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EPA/635/R-10/006C

www.epa.gov/iris

Toxicological Review of Benzo[a]pyrene

(CASRN 50-32-8)

In Support of Summary Information on the

Integrated Risk Information System (IRIS)

Supplemental Information

June 2012

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.

National Center for Environmental Assessment

Office of Research and Development

U.S. Environmental Protection Agency

Washington, DC
Toxicological Review of benzo[a]pyrene

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

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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 of 

recommendation for use.
Toxicological Review of benzo[a]pyrene

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

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CONTENTS

APPENDIX A. OTHER AGENCY AND INTERNATIONAL ASSESSMENTS........................................A-1

APPENDIX B. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSEREPONSE ANALYSIS.............................................................................................. B-3

TOXICOKINETICS................................................................................................... B-3

HUMAN STUDIES................................................................................................ B-16

ANIMAL BIOASSAYS ........................................................................................... B-36

OTHER PERTINENT TOXICITY INFORMATION .................................................... B-88

APPENDIX C. DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE 

VALUES FOR EFFECTS OTHER THAN CANCER AND THE DERIVATION OF 

CANCER RISK ESTIMATES ..................................................................................... C-1

DOSE-RESPONSE MODELING FOR DERVIATION OF RFD...................................... C-1

INHALATION DOSIMETRY MODELING FOR RFC DERIVATION............................ C-23

DOSE-RESPONSE MODELING FOR CANCER RISK VALUES.................................. C-26

DOSE-RESPONSE MODELING FOR THE INHALATION UNIT RISK........................ C-58

DOSE-RESPONSE MODELING FOR THE DERMAL SLOPE FACTOR ...................... C-65

ALTERNATIVE APPROACHES FOR CROSS-SPECIES SCALING OF THE 

DERMAL SLOPE FACTOR .................................................................................... C-97

APPENDIX D. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND 

EPA’S DISPOSITION ..............................................................................................D-1

REFERENCES FOR APPENDICES ....................................................................................................... 1
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TABLES AND FIGURES

Table A-1. Health assessments and regulatory limits by other national and international agenciesA1

Figure B-1. Metabolic pathways for benzo[a]pyrene. .........................................................................................B-8

Figure B-2. The stereospecific activation of benzo[a]pyrene. .........................................................................B-9

Table B-1. Exposure to benzo[a]pyrene and mortality from cardiovascular diseases in a European 

cohort of asphalt paving workers .............................................................................................................. B-17

Table B-2. Exposure to benzo[a]pyrene and mortality from cardiovascular diseases in a Canadian 

cohort of male aluminum smelter workers ........................................................................................... B-19

Table B-3. Exposure-related effects in Chinese coke oven workers or warehouse controls exposed 

to benzo[a]pyrene in the workplace......................................................................................................... B-25

Table B-4. Exposure-related effects in Chinese coke oven workers or warehouse controls exposed 

to benzo[a]pyrene in the workplace, stratified by urinary metabolite levels ......................... B-26

Table B-5. Background information on Chinese coke oven workers or warehouse controls exposed 

to benzo[a]pyrene in the workplace......................................................................................................... B-27

Table B-6. Exposure-related effects in male Wistar rats exposed to benzo[a]pyrene by gavage 5 

days/week for 5 weeks .................................................................................................................................. B-36

Table B-7. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene by gavage 5 

days/week for 5 weeks .................................................................................................................................. B-40

Table B-8. Means ± SDa for liver and thymus weights in Wistar rats exposed to benzo[a]pyrene by 

gavage 5 days/week for 90 days ................................................................................................................ B-42

Table B-9. Incidences of exposure-related neoplasms in Wistar rats treated by gavage with 

benzo[a]pyrene, 5 days/week, for 104 weeks...................................................................................... B-44

Table B-10. Incidences of alimentary tract tumors in Sprague-Dawley rats chronically exposed to 

benzo[a]pyrene in the diet or by gavage in caffeine solution ........................................................ B-47

Table B-11. Incidence of nonneoplastic and neoplastic lesions in female B6C3F1 mice fed 

benzo[a]pyrene in the diet for up to 2 years......................................................................................... B-49

Table B-12. Other oral exposure cancer bioassays in mice............................................................................. B-51

Table B-13. Incidence of respiratory and upper digestive tract tumors in male hamsters treated for 

life with benzo[a]pyrene by inhalation ................................................................................................... B-56

Table B-14. Number of animals with pharynx and larynx tumors in male hamsters exposed by 

inhalation to benzo[a]pyrene for life........................................................................................................ B-57

Table B-15. Skin tumor incidence and time of appearance in male C57L mice dermally exposed to 

benzo[a]pyrene for up to 103 weeks........................................................................................................ B-59

Table B-16. 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................. B-60

Table B-17. Tumor incidence in female Swiss mice dermally exposed to benzo[a]pyrene for up to 

93 weeks............................................................................................................................................................... B-61

Table B-18. Skin tumor incidence in female NMRI and Swiss mice dermally exposed to 

benzo[a]pyrene ................................................................................................................................................. B-62

Table B-19. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene .... B-62

Table B-20. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene .... B-63

Table B-21. Skin tumor incidence and time of appearance in female CFLP mice dermally exposed to 

benzo[a]pyrene for 104 weeks ................................................................................................................... B-64

Table B-22. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene for life

.................................................................................................................................................................................. B-65

Table B-23. Skin tumor incidence in male C3H/HeJ mice dermally exposed to benzo[a]pyrene for 
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24 months ............................................................................................................................................................ B-66

Table B-24. Mortality and cervical histopathology incidences in female ICR mice exposed to 

benzo[a]pyrene via gavage for 14 weeks ............................................................................................... B-70

Table B-25. Means ± SD for ovary weight in female Sprague-Dawley rats ............................................. B-72

Table B-26. Reproductive effects in male and female CD-1 F1 mice exposed in utero to 

benzo[a]pyrene ................................................................................................................................................. B-74

Table B-27. Effect of prenatal exposure to benzo[a]pyrene on indices of reproductive performance 

in F1 female NMRI mice ................................................................................................................................. B-76

Table B-28. Exposure-related effects in Long Evans Hooded rats exposed to benzo[a]pyrene by 

gavage daily in utero from GD14 – GD17................................................................................................ B-80

Table B-29. Exposure-related effects in Swiss Albino OF1 mice exposed as pups to benzo[a]pyrene 

in breast milk from dams treated by gavage daily from PND1 – PND14................................... B-82

Table B-30. Pregnancy outcomes in female F344 rats treated with benzo[a]pyrene on GDs 11–21 

by inhalation....................................................................................................................................................... B-84

Table B-31. In vitro genotoxicity studies of benzo[a]pyrene in non-mammalian cells ..................... B-88

Table B-32. In vitro genotoxicity studies of benzo[a]pyrene in mammalian cells ............................... B-89

Table B-33. In vivo genotoxicity studies of benzo[a]pyrene ............................................................................B-1

Table C-1. Means ± SDa for thymus weight in male Wistar rats exposed to benzo[a]pyrene by 

gavage 5 days/week for 90 days ...................................................................................................................C-2

Table C-2. Model predictions for decreased thymus weight in male Wistar rats—90 days...............C-2

Figure C-1. Fit of linear model (nonconstant variance) to data on decreased thymus weight in male 

Wistar rats—90 days. ........................................................................................................................................C-3

Table C-3. Means ± SDa for thymus weight in female Wistar rats exposed to benzo[a]pyrene by 

gavage 5 days/week for 90 days ...................................................................................................................C-6

Table C-4. Model predictions for decreased thymus weight in female Wistar rats—90 days ...........C-6

Figure C-2. Fit of linear model (constant variance) to data on decreased thymus weight in female 

Wistar rats—90 days. ........................................................................................................................................C-7

Table C-5. Means ± SDs for ovary weight in female Sprague-Dawley rats .............................................. C-10

Table C-6. Model predictions for decreased ovary weight in female Sprague-Dawley rats............. C-10

Figure C-3. Fit of linear/polynomial (1°) model to data on decreased ovary weight. ........................ C-11

Table C-7. Means ± SDs for Escape Latency and Time Spent in Target Quadrant................................ C-14

Table C-8. Model predictions for increase in Morris water maze test for escape latency, male and 

female rats ........................................................................................................................................................... C-14

Figure C-4. Fit of Hill model to data on Morris water maze test escape latency. .................................. C-15

Table C-9. Model predictions for decrease in Morris water maze test for time spent in target 

quadrant, male and female rats .................................................................................................................. C-18

Figure C-5. Fit of Exponential 4 model to data on Morris water maze time spent in target quadrant.

.................................................................................................................................................................................. C-18

Table C-10. Incidence of cervical epithelial hyperplasia.................................................................................. C-21

Table C-11. Model predictions for increased incidence of epithelial hyperplasia in female ICR miceC21

Figure C-6. Human fractional deposition............................................................................................................... C-23

Figure C-7. Rat fractional deposition....................................................................................................................... C-24

Table C-12. Tumor incidence data, with time to death with tumor; male rats exposed by gavage to 

benzo[a]pyrene—Kroese et al. (2001).................................................................................................... C-29

Table C-13. Tumor incidence data, with time to death with tumor; female rats exposed by gavage to 

benzo[a]pyrene—Kroese et al. (2001).................................................................................................... C-31

Table C-14. Tumor incidence, with time to death with tumor; female mice exposed to 

benzo[a]pyrene via diet—Beland and Culp (1998)........................................................................... C-33

Table C-15. Derivation of HEDs to use for BMD modeling of Wistar rat tumor incidence data from 
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Kroese et al. (2001).......................................................................................................................................... C-34

Table C-16. Derivation of HEDs for dose-response modeling of B6C3F1 female mouse tumor 

incidence data from Beland and Culp (1998)....................................................................................... C-35

Table C-17. Summary of model selection and modeling results for best-fitting multistage-Weibull 

models, using time-to-tumor data for rats from Kroese et al. (1981)........................................ C-36

Table C-18. Summary of human equivalent overall oral slope factors, based on male and female rat 

tumor incidence................................................................................................................................................. C-55

Table C-19. Summary of model selection among multistage-Weibull models fit to alimentary tract 

tumor data for female mice .......................................................................................................................... C-55

Table C-20. Individual pathology and tumor occurrence data for male Syrian hamsters exposed to 

benzo[a]pyrene via inhalation for lifetime—Thyssen et al. (1981). ........................................... C-58

Table C-21. Summary of model selection among multistage-Weibull models fit to tumor data for 

male hamsters.................................................................................................................................................... C-60

Table C-22. Skin tumor incidence, benign or malignant in female Swiss or NMRI mice dermally 

exposed to benzo[a]pyrene .......................................................................................................................... C-68

Table C-23. Skin tumor incidence, benign or malignant, in C57L male mice dermally exposed to 

benzo[a]pyrene ................................................................................................................................................. C-69

Table C-24. Skin tumor incidence, benign or malignant, in female CFLP mice dermally exposed to 

benzo[a]pyrene ................................................................................................................................................. C-69

Table C-25. Skin tumor incidence, benign or malignant, in male C3H/HeJ mice dermally exposed to 

benzo[a]pyrene ................................................................................................................................................. C-70

Table C-26. 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....C71

Figure C-8. Fit of multistage model to skin tumors in C57L mice exposed dermally to 

benzo[a]pyrene (Poel, 1959); graph and model output................................................................... C-72

Figure C-9. 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. ........................................................ C-75

Figure C-10. 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. ............................................... C-77

Figure C-11. 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. ............................................... C-79

Figure C-12. 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................................................ C-81

Figure C-13. 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....................................................... C-83

Figure C-14. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to 

benzo[a]pyrene (Habs et al., 1984); graph and model output....................................................... C-85

Figure C-15. 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. ............................................. C-87

Figure C-16. 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. ............................................. C-89

Figure C-17. 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. ............................................. C-91

Figure C-18. 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.C-93

Figure C-19. Fit of multistage model to skin tumors in male CeH/HeJ mice exposed dermally to 

benzo[a]pyrene (Sivak et al., 1997); graph and model output..................................................... C-95

