically significant differences in performance in several neuromotor 

5 and behavioral tests which indicated decreased righting reflex and disinhibition behavior 

6 (Bouayed et al., 2009). 

7 

8 Reproductive effects 

9 Epidemiological studies indicate that exposure to complex mixtures of PAHs, such as 

10 through cigarette smoke, is associated with measures of decreased fertility in humans (El Nemr 

11 et al., 1998; Neal et al., 2005) and that prenatal exposure to cigarette smoking is associated with 

12 reduced fertility of women later in life (Weinberg et al., 1989). A case-control study in a 

13 Chinese population has also indicated that women with elevated levels of benzo[a]pyrene-DNA 

14 adducts in maternal blood were four times more likely to have experienced a missed abortion 

15 (Wu et al., 2010). 

16 Oral multigenerational studies of benzo[a]pyrene exposure in mice demonstrate effects 

17 on fertility and the development of reproductive organs in male and female offspring exposed to 

18 benzo[a]pyrene during development at levels in which no overt toxicity or depression in fertility 

19 is seen in the parental animals (Mackenzie and Angevine 1981; Kristensen et al., 1995). 

20 MacKenzie and Angevine (1981) exposed groups of mice to benzo[a]pyrene on GDs 7–

21 16 and reproductive outcomes of the offspring were investigated. Fertility of the F1 generation 

22 was decreased in a dose dependant manner. At maturity, the fertility of these animals was tested. 

23 The F1 male and F1 female fertility indices were significantly decreased in each exposure group. 

24 These reductions in fertility indices were associated with decreased testes and ovary weight in 

25 the F1 animals. Male offspring showed histological damage of the seminiferous tubules and 

26 female offspring had hypoplastic ovaries with few follicles and corpora lutea. Similar results 

27 were reported in a study in which female mice were exposed by gavage to benzo[a]pyrene on 

28 GDs 7–16 (Kristensen et al., 1995). F1 females had decreased mean ovary weight and reduced 

29 fertility. At necropsy, the F1 females had reduced ovary weights with decreased numbers of 

30 small, medium, or large follicles and corpora lutea. Inhalation exposure of pregnant female rats 

31 to benzo[a]pyrene:CB aerosols during gestation has also been associated with decreased fetal 

32 survival and number of pups per litter associated with decreased levels of plasma progesterone, 

33 estradiol, and prolactin (Archibong et al., 2002). 

34 These reductions in fertility observed in animal models are supported by a large database 

35 of animal studies in adult animals indicating that benzo[a]pyrene is ovotoxic with effects 

36 including decreased ovary weight, decreased primordial follicles, and reduced fertility (Mattison 

37 et al., 1980; Swartz and Mattison 1985; Miller et al., 1992; Borman et al, 2000). 

38 
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1 Developmental Immune effects 

2 The severity and persistence of immune effects observed during in utero studies suggests 

3 that immunotoxicity may be greater during gestation than adulthood (Dietert and Pieperbrink, 

4 2006; Holladay and Smialowicz, 2000). Urso and Gengozian (1982) provide experimental 

5 support demonstrating immunosuppression from benzo[a]pyrene exposure during gestation was 

6 greater than for mice exposed after birth to a 25-fold higher dose. There is also substantial 

7 general literature indicating that disruption of the immune system during certain critical periods 

8 of development (e.g., initiation of hematopoiesis; migration of stem cells; expansion of 

9 progenitor cells) may have significant and lasting impacts on lifetime immune function (e.g. 

10 Burns-Naas et al., 2008; Dietert, 2008; Landreth et al., 2002; Dietert et al., 2000), as well as 

11 more specific studies showing increased dose sensitivity and disease persistence from 

12 developmental versus adult chemical exposure (reviewed in Luebke et al., 2006). 

13 Thymus toxicity is a sensitive and specific effect of benzo[a]pyrene and has been 

14 observed in both prenatal and adult exposure studies. The thymus serves as a major site of 

15 thymocyte proliferation and selection for maturation, and impairment can lead to cell-mediated 

16 immune suppression (Kuper 2002, 1992; De Waal et al., 1997). The thymus is believed to be 

17 critical for T lymphocyte production during early life and not in adulthood (Hakim et al., 2005; 

18 Schonland et al., 2003; Petrie et al., 2002; Mackall et al., 1995). Therefore, the decreases in 

19 thymus weight observed in studies of adult animals exposed to benzo[a]pyrene suggest that 

20 immunosuppression may be a heightened concern for individuals developmentally exposed to 

21 benzo[a]pyrene. 

22 

23 Cancer 

24 As mentioned above in section 4.7.3.4, investigations in young animals exposed to 

25 benzo[a]pyrene provide evidence that early life exposure may present increased risk of cancer. 

26 Comparisons of cancer responses in newborn (1 day old), infant (15 days old), and young adult 

27 (42 days old) mice indicate that exposure to benzo[a]pyrene during early life stages can present 

28 additional risk for cancer, compared with exposure during young adulthood (Vesselinovitch et 

29 al., 1975), but studies designed to compare risks of cancer from early-life (including gestational 

30 and pre-weaning) plus chronic adulthood exposures with risks from chronic adulthood exposure 

31 alone were not located. Following i.p. injection of single doses of 75 or 150 mg/kg 

32 benzo[a]pyrene to newborn (1 day old), infant (15 days old), or young adult (42 days old), 

33 newborn and infant mice more readily developed tumors than young adult mice in the liver and 

34 lung, the most predominant tissue sites of cancer development under these exposure conditions 

35 (Vesselinovitch et al., 1975). The benzo[a]pyrene-exposed groups also displayed increased 

36 incidences of stomach and lymphoreticular tumors, but the data indicated that these tumors 

37 developed more readily with exposure at 42 or 15 days, compared with exposure on PND 1 . 

38 
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1 4.8.2. Possible Gender Differences 

2 Cheng et al. (2007) had conducted a study in which they found that lung tumor tissue 

3 from nonsmoking females contained higher benzo[a]pyrene-DNA adduct levels than that from 

4 nonsmoking males. The adduct levels were associated with CYP1A1 protein levels in the same 

5 tissues. Female lung cancer tissue contains higher levels of DHH activity than male lung cancer 

6 tissue. DHH is an enzyme that can divert benzo[a]pyrene-7,8-dihydrodiol into the quinone 

7 pathway, thus preventing BPDE and DNA adduct formation. It is highly expressed in liver, but 

8 only weakly in lung. Cheng et al. (2007) decided to investigate benzo[a]pyrene-DNA adduct 

9 formation in several lung cancer cell lines to elucidate the roles of CYP1A1 and DHH in this 

10 process. They found that DNA adduct levels were increased in cell lines that contain elevated 

11 CYP1A1 and DHH isoform 1 activities. When DHH1 activity was blocked in these cells, DNA 

12 adduct levels were increased. Benzo[a]pyrene-DNA adduct levels in 120 lung tumor samples 

13 were associated with the protein levels of CYP1A1, but not DHH1. Comparing tumor tissues 

14 from both genders lung cancer patients they observed that a significantly higher percentage of 

15 female lung tumors had measurable CYP1A1 levels, but were negative for DHH1, compared 

16 with male tumors. The authors suggested that a gender difference in DHH1 activity was in part 

17 responsible for the increased lung tumor incidence in females. 

18 Chang et al. (2007) conducted a study also based in the increased incidence of lung 

19 cancer in human females, but focused on the benzo[a]pyrene interaction with estrogen that result 

20 in elevated COX-2 expression. COX-2 (aka PHS-2) can activate the procarcinogen 

21 benzo[a]pyrene-7,8-dihydrodiol to BPDE. Human bronchial epithelial cells were treated with 

22 benzo[a]pyrene and/or 17β-estradiol. The combined, but not the individual treatments induced 

23 COX-2 expression. The authors considered their findings as mechanistic evidence towards 

24 understanding the gender difference in susceptibility towards benzo[a]pyrene. 

25 Taioli et al. (2007) reviewed the evidence for a connection between MPO polymorphism 

26 and lung cancer (a more detailed overview of this study is given in Section 4.8.3.3). MPO 

27 converts benzo[a]pyrene metabolites into highly reactive epoxides and a known polymorphism 

28 in its promoter region is said to afford some protection from lung cancer. Several genetic 

29 variants of MPO are known, most of which result in deficiency of the enzyme. The MPO-G/G 

30 genotype (WT) is said to be associated, among others, with acute promyelocytic leukemia, 

31 aerodigestive tract cancer, coronary artery disease, early-onset multiple sclerosis, and an 

32 increased incidence of Alzheimer disease. A multi-study analysis was conducted after several 

33 epidemiologic studies had suggested an association between this gene polymorphism and lung 

34 cancer incidence. The data were stratified for ethnicity, age, gender, and smoking status but 

35 neither age nor gender showed any association for MPO polymorphism and lung cancer risk. 

36 The authors hypothesized that age- and gender-related associations with MPO genotype and lung 

37 cancer incidence, as had been observed in other studies, may be related to age- and gender38 dependent smoking habits rather than to the gene polymorphism itself. 
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1 Mammary epithelium and tissues from the female genital tract have the ability to activate 

2 benzo[a]pyrene. Morris and Seifter (1992) made a strong case for benzo[a]pyrene as a potential 

3 causative agent in breast cancer, not only based on the anti-estrogenic action of benzo[a]pyrene 

4 but also on its tendency to accumulate in adipose and hence breast tissue. Jeffy et al. (2002) also 

5 pointed out that PAHs are risk factors for breast cancer. Because the populations in the studies 

6 presented in Section 4.1.4 were predominantly, if not exclusively males, data for breast or 

7 cervical cancers in relation to benzo[a]pyrene exposure are not available. 

8 Gender differences in the response to benzo[a]pyrene have been demonstrated in 

9 numerous animal studies (Knuckles et al., 2001; Kroese et al., 2001; Ramesh et al., 2001a, 2000; 

10 Hood et al., 2000; Rodriguez et al., 1997; Weyand et al., 1994; Turusov et al., 1990; Weyand and 

11 Bevan, 1987). The differences ranged from variations in feed intake, with or without effects on 

12 body weight, to differences in disposition, all the way to different susceptibility towards 

13 benzo[a]pyrene-induced cancers. In some studies, females and males were dosed differently; in 

14 general, females were more resistant to benzo[a]pyrene toxicity than males. None of the studies 

15 presented cogent explanations for the observed differences. 

16 In the 2-year bioassay by Kroese et al. (2001), female rats had fewer tumors of the 

17 forestomach and auditory canal, but more tumors of the liver, compared with males. There was a 

18 rather striking negative dose response for pituitary tumors in females (eight to zero tumors from 

19 control to highest dose), but not in males. Brune et al. (1981) used both sexes of animals in their 

20 study, but did not report their findings for the sexes separately, allowing the conclusion that no 

21 obvious sex differences were observed. 

22 Soyka (1980) found that prenatal treatment of mice with benzo[a]pyrene affected 

23 response to an enzyme-inducing challenge with 3-MC in a gender-specific way when the animals 

24 were 3 months old. Female offspring of benzo[a]pyrene-treated mice had significantly elevated 

25 hepatic microsomal aminopyrine demethylase activity, while CYP450 levels were significantly 

26 lower in male offspring. 

27 Sharma et al. (1997) specifically attempted to resolve the gender difference in cancer 

28 susceptibility of CD-1 mice. They focused on glutathione S-transferase π (GSTP) because it 

29 detoxifies BPDE. They noted that constitutive expression of GSTP in the liver of the male CD-1 

30 mouse was higher than in the female and that GSTP activity was much more inducible by the 

31 antioxidant butylhydroxyanisole in the female than in the male mouse. They reported that 

32 female mice were more susceptible to the carcinogenic effect of benzo[a]pyrene than males, but 

33 only females could be partially protected from benzo[a]pyrene-induced carcinogenesis by the co34 administration of butylhydroxyanisole. This is an indication that Phase II enzymes may play a 

35 role in the gender difference towards benzo[a]pyrene toxicity. 

36 Martin et al. (2004) used a transgenic mouse model to address the question of gender 

37 differences. They used p53 heterozygous Tg.AC (v-Ha-ras) mice, a strain possessing a 

38 carcinogen-inducible ras oncogene, but only one functional p53 tumor suppressor gene. The 
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1 authors emphasized that these two mutations have been observed frequently in human tumors. 

2 Female and male animals received 20 mg/kg benzo[a]pyrene by gavage in corn oil twice weekly 

3 for 10 weeks. Eighteen weeks after termination of dosing, tissues were collected for histologic 

4 processing. There were evident differences in gender response (percent of total, female 

5 control/male control:female treated/male treated): mortality (60/60:70/40), thymus hyperplasia 

6 (9/30:27/10), bladder papilloma (0/0:0/13), hepatocyte vacuolization (54/92:86/55), 

7 hematopoietic cell proliferation in spleen (57/8:82/27), lymph node hyperplasia (0/0:0/33), and 

8 malignant lymphoma (0/0:7/36). Neoplasia of the forestomach did not show a gender difference. 

9 The focus of this study was evaluation of feed modifications in carcinogenesis studies (some of 

10 the animals were given N-acetyl cysteine in the feed); the authors did not speculate on the 

11 reasons for the gender differences. Wei et al. (2000) (see Section 4.1.4) observed that female 

12 lung cancer patients displayed less DNA repair capacity than males. 

13 benzo[a]pyrene has also been described as an anti-androgen (see Sections 4.4.1.3 and 

14 4.5.3) in animal studies. The AhR mediates anti-estrogenic effects of its ligands (see Section 

15 4.6.3.2). The findings in animals—anti-estrogenic and potential protection from breast cancer—

16 are at odds with postulates made for humans that benzo[a]pyrene may advance the development 

17 of breast cancer (Jeffy et al., 2002; Morris and Seifter, 1992). Li et al. (1999) reported that 41% 

18 of the samples of noncancerous breast tissue from breast cancer patients contained 

19 benzo[a]pyrene-like DNA adducts, while no such adducts were detected in tissues obtained from 

20 breast reduction surgery patients. Gender-specific expression of glycine N-methyltransferase 

21 (GNMT) has been shown for mice (Section 4.5.2) and might help to explain gender-related 

22 effects, including cancer formation, in this species. Applicability to human populations, 

23 however, has not been addressed. 

24 

25 4.8.3. Genetic Polymorphisms 

26 The metabolic formation and subsequent binding to critical positions in DNA of ultimate 

27 carcinogenic forms of PAH are recognized as key mechanistic events in tumorigenesis. 

28 Increased PAH exposure concentrations are associated with increased levels of DNA adducts and 

29 other biomarkers in both target and surrogate tissues. Observed biomarker levels vary 

30 considerably even among persons with apparently comparable exposures (Garte et al., 2007; 

31 Perera and Weinstein, 2000). While laboratory variation and uncertain exposure estimates 

32 contribute to the differences, inter-individual variation in PAH metabolism (activation and 

33 detoxification) may be especially important. In particular, heritable metabolic gene (single 

34 nucleotide polymorphisms [SNPs]) appear in some cases to influence individual susceptibility to 

35 specific types of cancer, in addition to factors such as ethnicity, age, gender, nutrition, hormonal 

36 and immune status, and preexisting health impairment. 

37 In humans, benzo[a]pyrene is metabolized to the highly DNA-reactive BPDE by 

38 microsomal CYP Phase I enzymes, mediated primarily by the CYP1A1 and CYP1B1 genes. 
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1 Polymorphic variations in the human population that result in high inducibility of CYP1A1, 

2 CYP1B1, and other CYPs have been implicated as determinants of increased lung cancer risk in 

3 some studies (Aklillu et al., 2005; Alexandrov et al., 2002; Perera and Weinstein, 2000). The 

4 Phase II cytosolic GST, mediated by variants of the GSTM1 and GSTT1 genes, prevent the 

5 formation of BPDE-DNA adducts. Some evidence suggests that humans lacking a functional 

6 GST gene have higher PAH(BPDE)-DNA adduct levels and thus be at greater risk for cancer 

7 (Vineis et al., 2007a; Pavanello et al., 2004; Alexandrov et al., 2002; Perera and Weinstein, 

8 2000). Similarly, acquired deficiencies or inherited gene polymorphisms that affect the 

9 efficiency or fidelity of DNA repair may also influence individual susceptibility to cancer 

10 (Matullo et al., 2003; Shen et al., 2003; Cheng et al., 2000; Perera and Weinstein, 2000; Wei et 

11 al., 2000; Amos et al., 1999). In general, however, support for the role of SNP in significantly 

12 modulating PAH cancer risk is relatively weak (i.e., small increases in cancer risk) or 

13 inconsistent, possibly due in part to small study size and the use of DNA-adduct detection 

14 methods with low specificity (i.e., bulky DNA-adducts) and sensitivity. Combinations of 

15 metabolic polymorphisms, on the other hand, are receiving increased attention as critical 

16 determinants of cumulative DNA-damaging dose, and thus individual cancer risk. 

17 Following a report from Japan that the GSTM1 null genotype combined with a mutated 

18 CYP1A1 genotype was associated with increased lung cancer risk, Rojas and coworkers (2000) 

19 measured specific BPDE-DNA adducts in leukocytes (HPLC with fluorometric detection) to 

20 evaluate the impact of CYP1A1, GSTM1, and GSTT1 genotype combinations. The human 

21 subjects were 89 PAH-exposed coke oven workers (smokers and nonsmokers) and 44 power 

22 plant workers (all smokers) not occupationally exposed to PAH. Increased adduct levels were 

23 significantly correlated with CYP1A1 polymorphism, occupational PAH exposure, and smoking. 

24 Combinations of genotypes were observed to have a significant impact on BPDE-DNA adducts, 

25 ranging from the absence of adducts in subjects with the active CYP1A1/GSTM1 genotypes to 

26 the highest BPDE-DNA adduct level in the most susceptible combination of mutated CYP1A1 

27 with null GSTM1 genotype. The results provide mechanistic support for distinguishing high28 susceptibility benzo[a]pyrene-exposed subgroups, and for understanding their association with 

29 increased cancer rates. 

30 Pavanello et al. (2005) studied associations between xeroderma pigmentosum 

31 (XP)-linked gene polymorphisms, the GSTM1 polymorphism, and bulky BPDE-type DNA 

32 adduct formation in peripheral lymphocytes from 67 highly PAH-exposed male Polish coke oven 

33 workers. The four XP genotypes studied impart low NER capacity, while the GSTM1 active or 

34 null genotypes are associated with effective or ineffective removal of biologically active 

35 benzo[a]pyrene metabolites via GSH conjugation. Workers were questioned for smoking habits, 

36 charbroiled meat consumption, and other factors that might have affected their PAH exposure. 

37 PAH exposure was assessed via urinary 1-OH-pyrene levels. There was a statistically significant 

38 difference in the number of DNA adducts between the GSTM1 active and null carriers (3.37 ± 
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2.20 vs. 6.73 ± 6.61 adducts per 1081 nucleotides, respectively). For the DNA repair gene 

2 polymorphisms, there was an increase in DNA adduct numbers from homozygous WT carriers to 

3 low-DNA-repair homozygous variant carriers. This difference was statistically significant for 

4 homozygous carriers of the XPC-PAT and XPA-A23G variants, but not for the homozygous 

5 XPD-Lys751Gln and XPD-Asp312Asn variants. Individuals with a combination of DNA repair6 unfavorable XPC or XPA genotypes and the GSTM1-null variant generally fell into the highest 

7 tertile of DNA adduct numbers. Smoking status and diet did not influence urinary 1-OH-Py or 

8 BPDE-DNA adduct levels. These results support the conclusion that certain gene polymorphism 

9 combinations affecting DNA repair or detoxification capacities may increase health risks 

10 resulting from PAH exposure. 

11 Porter et al. (2005) also evaluated the influence of XPA gene variants involved in NER 

12 on BPDE-induced cytotoxicity. SV40-transformed human skin fibroblasts from an XP patient 

13 with a nonsense XPA mutation were stably transfected with the WT XPA gene or either of two 

14 rare XPA variants; the transfected genes could be overinduced with ponasterone A. The WT 

15 XPA and both variants had greatly improved survival compared to XPA-free cells. Survival was 

16 even more improved by ponasterone A induction in the variant, but not the WT cells. These 

17 findings indicate that the polymorphic XPAs show greater ability to repair BPDE-induced DNA 

18 damage, and thus may offer some protection from benzo[a]pyrene-induced genotoxicity, while 

19 the nonsense mutation is likely to increase genotoxic risk.
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1 

2 5. DOSE-RESPONSE ASSESSMENTS 

3 

4 

5 5.1. ORAL REFERENCE DOSE (RfD) 

6 5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

7 There are limited data establishing associations between increased risk for noncancer 

8 health effects in humans and exposure to benzo[a]pyrene. Several epidemiology studies have 

9 reported associations between adverse birth outcomes including reduced birth weight, postnatal 

10 body weight, and head circumference and internal biomarkers of exposure to benzo[a]pyrene 

11 (BPDE-DNA adducts) via exposure to complex PAH mixtures (Tang et al., 2008, 2006; Perera et 

12 al., 2005a, b). However, extrapolations from these studies are complicated by the concomitant 

13 exposure to multiple PAHs and other components in the mixture. Thus, studies in humans were 

14 not selected to serve as the basis of the RfD. 

15 The subchronic and chronic oral exposure animal database includes a 2-year gavage 

16 cancer bioassay with male and female Wistar rats (Kroese et al. 2001), a 2-year dietary cancer 

17 bioassay with female B6C3F1 mice (Beland and Culp, 1998; Culp et al., 1998), a 90-day gavage 

18 study with male and female Wistar rats (Kroese et al., 2001), a 90-day dietary study with male 

19 and female F344 rats (Knuckles et al., 2001), and a 35-day study in male Wistar rats evaluating 

20 immune endpoints (De Jong et al., 1999). Also available are five reproductive/developmental 

21 toxicity studies in rodents examining reproductive endpoints in male Sprague-Dawley rats 

22 (Zheng et al., 2010) and C57BL/6 mice (Mohamed et al., 2010), in offspring of treated CD-1 and 

23 NMRI female mice (Kristensen et al., 1995; MacKenzie and Angevine, 1981), and in female 

24 Sprague-Dawley rats (Xu et al., 2010). 

25 Kroese et al. (2001) exposed Wistar rats to benzo[a]pyrene in soybean oil by gavage at 

26 doses of 0, 3, 10, or 30 mg/kg-day, 5 days/week, for 2 years. This study was primarily designed 

27 as a cancer bioassay and did not evaluate other endpoints. An increase in the incidence of 

28 animals with forestomach hyperplasia, compared with the control incidence, occurred at the low 

29 and mid-dose but not the high-dose level; an dose-related, increased incidence of forestomach 

30 tumors was observed at doses ≥ 3 mg/kg-day. An increased incidence of hepatic clear cell foci 

31 of cellular alteration was also observed at the 3 mg/kg-day, but not at the 10 or 30 mg/kg-day. 

32 At the two highest exposure levels, elevated incidences of liver tumors were observed. 

33 Female B6C3F1 mice were administered benzo[a]pyrene in the diet at average daily doses 

34 of 0, 0.7, 3.3, and 16.5 mg/kg-day for 2 years (Beland and Culp, 1998; Culp et al., 1998). An 

35 increase in the incidence of mice with forestomach hyperplasia, compared with the control 

36 incidence, occurred at the lowest exposure level (23/47 at 0.7 mg/kg-day versus 13/48 in 

37 controls). Similar to the rat bioassay (Kroese et al., 2001), forestomach hyperplasia was 

38 observed with increasing incidence of animals with forestomach tumors (squamous cell 
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1 papillomas or carcinomas) with increasing dose. No other dose-related effects were reported in 

2 this cancer bioassay. 

3 A 90 day study also reported by Kroese et al. (2001) treated animals by gavage 5 

4 days/week with 0, 3, 10, or 30 mg/kg benzo[a]pyrene in corn oil. The most sensitive effects 

5 observed included increased liver weight and decreased thymus weight. Increases in liver weight 

6 greater than 10% of controls were observed at 10 and 30 mg/kg-day in males only, and were 

7 statistically significant. However, there were no statistically significant elevations in liver 

8 enzymes screened in serum (ALT, AST, LDH, and GGT). The biological significance of 

9 increased liver weight in males in the absence of elevated liver enzymes in the serum is unclear. 

10 A decrease in thymus weight was observed in both sexes at 30 mg/kg-day, at 17 and 33% in 

11 females and males, respectively, compared with controls. At 10 mg/kg-day, thymus weight in 

12 males was decreased by 15% (not statistically significant). An increase in the incidence of 

13 thymus atrophy was also observed in the 30 mg/kg-day males that showed a reduction of thymus 

14 weight. Incidences for thymus atrophy (categorized in severity as slight) for the control through 

15 high-dose groups were 0/10, 0/10, 0/10, and 3/10 for females and 0/10, 2/10, 1/10, and 6/10 for 

16 males. The thymus is an organ involved in the maturation of immune cells especially early in 

17 development. A change in thymus weight in the adult animal may be accompanied by alterations 

18 of the immune system in functional assays, but the significance of thymus weight changes alone 

19 is unknown. 

20 Knuckles et al. (2001) exposed male and female F344 rats (6-8 per group) to 

21 benzo[a]pyrene at doses of 0, 5, 50, or 100 mg/kg-day in the diet for 90 days. Statistically 

22 significant decreases in RBC counts and hematocrit level (decreases as much as 10 and 12%, 

23 respectively) were observed in males at doses ≥50 mg/kg-day and in females at 100 mg/kg-day. 

24 The effect observed in this study at the lowest dose was an increase in abnormal tubular casts in 

25 the kidney in males in which increases were observed at 5 mg/kg-day (40%), 50 mg/kg-day 

26 (80%) and 100 mg/kg-day (100%), compared to 10% in the controls. In females, only 10% 

27 showed significant kidney tubular changes at the two high dose levels compared to zero 

28 incidence in controls. The incidences for kidney lesions were not provided; instead the data are 

29 reported graphically as rounded percent incidences. Several reporting gaps in Knuckles et al 

30 (2001) make interpretation of the results difficult. Specifically, the authors do not provide 

31 statistical analysis of the renal endpoint nor do they provide the incidence data which would 

32 allow for independent statistical analysis. The study author was contacted, but additional 

33 clarification of the study data was not provided. Therefore, due to reporting gaps and resulting 

34 reduced confidence, this study was not considered further in selecting the principal study. 

35 De Jong et al. (1999) treated male Wistar rats (eight/dose group) with benzo[a]pyrene by 

36 gavage 5 days/week for 35 days at doses of 0, 3, 10, 30, and 90 mg/kg-day. Hematological and 

37 immunological changes were reported. Small, but statistically significant, dose-related decreases 

38 in RBC count (5%) and associated measures (hemoglobin, and hematocrit) were observed at 
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1 ≥10 mg/kg-day. In addition, a dose-related and statistically significant decrease in the relative 

2 number of B cells (13%) in the spleen was observed at ≥10 mg/kg-day compared to controls. 

3 Dose-related decreases in thymus weight were statistically significant at ≥10 mg/kg-day. 

4 Decreases in heart weight at 3 mg/kg-day and in kidney weight at 3 and 30 mg/kg-day were also 

5 observed, but these changes did not show dose-dependent responses. At doses above 10 mg/kg6 day, significant decreases were observed in absolute number of cells harvested in the spleen, in 

7 the number of B cells in the spleen, and in NK cell activity in the spleen, as well as a decrease in 

8 serum IgM and IgA in rats. 

9 Zheng et al., (2010) treated male Sprague-Dawley rats (8/group) to 0, 1, or 5 mg/kg-day 

10 benzo[a]pyrene by daily corn oil gavage for a duration of 30 or 90 days. Testicular testosterone 

11 was statistically significantly decreased in the high dose group (approximately 15%) following 

12 90 days of exposure. The low dose group also appeared to have a similar average depression of 

13 testosterone levels; however, the change did not reach statistical significance. 

14 Mohamed et al. (2010) investigated multi-generational effects in male mice following 

15 exposure of six-week old C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day 

16 benzo[a]pyrene for 6 weeks by daily corn oil gavage. Following final treatment, male mice were 

17 mated with two untreated female mice to produce an F1 generation; F1 and F2 males were also 

18 mated with untreated female mice. The mice of the F1, F2, and F3 generations were not exposed 

19 to benzo[a]pyrene. Statistically significant reductions of approximately 50% were observed in 

20 epididymal sperm counts of F0 and F1 generations treated with the low dose of benzo[a]pyrene. 

21 For F0 and F1 generations of the high dose group, epididymal sperm counts were reduced 

22 approximately 70%. Means and variances were not reported but were presented graphically. 

23 This study indicates that exposure to benzo[a]pyrene may have transgenerational effects on 

24 sperm count. However, due to incomplete reporting, this study was not considered further for 

25 selection as the principal study but was considered to be supportive of low dose male 

26 reproductive effects following benzo[a]pyrene exposure. 

27 MacKenzie and Angevine (1981) exposed groups of 30–60 female CD-1 mice to 0, 10, 

28 40, or 160 mg/kg-day benzo[a]pyrene on GDs 7–16. Crossover mating studies were then 

29 conducted in which F1 offspring were mated continuously with untreated mice to determine 

30 effects on fertility. Benzo[a]pyrene did not appear to be overtly toxic to mothers or offspring. 

31 However, statistically significant decreased pup weight was observed at all dose levels at day 42. 

32 At the lowest dose tested, pup weight was decreased 6% compared to control. At maturity, the 

33 fertility of these animals was tested. The F1 male and F1 female fertility indices (i.e., percent of 

34 mated animals that were pregnant) were significantly decreased in each exposure group as 

35 follows (control through high-dose groups): F1 males: 80.4, 52.0, 4.7, and 0.0; F1 females 100, 

36 65.7, 0.0, and 0.0. After six months on the breeding study, 34% of the gestationally treated 

37 females in the 10 mg/kg-day dose group failed to produce any litters, and the the F1 females in 

38 this dose group that did litter produced statistically significantly smaller litter sizes (19% 
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1 reduction in mean litter size). These reductions in fertility indices were associated with 

2 decreased testes and ovary weight in the F1 animals. Testes weight was decreased 40 and 88% 

3 at 10 and 40 mg/kg-day, respectively, and was associated with histologic evidence of injury to 

4 the seminiferous tubules. In female F1 animals, severe reductions in ovarian tissues were 

5 observed at all dose levels such that ovary weight measurements were difficult to obtain. 

6 Examination of available tissue in these females revealed hypoplastic ovaries with few follicles 

7 and corpora lutea (10 mg/kg-day) or with no evidence of folliculogenesis at the higher dose 

8 (40 mg/kg-day). 

9 Similar results were reported in a study in which groups of nine NMRI F0 female mice 

10 were exposed by gavage to 0 or 10 mg/kg-day benzo[a]pyrene on GDs 7–16 (Kristensen et al., 

11 1995). F1 females were continuously bred with an untreated male for 6 months. F1 females had 

12 decreased mean ovary weight (30% decreased, compared with controls) and reduced fertility as 

13 reflected by decreased mean number of F2 litters (three compared with eight for control F1 

14 females). F1 females had statistically significantly lower median numbers of offspring, number 

15 of litters, and litter sizes and a statistically significantly greater median number of days between 

16 litters as compared with the controls. At necropsy, the F1 females had statistically significantly 

17 reduced ovary weight with histologic examination revealing decreased numbers of small, 

18 medium, or large follicles and corpora lutea. 

19 Xu et al., (2010) treated female Sprague-Dawley rats (6/group) to 0, 5, or 10 mg/kg-day 

20 benzo[a]pyrene by corn oil gavage every other day for a duration of 60 days. This resulted in 

21 time weighted average doses of 0, 2.5, and 5 mg/kg-day over the study period of 60 days. 

22 Absolute ovary weight was statistically significantly reduced in the both the low and high 

23 benzo[a]pyrene dose groups (11 and 15%, respectively; see Table 5-1). Animals in the high dose 

24 group also had statistically significantly depressed levels of estradiol (by approximately 25%) 

25 and decreased numbers of primordial follicles (by approximately 20%) compared to controls. 

26 Statistically significantly altered estrus cyclicity was also evident in the high dose of 

27 benzo[a]pyrene. 

28 

Table 5-1. Means ± SD for ovary weight in female SD-rats 

Dose (mg/kg-d)a

0 2.5 5 
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Ovary weight (g) 0.160 ± 0.0146 0.143 ± 0.0098b

0.136 ± 0.0098b

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) using one-way ANOVA 

Source: Xu et al. (2010). 

1 

2 5.1.2. Methods of Analysis 

3 A number of noncancer effects observed following chronic or subchronic administration 

4 of benzo[a]pyrene were modeled with U.S. EPA’s Benchmark Dose (BMD) Modeling Software 

5 (BMDS) where data were amenable. These endpoints included increased liver weight, decreased 

6 thymus weight, decreased percent of splenic B-cells, increased forestomach hyperplasia, and 

7 decreased ovary weight (Xu et al., 2010; Kroese et al., 2001; De Jong et al., 1999). Zheng et al. 

8 (2010) did not provide enough information (i.e., incidences or means and variances) to allow for 

9 BMD modeling. Data from other studies could not be modeled due study design utilizing only 

10 one dose (Kristensen et al., 1995) or a highly elevated magnitude of response at the lowest dose 

11 (MacKenzie and Angevine 1981) in which extrapolation down to a suitable benchmark response 

12 would be unsupported by the available models. 

13 In accordance with U.S. EPA’s Benchmark Dose Technical Guidance Document (U.S. 

14 EPA, 2000b), the BMD and the 95% lower confidence limit on the BMD (BMDL) were 

15 estimated using a benchmark response (BMR) of 1 standard deviation (SD) from the control 

16 mean for continous data or a BMR of 10% extra risk for dichotomous data in the absence of 

17 information regarding what level of change is considered biologically significant, and also to 

18 facilitate a consistent basis of comparison across endpoints and assessments. A summary of 

19 modeling results for each endpoint is listed below in Table 5-2. Further details including the 

20 output and graph for the best fit model can be found in Appendix B. In general, model fit was 

21 assessed by a chi-square goodness-of-fit test (i.e., models with p < 0.1 failed to meet the 

22 goodness-of-fit criterion) and the Akaike Information Criterion (AIC) value (i.e., a measure of 

23 the deviance of the model fit that allows for comparison across models for a particular endpoint). 

24 Of the models exhibiting adequate fit, the model yielding the lowest AIC value was selected as 

25 the best-fit model. (U.S. EPA, 2000b). 

26 For the forestomach hyperplasia endpoint, all data sets provided adequate descriptions of 

27 the dose-response relationship from chronic oral exposure to benzo[a]pyrene, but at the highest 

28 dose level for the rats (Kroese et al., 2001), the incidence of forestomach hyperplasia was not 

29 increased relative to controls. It is possible that the forestomach hyperplasia observed following 

30 benzo[a]pyrene exposure may be a precursor to the development of forestomach tumors, but 

31 specific data supporting this conclusion are unavailable. Regardless, the male and female data 
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1 sets in rats (Kroese et al., 2001) were modeled without the data from the highest dose group due 

2 to the nonmontonic increase in response to increasing dose (Kroese et al., 2001). 

3 Points of departure (PODs) for endpoints that were not amenable to BMD modeling were 

4 identified using a NOAEL/LOAEL approach (Zheng et al., 2010; Kristensen et al., 1995; 

5 MacKenzie and Angevine, 1981). A LOAEL of 5 mg/kg-day was identified for Zheng et al. 

6 (2010) for significantly descreased testicular testosterone. A LOAEL of 10 mg/kg-day was 

7 identified as the POD for Mackenzie and Angevine for decreased postnatal body weight and 

8 decreased fertility of male and female mice treated during gestation. A POD based on the 

9 LOAEL of 10 mg/kg/day was established from Kristensen et al. (1995) based on decreased 

10 fertility and decreased ovary weight in female mice treated during gestation.
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Table 5-2. Summary of BMDs and BMDLs for modeled noncancer effects following oral ex

Endpoint/data Exposure 

duration 

BMR Fitted model Goodness-offit 

p-value 

AIC BMD

(mg/kg-d)

BM

(mg/k

Increased Liver Weight in Male Wistar Mice 

90 d 10% Linear (1

◦ polynomial), 

Power 

0.58 49.51 8.11 5

Polynomial (2

◦) 0.74 50.53 4.53 2.