Table C-27. Alternative approaches to cross-species scaling .....................................................................C-100
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ABBREVIATIONS

3-MC 3-methylcholanthrene

8-OHdG 8-hydroxydeoxyguanosine

ADAF age-dependent adjustment factor

Ah aryl hydrocarbon

AHH aryl hydrocarbon hydroxylase

AhR Ah receptor

AIC Akaike’s Information Criterion

AKR aldo-keto reductase

ALT alanine aminotransferase

ANOVA analysis of variance

ATSDR Agency for Toxic Substances and 

Disease Registry

AUC area under the curve

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

BrdU bromodeoxyuridine

BSM benzene-soluble matter

BUN blood urea nitrogen

CA chromosomal aberration

CASRN Chemical Abstracts Service Registry 

Number

CHO Chinese hamster ovary

CI confidence interval

CNS central nervous system

CONSAAM Conversational SAAM

COX cyclooxygenase

CYP cytochrome

CYP450 cytochrome P450

dG-N2-BPDE 10β-(deoxyguanosin-N2-yl)-

7β,8α,9α-trihydroxy7,8,9,10-tetrahydro-benzo[a]pyrene

DHH dihydrodiol dehydrogenase

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

DNCB 2,4-dinitrochlorobenzene

DSF dermal slope factor

ED effective dose

EH epoxide hydrolase

EROD 7-ethoxyresorufin-O-deethylase

ETS environmental tobacco smoke

Fe2O3 ferrous oxide

FOB functional observational battery

Ga2O3 gallium oxide

GD gestational day

GGT -glutamyl transferase

GI gastrointestinal

GJIC gap junctional intercellular 

communication

GNMT glycine N-methyltransferase

GSH reduced glutathione

GST glutathione-S-transferase

HEC human equivalent concentration

HED human equivalent dose

HFC high-frequency cells

HPLC high-performance liquid 

chromatography

hprt hypoxanthine guanine phosphoribosyl 

transferase

IFN interferon

Ig immunoglobulin

IHD ischemic heart disease

IL interleukin

i.p. intraperitoneal

IRIS Integrated Risk Information System

i.v. intravenous

LC liquid chromatography

LH luteinizing hormone

LOAEL lowest-observed-adverse-effect level

MLE maximum likelihood estimate

MMAD mass median aerodynamic diameter

MN micronucleus

MOA mode of action

MPO myeloperoxidase

mRNA messenger ribonucleic acid

MS mass spectrometry

NF naphthoflavone

NK natural-killer

NMDA N-methyl-D-aspartate

NOAEL no-observed-adverse-effect level

NQO NADPH:quinone oxidoreductase

NTP National Toxicology Program

OR odds ratio

PAH polycyclic aromatic hydrocarbon

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

QSAR quantitative structure activity 

relationship

RBC red blood cell

RfC reference concentration
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RfD reference dose

RN reaction network

RNA ribonucleic acid

ROS reactive oxygen species

RR relative risk

s.c. subcutaneous

SAAM Simulation, Analysis and Modeling

SAM S-adenosylmethionine

SCC squamous cell carcinoma

SCE sister chromatid exchange

SD standard deviation

SE standard error

SEM standard error of the mean

SIR standardized incidence 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

TPA 12-O-tetradecanoylphorbol-13-acetate

TWA time-weighted average

UCL upper confidence limit

UDP uridine diphosphate

UF uncertainty factor

WBC white blood cells

WT wild type

WTC World Trade Center

XP xeroderma pigmentosum

XPA xeroderma pigmentosum group A
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1 APPENDIX A. OTHER AGENCY AND 

2 INTERNATIONAL ASSESSMENTS

3 Table A-1. Health assessments and regulatory limits by other national 

4 and international agencies

Organization Toxicity value or determination

Non-cancer: oral value

CalEPA (2010) The concentration of 4 μg/L (ADD = 1.7 x 10-3 mg/kg-day) for benzo[a]pyrene in 

water for noncarcinogenic effects was derived from a LOAEL of 5 mg/kg-day for 

renal toxicity from Knuckles et al. (2001), a UF of 3,000.

Non-cancer: inhalation value

WHO

(1996, 2003)

The guideline value for benzo[a]pyrene in drinking water of 0.7 μg/L was based on a 

cancer slope factor of 0.46 (mg/kg-day)-1derived from Neal and Rigdon (1967) and a 

lifetime excess cancer risk of 10-5. 

Health Canada

(1986, 2005)

The Maximum Acceptable Concentration (MAC) for benzo[a]pyrene in drinking 

water of 0.01 μg/L was derived from Neal and Rigdon (1967) using a drinking water 

consumption rate of 1.5 L/day, body weight of 70 kg, and a lifetime cancer risk of 5 x 

10-7. The concentrations of 2, 0.2, and 0.02 μg/L benzo[a]pyrene correspond to 

lifetime excess cancer risks of 10-4, 10-5, and 10-6.

Cancer: Oral value

CalEPA (2010) Cancer slope factor of 2.9 (mg/kg-day)-1

derived from Culp et al. (1998). This 

includes an age sensitivity factor of 1.7.

Cancer: Inhalation value

WHO

(2000, 2010)

Does not recommend specific guideline values for PAHs in air. A unit risk of 87 

(mg/m3)

-1

for benzo[a]pyrene, as an indicator a PAH mixtures, was derived from U.S. 

EPA’s IUR from coke oven emissions. The concentrations 0.0012, 1.2 x 10-4, and 1.2 

x 10-5μg/m3benzo[a]pyrene correspond to lifetime excess cancer risks of 10-4, 10-5, 

and 10-6.

CalEPA (1994) The inhalation unit risk of 1.1 (mg/m3)

-1 was derived based on Thyssen et al. (1981).

EU (2005)

Target value of 1 ng/m3benzo[a]pyrene (averaged over one calendar year) as a 

marker of PAH carcinogenic risk. Does not include information for how target value 

was derived.
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Cancer characterization

IARC (2010) Carcinogenic to humans (Group 1) (based on mechanistic data)

NTP (2011) “reasonably anticipated to be a human carcinogen”

CalEPA (2000) “Sufficient reason for concern regarding the carcinogenic potential of this toxicant in 

humans.”

Health Canada

(1986, 1988) Probably carcinogenic to man

1
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1 APPENDIX B. INFORMATION IN SUPPORT OF 

2 HAZARD IDENTIFICATION AND DOSE-REPONSE 

3 ANALYSIS

4 TOXICOKINETICS

5 Overview

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

7 rate and extent of absorption are dependent upon the exposure medium. The presence of 

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

9 metabolites in serum and excreta demonstrate wide systemic tissue distribution. Benzo[a]pyrene 

10 metabolism occurs in essentially all tissues, with high metabolic capacity in the liver and significant 

11 metabolism in tissues at the portal of entry (lung, skin, and gastrointestinal [GI] tract) and in 

12 reproductive tissues. Stable metabolic products identified in body tissues and excreta are very 

13 diverse and include phenols, quinones, and dihydrodiols. These classes of metabolites are typically 

14 isolated as glucuronide or sulfate ester conjungates in the excreta, but can also include glutathione 

15 conjugates formed from quinones or intermediary epoxides. The primary route of metabolite 

16 elimination is in the feces via biliary excretion, particularly following exposure by the inhalation 

17 route. To a lesser degree, benzo[a]pyrene metabolites are eliminated via urine. Overall, 

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

19 Absorption

20 The absorption of benzo[a]pyrene has been studied in humans and laboratory animals for 

21 inhalation, ingestion and dermal exposure. Studies of workers occupationally exposed to 

22 benzo[a]pyrene have qualitatively demonstrated absorption via inhalation by correlating 

23 concentrations of benzo[a]pyrene in the air and benzo[a]pyrene metabolites in the exposed 

24 worker’s urine. Occuational exposures to benzo[a]pyrene measured with personal air samplers 

25 were correlated to urine concentrations of benzo[a]pyrene-9,10-dihydrodiol, a specific metabolite 

26 of benzo[a]pyrene, in 24 hour aggregate urine samples by Grimmer et al., 1994. The amount of 

27 benzo[a]pyrene extracted- from personal air monitoring devices (a surrogate for ambient PAHs) of 

28 coke oven workers were correlated with r-7,t-8,9,c 10 tetrahydroxy-7,8,9,10-

29 tetrahydrobenzo[a]pyrene (trans-anti-benzo[a]pyrene-tetrol, a specific metabolite of 

30 benzo[a]pyrene) in the worker’s urine by Wu et al. (2002). In both of these studies only a very 

31 small fraction (< 1%) of the inhaled benzo[a]pyrene was recovered from urine, consistent with 

32 studies in animals that find urine is not a major route of elimination for benzo[a]pyrene (as 

33 described in the excretion section below). These occupational studies cannot be used to quantify 
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1 absorption through inhalation-only exposure in humans because the persistence of 

2 benzo[a]pyrene-contaminated particulate matter on surfaces and food may lead to exposures via 

3 additional routes (Bostrum et al., 2002). Nevertheless, the observation of benzo[a]pyrene 

4 metabolites in excreta of exposed humans provides qualitative evidence for benzo[a]pyrene 

5 absorption, at least some of which is likely to occur via inhalation.

6 Results from studies of animals following intratracheal instillation of benzo[a]pyrene 

7 provide supporting, quantitative evidence that absorption by the respiratory tract is rapid (Bevan 

8 and Ulman, 1991; Gerde et al. 1993 b; Weyand and Bevan, 1986; 1987). Following intratracheal 

9 instillation of 1 μg 3H-labeled benzo[a]pyrene/kg dissolved in triethylene glycol to Sprague-Dawley 

10 rats, radioactivity rapidly appeared in the liver (reaching a maximum of about 21% of the 

11 administered dose within 10 minutes). Elimination of radioactivity from the lung was biphasic, 

12 with elimination half-times of 5 and 116 minutes (Weyand and Bevan, 1986). In bile-cannulated 

13 rats, bile collected for 6 hours after instillation accounted for 74% of the administered radioactivity 

14 (Weyand and Bevan, 1986). The results are consistent with rapid and extensive absorption by the 

15 respiratory tract and rapid entry into hepatobiliary circulation following intratracheal instillation. 

16 The respiratory tract absorption may also be affected by the vehicle, since higher amounts of 

17 benzo[a]pyrene were excreted in bile when administered with hydrophilic triethylene glycol than 

18 with lipophilic solvents ethyl laurate or tricaprylin (Bevan and Ulman, 1991). Particle-bound 

19 benzo[a]pyrene deposited in the respiratory tract is absorbed and cleared more slowly than the 

20 neat compound (Gerde et al., 2001).

21 Studies conducted to assess levels of benzo[a]pyrene metabolites or benzo[a]pyrene-DNA 

22 adduct levels in humans exposed to benzo[a]pyrene by the oral route are not adequate to develop 

23 quantitative estimates of oral bioavailability. The concentration of benzo[a]pyrene was below 

24 detection limits (<0.1 μg/person) in the feces of eight volunteers who had ingested broiled meat 

25 containing approximately 8.6 μg of benzo[a]pyrene (Hecht et al., 1979). However, studies in 

26 laboratory animals demonstrate benzo[a]pyrene is absorbed via ingestion. Studies of rats and pigs 

27 measured the oral bioavailability of benzo[a]pyrene in the range from 10 to 40% (Ramesh et al., 

28 2001b; Foth et al., 1988; Cavret et al., 2003; Hecht et al., 1979). The absorption of benzo[a]pyrene 

29 may depend on the vehicle. Intestinal absorption of benzo[a]pyrene was enhanced in rats when the 

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

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

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

33 benzo[a]pyrene, while lipid media such as corn oil increased it (Stavric and Klassen, 1994). The 

34 addition of wheat bran to the benzo[a]pyrene containing diets increased fecal excretion of 

35 benzo[a]pyrene (Mirvish et al., 1981).