Hill 0.82 50.48 4.1 1.

Decreased Thymus Weight in Male Wistar Mice 

90 d 1 SD Linear , Polynomial 

(2

◦), Power 

(nonconstant variance)

0.23 380.71 16.40 11

Hill (nonconstant variance) 

NA 

Decreased Thymus Weight in Female Wistar rats 

90 d 1 SD Linear 0.81 349.12 10.52 7.

Hill NA Polynomial (2

◦) 0.77 350.80 13.29 7.

Power NA 

Decreased Thymus Weight in Male Wistar Mice 

35 d 1 SD Linear , Polynomial (2

◦

) 0.52 381.41 14.41 11

Hill 0.42 382.91 11.15 6.Power NA 

Decreased Splenic Bcells in Male Wistar rats 35 d 1 SD Linear, Polynomial (2

◦), 

Power (constant variance) 

0.21 145.28 15.58 12

Hill (constant variance) 0.18 146.18 10.24 5.

Increased Forestomach Hyperplasia

a in Male 

Wistar Rats 

2 yrs 10% Log-logistic 0.13 112.27 5.31 2.

Gamma, Multistage, Weibull 

0.12 112.37 5.63 2.

Logistic 0.09 112.93 7.25 4.LogProbit 0.06 113.88 8.36 4.Probit 0.10 112.87 7.09 4.

Increased Forestomach Hyperplasia

a in Female 

2 yrs 10% Log-logistic 0.32 117.04 2.15 1.

Logistic 0.06 120.02 4.23 3.
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Wistar Rats LogProbit 0.02 121.13 3.91 2.

Gamma, Multistage, Weibull 

0.24 117.42 2.40 1.

Probit 0.06 119.74 3.99 3.

Increased Forestomach Hyperplasia in Female B6C3F1 mice 

2 yrs 10% Log-logistic 0.21 193.3 0.329 0.1

Logistic 0.06 194.7 0.757 0.5LogProbit 0.29 192.1 0.670 0.4Gamma, Multistage, Weibull 

0.42 191.3 0.421 0.2

Probit 0.03 196.6 0.946 0.7

Decreased Ovary Weight in Female Sprague-Dawley Rats 

60 d 1 SD Linear , Polynomial (1

◦

) 0.39 -138.67 2.3 1

Power NA 

a The best fit of each model considered is summarized. For continuous models (linear, polynomial, power, Hill), if an 

adequate required including a variance model, only the results including modeled variance are summarized and the use of 

nonconstant variance is indicated; otherwise constant variance was assumed. Details in Appendix B. 

bData for the high-dose group were excluded from the modeled dataset due to a decreased incidence judged to be due to 

competing effects masking the response. NA = not applicable, model failed 
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1 Several candidate principal studies (Kroese et al., 2001; De Jong et al., 1999; Zheng et 

2 al., 2010; Kristensen et al., 1995; MacKenzie and Angevine, 1981) reported liver, thymus, 

3 immune, and reproductive effects at higher doses relative to the ovary and forestomach effects 

4 (reported by Kroese et al., 2001; Beland and Culp, 1998) and were considered less sensitive 

5 measures of benzo[a]pyrene effects. Forestomach hyperplasia was not selected as the critical 

6 effect, even though it was observed at lower doses compared with other effects, based on the 

7 consideration that the reproductive and fertility effects, observed in animals and supported by 

8 human data, appear to better characterize noncancer low dose effects of BaP. Specifically, the 

9 Xu et al., (2010) study was chosen as the principal study and decreased ovarian weight as the 

10 critical effect for the derivation of the RfD. This study identified biologically and statistically 

11 significant decreases in ovary weight, estrogen, and primordial follicles, and altered estrus 

12 cycling in treated animals. These reductions in female reproductive parameters are supported by 

13 a large database of animal studies indicating that benzo[a]pyrene is ovotoxic with effects 

14 including decreased ovary weight, decreased primordial follicles, and reduced fertility (Mattison 

15 et al., 1980; MacKenzie and Angevine 1981; Swartz and Mattison 1985; Miller et al., 1992; 

16 Kristensen et al., 1995; Borman et al, 2000). Additionally, studies indicate that exposure to 

17 complex mixtures of PAHs, such as through cigarette smoke, is associated with measures of 

18 decreased fertility in humans (El Nemr et al., 1998; Neal et al., 2005). Specific associations have 

19 also been made between infertility and increased levels of benzo[a]pyrene in follicular fluid in 

20 women undergoing in vitro fertilization (Neal et al., 2008). 

21 

22 5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 

23 Of the endpoints discussed in section 5.1.1., decreased ovary weight in female rats 

24 (BMDL1SD of 1.5 mg/kg-day) reported by Xu et al. (2010) was selected to serve as the critical 

25 effect for the RfD. A total UF of 3000 was applied to the POD of 1.5 mg/kg-day to account for 

26 several areas of uncertainty. 

27 An UFA of 10 was applied to account for toxicokinetic and toxicodynamic differences 

28 associated with extrapolation from animals to humans. The available data do not provide 

29 quantitative information on the difference in susceptibility to benzo[a]pyrene between rats and 

30 humans. 

31 An UFH of 10 was applied to account for variability in susceptibility among members of 

32 the human population (i.e., interindividual variability). Insufficient information is available to 

33 quantitatively estimate variability in human susceptibility to benzo(a)pyrene. 

34 An UFS of 10 was applied for the extrapolation of subchronic-to-chronic exposure 

35 duration. The 60-day study by Xu et al. (2010) falls well short of a lifetime duration. 

36 Therefore, it is unknown whether effects would be more severe or would be observed at lower 

37 doses with a longer exposure duration. 
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1 An UFL of 1 was applied for LOAEL-to-NOAEL extrapolation because the current 

2 approach is to address this factor as one of the considerations in selecting a BMR for BMD 

3 modeling. In this case, a BMR of a 1 SD change from the control mean in ovary weight was 

4 selected under an assumption that it represents a minimal biologically significant response level. 

5 An UFD of 3 was applied to account for deficiencies in the benzo[a]pyrene toxicity 

6 database. Limited observational studies in humans have suggested associations between 

7 biomarkers of internal dose of benzo[a]pyrene and adverse birth outcomes (including reduced 

8 birth weight, postnatal body weight, head circumference, and neurodevelopment) and decreased 

9 fertility (Edwards et al., 2010; Neal et al., 2008; Tang et al., 2008, 2006; Perera et al., 2009; 

10 2005a, b). However, the likely contribution of multiple exposure routes in these studies make 

11 extrapolation to exposure concentrations uncertain. Several animal studies exist for 

12 benzo[a]pyrene to inform noncancer effects, including subchronic oral toxicity studies in rats and 

13 mice, and two developmental studies and several reproductive studies in mice and rats. The lack 

14 of a standard multigenerational study (specifically, one which includes exposure from pre-mating 

15 to lactation) is a data gap, especially considering benzo[a]pyrene has been shown to affect 

16 fertility in adult male and female animals by multiple routes of exposure (Mohamed et al., 2010; 

17 MacKenzie and Angevine 1981; Kristensen et al., 1995; Archibong et al., 2008; Borman et al., 

18 2000; Swartz and Mattison 1985). In addition, the lack of a study examining functional 

19 neurological endpoints following in utero exposure is also a data gap considering the available 

20 epidemiological evidence showing the association of in utero PAH exposure and indicators of 

21 decreased neurological development (Edwards et al., 2010, Perera et al., 2009, Tang et al., 2008). 

22 Therefore, an UF of 3 was applied to the POD for the lack of a standard multigenerational 

23 reproductive toxicity study and a neurodevelopmental study. 

24 

25 The RfD for benzo[a]pyrene was calculated as follows: 

26 

27 RfD = BMDL1SD ÷ UF 

28 = 1.5 mg/kg-day ÷ 3000 

29 = 0.0005 mg/kg-day 

30 

31 

32 5.1.4. Previous RfD Assessment 

33 No RfD was derived in the previous IRIS assessment.

34 

35 5.2. INHALATION REFERENCE CONCENTRATION (RfC) 

36 5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

37 The only chronic inhalation study available for benzo[a]pyrene was designed as a cancer 

38 bioassay and did not report noncancer endpoints (Thyssen et al.1981). However, several repeat 
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1 dose reproductive and developmental toxicity studies are available in which effects on fetal 

2 survival and the male reproductive system have been observed. 

3 Reproductive system variables were adversely impacted in male F344 rats (10/group) 

exposed to benzo[a]pyrene aerosols at 75 μg/m34 for 60 days (Archibong et al., 2008; Ramesh et 

5 al., 2008). The testes from exposed rats weighed 34% less than those from unexposed controls. 

6 Treatment with benzo[a]pyrene was also associated with reductions (compared with control 

7 values) in total tubular volume and weight (~20%) and tubular length (~40%); total weight and 

8 volume of interstitium per paired testis (12%); and percentage of progressively motile stored 

9 spermatozoa, stored sperm density, and percentage of morphologically normal sperm (~70–

10 80%). Daily sperm production and levels of circulating and intratesticular testosterone were also 

11 decreased in treated rats (by about 50, 70, and 80% respectively), compared with controls. 

12 Luteinizing hormone levels were 50–60% higher in treated rats than controls 72 hours after 

13 exposure. 

14 In a developmental study, timed-pregnant F344 rats (10/group) were exposed to 

benzo[a]pyrene on GDs 11–21 as a carbon black aerosol at 100 μg/m315 to assess neurological 

16 endpoints in offspring during PNDs 60–70 (Wormley et al., 2004). Pups of the F1 generation 

17 were weaned on PND 30 and tested for long-term potentiation electrophysiological responses in 

18 the hippocampus during PNDs 60–70. Although the number of implantation sites in treated rats 

19 was within 1% of unexposed controls, the percentage of pups born relative to recorded 

20 implantation sites in each dam (the birth index) was reduced by 65% in treated rats compared 

21 with unexposed controls. In addition, protein levels of NMDA receptor subunit 1 were down22 regulated (by 18% on PND 10 and 67% on PND 30) in the hippocampus of benzo[a]pyrene23 exposed F1 pups, and the magnitude of the long-term potentiation response across the perforant 

24 path-granular cells in the hippocampus of F1 rats was consistently weaker than the response 

25 observed for the controls (about 25% weaker), suggesting that exposure to benzo[a]pyrene via 

26 the inhalation route attenuates the capacity for long-term potentiation in the F1 generation. 

27 However, no functional tests to assess neurotoxicity were conducted in this study. 

28 In another developmental toxicity study, timed-pregnant F344 rats (10/group) were 

exposed to benzo[a]pyrene aerosols at concentrations of 25, 75, or 100 μg/m329 on GDs 11–20 and 

30 evaluated for post-implantation fetal survival and hormone levels associated with pregnancy 

31 (Archibong et al., 2002). The total number of implantation sites in treated rats was within 5% of 

32 the values obtained for sham-exposed and unexposed controls. However, dose-dependent trends 

33 were observed for decreased numbers of pups per litter and percent fetal survival per litter with 

34 increasing benzo[a]pyrene concentrations. The number of pups/litter was decreased by 

approximately14, 50, and 65% at 25, 75, and 100 μg/m

3

35 , respectively, compared with sham36 exposed and unexposed controls. Percent survival/was similarly reduced by about 20, 60, and 

37 65%, respectively, at the same exposure concentrations. In addition, biologically significant 

decreases in pup weights (presented as g/litter) were observed at concentrations ≥75 μg/m3 38 (14 
212 DRAFT - DO NOT CITE OR QUOTE 

and 16% decreases at 75 and 100 μg/m31 , respectively). Levels of plasma progesterone, estradiol17β, and prolactin on GD 17 were decreased by about 12, 14, and 35%, respectively, at 25 μg/m3

2 

and 17, 60, and 70%, respectively, at 75 μg/m33 compared with respective controls. 

4 The study by Archibong et al. (2002) was selected as the principal study as it observed 

5 biologically significant effects at the lowest dose tested by the inhalation route. This study 

6 indicates that the developing fetus is a sensitive target following inhalation exposure to 

benzo[a]pyrene. A LOAEL of 25 μg/m37 was identified based on exposure to benzo[a]pyrene on 

8 GDs 11–20 that caused biologically significant reductions in fetal survival and body weight 

9 decreases in the surviving pups (see Table 5-3). The observed decrease in pup weight and fetal 

10 survival were selected as critical effects as they are the most sensitive noncancer effects observed 

11 following inhalation exposure to benzo[a]pyrene. Though only a few studies exist which 

12 evaluate benzo[a]pyrene by the inhalation route, additional support for this endpoint can be 

13 found from oral studies of benzo[a]pyrene. A developmental/reproductive study conducted via 

14 the oral route in mice observed decreased survival of litters, decreased pup weight, and decreased 

15 reproductive organ weight following in utero exposure to benzo[a]pyrene on GD 7-16 

16 (MacKenzie and Angevine, 1981). 

17 

18 5.2.2. Methods of Analysis- Adjustment to a Human Equivalent Concentration (HEC) 

19 By definition, the RfC is intended to apply to continuous lifetime exposures for humans 

20 (U.S. EPA, 1994). EPA recommends that adjusted continuous exposures be used for inhalation 

21 developmental toxicity studies as well as for studies of longer durations (U.S. EPA, 2002). The 

LOAEL of 25 μg/m322 based on decreased pup weight and fetal survival reported in the 

23 developmental study by Archibong et al. (2002) was selected to serve as the POD. 

24 

Table 5-3. Pregnancy outcomes in female F344 rats treated with 

benzo[a]pyrene on GDs 11–21 by inhalation 

Parametera

Administered concentration of benzo[a]pyrene (μg/m3) 

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 ± 0.5b 4.2 ± 0.1b 3.0 ± 0.2b

Survival (litter %) 98.9 ± 1.1 96.7 ± 1.7 78.3 ± 4.1

b

 38.0 ± 2.1b 33.8 ± 1.3b

Pup weight (g/litter) 10.6 ± 0.1 8.8 ± 0.1 10.5 ± 0.2 9.1 ± 0.2b 8.9 ± 0.1b

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. 

b

Significantly different from controls at p < 0.05 by one-tailed post-hoc t-testing following ANOVA. 

Source: Archibong et al. (2002). 

25 
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1 Data for decreased pup survival were not amenable to BMD modeling due to the pattern 

2 of variability (heterogeneous variances) in the data set. Therefore, the LOAEL from this study 

3 was used as the POD. The LOAEL from this study is based on a 4 hour exposure of pregnant 

rats to 25 μg/m34 benzo[a]pyrene on GDs 11-20. This concentration was adjusted to account for 

5 the discontinuous daily exposure as follows: 

6 

7 PODADJ = POD x hours exposed per day/24 hours 

8 = LOAEL x (4 hr/24 hr) 

= 25 μg/m39 x 4/24 

= 4.2 μg/m310 

11 

12 The human equivalent concentration (HEC) was calculated from the PODADJ by 

13 multiplying by a dosimetric adjustment factor (DAF), which, in this case, was the regional 

14 deposited dose ratio (RDDRER) for extrarespiratory (i.e. systemic) effects as described in 

15 Methods for Derivation of Inhalation Reference Concentrations and Application of Inhalation 

16 Dosimetry (U.S. EPA, 1994b). The observed developmental effects are considered systemic in 

17 nature (i.e., extrarespiratory) and the current normalizing factor for extrarespiratory effects of 

18 particles is body weight. In the case of benzo[a]pyrene, the RDDRER was calculated as follows: 

19 

TOT H

TOT A

E H

E A

A

H

ER (F )

(F )

(V )

(V )

BW

BW

20 RDDR = × ×

21 where: 

22 BW = body weight (kg) 

23 VE = ventilation rate (L/min) 

24 FTOT = total fractional deposition 

25 

26 The total fractional deposition (FTOT) includes particle deposition in the nasal-pharyngeal 

27 region, the tracheobronchial region, and the pulmonary region. FTOT for both animals and 

28 humans was calculated using the Multi-Path Particle Dosimetry model, a computational model 

29 that can be used for estimating human and rat airway particle deposition and clearance (MPPD; 

30 Version 2.0 © 2006, publicly available through the Hamner Institute). The FTOT was calculated 

31 based on the average particle size of 1.7 ± 0.085 (mass median aerodynamic diameter ± 

32 geometric standard deviation) as reported in the description of particle generation methods in 

33 Ramesh et al. (2000). For the model runs, the Yeh-Schum 5-lobe model was used for the human 

34 and the asymmetric multiple path model was used for the rat (see Appendix C for MPPD model 

35 output). Both models were run under nasal breathing scenarios with the inhalability adjustment 

36 selected. A geometric standard deviation (GSD) of 1 was used as the default by the model 

37 because the reported GSD of 0.085 ≤ 1.05. 
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1 The human parameters used in the model for calculating FTOT and in the subsequent 

2 calculation of the PODHEC were as follows: human - BW, 70 kg; VE, 13.8 L/min; breathing 

3 frequency, 16 per minute; tidal volume, 860 mL; FRC (functional residual capacity), 3300 mL; 

4 and URT (upper respiratory tract) volume, 50 mL. Although the most sensitive population in the 

5 principal study is the developing fetus, the adult rat dams were exposed. Thus, adult human 

6 parameters were used in the calculation of the HEC to extrapolate from a pregnant rat to a 

7 pregnant human. The parameters used for the rat were BW, 0.25 kg (based on the approximate 

8 weight of a 100 day-old, female timed-pregnant Sprague-Dawley rat); VE, 0.18 L/min; breathing 

9 frequency, 102 per minute; tidal volume, 1.8 mL; FRC (functional residual capacity), 4 mL; and 

10 URT (upper respiratory tract) volume, 4.42 mL. All other parameters were set to the default 

11 value (see Appendix C).

12 Under these conditions, the MPPD model calculated FTOT values of 0.621 for the human 

13 and 0.181 for the rat. Using the above equation, the RDDRER was calculated to be 1.06. 

14 From this, the PODHEC was calculated as follows: 

15 

16 PODHEC = PODADJ × RDDRER 

 PODHEC = 4.2 μg/m317 × 1.1 

PODHEC = 4.6 μg/m318 

19 

20 

21 5.2.3. RfC Derivation- Including Application of Uncertainty Factors (UFs)

22 The critical effect for the derivation of the RfC was identified as decreased fetal survival 

23 and decreased pup weight associated with inhalation exposure to pregnant rats on GDs 11–20. 

24 The LOAEL for decreased fetal survival was adjusted to a continuous human equivalent 

25 concentration and used as the POD for the derivation of the RfC. A total UF of 1000 was 

26 applied to the PODHEC to account for four main areas of uncertainty: 

27 A UFA of 3 was applied to account for uncertainties in extrapolating from rats to humans. 

28 Application of a UF of 10 encompasses two areas of uncertainty: toxicokinetic and 

29 toxicodynamic uncertainties. In this assessment, the toxicokinetic component is mostly 

30 addressed by the determination of a HEC as described in the RfC methodology (U.S. EPA, 

31 1994b). Therefore, a UF of 3 was applied to account for the remaining toxicodynamic 

32 uncertainties in the extrapolation from rats and humans. 

33 A UFH of 10 was applied to account for variability in susceptibility among members of 

34 the human population (i.e., interindividual variability). Insufficient information is available to 

35 quantitatively estimate variability in human susceptibility to benzo(a)pyrene. 

36 A UFL of 10 was applied to account for the use of a LOAEL. A NOAEL was not 

37 identified for decreased fetal survival observed by Archibong et al (2002). At the lowest dose, 

38 benzo[a]pyrene treated dams gave birth to 15% fewer pups compared to dams treated with 
215 DRAFT - DO NOT CITE OR QUOTE 

1 vehicle alone (carbon black particles). Due to the lack of a NOAEL and the inability to model 

2 the data set for decreased fetal survival, a UF of 10 was applied to extrapolate to a NOAEL. 

3 A UFS of 1 was applied to account for extrapolation from subchronic to chronic exposure 

4 because developmental toxicity resulting from a narrow period of exposure was used as the 

5 critical effect. The developmental period is recognized as a susceptible life stage when exposure 

6 during a time window of development is more relevant to the induction of developmental effects 

7 than lifetime exposure (U.S. EPA, 1991a). 

8 A UFD of 3 was applied to account for deficiencies in the benzo[a]pyrene toxicity 

9 database. One developmental study exists for benzo[a]pyrene by the inhalation route. The 

10 developmental study by Archibong et al., 2002, which was used as the basis for the RfC, 

11 observed decreased fetal survival and decreased litter size following gestational treatment on 

12 GDs 11-20. Limited observational studies in humans have suggested associations between 

13 biomarkers of internal doses of benzo[a]pyrene and adverse birth outcomes (including reduced 

14 birth weight and postnatal weight, decreased head circumference, and impaired 

15 neurodevelopment) and decreased fertility (Neal et al., 2008; Tang et al., 2008, 2006; Perera et 

16 al., 2005a, b). A multigenerational reproductive study examining these types of effects in 

17 animals does not exist for the inhalation route. However, oral multigenerational studies indicate 

18 that effects on fertility would be expected in male and female offspring exposed to 

19 benzo[a]pyrene during development (Mackenzie and Angevine 1981; Kristensen et al., 1995). In 

20 addition, the lack of a study examining functional neurological endpoints following in utero 

21 exposure is also a data gap considering the available epidemiological evidence showing the 

22 association of in utero PAH exposure and indicators of decreased neurological development 

23 (Edwards et al., 2010, Perera et al., 2009, Tang et al., 2008). Therefore, a UF of 3 was applied 

24 to the POD for the lack of a multigenerational reproductive toxicity study and a 

25 neurodevelopmental study. 

26 

27 

28 The RfC for benzo[a]pyrene was calculated as follows: 

29 

30 RfC = LOAELADJ[HEC] ÷ UF 

 = 4.6 μg/m331 -day ÷ 1000 

 = 4.6 x 10-3 μg/m3-day or 5 x 10-6 mg/m332 

33 

34 

35 5.2.4. Previous RfC Assessment 

36 

37 An RfC was not derived in the previous IRIS assessment. 

38 

39 
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1 5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION 

2 REFERENCE CONCENTRATION 

3 The following discussion identifies uncertainties associated with the RfD and RfC for 

4 benzo[a]pyrene. To derive the RfD, the UF approach (U.S. EPA, 2000, 1994b) was applied to a 

5 POD based on decreased ovary weight in female rats exposed to benzo[a]pyrene. To derive the 

6 RfC, this same approach was applied to a POD from a developmental study for the effect of 

7 decreased fetal survival. Uncertainty factors were applied to the POD to account for 

8 extrapolating from an animal bioassay to human exposure, the likely existence of a diverse 

9 population of varying susceptibilities, and for database deficiencies. These extrapolations are 

10 carried out with default approaches given the lack of data to inform individual steps. 

11 The database for benzo[a]pyrene contains limited human data. The observation of effects 

12 associated with benzo[a]pyrene exposure in humans is complicated by several factors including 

13 the existence of benzo[a]pyrene in the environment as one component of complex mixtures of 

14 PAHs, exposure to benzo[a]pyrene by multiple routes of exposure, and the difficulty in obtaining 

15 accurate exposure information. Data on the effects of benzo[a]pyrene alone are derived from a 

16 large database of studies in animal models. The database for oral benzo[a]pyrene exposure 

17 includes two chronic bioassays in rats and mice, two developmental studies in mice, and several 

18 subchronic studies in rats. 

19 Although the database is adequate for RfD derivation, there is uncertainty associated with 

20 the database, because a NOAEL was not identified in the reproductive and developmental oral 

21 toxicity studies, comprehensive two-generation reproductive/developmental toxicity studies are 

22 not available, and immune system endpoints affected in the subchronic-duration studies were not 

23 evaluated in the chronic-duration toxicity studies. Additionally, the only available chronic 

24 studies of oral exposure to benzo[a]pyrene focused primarily on neoplastic effects. These studies 

25 identify forestomach hyperplasia as one of the more sensitive histological effects following 

26 repeated oral exposure to benzo[a]pyrene. However, data from chronic cancer bioassays for 

27 benzo[a]pyrene show no increase in this endpoint at the high dose in rats. An increased 

28 incidence of forestomach tumors is observed at similar doses; suggesting that this effect may be 

29 pre-neoplastic in nature. 

30 The only chronic inhalation study of benzo[a]pyrene was designed as a lifetime 

31 carcinogenicity study and did not examine noncancer endpoints (Thyssen et al., 1981). However 

32 subchronic and short term inhalation studies are available which examine developmental and 

33 reproductive endpoints in rats. Developmental studies by the inhalation route identified 

34 biologically significant reductions in the number of pups/litter and percent fetal survival and 

35 possible neurodevelopmental effects (e.g., diminished electrophysiological responses to stumuli 

36 in the hippocampus) following gestational exposures. Additionally, a 60 day oral study in male 

37 rats reported male reproductive effects (e.g., decreased testes weight and sperm production and 

38 motility), but provides limited information to characterize dose-response relationships with 
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1 chronic exposure scenarios. One area of uncertainty pertains to the lack of information regarding 

2 fertility in animals exposed gestationally to benzo[a]pyrene, especially in light of developmental 

3 studies by the oral route indicating reduced fertility in the F1 generation and decreased 

4 reproductive organ weights. The database also lacks a multigenerational reproductive study via 

5 the inhalation route. Areas of uncertainty include the lack of chronic inhalation studies focusing 

6 on noncancer effects, limited data on dose-response relationships for impaired male or female 

7 fertility with gestational exposure or across several generations, and limited data on immune 

8 system endpoints with chronic exposure to benzo[a]pyrene.

9 The toxicokinetic and toxicodynamic differences for benzo[a]pyrene between the animal 

10 species in which the POD was derived and humans are unknown. PBPK models can be useful 

11 for the evaluation of interspecies toxicokinetics; however, the benzo[a]pyrene database lacks an 

12 adequate model that would inform potential differences. There is some evidence from the oral 

13 toxicity data that mice may be more susceptible than rats to some benzo[a]pyrene effects (such 

14 as ovotocity [Borman et al., 2000]), though the underlying mechanistic basis of this apparent 

15 difference in not understood. Most importantly, it is unknown which animal species may be 

16 more comparable to humans. 

17 

18 

19 5.4. CANCER ASSESSMENT 

20 As discussed in Section 4.7, benzo[a]pyrene is “carcinogenic to humans” based on 

21 evidence of carcinogenicity in humans exposed to different PAH mixtures containing 

22 benzo[a]pyrene, extensive and consistent evidence of carcinogenicity in laboratory animals 

23 exposed to benzo[a]pyrene via several routes of administration, and extensive and consistent 

24 evidence that the mode of action of carcinogenesis in laboratory animals also occurs in humans 

25 exposed to PAH mixtures containing benzo[a]pyrene.

26 

27 5.4.1. Oral Exposure—Oral Slope Factor 

28 5.4.1.1. Choice of Study/Data—with Rationale and Justification—Oral Exposure 

29 Numerous cancer bioassays exist which identify tumors, primarily of the alimentary tract, 

30 following oral exposure in rodents (see Table 4-8 for references). These studies provide support 

31 for the carcinogenic hazard for benzo[a]pyrene, however, are not suitable for dose-response 

32 analysis due to limitations in study design, methods, and/or reporting. Specifically, several of 

33 these studies 1) lack a vehicle control group 2) use only one benzo[a]pyrene dose group or 3) use 

34 a single one-time exposure to benzo[a]pyrene (Benjamin et al., 1988; Robinson et al., 1987; El 

35 Bayoumy, 1985; Wattenberg, 1974; Roe et al., 1970; Biancifiori et al. 1967; Chouroulinkov et 

36 al., 1967; Field and Roe, 1970; Berenblum and Haran 1955). Of the controlled, multiple dose37 group, repeat-dosing studies that remain, most treated animals for less than a year, which is less 
218 DRAFT - DO NOT CITE OR QUOTE 

1 optimal for extrapolating to a lifetime exposure (Weyand et al., 1995; Triolo et al., 1977; 

2 Fedorenko et al., 1967; Neal and Rigdon 1967). 

3 Three 2-year oral bioassays remain which associate lifeftime benzo[a]pyrene exposure 

4 with forestomach, liver, oral cavity, jejunum, kidney, auditory canal (Zymbal’s gland) tumors, 

5 and skin or mammary gland tumors in male and female Wistar rats (Kroese et al., 2001); 

6 forestomach tumors in male and female Sprague-Dawley rats (Brune et al., 1981); and 

7 forestomach, esophagus, tongue, and larynx tumors in female B6C3F1 mice (male mice were not 

8 tested; Beland and Culp, 1998; Culp et al., 1998). Brune et al. (1981) dosed rats (32/sex/group) 

9 with several concentration of benzo[a]pyrene dissolved a 1.5% caffeine solution, sometimes as 

infrequently as once every 9th 10 day, for up to two years and observed increased forestomach 

11 tumors. This study was not selected for quantitation due to the non-standard treatment protocol. 

12 The rat bioassay by Kroese et al. (2001) and the mouse bioassay by Beland and Culp (1998) 

13 were conducted in accordance to Good Laboratory Practice (GLP) principles as established by 

14 OECD. These studies included histological examinations for tumors in many different tissues, 

15 contained three exposure levels and controls, contained adequate numbers of animals per dose 

16 group (~50/sex/group), treated animals for two years or until death, and included detailed 

17 reporting of methods and results (including individual animal data). 

18 Therefore, the Kroese et al. (2001) and Beland and Culp s(1998) tudies were selected as 

19 the best available studies for dose-response analysis and extrapolation to lifetime cancer risk 

20 following oral exposure to benzo[a]pyrene. 

21 

22 5.4.1.2. Dose-response Data—Oral Exposure 

23 Details of the rat (Kroese et al., 2001) and female mouse (Beland and Culp, 1998) study 

24 designs are provided in Section 4.2.1.2. Dose-related, statistically significant increasing trends 

25 in tumors were noted at the following sites: 

26 • Squamous cell carcinomas or papillomas of the forestomach or oral cavity in male and 

27 female rats; 

28 • Squamous cell carcinomas or papillomas of the forestomach, tongue, larynx, or 

29 esophagus in female mice; 

30 • Auditory canal carcinomas in male and female rats; 

31 • Kidney urothelial carcinomas in male rats; 

32 • Jejunum adenocarcinomas in female and male rats; 

33 • Hepatocellular adenomas or carcinomas in male and female rats; 

34 • Squamous cell carcinomas or basal cell tumors of the skin or mammary gland in male 

35 rats. 

36 

37 These tumors were generally observed earlier during the study with increasing exposure 

38 levels, and showed statistically significantly increasing trends in incidence with increasing 
219 DRAFT - DO NOT CITE OR QUOTE 

1 exposure level (Cochran-Armitage trend test, p≤0.001). These data are summarized in Tables 5-

2 4 (male and female rats) and 5-5 (female mice). As recommended by the NTP (McConnell et al., 

3 1986), etiologically similar tumor types, i.e., benign and malignant tumors of the same cell type, 

4 were combined for these tabulations when it was judged that the benign tumors could progress to 

5 the malignant form, as outlined in the Cancer Guidelines (U.S. EPA, 2005a). In addition, when 

6 one tumor type occurred across several functionally related tissues, as with squamous cell tumors 

7 in the tongue, esophagus, larynx and forestomach, or adenocarcinomas of the jejunum or 

8 duodenum, these incidences were also aggregated as counts of tumor-bearing animals. 

9 In the rat study (Kroese et al., 2001), the oral cavity and auditory canal were examined 

10 histologically only if a lesion or tumor was observed grossly at necropsy. Consequently, dose11 response analysis for these sites was not straightforward. Use of the number of tissues examined 

12 histologically as the number at risk would tend to overestimate the incidence, because the 

13 unexamined animals were much less likely to have a tumor. On the other hand, use of all 

14 animals in a group as the number at risk would tend to underestimate if any of the unexamined 

15 animals had tumors which could only be detected microscopically. The oral cavity squamous 

16 cell tumors were combined with those in the forestomach because both are part of the alimentary 

17 tract, recognizing that there was some potential for underestimating this cancer risk. 

18 The auditory canal tumors from the rat study were not considered for dose-response 

19 separately or combined with another site. First, very few tissues were examined in the control 

20 and lower dose groups (see Table 4-4). Also, the tumors were not clearly related to any other 

21 site or incidence type, as they were described as a mixture of squamous and sebaceous cells 

22 derived from pilosebaceous units. The tumors found were observed mainly in the high dose 

23 groups and were highly coincident with the oral cavity and forestomach tumors. That is, only 

24 one mid-dose male with an auditory canal tumor did not also have a forestomach or oral cavity 

25 squamous cell tumor. No low-dose male or female rats were found with auditory canal tumors. 

26 While the investigators did not suggest that these tumors were metastases from other sites (in 

27 which the auditory canal tumors could be reflections of other tumor types), it is difficult to 

28 conclude that they are independent on a purely statistical basis without sufficient low-dose data. 

29 Therefore dose-response analysis was not pursued for this site. 

30 
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Table 5-4. Incidence data for tumors in Wistar rats exposed to 

benzo[a]pyrene by gavage, 5 days/week for 104 weeks

Tumor site/typea

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 papilloma 

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 39/47 51/51 

Jejunum/duodenum: adenocarcinoma 0/50 0/46 0/45 4/42 

a

For each tissue site, the numerator of the tumor incidence value is the number of animals bearing the specified 

tumors. The denominators are the number of animals examined histologically, minus the number of animals who 

died before the earlier of the first occurrence of the tumor type in each group or Week 52. 

bHEDs for continuous exposure were calculated using the animal to human scaling factor for each dose group × 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 TWA 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 B6C3F1 mice exposed to 

benzo[a]pyrene in the diet for 104 weeks 

Tumor site/typea

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 the number of 

animals who died before the earlier of the first occurrence of the tumor type in each group or Week 52. 

bAdministered doses were calculated using TWA body weight for mice and reported food intakes. 

cHEDs were calculated using the animal to human scaling factor for each dose group × the administered dose. 

Scaling factors were calculated using U.S. EPA (1988) reference body weights for humans (70 kg), and the TWA 

body weight for each dose group: (TWA body weight/70)0.25 × dose = HED. See Table D-5 for more information. 

Source: Beland and Culp (1998). 

3 

4 5.4.1.3. Dose Adjustments and Extrapolation Method(s)—Oral Exposure 
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1 The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend 

2 that the method used to characterize and quantify cancer risk from a chemical is determined by 

3 what is known about the mode of action of the carcinogen and the shape of the cancer dose4 response curve. The dose response is assumed to be linear in the low dose range, when evidence 

5 supports a mutagenic mode of action because of DNA reactivity, or if another mode of action 

6 that is anticipated to be linear is applicable. In this assessment, EPA concluded that 

7 benzo[a]pyrene causes cancer via a mutagenic MOA (as discussed in Section 4.7.3). Thus, a 

8 linear approach to low-dose extrapolation was used.

9 The high-dose groups of both the rat and mouse studies were dead or moribund by week 

10 79 for female mice, week 72 for female rats, and week 76 for male rats. Due to the occurrence of 

11 multiple tumor types, earlier occurrence with increasing exposure, and early termination of the 

12 high-dose group in each study, methods that can reflect the influence of competing risks and 

13 intercurrent mortality on site-specific tumor incidence rates are preferred. EPA has generally 

14 used a model which incorporates the time at which death-with-tumor occurred as well as the 

15 dose; the multistage-Weibull model is multistage in dose and Weibull in time, and has the form: 

16 

P(d, t) = 1 - exp[-(q0 + q1d + q2d

2

 + ... + qkd

k

) × (t ± t0)

z

17 ], 

18 

19 where P(d, t) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent 

20 exposure in this case) and age t (in bioassay weeks); parameters qi ≥ 0, for i = 0, 1, ..., k; t is the 

21 time at which the tumor was observed; and z is a parameter which characterizes the change in 

22 response with age. The parameter t0 represents the time between when a potentially fatal tumor 

23 becomes observable and when it causes death, and is generally set to 0 either when all tumors are 

24 considered incidental or because of a lack of data to estimate the time reliably. The dose25 response analyses were conducted using the computer software program MultiStage-Weibull 

26 (U.S. EPA, 2010), which is based on Weibull models drawn from Krewski et al. (1983). 

27 Parameters were estimated using the method of maximum likelihood. 

28 Two general characteristics of the observed tumor types were considered prior to 

29 modeling; allowance for different, although unidentified modes of action, and allowance for 

30 relative severity of tumor types. First, etiologically different tumor types were not combined 

31 across sites prior to modeling (that is, overall counts of tumor-bearing animals were not 

32 tabulated), in order to allow for the possibility that different tumor types could have different 

33 dose-response relationships due to different underlying mechanisms or factors, such as latency. 

34 Consequently, all of the tumor types listed separately in Tables 5-4 and 5-5 were also modeled 

35 separately. 

36 Additionally, the multistage-Weibull model can address relative severity of tumor types 

37 by distinguishing between tumors as being either fatal or incidental to the death of an animal, in 

38 order to adjust partially for competing risks. Incidental tumors are those tumors thought not to 
222 DRAFT - DO NOT CITE OR QUOTE 

1 have caused the death of an animal, while fatal tumors are thought to have resulted in animal 

2 death. Cause of death information for most early animals deaths was provided by the 

3 investigators of both of the bioassays. In the rat study, tumors of the forestomach or liver were 

4 the principal cause of death for most animals dying or sacrificed (due to moribundity) before the 

5 end of the study, while tumors of the forestomach were the most common cause of early deaths 

6 in the mouse study. 

7 Adjustments for approximating human equivalent slope factors applicable for continuous 

8 exposure were applied prior to dose-response modeling. First, continuous daily exposure for the 

9 gavage study in rats (Kroese et al, 2001) was estimated by multiplying each administered dose 

10 by (5 days)/(7 days) = 0.71, under the assumption of equal cumulative exposure yielding 

11 equivalent outcomes. Dosing was continuous in the mouse diet study (Beland and Culp, 1998), 

12 so no continuous adjustment was necessary. Next, consistent with the Guidelines for 

13 Carcinogen Risk Assessment (U.S. EPA, 2005a), an adjustment for cross-species scaling was 

14 applied to address toxicological equivalence across species. Following EPA’s cross-species 

15 scaling methodology, the time-weighted daily average doses were converted to human equivalent 

doses on the basis of (body weight)3/4 16 (U.S. EPA, 1992). This was accomplished by multiplying 

administered doses by (animal body weight(kg)/70 kg)

0.25 17 (U.S.EPA, 1992), where the animal 

18 body weights were time-weighted averages from each group (see Tables D-4, D-5), and the U.S. 

19 EPA (1988) reference body weight for humans is 70 kg. It was not necessary to adjust the 

20 administered doses for lifetime equivalent exposure prior to modeling for the groups terminated 

21 early, because the multistage-Weibull model characterizes the tumor incidence as a function of 

22 time, from which it provides an extrapolation to lifetime exposure.

23 The multistage-Weibull model was applied to the datasets. Specific n-stage Weibull 

24 models were selected for each tumor dataset based on the values of the log-likelihoods according 

25 to the strategy used by EPA (U.S. EPA, 2002). If twice the difference in log-likelihoods was less 

than a χ

2

26 with degrees of freedom equal to the difference in the number of stages included in the 

27 models being compared, the models were considered comparable and the most parsimonious 

28 model (i.e., the lowest-stage model) was selected. This method generally led to the same 

29 conclusion as selecting the model fit with the lowest AIC. If a model with one more stage fitted 

30 the low-dose data better than the most parsimonious model, then the model with one higher stage 

31 was selected. 

32 PODs for estimating low-dose risk were identified at doses at the lower end of the 

33 observed data, generally corresponding to 10% extra risk, where extra risk is defined as [P(d) - 

34 P(0)]/[1 - P(0)]. The lifetime oral cancer slope factor for humans is defined as the slope of the 

35 line from the lower 95% bound on the exposure at the POD to the control response (slope factor 

36 = 0.1/BMDL10). This slope, a 95% upper confidence limit (UCL) represents a plausible upper 

37 bound on the true risk. 

38 
223 DRAFT - DO NOT CITE OR QUOTE 

1 5.4.1.4. Oral Slope Factor Derivation 

2 The PODs estimated for each tumor site are summarized in Table 5-5. Details of the 

3 model selection process are provided in Table D-6. Using linear extrapolation from the 

4 BMDL10, human equivalent oral slope factors were derived for each gender/tumor site 

5 combination and are listed in Table 5-6. 

6 

Table 5-6. Human equivalent PODs and oral slope factors derived from 

multistage-Weibull modeling of tumor incidence data at multiple tissue sites 

in Wistar rats and B6C3F1 mice exposed to benzo[a]pyrene orally for 2 years 

Species Sex Tumor 

BMD10 

(mg/kg-d) 

BMDL10

(mg/kg-d) 

Slope Factora 

(mg/kg-d)-1 

Rats Male Forestomach, oral cavity: squamous cell 

tumors 

0.453 0.281 0.4 

0.5b 

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 

0.3b 

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 

aHuman equivalent slope factor = 0.1/BMDL10HED; see Appendix D for details of modeling results.

b

 Estimates of risk of incurring at least one of the tumor types listed. 

7 

8 Oral slope factors derived from rat bioassay data varied by gender and tumor site 

9 (Table 5-6). Values ranged from 0.04 per mg/kg-day, based on kidney tumors in males, to 

10 0.4 per mg/kg-day, based on alimentary tract tumors in males. Slope factors based on liver 

11 tumors in male and female rats (approximately 0.2 per mg/kg-day) were only slightly lower than 

12 slope factors based on alimentary tract tumors. The oral slope factor for female mice was 

13 highest, at 1 per mg/kg-day for alimentary tract tumors (Table 5-6), approximately fourfold 

14 higher than the oral slope factor derived from the alimentary tract tumors in male rats. 

15 Although the time-to-tumor modeling helps account for competing risks associated with 

16 decreased survival times and other tumors, considering the tumor sites individually still does not 

17 convey the total amount of risk potentially arising from the sensitivity of multiple sites—that is, 

18 the risk of developing any combination of the increased tumor types, not just the risk of 

19 developing all simultaneously. One approach suggested in the Guidelines for Carcinogen Risk 

20 Assessment (U.S. EPA, 2005a) would be to estimate cancer risk from tumor-bearing animals. 

21 EPA traditionally used this approach until the National Resource Council (NRC) document 

22 Science and Judgment (NRC, 1994) made a case that this approach would tend to underestimate 

23 overall risk when tumor types occur in a statistically independent manner. In addition, 
224 DRAFT - DO NOT CITE OR QUOTE 

1 application of one model to a composite data set does not accommodate biologically relevant 

2 information that may vary across sites or may only be available for a subset of sites. For 

3 instance, the time courses of the multiple tumor types evaluated varied, as is suggested by the 

4 variation in estimates of z (see Table 5-6), from 1.5 (e.g., male rat skin or mammary gland basal 

5 cell tumors), indicating relatively little effect of age on tumor incidence, to 3.7 (e.g., male mouse 

6 alimentary tract tumors), indicating a more rapidly increasing response with increasing age (in 

7 addition to exposure level). The result of fitting a model with parameters which can reflect 

8 underlying mechanisms, such as z in the multistage-Weibull model, would be difficult to 

9 interpret with composite data (i.e., counts of tumor-bearing animals). A simpler model, such as 

10 the multistage model, could be used for the composite data but relevant biological information 

11 would then be ignored. 

12 Following the recommendations of the NRC (1994) regarding combining risk estimates, 

13 statistical methods which can accommodate the underlying distribution of slope factors are 

14 optimal, such as through maximum likelihood estimation or through bootstrapping or Bayesian 

15 analysis. However, these methods have not yet been extended to models such as the multistage16 Weibull model. A method involving the assumption that the variability in the slope factors could 

17 be characterized by a normal distribution is detailed below (U.S. EPA, 2010). Using the results 

18 in female rats to illustrate, the overall risk estimate involved the following steps: 

19 

20 1) It was assumed that the tumor groupings modeled above were statistically 

21 independent—that is, that the occurrence of a liver tumor was not dependent 

22 upon whether there was a forestomach tumor. This assumption cannot currently 

23 be verified, and if not correct could lead to an overestimate of risk from 

24 summing across tumor sites. However, NRC (1994) argued that a general 

25 assumption of statistical independence of tumor-type occurrences within 

26 animals was not likely to introduce substantial error in assessing carcinogenic 

27 potency from rodent bioassay data. 

28 

29 2) The models previously fitted to estimate the BMDs and BMDLs were used to 

30 extrapolate to a lower level of risk (R), in order to reach the region of each 

31 estimated dose-response function where the slope was reasonably constant and 

upper bound estimation was still numerically stable. For these data, a 10-3 32 risk 

33 was generally the lowest risk necessary. The oral slope factor for each site was 

34 then estimated by R/BMDLR, as for the estimates for each tumor site above. 

35 

36 3) The maximum likelihood estimates (MLE) of unit potency (that is, risk per unit 

37 of exposure) estimated by R/BMDR, were summed across the alimentary tract, 

38 liver, and jejunum/duodenum in female rats. 

39 

40 4) An estimate of the 95% (one-sided) upper bound on the summed oral slope 

41 factor was calculated by assuming a normal distribution for the individual risk 

42 estimates, and deriving the variance of the risk estimate for each tumor site from 

43 its 95% upper confidence limit (UCL) according to the formula: 
225 DRAFT - DO NOT CITE OR QUOTE 

1 

2 95% UCL = MLE + 1.645 × s.d., 

3 rearranged to: 

4 s.d. = (UCL – MLE ) / 1.645, 

5 

6 where 1.645 is the t-statistic corresponding to a one-sided 95% confidence 

7 interval and >120 degrees of freedom, and the standard deviation (s.d.) is the 

square root of the variance of the MLE. The variances (variance = s.d.28 ) for each 

9 site-specific estimate were summed across tumor sites to obtain the variance of 

10 the sum of the MLEs. The 95% UCL on the sum of MLEs was calculated from 

11 the expression above for the UCL, using the variance of the sum of the MLE to 

obtain the relevant s.d (s.d. = variance1/2 12 ). 

13 

14 The resulting composite slope factor for all tumor types for male rats was 0.5 per mg/kg15 d, about 25% higher than the slope factor based on the most sensitive tumor site, oral cavity and 

16 forestomach, while for female rats the composite slope factor was equivalent to that for the most 

17 sensitive site (Table 5-6; see Table D-7 for details of the composite slope factor estimates). 

18 The risk estimates from rats and mice spanned a nearly five-fold range. As there are no 

19 data to support any one result as most relevant for extrapolating to humans, the most sensitive 

20 result was used to derive the oral slope factor. The recommended slope factor for assessing 

21 human cancer risk associated with chronic oral exposure to benzo[a]pyrene is 1 per mg/kg-day,

22 based on the alimentary tract tumor response in female B6C3F1 mice. 

23 

24 5.4.2. Inhalation Exposure—Inhalation Unit Risk 

25 5.4.2.1. Choice of Study/Data—with Rationale and Justification—Inhalation Exposure 

26 Inhalation exposure to benzo[a]pyrene was associated with nasal adenocarcinomas and 

27 squamous cell tumors in the larynx, pharynx, trachea, esophagus, and forestomach, of male 

28 Syrian golden hamsters exposed to benzo[a]pyrene:NaCl aerosols at concentrations of 10 or 50 

mg/m329 until natural death (up to 133 weeks) for 3–4.5 hours/day, 5-7 days/week (Thyssen et al., 

30 1981). Supportive evidence for the carcinogenicity of inhaled benzo[a]pyrene comes from 10 

31 additional studies with hamsters exposed to benzo[a]pyrene via intratracheal instillation (see 

32 Section 4.2.2.2 for references). However, the use of intratracheal dosing alters the deposition, 

33 clearance, and retention of substances and therefore studies utilizing this exposure technique are 

34 not as useful for the quantitative extrapolation of cancer risk from the inhalation of 

35 benzo[a]pyrene in the environment (Driscoll et al., 2000). 

36 The Thyssen et al. (1981) bioassay represents the only lifetime inhalation cancer bioassay 

37 available for describing dose-response relationships for cancer from inhaled benzo[a]pyrene. 

38 Limitations of the study include the following: (1) only male animals were included; (2) particle 

39 analysis of aerosols was not reported [i.e., MMAD and geometric SD were not reported], and 

40 (3) benzo[a]pyrene exposure occurred through the inhalation of hygroscopic particles 

41 [benzo[a]pyrene was adsorbed onto NaCl aerosols] which may have a different deposition than 
226 DRAFT - DO NOT CITE OR QUOTE 

1 benzo[a]pyrene adsorbed onto non-hygroscopic particles in the environment. Strengths of the 

2 study include exposure to hamsters for life, histological tumor examination of organs, use of 

3 multiple exposure groups, including approximately 30 male hamsters per group, and the 

4 availability of individual animal pathology reports with time of death and tumor detection data. 

5 Although the study has a few limitations, the strengths of the study support use of the data to 

6 derive an inhalation unit risk for benzo[a]pyrene. 

7 

8 5.4.2.2. Dose-response Data—Inhalation Exposure 

9 Survival was decreased relative to control only in the high-dose exposure group; mean 

survival times in the 0, 2, and 10 mg/m310 concentration groups were 96.4, 95.2, and 96.4 weeks, 

respectively, and 59.5 weeks in the 50 mg/m311 group animals. Overall, tumors occurred earlier in 

12 the highest benzo[a]pyrene exposure group than in the mid-exposure group. Increased 

13 incidences of benign and malignant tumors of the larynx, trachea, pharynx, esophagus and 

14 forestomach were seen with increasing exposure concentration. Benign tumors—papillomas, 

15 polyps and papillary polyps—were considered by the study authors as early stages of the 

16 squamous cell carcinomas in these tissues. 

17 Nasal cavity tumors were also observed in the mid- and high-dose groups. Consideration 

18 of early mortality (using the poly-3 approach; Bailer and Portier, 1988) suggested that an 

19 increasing dose-response was consistent with these data. However trend testing was not 

20 statistically significant (p=0.08), and the site was not considered further for unit risk derivation. 

21 Table E-1 in Appendix E summarizes the individual animal tumor data, noting the presence or 

22 absence of a tumor in these tissues, whether or not the tissue was available for examination by 

23 the pathologist, and the time of death. A summary of the incidence of these tumors is provided 

24 in Table 5-7. 

25 

Table 5-7. Incidence of tumors in male hamsters exposed by inhalation to 

benzo[a]pyrene for life 

Average 

continuous 

benzo[a]pyrene 

concentrationa 

(mg/m3) 

Number 

of 

hamsters 

in groupb Larynx Pharynx Trachea Esophagus Forestomach

Any 

Tumorc

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 

aCalculated from air monitoring data. 

bNumber of animals examined histologically, minus the 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 of the larynx, pharynx, trachea, esophagus, or forestomach. 

Source: Thyssen et al. (1981) and a reanalysis of this data by Clement Associates (1990). See Appendix E for 

more detailed incidence data. 
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1 

2 5.4.2.3. Dose Adjustments and Extrapolation Method(s)—Inhalation Exposure

3 A toxicokinetic model to assist in cross species scaling of benzo[a]pyrene inhalation 

4 exposure was not available. In addition, default dosimetry adjustments utilized in the 

5 benzo[a]pyrene RfC calculation could not be applied because aerosol particle distribution data 

6 were not available for the hamster inhalation bioassay by Thyssen et al. (1981). The carrier 

7 particle used in Thyssen et al. (1981) was sodium chloride, a soluble hygroscopic particle, and 

8 the approaches presented in the RfC methodology guidelines (US EPA 1994b) were developed 

9 for insoluble and nonhygroscopic particles. 

10 The availability of the raw chamber air monitoring data and individual times on study 

11 allowed the calculation of time-weighted average (TWA) continuous exposure rates for each 

12 hamster. Group averages of individual TWA continuous exposure concentrations were 0, 0.25, 

1.01, and 4.29 mg/m3, respectively, for the 0, 2, 10, and 50 mg/m313 study concentrations. 

14 A time-to-tumor dose-response model was fit to the time-weighted average exposure 

15 concentrations and the individual animal occurrence data for tumors in the larynx, pharynx, 

16 trachea, esophagus, and forestomach (Table E-1 in Appendix E) using the computer software 

17 program multistage-Weibull (U.S. EPA, 2010) as described in Section 5.4.1.3. The 

18 investigators did not determine cause of death for any of the animals. Since in the available oral 

19 bioassays the investigators considered these same tumors to be fatal at least some of the time, 

20 bounding estimates for the Thyssen et al. data were developed by treating the tumors alternately 

21 as either all incidental or all fatal. In either case, therefore, an estimate of t0 (the time between a 

22 tumor first becoming observable and causing death) could not be estimated. 

23 Because benzo[a]pyrene is expected to cause cancer via a mutagenic MOA, a linear 

24 approach to low dose extrapolation from the BMCL10 was used (U.S. EPA, 2005a). 

25 

26 5.4.2.4. Inhalation Unit Risk Derivation 

Modeling results are provided in Appendix E. The BMC (0.28 mg/m327 ) and BMCL (0.20 

mg/m328 ) associated with an extra risk of 10% were calculated based on the occurrence of upper 

29 respiratory and upper digestive tract tumors in male hamsters exposed to aerosols of 

30 benzo[a]pyrene for 104 weeks using the multistage-Weibull model Using linear extrapolation 

from the BMCL10 of 0.20 mg/m3, an inhalation unit risk of 0.5 per mg/m3 or 5 x 10-4 per μg/m331 

32 was calculated. 

33 

34 5.4.3. Dermal Exposure—Dermal Slope Factor 

35 5.4.3.1. Choice of Study/Data—with Rationale and Justification—Dermal Exposure 

36 Skin cancer in humans has been documented to result from occupational exposure to 

37 complex mixtures of PAHs including benzo[a]pyrene such as coal tar, coal tar pitches, non38 refined mineral oils, shale oils and soot (IARC, 2010; Baan et al., 2009; Boffetta et al., 1997; 
228 DRAFT - DO NOT CITE OR QUOTE 

1 WHO, 1998; ATSDR, 1995), but no studies of human exposures to benzo[a]pyrene alone are 

2 known to exist. In animal models, numerous dose response studies have demonstrated an 

3 increased incidence of skin tumors with increasing dermal exposure to benzo[a]pyrene, in all 

4 species tested (mice, rabbits, rats, and guinea pigs), though most benzo[a]pyrene chronic dermal 

5 bioassays which provide quantitative information have been conducted in mice. In addition, 

6 mice appear to be the most sensitive laboratory model of carcinogenesis following dermal 

7 benzo[a]pyrene exposure. Therefore, this analysis focuses on chronic carcinogenicity bioassays 

8 in several strains of mice demonstrating increasing incidence of benign and malignant skin 

9 tumors, and earlier occurrence of tumors with increasing exposure, following repeated dermal 

10 exposure to benzo[a]pyrene for the animals’ lifetime. These studies involved 2- or 3-times/week 

11 exposure protocols, at least two exposure levels plus controls, and included histopathological 

12 examinations of the skin and other tissues (Sivak et al., 1997; Grimmer et al., 1984; 1983; Habs 

13 et al., 1980; 1984; Schmähl et al., 1977; Schmidt et al., 1973; Roe et al., 1970; Poel, 1960; 1959). 

14 These data sets are described in greater detail in Section 4.2.3.2. 

15 Because of the availability of the lifetime studies listed above, other carcinogenicity 

16 studies were not considered for this assessment. The other studies included: 1) early “skin 

17 painting” studies of benzo[a]pyrene carcinogenicity in mouse skin which did not report sufficient 

18 information to estimate the doses applied (e.g., Wynder and Hoffman 1959; Wynder et al, 1957); 

19 2) initiation-promotion studies utilizing acute dosing of benzo[a]pyrene followed by repeated 

20 exposure to a potent tumor promoter (sometimes benzo[a]pyrene at a lower dose than the 

21 initiation step), because they are not as relevant for calculating risks from constant 

22 benzo[a]pyrene exposure alone; 3) bioassays with one benzo[a]pyrene dose level or with only 

23 dose levels inducing 90–100% incidence of mice with tumors, because they provide relatively 

24 little information about the shape of the dose-response relationship (e.g., Wilson and Holland, 

25 1988); 4) studies with shorter exposure and observation periods (i.e., less than one year; Levin et 

26 al., 1977; Nesnow et al, 1983; Albert et al., 1991; Emmett et al, 1981; Higginbotham et al., 1993) 

27 which are less relevant for characterizing lifetime risk; and 5) studies involving vehicles 

28 expected to interact with or enhance benzo[a]pyrene carcinogenicity (e.g., Bingham and Falk, 

29 1969) which precludes assessment of carcinogenic risks of benzo[a]pyrene alone. 

30 

31 5.4.3.2. Dose-response Data—Dermal Exposure 

32 Several studies were considered for dose-response modeling for derivation of the dermal 

33 slope factor for benzo[a]pyrene, reflecting a relatively large database. Study designs and the 

34 extent of data reported varied across the studies, with no individual animal data available. All of 

35 the studies identified in the previous section (Sivak et al., 1997; Grimmer et al., 1984; 1983; 

36 Habs et al., 1980; 1984; Schmähl et al., 1977; Schmidt et al., 1973; Roe et al., 1970; Poel, 1960; 

37 1959) were considered further in order to evaluate overall consistency of the available database. 

38 These data sets are presented in Tables 5-8 through 5-12, and are grouped by study 
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1 characteristics such as mouse strain and vehicle in order to facilitate qualitative comparisons 

2 where possible. 

3 Each data set was examined for study design and both strengths and limitations that could 

4 potentially impact the dose-response evaluation, including the potential of early mortality to 

5 impact the number at risk for developing tumors, and the length of exposure. Although nearly all 

6 studies reported lifetime exposures, this term most often indicated that exposure continued until 

7 natural death, not a scheduled sacrifice at 104 weeks. 

8 The studies by Poel (1959, 1960) were conducted in male mice and used toluene as the 

9 vehicle (see Tables 4-11, 4-12, respectively). In addition to a control group, the 1959 study 

10 included nine dose groups of one mouse strain (C57L) and the 1960 study included seven dose 

11 groups of 3 other mouse strains. Both studies demonstrated high mortality and tumor incidence 

12 at higher exposure levels. As noted in Table 4-11, all C57L mice in dose groups with >3.8 

13 μg/application died by Week 44 of the study (Poel, 1959). Therefore, these five dose groups 

14 were omitted prior to dose-response modeling because of the relatively large uncertainty in 

15 characterizing cancer risk in relation to lifetime exposure. Four dose groups in addition to 

16 control remained. Among these groups mice survived and were exposed until weeks 83–103. 

17 According to the lifespan ranges provided, at least one mouse in each dose group died before the 

18 first appearance of tumor, but insufficient information was available to determine how many; 

19 consequently the incidence denominators were not adjusted. The dose-response data are 

20 summarized in Table 5-7. 

21 For the Poel (1960) studies, all tumors in the highest three dose groups for each of the 

22 three mouse strains had occurred by Week 40 (see Table 4-12). While these observations 

23 support concern for cancer risk, as noted above such results are relatively uncertain for 

24 estimating lifetime cancer risk. In addition, there was no information indicating duration of 

25 exposure for the mice without tumors; although exposure was for lifetime, it might have been as 

26 short as for the mice with tumors. Overall, these datasets did not provide sufficient information 

27 to estimate the extent of exposure associated with the observed tumor incidence. Consequently 

28 the experiments reported by Poel (1960) were not used for dose-response modeling. 

29 The studies listed in Table 5-9 all used acetone as the vehicle and either Swiss or NMRI, 

30 female mice (Roe et al., 1970; Schmidt et al., 1973; Schmähl et al., 1977; Habs et al., 1980, 

31 1984). Roe et al. (1970) applied benzo[a]pyrene dermally for 93 weeks or until natural death; 

32 with the exception of the highest dose group, each group still had approximately 20 animals at 86 

33 weeks (Table 4-14). The tumors were first observed in the lowest and highest dose groups 

34 during the interval of weeks 29-43. Mice that died before week 29 were likely not at risk of 

35 tumor development. However because tumor incidence and mortality were reported in 100-day 

36 intervals, mice that had not been on study long enough to develop tumors were not easily 

37 identifiable. Incidence denominators reflect the number of animals alive at Week 29, and thus 
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1 may tend to lead to underestimates of tumor risk if the number of animals at risk have been 

2 overestimated. 

3 Schmidt et al. (1973) did not report survival information, instead the authors provided 

4 incidences based on the numbers of mice initially included in each dose group at the start of the 

5 study. Overall latency was reported for the two high dose groups in each series, but these data 

6 only describe the survival of mice with tumors (animals were removed from study when a tumor 

7 appeared). It is not clear how long exposures lasted overall in each dose group, or whether some 

8 mice may have died on study from other causes before tumors appeared. While it is possible that 

9 no mice died during the study, all of the other studies considered here demonstrate mortality. 

10 However, the data were modeled as reported, recognizing the possibility of underestimating risk 

11 associated with incidences reported and lack of duration of exposure. 

12 Schmähl et al. (1977) reported that reduced numbers of animals at risk (77−88 mice per 

13 dose group compared with the initial group sizes of 100) resulted from varying rates of autolysis. 

14 No other survival or latency information was provided, so all exposures were assumed to have 

15 lasted for 104 weeks and were modeled as reported. Given the results of the other studies, it 

16 seems possible that the numbers at risk in each group may be overestimated, which could lead to 

17 an underestimate of lifetime risk. 

18 Habs et al. (1980) reported age-standardized skin tumor incidence rates, indicating earlier 

19 mortality in the two highest dose groups (2.8 and 4.6 μg/application). These rates were used to 

20 estimate the number at risk in the dose-response modeling, by dividing the number of mice with 

21 tumors by the age-standardized rates (see Table 5-9). Exposure lasted longer than 104 weeks in 

22 the two lower exposure groups, at about 120 and 112 weeks, and until about 88 weeks in the 

23 highest exposure group. Incidence in the two lower exposure groups may be higher than if the 

24 exposure had lasted just 104 weeks. There was mortality in the first 52 weeks of exposure, about 

25 10−15% in the three exposure groups, but because there was no information concerning when 

26 tumors first appeared it is not possible to determine how much the early mortality may have 

27 impacted the number of mice at risk in each group. 

28 Habs et al. (1984) reported mean survival times (with 95% confidence intervals) for each 

29 dose group. The confidence intervals supported the judgment that the control and lower dose 

30 groups were treated for 104 weeks. The higher dose group (4 μg/application) was probably 

31 treated for less than 104 weeks, because the upper 95% confidence limit for the mean survival 

32 was approximately 79 weeks (Table 4-20). However, since it was not possible to estimate a 

33 more realistic duration for this group, an estimate of 104 weeks was used. 

34 Grimmer et al. (1983 and 1984), studied female CFLP mice, using acetone:DMSO (1:3) 

35 as the vehicle (see Table 5-10). Mean or median latency times were reported (as well as 

36 measures of variability), but no information concerning overall length of exposure or survival 

37 was included in the results. The total of tumor-bearing mice and the reported percentages of 

38 mice with any skin tumors was reported and varied at most one animal from the number of 
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1 animals initially placed on study. The decreasing latency and variability and increasing tumor 

2 incidence with increasing benzo[a]pyrene exposure suggests that exposure probably did not last 

3 for 104 weeks in at least the high dose group, but the available information did not provide 

4 duration of exposure. The data reported were modeled under the assumption that at least some 

5 animals in each group were treated and survived until Week 104. 

6 The study listed in Table 5-11, Sivak et al. (1997) exposed male C3H /HeJ mice dermally 

7 to benzo[a]pyrene in cyclohexanone/acetone (1:1) for 24 months, and reported mean survival 

8 times for each group (see Table 4-21). All high dose mice died before the final sacrifice. From 

9 the information provided it is apparent that the animals in the control and lower two dose groups 

10 survived until study termination at Week 104. The study authors did not report how long 

11 treatment in the highest dose group lasted, but estimation of the figure from the publication 

12 suggest that exposure duration was 74 weeks. The tumor incidences and estimated duration of 

13 exposure for each dose group are presented in Table 5-11. 

14 
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Table 5-8. Skin tumor incidence, benign or malignant, in C57L male mice 

dermally exposed to benzo[a]pyrene 

Study 

Mouse 

strain Dose (μg)a

Average 

daily 

dose 

(μg/d) 

First 

appearance 

of tumor 

(weeks) 

Length 

of 

Exposure 

(weeks) 

Lifetime 

Average 

Daily Doseb 

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%) 

aDoses 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 five highest dose groups (19-752 μg) 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 extrapolation to lifetime average daily doses. 

1 

2 
233 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 5-9. Skin tumor incidence, benign or malignant in female Swiss or 

NMRI mice dermally exposed to benzo[a]pyrene 

Study 

Mouse 

strain Dose (μg) 

Average 

daily 

dose 

(μg/d) 

First 

appearance 

of tumor 

(weeks) 

Length of 

exposure 

(weeks) 

Lifetime 

average daily 

dose 

(μg/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 

— 

— 

— 

53e 

76e 

104d

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 

— 

— 

— 

58e 

61e 

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%) 

aDoses were applied 3 times/week for up to 93 wks to shaved dorsal skin. 

bNumerator: number of mice detected with a skin tumor. Denominator: number of mice surviving to 29 weeks 

(200 days). 

cDoses were applied 2 times/week to shaved skin of the back. Mice were exposed until natural death or until they 

developed a carcinoma at the site of application. 

dExposure periods not reported were assumed to be 104 weeks; indicated in italics. 

eCentral tendency estimates; range or other variability measure not reported. 

fThe percentages were reported by the authors as age-standardized tumor incidences, derived using mortality data 

from the entire study population. The incidences reflect reported counts of tumor-bearing animals and

denominators estimated from the reported age-standardized rates. 

NR=not reported. 

2 

3 

4 
234 DRAFT - DO NOT CITE OR QUOTE 

Table 5-10. Skin tumor incidence, benign or malignant, in female CFLP mice 

dermally exposed to benzo[a]pyrene 

Study Dose (μg)a

Average 

daily dose 

(μg/d) 

Mean or 

median time 

of tumor 

appearance 

(weeks) 

Length of 

exposure 

(weeks)d

Lifetime 

average daily 

dose 

(μg/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 ± 16.8b 

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 skin of the back for up to 104 weeks. 

bMean ± SD. 

c

 Median and 95% confidence limit. 

dAssumed exposure period is indicated in italics. 

e

Incidence denominators were calculated from reported tumor-bearing animals and reported percentages. 

1 

Table 5-11. Skin tumor incidence, benign or malignant, in male C3H /HeJ 

mice dermally exposed to benzo[a]pyrene(Sivak et al., 1997) 

Dose (μg)a

Average 

daily 

dose 

(μg/d)

First 

appearance 

of tumor 

(weeks)

Length of 

exposure 

(weeks)b

Lifetime 

average 

daily dose 

(μg/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 dorsal skin. 

bAssumed exposure period is indicated in italics. 

NR=not reported. 

2 

3 

4 5.4.3.3. Dose Adjustments and Extrapolation Method(s)—Dermal Exposure

5 As with the oral and inhalation benzo[a]pyrene carcinogenicity data (see sections 5.4.1.3 

6 and 5.4.2.3), benzo[a]pyrene’s dermal exposure carcinogenicity data were generally 

7 characterized by earlier occurrence of tumors and increased mortality with increasing exposure 

8 level. However, individual animal data were not available for any of the identified studies. 

9 Therefore, time to tumor modeling was not possible. Each of the dermal data sets was modeled 

10 using the multistage model, incorporating adjustments for early mortality, when data were 

11 available. 
235 DRAFT - DO NOT CITE OR QUOTE 

1 

2 First, for all studies, administered doses were converted to average daily doses using the 

3 equation: 

4 

5 Average dose/day = (μg/application) × (number of exposures/week ÷ 7 days/week). 

6 

7 Next, lifetime equivalent doses were estimated for study groups that were reported to end 

before 104 weeks by multiplying the relevant average daily doses by (Le/104)38 , where Le is the 

9 length of exposure, based on observations that tumor incidence tends to increase with age (Doll, 

10 1971). Note that exposure periods less than 52 weeks would lead to a relatively large adjustment 

[i.e., (52/104)311 = 0.125, or an eightfold lower dose than administered], reflecting considerable 

12 uncertainty in lifetime equivalent dose estimates generated from relatively short studies. 

13 The multistage-cancer model in the EPA BMDS (version 2.1) was fit to each data set in 

14 Tables 5-8 through 5-11. The multistage model with the most parsimonious fit (fewest 

15 parameters yielding an adequate fit) was selected to calculate the potential POD from each data 

16 set (see Appendix F for details). Because the multistage model is preferred for cancer modeling, 

17 the conventional α-level of 0.05 was used to judge goodness-of-fit. If there was no adequate fit 

18 using the multistage-cancer model, then other dichotomous models were considered. If there 

19 was still no adequate fit, high doses were dropped incrementally and the multistage-cancer model 

20 was considered before attempting other models. BMDs and BMDLs associated with an extra 

21 risk of 10% were calculated. Because benzo[a]pyrene is expected to cause cancer via a 

22 mutagenic MOA, a linear approach to low dose extrapolation from the PODs (i.e., BMDL10) was 

23 used (U.S. EPA, 2005a) for candidate dermal slope factors. 

24 

25 5.4.3.4. Dermal Slope Factor Derivation 

26 Adequate model fits were found using the multistage model for all but one of the mouse 

27 skin tumor incidence data sets in Tables 5-8 to 5-11, as described in Appendix F. In one case, 

28 the data from Grimmer et al. (1984) could not be adequately fit by the multistage model initially, 

29 and the other dichotomous models available in BMDS were considered. Due to the supralinear 

30 shape of the dose-response data, only the log-logistic and dichotomous Hill models provided 

31 adequate fits. Also due to the supralinear dose-response shape, the point of departure for slope 

32 factor derivation was identified near the lowest response of ~70%, in order to avoid excessive 

33 extrapolation of the fitted model. 

34 Dermal slope factors, calculated in units of risk per (μg/day) using linear extrapolation 

35 from the BMDL10 values, ranged from 0.25 to 1.8 per μg/day, a roughly 7-fold range (see Table 

36 5-12). A number of differences among studies contribute to this range, including solvent choice, 

37 sex and strain of mice studied, dose ranges and the level of detail reported. Mouse strains were 

38 not repeated across sexes among these studies, so it cannot be established whether male mice are 
236 DRAFT - DO NOT CITE OR QUOTE 

1 generally more or less sensitive than female mice to benzo[a]pyrene dermal carcinogenicity, or 

2 whether Swiss or NRMI mice are more or less sensitive than other strains. In addition, different 

3 solvents were used in the various studies with varying strain and sex combinations tested. For 

4 example, toluene was used in one male study only and all of the female studies used acetone. 

5 Thus, any possible impact of the solvents used is not clear. The estimates derived from the two 

6 studies in males were at the higher end of the range of slope factors derived, but the available 

7 information is too limited to conclude that males are more sensitive than females. Also, as noted 

8 earlier, incomplete mortality information in several of the female mouse studies (Schmidt et al., 

9 1973; Schmähl et al., 1977; Grimmer et al., 1983, 1984; and Habs et al., 1980, 1984) suggests 

10 that the derived dermal slope factors may underestimate cancer risk. 

11 The BMDL10 of 0.066 μg/animal-day, based on the tumor response in C3H/HeJ male 

12 mice (Sivak et al., 1997), is recommended for developing a human dermal slope factor because it 

13 is the lowest POD among studies with lower doses where intercurrent mortality was less likely to 

14 impact the number at risk and represented a chronic duration of exposure. 