36 Studies of benzo[a]pyrene metabolites or DNA adducts measured in humans exposed 

37 dermally to benzo[a]pyrene-containing mixtures demonstrate that benzo[a]pyrene is absorbed 

38 dermally. One study of dermal absorption in human volunteers found absorption rate constants 
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1 ranging from 0.036 to 0.135/hour over a 45 minute exposure, suggesting 20–56% of the dose 

2 would be absorbed within 6 hours (Van Rooij et al., 1993). Dermal absorption rates varied 69% 

3 between different anatomical sites (forehead, shoulder, volar forearm, palmar side of the hand, 

4 groin, and ankle) and only 7% between different individual volunteers (Van Rooij et al., 1993). The 

5 overall absorbed amount of benzo[a]pyrene in explanted viable skin samples from tissue donors 

6 (maintained in short-term organ cultures) exposed for 24 hours ranged from 0.09 to 2.6% of the 

7 dose (Kao et al., 1985; Wester et al., 1990). Similar amounts of penetration were measured in skin 

8 samples from other species including marmosets, rats, and rabbits (Kao et al., 1985). Skin from 

9 mice allowed more of the dose to penetrate (more than 10%), while that of guinea pig let only a 

10 negligible percentage of the dose penetrate (Kao et al., 1985). The vehicle for benzo[a]pyrene 

11 exposure is an important factor in skin penetration. Exposure of female Sprague-Dawley rats and 

12 female rhesus monkeys topically to benzo[a]pyrene in crude oil or acetone caused approximately 4-

13 fold more extensive absorption than benzo[a]pyrene in soil (Wester et al., 1990; Yang et al., 1989). 

14 The viscosity of oil product used as a vehicle also changed skin penetration with increased uptake 

15 of benzo[a]pyrene for oils with decreased viscosity (Potter et al., 1999). Metabolism is also an 

16 important determinant of permeation, with very low rates observed in nonviable skin (Kao et al., 

17 1985).

18 Distribution

19 No adequate quantitative studies of benzo[a]pyrene tissue distribution in exposed humans 

20 were identified. Obana et al. (1981) observed low levels of benzo[a]pyrene in liver and fat tissues 

21 from autopsy samples. However, prior exposure histories were not available for the donors. 

22 Nevertheless, the identification of benzo[a]pyrene metabolites or DNA adducts in tissues and 

23 excreta of PAH-exposed populations suggest that benzo[a]pyrene is widely distributed.

24 Distribution of benzo[a]pyrene has been studied in laboratory animals for multiple routes 

25 of exposure, including inhalation, ingestion, dermal and intravenous. Exposure to benzo[a]pyrene 

26 in various species (Sprague-Dawley rats, Gunn rats, guinea pigs, and hamsters) results in wide 

27 distribution throughout the body and rapid uptake into well-perfused tissues (i.e. lung, kidney, and 

28 liver) (Weyand and Bevan, 1987; Weyand and Bevan, 1986). Route of administration of 

29 benzo[a]pyrene has little influence on the tissue distribution with similar results from studies of 

30 inhalation (or intratracheal instillation), oral, i.v. and dermal exposures (Weyand and Bevan, 1987; 

31 Weyand and Bevan, 1986; Morse and Carlson, 1985; Saunders et al., 2002; Neubert and Tapken 

32 1988; Moir et al., 1998). Intratracheal instillation of radiolabeled benzo[a]pyrene in mice resulted 

33 in increased radioactivity in lung-associated lymph nodes, suggesting distribution of 

34 benzo[a]pyrene or its metabolites via the lymph (Schnizlein et al. 1987). Rats with biliary cannulas 

35 had high excretion of benzo[a]pyrene and benzo[a]pyrene metabolites in bile. The benzo[a]pyrene 

36 thioether and glucuronic acid-conjugated metabolites in intestines indicated enterohepatic 

37 recirculation of benzo[a]pyrene and benzo[a]pyrene metabolites (Weyand and Bevan, 1986). The 

38 vehicle for delivery of inhalated benzo[a]pyrene impacts the distribution with aerosolized 
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1 benzo[a]pyrene more readily absorbed directly in the respiratory tract than particle-adsorbed 

2 benzo[a]pyrene (which is cleared by the mucociliary and then ingested) (Sun et al., 1982). 

3 Exposure of pregnant rats and mice to benzo[a]pyrene via inhalation and ingestion showed a wide 

4 tissue distribution of benzo[a]pyrene, consistent with other studies and demonstrated placental 

5 transfer of benzo[a]pyrene and its metabolites (Withey et al., 1993; Neubert and Tapken 1988; 

6 Shendrikova and Aleksandrov, 1974). The reactive metabolites of benzo[a]pyrene are also 

7 transported in the blood and may be distributed to tissues incapable of benzo[a]pyrene 

8 metabolism, such. Serum of benzo[a]pyrene-treated mice incubated with splenocytes or salmon 

9 sperm DNA resulted in adduct formation, suggesting that reactive benzo[a]pyrene metabolites 

10 were systemically distributed and available for interaction with target tissues (Ginsberg and 

11 Atherholt, 1989).

12 Metabolism

13 The metabolic pathways of benzo[a]pyrene (Figure B-1) and variation in species, strains, 

14 organ system, age and sex have been studied extensively with in vitro and in vivo experiments. In 

15 addition, there have been numerous studies of exposed humans or animals with subsequent 

16 detection of benzo[a]pyrene metabolites in tissues or excreta. For example, elevated frequency of a 

17 detected urinary metabolite (7,8,9,10-tetrol) was observed in patients treated with coal tar 

18 medication (Bowman et al., 1997), demonstrating extensive metabolism of benzo[a]pyrene in 

19 humans. 

20 Phase I metabolism results in a number of reactive metabolites such as epoxide 

21 intermediates, dihydrodiols, phenols, quinones, and their various combinations that are likely to 

22 contribute to the toxic effects of benzo[a]pyrene (e.g. dihydrodiol epoxides and quinones). The 

23 Phase II metabolism of benzo[a]pyrene metabolites protects cellular macromolecules from binding 

24 with reactive benzo[a]pyrene diolepoxides and radical cations. These metabolic process include 

25 glutathione conjugation of diol epoxides, sulfation and glucuronidation of phenols, and reduction of 

26 quinones by NADPH:quinone oxidoreductase (NQO). Numerous reviews on the metabolism of 

27 benzo[a]pyrene are available (Miller and Ramos, 2001; WHO, 1998; ATSDR, 1995; Conney et al., 

28 1994; Grover, 1986; Levin et al., 1982; Gelboin, 1980). Key concepts have been adapted largely 

29 from these reviews and supplemented with recent findings.

30 Phase I metabolism

31 Phase I reactions of benzo[a]pyrene are catalyzed primarily by CYP450 and produce 

32 metabolites including epoxides, dihydrodiols, phenols and quinones (Figure B-2). The first step of 

33 Phase I metabolism is reaction of benzo[a]pyrene into epoxides, the four major forms of which are 

34 the 2,3-, 4,5-, 7,8-, and 9,10-isomers (Gelboin, 1980). Once formed, these epoxides may undergo 

35 three different routes of metabolism: (1) spontaneous rearrangement to phenols, (2) hydration to 

36 trans-dihydrodiols catalyzed by microsomal epoxide hydrolase, or (3) the Phase II detoxification of 

37 binding with glutathione (either spontaneously or catalyzed by cytosolic glutathione-S-transferases 
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1 (IARC 1983)). The metabolism of benzo[a]pyrene to phenols occurs for 5 phenol isomers (1-, 3-, 6-, 

2 7, and 9-OH benzo[a]pyrene) (Pelkonen et al. 1982). The hydration of benzo[a]pyrene epoxides to 

3 trans-dihydrodiols occurs for all four major epoxide isomers (2,3-, 4,5-, 7,8-, and 9,10-). The 

4 7,8-oxide is the focus of much of the study of benzo[a]pyrene metabolism, since it is a precursor to 

5 the potent DNA-binding metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). BPDE is formed 

6 from benzo[a]pyrene 7,8-transdiol by multiple mechanisms including catalysis by CYPs (Deutsch 

7 1979; Grover 1986), myeloperoxidase (MPO) (Mallet 1991), or prostaglandin h synthase (PHS, also 

8 known as cyclooxygenase COX) (Marnett 1990), and lipid peroxidation (Byczkowski 1990). The 

9 diolepoxides can react further by spontaneously hydrolyzing to tetrols (Hall and Grover 1988).
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1

2 Source: Miller and Ramos (2001).

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

4 The metabolism of benzo[a]pyrene, proceeds with a high degree of stereoselectivity. Liver 

5 microsomes from rats stereospecifically oxidize the 7,8-bond of benzo[a]pyrene to yield almost 

6 exclusively the (+)-benzo[a]pyrene-(7,8)-oxide (see Figure B-2). Each enantiomer of the 7,8-oxide 

7 is stereospecifically converted by epoxide hydrolase (EH) to a different dihydrodiol and further 

8 metabolism of the (–)-benzo[a]pyrene-7,8-dihydrodiol enantiomer by rat CYP enzymes 

9 preferentially yields (+)-benzo[a]pyrene-7R,8S-diol-9S,10R-epoxide [(+)-anti- benzo[a]pyrene-7,8-

10 diol-9,10-epoxide (BPDE)], which is believed to be the most potent carcinogen among the four 

11 stereoisomers (Figure B-2). Formation of these stereoisomers does not occur at equimolar ratios, 

12 and the ratios differ between biological systems. For example a study in rabbit livers demonstrated 
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1 that purified microsomes oxidized the (-)-benzo[a]pyrene-7,8-dihydrodiol to isomeric diol epoxides 

2 in a ratio ranging from 1.8:1 to 11:1 in favor of the (+)-anti-BPDE isomer (Deutsch et al., 1979).

3

4

5 Source: Grover (1986).

6 Figure B-2. The stereospecific activation of benzo[a]pyrene.

7 Several studies have attempted to determine which CYP isozyme is predominantly 

8 responsible for the metabolism of benzo[a]pyrene. Dermal administration of [3H]-benzo[a]pyrene 

9 to mice that have an Ah receptor (AhR) knock-out (AhR–/–) had significantly decreased formation 

10 of (+)-anti-BPDE-DNA adducts compared to WT and 1B1–/– mice (Kleiner et al. 2004). Gavage 

11 administration of benzo[a]pyrene in AhR knock-out mice found the AhR–/– mice (with lower levels 

12 of CYP1A1) had higher levels of protein adducts and unmetabolized benzo[a]pyrene than the 

13 AhR+/+ or +/– mice (Sagredo et al., 2006). Similarly, CYP1A1 (–/–) knock-out mice administered 

14 benzo[a]pyrene in feed for 18 days had higher steady-state blood levels of benzo[a]pyrene and 

15 benzo[a]pyrene–DNA adducts (Uno et al. 2006). DNA post-labeling studies of mice administered by 

16 gavage demonstrated higher benzo[a]pyrene-DNA adduct levels in CYP1A1(–/–) than CYP1A1(+/+) 

17 mice in liver, small intestines, spleen and bone marrow (Uno et al., 2004). These findings establish 

18 important roles in benzo[a]pyrene metabolism for CYP1A1, but the relationship is not clear 

19 between the CYP enzymes and biological activation or detoxification.

20 Another important factor in evaluating variability in the metabolic activation of 

21 benzo[a]pyrene by CYP P450s is the effect of functional polymorphisms, which has been the subject 

22 of numerous reviews (e.g., Wormhoudt et al., 1999). Recombinant CYP1A1 allelic variants 

23 produced BPDE with generally lower catalytic activity and Km values than the WT allele (Schwarz 

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
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1 et al., 2001). However, the formation of diol epoxides is stereospecific, with the allelic variants 

2 producing about three times the amount of (±)-anti-BPDE isomers as compared to the 

3 stereoisomers, (±)-syn-BPDE (Schwarz et al., 2001). In a study of occupational exposures to 

4 benzo[a]pyrene, no relationship was observed between benzo[a]pyrene metabolite formation and 

5 the CYP1A1 MspI polymorphism (Wu et al., 2002).