15 

16 Table 5-12. PODs derived from skin tumor incidence data in mice exposed to 

benzo[a]pyrene by the dermal route of exposurea 17 

18 

Reference Mouse strain Solvent 

BMD10 

(μg/animal-d) 

BMDL10

(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 1.07b 0.48b

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

19 See Appendix F for details of modeling results. 

b

20 BMR=70% for this dataset, in order to avoid excessive extrapolation via the fitted model. 

21 

22 5.4.3.5. Dermal Slope Factor Cross Species Scaling 

23 Different methodologies have been established for interspecies scaling of points of 

24 departure (PODs) used to derive oral slope factors and inhalation unit risks. Cross-species 

25 adjustment of oral doses is based on allometric scaling using the three-fourths power of body 

26 weight. This adjustment accounts for more rapid distribution, metabolism, and clearance in 

27 small animals (US EPA 2005). Cross-species extrapolation of inhalation exposures is based on 

28 standard dosimetry models that consider factors such as solubility, reactivity, and persistence 
237 DRAFT - DO NOT CITE OR QUOTE 

1 (US EPA 1994). However, no established methodology exists to adjust for interspecies 

2 differences in dermal toxicity at the point of contact. Because there is no established 

3 methodology for cross-species extrapolation of dermal toxicity, several alternative approaches 

4 were evaluated (see Appendix H). Among the alternative described in Appendix H, cross5 species adjustment based on allometric scaling using body weight to the 3/4 power was selected. 

6 Under this approach, rodents and humans exposed to the same daily dose of a carcinogen, 

adjusted for BW3/4 7 , would be expected to have equal lifetime risks of cancer. 

8 

9 The PODM derived from the mouse study by Sivak et al., (1997) is adjusted to a human 

10 equivalent dose (HED) as follows: 

11 

POD HED(μg/day) = PODM (μg/day) × (BWH / BWM)

3/4 12 

13 

PODHED (μg/day) = 0.066 μg/day × (70 kg / 0.035 kg)3/4 14 

15 = 19.7 μg/day 

16 

17 

18 The resulting PODHED is used to calculate the dermal slope factor for benzo[a]pyrene: 

19 

20 DSF = 0.1/ PODHED 

21 

DSF = 0.1/(19.7 μg/day) = 0.005 (μg/day)-1 22 

23 

24 Note that the DSF should only be used with lifetime human exposures < 20 μg/day, the 

25 human equivalent of the bioassay POD, because above this level the dose-response relationship 

26 may not be proportional to mass of the compound applied. 

27 Several assumptions are made in the use of this scaling method. First, it is assumed that 

28 the toxicokinetic processes in the skin will scale with interspecies differences in whole body 

29 toxicokinetics. Secondly, it is assumed that the risk at low doses of benzo[a]pyrene is linear; 

30 however, one study indicates that at high doses of benzo[a]pyrene, carcinogenic potency is 

31 related to mass applied per unit skin and not to total mass (Davies 1967). However, this may be 

32 due to promotional effects, such as inflammation, that are observed at high doses of 

33 benzo[a]pyrene. 

34 This slope factor has been developed for a local effect and it is not intended to estimate 

35 systemic risk of cancer following dermal absorption of benzo[a]pyrene into the systemic 

36 circulation. Although some information suggests that benzo[a]pyrene metabolites can enter 

37 systemic circulation following dermal exposure (Godschalk et al 1998), lifetime skin cancer 

38 bioassays which have included pathological examination of other organs, have not found 
238 DRAFT - DO NOT CITE OR QUOTE 

1 elevated incidences of tumors at distal sites (Poel 1959; Roe et al., 1970; Schmidt et al., 1973; 

2 Schmahl et al., 1977; Habs et al., 1980; Higginbotham et al.,1993). In addition, benzo[a]pyrene 

3 tends to bind to targets within the skin rather than enter the plasma receptor fluid (a surrogate 

4 measure of systemic absorption) in in vitro human skin experiments. These data are consistent 

5 with benzo[a]pyrene’s metabolism to reactive metabolites within the viable layers of the skin 

6 (Wester et al., 1990). Some studies indicate that the fraction of benzo[a]pyrene left within the 

7 viable layers of the skin is a large portion of the applied dose (Moody et al., 2007; 1995). Taken 

8 together, these data support the conclusion that the risk of skin cancer following dermal exposure 

9 likely outweighs cancer risks at distal organs. 

10 

11 5.4.4. Application of Age-Dependent Adjustment Factors 

12 Based on sufficient support in laboratory animals and relevance to humans (see Section 

13 4.7.3) benzo[a]pyrene is determined to be carcinogenic by a mutagenic MOA. According to the 

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

15 (“Supplemental Guidance”) (U.S. EPA, 2005b), individuals exposed during early life to 

16 carcinogens with a mutagenic MOA are assumed to have increased risk for cancer. The oral 

slope factor of 1.4 per mg/kg-day, inhalation unit risk of 0.5 per mg/m317 , and dermal slope factor 

18 of 0.0051 per μg/day for benzo[a]pyrene, calculated from data applicable to adult exposures, do 

19 not reflect presumed early life susceptibility to this chemical. Though some chemical specific 

20 data exist for benzo[a]pyrene which demonstrate increased early life susceptibility to cancer 

21 (Vesselinovitch et al. 1984), these data were not considered sufficient to develop separate risk 

22 estimates for childhood exposure, as they used acute, i.p. exposures (U.S. EPA, 2005b). In the 

23 absence of adequate chemical-specific data to evaluate differences in age-specific susceptibility, 

24 the Supplemental Guidance (U.S. EPA, 2005b) recommends that age-dependent adjustment 

25 factors (ADAFs) be applied in estimating cancer risk. 

26 The Supplemental Guidance (U.S. EPA, 2005b) establishes ADAFs for three specific age 

27 groups. These ADAFs and their corresponding age groupings are: 10 for individuals exposed 

28 <2 years, 3 for exposed individuals 2 to <16 years, and 1 for exposed individuals ≥16 years. The 

29 10- and 3-fold adjustments are combined with age specific exposure estimates when estimating 

30 cancer risks from early life (<16 years age) exposures to benzo[a]pyrene To illustrate the use of 

31 the ADAFs established in the Supplemental Guidance (U.S. EPA, 2005b), sample calculations 

32 are presented for three exposure duration scenarios, including full lifetime, assuming a constant 

33 benzo[a]pyrene exposure of 0.001 mg/kg-day (Table 5-13). 

34 Calculations for the application of ADAFs to oral exposures are presented in Table 5-13; 

35 calculations for exposures by the inhalation and dermal routes follow the same procedure (Table 

36 5-13 and 5-14). Exposure duration scenarios include full lifetime exposure (assuming a 70-year 

37 lifespan), and two 30-year exposures at ages 0–30 and ages 20–50. Table 5-13 lists the four 

38 factors (ADAFs, cancer risk estimate, assumed exposure, and duration adjustment) that are 
239 DRAFT - DO NOT CITE OR QUOTE 

1 needed to calculate the partial cancer risk based on the early age-specific group. The cancer risk 

2 for each age group is the product of the four factors in columns 2–5. Therefore, the cancer risk 

3 following daily benzo[a]pyrene oral exposure in the age group 0 to <2 years is the product of the 

values in columns 2–5 or 10 × 1× 0.001 × 2/70 = 4 × 10-4 4 . The cancer risk for specific exposure 

5 duration scenarios that are listed in the last column are added together to get the total risk. Thus,

6 a 70-year (lifetime) risk estimate for continuous exposure to 0.001 mg/kg-day benzo[a]pyrene is 

2 × 10-3 7 , which is adjusted for early-life susceptibility and assumes a 70-year lifetime and 

8 constant exposure across age groups. 

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 

12 In calculating the cancer risk for a 30-year constant exposure to benzo[a]pyrene at an 

13 exposure level of 0.001 mg/kg-day from ages 0–30, the duration adjustments would be 2/70, 

14/70, and 14/70, and the partial risks for the three age groups would be 3 × 10-4, 6 × 10-4 14 , and 

2 × 10-4, which would result in a total risk estimate of 1 × 10-3 15 . 

16 In calculating the cancer risk for a 30-year constant exposure to benzo[a]pyrene at an 

17 exposure level of 0.001 mg/kg-day from ages 20–50, the duration adjustments would be 0/70, 

0/70, and 30/70. The partial risks for the three groups are 0, 0, and 4 × 10-4 18 , which would result 

in a total risk estimate of 4 × 10-4 19 . 

20 Consistent with the approaches for the oral route of exposure, the ADAFs should also be 

21 applied when assessing cancer risks for subpopulations with early life exposures to 

22 benzo[a]pyrene via the inhalation and dermal routes are presented in Tables 5-14 and 5-15. 

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/m3) (μg/m3) 
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 

3 Table 5-15. Application of ADAFs to benzo[a]pyrene cancer risk following a 

4 lifetime (70-year) dermal exposure 

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 

8 5.4.5. Uncertainties in Cancer Risk Values 

9 5.4.5.1. Oral Slope Factor 

10 Uncertainty in the recommended oral slope factor is reflected in the range of slope factors 

11 among tumors sites and species; the lowest and highest slope factors listed in Table 5-6 show 

12 about a 35-fold difference. While the highest risk estimates were derived from the incidence 

13 data for forestomach tumors in both rats and mice, the oral slope factor based on the mouse 

14 forestomach data was about threefold higher than the oral slope factor based on male rat data 

15 (Table 5-6). These comparisons show that the selection of target organ, animal species, and 

16 dosimetric extrapolation can impact the oral cancer risk estimate. However, all of the activation 

17 pathways implicated in benzo[a]pyrene carcinogenicity have been observed in human tissues and 

18 associations have been made between the spectra of mutations in tumor tissues from 

19 benzo[a]pyrene-exposed animals and humans exposed to complex PAH mixtures containing 

20 benzo[a]pyrene (see Section 4.7.3). 

21 

22 5.4.5.2. Inhalation Unit Risk 

23 Only one animal cancer bioassay by the inhalation route is available which describes the 

24 dose-response relationship for respiratory tract tumors with chronic inhalation exposure to 

25 benzo[a]pyrene (Thyssen et al., 1981). Although corroborative information on dose-response 

26 relationships in other animal species is lacking, the findings for upper respiratory tract tumors are 

27 consistent with findings in other hamster studies with intratracheal administration of 

28 benzo[a]pyrene. This study is adequate for dose-response analysis and derivation of an 

29 inhalation unit risk estimate, but some associated uncertainty includes the inability to apply U.S. 
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1 EPA (1994b) dosimetry approaches to extrapolate inhaled doses from animals to humans, due to 

2 the use of a soluble hygroscopic carrier particle (NaCl) for the delivery of benzo[a]pyrene. One 

3 likely consequence of the use of hygroscopic carrier particles would be the growth of 

4 benzo[a]pyrene-NaCl particles in the humid environment of the respiratory tract resulting in 

5 increased particle diameter and resulting changes in particle deposition, specifically, increased 

6 impaction in the upper respiratory tract (Xu and Yu 1985; Ferron 1994; Asgharian 2004; 

7 Varghese and Gangamma 2009). Exposure to benzo[a]pyrene in the environment predominantly 

8 occurs via non-soluble, non-hygroscopic particles. The potential impact of differences in carrier 

9 particle on the magnitude of the inhalation unit risk is unknown. 

10 

11 5.4.5.3 Dermal Slope Factor 

12 Uncertainty in the recommended dermal slope factor is partly reflected in the range of 

13 slope factors derived from the modeled mouse skin tumor data sets: the lowest and highest 

14 dermal slope factors listed in Table 5-12 show a 7-fold difference (0.25-1.8 ug/day) in 

15 magnitude. There is some indication that the recommended dermal slope factor may 

16 underestimate cancer risk, due to inadequate data to take the observed decreasing tumor latency 

17 with increasing exposure level into account. Reliance on studies with the lowest exposure levels 

18 where early mortality due to benzo[a]pyrene exposure was low and exposures continued for 

19 approximately 104 weeks may minimize this source of uncertainty. 

20 Human dermal exposure to benzo[a]pyrene in the environment likely occurs 

21 predominantly through soil contact. The available mouse dermal bioassays of benzo[a]pyrene 

22 relied on delivery of benzo[a]pyrene to the skin in a solvent solution (typically acetone or 

23 toluene). The use of a volatile solvent likely results in a larger dose of benzo[a]pyrene available 

24 for uptake into the skin (compared to soil). Reliance on these studies may overestimate the risk 

25 of skin tumors from benzo[a]pyrene contact through soil; however, cancer bioassays delivering 

26 benzo[a]pyrene through a soil matrix are not available. 

27 There is uncertainty in extrapolating from the intermittent exposures in the mouse assays 

28 to daily exposure scenarios. This assessment makes the assumption that risk is proportional to 

29 total cumulative exposure. The extent to which this assumption under- or overestimates risk is 

30 unknown. 

31 The available data were not useful to determine which animal species may be the best 

32 surrogate for human dermal response to benzo[a]pyrene. In extrapolation of the animal dermal 

33 information to humans the inherent assumption is that equal area of skin from a mouse or human 

34 would have equal probability of developing a tumor upon benzo[a]pyrene exposure. 

35 Qualitatively, the toxicokinetics and toxicodynamics in mouse and human skin appear to be 

36 similar (Knafla et al., 2010; Bickers et al., 1984). Specifically, all of the activation pathways 

37 implicated in benzo[a]pyrene carcinogenicity have been observed in mouse and human skin and 

38 associations have been made between the spectra of mutations in tumor tissues from 
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1 benzo[a]pyrene-exposed animals and humans exposed to complex PAH mixtures containing 

2 benzo[a]pyrene (see Section 4.7.3). 

3 This dermal slope factor for benzo[a]pyrene is based on skin cancer and it is not 

4 developed to represent systemic cancer risk from dermal exposure. It is unclear whether dermal 

5 exposure to benzo[a]pyrene would result in elevated risk of systemic tumors. Some studies in 

6 humans suggest that although the skin may be responsible for a “first pass” metabolic effect, 

7 benzo[a]pyrene-specific adduct levels have been detected in WBC following dermal exposure to 

8 benzo[a]pyrene, indicating that dermally applied benzo[a]pyrene enters systemic circulation 

9 (Godschalk et al., 1998). Although none of the lifetime dermal bioassays in mice, which 

10 included macroscopic examination of internal organs, reported an elevation of systemic tumors 

11 in benzo[a]pyrene-treated mice compared to controls (Poel 1959; Roe et al., 1970; Schmidt et al., 

12 1973; Schmahl et al., 1977; Habs et al., 1980; Higginbotham et al.,1993), most of these studies 

13 attempted to remove animals with grossly observed tumors of the skin from the study before the 

14 death of the animal, possibly minimizing the development of more distant tumors with longer 

15 latency. The risk of benzo[a]pyrene-induced point of contact tumors in the skin likely competes 

16 with systemic risk of tumors. Currently, the potential contribution of dermally absorbed 

17 benzo[a]pyrene to systemic cancer risk is unclear. 

18 

19 5.5.5. Previous Cancer Assessment 

20 The previous cancer assessment for benzo[a]pyrene was posted on the IRIS database in 

21 1987. At that time, benzo[a]pyrene was classified as a probable human carcinogen (Group B2) 

22 based on inadequate data in humans and sufficient data in animals via several routes of exposure. 

23 An oral slope factor was derived from the geometric mean of four slope factor estimates based 

24 on studies in Sprague-Dawley rats (Brune et al., 1981) and CFW-Swiss mice (Neal and Rigdon, 

25 1967). Brune et al. (1981) administered 0.15 mg/kg benzo[a]pyrene in the diet every 9th day or 

26 5 days/week in a 1.5% caffeine solution until rats were moribund or dead. A single slope factor 

27 estimate of 11.7 per mg/kg-day, based on a linearized multistage model applied to the combined 

28 incidence of forestomach, esophageal, and laryngeal tumors, was derived. In the Neal and 

29 Rigdon (1967) bioassay, mice administered benzo[a]pyrene in the diet at concentrations ranging 

30 from 1 to 250 ppm for up to 197 days developed significantly increased incidences of 

31 forestomach tumors. This study utilized mixed sex dose groups with mice from 3 weeks to 6 

32 months old at the start of dosing. This study did not include concurrent controls. This 

33 necessitated the use of historical controls (from SWR/J mice) for the incidence of forestomach 

34 tumors from a study by (Rabstein et al., 1973). Three modeling procedures were used to derive 

35 risk estimates from these data. For one risk estimate, Clement Associates (1990) fit a two-stage 

36 response model, based on exposure-dependent changes in both transition rates and growth rates 

37 of preneoplastic cells, to derive a value of 5.9 per mg/kg-day. In a U.S. EPA report (1991b), a 

38 value of 9.0 per mg/kg-day, derived by linear extrapolation from the 10% response point to the 
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1 background of an empirically fitted dose-response curve, was identified. Finally, using a 

2 Weibull-type model to reflect less-than-lifetime exposure to benzo[a]pyrene, the same U.S. EPA 

3 report (1991b) derived an upper-bound slope factor estimate of 4.5 per mg/kg-day. Since the 

4 variance for the four slope factor estimates was low, and in order to consider all of the available 

5 data, the geometric mean of these four estimates, 7.3 per mg/kg-day, was recommended as the 

6 oral slope factor. 

7 An inhalation unit risk and dermal slope factor were not previously available on IRIS. 

8 

9 
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1 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE 

2 RESPONSE 

3 

4 

5 6.1. HUMAN HAZARD POTENTIAL 

6 benzo[a]pyrene is a five-ring nonsubstituted PAH, which is produced through natural and 

7 anthropogenic processes involving the incomplete combustion or pyrrolysis of carbon-containing 

8 materials. Benzo[a]pyrene exists in the environment in complex mixtures, which may consist of 

9 numerous PAHs, including heterocyclic and nonheterocyclic forms as well as aza arenes and 

10 nitro-substituted PAHs. The magnitude of human exposure to benzo[a]pyrene and other PAHs 

11 depends on several factors related to lifestyle (e.g., diet, tobacco smoking), occupation, and 

12 living conditions (e.g., urban versus rural setting, domestic heating, and cooking methods). 

13 There are limited data establishing associations between increased risk for noncancer 

14 health effects in humans and exposure to benzo[a]pyrene. Several epidemiology studies have 

15 reported associations between adverse birth outcomes including reduced birth weight, postnatal 

16 body weight, and head circumference with internal biomarkers of exposure to benzo[a]pyrene 

17 (BPDE-DNA adducts) via exposure to complex PAH mixtures (Tang et al., 2008, 2006; Perera et 

18 al., 2005a, b). However, extrapolations from these studies are complicated by the concomitant 

19 exposure to multiple PAHs and other components in the mixture. 

20 There is evidence of human carcinogenicity for several PAH mixtures containing 

21 benzo[a]pyrene, such as soot, coal tars, coal-tar pitch, mineral oils, and shale oils (IARC, 2010; 

22 Baan et al., 2009; Straif et al., 2005). There is also evidence of carcinogenicity in occupations 

23 involving exposure to PAH mixtures containing benzo[a]pyrene, such as aluminum production, 

24 chimney sweeping, coal gasification, coal-tar distillation, coke production, iron and steel 

25 founding, and paving and roofing with coal tar pitch (IARC, 2010; Baan et al., 2009; Straif et al., 

26 2005). Benzo[a]pyrene is also a notable constituent of tobacco smoke (IARC 2004). An 

27 increasing number of studies report exposure biomarkers such as benzo[a]pyrene- or PAH-DNA 

28 adducts, and several cohort studies demonstrate a positive exposure-response relationship with 

29 cumulative PAH exposure using benzo[a]pyrene as an indicator substance. Because 

30 benzo[a]pyrene is only one of many PAHs that could contribute to these observed increases in 

31 cancer, the epidemiologic studies provide credible but limited support for a causative role of 

32 benzo[a]pyrene in human cancer. Studies in multiple species of laboratory animals indicate that 

33 benzo[a]pyrene is carcinogenic by all routes of exposure. 

34 

35 6.2. DOSE RESPONSE 

36 6.2.1. RfD 

37 Limited human data are available to inform noncancer health effects following chronic 

38 oral exposure to benzo[a]pyrene. Animal studies reporting effects of benzo[a]pyrene include 
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1 several chronic cancer bioassays (which report limited noncancer endpoints; Kroese et al. 2001; 

2 Beland and Culp, 1998; Culp et al., 1998), several subchronic studies (Knuckles et al., 2001; De 

3 Jong et al., 1999), developmental toxicity studies (Kristensen et al., 1995; MacKenzie and 

4 Angevine, 1981) and several reproductive toxicity studies (Mohamed et al., 2010; Xu et al., 

5 2010; Zheng et al., 2010). 

6 In consideration of the available studies reporting low-dose effects of chronic and 

7 subchronic oral exposure to benzo(a)pyrene in animals, the Xu et al., (2010) study was chosen as 

8 the principal study. This study identified biologically and statistically significant decrements in 

9 ovary weight, number of primordial follicles, estrogen levels, and estrus cyclicity. These 

10 reductions in female reproductive endpoints observed in rats are supported by a large database of 

11 animal studies indicating that benzo[a]pyrene, administered by multiple routes of exposure, is 

12 ovotoxic with effects including decreased ovary weight, decreased primordial follicles, and 

13 reduced fertility (Mattison et al., 1980; MacKenzie and Angevine, 1981; Swartz and Mattison 

14 1985; Miller et al., 1992; Kristensen et al.,1995; Borman et al, 2000; Archibong et al., 2002). 

15 Additionally, studies indicate that exposure to complex mixtures of PAHs, such as through 

16 cigarette smoke, is associated with measures of decreased fertility in humans (El Nemr et al., 

17 1998; Neal et al., 2005). Specific associations have also been made between infertility and 

18 increased levels of benzo[a]pyrene in follicular fluid in women undergoing in vitro fertilization 

19 (Neal et al., 2008). 

20 The RfD of 0.0005 mg/kg-day (0.5 μg/kg-day) was derived using a BMDL1SD of 

21 1.5 mg/kg-day for reduced ovary weight in SD rats exposed to benzo[a]pyrene via gavage for 60 

22 days (Xu et al., 2010). To derive the RfD, this POD was divided by a total UF of 3000 (factors 

23 of 10 for animal-to-human extrapolation, human interindividual variability in susceptibility, and 

24 subchronic to chronic extrapolation, and 3 for database deficiencies). The default animal-to25 human extrapolation and human variability factors were applied because of the lack of 

26 quantitative information to assess toxicokinetic or toxicodynamic differences between animals 

27 and humans and the range of susceptibilities in human populations. A subchronic to chronic 

28 extrapolation factor was applied because the POD was chosen from a study with a less than 

29 lifetime exposure duration. In addition, a database uncertainty factor of 3 was applied to account 

30 for deficiencies in the benzo[a]pyrene toxicity database, primarily the lack of a standard 

31 multigenerational reproductive study and the lack of a neurodevelopmental study. 

32 The overall confidence in the RfD is low-to-medium. Confidence in the principal study 

33 (Xu et al., 2010) is medium. The design, conduct, and reporting of this subchronic toxicity study 

34 were adequate; however, the number of dose groups and number of animals per group were low. 

35 Confidence in the database is low-to-medium primarily due to the lack of a multigeneration 

36 reproductive toxicity study (with exposure from pre-mating to sexual maturity) and the lack of a 

37 neurodevelopmental study. Reflecting medium confidence in the principal study and low-to38 medium confidence in the database, confidence in the RfD is low-to-medium. 
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1 

2 6.2.2. RfC 

3 The only chronic inhalation study available for benzo[a]pyrene was designed as a cancer 

4 bioassay and did not report noncancer endpoints (Thyssen et al.1981). However, several 

5 repeated dose reproductive and developmental toxicity studies are available in which effects on 

6 fetal survival and the male reproductive system have been observed following inhalation 

7 exposure. 

8 Archibong et al. (2002) was selected as the principal study as it observed biologically 

9 significant effects in F344 rats at the lowest dose tested by the inhalation route. This study 

10 indicates that the developing fetus is a sensitive target following inhalation exposure to 

benzo[a]pyrene. Exposure to benzo[a]pyrene at 25 μg/m311 on GDs 11–20 caused biologically 

12 significant reductions in fetal survival and body weight decreases in the surviving pups. The 

13 observed decrease in pup weight and fetal survival were selected as critical effects as they are the 

14 most sensitive noncancer effects observed following inhalation exposure to benzo[a]pyrene. 

15 Additional support for this endpoint can be found from an oral study of benzo[a]pyrene in mice 

16 which observed decreased survival of litters, decreased pup weight, and decreased reproductive 

17 organ weight following in utero exposure to benzo[a]pyrene on GD 7-16 (MacKenzie and 

18 Angevine, 1981). Though only a few studies exist that evaluate benzo[a]pyrene by the 

19 inhalation route, the oral studies support the reproductive and developmental effects observed in 

20 the available inhalation studies. 

The RfC of 5 x 10-6 mg/m3-day was derived using a LOAELADJ[HEC] of 4.6 μg/m321 -day for 

22 decrease in pup weight and fetal survival in F344 rats exposed to benzo[a]pyrene aerosols on 

23 GDs 11-20 (Archibong et al., 2002). To derive the RfC, the POD was divided by a total UF of 

24 1000 (factors of 3 for animal-to-human extrapolation, 10 for human interindividual variability in 

25 susceptibility and LOAEL-to-NOAEL extrapolation, and 3 for database deficiencies). The 

26 default animal-to-human extrapolation and human variability factors were applied because of the 

27 lack of quantitative information to assess toxicodynamic differences between animals and 

28 humans, whereas the toxicokinetic component is addressed by the determination of a HEC as 

29 described in the RfC methodology (U.S. EPA, 1994b). The default human variability factor was 

30 applied because of the lack of information regarding the range of susceptibilities in human 

31 populations. A LOAEL-to-NOAEL extrapolation factor was applied because a NOAEL was not 

32 identified for decreased fetal survival observed by Archibong et al (2002). In addition, a 

33 database uncertainty factor of 3 was applied to account for deficiencies in the benzo[a]pyrene 

34 toxicity database, primarily the lack of a standard multigenerational reproductive study. 

35 The overall confidence in the RfC is low-to-medium. Confidence in the principal study 

36 (Archibong et al., 2002) is medium. The conduct, and reporting of this developmental dietary 

37 study were adequate, however, a NOAEL was not identified. Confidence in the database is low38 to-medium due to the lack of a multigeneration reproductive toxicity study, the lack of studies on 
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1 immune endpoints, and the lack of information regarding subchronic and chronic inhalation 

2 exposure. Reflecting medium confidence in the principal study and low-to-medium confidence 

3 in the database, confidence in the RfC is low-to-medium. 

4 

5 6.2.3. Cancer 

6 Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 

7 benzo[a]pyrene is “carcinogenic to humans” based on evidence of carcinogenicity in humans 

8 exposed to different PAH mixtures containing benzo[a]pyrene, extensive and consistent evidence 

9 of carcinogenicity in laboratory animals exposed to benzo[a]pyrene via several routes of 

10 administration, and strong evidence that mechanisms of carcinogenesis in laboratory animals 

11 also occur in humans exposed to PAH mixtures containing benzo[a]pyrene. Strong evidence 

12 links the metabolism of benzo[a]pyrene to DNA-reactive agents with key mutational events in 

13 genes that can lead to tumor initiation. Specifically, the metabolic activation of benzo[a]pyrene 

14 occurs in human tissues, and associations have been made between spectra of mutations in the 

15 p53 tumor suppressor gene or ras oncogenes induced by benzo[a]pyrene metabolites and the 

16 spectra of mutations in these genes in tumor tissue from benzo[a]pyrene-exposed animals and 

17 humans. 

18 Several lines of evidence relating to mutagenicity and tumor initiation are available for 

19 benzo[a]pyrene including: in vitro evidence of DNA adducts, mutations, cytogenetic damage, 

20 and primary DNA damage; in vivo DNA adducts, gene mutations, cytogenetic damage, and other 

21 measures of primary DNA damage; detection of DNA adducts in target tissue in vivo; and 

22 detection of cancer-relevant oncogene/tumor suppressor gene mutations in target tissue in vivo. 

23 Taken together, these data provide support for a mutagenic MOA for benzo[a]pyrene-induced 

24 cancer. Because benzo[a]pyrene is expected to cause cancer via a mutagenic MOA, a linear 

25 approach to low-dose extrapolation was used in the derivation of the cancer risk estimates. 

26 In the absence of appropriate benzo[a]pyrene-specific data to adjust cancer risk values for 

27 early life exposure, ADAFs combined with age-specific exposure estimates should be applied to 

28 the cancer risk values (oral slope factor, inhalation unit risk, and dermal slope factor) when 

29 assessing cancer risks for individuals exposed during early life periods, as per U.S. EPA (2005b) 

30 Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens. 

31 

32 6.2.3.1. Cancer—Oral 

33 Lifetime oral exposure to benzo[a]pyrene has been associated with forestomach, liver, 

34 oral cavity, jejunum or duodenum, and auditory canal tumors in male and female Wistar rats 

35 (Kroese et al., 2001), with forestomach tumors in male and female Sprague-Dawley rats (Brune 

36 et al., 1981), and with forestomach, esophagus, tongue, and larynx tumors in female B6C3F1 

37 mice (male mice were not tested); (Beland and Culp, 1998; Culp et al., 1998). Less-than-lifetime 

38 oral exposure to benzo[a]pyrene is also associated with forestomach tumors in more than 10 
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1 additional bioassays with several strains of mice (see Section 4.2.1.2.). Both the rat bioassay by 

2 Kroese et al. (2001) and the mouse bioassay by Beland and Culp (1998) included histological 

3 examinations for tumors, three exposure levels and controls, and 50 animals per dose group. The 

4 chronic studies by Kroese et al. and Beland and Culp studies were therefore selected for dose5 response analysis. 

6 EPA used the multistage-Weibull model in the derivation of the oral slope factor because 

7 it incorporates the time at which death-with-tumor occurred and can account for differences in 

8 mortality observed between the exposure groups in the rat bioassay. Using linear extrapolation 

9 from the BMDL10, human equivalent oral slope factors were derived for each gender/tumor site 

10 combination (slope factor = 0.1/BMDL10). The oral slope factor of 1 per mg/kg-day is based on 

11 the tumor response in the alimentary tract (forestomach, esophagus, tongue, larynx) of female 

12 B6C3F1 mice exposed to benzo[a]pyrene in the diet for 2 years (Beland and Culp, 1998). The 

13 slope factor was derived by linear extrapolation from a human equivalent BMDL10 of 0.07 

14 mg/kg-day for forestomach, esophagus, tongue, and larynx papillomas or carcinomas. The 

15 recommended slope factor was selected as the factor with the highest value among a range of 

16 slope factors derived from tumor responses at several sites in the 2-year male and female Wistar 

17 rat bioassay by Kroese et al. (2001) and the 2-year female B6C3F1 mouse bioassay by Beland 

18 and Culp (1998). 

19 

20 6.2.3.2. Cancer—Inhalation 

21 Inhalation exposure to benzo[a]pyrene was associated with squamous cell neoplasia in 

22 the larynx, pharynx, trachea, esophagus, and forestomach, of male Syrian golden hamsters 

23 exposed to benzo[a]pyrene condensed onto NaCl particles (Thyssen et al., 1981). Supportive 

24 evidence for the carcinogenicity of inhaled benzo[a]pyrene comes from 10 additional studies 

25 with hamsters exposed to benzo[a]pyrene via intratracheal instillation (see Section 4.2.2.2 for 

26 references). The Thyssen et al. (1981) bioassay represents the best available data for describing 

27 dose-response relationships for cancer from inhaled benzo[a]pyrene. 

28 A time-to-tumor dose-response model was fit to the time-weighted average exposure 

29 concentrations and the individual animal occurrence data for tumors in the larynx, pharynx, 

trachea, esophagus, and forestomach. The inhalation unit risk of 5 x 10-4 per μg/m330 was 

calculated by linear extrapolation (slope factor = 0.1/BMDL10) from a BMDL10 of 0.20 mg/m3 31 

32 for the occurrence of upper respiratory and upper digestive tract tumors in male hamsters 

33 chronically exposed by inhalation to benzo[a]pyrene (Thyssen et al., 1981). 

34 

35 6.2.3.3. Cancer—Dermal 

36 Skin cancer in humans has been documented to result from occupational exposure to 

37 complex mixtures of PAHs including benzo[a]pyrene such as coal tar, coal tar pitches, non38 refined mineral oils, shale oils and soot (Boffetta et al., 1997; WHO, 1998; ATSDR, 1995). No 
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1 human studies of exposure to benzo[a]pyrene alone are known to exist. In animal models, 

2 numerous dose response studies have demonstrated the increased incidence of skin tumors with 

3 increasing dermal exposure of benzo[a]pyrene, in all species tested (mice, rabbits, rats, and 

4 guinea pigs), though most benzo[a]pyrene chronic dermal bioassays have been conducted in 

5 mice. This analysis focuses on chronic carcinogenicity bioassays in several strains of mice 

6 demonstrating increasing incidence of benign and malignant skin tumors following repeated 

7 dermal exposure to benzo[a]pyrene for the animals’ lifetime. 

8 As with the oral and inhalation benzo[a]pyrene carcinogenicity data (see Sections 5.4.1.3 

9 and 5.4.2.3), benzo[a]pyrene’s dermal exposure carcinogenicity data were generally 

10 characterized by earlier occurrence of tumors with increasing exposure and increased mortality 

11 with increasing exposure level. Each of the dermal data sets was modeled using the multistage 

12 model, incorporating adjustments for early mortality, when data were available, prior to 

13 modeling. 

14 The POD of 0.0066 μg/day, based on the tumor response in C3H/HeJ male mice (Sivak 

15 et al., 1997), is recommended for developing a human dermal slope factor because it is the 

16 highest POD among studies with low observed tumor response to benzo[a]pyrene (20%). 

17 Following the modeling, this POD from the Sivak et al. (1997) dataset in male C3H/HeJ mice 

18 was adjusted by allometric scaling. The dermal slope factor of 0.005 per μg/day was calculated 

19 by linear extrapolation (slope factor = 0.1/BMDL10-HED) from the human equivalent POD (19.7 

20 μg/day) for the occurrence of skin tumors in male mice chronically exposed dermally to 

21 benzo[a]pyrene (Sivak et al., 1997). 

22 This dermal slope factor has been calculated based on the risk of skin tumors in mice 

23 following dermal exposure to benzo[a]pyrene. As this slope factor has been developed for a 

24 local effect, it is not intended to estimate systemic risk of cancer following dermal absorption of 

25 benzo[a]pyrene into the systemic circulation. 

26 
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1 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

2 COMMENTS AND DISPOSITION 

3 

4 

5 [THIS PAGE INTENTIONALLY LEFT BLANK] 
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1 APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR NONCANCER 

2 

3 Increased liver weight (Kroese et al, 2001) male 

4 

5 

Table B-1. Liver weight (±SD)a6 in male F344 rats administered 

7 benzo[a]pyrene by gavage for 90 days 

8 

Organ 

Dose (mg/kg-d) 

0 3 10 30 

Liver weight (g) 

Males 7.49 ± 0.97 8.00 ± 0.85 8.62 ± 1.30b

9.67 ± 1.17b

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-test (unpaired, two-tailed); n = 10/sex/group. 

9 

10 

11 

12 

13 Table B-2. BMD modeling results for increased liver weight in male rats, 

14 with BMR=10% 

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°)a50.53 0.74 4.53 2.29 

Hill 50.48 0.82 4.1 1.24 

16 

a

17 In order to consider apparent curvature in the dose-response data, the polynomial coefficients were allowed to be 

18 negative; a satisfactory fit was achieved, with monotonically increasing predictions within the observed data range. 

19 Since the AIC was higher than for the linear model, the 2-degree polynomial was not selected as the best fit. 

20 
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1 

2 

3 

4 Figure B-1. Fit of polynomial model to data on increased liver weight in 

5 male Wistar rats—90 days. 

6 

7 

8 Model output: 

9 

10 ==================================================================== 

11 Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

12 Input Data File: C:\USEPA\BMDS21\Data\linLiverwtKroeseLinearDefault.(d) 

13 Gnuplot Plotting File: C:\USEPA\BMDS21\Data\linLiverwtKroeseLinearDefault.plt 

14 Tue Jan 12 13:09:58 2010 

15 ==================================================================== 

16 

17 BMDS Model Run 

18 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

19 

20 The form of the response function is: 

21 

22 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 

23 

24 

25 Dependent variable = Liver_wt 

26 Independent variable = Dose 

27 rho is set to 0 

28 Signs of the polynomial coefficients are not restricted 

29 A constant variance model is fit 

30 

31 Total number of dose groups = 4 

32 Total number of records with missing values = 0 

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 alpha = 1.18058 

 6.5

 7

 7.5

 8

 8.5

 9

 9.5

 10

 10.5

 0 5 10 15 20

Mean Response

dose

Linear Model with 0.95 Confidence Level

13:09 01/12 2010

BMDL BMD

 

Linear
290 DRAFT - DO NOT CITE OR QUOTE 

1 rho = 0 Specified 

2 beta_0 = 7.71695 

3 beta_1 = 0.0951703 

4 

5 

6 Asymptotic Correlation Matrix of Parameter Estimates 

7 

8 ( *** The model parameter(s) -rho 

9 have been estimated at a boundary point, or have been specified by the user, 

10 and do not appear in the correlation matrix ) 

11 

12 alpha beta_0 beta_1 

13 

14 alpha 1 -3.6e-011 1e-010 

15 

16 beta_0 -3.6e-011 1 -0.68 

17 

18 beta_1 1e-010 -0.68 1 

19 

20 

21 

22 Parameter Estimates 

23 

24 95.0% Wald Confidence Interval 

25 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

26 alpha 1.09179 0.244132 0.613302 1.57028 

27 beta_0 7.71695 0.224102 7.27772 8.15618 

28 beta_1 0.0951703 0.0197929 0.0563769 0.133964 

29 

30 

31 

32 Table of Data and Estimated Values of Interest

33 

34 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

35 ------ --- -------- -------- ----------- ----------- ---------- 

36 

37 0 10 7.49 7.72 0.97 1.04 -0.687 

38 2.1 10 8 7.92 0.85 1.04 0.252 

39 7.1 10 8.62 8.39 1.3 1.04 0.688 

40 21.4 10 9.67 9.75 1.17 1.04 -0.253 

41 

42 

43 

44 Model Descriptions for likelihoods calculated 

45 

46 

47 Model A1: Yij = Mu(i) + e(ij) 

48 Var{e(ij)} = Sigma^2 

49 

50 Model A2: Yij = Mu(i) + e(ij) 

51 Var{e(ij)} = Sigma(i)^2 

52 

53 Model A3: Yij = Mu(i) + e(ij) 

54 Var{e(ij)} = Sigma^2 

55 Model A3 uses any fixed variance parameters that 

56 were specified by the user 

57 

58 Model R: Yi = Mu + e(i) 

59 Var{e(i)} = Sigma^2 

60 

61 

62 Likelihoods of Interest 

63 

64 Model Log(likelihood) # Param's AIC 

65 A1 -21.212822 5 52.425644 

66 A2 -20.156688 8 56.313377 

67 A3 -21.212822 5 52.425644 

68 fitted -21.756413 3 49.512826 

69 R -30.879511 2 65.759022 

70 

71 

72 Explanation of Tests 

73 

74 Test 1: Do responses and/or variances differ among Dose levels? 

75 (A2 vs. R) 

76 Test 2: Are Variances Homogeneous? (A1 vs A2) 

77 Test 3: Are variances adequately modeled? (A2 vs. A3) 

78 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

79 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

80 

81 Tests of Interest 

82 

83 Test -2*log(Likelihood Ratio) Test df p-value 

84 
291 DRAFT - DO NOT CITE OR QUOTE 

1 Test 1 21.4456 6 0.001525 

2 Test 2 2.11227 3 0.5494 

3 Test 3 2.11227 3 0.5494 

4 Test 4 1.08718 2 0.5807 

5 

6 The p-value for Test 1 is less than .05. There appears to be a 

7 difference between response and/or variances among the dose levels 

8 It seems appropriate to model the data 

9 

10 The p-value for Test 2 is greater than .1. A homogeneous variance 

11 model appears to be appropriate here 

12 

13 

14 The p-value for Test 3 is greater than .1. The modeled variance appears 

15 to be appropriate here 

16 

17 The p-value for Test 4 is greater than .1. The model chosen seems 

18 to adequately describe the data 

19 

20 

21 Benchmark Dose Computation 

22 

23 Specified effect = 0.1 

24 

25 Risk Type = Relative risk 

26 

27 Confidence level = 0.95 

28 

29 BMD = 8.10857 

30 

31 

32 BMDL = 5.80436 

33 

34 

35 

36 

37 
292 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Decreased thymus weight (males) Kroese et al., 2001

3 

4 

Table B-3. Means ± SDa 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 ± 40b

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-test (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-valuea

Goodness-of-fit 

p-value AIC 

BMD1SD

(mg/kg-d) 

BMDL1SD

(mg/kg-d) 

Constant variance 

Linear 0.01 0.74 384.84 12.97 8.97 

Nonconstant variance 

Hillc NA 

Linear, Polynomial (2-degree), 

Powerc

0.30 0.23 380.71 16.40 11.30 

NA = not applicable, model failed; 

5 
293 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 BMDs and BMDLs indicated are associated with a change of 1 SD from the control, and are in 

4 units of mg/kg-day. 

5 

6 Source: Kroese et al. (2001). 

7 

8 Figure B-2. Fit of linear model (nonconstant variance) to data on decreased 

9 thymus weight in male Wistar rats—90 days. 

10 

11 Linear (nonconstant variance)

12 

13 

14 

15 ==================================================================== 

16 Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

17 Input Data File: 

18 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\male\durationadjusted\2

19 Linkrolin.(d) 

20 Gnuplot Plotting File: 

21 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\male\durationadjusted\2

22 Linkrolin.plt 

23 Thu Oct 15 15:33:37 2009 

24 ==================================================================== 

25 

26 BMDS Model Run 

27 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

28 

29 The form of the response function is: 

30 

31 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 

 250

 300

 350

 400

 450

 0 5 10 15 20

Mean Response

dose

Linear Model with 0.95 Confidence Level

15:33 10/15 2009

BMDL BMD

 

Linear
294 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 Dependent variable = mean 

4 Independent variable = dose 

5 The polynomial coefficients are restricted to be negative 

6 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho) 

7 

8 Total number of dose groups = 4 

9 Total number of records with missing values = 0 

10 Maximum number of iterations = 250 

11 Relative Function Convergence has been set to: 1e-008 

12 Parameter Convergence has been set to: 1e-008 

13 

14 

15 

16 Default Initial Parameter Values 

17 lalpha = 8.56121 

18 rho = 0 

19 beta_0 = 380.763 

20 beta_1 = -5.3285 

21 

22 

23 Asymptotic Correlation Matrix of Parameter Estimates 

24 

25 lalpha rho beta_0 beta_1 

26 

27 lalpha 1 -1 0.048 -0.061 

28 

29 rho -1 1 -0.048 0.061 

30 

31 beta_0 0.048 -0.048 1 -0.84 

32 

33 beta_1 -0.061 0.061 -0.84 1 

34 

35 

36 

37 Parameter Estimates 

38 

39 95.0% Wald Confidence 

40 Interval 

41 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

42 Limit 

43 lalpha -18.8293 9.75429 -37.9473 

44 0.288754 

45 rho 4.66515 1.67581 1.38062 

46 7.94967 

47 beta_0 378.954 16.5291 346.558 

48 411.351 

49 beta_1 -5.14219 1.00497 -7.11189 -

50 3.17249 

51 

52 

53 

54 Table of Data and Estimated Values of Interest

55 

56 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

57 ------ --- -------- -------- ----------- ----------- ---------- 

58 

59 0 10 380 379 60 84.3 0.0392 

60 2.1 10 380 368 110 78.8 0.475 

61 7.1 10 330 342 60 66.6 -0.591 

62 21.4 10 270 269 40 37.9 0.0908 

63 

64 

65 

66 Model Descriptions for likelihoods calculated 
295 DRAFT - DO NOT CITE OR QUOTE 

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)} = exp(lalpha + rho*ln(Mu(i)))

11 Model A3 uses any fixed variance parameters that 

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 -189.116991 5 388.233982 

22 A2 -183.673279 8 383.346558 

23 A3 -184.883626 6 381.767253 

24 fitted -186.353541 4 380.707081 

25 R -196.353362 2 396.706723 

26 

27 

28 Explanation of Tests 

29 

30 Test 1: Do responses and/or variances differ among 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 25.3602 6 0.0002928 

42 Test 2 10.8874 3 0.01235 

43 Test 3 2.42069 2 0.2981 

44 Test 4 2.93983 2 0.2299 

45 

46 The p-value for Test 1 is less than .05. There appears 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 less than .1. A non-homogeneous variance 

51 model appears to be appropriate 

52 

53 The p-value for Test 3 is greater than .1. The modeled variance appears 

54 to be appropriate here 

55 

56 The p-value for Test 4 is greater than .1. The model chosen seems 

57 to adequately describe the data 

58 

59 

60 Benchmark Dose Computation 

61 

62 Specified effect = 1 

63 

64 Risk Type = Estimated standard deviations from the control mean 

65 

66 Confidence level = 0.95 
296 DRAFT - DO NOT CITE OR QUOTE 

1 

2 BMD = 16.4008 

3 

4 

5 BMDL = 11.2965 

6 

7 

8 

9 

10 

11 

12 
297 DRAFT - DO NOT CITE OR QUOTE 

1 Decreased thymus weight females (Kroese et al., 2001) 

2 

3 

Table B-5. Means ± SDa 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 ± 30b

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-test (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-valuea

Means 

p-valuea AIC 

BMD1SD

(mg/kg-d) 

BMDL1SD

(mg/kg-d) 

Hillb NA 

Linearc 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 

Powerb NA 

aValues <0.10 fail to meet conventional goodness-of-fit criteria. 

b

Power restricted to ≥1. 

cCoefficients restricted to be negative. 

dLowest degree polynomial with an adequate fit is reported. 

BMD/BMC = maximum likelihood estimate of the dose/concentration 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 

2 BMDs and BMDLs indicated are associated with a change 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-3. Fit of linear model (constant variance) to data on decreased 

8 thymus weight in female Wistar rats—90 days. 

9 

10 

11 

12 ==================================================================== 

13 Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

14 Input Data File: 

15 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\female\durationadjusted

16 \2Linkrolin.(d) 

17 Gnuplot Plotting File: 

18 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\female\durationadjusted

19 \2Linkrolin.plt 

20 Thu Oct 15 16:27:44 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 

31 Dependent variable = mean 

 200

 220

 240

 260

 280

 300

 320

 340

 360

 0 5 10 15 20

Mean Response

dose

Linear Model with 0.95 Confidence Level

16:27 10/15 2009

BMDL BMD

 

Linear
299 DRAFT - DO NOT CITE OR QUOTE 

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 = 4 

7 Total number of records with missing values = 0 

8 Maximum number of iterations = 250 

9 Relative Function Convergence has been set to: 1e-008 

10 Parameter Convergence has been set to: 1e-008 

11 

12 

13 

14 Default Initial Parameter Values 

15 alpha = 1 

16 rho = 0 Specified 

17 beta_0 = 322.144 

18 beta_1 = -4.2018 

19 

20 

21 Asymptotic Correlation Matrix of Parameter 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 correlation matrix ) 

27 

28 alpha beta_0 beta_1 

29 

30 alpha 1 2.4e-008 -2.3e-008 

31 

32 beta_0 2.4e-008 1 -0.68 

33 

34 beta_1 -2.3e-008 -0.68 1 

35 

36 

37 

38 Parameter Estimates 

39 

40 95.0% Wald Confidence 

41 Interval 

42 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

43 Limit 

44 alpha 1954.92 437.134 1098.16 

45 2811.69 

46 beta_0 322.144 9.48287 303.558 

47 340.73 

48 beta_1 -4.2018 0.837537 -5.84334 -

49 2.56026 

50 

51 

52 

53 Table of Data and Estimated Values of Interest

54 

55 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

56 ------ --- -------- -------- ----------- ----------- ---------- 

57 

58 0 10 320 322 60 44.2 -0.153 

59 2.1 10 310 313 50 44.2 -0.237 

60 7.1 10 300 292 40 44.2 0.55 

61 21.4 10 230 232 30 44.2 -0.159 

62 

63 

64 

65 Model Descriptions for likelihoods calculated 

66 
300 DRAFT - DO NOT CITE OR QUOTE 

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)} = Sigma^2 

10 Model A3 uses any fixed variance parameters that 

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 -171.357252 5 352.714504 

21 A2 -168.857234 8 353.714467 

22 A3 -171.357252 5 352.714504 

23 fitted -171.562118 3 349.124237 

24 R -181.324151 2 366.648303 

25 

26 

27 Explanation of Tests 

28 

29 Test 1: Do responses and/or variances differ among 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 24.9338 6 0.0003512 

41 Test 2 5.00004 3 0.1718 

42 Test 3 5.00004 3 0.1718 

43 Test 4 0.409733 2 0.8148 

44 

45 The p-value for Test 1 is less than .05. There appears 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 greater than .1. A homogeneous variance 

50 model appears to be appropriate here 

51 

52 

53 The p-value for Test 3 is greater than .1. The modeled variance appears 

54 to be appropriate here 

55 

56 The p-value for Test 4 is greater than .1. The model chosen seems 

57 to adequately describe the data 

58 

59 

60 Benchmark Dose Computation 

61 

62 Specified effect = 1 

63 

64 Risk Type = Estimated standard deviations from the control mean 

65 

66 Confidence level = 0.95 
301 DRAFT - DO NOT CITE OR QUOTE 

1 

2 BMD = 10.5228 

3 

4 

5 BMDL = 7.64037 

6 

7 

8 
302 DRAFT - DO NOT CITE OR QUOTE 

1 Decreased thymus weight males (DeJong et al., 1999)

2 

3 

Table B-7. Means ± SD for thymus weight in male Wistar 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 ± 64a

388 ± 71a198 ± 65a

5 

6 

Table B-8. Model predictions for decreased thymus weight in male Wistar 

rats—35 days 

Model (constant variance) 

Variance 

p-valueb

Means 

p-valueb AIC 

BMD1SD

(mg/kg-d) 

BMDL1SD

(mg/kg-d) 

Hillc 0.50 0.42 382.91 11.15 6.19 

Lineard, Polynomial (2-degree)d,e 0.50 0.52 381.41 14.41 11.58 

Powerd NA 

aNumber of animals was reported to be 7–8 per dose group, and was not specified for each group; forBMD 

modeling purposes, n = 8 was used. 

bValues <0.10 fail to meet conventional goodness-of-fit criteria. 

c

Power restricted to ≥1. 

dCoefficients restricted to be negative. 

eLowest degree polynomial with an adequate fit is reported. 

BMD/BMC = maximum likelihood estimate of the dose/concentration 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 

3 BMDs and BMDLs indicated are associated with a change of 1 SD from the control, and are in 

4 units of mg/kg-day. 

5 

6 Source: De Jong et al. (1999). 

7 

8 Figure B-4. Fit of linear model (constant variance) to data on decreased 

9 thymus weight in male Wistar rats—35 days. 

10 

11 Model Output: 

12 

13 ==================================================================== 

14 Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

15 Input Data File: 

16 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\thymusweightmale\durationadjusted\2L

17 indejlin.(d) 

18 Gnuplot Plotting File: 

19 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\thymusweightmale\durationadjusted\2L

20 indejlin.plt 

21 Mon Oct 19 04:31:24 2009 

22 ==================================================================== 

23 

24 BMDS Model Run 

25 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

26 

27 The form of the response function is: 

28 

29 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 

30 

 150

 200

 250

 300

 350

 400

 450

 500

 550

 0 10 20 30 40 50 60

Mean Response

dose

Linear Model with 0.95 Confidence Level

04:31 10/19 2009

BMDL BMD

 

Linear
304 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Dependent variable = mean 

3 Independent variable = dose 

4 rho is set to 0 

5 The polynomial coefficients are restricted to be negative 

6 A constant variance model is fit 

7 

8 Total number of dose groups = 5 

9 Total number of records with missing values = 0 

10 Maximum number of iterations = 250 

11 Relative Function Convergence has been set to: 1e-008 

12 Parameter Convergence has been set to: 1e-008 

13 

14 

15 

16 Default Initial Parameter Values 

17 alpha = 1 

18 rho = 0 Specified 

19 beta_0 = 489.769 

20 beta_1 = -4.5927 

21 

22 

23 Asymptotic Correlation Matrix of Parameter Estimates 

24 

25 ( *** The model parameter(s) -rho 

26 have been estimated at a boundary point, or have been specified by 

27 the user, 

28 and do not appear in the correlation matrix ) 

29 

30 alpha beta_0 beta_1 

31 

32 alpha 1 -3.1e-009 -3.2e-009 

33 

34 beta_0 -3.1e-009 1 -0.62 

35 

36 beta_1 -3.2e-009 -0.62 1 

37 

38 

39 

40 Parameter Estimates 

41 

42 95.0% Wald Confidence 

43 Interval 

44 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

45 Limit 

46 alpha 4382.3 979.911 2461.71 

47 6302.89 

48 beta_0 489.769 13.3751 463.555 

49 515.984 

50 beta_1 -4.5927 0.438716 -5.45257 -

51 3.73283 

52 

53 

54 

55 Table of Data and Estimated Values of Interest

56 

57 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

58 ------ --- -------- -------- ----------- ----------- ---------- 

59 

60 0 8 517 490 47 66.2 1.16 

61 2.1 8 472 480 90 66.2 -0.347 

62 7.1 8 438 457 64 66.2 -0.819 

63 21.4 8 388 391 71 66.2 -0.149 

64 64.3 8 198 194 65 66.2 0.151 

65 

66 
305 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Model Descriptions for likelihoods calculated 

3 

4 

5 Model A1: Yij = Mu(i) + e(ij) 

6 Var{e(ij)} = Sigma^2 

7 

8 Model A2: Yij = Mu(i) + e(ij) 

9 Var{e(ij)} = Sigma(i)^2 

10 

11 Model A3: Yij = Mu(i) + e(ij) 

12 Var{e(ij)} = Sigma^2 

13 Model A3 uses any fixed variance parameters that 

14 were specified by the user 

15 

16 Model R: Yi = Mu + e(i) 

17 Var{e(i)} = Sigma^2 

18 

19 

20 Likelihoods of Interest 

21 

22 Model Log(likelihood) # Param's AIC 

23 A1 -186.580733 6 385.161466 

24 A2 -184.896632 10 389.793264 

25 A3 -186.580733 6 385.161466 

26 fitted -187.706565 3 381.413130 

27 R -214.086904 2 432.173809 

28 

29 

30 Explanation of Tests 

31 

32 Test 1: Do responses and/or variances differ among Dose levels? 

33 (A2 vs. R) 

34 Test 2: Are Variances Homogeneous? (A1 vs A2) 

35 Test 3: Are variances adequately modeled? (A2 vs. A3) 

36 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

37 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

38 

39 Tests of Interest 

40 

41 Test -2*log(Likelihood Ratio) Test df p-value 

42 

43 Test 1 58.3805 8 <.0001 

44 Test 2 3.3682 4 0.4982 

45 Test 3 3.3682 4 0.4982 

46 Test 4 2.25166 3 0.5218 

47 

48 The p-value for Test 1 is less than .05. There appears to be a 

49 difference between response and/or variances among the dose levels 

50 It seems appropriate to model the data 

51 

52 The p-value for Test 2 is greater than .1. A homogeneous variance 

53 model appears to be appropriate here 

54 

55 

56 The p-value for Test 3 is greater than .1. The modeled variance appears 

57 to be appropriate here 

58 

59 The p-value for Test 4 is greater than .1. The model chosen seems 

60 to adequately describe the data 

61 

62 

63 Benchmark Dose Computation 

64 

65 Specified effect = 1 

66 
306 DRAFT - DO NOT CITE OR QUOTE 

1 Risk Type = Estimated standard deviations from the control mean 

2 

3 Confidence level = 0.95 

4 

5 BMD = 14.4139 

6 

7 

8 BMDL = 11.577 

9 
307 DRAFT - DO NOT CITE OR QUOTE 

1 Decreased Splenic B cells 

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 ± 3a

40 ± 9 

24 ± 5 

24 ± 7 

32 ± 4a

36 ± 2 

22 ± 4 

19 ± 2 

23 ± 4a

44 ± 6 

26 ± 4 

27 ± 5 

a

4 Significantly (p < 0.05) different from control mean. 

5 

6 Source: De Jong et al. (1999). 

7 

8 

9 

Table B-10. Model predictions for decreased spleen B-cells in male Wistar 

rats—35 days 

Model 

Variance 

p-valuea

Means 

p-valuea AIC 

BMD1SD

(mg/kg-d) 

BMDL1SD

(mg/kg-d) 

Constant variance

Hillb 0.30 0.18 146.18 10.24 5.31 

Linearc, Polynomial (2-degree)c,d, 

Powerb

0.30 0.21 145.28 15.58 12.43 

aValues <0.10 fail to meet conventional goodness-of-fit criteria. 

b

Power restricted to ≥1. 

cCoefficients restricted to be negative. 

dLowest degree polynomial with an adequate fit is reported. 

BMD/BMC = maximum likelihood estimate of the dose/concentration 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 

2 BMDs and BMDLs indicated are associated with a change 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-5. Fit of linear model to data on decreased spleen B-cells in male 

8 Wistar rats—35 days. 

9 

10 Model Output: 

11 

12 ==================================================================== 

13 Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

14 Input Data File: 

15 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\spleenBcell\durationadjusted\2Lindej

16 lin.(d) 

17 Gnuplot Plotting File: 

18 C:\USEPA\IRIS\benzo[a]pyrene\RfD\dejong1999\35day\spleenBcell\durationadjusted\2Lindej

19 lin.plt 

20 Mon Oct 19 05:06:33 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 

 20

 25

 30

 35

 40

 0 10 20 30 40 50 60

Mean Response

dose

Linear Model with 0.95 Confidence Level

05:06 10/19 2009

BMDL BMD

 

Linear
309 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: 1e-008 

11 Parameter Convergence has been set to: 1e-008 

12 

13 

14 

15 Default Initial Parameter Values 

16 alpha = 12.2 

17 rho = 0 Specified 

18 beta_0 = 37.0148 

19 beta_1 = -0.222068 

20 

21 

22 Asymptotic Correlation Matrix of Parameter 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 correlation matrix ) 

28 

29 alpha beta_0 beta_1 

30 

31 alpha 1 -2.4e-009 3.8e-009 

32 

33 beta_0 -2.4e-009 1 -0.62 

34 

35 beta_1 3.8e-009 -0.62 1 

36 

37 

38 

39 Parameter Estimates 

40 

41 95.0% Wald Confidence 

42 Interval 

43 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

44 Limit 

45 alpha 11.9647 2.6754 6.72106 

46 17.2084 

47 beta_0 37.0148 0.698873 35.6451 

48 38.3846 

49 beta_1 -0.222068 0.0229237 -0.266997 -

50 0.177138 

51 

52 

53 

54 Table of Data and Estimated Values of Interest

55 

56 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

57 ------ --- -------- -------- ----------- ----------- ---------- 

58 

59 0 8 39 37 4 3.46 1.62 

60 2.1 8 36 36.5 2 3.46 -0.449 

61 7.1 8 34 35.4 3 3.46 -1.18 

62 21.4 8 32 32.3 4 3.46 -0.215 

63 64.3 8 23 22.7 4 3.46 0.216 

64 

65 

66 
310 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 that 

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 -67.358091 6 146.716182 

23 A2 -64.934513 10 149.869025 

24 A3 -67.358091 6 146.716182 

25 fitted -69.639287 3 145.278575 

26 R -93.795081 2 191.590163 

27 

28 

29 Explanation of Tests 

30 

31 Test 1: Do responses and/or variances differ among 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 57.7211 8 <.0001 

43 Test 2 4.84716 4 0.3033 

44 Test 3 4.84716 4 0.3033 

45 Test 4 4.56239 3 0.2068 

46 

47 The p-value for Test 1 is less than .05. There appears 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 homogeneous variance 

52 model appears to be appropriate here 

53 

54 

55 The p-value for Test 3 is greater than .1. The modeled variance appears 

56 to be appropriate here 

57 

58 The p-value for Test 4 is greater than .1. The model chosen seems 

59 to adequately describe the data 

60 

61 

62 Benchmark Dose Computation 

63 

64 Specified effect = 1 

65 

66 Risk Type = Estimated standard deviations from the control mean 
311 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Confidence level = 0.95 

3 

4 BMD = 15.5764 

5 

6 

7 BMDL = 12.4286 

8 

9 
312 DRAFT - DO NOT CITE OR QUOTE 

1 Forestomach hyperplasia 

2 

3 All available dichotomous models in the EPA BMDS (version 2.1) were fit to the 

4 incidence data shown in Table B-9 for forestomach hyperplasia in rats and mice orally exposed 

5 to benzo[a]pyrene for 2 years (Kroese et al., 2001; Beland and Culp, 1998). In accordance with 

6 U.S. EPA (2000) guidance, BMDs and BMDLs associated with an extra risk of 10% are 

7 calculated for all models. 

8 Adequate model fit was judged by three criteria: goodness-of-fit p-value (p > 0.1), visual 

9 inspection of the dose-response curve, and scaled residual at the data point (except the control) 

10 closest to the predefined benchmark response (BMR). Among all of the models providing 

11 adequate fit to the data, the lowest BMDL is selected as the POD when the difference between 

12 the BMDLs estimated from these models are more than threefold; otherwise, the BMDL from 

13 the model with the lowest Akaike’s Information Criterion (AIC) is chosen. If an adequate fit to 

14 the data was not achieved using the protocol above for the full dataset, doses were dropped 

15 (starting with the highest dose) until an adequate fit was achieved. 

16 

Table B-11. Dose-response data for forestomach hyperplasia in Wistar rats 

and B6C3F1 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 

B6C3F1 mice, female (Beland and Culp, 1998) 13/48 23/47 33/46 46/47 

17 

18 

19 All data sets provided adequate descriptions of the dose-response relationship for 

20 forestomach hyperplasia from chronic oral exposure to benzo[a]pyrene, but at the highest dose 

21 level in rats, the incidence of hyperplasia was not increased. It is possible that the forestomach 

22 hyperplasia observed following benzo[a]pyrene exposure may be a precursor to the development 

23 of forestomach tumors, but specific data supporting this conclusion are unavailable. Regardless, 

24 the male and female data sets in rats (Kroese et al., 2001) were modeled without the data from 

25 the highest dose group due to the nonmontonic increase in response to increasing dose (Kroese et 

26 al., 2001). 

27 

28 

29 
313 DRAFT - DO NOT CITE OR QUOTE 

Table B-12. Summary of BMDs and BMDLs from the best 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 Kroese 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 

B6C3F1 mouse 

(female) 

2 yrs Log-logistic 0.33 0.12 Beland and Culp, 

1998 

a

1 Data for the high-dose group were excluded in the modeled datset due to the absence of an increase in incidence at 

2 the high dose. 

3 

4 

5 

6 

7 Forestomach hyperplasia- Male Wistar Rats, 2 yrs (Kroese et al., 2001) 

8 

9 

Table B-13. Model predictions for forestomach hyperplasia in male Wistar 

rats in a 2-year study 

Model 

Degrees of 

freedom χ

2

χ

2

 Goodnessof-fit p-valuea AIC BMD10 

(mg/kg-d) 

BMDL10

(mg/kg-d) 

Highest dose excludedb

Gammac, Multistaged, 

Weibullc

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 

aValues <0.10 fail to meet conventional goodness-of-fit criteria. 

b Data for the high-dose group were excluded in the modeled datset due to the absence of an increase in incidence at 

the high dose; likely related to the statistically significantly increased incidence of forestomach tumors in these 

animals. 

c

Power restricted to ≥1. 

dBetas 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 

4 Figure B-6. Fit of log logistic model to data on forestomach hyperplasia in 

5 male Wistar rats in a 2-year study. 

6 

7 

8 

9 Model output: 

10 

11 ==================================================================== 

12 Logistic Model. (Version: 2.12; Date: 05/16/2008) 

13 Input Data File: 

14 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic\forestomachhyperplasia\male\durati

15 onadjusted\3Logkrolog.(d) 

16 Gnuplot Plotting File: 

17 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic\forestomachhyperplasia\male\durati

18 onadjusted\3Logkrolog.plt 

19 Tue Oct 13 21:42:15 2009 

20 ==================================================================== 

21 

22 BMDS Model Run 

23 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

24 

25 The form of the probability function is: 

26 

27 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 

28 

29 

30 Dependent variable = incidence 

31 Independent variable = dose 

32 Slope parameter is restricted as slope >= 1 

33 

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0 1 2 3 4 5 6 7

Fraction Affected

dose

Log-Logistic Model with 0.95 Confidence Level

08:57 10/14 2009

BMDL BMD

 

Log-Logistic
315 DRAFT - DO NOT CITE OR QUOTE 

1 Total number of observations = 3 

2 Total number of records with missing values = 0 

3 Maximum number of iterations = 250 

4 Relative Function Convergence has been set to: 1e-008 

5 Parameter Convergence has been set to: 1e-008 

6 

7 

8 

9 User has chosen the log transformed model 

10 

11 

12 Default Initial Parameter Values 

13 background = 0.04 

14 intercept = -3.27842 

15 slope = 1 

16 

17 

18 Asymptotic Correlation Matrix of Parameter Estimates 

19 

20 ( *** The model parameter(s) -slope 

21 have been estimated at a boundary point, or have been specified by 

22 the user, 

23 and do not appear in the correlation matrix ) 

24 

25 background intercept 

26 

27 background 1 -0.7 

28 

29 intercept -0.7 1 

30 

31 

32 

33 Parameter Estimates 

34 

35 95.0% Wald Confidence 

36 Interval 

37 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

38 Limit 

39 background 0.0623861 * * * 

40 intercept -3.86644 * * * 

41 slope 1 * * * 

42 

43 * - Indicates that this value is not calculated. 

44 

45 

46 

47 Analysis of Deviance Table 

48 

49 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

50 Full model -53.0468 3 

51 Fitted model -54.1335 2 2.17337 1 0.1404 

52 Reduced model -55.5429 1 4.99229 2 0.0824 

53 

54 AIC: 112.267 

55 

56 

57 Goodness of Fit 

58 Scaled 

59 Dose Est._Prob. Expected Observed Size Residual 

60 ------------------------------------------------------------------------ 

61 0.0000 0.0624 3.119 2.000 50 -0.654 

62 2.1000 0.1019 5.297 8.000 52 1.239 

63 7.1400 0.1843 9.584 8.000 52 -0.566 

64 

65 Chi^2 = 2.28 d.f. = 1 P-value = 0.1306

66 
316 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Benchmark Dose Computation 

3 

4 Specified effect = 0.1 

5 

6 Risk Type = Extra risk 

7 

8 Confidence level = 0.95 

9 

10 BMD = 5.308 

11 

12 BMDL = 2.38692 

13 

14 

15 

16 Forestomach hyperplasia- Female Wistar Rats, 2 yrs (Kroese et al., 2001) 

17 

Table B-14. Model predictions for forestomach hyperplasia in female 

Wistar rats in a 2-year study 

Model 

Degrees of 

freedom χ

2

χ

2

 Goodnessof-fit p-valuea AIC BMD10 

(mg/kg-d) 

BMDL10

(mg/kg-d) 

Highest dose excludedb

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 

Gammac, Multistaged, 

Weibullc

1 1.40 0.24 117.42 2.40 1.59 

Probit 1 3.47 0.06 119.74 3.99 3.06 

aValues <0.10 fail to meet conventional goodness-of-fit criteria. 

bData for the high-dose group were excluded in the modeled datset due to the absence of an increase in incidence at 

the high dose; likely related to the statistically significantly increased incidence of forestomach tumors in these 

animals. 

c

Power restricted to ≥1. 

dBetas restricted to ≥0; lowest degree polynomial with an adequate fit is reported (1-degree polynomial). 

BMD = maximum likelihood estimate of the dose/concentration associated with the selected BMR 

Source: Kroese et al. (2001). 

18 
317 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 Figure B-7. Fit of log logistic model to data on forestomach hyperplasia in 

4 female Wistar rats in a 2-year study. 

5 

6 

7 Model output: 

8 ==================================================================== 

9 Logistic Model. (Version: 2.12; Date: 05/16/2008) 

10 Input Data File: 

11 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic\forestomachhyperplasia\female\dura

12 tionadjusted\3Logkrolog.(d) 

13 Gnuplot Plotting File: 

14 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\chronic\forestomachhyperplasia\female\dura

15 tionadjusted\3Logkrolog.plt 

16 Wed Oct 14 08:59:23 2009 

17 ==================================================================== 

18 

19 BMDS Model Run 

20 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

21 

22 The form of the probability function is: 

23 

24 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 

25 

26 

27 Dependent variable = incidence 

28 Independent variable = dose 

29 Slope parameter is restricted as slope >= 1 

30 

31 Total number of observations = 3 

32 Total number of records with missing values = 0 

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 

 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0.35

 0.4

 0 1 2 3 4 5 6 7

Fraction Affected

dose

Log-Logistic Model with 0.95 Confidence Level

08:59 10/14 2009

BMDL BMD

 

Log-Logistic
318 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 

4 User has chosen the log transformed model 

5 

6 

7 Default Initial Parameter Values 

8 background = 0.0192308 

9 intercept = -2.7983 

10 slope = 1 

11 

12 

13 Asymptotic Correlation Matrix of Parameter Estimates 

14 

15 ( *** The model parameter(s) -slope 

16 have been estimated at a boundary point, or have been specified by 

17 the user, 

18 and do not appear in the correlation matrix ) 

19 

20 background intercept 

21 

22 background 1 -0.46 

23 

24 intercept -0.46 1 

25 

26 

27 

28 Parameter Estimates 

29 

30 95.0% Wald Confidence 

31 Interval 

32 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

33 Limit 

34 background 0.0238694 * * * 

35 intercept -2.96044 * * * 

36 slope 1 * * * 

37 

38 * - Indicates that this value is not calculated. 

39 

40 

41 

42 Analysis of Deviance Table 

43 

44 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

45 Full model -56.048 3 

46 Fitted model -56.5181 2 0.940072 1 0.3323 

47 Reduced model -63.1579 1 14.2198 2 0.000817 

48 

49 AIC: 117.036 

50 

51 

52 Goodness of Fit 

53 Scaled 

54 Dose Est._Prob. Expected Observed Size Residual 

55 ------------------------------------------------------------------------ 

56 0.0000 0.0239 1.241 1.000 52 -0.219 

57 2.1000 0.1196 6.101 8.000 51 0.819 

58 7.1400 0.2874 14.658 13.000 51 -0.513 

59 

60 Chi^2 = 0.98 d.f. = 1 P-value = 0.3216

61 

62 

63 Benchmark Dose Computation 

64 

65 Specified effect = 0.1 

66 
319 DRAFT - DO NOT CITE OR QUOTE 

1 Risk Type = Extra risk 

2 

3 Confidence level = 0.95 

4 

5 BMD = 2.14515 

6 

7 BMDL = 1.34776 

8 

9 

10 Forestomach hyperplasia- female mice, 2 yrs (Beland and Culp, 1998) 

Table B-15. Model predictions for forestomach hyperplasia in female 

B6C3F1 mice in a 2-year study 

Model 

Degrees of 

freedom χ

2

χ

2

 Goodnessof-fit p-valuea AIC BMD10 

(mg/kg-d) 

BMDL10

(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 

Multistagec, Weibullb,

Gammab

2 1.74 0.42 191.3 0.421 0.295 

Probit 2 7.04 0.03 196.6 0.946 0.711 

aValues <0.10 fail to meet conventional goodness-of-fit criteria. 

b

Power restricted to ≥1. 

cBetas restricted to ≥0; lowest degree polynomial with an adequate fit is reported (1-degree polynomial). 

BMD = maximum likelihood estimate of the dose/concentration associated with the selected BMR 

Source: Beland and Culp (1998). 
320 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Figure B-8. Fit of log logistic model to data on forestomach hyperplasia in 

3 female B6C3F1 mice in a 2-year study. 