6 Another metabolic pathway of benzo[a]pyrene metabolism is the conversion of 

7 transdihydrodiol-benzo[a]pyrene or 6-OH benzo[a]pyrene into quinones, primarily the 1,6-, 3,6-, 

8 7,8- and 6,12- isomers. Transdihydrodiol-benzo[a]pyrene such as (+/-)-anti-BPDE can be 

9 converted in a redox cycling reaction into benzo[a]pyrene-7,8-quinone (BPQ) catalyzed by 

10 dihydrodiol dehydrogenase (DD). This reaction pathway produces peroxide anion radicals, 

11 benzo[a]pyrene semiquinone radicals, hydroxyl radicals, and H2O2 which in turn can causes 

12 extensive DNA fragmentation (Penning 1999; Flowers et al., 1996; 1997). 

13 6-Hydroxybenzo[a]pyrene can be oxidized into 6-oxo-benzo[a]pyrene semi-quinone radical and 

14 further metabolized into 1,6-, 3,6-, or 6,12-quinones spontaneously, or catalytically by 

15 prostaglandin endoperoxide synthetase (Eling, et al 1983).

16 Phase II metabolism

17 The reactive products of phase I metabolism are subject to the action of several phase II 

18 conjugation and detoxification enzyme systems that display preferential activity for specific 

19 oxidation products of benzo[a]pyrene. These phase II reactions play a critical role in protecting 

20 cellular macromolecules from binding with reactive benzo[a]pyrene diolepoxides, radical cations, 

21 or ROS. Therefore, the balance between Phase I activation of benzo[a]pyrene and its metabolites 

22 and detoxification by Phase II processes is an important determinant of toxicity.

23 The diol epoxides formed from benzo[a]pyrene metabolism by Phase I reactions are not 

24 usually found as urinary metabolites. Rather, they are detected as adducts of nucleic acids or 

25 proteins o further metabolized by glutathione (GSH) conjugation, glucuronidation, and sulfation. 

26 These metabolites make up a significant portions of total metabolites in excreta or tissues For 

27 example, the identified metabolites in bile 6 hours after a 2 μg/kg benzo[a]pyrene dose by 

28 intratracheal instillation to male Sprague-Dawley rats were 49% glucuronides (quinol 

29 diglucuronides or monglucuronides), 30.4% thioether conjugates, 6.2% sulfate conjugates, and 

30 14.4% unconjugated metabolites (Bevan and Sadler, 1992).

31 Conjugation of benzo[a]pyrene with GSH is catalyzed by GSTs. Numerous studies using 

32 human GSTs expressed in mammalian cell lines have demonstrated the ability of GST to metabolize 

33 benzo[a]pyrene diol epoxides. Isolated human GST have significant catalytic activity toward 

34 benzo[a]pyrene-derived diol epoxides and (±)anti-BPDE with variation in activity across GST 

35 isoforms (Dreij et al. 2002; Robertson et al. 1986; Rojas et al. 1998). Benzo[a]pyrene quinones can 

36 also be conjugated with glutathione (Agarwal et al. 1991; IARC 1983). This compelling evidence for 

37 a role of GSTs in the metabolism of reactive benzo[a]pyrene metabolites has triggered several 

38 molecular epidemiology studies. However, recent studies on the impact of polymorphism on 
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1 adduct levels in PAH-exposed human populations did not show a clear relationship between the 

2 Phase I (CYP1A1, EH), or Phase II (GST) enzyme polymorphisms and formation of DNA adducts 

3 (Hemminki et al., 1997) or blood protein adducts (Pastorelli et al., 1998).

4 Conjugation with UDP-glucuronide catalyzed by UGT enzymes is another important 

5 detoxification mechanism for oxidative benzo[a]pyrene metabolites. UGT isoforms, as well as their 

6 allelic variants, are expressed and have glucuronidation activity toward benzo[a]pyrene-derived 

7 phenols and diols in the aerodigestive tract (tongue, tonsil, floor of the mouth, larynx, esophagus), 

8 but not lung or liver (Zheng et al., 2002; Fang and Lazarus 2004). UGT activity also shows 

9 significant interindividual variability. Incubation of lymphocytes with benzo[a]pyrene resulted in 

10 covalent binding to protein with a 143-fold interindividual variability and a statistically significant 

11 inverse correlation between glucuronidation and protein binding (Hu and Wells, 2004).

12 Sulfotransferases can catalyze the formation of sulfates of benzo[a]pyrene metabolites. In 

13 rat or mouse liver, cytosolic sulfotransferase (in the presence of 3'-phosphoadenosine 5'-

14 phosphosulfate) catalyzes formation of sulfates of three benzo[a]pyrene metabolites: 

15 benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol, benzo[a]pyrene-7,8-dihydrodiol, and benzo[a]pyrene16 7,8,9,10-tetrol. The benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol-sulfate is able to form potentially 

17 damaging DNA adducts (Surh and Tannenbaum, 1995). In human lung tissue 3-

18 hydroxybenzo[a]pyrene conjugation to sulfate produces benzo[a]pyrene-3-yl-hydrogen sulfate, a 

19 very lipid soluble compound that would not be readily excreted in the urine (Cohen et al. 1976).

20 Although not specific for benzo[a]pyrene, there is now considerable evidence that genetic 

21 polymorphisms of the GST, UGT, and EH genes impart an added risk to humans for developing 

22 cancer. Of some significance to the assessment of benzo[a]pyrene may be that smoking, in 

23 combination with genetic polymorphism at several gene loci, increases the risk for bladder cancer 

24 (Moore et al., 2004; Choi et al., 2003; Park et al., 2003) and lung cancer (Alexandrie et al., 2004; Lin 

25 et al., 2003). Coke oven workers (who are exposed to PAHs, including benzo[a]pyrene) 

26 homozygous at the P187S site of the NQO1 gene (an inhibitor of benzo[a]pyrene-quinone adducts 

27 with DNA), or carrying the null variant of the GSTM1 gene, had a significantly increased risk of 

28 chromosomal damage in peripheral blood lymphocytes. Meanwhile, the risk was much lower than 

29 controls in subjects with a variant allele at the H113Y site of the EH gene (Leng et al., 2004).

30 Tissue-specific Metabolism

31 Benzo[a]pyrene metabolism has been demonstrated in vivo in laboratory animals for 

32 various tissues via multiple routes including inhalation, ingestion and dermal absorption. Nasal 

33 instillation or inhalation of benzo[a]pyrene in monkeys, dogs, rats and hamsters resulted in the 

34 formation of dihydrodiols, phenols, quinones, and tetrols in the nasal mucus and lung (Petridou35 Fischer et al. 1988; Weyand and Bevan 1986, 1987a, 1988; Dahl et al. 1985; Wolff et al. 1989b). In 

36 rats, the fractions of metabolites in the lung at 6 hours after instillation were: 20% unmetabolised 

37 benzo[a]pyrene, 16% conjugates or polyhydroxylated compounds, 10.7% 4,5-, 7,8-, and 9,10-

38 dihydrodiols, 9.3% 1,6-, 3,6-, 6,12- quinone, and 6.9% 3- and 9-hydroxybenzo[a]pyrene (Weyand 
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1 and Bevan 1986). In hamsters, approximately 50% of the benzo[a]pyrene instilled was 

2 metabolized in the nose (nasal tissues had the highest metabolic acitivity per-gram of the 

3 respiratory tract tissues), and the metabolites produced were similar to other species (Dahl et al. 

4 1985).

5 In vitro studies of human and laboratory cells and cell lines provide further quantitative and 

6 mechanistic details of the metabolism of benzo[a]pyrene in the cells of the respiratory tract, skin, 

7 liver and other tissues. Tracheobronchial tissues in culture of several species (including humans, 

8 mice, rats, hamsters, and bovines) were all found to metabolize benzo[a]pyrene extensively to 

9 phenols, diols, tetrols, quinones, and their conjugates (Autrup et al., 1980). The results show a high 

10 degree of interindividual variability (a 33-fold difference in human bronchus, a 5-fold variation in 

11 human trachea, and a 3-fold difference in bovine bronchus), but minimal variation among 

12 individuals of the laboratory animal species (Autrup et al., 1980). Human bronchial epithelial and 

13 lung tissue conjugated benzo[a]pyrene metabolites to glutathione and sulfates, but not with 

14 glucuronide (Autrup et al. 1978; Cohen et al. 1976; Kiefer et al. 1988). The binding of 

15 benzo[a]pyrene metabolites with DNA in primary human hepatocytes was associated with the 

16 amount of unconjugated 7,8-dihydrodiol (Monteith et al. 1987).

17 Human and animal skin is able to metabolize benzo[a]pyrene. Human skin samples 

18 maintained in short term organ culture (i.e., human epithelial tissue, samples from human hair 

19 follicles, and melanocytes isolated from adult human skin) can metabolize benzo[a]pyrene into 

20 dihydrodiols, phenolas, quinones and glucuronide and sulfate conjugates (Hall & Griver, 1988; Merk 

21 et al., 1987; Alexandrov et al., 1990; Agarwal et al., 1991). The permeation of benzo[a]pyrene in 

22 skin is linked to benzo[a]pyrene metabolism. Nonviable skin is unable to metabolize 

23 benzo[a]pyrene (the permeation into nonviable skin is lower than viable skin) as measured in a 

24 range of species including humans, rat, mouse, rabbit and marmoset (Kao et al., 1985). Viable 

human skin samples treated with 2 μg/cm2 [ 25 14C]-benzo[a]pyrene in acetone and incubated for 

26 24 hours produced the following proportions of benzo[a]pyrene metabolites; 52% water-soluble 

27 compounds, 8% polar compounds, 17% diols, 1% phenols, 2.5% quinones and 18% unmetabolized 

28 benzo[a]pyrene (Kao et al., 1985).

29 Benzo[a]pyrene is also metabolized by multiple reproductive tissues including prostate, 

30 endometrium, cervical epithelial and styromal, and testes (Williams et al., 2000; Bao et al., 2002; 

31 Melikian et al., 1999; Ramesh et al., 2003). Exposure of fetal tissues to reactive benzo[a]pyrene 

32 metabolites in utero is a concern. Transport of benzo[a]pyrene and benzo[a]pyrene metabolites to 

33 fetal tissues including plasma, liver, hippocampus and cerebral cortex has been demonstrated in 

34 multiple studies (McCabe and Flynn, 1990; Neubert and Tapken, 1988; Shendrikova and 

35 Aleksandrov, 1974), and benzo[a]pyrene is metabolized by human fetal esophageal cell culture 

36 (Chakradeo et al. 1993).
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1 Elimination

2 Benzo[a]pyrene metabolites have been detected in the urine of exposed humans, but the 

3 fecal excretion has not been investigated in any detail. Studies of benzo[a]pyrene elimination in 

4 animals following exposure via inhalation, ingestion and dermal routes have shown benzo[a]pyrene 

5 is excreted preferentially in the feces in multiple species of laboratory animals including rat, mice, 

6 hamsters, guinea pigs, monkeys and dogs (Petridou-Fischer et al., 1988; Wolff et al., 1989; Sun et al., 

7 1982; Wang et al., 2003; Weyand and Bevan, 1987; Yang et al., 1989; Hecht et al., 1979; Likhachev 

8 et al., 1992). The metabolites in bile are primarily benzo[a]pyrene conjugates, predominately 

9 thioether conjugates of varying extent in different species (Weyand and Bevan, 1987). Six hours 

10 after a single intratracheal instillation of benzo[a]pyrene (2 μg/kg) to male Sprague-Dawley rats, 

11 relative metabolite levels were 31.2% diglucuronides, 30.4% thioether conjugates, 17.8% 

12 monoglucuronides, 6.2% sulfate conjugates, and 14.4% unconjugated metabolites (Bevan and 

13 Sadler, 1992). Rats administered benzo[a]pyrene via i.v. excrete a larger fraction in urine than via 

14 inhalation or oral exposure, suggesting an important role for enterohepatic circulation of 

15 benzo[a]pyrene metabolite conjugates (Moir et al., 1998; Weyand and Bevan, 1986; Hirom et al., 

16 1983). The vehicle impacts the amount of benzo[a]pyrene excreted and may in part be due to the 

elimination rate or to other factors such as the absorption rate. For [ 17 3H]-benzo[a]pyrene 

18 administered to Sprague-Dawley rats in hydrophilic triethylene glycol, 70.5% of the dose was 

19 excreted into bile within 6 hours. If lipophilic solvents ethyl laurate and tricaprylin were used as 

20 vehicles, 58.4 and 56.2% of the dose were excreted (Bevan and Ulman, 1991). In addition to 

21 benzo[a]pyrene and its metabolites, adducts of benzo[a]pyrene with nucleotides have also been 

22 identified as a small fraction of the administered dose in feces and urine of animals. The level of 

23 BPDE adducts with guanine detected in urine of male Wistar rats was dose-dependent. 48 hours 

24 after dosing with 100 μg/kg tritiated benzo[a]pyrene, 0.15% of the administered benzo[a]pyrene 

25 dose was excreted in the urine as an adduct with guanine (Autrup and Seremet, 1986). Overall, the 

26 data in humans and laboratory animals are sufficient to describe benzo[a]pyrene elimination 

27 qualitatively but to limited to estimate quantitative rates of elimination.

28 Physiologically based pharmacokinetic models

29 Several toxicokinetic or pharmacokinetic models of benzo[a]pyrene have been developed 

30 for rodents (rat and hamster). However, human models have only been developed via allometric 

31 scaling, and metabolic parameters in humans have not been calibrated against in vivo toxicokinetic 

32 data or in vitro experiments.

33 Bevan and Weyand (1988) performed compartmental pharmacokinetic analysis of 

34 distribution of radioactivity in male Sprague-Dawley rats, following the intratracheal instillation of 

35 benzo[a]pyrene to normal and bile duct-cannulated animals (Weyand and Bevan, 1987, 1986). 

36 However, implicit simulation approaches were used, as opposed to physiologically-based 
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1 approaches. The model calculated linear rate constants among compartments, and assumed the 

2 kinetics of benzo[a]pyrene and its metabolites were the same

3 Roth and Vinegar (1990) reviewed the capacity of the lung to impact the disposition of 

4 chemicals and used benzo[a]pyrene as a case study. A PBPK model was presented based on data 

5 from Wiersma and Roth (1983a, b) and was evaluated against tissue concentration data from 

6 Schlede et al. (1970). The model was structured with compartments for arterial blood, venous 

7 blood, lung, liver, fat, and slowly as well as rapidly perfused tissues. Metabolism in liver and lung 

8 was estimated using kinetic data from control rats and rats pretreated with 3-MC to induce 

9 benzo[a]pyrene metabolism. The results of PBPK simulations showed that induction of 

10 metabolizing enzymes increased the amount of benzo[a]pyrene cleared by the lungs relative to the 

11 liver. An adequate fit was obtained for some compartments; however tissue-level data for 

12 calibration and validation of this model were limited. 

13 Moir et al. (1998) conducted a pharmacokinetic study on benzo[a]pyrene to obtain data for 

model development. Rats were injected with varying doses of [ 14 14C]-benzo[a]pyrene to 15 mg/kg 

15 and blood, liver, fat, and richly perfused tissue were sampled varying time points after dosing. Moir 

16 (1999) then described a model for lung, liver, fat, richly and slowly perfused tissues, and venous 

17 blood, with saturable metabolism occurring in the liver. The fat and richly perfused tissues were 

18 modeled as diffusion-limited, while the other tissues were flow-limited. The model predicted the 

19 blood benzo[a]pyrene concentrations well, although it overestimated the 6 mg/kg results at longer 

20 times (>100 minutes). The model also produced a poor fit to the liver data. The model simulations 

21 were also compared to data of Schlede et al. (1970), who had injected rats with 0.056 mg/kg body 

22 weight of benzo[a]pyrene. The model predicted blood and fat benzo[a]pyrene concentrations well, 

23 but still poorly predicted liver benzo[a]pyrene concentrations. The model included only one 

24 saturable metabolic pathway, and only parent chemical concentrations were used to establish the 

25 model. No metabolites were included in the model. This model was re-calibrated by Crowell et al. 

26 (2011) by optimizing against additional rodent data and altering partition coefficient derivation. 

27 However, it still did not incorporate metabolites, and some tissues continued to exhibit poor model 

28 fits. 

29 An attempt to scale the Moir et al. (1998) rodent PBPK model to humans, relevant to risk 

30 assessment of oral exposures to benzo[a]pyrene, was presented by Zeilmaker et al. (1999a, b). The 

31 PBPK model for benzo[a]pyrene was derived from an earlier model for TCDD in rats (Zeilmaker and 

32 van Eijkeren, 1997). Most compartments were perfusion-limited, and tissues modeled included 

33 blood, adipose (with diffusion limitation), slowly and richly perfused tissues, and the liver. 

34 However, there was no separate compartment for the lung. The liver compartment featured the 

35 AhR-dependent CYP450 induction mechanism and DNA adduct formation as a marker for 

36 formation of genotoxic benzo[a]pyrene metabolites. It was assumed that DNA adduct formation 

37 and the bulk benzo[a]pyrene metabolism were mediated by two different metabolic pathways. The 

38 model was experimentally calibrated in rats with the data for EROD and formation of DNA adducts 
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1 in the liver after i.v. administration of a single dose and per os (p.o.) administration of a single or 

2 repeated doses of benzo[a]pyrene (Zeilmaker et al., 1999a).

3 Zeilmaker et al. (1999b) assumed identical values for several parameters in rats and 

4 humans (i.e. benzo[a]pyrene tissue partition coefficients, AhR concentration in liver, rate constant 

5 for the decay of the benzo[a]pyrene-CYP450 complex, half-life of the CYP450 protein, fraction and 

6 rate of GI absorption of benzo[a]pyrene, and rates of formation and repair of DNA adducts in liver). 

7 The basal CYP450 activity in humans was assumed to be lower than that in rat liver. The 

8 mechanism of AhR-dependent induction of CYP450 dominated the simulated benzo[a]pyrene-DNA 

9 adduct formation in the liver. The results of PBPK model simulations indicated that the same dose 

10 of benzo[a]pyrene administered to rats or humans might produce one order of magnitude higher 

11 accumulation of DNA adducts in human liver when compared with the rat (Zeilmaker et al., 1999b).

12 Even though the model of Zeilmaker et al. (1999b) represents a major improvement in 

13 predictive modeling of benzo[a]pyrene toxicokinetics, the interspecies extrapolation introduce 

14 significant uncertainties. As emphasized by the authors, the conversion of benzo[a]pyrene to its 

15 mutagenic and carcinogenic metabolites could not be explicitly modeled in human liver because no 

16 suitable experimental data were available. According to the authors, improvement of the model 

17 would require direct measurements of basal activities of CYP1A1 and CYP1A2 and formation of 

18 benzo[a]pyrene-DNA adducts in human liver. Metabolic clearance of benzo[a]pyrene in the lungs 

19 was also not addressed. Additionally, the toxicokinetic modeling by Zeilmaker et al. (1999b) 

20 addressed only one pathway of benzo[a]pyrene metabolic activation, a single target organ (the 

21 liver), and one route of administration (oral). In order to model health outcomes of exposures to 

22 benzo[a]pyrene, the PBPK model needs to simulate rate of accumulation of benzo[a]pyrene-DNA 

23 adducts and/or the distribution and fate of benzo[a]pyrene metabolites (e.g., BPDE) that bind to 

24 DNA and other macromolecules. Alternatively, stable toxic metabolites (e.g., trans-anti-tetrol25 benzo[a]pyrene) may be used as an internal dose surrogate. While the metabolic pattern of 

26 benzo[a]pyrene has been relatively well characterized qualitatively in animals, the quantitative 

27 kinetic relationships between the more complex metabolic reactions in potential target organs are 

28 not yet well defined.

29 Recommendations for the use of PBPK models in toxicity value derivation

30 PBPK models for benzo[a]pyrene were evaluated to determine the capability to extrapolate 

31 from rats to humans, or between oral and inhalation exposure routes. Due to significant 

32 uncertainties with respect to the inter-species scaling of the metabolic parameters between rats 

33 and humans, these models were not used for cross-species extrapolation. Furthermore, no 

34 complete mechanistic PBPK model for the inhalation route was identified, nor was there a model 

35 for humans that simulates the typical inhalation exposure to benzo[a]pyrene on poorly soluble 

36 carbonaceous particles. This precluded the model’s use for cross-route extrapolation to the 

37 inhalation pathway.
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1 HUMAN STUDIES

2 Non-Cancer Endpoints

3 Cardiovascular Endpoints

4 Burystn et al. (2005) reported the association of death from cardiovascular disease with 

5 B[a]P exposure in a cohort of 12,367 male European asphalt workers (Table B-1). These workers 

6 were first employed in asphalt paving between 1913 and 1999, and worked at least one season. 

7 Average duration of follow-up was 17 ± 9 years (mean ± SD), encompassing 193,889 person-years 

8 of observation. Worker exposure to coal tar was estimated using industrial process and hygiene 

9 information and modeling (presented in a previous report), and coal tar exposure was found to be 

10 the strongest determinant of exposure to B[a]P. Benzo[a]pyrene exposure was assessed 

11 quantitatively using measurement-driven mixed effects exposure models, using data collected from 

12 other asphalt industry workers, and this model was constructed and validated previously. Due to 

13 limited data availability, only information regarding the primary cause of death was collected, and 

14 this analysis was limited to diseases of the circulatory system (ICD codes 390 – 459), specifically 

15 ischemic heart disease (IHD: ICD codes 410 – 414). Diesel exhaust exposure was also assessed in 

16 this cohort, but varied little among the asphalt pavers, and was not associated with risk of death 

17 from cardiovascular disease. 0.25% of the cohort was lost to follow-up, and 0.38% emigrated 

18 during the course of observation. Relative risks and associated 95% confidence intervals were 

19 estimated using Poisson regression, and all models included exposure index for agent of interest 

20 (coal tar or B[a]P), age, calendar period of exit from cohort, total duration of employment and 

21 country, using the category of lowest exposure as the reference. Confounding by tobacco smoke 

22 exposure was considered in relation to the strength of its association with cardiovascular disease 

23 and the smoking prevalence in the population. The RR attributed to cigarette smoking in former 

24 and current smokers was assumed to be 1.2 and 2, respectively, based upon literature reports. 

25 From analysis of smoking incidence in a sub-cohort, the following smoking distribution was 

26 proposed: in the lowest exposure group, 40% never smokers, 30% former smokers and 30% 

27 current smokers; among the highest exposed, the proportion shifted to 20/30/50%, respectively.

28 Exposed subjects were stratified into quintiles based upon IHD mortality, with 83 – 86 

29 deaths per exposure category, composing approximately 2/3 of the 660 cardiovascular disease30 related deaths. Both cumulative and average exposure indices for B[a]P were positively associated 

31 with IHD mortality, with a RR of approximately 1.6 in the highest exposure quintile from both 

32 metrics, independent of total employment duration. Similar monotonic trends were observed for 

33 all cardiovascular diseases (combined), although a dose-response relationship was evident only for 

34 IHD and not hypertension or other individual heart disease categories. Similar trends were also 

35 observed for coal tar exposure and IHD. Adjusting the RR to account for possible confounding by 

36 smoking yields a RR of 1.39 under the assumptions mentioned above, and is still elevated (1.21) if 

37 the contribution of smoking to cardiovascular disease etiology was greater than the original 
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1 assumptions. Furthermore, the RR for the high vs. low exposure quintile is 1.24 even if the 

2 distribution of non-smokers/former smokers/current smokers shifts to 0/30/70%, using the 

3 original assumptions of cigarette smoke casual potency.