4 

5 

6 Model output: 

7 

8 ==================================================================== 

9 Logistic Model. (Version: 2.12; Date: 05/16/2008) 

10 Input Data File: 

11 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_BelandCulp_mice_4s_hyperplasia_generic_dich_10

12 .(d) 

13 Gnuplot Plotting File: 

14 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_BelandCulp_mice_4s_hyperplasia_generic_dich_10

15 .plt 

16 

17 ==================================================================== 

18 

19 BMDS Model Run 

20 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

21 

22 The form of the probability function is: 

23 

24 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 

25 

26 

27 Dependent variable = NumAff 

28 Independent variable = dose 

29 Slope parameter is restricted as slope >= 1 

30 

31 Total number of observations = 4 

32 Total number of records with missing values = 0 

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 User has chosen the log transformed model 

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10 12 14 16

Fraction Affected

dose

Log-Logistic Model with 0.95 Confidence Level

BMDL BMD

Log-Logistic
321 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 Default Initial Parameter Values 

4 background = 0.270833 

5 intercept = -0.637972 

6 slope = 1.38091 

7 

8 

9 Asymptotic Correlation Matrix of Parameter Estimates 

10 

11 background intercept slope 

12 

13 background 1 -0.66 0.46 

14 

15 intercept -0.66 1 -0.8 

16 

17 slope 0.46 -0.8 1 

18 

19 

20 

21 Parameter Estimates 

22 

23 95.0% Wald Confidence 

24 Interval 

25 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

26 Limit 

27 background 0.286381 * * * 

28 intercept -0.789676 * * * 

29 slope 1.26641 * * * 

30 

31 * - Indicates that this value is not calculated. 

32 

33 

34 

35 Analysis of Deviance Table 

36 

37 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

38 Full model -92.8312 4 

39 Fitted model -93.6497 3 1.63686 1 0.2008 

40 Reduced model -125.58 1 65.4982 3 <.0001 

41 

42 AIC: 193.299 

43 

44 

45 Goodness of Fit 

46 Scaled 

47 Dose Est._Prob. Expected Observed Size Residual 

48 ------------------------------------------------------------------------ 

49 0.0000 0.2864 13.746 13.000 48 -0.238 

50 0.7000 0.4464 20.979 23.000 47 0.593 

51 3.3000 0.7667 35.270 33.000 46 -0.791 

52 16.5000 0.9575 45.005 46.000 47 0.720 

53 

54 Chi^2 = 1.55 d.f. = 1 P-value = 0.2127

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 = 0.329083 

66 
322 DRAFT - DO NOT CITE OR QUOTE 

1 BMDL = 0.115446
323 DRAFT - DO NOT CITE OR QUOTE 

1 Decreased ovary weight- female rats, 60 days (Xu et al., 2010)

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 ± 0.0098b

0.136 ± 0.0098b

a

 TWA doses over the 60 day study period 

b

 Statistically different (p < 0.05) from controls using one-way ANOVA 

Table B-17. Model predictions for decreased ovary weight in female SDrats—60 days 

Model 

Goodness-of-fit 

p-value AIC 

BMD1SD

(mg/kg-d) 

BMDL1SD

(mg/kg-d) 

Power N/A 

Linear, Polynomial (1°) 

0.39 -138.67 2.27 1.49 

NA = not applicable, model failed; 

4 

5 
324 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 Figure B-9. Fit of linear/polynomial (1°) model to data on decreased ovary 

4 weight 

5 

6 

7 

8 Model Output: 

9 

10 ==================================================================== 

11 Polynomial Model. (Version: 2.16; Date: 05/26/2010) 

12 Input Data File: 

13 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_1

14 SD.(d) 

15 Gnuplot Plotting File: 

16 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_1

17 SD.plt 

18 Tue Dec 14 13:51:32 2010 

19 ==================================================================== 

20 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

21 

22 The form of the response function is: 

23 

24 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 

25 

26 

27 Dependent variable = Mean 

28 Independent variable = Dose 

29 rho is set to 0 

30 Signs of the polynomial coefficients are not restricted 

31 A constant variance model is fit 

32 

33 Total number of dose groups = 3 

 0.13

 0.14

 0.15

 0.16

 0.17

 0.18

 0 1 2 3 4 5

Mean Response

dose

Linear Model with 0.95 Confidence Level

16:03 12/14 2010

BMDL BMD

 

Linear
325 DRAFT - DO NOT CITE OR QUOTE 

1 Total number of records with missing values = 0 

2 Maximum number of iterations = 250 

3 Relative Function Convergence has been set to: 1e-008 

4 Parameter Convergence has been set to: 1e-008 

5 

6 

7 

8 Default Initial Parameter Values 

9 alpha = 0.000136 

10 rho = 0 Specified 

11 beta_0 = 0.158333 

12 beta_1 = -0.0048 

13 

14 

15 Asymptotic Correlation Matrix of Parameter Estimates 

16 

17 ( *** The model parameter(s) -rho 

18 have been estimated at a boundary point, or have been specified by 

19 the user, 

20 and do not appear in the correlation matrix ) 

21 

22 alpha beta_0 beta_1 

23 

24 alpha 1 4e-010 -4.5e-010 

25 

26 beta_0 4e-010 1 -0.77 

27 

28 beta_1 -4.5e-010 -0.77 1 

29 

30 

31 

32 Parameter Estimates 

33 

34 95.0% Wald Confidence Interval 

35 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

36 alpha 0.000118889 3.96296e-005 4.12162e-005 0.000196562 

37 beta_0 0.158333 0.00406354 0.150369 0.166298 

38 beta_1 -0.0048 0.00125904 -0.00726768 -0.00233232 

39 

40 

41 

42 Table of Data and Estimated Values of Interest

43 

44 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

45 ------ --- -------- -------- ----------- ----------- ---------- 

46 

47 0 6 0.16 0.158 0.0147 0.0109 0.374 

48 2.5 6 0.143 0.146 0.0098 0.0109 -0.749 

49 5 6 0.136 0.134 0.0098 0.0109 0.374 

50 

51 

52 

53 Model Descriptions for likelihoods calculated 

54 

55 

56 Model A1: Yij = Mu(i) + e(ij) 

57 Var{e(ij)} = Sigma^2 

58 

59 Model A2: Yij = Mu(i) + e(ij) 

60 Var{e(ij)} = Sigma(i)^2 

61 

62 Model A3: Yij = Mu(i) + e(ij) 

63 Var{e(ij)} = Sigma^2 

64 Model A3 uses any fixed variance parameters that 

65 were specified by the user 

66 

67 Model R: Yi = Mu + e(i) 

68 Var{e(i)} = Sigma^2 
326 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 Likelihoods of Interest 

4 

5 Model Log(likelihood) # Param's AIC 

6 A1 72.766595 4 -137.533190 

7 A2 73.468565 6 -134.937129 

8 A3 72.766595 4 -137.533190 

9 fitted 72.335891 3 -138.671782 

10 R 67.008505 2 -130.017010 

11 

12 

13 Explanation of Tests 

14 

15 Test 1: Do responses and/or variances differ among Dose levels? 

16 (A2 vs. R) 

17 Test 2: Are Variances Homogeneous? (A1 vs A2) 

18 Test 3: Are variances adequately modeled? (A2 vs. A3) 

19 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

20 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

21 

22 Tests of Interest 

23 

24 Test -2*log(Likelihood Ratio) Test df p-value 

25 

26 Test 1 12.9201 4 0.01167 

27 Test 2 1.40394 2 0.4956 

28 Test 3 1.40394 2 0.4956 

29 Test 4 0.861408 1 0.3533 

30 

31 The p-value for Test 1 is less than .05. There appears to be a 

32 difference between response and/or variances among the dose levels 

33 It seems appropriate to model the data 

34 

35 The p-value for Test 2 is greater than .1. A homogeneous variance 

36 model appears to be appropriate here 

37 

38 

39 The p-value for Test 3 is greater than .1. The modeled variance appears 

40 to be appropriate here 

41 

42 The p-value for Test 4 is greater than .1. The model chosen seems 

43 to adequately describe the data 

44 

45 

46 Benchmark Dose Computation 

47 

48 Specified effect = 1 

49 

50 Risk Type = Estimated standard deviations from the control mean 

51 

52 Confidence level = 0.95 

53 

54 BMD = 2.27159 

55 

56 

57 BMDL = 1.49968 

58 

59 
1 APPENDIX C. ADDITIONAL CALCULATIONS FOR THE RfC

2 

3 

4 

5 

6 

7 

8 Figure C-1. Human Fractional Deposition

9 

10 

11 

12 Species = humanlimited 

13 FRC = 3300.0 

14 Head volume = 50.0 

15 Density = 1.0 

16 Number of particles calculated = single

17 Diameter = 1.7000000000000002 μm MMAD

18 Inhalability = yes 

19 GSD = 1.0 

20 Breathing interval: One single breath

21 Concentration = 4.2 

22 Breathing Frequency = 16.0

23 Tidal Volume = 860.0 

24 Inspiratory Fraction = 0.5

25 Pause Fraction = 0.0 

26 Breathing Route = nasal 

27 

28 Region: Entire Lung 

29 Region: Entire Lung 

30 Region Deposition Fraction 

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
328 DRAFT - DO NOT CITE OR QUOTE 

1 -- -- 

2 Head 0.449 

3 TB 0.045 

4 P 0.127 

5 Total 0.621 

6 
1 

2 

3 Figure C-2. Rat Fractional Deposition

4 

5 

6 Species = rat 

7 FRC = 4.0 

8 Head volume = 0.42 

9 Density = 1.0 

10 Number of particles calculated = single

11 Diameter = 1.7000000000000002 μm MMAD

12 Inhalability = yes 

13 GSD = 1.0 

14 Breathing interval: One single breath

15 Concentration = 4.2 

16 Breathing Frequency = 102.0

17 Tidal Volume = 1.8 

18 Inspiratory Fraction = 0.5

19 Pause Fraction = 0.0 

20 Breathing Route = nasal 

21 

22 Region: Entire Lung 

23 Region: Entire Lung 

24 Region Deposition Fraction

25 -- -- 

26 Head 0.072 

27 TB 0.041 

28 P 0.068 

29 Total 0.181

329 DRAFT - DO NOT CITE OR QUOTE

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
330 DRAFT - DO NOT CITE OR QUOTE 

APPENDIX D. TIME-TO-TUMOR MODELING FOR THE ORAL SLOPE 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 Fatala 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 

 
331 DRAFT - DO NOT CITE OR QUOTE 

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 Fatala 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 to have caused death of particular animals. “Fatal” denotes incidence of tumors reported by 

the study investigators to have caused death of particular animals. 
332 DRAFT - DO NOT CITE OR QUOTE 

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 

Incidentala Fatala 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 

 
333 DRAFT - DO NOT CITE OR QUOTE 

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 

Incidentala Fatala 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 particular animals. 

 
334 DRAFT - DO NOT CITE OR QUOTE 

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 

Fatala 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 

 
335 DRAFT - DO NOT CITE OR QUOTE 

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 

Fatala 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 particular 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 factora

HEDb

(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 (1988) reference body weights for 

humans (70 kg), and the TWA body weights for each dose 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 (μg/d) 

TWA body 

weight average 

(kg) 

Administered 

Dosea (mg/kg-d) Scaling factorb HEDc (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 from dietary concentrations of benzo[a]pyrene using the TWA 

body weight and reported food intakes for mice. 

b

 Scaling factors were calculated using U.S. EPA (1988) 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 factor 
337 DRAFT - DO NOT CITE OR QUOTE 

Table D-6. Summary of Model Selection and Modeling Results for best-fitting multistage-Weibull modtime-to-tumor data for rats (Kroese et al., 2001) 

Sex EndPoints

Model 

stages LLa χ2 b

Number 

of 

param. AIC BMD10

Responses at mg/kg-d levelsc 

Selected model 

parameter 

estimates 

0 0.54 1.8 5.2 c t0 Model Selection Rational

Males Oral Cavity and 

Forestomach: Squam. Cell Tumors 

0 8 45 52 

1 -284.891 NR 4 577.8 0.104 0.0 21.1 39.8 38.7 2.2 442 -198.795 172.2 5 407.6 0.678 0.0 3.3 25.4 48.0 1.3 443 -108.512 180.6 6 229.0 0.453 0.0 6.8 41.7 50.0 3.7 41 Lowest AIC, best fit to lo

Hepatocellular Tumors 

0 4 38 49 

1 -179.664 NR 4 367.3 0.181 0 13.6 32.1 42 1 522 -145.749 67.8 5 301.5 0.472 0.0 6.5 37.2 49 2.3 48.43 -138.544 14.4 6 289.1 0.651 0.0 3.4 36.8 49.6 3.5 40.2 Lowest AIC, best fit to lo

Duodenum and Jejunum Tumors 

0 0 1 9 

1 -31.781 NR 3 69.6 2.64 0.0 1.1 3.4 5.7 1 NR 2 -28.941 5.7 4 65.9 3.04 0.0 0.2 1.8 8.2 1 NR 3 -28.439 1.0 5 66.9 3.03 0.0 0.0 1.0 9.0 1.8 NR Best fit to data 

Kidney: Uroethelial Carcinoma 

0 0 0 3 

1 -12.956 NR 3 31.9 9.16 0.0 0.3 1 1.7 1 NR 2 -11.837 2.2 4 31.7 5.71 0.0 0.1 0.5 2.5 1 NR 3 -11.398 0.9 5 32.8 4.65 0.0 0.0 0.3 2.7 1.7 NR Best fit to data 

Skin and Mammary Gland: Basal Cell Tumors 

2 1 1 13 

1 -52.314 NR 3 110.6 1.88 1.0 2.5 5.5 8.3 1 NR 2 -48.570 7.5 4 105.1 2.58 1.1 1.3 3.4 11.4 1 NR 3 -47.362 2.4 5 104.7 2.86 1.2 1.2 2.3 12.5 1.4 NR Lowest AIC, best fit to lo

Skin and Mammary Gland: Squam. Cell Tumors 

0 1 1 6 

1 -28.745 NR 3 63.5 3.36 0.0 0.9 2.7 4.5 1 NR Lowest AIC, best fit to lo2 -28.145 1.2 4 64.3 2.75 0.0 0.4 2.4 5.2 1.9 NR 3 -27.652 1.0 5 65.3 2.64 0.0 0.4 2.1 5.5 3.0 NR 

Females Oral Cavity and 

Forestomach: Squam. Cell Tumors 

1 6 30 50 

1 -134.532 NR 4 277.1 0.245 1.0 10.1 23.7 35.1 1 582 -100.809 67.4 5 211.6 0.428 0.8 6.8 33.2 47.9 2.5 523 -94.512 12.6 6 201.0 0.539 1.1 4.9 31.8 49.4 3.5 47 Lowest AIC, best fit to lo

Hepatocellular Tumors 

0 1 39 51 

1 -293.771 NR 4 595.5 0.146 0.0 14.6 32.6 43.8 1 442 -382.470 177.4 5 774.9 0.370 0.0 8.1 38.5 50.1 2.2 443 -228.170 308.6 6 468.3 0.575 0.0 3.0 38.4 51.4 3.1 39 Lowest AIC, best fit to lo

Duodenum and Jejunum Tumors 

0 0 0 4 

1 -15.948 NR 3 37.9 6.00 0.0 0.4 1.3 2.4 1 NR 2 -14.518 2.9 4 37.0 4.33 0.0 0.0 0.7 3.3 1.1 NR 3 -13.878 1.3 5 37.8 3.43 0.0 0.0 0.4 3.6 2.3 NR Best fit to low dose data 

a LL=log-likelihood. b χ2 = chi-squared statistic for testing the difference between 2 model fits, from 2 × |(LLi – LLj)| evaluated for i-j degrees of freedom. In all cases the difference was evaluated fo

stages; i-j = 1, and χ2 at α = 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 de

with the observed data. 
338 DRAFT - DO NOT CITE OR QUOTE 

Table D-6. Summary of Model Selection and Modeling Results for best-fitting multistage-Weibull mod

time-to-tumor data for rats (Kroese et al., 2001) 

Sex EndPoints

Model 

stages LLa χ2 b

Number 

of 

param. AIC BMD10

Responses at mg/kg-d levelsc 

Selected model 

parameter 

estimates 

0 0.54 1.8 5.2 c t0 Model Selection Rational

Males Oral Cavity and 

Forestomach: Squam. Cell Tumors 

0 8 45 52 

1 -284.891 NR 4 577.8 0.104 0.0 21.1 39.8 38.7 2.2 442 -198.795 172.2 5 407.6 0.678 0.0 3.3 25.4 48.0 1.3 443 -108.512 180.6 6 229.0 0.453 0.0 6.8 41.7 50.0 3.7 41 Lowest AIC, best fit to lo

Hepatocellular Tumors 

0 4 38 49 

1 -179.664 NR 4 367.3 0.181 0 13.6 32.1 42 1 522 -145.749 67.8 5 301.5 0.472 0.0 6.5 37.2 49 2.3 48.43 -138.544 14.4 6 289.1 0.651 0.0 3.4 36.8 49.6 3.5 40.2 Lowest AIC, best fit to lo

Duodenum and Jejunum Tumors 

0 0 1 9 

1 -31.781 NR 3 69.6 2.64 0.0 1.1 3.4 5.7 1 NR 2 -28.941 5.7 4 65.9 3.04 0.0 0.2 1.8 8.2 1 NR 3 -28.439 1.0 5 66.9 3.03 0.0 0.0 1.0 9.0 1.8 NR Best fit to data 

Kidney: Uroethelial Carcinoma 

0 0 0 3 

1 -12.956 NR 3 31.9 9.16 0.0 0.3 1 1.7 1 NR 2 -11.837 2.2 4 31.7 5.71 0.0 0.1 0.5 2.5 1 NR 3 -11.398 0.9 5 32.8 4.65 0.0 0.0 0.3 2.7 1.7 NR Best fit to data 

Skin and Mammary Gland: Basal Cell Tumors 

2 1 1 13 

1 -52.314 NR 3 110.6 1.88 1.0 2.5 5.5 8.3 1 NR 2 -48.570 7.5 4 105.1 2.58 1.1 1.3 3.4 11.4 1 NR 3 -47.362 2.4 5 104.7 2.86 1.2 1.2 2.3 12.5 1.4 NR Lowest AIC, best fit to lo

Skin and Mammary Gland: Squam. Cell Tumors 

0 1 1 6 

1 -28.745 NR 3 63.5 3.36 0.0 0.9 2.7 4.5 1 NR Lowest AIC, best fit to lo2 -28.145 1.2 4 64.3 2.75 0.0 0.4 2.4 5.2 1.9 NR 3 -27.652 1.0 5 65.3 2.64 0.0 0.4 2.1 5.5 3.0 NR 

Females Oral Cavity and 

Forestomach: Squam. Cell Tumors 

1 6 30 50 

1 -134.532 NR 4 277.1 0.245 1.0 10.1 23.7 35.1 1 582 -100.809 67.4 5 211.6 0.428 0.8 6.8 33.2 47.9 2.5 523 -94.512 12.6 6 201.0 0.539 1.1 4.9 31.8 49.4 3.5 47 Lowest AIC, best fit to lo

Hepatocellular Tumors 

0 1 39 51 

1 -293.771 NR 4 595.5 0.146 0.0 14.6 32.6 43.8 1 442 -382.470 177.4 5 774.9 0.370 0.0 8.1 38.5 50.1 2.2 443 -228.170 308.6 6 468.3 0.575 0.0 3.0 38.4 51.4 3.1 39 Lowest AIC, best fit to lo

Duodenum and Jejunum Tumors 

0 0 0 4 

1 -15.948 NR 3 37.9 6.00 0.0 0.4 1.3 2.4 1 NR 2 -14.518 2.9 4 37.0 4.33 0.0 0.0 0.7 3.3 1.1 NR 3 -13.878 1.3 5 37.8 3.43 0.0 0.0 0.4 3.6 2.3 NR Best fit to low dose data 

NR = not relevant.
339 DRAFT - DO NOT CITE OR QUOTE 

1 Male Rat (Kroese et al., 2001): Squamous Cell Papilloma or Carcinoma in Oral Cavity or 

2 Forestomach 

3 

4 ======================================================================= 

5 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

6 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

7 Input Data File: OralForstKroeseM3.(d) 

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 positive 

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 Maximum number of iterations = 64 

25 Relative Function Convergence has been set to: 2.22045e-016 

26 Parameter Convergence has been set to: 1.49012e-008 

27 

28 

29 Default Initial Parameter Values 

30 c = 3.6 

31 t_0 = 39.1111 

32 beta_0 = 0 

33 beta_1 = 8.8911e-009 

34 beta_2 = 1.60475e-031 

35 beta_3 = 1.95818e-008 

36 

37 

38 Asymptotic Correlation Matrix of Parameter 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 correlation matrix ) 

42 

43 c t_0 beta_1 beta_3 

44 

45 c 1 -0.53 -0.93 -0.99 

46 

47 t_0 -0.53 1 0.47 0.57 

48 

49 beta_1 -0.93 0.47 1 0.9 

50 

51 beta_3 -0.99 0.57 0.9 1 

52 

53 

54 Parameter Estimates 

55 95.0% Wald Confidence Interval 

56 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

57 c 3.74559 0.447309 2.86888 4.6223 

58 t_0 41.4581 2.14975 37.2447 45.6716 

59 beta_0 0 NA 

60 beta_1 4.37816e-009 1.07528e-008 -1.6697e-008 2.54533e-008 

61 beta_2 0 NA 

62 beta_3 1.01904e-008 1.94164e-008 -2.78651e-008 4.82458e-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 -108.512 6 229.024 

70 

71 

72 Data Summary 

73 CONTEXT 

74 C F I U Total Expected Response 

75 DOSE 

76 0 52 0 0 0 52 0.00 

77 0.54 44 0 8 0 52 6.77 

78 1.8 7 0 45 0 52 41.69 

79 5.2 0 9 43 0 52 49.97 

80 

81 Minimum observation time for F tumor context = 44 

82 

83 
340 DRAFT - DO NOT CITE OR QUOTE 

1 Benchmark Dose Computation 

2 Risk Response = Incidental 

3 Risk Type = Extra 

4 Confidence level = 0.9 

5 Time = 104 

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 

1 Male Rat (Kroese et al., 2001): Hepatocellular Adenoma or Carcinoma 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: LiverKroeseM3.(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 positive 

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: 2.22045e-016 

27 Parameter Convergence has been set to: 1.49012e-008 

28 

29 

30 Default Initial Parameter Values 

31 c = 3.6 

32 t_0 = 34.6667 

33 beta_0 = 0 

34 beta_1 = 2.73535e-009 

35 beta_2 = 8.116e-028 

36 beta_3 = 1.43532e-008 

37 

38 

39 Asymptotic Correlation Matrix of Parameter Estimates 

40 ( *** The model parameter(s) -beta_0 -beta_2 

41 have been estimated at a boundary point, or have been specified by the user, 

42 and do not appear in the correlation matrix ) 

43 

44 c t_0 beta_1 beta_3 

45 

46 c 1 -0.84 -0.88 -1 

47 

48 t_0 -0.84 1 0.71 0.86 

49 

50 beta_1 -0.88 0.71 1 0.86 

51 

52 beta_3 -1 0.86 0.86 1 

53 

54 

55 Parameter Estimates 

56 95.0% Wald Confidence Interval 

57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

58 c 3.49582 0.629257 2.26249 4.72914 

59 t_0 40.2211 5.65421 29.1391 51.3032 

60 beta_0 0 NA 

61 beta_1 4.43906e-009 1.76051e-008 -3.00664e-008 3.89445e-008 

62 beta_2 0 NA 

63 beta_3 2.35065e-008 6.47999e-008 -1.03499e-007 1.50512e-007 

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 -138.544 6 289.088 

71 

72 

73 Data Summary 

74 CONTEXT 

75 C F I U Total Expected Response 

76 DOSE 

77 0 52 0 0 0 52 0.00 

78 0.54 48 0 4 0 52 3.38 

79 1.8 14 2 36 0 52 36.81 

80 5.2 3 17 32 0 52 49.55 

81 

82 Minimum observation time for F tumor context = 52 

83 

84 
342 DRAFT - DO NOT CITE OR QUOTE 

1 Benchmark Dose Computation 

2 Risk Response = Incidental 

3 Risk Type = Extra 

4 Confidence level = 0.9 

5 Time = 104 

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 80 100

0.0 0.4 0.8

Dose = 0.00

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 0.54

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 1.81

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 5.17

Time

Probability

Incidental Risk: Hepatocellular_Kroese_M3

points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
343 DRAFT - DO NOT CITE OR QUOTE 

1 Male Rat (Kroese et al., 2001): Duodenum or Jejunum Adenocarcinoma 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: DuoJejKroeseM3.(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 positive 

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 = 1 

22 Degree of polynomial = 3 

23 

24 

25 

26 User specifies the following parameters: 

27 t_0 = 0 

28 

29 Maximum number of iterations = 64 

30 Relative Function Convergence has been set to: 2.22045e-016 

31 Parameter Convergence has been set to: 1.49012e-008 

32 

33 

34 Default Initial Parameter Values 

35 c = 1.63636 

36 t_0 = 0 Specified 

37 beta_0 = 4.31119e-027 

38 beta_1 = 2.96347e-025 

39 beta_2 = 0 

40 beta_3 = 1.76198e-006 

41 

42 

43 Asymptotic Correlation Matrix of Parameter Estimates 

44 ( *** The model parameter(s) -t_0 -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 correlation matrix ) 

47 

48 c beta_3 

49 

50 c 1 -1 

51 

52 beta_3 -1 1 

53 

54 

55 Parameter Estimates 

56 95.0% Wald Confidence Interval 

57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

58 c 1.77722 2.03042 -2.20233 5.75677 

59 beta_0 0 NA 

60 beta_1 0 NA 

61 beta_2 0 NA 

62 beta_3 9.82635e-007 8.29355e-006 -1.52724e-005 1.72377e-005 

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 -28.4387 5 66.8773 

70 

71 

72 Data Summary 

73 CONTEXT 

74 C F I U Total Expected Response 

75 DOSE 

76 0 52 0 0 0 52 0.00 

77 0.54 52 0 0 0 52 0.03 

78 1.8 51 0 1 0 52 1.04 

79 5.2 43 0 9 0 52 8.96 

80 

81 

82 Benchmark Dose Computation 

83 Risk Response = Incidental 
344 DRAFT - DO NOT CITE OR QUOTE 

1 Risk Type = Extra 

2 Specified effect = 0.1 

3 Confidence level = 0.9 

4 Time = 104 

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 80 100

0.00 0.15

Dose = 0.00

Time

Probability

0 20 40 60 80 100

0.00 0.15

Dose = 0.54

Time

Probability

0 20 40 60 80 100

0.00 0.15

Dose = 1.81

Time

Probability

0 20 40 60 80 100

0.00 0.15

Dose = 5.17

Time

Probability

Incidental Risk: DuoJej_Kroese_M3
345 DRAFT - DO NOT CITE OR QUOTE 

1 Male Rat (Kroese et al., 2001): Skin or Mammary Gland Basal Cell Tumors 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: SKinMamBasalKroeseM3.(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 positive 

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 = 1 

22 Degree of polynomial = 3 

23 

24 

25 

26 User specifies the following parameters: 

27 t_0 = 0 

28 

29 Maximum number of iterations = 64 

30 Relative Function Convergence has been set to: 2.22045e-016 

31 Parameter Convergence has been set to: 1.49012e-008 

32 

33 

34 Default Initial Parameter Values 

35 c = 1.38462 

36 t_0 = 0 Specified 

37 beta_0 = 3.84298e-005 

38 beta_1 = 1.06194e-028 

39 beta_2 = 0 

40 beta_3 = 6.84718e-006 

41 

42 

43 Asymptotic Correlation Matrix of Parameter Estimates 

44 ( *** The model parameter(s) -t_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 correlation matrix ) 

47 

48 c beta_0 beta_3 

49 

50 c 1 -1 -1 

51 

52 beta_0 -1 1 0.99 

53 

54 beta_3 -1 0.99 1 

55 

56 

57 Parameter Estimates 

58 95.0% Wald Confidence Interval 

59 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

60 c 1.47227 1.76686 -1.9907 4.93525 

61 beta_0 2.54786e-005 0.000211261 -0.000388585 0.000439542 

62 beta_1 0 NA 

63 beta_2 0 NA 

64 beta_3 4.81611e-006 3.49e-005 -6.35866e-005 7.32188e-005 

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 -47.3623 5 104.725 

72 

73 

74 Data Summary 

75 CONTEXT 

76 C F I U Total Expected Response 

77 DOSE 

78 0 50 0 2 0 52 1.18 

79 0.54 51 0 1 0 52 1.22 

80 1.8 51 0 1 0 52 2.32 

81 5.2 39 0 13 0 52 12.54 

82 

83 
346 DRAFT - DO NOT CITE OR QUOTE 

1 Benchmark Dose Computation 

2 Risk Response = Incidental 

3 Risk Type = Extra 

4 Confidence level = 0.9 

5 Time = 104 

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 80

0.0 0.4 0.8

Dose = 0.54

Time

Probability

0 20 40 60 80

0.0 0.4 0.8

Dose = 1.81

Time

Probability

0 20 40 60 80

0.0 0.4 0.8

Dose = 5.17

Time

Probability

Incidental Risk: Skin_Mam_Basal_Kroese_M3
347 DRAFT - DO NOT CITE OR QUOTE 

1 Male Rat (Kroese et al., 2001): Skin or Mammary Gland Squamous Cell Tumors 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: SKinMamSCCKroeseM3.(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 positive 

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 = 1 

22 Degree of polynomial = 3 

23 

24 

25 

26 User specifies the following parameters: 

27 t_0 = 0 

28 

29 Maximum number of iterations = 64 

30 Relative Function Convergence has been set to: 2.22045e-016 

31 Parameter Convergence has been set to: 1.49012e-008 

32 

33 

34 Default Initial Parameter Values 

35 c = 3 

36 t_0 = 0 Specified 

37 beta_0 = 0 

38 beta_1 = 1.25256e-008 

39 beta_2 = 1.25627e-030 

40 beta_3 = 3.34696e-009 

41 

42 

43 Asymptotic Correlation Matrix of Parameter Estimates 

44 ( *** The model parameter(s) -t_0 -beta_0 -beta_2 

45 have been estimated at a boundary point, or have been specified by the user, 

46 and do not appear in the correlation matrix ) 

47 

48 c beta_1 beta_3 

49 

50 c 1 -0.99 -1 

51 

52 beta_1 -0.99 1 0.99 

53 beta_3 -1 0.99 1 

54 

55 

56 Parameter Estimates 

57 95.0% Wald Confidence Interval 

58 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

59 c 2.96213 2.591 -2.11613 8.04039 

60 beta_0 0 NA 

61 beta_1 1.50104e-008 1.86972e-007 -3.51447e-007 3.81468e-007 

62 beta_2 0 NA 

63 beta_3 3.9084e-009 4.15374e-008 -7.75033e-008 8.53201e-008 

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 -27.652 5 65.304 

71 

72 

73 Data Summary 

74 CONTEXT 

75 C F I U Total Expected Response 

76 DOSE 

77 0 52 0 0 0 52 0.00 

78 0.54 51 0 1 0 52 0.42 

79 1.8 51 0 1 0 52 2.12 

80 5.2 46 0 6 0 52 5.51 

81 

82 

83 Benchmark Dose Computation 

84 Risk Response = Incidental 
348 DRAFT - DO NOT CITE OR QUOTE 

1 Risk Type = Extra 

2 Confidence level = 0.9 

3 Time = 104 

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 

9 Dose Response plot 

10 

11 
349 DRAFT - DO NOT CITE OR QUOTE 

1 Male Rat (Kroese et al., 2001): Kidney Urothelial Carcinomas 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: KidneyUrothelialCarKroeseM3.(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 positive 

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 = 1 

22 Degree of polynomial = 3 

23 

24 

25 

26 User specifies the following parameters: 

27 t_0 = 0 

28 

29 Maximum number of iterations = 64 

30 Relative Function Convergence has been set to: 2.22045e-016 

31 Parameter Convergence has been set to: 1.49012e-008 

32 

33 

34 Default Initial Parameter Values 

35 c = 1.63636 

36 t_0 = 0 Specified 

37 beta_0 = 3.78734e-027 

38 beta_1 = 1.59278e-027 

39 beta_2 = 2.718e-024 

40 beta_3 = 4.96063e-007 

41 

42 

43 Asymptotic Correlation Matrix of Parameter Estimates 

44 ( *** The model parameter(s) -t_0 -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 correlation matrix ) 

47 

48 c beta_3 

49 

50 c 1 -1 

51 

52 beta_3 -1 1 

53 

54 

55 Parameter Estimates 

56 95.0% Wald Confidence Interval 

57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

58 c 1.74897 3.79403 -5.68719 9.18512 

59 beta_0 0 NA 

60 beta_1 0 NA 

61 beta_2 0 NA 

62 beta_3 3.11107e-007 4.90313e-006 -9.29885e-006 9.92107e-006 

63 

64 NA - Indicates that this parameter has hit a 

65 bound implied by some inequality constraint 

66 and thus has no standard error. 