4 Table B-1. Exposure to benzo[a]pyrene and mortality from 

5 cardiovascular diseases in a European cohort of asphalt paving workers

Effect measured

Cumulative exposure (ng/m3– years) P for 

0 – 189 trend a189 – 501 502 – 931 932 – 2012 ≥2013

Diseases of the circulatory system

Deaths

RR 

95% CI

137

1.00

145

1.08

0.85-1.38

118

1.06

0.80-1.42

132

1.24

0.89-1.71

128

1.42

0.96-2.09

0.09

Ischemic heart disease

Deaths

RR 

95% CI

83

1.00

83

0.99

0.72-1.36

84

1.22

0.86-1.74

83

1.24

0.82-1.85

85

1.58

0.98-2.55

0.06

Effect measured

Average exposure (ng/m3) P for 

trend 0 – 68a

68 – 105 106 – 146 147 – 272 ≥273

Diseases of the circulatory system

Deaths

RR 

95% CI

128

1.00

142

1.30

1.01-1.67

143

1.55

1.18-2.05

139

1.45

1.09-1.93

108

1.58

1.16-2.15

<0.001

Ischemic heart disease

Deaths

RR 

95% CI

83

1.00

83

1.13

0.82-1.55

83

1.33

0.94-1.90

86

1.20

0.84-1.71

83

1.64

1.13-2.38

0.02

a

Reference category

Source: Burstyn et al. (2005).

6

7 Friesen et al. (2010) examined the association between B[a]P exposure and deaths from 

8 chronic non-malignant disease in a cohort of 6.423 male and 603 female Canadian aluminum 

9 smelter workers (Table B-2). Inclusion criteria required at least 3 years of continuous employment 

10 in either the smelter facility or power-generating station from 1954 – 1997, with worker history 

11 collected up through 1999. This cohort was probabilistically linked to the Canadian national 

12 mortality database for external comparison to the British Columbia population and calculation of 

13 standardized mortality ratios, which were adjusted for age, sex and time period. Ninety-five % 

14 confidence intervals were calculated for the SMRs assuming a Poisson distribution. Internal 

15 comparisons were also made during the analysis of IHD mortality in male workers, calculating 

16 hazard ratios (HR) for IHD with or without acute myocardial infarction (AMI) after 1969, as AMI 

17 could not be differentiated from other IHD on death certificates issued previously. HRs were 
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1 calculated using Cox regression models, with age as a metamarker of time, also including smoking 

2 status, time since 1st employed and work location status. Smoking information for 77% of this 

3 updated cohort was collected by questionnaire, and workers categorized as 75% ever-smokers and 

4 25% never-smokers. Quantitative exposure to coal tar pitch volatiles were estimated by B[a]P 

5 measurements, calculated by a job classification and time-based exposure matrix, as described in a 

6 previous report; annual arithmetic mean values were calculated for exposures from 1977 – 2000, 

7 while pre-1977 levels were backwards-extrapolated from 1977 values, incorporating major 

8 technological changes in time periods as appropriate.

9 Cumulative exposure metrics were highly skewed. Cumulative B[a]P with a 5-year lag (past 

10 B[a]P exposure) and cumulative B[a]P in the most recent 5 years (recent B[a]P exposure) were only 

11 slightly positively correlated (r = 0.10, P < 0.001). Current B[a]P exposure was highly correlated 

12 with cumulative exposure for the most recent 5 years of exposure (r = 0.86, P < 0.001), but not with 

13 5-year lagged cumulative exposure (r = 0.03, P < 0.001). Lagged cumulative exposure metrics (0 –

14 10 years) were all highly correlated with each other (r = 0.96, all P’s < 0.001); lagged metrics for 

15 cumulative exposure were used to distinguish between effects of current versus long-term 

16 exposure.

17 When exposed workers were pooled and compared externally to non-exposed referents, the 

18 IHD and AMI standardized mortality ratios were all ≤ 1.00 for males, and the only significant 

19 association in females was an SMR of 1.27 for AMI. For internal comparisons, exposed males were 

20 stratified into quintiles based upon IHD mortality, with approximately 56 deaths per exposure 

21 category. 5-year lagged cumulative B[a]P exposure was significantly associated with elevated risk 

22 of IHD mortality, HR = 1.62 (95% CI: 1.06, 2.46) in the highest exposure quintile, while no 

23 association was observed between most recent (5 years) exposure and mortality. Restricting IHD 

24 events to only AMI (1969 onward) resulted in similar monotonic trends, albeit of lower statistical 

25 significance. No association was observed between B[a]P exposure and non-AMI IHD. While there 

26 was little difference in the exposure-response association among 0, 2 and 5-year lagged data, 10-

27 year lagged data resulted in a weaker association. All risk estimates were strengthened by the 

28 incorporation of work status and time-since-hire to account for the healthy worker effect, as 

29 evidenced by the SMR of 0.87 (95% CI: 0.82, 0.92) for all chronic non-malignant diseases combined 

30 in male exposed workers versus external referents. Using a continuous variable, the authors 

calculated that the risk of death from IHD to be 1.002 (95% CI: 1.000, 1.005) per μg/m3 31 from 

32 cumulative B[a]P exposure; however, visual inspection of the categorical relationships indicated 

33 that the association is nonlinear, suggesting that this value may be an underestimate. Restricting 

34 the cohort to only members who died within 30 days of active employment at the worksite, 

35 cumulative B[a]P exposure was not significantly associated with IHC or AMI, although the HR for 

36 the highest exposure group was 2.39 (95% CI: 0.95, 6.05). Exposure-response relationships were 

37 similarly examined in male smelter workers for chronic obstructive pulmonary disease (COPD) and 
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1 cerebrovascular disease, but neither was significantly associated with cumulative B[a]P exposure in 

2 either internal or external comparisons.

3 Table B-2. Exposure to benzo[a]pyrene and mortality from 

4 cardiovascular diseases in a Canadian cohort of male aluminum smelter 

5 workers

Effect measured

Categorical cumulative exposure with a 5-year lag (μg/m3– year) P for 

trenda

Continuousb

0 0 – 7.79 7.79 – 24.3 24.3 – 66.7 ≥66.7

All ischemic heart disease (1957 onward)

Deaths

P-Y

c

of followup

HR 

95% CI

56

33,111

1

referent

56

37,581

1.11

0.76-1.62

57

34,838

1.48

1.01-2.17

56

31,533

1.28

0.86-1.91

56

13,688

1.62

1.06-2.46

0.053

281

150,751

1.002

1.000, 

1.005

Acute myocardial infarction (1969 onward)

0 0 – 7.51 7.51 – 27.7 27.7 – 67.4 ≥67.4

Deaths

P-Y

c

of followup

HR 

95% CI

35

25,071

1

referent

37

30,454

1.14

0.71, 1.82

37

34,621

1.21

0.75, 1.96

38

24,081

1.36

0.84, 2.45

37

13,261

1.46

0.87, 2.45

0.19

184

127,488

1.001

0.997, 

1.005

a

Two-sided test for trend using the person-year-weighted mean value for each category as a linear, 

continuous variable.

b

Exposure variable was entered as a continuous, linear variable in the model

c

P-Y, person-years

Source: Friesen et al. (2010).

6 Reproductive and Developmental Endpoints

7 Wu et al. (2010) conducted a study of benzo[a]pyrene-DNA adduct levels in relation to risk 

8 of fetal death in Tianjin, China. This case-control study included women who experienced a missed 

9 abortion before 14 weeks gestational age (i.e., a fetal death that remained in utero and therefore 

10 required surgical intervention). Cases were matched by age and gravidity to controls (women 

11 undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded 

12 women who smoked, women with chronic disease and pregnancy complications, and women with 

13 occupational exposures to PAHs. Residency within Tianjin for at least 1 year was also an eligibility 

14 criterion. The participation rate was high: 81/84 eligible cases participated and 81/89 eligible 

15 controls participated. Data pertaining to demographic characteristics, reproductive history, and 

16 factors relating to potential PAH exposure were collected using a structured interview, and samples 

17 from the aborted tissue were obtained. In two of the four hospitals used in the study, blood 

18 samples from the women (n = 51 cases and 51 controls) were also collected. The presence of 

19 benzo[a]pyrene-BPDE adducts was assessed in the blood and tissue samples using HPLC. There 
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1 was no correlation between blood and aborted tissue levels of benzo[a]pyrene adducts (r = -0.12 

2 for the 102 blood-tissue pairs, r = -0.02 for the 51 case pairs and r = -0.21 for the 51 control pairs). 

3 (The authors noted that there was little difference between women with and without blood 

4 samples in terms of the interview-based measures collected or in terms of the DNA-adduct levels in 

5 aborted tissue.) Benzo[a]pyrene-adduct levels were similar but slightly lower in the aborted tissue 

6 of cases compared with controls (mean ± SD 4.8 ± 6.0 in cases and 6.0 ± 7.4 in controls, p = 0.29). In 

7 the blood samples, however, benzo[a]pyrene-adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ± 

8 2.2 in cases and controls, respectively, p < 0.001). In logistic regression analyses using a continuous 

9 adduct measure, the OR was 1.35 (95% CI 1.11–1.64) per adduct/108 nucleotide. These results 

10 were adjusted for education and household income, but were very similar to the unadjusted results. 

11 Categorizing exposure at the median value resulted in an adjusted OR of 4.27 (95% CI 1.41–12.99) 

12 in the high compared with low benzo[a]pyrene-adduct group. There was no relation between 

13 benzo[a]pyrene-adduct levels in the aborted tissue and missed abortion in the logistic regression 

14 analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93–1.02) or dichotomous 

15 exposure measure (adjusted OR 0.76, 95% CI 0.37–1.54). Associations between missed abortion 

16 and several interview-based measures of potential PAH exposure were also seen: adjusted OR 3.07 

17 (95% CI 1.31–7.16) for traffic congestion near residence, 3.52 (95% CI 1.44–8.57) for commuting 

18 by walking, 3.78 (95% CI 1.11–12.87) for routinely cooked during pregnancy, and 3.21 (95% CI 

19 0.98–10.48) for industrial site or stack near residence, but there was no association with other 

20 types of commuting (e.g., by bike, car, or bus).

21 Perera et al. (2005a) studied 329 nonsmoking pregnant women (30 ± 5 years old) possibly 

22 exposed to PAHs from fires during the 4 weeks after 09/11/2001. Maternal and umbilical cord 

23 blood levels of benzo[a]pyrene (BPDE)-DNA adducts were highest in study participants who lived 

24 within 1 mile of the WTC, with an inverse correlation between cord blood levels and distance from 

25 the WTC. Neither cord blood adduct level nor ETS alone was positively correlated with adverse 

26 birth outcomes. However, the interaction between ETS exposure and cord blood adducts was 

27 significantly associated with reduced birth weight and head circumference. Among babies exposed 

28 to ETS in utero, a doubling of cord blood benzo[a]pyrene-DNA adducts was associated with an 8% 

29 decrease in birth weight (p = 0.03) and a 3% decrease in head circumference (p = 0.04).

30 Perera et al. (2005b) compared various exposures—ETS, nutrition, pesticides, material 

31 hardship—with birth outcomes (length, head circumference, cognitive development). ETS 

32 exposure and intake of PAH-rich foods by pregnant women were determined by questionnaire. 

33 Levels of benzo[a]pyrene diol epoxide (BPDE)-DNA adducts were determined in umbilical cord 

34 blood collected at delivery. The study population consisted of Dominican or African-American 

35 nonsmoking pregnant women (n = 529; 24 ± 5 years old) free of diabetes, hypertension, HIV, and 

36 drug or alcohol abuse. Benzo[a]pyrene adducts, ETS, and dietary PAHs were not significantly 

37 correlated with each other. However, the interaction between benzo[a]pyrene-DNA adducts and 
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1 ETS exposure was significantly associated with reduced birth weights (-6.8%; p = 0.03) and 

2 reduced head circumference (-2.9%; p = 0.04).

3 Tang et al. (2006) measured benzo[a]pyrene diol epoxide (BPDE)-DNA adducts in maternal 

4 and umbilical cord blood obtained at delivery from a cohort of 150 nonsmoking women and their 

5 newborns in China. Exposure assessment was related to the seasonal operation of a local, coal-fired 

6 power plant; however, airborne PAH concentrations were not measured. Dietary PAH intake was 

7 not included as a covariate because it did not significantly contribute to the final models, but ETS, 

8 sex, and maternal height and weight were considered as covariates. DNA adduct levels were 

9 compared to several birth outcomes and physical development parameters, such as gestational age 

10 at birth; infant sex, birth weight, length, head circumference, and malformations; maternal height 

11 and pregnancy weight total weight gain; complications of pregnancy and delivery; and medications 

12 used during pregnancy.