67 

68 

69 Log(likelihood) # Param AIC 

70 Fitted Model -11.3978 5 32.7956 

71 

72 

73 Data Summary 

74 CONTEXT 

75 C F I U Total Expected Response 

76 DOSE 

77 0 52 0 0 0 52 0.00 

78 0.54 52 0 0 0 52 0.01 

79 1.8 52 0 0 0 52 0.29 

80 5.2 49 0 3 0 52 2.71 

81 

82 

83 Benchmark Dose Computation 
350 DRAFT - DO NOT CITE OR QUOTE 

1 Risk Response = Incidental 

2 Risk Type = Extra 

3 Confidence level = 0.9 

4 Time = 104 

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 80 100

0.00 0.05 0.10 0.15

Dose = 0.00

Time

Probability

0 20 40 60 80 100

0.00 0.05 0.10 0.15

Dose = 0.54

Time

Probability

0 20 40 60 80 100

0.00 0.05 0.10 0.15

Dose = 1.81

Time

Probability

0 20 40 60 80 100

0.00 0.05 0.10 0.15

Dose = 5.17

Time

Probability

Incidental Risk: Kidney_Kroese_M3
351 DRAFT - DO NOT CITE OR QUOTE 

1 Female Rat (Kroese et al., 2001): Oral Cavity or Forestomach, Squamous Cell Papilloma 

2 or Carcinoma 

3 

4 ======================================================================= 

5 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

6 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

7 Input Data File: OralForstKroeseF3.(d) 

8 ======================================================================= 

9 

10 The form of the probability function is: 

11 P[response] = 1-EXP{-(t - t_0)^c * 

12 (beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

13 

14 The parameter betas are restricted to be positive 

15 

16 Dependent variable = CONTEXT 

17 Independent variables = DOSE, TIME 

18 

19 Total number of observations = 208 

20 Total number of records with missing values = 0 

21 Total number of parameters in model = 6 

22 Total number of specified parameters = 0 

23 Degree of polynomial = 3 

24 

25 

26 Maximum number of iterations = 64 

27 Relative Function Convergence has been set to: 2.22045e-016 

28 Parameter Convergence has been set to: 1.49012e-008 

29 

30 

31 Default Initial Parameter Values 

32 c = 3.6 

33 t_0 = 45.1111 

34 beta_0 = 1.11645e-009 

35 beta_1 = 4.85388e-009 

36 beta_2 = 0 

37 beta_3 = 1.95655e-008 

38 

39 Asymptotic Correlation Matrix of Parameter Estimates 

40 ( *** The model parameter(s) -beta_2 

41 have been estimated at a boundary point, or have been specified by the user, 

42 and do not appear in the correlation matrix ) 

43 

44 c t_0 beta_0 beta_1 beta_3 

45 

46 c 1 -0.79 -0.92 -0.93 -1 

47 

48 t_0 -0.79 1 0.73 0.72 0.8 

49 

50 beta_0 -0.92 0.73 1 0.79 0.92 

51 

52 beta_1 -0.93 0.72 0.79 1 0.91 

53 

54 beta_3 -1 0.8 0.92 0.91 1 

55 

56 

57 Parameter Estimates 

58 95.0% Wald Confidence Interval 

59 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

60 c 3.52871 0.701117 2.15454 4.90287 

61 t_0 46.553 5.93306 34.9244 58.1816 

62 beta_0 1.53589e-009 5.40523e-009 -9.05817e-009 1.21299e-008 

63 beta_1 7.57004e-009 2.9647e-008 -5.05369e-008 6.5677e-008 

64 beta_2 0 NA 

65 beta_3 2.53126e-008 7.66404e-008 -1.249e-007 1.75525e-007 

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 -94.5119 6 201.024 

73 

74 

75 Data Summary 

76 CONTEXT 

77 C F I U Total Expected Response 

78 DOSE 

79 0 51 0 1 0 52 1.14 

80 0.49 46 0 6 0 52 4.90 

81 1.6 22 0 30 0 52 31.81 

82 4.6 2 7 43 0 52 49.43 
352 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Minimum observation time for F tumor context = 58 

3 

4 Benchmark Dose Computation 

5 Risk Response = Incidental 

6 Risk Type = Extra 

7 Confidence level = 0.9 

8 Time = 104 

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 80 100

0.0 0.4 0.8

Dose = 0.00

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 0.49

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 1.62

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 4.58

Time

Probability

Incidental Risk: OralForstKroeseF3

points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
353 DRAFT - DO NOT CITE OR QUOTE 

1 Female Rat (Kroese et al., 2001): Hepatocellular Adenoma or Carcinoma 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: LiverKroeseF3.(d) 

7 Fri Apr 16 09:08:03 2010 

8 ======================================================================= 

9 

10 Timer to Tumor Model, Liver Hepatocellular Tumors, Kroese et al, Female 

11 

12 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

13 

14 The form of the probability function is: 

15 P[response] = 1-EXP{-(t - t_0)^c * 

16 (beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

17 

18 The parameter betas are restricted to be positive 

19 

20 Dependent variable = CONTEXT 

21 Independent variables = DOSE, TIME 

22 

23 Total number of observations = 208 

24 Total number of records with missing values = 0 

25 Total number of parameters in model = 6 

26 Total number of specified parameters = 0 

27 Degree of polynomial = 3 

28 

29 

30 Maximum number of iterations = 64 

31 Relative Function Convergence has been set to: 2.22045e-016 

32 Parameter Convergence has been set to: 1.49012e-008 

33 

34 

35 Default Initial Parameter Values 

36 c = 3.6 

37 t_0 = 31.7778 

38 beta_0 = 0 

39 beta_1 = 4.9104e-031 

40 beta_2 = 5.45766e-030 

41 beta_3 = 3.44704e-008 

42 

43 

44 Asymptotic Correlation Matrix of Parameter Estimates 

45 ( *** The model parameter(s) -beta_0 -beta_1 -beta_2 

46 have been estimated at a boundary point, or have been specified by the user, 

47 and do not appear in the correlation matrix ) 

48 

49 c t_0 beta_3 

50 

51 c 1 -0.9 -1 

52 

53 t_0 -0.9 1 0.92 

54 

55 beta_3 -1 0.92 1 

56 

57 

58 Parameter Estimates 

59 95.0% Wald Confidence Interval 

60 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

61 c 3.11076 0.549208 2.03434 4.18719 

62 t_0 38.6965 5.21028 28.4846 48.9085 

63 beta_0 0 NA 

64 beta_1 0 NA 

65 beta_2 0 NA 

66 beta_3 2.94354e-007 7.19418e-007 -1.11568e-006 1.70439e-006 

67 

68 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 

69 and thus has no standard error. 

70 

71 

72 Log(likelihood) # Param AIC 

73 Fitted Model -228.17 6 468.34 

74 

75 

76 Data Summary 

77 CONTEXT 

78 C F I U Total Expected Response 

79 DOSE 

80 0 52 0 0 0 52 0.00 

81 0.49 51 0 1 0 52 3.02 

82 1.6 13 12 27 0 52 38.36 

83 4.6 1 38 13 0 52 51.36 
354 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Minimum observation time for F tumor context = 44 

3 

4 

5 Benchmark Dose Computation 

6 Risk Response = Incidental 

7 Risk Type = Extra 

8 Confidence level = 0.9 

9 Time = 104 

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 80 100

0.0 0.4 0.8

Dose = 0.00

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 0.49

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 1.62

Time

Probability

0 20 40 60 80 100

0.0 0.4 0.8

Dose = 4.58

Time

Probability

Incidental Risk: Hepatocellular_Kroese_F3

points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
355 DRAFT - DO NOT CITE OR QUOTE 

1 Female Rat (Kroese et al., 2001): Duodenum or Jejunum Adenocarcinoma 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: DuoJejKroeseF3.(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 positive 

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 User specifies the following parameters: 

25 t_0 = 0 

26 

27 Maximum number of iterations = 64 

28 Relative Function Convergence has been set to: 2.22045e-016 

29 Parameter Convergence has been set to: 1.49012e-008 

30 

31 

32 Default Initial Parameter Values 

33 c = 2.25 

34 t_0 = 0 Specified 

35 beta_0 = 0 

36 beta_1 = 0 

37 beta_2 = 0 

38 beta_3 = 7.289e-008 

39 

40 

41 Asymptotic Correlation Matrix of Parameter Estimates 

42 ( *** The model parameter(s) -t_0 -beta_0 -beta_1 -beta_2 

43 have been estimated at a boundary point, or have been specified by the user, 

44 and do not appear in the correlation matrix ) 

45 

46 c beta_3 

47 

48 c 1 -1 

49 

50 beta_3 -1 1 

51 

52 

53 Parameter Estimates 

54 95.0% Wald Confidence Interval 

55 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

56 c 2.32531 3.58729 -4.70565 9.35626 

57 beta_0 0 NA 

58 beta_1 0 NA 

59 beta_2 0 NA 

60 beta_3 5.32209e-008 7.98487e-007 -1.51178e-006 1.61823e-006 

61 

62 NA - Indicates that this parameter has hit a bound implied by some inequality constraint 

63 and thus has no standard error. 

64 

65 

66 Log(likelihood) # Param AIC 

67 Fitted Model -13.8784 5 37.7569 

68 

69 

70 Data Summary 

71 CONTEXT 

72 C F I U Total Expected Response 

73 DOSE 

74 0 52 0 0 0 52 0.00 

75 0.49 52 0 0 0 52 0.01 

76 1.6 52 0 0 0 52 0.44 

77 4.6 48 0 4 0 52 3.57 

78 

79 

80 Benchmark Dose Computation 

81 Risk Response = Incidental 

82 Risk Type = Extra 

83 Confidence level = 0.9 
356 DRAFT - DO NOT CITE OR QUOTE 

1 Time = 104 

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 80 100

0.00 0.05 0.10 0.15

Dose = 0.00

Time

Probability

0 20 40 60 80 100

0.00 0.05 0.10 0.15

Dose = 0.49

Time

Probability

0 20 40 60 80 100

0.00 0.05 0.10 0.15

Dose = 1.62

Time

Probability

0 20 40 60 80 100

0.00 0.05 0.10 0.15

Dose = 4.58

Time

Probability

Incidental Risk: DuoJej_Kroese_F3
357 DRAFT - DO NOT CITE OR QUOTE 

Table D-7. Summary of human equivalent overall cancer risk values, based on male and 

female rat tumor incidence (Kroese et al., 2001) 

Data set Tumor Site BMD001 BMDL001

Risk valuea

 at 

SD SD2

Prop. of 

total 

BMD001 BMDL001 variance 

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 BMD001: 2.25E-01 Sum, SD2: 2.17E-02 

Overall SDb: 1.47E-01 

Upper bound on sum of risk estimatesc: 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 BMD001: 1.09E-01 Sum, SD2: 1.48E-02 

Overall SD: 1.22E-01 

Upper bound on sum of risk estimatesc: 3.09E-01 

a

 Risk value=0.001/BMDL001 

b

 Overall SD = (Sum, SD2)

0.5 

c

 Upper bound on the overall risk estimate = Sum of BMD001 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 LLaχ

2 b

Number 

of 

param. AIC BMD10

Responses @ mg/kg-d levelsc 

Selected model 

parameter 

estimates 

Model Selection 

0 0.1 0.48 2.3 c t0 Rationale

 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 × |(LLi – LLj)| 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 each tumor type; observed responses are in italics, and 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 

1 Female Mice (Beland and Culp, 1998): Alimentary Tract Squamous Cell Tumors 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: C:\msw10-09\benzo[a]pyrene_FemaleSquamF3i.(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 positive 

14 

15 Dependent variable = Class 

16 Independent variables = Dose, time 

17 

18 Total number of observations = 191 

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: 2.22045e-016 

27 Parameter Convergence has been set to: 1.49012e-008 

28 

29 

30 

31 User Inputs Initial Parameter Values 

32 c = 2 

33 t_0 = 15 

34 beta_0 = 1.6e-014 

35 beta_1 = 0 

36 beta_2 = 5.5e-012 

37 beta_3 = 4.4e-012 

38 

39 

40 Asymptotic Correlation Matrix of Parameter Estimates 

41 c t_0 beta_0 beta_1 beta_2 beta_3 

42 

43 c 1 -0.78 -0.97 -0.42 -0.99 -0.99 

44 

45 t_0 -0.78 1 0.76 0.39 0.74 0.84 

46 

47 beta_0 -0.97 0.76 1 0.33 0.97 0.96 

48 

49 beta_1 -0.42 0.39 0.33 1 0.31 0.46 

50 

51 beta_2 -0.99 0.74 0.97 0.31 1 0.97 

52 

53 beta_3 -0.99 0.84 0.96 0.46 0.97 1 

54 

55 

56 Parameter Estimates 

57 95.0% Wald Confidence Interval 

58 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

59 c 6.92317 1.33874 4.29929 9.54705 

60 t_0 13.9429 4.96646 4.20881 23.677 

61 beta_0 2.46916e-016 1.47619e-015 -2.64636e-015 3.14019e-015 

62 beta_1 0 1.30525e-014 -2.55825e-014 2.55825e-014 

63 beta_2 5.85452e-014 3.75144e-013 -6.76723e-013 7.93813e-013 

64 beta_3 9.76542e-014 5.62017e-013 -1.00388e-012 1.19919e-012 

65 

66 

67 Log(likelihood) # Param AIC 

68 Fitted Model -306.265 6 624.53 

69 

70 

71 Data Summary 

72 Class 

73 C F I U Total Expected Response 

74 Dose 

75 0 47 0 1 0 48 0.93 

76 0.1 45 0 3 0 48 3.21 

77 0.48 8 23 15 1 47 30.82 

78 2.3 1 46 0 1 48 41.91 

79 

80 Minimum observation time for F tumor context = 39 

81 

82 

83 Benchmark Dose Computation 

84 Risk Response = Incidental 
359 DRAFT - DO NOT CITE OR QUOTE 

1 Risk Type = Extra 

2 Specified effect = 0.1 

3 Confidence level = 0.9 

4 

5 Time = 104 

6 

7 BMD = 0.126983 

8 BMDL = 0.0706103 

9 BMDU = 0.179419 

10 

11 

12 

13 

14 

15 
360 DRAFT - DO NOT CITE OR QUOTE 

1 APPENDIX E. TIME-TO-TUMOR MODELING FOR THE INHALATION UNIT RISK 

2 

3 

Table E-1. Individual pathology and tumor occurrence data for male Syrian 

hamsters exposed to benzo[a]pyrene via inhalation for lifetime (Thyssen et al., 1981) 

Admin. 

Exposure 

Conc. 

(mg/m3

) 

Time 

on 

Study 

Number 

Examined Larynx Pharynx Trachea Esophagus Forestomach 

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 occurrence data for male Syrian 

hamsters exposed to benzo[a]pyrene via inhalation for lifetime (Thyssen et al., 1981) 

Admin. 

Exposure 

Conc. 

(mg/m3

) 

Time 

on 

Study 

Number 

Examined Larynx Pharynx Trachea Esophagus Forestomach 

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 2 Tissue was not examined for one animal of total examined.

b 3 Tissue was not examined for two animals of total examined.
362 DRAFT - DO NOT CITE OR QUOTE 

Table E-2. Summary of model selection among multistage-Weibull models fit to tumor 

data for male hamstersa

 (Thyssen et al., 1981)

Model 

stages LLbχ

2 c

Number 

of 

param. AIC BMD10

Responses @ mg/kg-d levelsd 

Selected model 

parameter 

estimates Model Selection 

0 0.25 1.01 4.29 c t0 Rationale

Oral tract tumors: all tumors considered incidental 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 NR 

Lowest AIC, best fit 

to data; maximum 

number of stages that 

could be fit

Oral tract tumors: all tumors considered to be cause 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 × |(LLi – LLj)| evaluated for |i-j| degrees of freedom (df). In

all cases the difference was evaluated for consecutive 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 each tumor type; observed responses are in italics, and 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 

1 Output for Oral tract tumors: all tumors considered incidental to cause of death 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: C:\msw\benzo[a]pyrene-Thyssen_inc2st.(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)} 

12 

13 The parameter betas are restricted to be positive 

14 

15 Dependent variable = Class 

16 Independent variables = Conc, Time 

17 

18 Total number of observations = 96 

19 Total number of records with missing values = 0 

20 Total number of parameters in model = 5 

21 Total number of specified parameters = 1 

22 Degree of polynomial = 2 

23 

24 

25 

26 User specifies the following parameters: 

27 t_0 = 0 

28 

29 Maximum number of iterations = 32 

30 Relative Function Convergence has been set to: 1e-008 

31 Parameter Convergence has been set to: 1e-008 

32 

33 

34 Default Initial Parameter Values 

35 c = 3.6 

36 t_0 = 0 Specified 

37 beta_0 = 1.18657e-031 

38 beta_1 = 1.49e-030 

39 beta_2 = 6.10362e-008 

40 

41 

42 Asymptotic Correlation Matrix of Parameter Estimates 

43 ( *** The model parameter(s) -t_0 -beta_0 -beta_1 

44 have been estimated at a boundary point, or have been specified by the user, 

45 and do not appear in the correlation matrix ) 

46 

47 c beta_2 

48 

49 c 1 -1 

50 

51 beta_2 -1 1 

52 

53 

54 Parameter Estimates 

55 95.0% Wald Confidence Interval 

56 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

57 c 4.21938 0.840997 2.57105 5.8677 

58 beta_0 0 NA 

59 beta_1 0 NA 

60 beta_2 4.00402e-009 1.495e-008 -2.52974e-008 3.33054e-008 

61 

62 NA - Indicates that this parameter has hit a 

63 bound implied by some inequality constraint 

64 and thus has no standard error. 

65 

66 

67 Log(likelihood) # Param AIC 

68 Fitted Model -19.967 4 47.9339 

69 

70 

71 Data Summary 

72 Class 

73 C F I U Total Expected Response 

74 Conc 

75 0 23 0 0 0 23 0.00 

76 0.25 24 0 0 0 24 1.92 

77 1 8 0 18 0 26 16.04 

78 4.3 5 0 18 0 23 18.22 

79 

80 

81 Benchmark Dose Computation 

82 Risk Response = Incidental 

83 Risk Type = Extra 
364 DRAFT - DO NOT CITE OR QUOTE 

1 Specified effect = 0.1 

2 Confidence level = 0.9 

3 

4 Time = 104 

5 

6 BMD = 0.284958 

7 BMDL = 0.197807 

8 BMDU = 0.350247 

9 

10 

11 

12 

0 20 60 100

0.0 0.4 0.8

Dose = 0.00

Time

Probability

0 20 60 100

0.0 0.4 0.8

Dose = 0.25

Time

Probability

0 20 60 100

0.0 0.4 0.8

Dose = 1.00

Time

Probability

0 20 60 100

0.0 0.4 0.8

Dose = 4.29

Time

Probability

Incidental Risk: BaP-Thyssen_inc2st
365 DRAFT - DO NOT CITE OR QUOTE 

1 Output for Oral tract tumors: all tumors considered to be cause of death 

2 

3 ======================================================================= 

4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci 

6 Input Data File: C:\msw\benzo[a]pyrene-Thyssen_allfatal_noU_3st.(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 positive 

14 

15 Dependent variable = Class 

16 Independent variables = Conc, Time 

17 

18 Total number of observations = 96 

19 Total number of records with missing values = 0 

20 Total number of parameters in model = 6 

21 Total number of specified parameters = 1 

22 Degree of polynomial = 3 

23 

24 

25 

26 User specifies the following parameters: 

27 t_0 = 0 

28 

29 Maximum number of iterations = 32 

30 Relative Function Convergence has been set to: 1e-008 

31 Parameter Convergence has been set to: 1e-008 

32 

33 

34 Default Initial Parameter Values 

35 c = 4.5 

36 t_0 = 0 Specified 

37 beta_0 = 0 

38 beta_1 = 1.37501e-010 

39 beta_2 = 2.84027e-010 

40 beta_3 = 1.44668e-037 

41 

42 

43 Asymptotic Correlation Matrix of Parameter Estimates 

44 ( *** The model parameter(s) -t_0 -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 correlation matrix ) 

47 

48 c beta_3 

49 

50 c 1 -1 

51 

52 beta_3 -1 1 

53 

54 

55 Parameter Estimates 

56 95.0% Wald Confidence Interval 

57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

58 c 8.95016 0.896607 7.19284 10.7075 

59 beta_0 0 NA 

60 beta_1 0 NA 

61 beta_2 0 NA 

62 beta_3 3.43452e-019 1.39727e-018 -2.39515e-018 3.08205e-018 

63 

64 NA - Indicates that this parameter has hit a 

65 bound implied by some inequality constraint 

66 and thus has no standard error. 

67 

68 

69 Log(likelihood) # Param AIC 

70 Fitted Model -144.522 5 299.043 

71 

72 

73 Data Summary 

74 Class 

75 C F I U Total 

76 Conc 

77 0 23 0 0 0 23 

78 0.25 24 0 0 0 24 

79 1 8 18 0 0 26 

80 4.3 5 18 0 0 23 

81 

82 Minimum observation time for F tumor context = 40 

83 
366 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Benchmark Dose Computation 

3 Risk Response = Fatal 

4 Risk Type = Extra 

5 Specified effect = 0.1 

6 Confidence level = 0.9 

7 

8 Time = 104 

9 

10 BMD = 0.647659 

11 BMDL = 0.461415 

12 BMDU = 0.719325 

13 

14 

15 

16 

17 

18 

19 
367 DRAFT - DO NOT CITE OR QUOTE 

1 APPENDIX F. BENCHMARK MODELING FOR THE DERMAL SLOPE FACTOR 

2 

Table F-1. Summary of model selection and modeling results for bestfitting multistage models, for multiple data sets of 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 LLb χ

2 c

BMD10 

(μg/d) 

BMDL10

(μg/d) 

Model selection rationale, with best 

fitting model in boldfacea 

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 0.003 205.3b 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 

droppedd

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 residuals <2.0, good fit near BMR, lack of extreme curvature 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 χ2

= 2 × |(LLi – LLj)|, 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 remaining suite of models were fit to 

the data. The POD for Grimmer et al. (1984) was based on the LogLogistic model. For comparison purposes, the multistage model was it 

fit to these data for Grimmer et al. (1984) with the highest dose dropped. 
368 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-1. Fit of multistage model to skin tumors in C57L mice exposed 

2 dermally to benzo[a]pyrene (Poel, 1959); graph and model output. 

3 

4 

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_1959_MultiCanc3_0.1.(d) 

8 Gnuplot Plotting File: 

9 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_1959_MultiCanc3_0.1.plt 

10 

11 ==================================================================== 

12 

13 [add notes here] 

14 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

15 

16 The form of the probability function is: 

17 

18 P[response] = background + (1-background)*[1-EXP( 

19 -beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

20 

21 The parameter betas are restricted to be positive 

22 

23 

24 Dependent variable = NumAff 

25 Independent variable = LADD 

26 

27 Total number of observations = 5 

28 Total number of records with missing values = 0 

29 Total number of parameters in model = 4 

30 Total number of specified parameters = 0 

31 Degree of polynomial = 3 

32 

33 

34 Maximum number of iterations = 250 

35 Relative Function Convergence has been set to: 1e-008 

36 Parameter Convergence has been set to: 1e-008 

37 

38 

39 

40 Default Initial Parameter Values 

41 Background = 0.0449589 

42 Beta(1) = 0.490451 

43 Beta(2) = 0 

44 Beta(3) = 2.68146 

45 

46 

47 Asymptotic Correlation Matrix of Parameter Estimates 

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 0.8

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
369 DRAFT - DO NOT CITE OR QUOTE 

1 ( *** The model parameter(s) -Beta(2) 

2 have been estimated at a boundary point, or have been specified by the user, 

3 and do not appear in the correlation matrix ) 

4 

5 Background Beta(1) Beta(3) 

6 

7 Background 1 -0.87 0.74 

8 

9 Beta(1) -0.87 1 -0.92 

10 

11 Beta(3) 0.74 -0.92 1 

12 

13 

14 

15 Parameter Estimates 

16 

17 95.0% Wald Confidence Interval 

18 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

19 Background 0.0176699 * * * 

20 Beta(1) 0.79766 * * * 

21 Beta(2) 0 * * * 

22 Beta(3) 2.17146 * * * 

23 

24 * - Indicates that this value is not calculated. 

25 

26 

27 

28 Analysis of Deviance Table 

29 

30 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

31 Full model -87.1835 5 

32 Fitted model -90.4265 3 6.48606 2 0.03905 

33 Reduced model -141.614 1 108.86 4 <.0001 

34 

35 AIC: 186.853 

36 

37 

38 Goodness of Fit 

39 Scaled 

40 Dose Est._Prob. Expected Observed Size Residual 

41 ------------------------------------------------------------------------ 

42 0.0000 0.0177 0.583 0.000 33 -0.770 

43 0.0500 0.0563 3.098 5.000 55 1.112 

44 0.1600 0.1430 7.866 11.000 55 1.207 

45 0.2400 0.2128 11.917 7.000 56 -1.605 

46 0.8000 0.8293 40.635 41.000 49 0.139 

47 

48 Chi^2 = 5.88 d.f. = 2 P-value = 0.0528

49 

50 

51 Benchmark Dose Computation 

52 

53 Specified effect = 0.1 

54 

55 Risk Type = Extra risk 

56 

57 Confidence level = 0.95 

58 

59 BMD = 0.126567 

60 

61 BMDL = 0.0777875 

62 

63 BMDU = 0.272961 

64 

65 Taken together, (0.0777875, 0.272961) is a 90 % two-sided confidence 

66 interval for the BMD 

67 

68 Multistage Cancer Slope Factor = 1.28555 

69 

70 

71 
370 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Figure F-2. Fit of multistage model to skin tumors in female Swiss mice 

3 exposed dermally to benzo[a]pyrene (Roe et al., 1970); graph and model 

4 output. 

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 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

31 Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Roe_1970_Setting.(d) 

32 Gnuplot Plotting File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Roe_1970_Setting.plt 

33 

34 ==================================================================== 

35 

36 BMDS Model Run 

37 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

38 

39 The form of the probability function is: 

40 

41 P[response] = background + (1-background)*[1-EXP( 

42 -beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^4-beta5*dose^5)] 

43 

44 The parameter betas are restricted to be positive 

45 

46 

47 Dependent variable = tumors 

48 Independent variable = LADD 

49 

50 Total number of observations = 6 

51 Total number of records with missing values = 0 

52 Total number of parameters in model = 6 

53 Total number of specified parameters = 0 

54 Degree of polynomial = 5 

55 

56 

57 Maximum number of iterations = 250 

58 Relative Function Convergence has been set to: 1e-008 

59 Parameter Convergence has been set to: 1e-008 

60 

61 

62 

63 Default Initial Parameter Values 

64 Background = 0 

0

0.2

0.4

0.6

0.8

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BMDL BMD

Multistage Cancer

Linear extrapolation

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Linear extrapolation
371 DRAFT - DO NOT CITE OR QUOTE 

1 Beta(1) = 0.0962491 

2 Beta(2) = 0.141689 

3 Beta(3) = 0 

4 Beta(4) = 0 

5 Beta(5) = 0 

6 

7 

8 Asymptotic Correlation Matrix of Parameter Estimates 

9 

10 ( *** The model parameter(s) -Beta(3) -Beta(4) -Beta(5) 

11 have been estimated at a boundary point, or have been specified by the user, 

12 and do not appear in the correlation matrix ) 

13 

14 Background Beta(1) Beta(2) 

15 

16 Background 1 -0.57 0.45 

17 

18 Beta(1) -0.57 1 -0.94 

19 

20 Beta(2) 0.45 -0.94 1 

21 

22 

23 

24 Parameter Estimates 

25 

26 95.0% Wald Confidence Interval 

27 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

28 Background 0.00584893 * * * 

29 Beta(1) 0.0379152 * * * 

30 Beta(2) 0.166839 * * * 

31 Beta(3) 0 * * * 

32 Beta(4) 0 * * * 

33 Beta(5) 0 * * * 

34 

35 * - Indicates that this value is not calculated. 

36 

37 

38 

39 Analysis of Deviance Table 

40 

41 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

42 Full model -56.1835 6 

43 Fitted model -57.5694 3 2.77176 3 0.4282 

44 Reduced model -118.948 1 125.529 5 <.0001 

45 

46 AIC: 121.139 

47 

48 

49 Goodness of Fit 

50 Scaled 

51 Dose Est._Prob. Expected Observed Size Residual 

52 ------------------------------------------------------------------------ 

53 0.0000 0.0058 0.275 0.000 47 -0.526 

54 0.0300 0.0071 0.321 1.000 45 1.204 

55 0.0900 0.0106 0.444 0.000 42 -0.670 

56 0.3100 0.0331 1.423 1.000 43 -0.361 

57 0.9200 0.1664 6.821 8.000 41 0.494 

58 2.7600 0.7488 34.444 34.000 46 -0.151 

59 

60 Chi^2 = 2.57 d.f. = 3 P-value = 0.4626

61 

62 

63 Benchmark Dose Computation 

64 

65 Specified effect = 0.1 

66 

67 Risk Type = Extra risk 

68 

69 Confidence level = 0.95 

70 

71 BMD = 0.689131 

72 

73 BMDL = 0.393806 

74 

75 BMDU = 0.952365 

76 

77 Taken together, (0.393806, 0.952365) is a 90 % two-sided confidence 

78 interval for the BMD 

79 

80 Multistage Cancer Slope Factor = 0.253932 

81 

82 
372 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-3. Fit of multistage model to skin tumors in female NMRI mice 

2 exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph and model 

3 output. 

4 

5 

6 ==================================================================== 

7 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

8 Input Data File: 

9 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973femaleNMRI\2MulSchMS_.(d) 

10 Gnuplot Plotting File: 

11 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973femaleNMRI\2MulSchMS_.plt 

12 

13 ==================================================================== 

14 

15 BMDS Model Run 

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 positive 

24 

25 

26 Dependent variable = incidence 

27 Independent variable = dose 

28 

29 Total number of observations = 5 

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: 2.22045e-016 

38 Parameter Convergence has been set to: 1.49012e-008 

39 

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Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
373 DRAFT - DO NOT CITE OR QUOTE 

1 **** We are sorry but Relative Function and Parameter Convergence   

2 ** ** are currently unavailable in this model. Please keep checking   

3 ** ** the web sight for model updates which will eventually   

4 ** ** incorporate these convergence criterion. Default values used.   

5 

6 

7 

8 Default Initial Parameter Values 

9 Background = 0 

10 Beta(1) = 0 

11 Beta(2) = 1.11271 

12 

13 

14 Asymptotic Correlation Matrix of Parameter Estimates 

15 

16 ( ** * The model parameter(s) -Background -Beta(1) 

17 have been estimated at a boundary point, or have been specified by the user, 

18 and do not appear in the correlation matrix ) 

19 

20 Beta(2) 

21 

22 Beta(2) 1 

23 

24 

25 

26 Parameter Estimates 

27 

28 95.0% Wald Confidence Interval 

29 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

30 Background 0 * * * 

31 Beta(1) 0 * * * 

32 Beta(2) 0.970648 * * * 

33 

34 * - Indicates that this value is not calculated. 

35 

36 

37 

38 Analysis of Deviance Table 

39 

40 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

41 Full model -70.8903 5 

42 Fitted model -72.6831 1 3.58562 4 0.465 

43 Reduced model -118.917 1 96.054 4 <.0001 

44 

45 AIC: 147.366 

46 

47 

48 Goodness of Fit 

49 Scaled 

50 Dose Est._Prob. Expected Observed Size Residual 

51 ------------------------------------------------------------------------ 

52 0.0000 0.0000 0.000 0.000 100 0.000 

53 0.0100 0.0001 0.010 0.000 100 -0.099 

54 0.0600 0.0035 0.349 0.000 100 -0.592 

55 0.2300 0.0501 5.005 2.000 100 -1.378 

56 0.5700 0.2705 27.048 30.000 100 0.665 

57 

58 Chi^2 = 2.70 d.f. = 4 P-value = 0.6091

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 = 0.329464 

70 

71 BMDL = 0.286624 

72 

73 BMDU = 0.384046 

74 

75 Taken together, (0.286624, 0.384046) is a 90 % two-sided confidence 

76 interval for the BMD 

77 

78 Multistage Cancer Slope Factor = 0.348889 

79 

80 
374 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-4. Fit of multistage model to skin tumors in female Swiss mice 

2 exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph and model 

3 output. 

4 

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9 

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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\Schmidt1973swissmice\3MulSchMS_.(d) 

38 Gnuplot Plotting File: 

39 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973swissmice\3MulSchMS_.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-beta3*dose^3)] 

49 

50 The parameter betas are restricted to be positive 

51 

52 

53 Dependent variable = incidence 

54 Independent variable = dose 

55 

56 Total number of observations = 5 

57 Total number of records with missing values = 0 

58 Total number of parameters in model = 4 

59 Total number of specified parameters = 0 

60 Degree of polynomial = 3 

61 

62 

63 Maximum number of iterations = 250 

64 Relative Function Convergence has been set to: 2.22045e-016 

65 Parameter Convergence has been set to: 1.49012e-008 

66 

67 **** We are sorry but Relative Function and Parameter Convergence   

68 ** ** are currently unavailable in this model. Please keep checking   

69 ** ** the web sight for model updates which will eventually   

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BMDL BMD

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Linear extrapolation

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BMDL BMD

Multistage Cancer

Linear extrapolation
375 DRAFT - DO NOT CITE OR QUOTE 

1 ** ** incorporate these convergence criterion. Default values used.   

2 

3 

4 

5 Default Initial Parameter Values 

6 Background = 0 

7 Beta(1) = 0 

8 Beta(2) = 0.338951 

9 Beta(3) = 3.8728 

10 

11 

12 Asymptotic Correlation Matrix of Parameter Estimates 

13 

14 ( ** * The model parameter(s) -Background -Beta(1) 

15 have been estimated at a boundary point, or have been specified by the user, 

16 and do not appear in the correlation matrix ) 

17 

18 Beta(2) Beta(3) 

19 

20 Beta(2) 1 -0.99 

21 

22 Beta(3) -0.99 1 

23 

24 

25 

26 Parameter Estimates 

27 

28 95.0% Wald Confidence Interval 

29 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

30 Background 0 * * * 

31 Beta(1) 0 * * * 

32 Beta(2) 0.108125 * * * 

33 Beta(3) 4.31441 * * * 

34 

35 * - Indicates that this value is not calculated. 

36 

37 

38 

39 Analysis of Deviance Table 

40 

41 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

42 Full model -73.5285 5 

43 Fitted model -73.6628 2 0.268637 3 0.9658 

44 Reduced model -150.708 1 154.359 4 <.0001 

45 

46 AIC: 151.326 

47 

48 

49 Goodness of Fit 

50 Scaled 

51 Dose Est._Prob. Expected Observed Size Residual 

52 ------------------------------------------------------------------------ 

53 0.0000 0.0000 0.000 0.000 80 0.000 

54 0.0100 0.0000 0.001 0.000 80 -0.035 

55 0.0600 0.0013 0.106 0.000 80 -0.325 

56 0.2300 0.0566 4.524 5.000 80 0.230 

57 0.5700 0.5657 45.260 45.000 80 -0.059 

58 

59 Chi^2 = 0.16 d.f. = 3 P-value = 0.9833

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 = 0.282007 

71 

72 BMDL = 0.223401 

73 

74 BMDU = 0.309888 

75 

76 Taken together, (0.223401, 0.309888) is a 90 % two-sided confidence 

77 interval for the BMD 

78 

79 Multistage Cancer Slope Factor = 0.447626 

80 

81 

82 

83 
376 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-5. Fit of multistage model to skin tumors in female NMRI mice 

2 exposed dermally to benzo[a]pyrene (Schmähl et al., 1977); graph and model 

3 output. 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

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17 

18 

19 

20 

21 

22 

23 

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29 

30 

31 

32 

33 

34 

35 ==================================================================== 

36 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

37 Input Data File: 

38 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahl1977femaleNMRI\2MulschMS_.(d) 

39 Gnuplot Plotting File: 

40 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahl1977femaleNMRI\2MulschMS_.plt 

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-beta2*dose^2)] 

50 

51 The parameter betas are restricted to be positive 

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 = 3 

60 Total number of specified parameters = 0 

61 Degree of polynomial = 2 

62 

63 

64 Maximum number of iterations = 250 

65 Relative Function Convergence has been set to: 2.22045e-016 

66 Parameter Convergence has been set to: 1.49012e-008 

67 

68 **** We are sorry but Relative Function and Parameter Convergence   

69 ** ** are currently unavailable in this model. Please keep checking   

70 ** ** the web sight for model updates which will eventually   

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BMDL BMD

Multistage Cancer

Linear extrapolation

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Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
377 DRAFT - DO NOT CITE OR QUOTE 

1 ** ** incorporate these convergence criterion. Default values used. **** 

2 

3 

4 

5 Default Initial Parameter Values 

6 Background = 0.0115034 

7 Beta(1) = 0.284955 

8 Beta(2) = 0.750235 

9 

10 

11 Asymptotic Correlation Matrix of Parameter Estimates 

12 

13 Background Beta(1) Beta(2) 

14 

15 Background 1 -0.67 0.47 

16 

17 Beta(1) -0.67 1 -0.94 

18 

19 Beta(2) 0.47 -0.94 1 

20 

21 

22 

23 Parameter Estimates 

24 

25 95.0% Wald Confidence Interval 

26 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

27 Background 0.0123066 * * * 

28 Beta(1) 0.274413 * * * 

29 Beta(2) 0.764244 * * * 

30 

31 * - Indicates that this value is not calculated. 

32 

33 

34 

35 Analysis of Deviance Table 

36 

37 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

38 Full model -145.127 4 

39 Fitted model -145.13 3 0.00579898 1 0.9393 

40 Reduced model -184.158 1 78.0608 3 <.0001 

41 

42 AIC: 296.261 

43 

44 

45 Goodness of Fit 

46 Scaled 

47 Dose Est._Prob. Expected Observed Size Residual 

48 ------------------------------------------------------------------------ 

49 0.0000 0.0123 0.997 1.000 81 0.003 

50 0.2900 0.1446 11.137 11.000 77 -0.045 

51 0.4900 0.2813 24.756 25.000 88 0.058 

52 0.8600 0.5567 45.096 45.000 81 -0.022 

53 

54 Chi^2 = 0.01 d.f. = 1 P-value = 0.9393

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 = 0.232893 

66 

67 BMDL = 0.148895 

68 

69 BMDU = 0.320396 

70 

71 Taken together, (0.148895, 0.320396) is a 90 % two-sided confidence 

72 interval for the BMD 

73 

74 Multistage Cancer Slope Factor = 0.671616 

75 

76 

77 

78 
378 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-6. Fit of multistage model to skin tumors in female NMRI mice 

2 exposed dermally to benzo[a]pyrene (Habs et al., 1980); graph and model 

3 output. 

4 

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BMDL BMD

Multistage Cancer

Linear extrapolation
379 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 

4 

5 

6 ==================================================================== 

7 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

8 Input Data File: M:\_BMDS\msc_BAP_HABS1980_MultiCanc3_0.1.(d) 

9 Gnuplot Plotting File: M:\_BMDS\msc_BAP_HABS1980_MultiCanc3_0.1.plt 

10 

11 ==================================================================== 

12 

13 BMDS Model Run 

14 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

15 

16 The form of the probability function is: 

17 

18 P[response] = background + (1-background)*[1-EXP( 

19 -beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

20 

21 The parameter betas are restricted to be positive 

22 

23 

24 Dependent variable = NumAff 

25 Independent variable = LADD 

26 

27 Total number of observations = 4 

28 Total number of records with missing values = 0 

29 Total number of parameters in model = 4 

30 Total number of specified parameters = 0 

31 Degree of polynomial = 3 

32 

33 

34 Maximum number of iterations = 250 

35 Relative Function Convergence has been set to: 1e-008 

36 Parameter Convergence has been set to: 1e-008 

37 

38 

39 

40 Default Initial Parameter Values 

41 Background = 0 

42 Beta(1) = 0 

43 Beta(2) = 4.23649 

44 Beta(3) = 0 

45 

46 

47 Asymptotic Correlation Matrix of Parameter Estimates 

48 

49 ( *** The model parameter(s) -Background -Beta(1) -Beta(2) 

50 have been estimated at a boundary point, or have been specified 

51 by the user, 

52 and do not appear in the correlation matrix ) 

53 

54 Beta(3) 

55 

56 Beta(3) 1 

57 

58 

59 

60 Parameter Estimates 

61 

62 95.0% Wald Confidence 

63 Interval 

64 Variable Estimate Std. Err. Lower Conf. Limit Upper 

65 Conf. Limit 

66 Background 0 * * * 

67 Beta(1) 0 * * * 

68 Beta(2) 0 * * * 

69 Beta(3) 4.1289 * * * 

70 

71 * - Indicates that this value is not calculated. 

72 

73 

74 

75 Analysis of Deviance Table 

76 

77 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

78 Full model -34.8527 4 

79 Fitted model -37.3373 1 4.96903 3 0.1741 

80 Reduced model -82.5767 1 95.4478 3 <.0001 

81 

82 AIC: 76.6745 

83 

84 
380 DRAFT - DO NOT CITE OR QUOTE 

1 Goodness of Fit 

2 Scaled 

3 Dose Est._Prob. Expected Observed Size Residual 

4 ------------------------------------------------------------------------ 

5 0.0000 0.0000 0.000 0.000 35 0.000 

6 0.4900 0.3848 13.082 8.000 34 -1.791 

7 0.7400 0.8123 21.933 24.000 27 1.019 

8 0.8000 0.8792 21.102 22.000 24 0.563 

9 

10 Chi^2 = 4.56 d.f. = 3 P-value = 0.2067

11 

12 

13 Benchmark Dose Computation 

14 

15 Specified effect = 0.1 

16 

17 Risk Type = Extra risk 

18 

19 Confidence level = 0.95 

20 

21 BMD = 0.294407 

22 

23 BMDL = 0.215151 

24 

25 BMDU = 0.320955 

26 

27 Taken together, (0.215151, 0.320955) is a 90 % two-sided confidence 

28 interval for the BMD 

29 

30 Multistage Cancer Slope Factor = 0.46479 

31 

32 
381 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-7. Fit of multistage model to skin tumors in female NMRI mice 

2 exposed dermally to benzo[a]pyrene (Habs et al., 1984); graph and model 

3 output. 

4 

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30 

31 ==================================================================== 

32 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

33 Input Data File: C:\Usepa\BMDS21\mscDax_Setting.(d) 

34 Gnuplot Plotting File: C:\Usepa\BMDS21\mscDax_Setting.plt 

35 

36 ==================================================================== 

37 

38 BMDS Model Run 

39 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

40 

41 The form of the probability function is: 

42 

43 P[response] = background + (1-background)*[1-EXP( 

44 -beta1*dose^1)] 

45 

46 The parameter betas are restricted to be positive 

47 

48 

49 Dependent variable = tumors 

50 Independent variable = LADD 

51 

52 Total number of observations = 3 

53 Total number of records with missing values = 0 

54 Total number of parameters in model = 2 

55 Total number of specified parameters = 0 

56 Degree of polynomial = 1 

57 

58 

59 Maximum number of iterations = 250 

60 Relative Function Convergence has been set to: 1e-008 

61 Parameter Convergence has been set to: 1e-008 

62 

63 

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Multistage Cancer

Linear extrapolation

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Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
382 DRAFT - DO NOT CITE OR QUOTE 

1 

2 Default Initial Parameter Values 

3 Background = 0 

4 Beta(1) = 1.66414 

5 

6 

7 Asymptotic Correlation Matrix of Parameter Estimates 

8 

9 ( *** The model parameter(s) -Background 

10 have been estimated at a boundary point, or have been specified by the user, 

11 and do not appear in the correlation matrix ) 

12 

13 Beta(1) 

14 

15 Beta(1) 1 

16 

17 

18 

19 Parameter Estimates 

20 

21 95.0% Wald Confidence Interval 

22 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

23 Background 0 * * * 

24 Beta(1) 1.35264 * * * 

25 

26 * - Indicates that this value is not calculated. 

27 

28 

29 

30 Analysis of Deviance Table 

31 

32 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

33 Full model -22.217 3 

34 Fitted model -22.7878 1 1.14175 2 0.565 

35 Reduced model -41.0539 1 37.6739 2 <.0001 

36 

37 AIC: 47.5757 

38 

39 

40 Goodness of Fit 

41 Scaled 

42 Dose Est._Prob. Expected Observed Size Residual 

43 ------------------------------------------------------------------------ 

44 0.0000 0.0000 0.000 0.000 20 0.000 

45 0.5700 0.5375 10.749 9.000 20 -0.784 

46 1.1400 0.7860 15.721 17.000 20 0.697 

47 

48 Chi^2 = 1.10 d.f. = 2 P-value = 0.5765

49 

50 

51 Benchmark Dose Computation 

52 

53 Specified effect = 0.1 

54 

55 Risk Type = Extra risk 

56 

57 Confidence level = 0.95 

58 

59 BMD = 0.0778926 

60 

61 BMDL = 0.0558466 

62 

63 BMDU = 0.111853 

64 

65 Taken together, (0.0558466, 0.111853) is a 90 % two-sided confidence 

66 interval for the BMD 

67 

68 Multistage Cancer Slope Factor = 1.79062 

69 
383 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-8. Fit of multistage model to skin tumors in female CFLP mice 

2 exposed dermally to benzo[a]pyrene (Grimmer et al, 1983); graph and model 

3 output. 

4 

5 

6 

7 

8 

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31 

32 

33 

34 

35 ==================================================================== 

36 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

37 Input Data File: 

38 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmer1983CFLPmice\1MulGriMS_.(d) 

39 Gnuplot Plotting File: 

40 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmer1983CFLPmice\1MulGriMS_.plt 

41 

42 ==================================================================== 

43 

44 BMDS Model Run 

45 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

46 

47 The form of the probability function is: 

48 

49 P[response] = background + (1-background)*[1-EXP( 

50 -beta1*dose^1)] 

51 

52 The parameter betas are restricted to be positive 

53 

54 

55 Dependent variable = incidence 

56 Independent variable = dose 

57 

58 Total number of observations = 4 

59 Total number of records with missing values = 0 

60 Total number of parameters in model = 2 

61 Total number of specified parameters = 0 

62 Degree of polynomial = 1 

63 

64 

65 Maximum number of iterations = 250 

66 Relative Function Convergence has been set to: 2.22045e-016 

67 Parameter Convergence has been set to: 1.49012e-008 

68 

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Multistage Cancer

Linear extrapolation

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Multistage Cancer

Linear extrapolation
384 DRAFT - DO NOT CITE OR QUOTE 

1 **** We are sorry but Relative Function and Parameter Convergence   

2 ** ** are currently unavailable in this model. Please keep checking   

3 ** ** the web sight for model updates which will eventually   

4 ** ** incorporate these convergence criterion. Default values used.   

5 

6 

7 

8 Default Initial Parameter Values 

9 Background = 0 

10 Beta(1) = 0.478645 

11 

12 

13 Asymptotic Correlation Matrix of Parameter Estimates 

14 

15 ( ** * The model parameter(s) -Background 

16 have been estimated at a boundary point, or have been specified by the user, 

17 and do not appear in the correlation matrix ) 

18 

19 Beta(1) 

20 

21 Beta(1) 1 

22 

23 

24 

25 Parameter Estimates 

26 

27 95.0% Wald Confidence Interval 

28 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

29 Background 0 * * * 

30 Beta(1) 0.430366 * * * 

31 

32 * - Indicates that this value is not calculated. 

33 

34 

35 

36 Analysis of Deviance Table 

37 

38 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

39 Full model -108.532 4 

40 Fitted model -108.943 1 0.823537 3 0.8438 

41 Reduced model -186.434 1 155.805 3 <.0001 

42 

43 AIC: 219.887 

44 

45 

46 Goodness of Fit 

47 Scaled 

48 Dose Est._Prob. Expected Observed Size Residual 

49 ------------------------------------------------------------------------ 

50 0.0000 0.0000 0.000 0.000 80 -0.000 

51 1.1100 0.3798 24.687 22.000 65 -0.687 

52 2.2000 0.6120 39.169 39.000 64 -0.043 

53 4.4000 0.8495 54.366 56.000 64 0.571 

54 

55 Chi^2 = 0.80 d.f. = 3 P-value = 0.8496

56 

57 

58 Benchmark Dose Computation 

59 

60 Specified effect = 0.1 

61 

62 Risk Type = Extra risk 

63 

64 Confidence level = 0.95 

65 

66 BMD = 0.244816 

67 

68 BMDL = 0.208269 

69 

70 BMDU = 0.289606 

71 

72 Taken together, (0.208269, 0.289606) is a 90 % two-sided confidence 

73 interval for the BMD 

74 

75 Multistage Cancer Slope Factor = 0.480148 

76 

77 

78 
385 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-9. Fit of multistage model to skin tumors in female CFLP mice 

2 exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and 

3 model output. 

4 

0

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Multistage Cancer

Linear extrapolation
386 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_MultiCanc1_0.1.(d) 

7 Gnuplot Plotting File: 

8 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984_MultiCanc1_0.1.plt 

9 Wed Apr 27 17:11:28 2011 

10 

11 [add notes here] 

12 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

13 

14 The form of the probability function is: 

15 

16 P[response] = background + (1-background)*[1-EXP( 

17 -beta1*dose^1)] 

18 

19 The parameter betas are restricted to be positive 

20 

21 

22 Dependent variable = NumAff 

23 Independent variable = LADD 

24 

25 Total number of observations = 4 

26 Total number of records with missing values = 0 

27 Total number of parameters in model = 2 

28 Total number of specified parameters = 0 

29 Degree of polynomial = 1 

30 

31 

32 Maximum number of iterations = 250 

33 Relative Function Convergence has been set to: 1e-008 

34 Parameter Convergence has been set to: 1e-008 

35 

36 

37 

38 Default Initial Parameter Values 

39 Background = 0.311241 

40 Beta(1) = 0.502556 

41 

42 

43 Asymptotic Correlation Matrix of Parameter Estimates 

44 

45 ( *** The model parameter(s) -Background 

46 have been estimated at a boundary point, or have been specified 

47 by the user, 

48 and do not appear in the correlation matrix ) 

49 

50 Beta(1) 

51 

52 Beta(1) 1 

53 

54 

55 

56 Parameter Estimates 

57 

58 95.0% Wald Confidence 

59 Interval 

60 Variable Estimate Std. Err. Lower Conf. Limit Upper 

61 Conf. Limit 

62 Background 0 * * * 

63 Beta(1) 0.796546 * * * 

64 

65 * - Indicates that this value is not calculated. 

66 

67 

68 

69 Analysis of Deviance Table 

70 

71 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

72 Full model -95.8385 4 

73 Fitted model -101.643 1 11.61 3 0.008846 

74 Reduced model -175.237 1 158.797 3 <.0001 

75 

76 AIC: 205.287 

77 

78 

79 Goodness of Fit 

80 Scaled 

81 Dose Est._Prob. Expected Observed Size Residual 

82 ------------------------------------------------------------------------ 

83 0.0000 0.0000 0.000 0.000 65 0.000 
387 DRAFT - DO NOT CITE OR QUOTE 

1 0.9700 0.5382 34.446 43.000 64 2.145 

2 1.9100 0.7816 50.804 53.000 65 0.659 

3 3.9000 0.9552 62.091 57.000 65 -3.054 

4 

5 Chi^2 = 14.36 d.f. = 3 P-value = 0.0025

6 

7 

8 Benchmark Dose Computation 

9 

10 Specified effect = 0.1 

11 

12 Risk Type = Extra risk 

13 

14 Confidence level = 0.95 

15 

16 BMD = 0.132272 

17 

18 BMDL = 0.113427 

19 

20 BMDU = 0.154848 

21 

22 Taken together, (0.113427, 0.154848) is a 90 % two-sided confidence 

23 interval for the BMD 

24 

25 Multistage Cancer Slope Factor = 0.881621 

26 
388 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-9. Fit of log-logistic model to skin tumors in female CFLP mice 

2 exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and 

3 model output. 

4 

5 

6 ==================================================================== 

7 Logistic Model. (Version: 2.12; Date: 05/16/2008) 

8 Input Data File: 

9 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmer1984_Grimmer1984_0.70u.(d) 

10 Gnuplot Plotting File: 

11 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmer1984_Grimmer1984_0.70u.plt 

12 

13 ==================================================================== 

14 

15 BMDS Model Run 

16 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

17 

18 The form of the probability function is: 

19 

20 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 

21 

22 

23 Dependent variable = NumAff 

24 Independent variable = LADD 

25 Slope parameter is not restricted 

26 

27 Total number of observations = 4 

28 Total number of records with missing values = 0 

29 Maximum number of iterations = 250 

30 Relative Function Convergence has been set to: 1e-008 

31 Parameter Convergence has been set to: 1e-008 

32 

33 

34 

35 User has chosen the log transformed model 

36 

37 

38 Default Initial Parameter Values 

39 background = 0 

40 intercept = 0.799142 

41 slope = 0.894129 

42 

43 

44 Asymptotic Correlation Matrix of Parameter Estimates 

45 

46 ( *** The model parameter(s) -background 

47 have been estimated at a boundary point, or have been specified 

48 by the user, 

0

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Log-Logistic
389 DRAFT - DO NOT CITE OR QUOTE 

1 and do not appear in the correlation matrix ) 

2 

3 intercept slope 

4 

5 intercept 1 -0.68 

6 

7 slope -0.68 1 

8 

9 

10 

11 Parameter Estimates 

12 

13 95.0% Wald Confidence 

14 Interval 

15 Variable Estimate Std. Err. Lower Conf. Limit Upper 

16 Conf. Limit 

17 background 0 * * * 

18 intercept 0.783559 * * * 

19 slope 0.922655 * * * 

20 

21 * - Indicates that this value is not calculated. 

22 

23 

24 

25 Analysis of Deviance Table 

26 

27 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

28 Full model -95.8385 4 

29 Fitted model -95.9236 2 0.17031 2 0.9184 

30 Reduced model -175.237 1 158.797 3 <.0001 

31 

32 AIC: 195.847 

33 

34 

35 Goodness of Fit 

36 Scaled 

37 Dose Est._Prob. Expected Observed Size Residual 

38 ------------------------------------------------------------------------ 

39 0.0000 0.0000 0.000 0.000 65 0.000 

40 0.9700 0.6804 43.543 43.000 64 -0.146 

41 1.9100 0.7991 51.941 53.000 65 0.328 

42 3.9000 0.8849 57.516 57.000 65 -0.200 

43 

44 Chi^2 = 0.17 d.f. = 2 P-value = 0.9190

45 

46 

47 Benchmark Dose Computation 

48 

49 Specified effect = 0.7 

50 

51 Risk Type = Extra risk 

52 

53 Confidence level = 0.95 

54 

55 BMD = 1.07152 

56 

57 BMDL = 0.478669 

58 
390 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-11. Fit of multistage model to skin tumors in female CFLP mice 

2 exposed dermally to benzo[a]pyrene (Grimmer et al., 1984), highest dose 

3 dropped; graph and model output. 

4 

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BMDL BMD

Multistage Cancer

Linear extrapolation

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Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
391 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 

4 ==================================================================== 

5 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

6 Input Data File: 

7 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984_drophidose_Setting.(d) 

8 Gnuplot Plotting File: 

9 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984_drophidose_Setting.plt 

10 

11 ==================================================================== 

12 

13 BMDS Model Run 

14 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

15 

16 The form of the probability function is: 

17 

18 P[response] = background + (1-background)*[1-EXP( 

19 -beta1*dose^1-beta2*dose^2)] 

20 

21 The parameter betas are restricted to be positive 

22 

23 

24 Dependent variable = tumors 

25 Independent variable = LADD 

26 

27 Total number of observations = 3 

28 Total number of records with missing values = 0 

29 Total number of parameters in model = 3 

30 Total number of specified parameters = 0 

31 Degree of polynomial = 2 

32 

33 

34 Maximum number of iterations = 250 

35 Relative Function Convergence has been set to: 1e-008 

36 Parameter Convergence has been set to: 1e-008 

37 

38 

39 

40 Default Initial Parameter Values 

41 Background = 0.0806622 

42 Beta(1) = 0.88595 

43 Beta(2) = 0 

44 

45 

46 Asymptotic Correlation Matrix of Parameter Estimates 

47 

48 ( *** The model parameter(s) -Background -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 correlation matrix ) 

52 

53 Beta(1) 

54 

55 Beta(1) 1 

56 

57 

58 

59 Parameter Estimates 

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) 0.997118 * * * 

67 Beta(2) 0 * * * 

68 

69 * - Indicates that this value is not calculated. 

70 

71 

72 

73 Analysis of Deviance Table 

74 

75 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

76 Full model -71.5928 3 

77 Fitted model -72.2756 1 1.36568 2 0.5052 

78 Reduced model -134.46 1 125.735 2 <.0001 

79 

80 AIC: 146.551 

81 

82 

83 Goodness of Fit 

84 Scaled 
392 DRAFT - DO NOT CITE OR QUOTE 

1 Dose Est._Prob. Expected Observed Size Residual 

2 ------------------------------------------------------------------------ 

3 0.0000 0.0000 0.000 0.000 65 0.000 

4 0.9700 0.6199 39.671 43.000 64 0.857 

5 1.9100 0.8511 55.322 53.000 65 -0.809 

6 

7 Chi^2 = 1.39 d.f. = 2 P-value = 0.4992

8 

9 

10 Benchmark Dose Computation 

11 

12 Specified effect = 0.1 

13 

14 Risk Type = Extra risk 

15 

16 Confidence level = 0.95 

17 

18 BMD = 0.105665 

19 

20 BMDL = 0.0881529 

21 

22 BMDU = 0.149328 

23 

24 Taken together, (0.0881529, 0.149328) is a 90 % two-sided confidence 

25 interval for the BMD 

26 

27 Multistage Cancer Slope Factor = 1.13439 

28 

29 

30 

31 

32 

33 
393 DRAFT - DO NOT CITE OR QUOTE 

1 Figure F-12. Fit of multistage model to skin tumors in female CFLP mice 

2 exposed dermally to benzo[a]pyrene (Sivak et al., 1997); graph and model 

3 output. 

4 

5 

6 

7 

8 ==================================================================== 

9 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

10 Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivak1993_MultiCanc2_0.1.(d) 

11 Gnuplot Plotting File: 

12 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivak1993_MultiCanc2_0.1.plt 

13 

14 ==================================================================== 

15 

16 [add notes here] 

17 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

18 

19 The form of the probability function is: 

20 

21 P[response] = background + (1-background)*[1-EXP( 

22 -beta1*dose^1-beta2*dose^2)] 

23 

24 The parameter betas are restricted to be positive 

25 

26 

27 Dependent variable = NumAff 

28 Independent variable = LADD 

29 

30 Total number of observations = 4 

31 Total number of records with missing values = 0 

32 Total number of parameters in model = 3 

33 Total number of specified parameters = 0 

34 Degree of polynomial = 2 

35 

36 

37 Maximum number of iterations = 250 

38 Relative Function Convergence has been set to: 1e-008 

39 Parameter Convergence has been set to: 1e-008 

40 

41 

42 

43 Default Initial Parameter Values 

44 Background = 0 

45 Beta(1) = 0.0936505 

46 Beta(2) = 8.67239 

47 

48 

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Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
394 DRAFT - DO NOT CITE OR QUOTE 

1 Asymptotic Correlation Matrix of Parameter Estimates 

2 

3 ( *** The model parameter(s) -Background -Beta(1) 

4 have been estimated at a boundary point, or have been specified by the user, 

5 and do not appear in the correlation matrix ) 

6 

7 Beta(2) 

8 

9 Beta(2) 1 

10 

11 

12 

13 Parameter Estimates 

14 

15 95.0% Wald Confidence Interval 

16 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

17 Background 0 * * * 

18 Beta(1) 0 * * * 

19 Beta(2) 8.9375 * * * 

20 

21 * - Indicates that this value is not calculated. 

22 

23 

24 

25 Analysis of Deviance Table 

26 

27 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

28 Full model -23.2693 4 

29 Fitted model -23.3009 1 0.0631003 3 0.9959 

30 Reduced model -69.5898 1 92.641 3 <.0001 

31 

32 AIC: 48.6018 

33 

34 

35 Goodness of Fit 

36 Scaled 

37 Dose Est._Prob. Expected Observed Size Residual 

38 ------------------------------------------------------------------------ 

39 0.0000 0.0000 0.000 0.000 30 0.000 

40 0.0100 0.0009 0.027 0.000 30 -0.164 

41 0.1400 0.1607 4.821 5.000 30 0.089 

42 0.5100 0.9022 27.065 27.000 30 -0.040 

43 

44 Chi^2 = 0.04 d.f. = 3 P-value = 0.9982

45 

46 

47 Benchmark Dose Computation 

48 

49 Specified effect = 0.1 

50 

51 Risk Type = Extra risk 

52 

53 Confidence level = 0.95 

54 

55 BMD = 0.108575 

56 

57 BMDL = 0.058484 

58 

59 BMDU = 0.129641 

60 

61 Taken together, (0.058484, 0.129641) is a 90 % two-sided confidence 

62 interval for the BMD 

63 

64 Multistage Cancer Slope Factor = 1.70987 

65 
395 DRAFT - DO NOT CITE OR QUOTE 

1 APPENDIX G. Additional Information in Support of the Dermal Slope factor 

2 

3 Mouse Dermal Carcinogenesis Exposure Methods 

4 Studies which macroscopically examined systemic organs for tumors include Roe et al., 

5 1970; Schmidt et al., 1973; Schmahl et al., 1977; Habs et al., 1980, 1984; Grimmer et al., 1983, 

6 1984. The studies by Roe et al. 1970 and Habs et al. 1984 observed systemic tumors which the 

7 authors did not consider to be treatment related. The other studies which conducted post mortem 

8 macroscopic examinations of abnormal tissues, did not report any treatment related systemic 

9 effects. 
396 DRAFT - DO NOT CITE OR QUOTE 

Table G-1: Exposure methods for selected lifetime dermal exposure mouse cancer bioassays for benzo

induced skin tumors

Mouse strain sex  n 

Applied dose of benzo[a]pyrene, times per week, vehicle 

Application method, volume Application region Duration 

(weeks) 

Comments 

CeH/HeJ male 30 

0, 0.05, 0.5, or 5 μg 

2x/wk cyclohexane/acetone (1:1) 

not specified 0.050 ml 

shaved dorsal skin 104 

C57L male 13-55 

0, 0.15, 0.38, 0.75, 3.8, 19, 94, 188, 376, or 752 μg 

3x/wk toluene 

calibrated needle dropper 0.0075 ml (solvent let dry between drops to limit spread) 

shaved interscapular skin 

103 mice were 18-20 

age at the start o“principal organexamined 

SWR male 14-25 

0, 0.15, 0.38, 0.75, 3.8, 19.0, 94.0, or 470 μg 

3x/wk toluene 

Calibrated needle pipette 0.0075 ml 

Shaved interscapular skin 

lifetime1 exposed until tu

development or 

C3HeB male 14-25 

0, 0.15, 0.38, 0.75, 3.8, 19.0, 94.0, or 470 μg 

benzo[a]pyrene 3x/wk toluene 

Calibrated needle pipette 0.0075ml 

Shaved interscapular skin 

104 lifetime1

exposed until tudevelopment or 

A/He male 14-25 

0, 0.15, 0.38, 0.75, 3.8, 19.0, 94.0, or 470 μg 

benzo[a]pyrene 3x/wk toluene 

Calibrated needle pipette 0.0075 ml 

Shaved interscapular skin 

104 lifetime1

exposed until tudevelopment or 

Swiss SPF female 50 

0, 0.1, 0.3, 1, 3, or 9 μg 

3x/wk acetone 

Calibrated pipette 0.25 ml 

Entire shaved dorsal area 

93 systemic post-m

exam 
397 DRAFT - DO NOT CITE OR QUOTE 

1 1 Treated until natural death or sacrifice following tumor formation

2 

NMRI female 100 

0, 0.05, 0.2, 0.8, or 2 μg 

2x/wk acetone 

Drop burette (dose delivered in single drop; volume not given) 

Shaved back 104 

lifetime1

macroscopic exof internal organ

Swiss SPF female 80 

0, 0.05, 0.2, 0.8, or 2 μg 

2x/wk acetone 

Drop burette (dose delivered in single drop; volume not given) 

Shaved back 104 

lifetime1

macroscopic exof internal organ

NMRI female 100 

0, 1, 1.7, or 3 μg 

2x/wk acetone 

Drop application with syringe 0.02 ml 

Shaved back 104 

lifetime1

macroscopic exof internal organ

NMRI female 65 

0, 1.7, 2.8, and 4.6 ug  2x/wk 

Drop application by calibrated syringe, 0.02 ml 

dorsal skin in interscapular area 

104 lifetime1

 macroscopic exof internal organ

CFLP female 65 

0, 3.9, 7.7 and 15.4 μg 

acetone/DMSO (1:3) 2x/wk 

Drop 0.1 ml 

Dorsal skin, interscapular area 

104 macroscopic ex

of internal organ

CFLP female 65 

0, 3.4, 6.7, and 13.5 μg 

acetone:DMSO (1:3) 2x/wk 

Drop 0.1 ml 

Dorsal skin, interscapular area 

104 macroscopic ex

of internal organ

NMRI female 20 

0, 2 or 4 μg 

acetone 2x/wk 

drop application by calibrated syringe 0.01 ml 

dorsal skin in interscapular area 

lifetime1 macroscopic ex

of internal organ
398 DRAFT - DO NOT CITE OR QUOTE 

1 APPENDIX H. Alternative Approaches for Cross-Species Scaling of the Dermal Slope 

2 Factor 

3 

4 Several publications which develop a dermal slope factor for benzo[a]pyrene are 

5 available in the peer reviewed literature (Knafla et al., 2010; 2006; Hussain et al., 1998; LaGoy 

6 and Quirk 1994; Sullivan et al., 1991). With the exception of the 2010 Knafla et al. publication, 

7 none of these approaches applied quantitative adjustments to account for interspecies differences, 

8 though the proposed slope factors were developed to account for human risk. Knafla et al. 

9 (2010) qualitatively discuss processes which could affect the extrapolation between mice and 

10 humans including skin metabolic activity adduct formation, stratum corneum thickness, 

11 epidermal thickness, etc. Ultimately, the authors apply an adjustment based on the increased 

12 epidermal thickness of human skin on the arms and hands compared to mouse interscapular 

13 epidermal thickness. They hypothesize that the carcinogenic potential of benzo[a]pyrene may be 

14 related to the thickness of the epidermal layer. 

15 Because there is no established methodology for cross-species extrapolation of dermal 

16 toxicity, several alternative approaches were evaluated. Each approach begins with the POD of 

17 0.066 μg/day that was based on a 10% extra risk for skin tumors in male mice (see Section 

18 5.4.3). Based on the assumptions of each approach, a dermal slope factor for humans is 

19 calculated. The discussion of these approaches uses the following abbreviations: 

20 

21 DSF = dermal slope factor 

22 PODM = point of departure (for 10% extra risk) from mouse bioassay, in μg/day 

23 BW M = mouse body weight = 0.035 kg (assumed) 

24 BWH = human body weight = 70 kg (assumed) 

SAH = total human surface area = 19,000 cm225 (assumed) 

SAM = total mouse surface area = 100 cm226 (assumed)

27 

28 Approach 1. No interspecies adjustment to daily applied dose (POD) in mouse model 

29 Under this approach, a given mass of benzo[a]pyrene, applied daily, would pose the same 

30 risk in an animal or in humans, regardless of whether it is applied to a small surface area or to a 

31 larger surface area at a proportionately lower concentration. 

32 

33 DSF = 0.1/ PODM 

34 

DSF= 0.1/0.066 μg/day = 2 (μg/day)-1 35 

36 

37 Assumptions: The same mass of benzo[a]pyrene, applied daily, would have same potency in 

38 mice as in human skin regardless of treatment area.

39 

40 

41 Approach 2. Cross-species adjustment based on whole body surface-area scaling

42 Under this approach, animals and humans are assumed to have equal lifetime cancer risk 

with equal average whole body exposures in loading units (μg/cm243 -day). As long as doses are 

44 low enough that risk is proportional to the mass of applied compound, the daily dermal dose of 

45 benzo[a]pyrene can be normalized over the total surface area. 

46 

POD (μg/cm2-day) = PODM/SA (μg/cm2-day) = PODM (μg/day) / SAM (cm247 ) 

48 

POD = (0.066 μg/day) / 100 cm249 
399 DRAFT - DO NOT CITE OR QUOTE 

 = 0.00066 μg/cm2

1 -day 

2 

DSF = 0.1/(0.00066 μg/cm2-day) ≈ 152 (μg/cm2-day)-1 3 

4 

5 Assumptions: Mouse and human slope factors are equipotent if total dermal dose is averaged 

6 over equal fractions of the entire surface area. Tumor potency of benzo[a]pyrene is assumed to 

7 be related to overall dose and not dose per unit area. For example, someone exposed to 0.01 

μg/day on 10 cm28 would be assumed to have the same potential to form a skin tumor as someone 

treated with 0.01 μg/day over 10,000 cm29 . 

10 

11 

12 Approach 3. Cross-species adjustment based on body weight 

13 Under this approach, a given mass of benzo[a]pyrene is normalized relative to the body 

14 weight of the animal or human. This approach has been used for oral doses for noncancer 

15 effects. 

16 

17 PODM/ BWM= 0.066 μg/0.035 kg-day = 1.9 μg/kg-day 

18 

DSF = 0.1/1.9 μg/kg-day = 0.05 (μg/kg-day)-1 19 

20 

21 Assumptions: The potency of point of contact skin tumors is related to bodyweight and humans 

22 and mice would have an equal likelihood of developing skin tumors based on a dermal dose per 

23 kg basis. 

24 

25 Issues: Skin cancer following benzo[a]pyrene exposure is a local effect and not likely dependent 

26 on body weight. 

27 

28 

29 Approach 4. Cross-species adjustment based on allometric scaling using body weight to 

30 the 3/4 power 

31 Under this approach, rodents and humans exposed to the same daily dose of a carcinogen, 

adjusted for BW3/4 32 , would be expected to have equal lifetime risks of cancer. That is, a lifetime 

dose expressed as μg/kg3/4 33 -day would lead to an equal risk in rodents and humans. This scaling 

34 reflects the empirically observed phenomena of more rapid distribution, metabolism, and 

35 clearance in smaller animals. The metabolism of benzo[a]pyrene to reactive intermediates is a 

36 critical step in the carcinogenicity of benzo[a]pyrene, and this metabolism occurs in the skin.

37 

POD (μg/day) = PODM (μg/day) × (BWH / BWM)

3/4 38 

39 

POD (μg/day) = 0.066 μg/day × (70 kg / 0.035 kg)3/4 40 

41 = 19.7 μg/day 

42 

DSF = 0.1/(19.7 μg/day) ≈ 0.005 (μg/day)-1 43 

44 

45 Assumptions: Risk at low doses of benzo[a]pyrene is dependent on absolute dermal dose and not 

46 dose per unit of skin, meaning a higher exposure concentration of benzo[a]pyrene contacting a 

47 smaller area of exposed skin could carry the same risk of skin tumors as a lower exposure 

48 concentration of benzo[a]pyrene that contacts a larger area of skin. 

49 
400 DRAFT - DO NOT CITE OR QUOTE 

1 Issues: It is unclear if scaling of doses based on bodyweight ratios will correspond to differences 

2 in metabolic processes in the skin of mice and humans. 

3 

4 

5 Synthesis of the alternative approaches to cross-species scaling

6 A comparison of the above approaches is provided in Table H-1 below. The lifetime risk 

7 from a nominal human dermal exposure to benzo[a]pyrene over a 5% area of exposed skin 

(approximately 950 cm2), estimated at 1 x 10 -4 8 μg/day*, is calculated for each of the approaches 

9 in order to judge whether the method yields risk estimates that are unrealistically high. 

10 

11 Other potential interspecies adjustments 

12 The above discussion presents several mathematical approaches that result from varying 

13 assumptions about what is the relevant dose metric for determining equivalence across species. 

14 Biological information (that is not presently comprehensive or detailed enough to develop robust 

15 models) that could be used in future biologically based models for cross-species extrapolation 

16 include: 

17 

18 a. Quantitative information on interspecies differences in partitioning from exposure 

19 medium to the skin and absorption through the skin 

20 b. Thickness of the stratum corneum between anatomical sites and between species 

21 c. Thickness of epidermal layer 

22 d. Skin permeability 

23 e. Metabolic activity of skin 

24 f. Formation of DNA adducts in skin

25 
401 DRAFT - DO NOT CITE OR QUOTE 

1 2 Table H-1. Alternative approaches to cross-species scaling 

Approach Assumptions Dose metric DSF 

1. Mass-perday scaling 

Equal mass per day (μg /d), if applied to equal areas of skin (cm2), will affect similar 

numbers of cells across species. Cancer risk is proportional to the area (cm2) exposed if 

the loading rate (μg /cm2-d) is the same. This approach assumes that risk is proportional 

to dose expressed as mass per day. This approach implies that any combination of loading rate (μg /cm2-day) and skin area exposed (cm2) that have the same product when 

multiplied, will result in the same risk. 

μg/day 1 per μg/day 

2. Surfacearea scaling 

Equal mass per day (μg /d), if applied to equal fractions of total skin surface (cm2) 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 similar cancer risks across species. This approach assumes that risk is proportional to dose expressed as mass per area per day. This approach implies that risk does not increase with area exposed as long as dose per area remains constant. 

μg/cm2-day 152 per μg/cm2-day 

3. Bodyweight scaling 

The skin is an organ with thickness and volume; benzo[a]pyrene is distributed within this 

volume of skin. Cancer risk is proportional to the concentration of benzo[a]pyrene in the exposed volume of skin. Equal mass per day (μg /d), if distributed within equal fractions 

of 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 similar 

cancer risks across species. This approach assumes that risk is proportional to dose expressed as mass per kg body weight per day. This approach implies that any combination of dose (μg /day) and body weight (kg) that have the same result when divided, will result in the same risk. 

μg/kg-day 0.05 per μg/kg-day 

4. Allometric scaling (BW3/4) 

Same as for body-weight scaling, except that benzo[a]pyrene distribution and metabolism

takes place within this volume of skin. Allometric scaling is generally regarded as describing the relative rate of toxicokinetic processes across species. This approach also is used by EPA to scale oral exposures. 

μg/day 0.005 per μg /day 

3 * Nominal exposure calculated as a geometric mean of average daily doses (μg/day) calculated from a range of benzo[a]pyrene soil concent

4 from non-contaminated rural/agricultural soils (ATSDR, 1995) and a range of standard exposure assumptions.