13 High cord blood adduct levels were significantly associated with reduced infant/child 

14 weight at 18 months (β = -0.048, p = 0.03), 24 months (β = -0.041, p = 0.027), and 30 months of age 

15 (β = -0.040, p = 0.049); decreased birth head circumference was marginally associated with DNA 

16 adduct levels (β = -0.011, p = 0.057). Maternal adduct levels were correlated neither with cord 

17 blood adduct levels nor with fetal and child growth. Among female infants, cord blood adduct levels 

18 were significantly associated with smaller birth head circumference (p = 0.022) and with lower 

19 weight at 18 months (p = 0.014), 24 months (p = 0.012), and 30 months of age (p = 0.033), and with 

20 decreased body length at 18 months of age (p = 0.033). Among male infants, the corresponding 

21 associations were also inverse but were not statistically significant.

22 Considerable evidence of a deleterious effect of smoking on male and female fertility has 

23 accumulated from epidemiological studies of time to pregnancy, ovulatory disorders, semen 

24 quality, and spontaneous abortion (reviewed in Waylen et al., 2009; Cooper and Moley, 2008; 

25 Soares and Melo, 2008). In addition, the effect of smoking, particularly during the time of the 

26 perimenopausal transition, on acceleration of ovarian senescence (menopause) has also been 

27 established (Midgette and Baron, 1990). More limited data are available pertaining specifically to 

28 measures of benzo[a]pyrene and reproductive outcomes.

29 Neal et al. (2008, 2007) examined levels of benzo[a]pyrene and other PAHs in follicular 

30 fluid and serum sample from 36 women undergoing in vitro fertilization at a clinic in Toronto, and 

31 compared the successful conception rate in relation to benzo[a]pyrene levels. The women were 

32 classified by smoking status, with 19 current cigarette smokers, 7 with passive or sidestream 

33 smoke exposure (i.e., nonsmoker with a partner who smoked), and 10 nonsmokers exposed. An 

34 early follicular phase blood sample and follicular fluid sample from the follicle at the time of ovum 

35 retrieval were collected and analyzed for the presence of benzo[a]pyrene, acenapthelene, 

36 phenanthrene, pyrene, and chrysene using gas chromatography/MS (detection limit 5 pg/mL). The 

37 frequency of nondectable levels of serum benzo[a]pyrene was highest in the nonsmoking group 

38 (60.0, 14.3, and 21.0% below detection limit in nonsmoking, sidestream smoke, and active smoking 
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1 groups, respectively). A similar pattern was seen with follicular fluid benzo[a]pyrene (30.0, 14.3, 

2 and 10.5% below detection limit in nonsmoking, sidestream smoke, and active smoking groups, 

3 respectively). In the analyses comparing mean values across groups, an assigned value of 0 was 

4 used for nondetectable samples. Follicular fluid benzo[a]pyrene levels were higher in the active 

5 smoking group (mean ± SE, 1.32 ± 0.68 ng/mL) than in the sidestream (0.05 ± 0.01 ng/mL) or 

6 nonsmoking (0.03 ± 0.01 ng/mL) groups (p = 0.04). The between-group differences in serum 

7 benzo[a]pyrene levels were not statistically significant (0.22 ± 0.15, 0.98 ± 0.56, and 0.40 ± 

8 0.13 ng/mL in nonsmoking, sidestream smoke, and active smoking groups, respectively), and there 

9 were no differences in relation to smoking status. Among active smokers, the number of cigarettes 

10 smoked per day was strongly correlated with follicular fluid benzo[a]pyrene levels (r = 0.7, p < 

11 0.01). Follicular fluid benzo[a]pyrene levels were significantly higher among the women who did 

12 not conceive (1.79 ng/mL ± 0.86) compared with women who did get pregnant (mean 

13 approximately 0.10 ng/mL, as estimated from graph) (p < 0.001), but serum levels of 

14 benzo[a]pyrene were not associated with successful conception.

15 A small case-control study conducted between August 2005 and February 2006 in Lucknow 

16 city (Uttar Pradesh), India examined PAH concentrations in placental tissues (Singh et al., 2008) in 

17 relation to risk of preterm birth. The study included 29 cases (delivery between 28 and <36 weeks 

18 of gestation) and 31 term delivery controls. Demographic data smoking history, reproductive 

19 history, and other information were collected by interview, and a 10 g sample of placental tissue 

20 was collected from all participants. Concentration of specific PAHs in placental tissue was 

21 determined using HPLC. In addition to benzo[a]pyrene, the PAHs assayed were naphthalene, 

22 acenapththylene, phenanthrene, fluorene, anthracene, benzo(a)anthracene, fluoranthene, pyrene, 

23 benzo(k)fluoranthene, benzo(b)fluoranthene, benzo(g,h,i)perylene, and dibenzo(a,h)anthracene. 

24 PAH exposure in this population was from environmental sources and from cooking. The age of 

25 study participants ranged from 20 to 35 years. There was little difference in birth weight between 

26 cases and controls (mean 2.77 kg and 2.75 kg in the case and control groups, respectively). 

27 Placental benzo[a]pyrene levels were lower than the levels of the other PAHs detected (mean 8.83 

28 ppb in controls for benzo[a]pyrene compared with 25–30 ppb for anthracene, 

29 benzo(k)fluoranthene, benzo(b)fluoranthene, and dibenzo(a,h)anthracene, 59 ppb for 

30 acenaphthylene, and 200–380 ppm for naphthalene, phenanthrene, fluoranthene, and pyrene; 

31 nondetectable levels of fluorine, benzo(a)anthracene, and benzo(g,h,i)perylene were found). There 

32 was little difference in benzo[a]pyrene levels between cases (mean ± SE 13.85 ± 7.06 ppb) and 

33 controls (8.83 ± 5.84 ppb), but elevated levels of fluoranthene (325.91 ± 45.14 and 208.6 ± 21.93 

34 ppb in cases and controls, respectively, p < 0.05) and benzo(b)fluoranthene (61.91 ± 12.43 and 

35 23.84 ± 7.01 ppb in cases and controls, respectively, p < 0.05) were seen.

36 Wu et al. (2010) conducted a study of benzo[a]pyrene-DNA adduct levels in relation to risk 

37 of fetal death in Tianjin, China. This case-control study included women who experienced a missed 

38 abortion before 14 weeks gestational age (i.e., a fetal death that remained in utero and therefore 
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1 required surgical intervention). Cases were matched by age and gravidity to controls (women 

2 undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded 

3 women who smoked, women with chronic disease and pregnancy complications, and women with 

4 occupational exposures to PAHs. Residency within Tianjin for at least 1 year was also an eligibility 

5 criterion. The participation rate was high: 81/84 eligible cases participated and 81/89 eligible 

6 controls participated. Data pertaining to demographic characteristics, reproductive history, and 

7 factors relating to potential PAH exposure were collected using a structured interview, and samples 

8 from the aborted tissue were obtained. In two of the four hospitals used in the study, blood 

9 samples from the women (n = 51 cases and 51 controls) were also collected. The presence of 

10 benzo[a]pyrene-BPDE adducts was assessed in the blood and tissue samples using HPLC. There 

11 was no correlation between blood and aborted tissue levels of benzo[a]pyrene adducts (r = -0.12 

12 for the 102 blood-tissue pairs, r = -0.02 for the 51 case pairs and r = -0.21 for the 51 control pairs). 

13 (The authors noted that there was little difference between women with and without blood 

14 samples in terms of the interview-based measures collected or in terms of the DNA-adduct levels in 

15 aborted tissue.) Benzo[a]pyrene-adduct levels were similar but slightly lower in the aborted tissue 

16 of cases compared with controls (mean ± SD 4.8 ± 6.0 in cases and 6.0 ± 7.4 in controls, p = 0.29). In 

17 the blood samples, however, benzo[a]pyrene-adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ± 

18 2.2 in cases and controls, respectively, p < 0.001). In logistic regression analyses using a continuous 

19 adduct measure, the OR was 1.35 (95% CI 1.11–1.64) per adduct/108 nucleotide. These results 

20 were adjusted for education and household income, but were very similar to the unadjusted results. 

21 Categorizing exposure at the median value resulted in an adjusted OR of 4.27 (95% CI 1.41–12.99) 

22 in the high compared with low benzo[a]pyrene-adduct group. There was no relation between 

23 benzo[a]pyrene-adduct levels in the aborted tissue and missed abortion in the logistic regression 

24 analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93–1.02) or dichotomous 

25 exposure measure (adjusted OR 0.76, 95% CI 0.37–1.54). Associations between missed abortion 

26 and several interview-based measures of potential PAH exposure were also seen: adjusted OR 3.07 

27 (95% CI 1.31–7.16) for traffic congestion near residence, 3.52 (95% CI 1.44–8.57) for commuting 

28 by walking, 3.78 (95% CI 1.11–12.87) for routinely cooked during pregnancy, and 3.21 (95% CI 

29 0.98–10.48) for industrial site or stack near residence, but there was no association with other 

30 types of commuting (e.g., by bike, car, or bus).

31 Neurotoxicity

32 Niu et al. (2010) studied 176 Chinese coke-oven workers with elevated B[a]P exposure and 

33 compared them against 48 referents (workers in a supply warehouse), matched by socioeconomic 

34 status, lifestyle and health. Blood levels of monoamine, amino acid and chloine neurotransmitters 

35 were measured, and the WHO Neurobehavioral Core Test Battery (NCTB) was administered to 

36 assess emotional state, learning, memory and hand-eye coordination. The authors self-designed a 

37 study questionnaire to gather information on worker education, vocational history, smoking and 

38 drinking habits, personal habits, personal and family medical history, as well as any current 
Toxicological Review of benzo[a]pyrene

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1 symptoms and medications used in the pervious several weeks. Workers were excluded from the 

2 study for any of the following criteria: reported feeling depressed at any point during the previous 

3 6 months; had taken medicine in the previous 2 weeks which could affect nervous system function; 

4 or if they reported undertaking vigorous exercise less than 48 hrs previously. “Smoking” was 

5 defined as ≥ 10 cigarettes/day during the past year. Similarly, “drinking” was defined as 

6 wine/beer/spirits consumed ≥ 3 times/week for the past 6 months. Workplace environmental 

7 sampling stations were established at each of the physical work locations, including the referent’s 

8 warehouse, and dual automatic air sampling pumps collected samples at personal breathing zone 

9 height for 6 hours/day, over 3 consecutive days. B[a]P content was determined by HPLC, and 

10 relative exposure was compared to post-shift urine levels of a B[a]P metabolite, 1-hydroxypyrene 

11 (1-OH-Py). Blood was collected in the morning before breakfast; monoamine (norepinephrine and 

12 dopamine) and amino acid (Glu, Asp, Gly, and GABA) neurotransmitter levels were determined by 

13 HPLC, acetylcholine (Ach) levels determined by hydroxyamine chromometry, and Ach esterase 

14 (AchE) levels measured in lysed RBCs using activity kits. 

15 B[a]P mean concentrations were 19.56 ± 13.2, 185.96 ± 38.6 and 1623.56 ± 435.8 ng/m3 at 

16 the bottom, side and top of the coke oven, respectively, all of which were higher than the mean at 

the referents’ warehouse (10.26 ± 7.6 ng/m3 17 ). The authors did not report stratified analysis by 

18 different levels of B[a]P exposure, and reported only comparisons between the referents and all 

19 exposed workers combined (Table B-3), or between workers grouped by urinary B[a]P metabolite 

20 1-OH-Py levels (Table B-4). There were no significant differences in age, education, smoking or 

21 alcohol use between the coke oven and warehouse workers. Urinary 1-OH-Py levels were 32% 

22 higher in coke oven workers compared to the referent group, corresponding to the higher levels of 

23 B[a]P detected in all coke oven workstation compared to the supply warehouse. Performance in 

24 two neurobehavioral function tests, digit span and forward digit span, were significantly decreased 

25 in the exposed oven workers versus control group; when stratified by urinary metabolite level, 

26 scores significantly decreased with increasing 1-OH-Py levels. Of the neurotransmitters assessed, 

27 norepinephrine, dopamine, Asp and GABA were significantly decreased in exposed versus control 

28 workers; norepinephrine and Asp were also significantly and inversely related with 1-OH-Py levels. 

29 Dopamine levels appeared to decrease with increased urinary metabolite levels, although the 

30 relationship was not statistically significant. GABA levels were highly variable, and appeared to 

31 increase with increasing 1-OH-Py levels, although this relationship was statistically significant. 

32 Acetylcholine levels were 4-fold higher in coke oven workers compared to referents, and AchE 

33 actiivty 30% lower; both Ach and AchE were significantly associated with urinary B[a]P metabolite 

34 levels, although Ach increased and AchE activity decreased with increasing 1-OH-Py. The authors 

35 reported results of correlation analysis, indicating that digit span scores correlated negatively with 

36 Ach and positively with AchE (coefficients of -0.230, -0.276 and 0.120, 0.170, respectively), 

37 although no indication of statistical significance was given. No other associations were reported.
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1 Table B-3. Exposure-related effects in Chinese coke oven workers or 

2 warehouse controls exposed to benzo[a]pyrene in the workplace

Effect measured

Exposure Group

P value

Controls (n=48) Exposed workers (n=176)

Background information (mean ± SD, incidence or %)

Age (yr)

Education (junior/senior)

Smoking

Drinking

39.71 ± 7.51

23/25

77%

27%

37.86 ± 6.51

110/66

64%

39%

0.098

0.068

0.093

0.140

Urine B[a]P metabolite (μmol/mol Cr; mean ± SD)

1-OH-Py 2.77 ± 1.45 3.66 ± 0.67 0.000

Neurobehavioral function tests (mean ± SD)

Simple reaction time

Digit span

Forward digit span

413.88 ± 95.40

17.31 ± 4.54

10.65 ± 2.42

437.39 ± 88.44

15.47 ± 4.08

9.25 ± 2.64

0.109

0.006

0.001

Neurotransmitter concentrations (mean ± SD)

Norepinephrine (ng/ml)

Dopamine (ng/ml)

Asp (μg/ml)

Glu (μg/ml)

GABA (μg/ml)

Ach (μg/ml)

AchE activity (U/mg protein)

62.54 ± 58.07

1566.28 ± 317.64

2.13 ± 1.66

11.21 ± 5.28

2.52 ± 5.16

172.60 ± 67.19

71.31 ± 46.18

40.62 ± 29.78

1425.85 ± 422.66

1.58 ± 0.99

9.68 ± 5.72

1.01 ± 2.21

704.00 ± 393.86

50.27 ± 34.02

0.000

0.029

0.004

0.074

0.004

0.000

0.012

Source: Niu et al. (2010).
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1 Table B-4. Exposure-related effects in Chinese coke oven workers or 

2 warehouse controls exposed to benzo[a]pyrene in the workplace, 

3 stratified by urinary metabolite levels

Effect measured

Exposure Group categoried by 1-OH-Py level

P value 0 – 3.09 

μmol/mol Cr 

3.09 – 3.90 

μmol/mol Cr 

3.90 – 5.53 

μmol/mol Cr 

Number of subjects 33 72 36

Neurobehavioral function tests (mean ± SD)

Digit span

Forward digit span

Backward digit span

Right dotting

18.24 ± 4.58

10.85 ± 2.12

7.20 ± 3.07

152.15 ± 35.43

16.04 ± 4.24

9.80 ± 2.86

6.38 ± 2.55

153.80 ± 31.55

15.78 ± 3.71

9.58 ± 2.33

6.20 ± 2.15

167.22 ± 59.21

0.003

0.019

0.089

0.094

Neurotransmitter concentrations (mean ± SD)

Norepinephrine (ng/ml)

Dopamine (ng/ml)

Asp (μg/ml)

Glu (μg/ml)

GABA (μg/ml)

Ach (μg/ml)

AchE activity (U/mg

protein)

67.31 ± 67.45

1614.45 ± 683.57

2.29 ± 2.13

11.56 ± 8.92

1.40 ± 3.59

334.66 ± 83.75

68.17 ± 9.28

 

36.97 ± 23.58

1482.30 ± 323.66

1.61 ± 0.71

9.93 ± 4.14

1.42 ± 3.44

483.71 ± 57.87

54.98 ± 4.23

46.75 ± 35.88

1405.06 ± 332.23

1.47 ± 0.58

9.06 ± 3.30

1.56 ± 3.24

665.85 ± 94.34

52.64 ± 4.60

0.002

0.134

0.001

0.070

0.964

0.030

0.043

Source: Niu et al. (2010).

4 Immunotoxicity

5 Zhang et al. (2012) studied 129 Chinese coke-oven workers with elevated B[a]P exposure 

6 and compared them against 37 referents (workers in a supply warehouse), matched by 

7 socioeconomic status, lifestyle and health. Area B[a]P levels were quantified in the various work 

8 areas, and the primary endpoint was the level of early and late apoptosis in PBMCs isolated from 

9 each worker sub-group the morning following an overnight fast. The authors self-designed a study 

10 questionnaire to gather information on worker education, vocational history, smoking and drinking 

11 habits, personal habits, personal and family medical history, as well as any current symptoms and 

12 medications used in the pervious several weeks. “Smoking” was defined as ≥ 10 cigarettes/day 

13 during the past year, with “smoking index” defined as cigarettes/day x years smoking. Similarly, 

14 “drinking” was defined as wine/beer/spirits consumed ≥ 3 times/week for the past 6 months, and 

15 “drinking index” defined as grams of alcohol consumed/day x years drinking. Exposed workers 

16 were categorized by physical worksite location and expected differences in B[a]P exposure: 34 

17 oven bottom workers, 48 oven side workers, and 47 oven top workers. Workplace environmental 

18 sampling stations were established at each of the physical work locations, including the referent’s 

19 warehouse, and dual automatic air sampling pumps collected samples at personal breathing zone 

20 height for 6 hours/day, over 3 consecutive days. B[a]P content was determined by HPLC, and 
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1 relative exposure was compared to post-shift urine levels of a B[a]P metabolite, 1-hydroxypyrene 

2 (1-OH-Py). Collected and purified PBMCs were incubated with Annexin-V and PI prior to analysis 

3 by flow cytometry; early apoptotic cells were considered to be Annexin V+/PI-, while late apoptotic 

4 cells were considered Annexin V+/PI+. 

5 All apoptosis data was displayed graphically, and in all groupings early:late apoptotic 

6 PBMCs occurred at an approximate 2:1 frequency. PBMC apoptosis was similar in each of the three 

7 coke oven worker groups, which were all statistically significantly higher than referents 

8 (approximately 2-fold) for both early and late apoptosis. While self-reported smoking incidence 

9 varied significantly among the 4 worker groups, stratification by smoking years or smoking index 

10 did not reveal any significant association with PBMC apoptosis. Multiple linear stepwise regression 

11 analysis suggested that urine 1-OH-Py levels and years of coke oven operation were positively 

12 associated with increased early and late PMBC apoptosis (Table B-5), and that years of ethanol 

13 consumption was negatively associated with only early apoptosis. These associations were tested 

14 by stratifying workers into three groups by urinary 1-OH-Py levels or coke oven operation years, 

15 and in both cases, the groups with the highest urinary metabolite levels or longest oven operating 

16 experience had statistically significantly higher levels of both early and late apoptotic PBMCs, vs. 

17 the lowest or shortest duration groups, respectively. Likewise, when sorted into groups based 

18 upon years of ethanol consumption, the highest ethanol “years of consumption” group had 

19 statistically significantly lower early apoptosis rates when compared to the lowest ethanol 

20 consuming group.

21 Table B-5. Background information on Chinese coke oven workers or 

22 warehouse controls exposed to benzo[a]pyrene in the workplace

Effect measured

Exposure Group (ng/m3; mean ± SD)

10.2 ± 7.6 19.5 ± 13.2 185.9 ± 38.6 1623.5 ± 435.8 P value

Number of subjects 37 34 48 47

Background information (mean ± SD or %)

Age (yr)

Working years (yr)

Smoking

Drinking

37.16 ± 6.00

17.35 ± 7.19

62.2

24.3

39.09 ± 5.53

18.58 ± 7.23

64.7

41.2

36.98 ± 6.40

16.78 ± 6.90

83.3

39.6

37.34 ± 6.78

17.26 ± 7.44

53.2

44.7

0.451

0.742

0.017

0.259

Urine B[a]P metabolite (μmol/mol Cr; mean ± SD)

1-OH-Py 2.78 ± 1.04 3.22 ± 0.81* 3.51 ± 0.55* 3.66 ± 0.58* 0.000

* p < 0.05 significantly different from control mean

Source: Zhang et al. (2012).

23
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1 Cancer-related Endpoints

2 Benzo[a]pyrene-Induced Cytogenetic Damage

3 Many studies measure cytogenetic damage as biomarkers of early biological effects which 

4 also reflect exposure to genotoxic chemicals. Standard cytogenetic end points include 

5 chromosomal aberration (CA), sister chromatid exchange (SCE), micronucleus (MN) formation, 

6 hypoxanthine guanine phosphoribosyl transferase (hprt) mutation frequency, and glycophorin A 

7 mutation frequency (Gyorffy et al., 2008). These biomarkers are often incorporated in multi8 endpoint studies with other biomarkers of exposure. Because they indicate related but different 

9 endpoints, there is often a lack of correlation between the different categories of biomarkers.

Merlo et al. (1997) evaluated DNA adduct formation (measured by [ 10 32P]-postlabelling) and 

11 MN in WBCs of 94 traffic policemen versus 52 residents from the metropolitan area of Genoa, Italy. 

12 All study subjects wore personal air samplers for 5 hours of one work shift, and levels of 

13 benzo[a]pyrene and other PAHs were measured. Policemen were exposed to 4.55 ng 

benzo[a]pyrene/m3 air, compared with urban residents who were exposed to 0.15 ng/m3 14 . DNA 

15 adduct levels in policemen were 35% higher than in urban residents (p = 0.007), but MN in urban 

16 residents were 20% higher than in policemen (p = 0.02). Linear regressions of DNA adducts and 

17 MN incidence, respectively, versus benzo[a]pyrene exposure levels did not reveal significant 

18 correlations.

19 Perera and coworkers assessed DNA damage in Finnish iron foundry workers in two 

20 separate studies and using three methodologies. Based on results from personal sampling and 

21 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/m3 22 ) (Perera et 

23 al., 1994, 1993). In the first study, involving 48 workers, several biomarkers were analyzed for 

24 dose-response and interindividual variability (Perera et al., 1993). PAH-DNA adducts were 

25 determined in WBCs using an immunoassay as described in Section 4.1.2.2.1 and enzyme-linked 

26 immunosorbent assay with fluorescence detection. Mutations at the hprt locus were also measured 

27 in WBC DNA. The latter assay is based on the fact that each cell contains only one copy of the hprt 

28 gene, which is located on the X-chromosome. While male cells have only one X-chromosome, 

29 female cells inactivate one of the two X-chromosomes at random. The gene is highly sensitive to 

30 mutations such that in the event of a crucial mutation in the gene, enzyme activity disappears 

31 completely from the cell. In addition, mutations at the glycophorin A gene locus were measured in 

32 red blood cells (RBCs). The glycophorin A mutation frequency was not correlated with either 

33 benzo[a]pyrene exposure or PAH-DNA adduct formation. However, both PAH-DNA adduct levels 

34 and hprt mutation frequency increased with increasing benzo[a]pyrene exposure. In addition, 

35 there was a highly significant correlation between incidence of hprt mutations and PAH-DNA 

36 adduct levels (p = 0.004).

37 In a second study, Perera et al. (1994) surveyed 64 iron foundry workers with assessments 

38 conducted in 2 successive years; 24 of the workers provided blood samples in both years. Exposure 
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1 to benzo[a]pyrene, collected by personal and area sampling in the first year of the study, ranged 

2 from <5 to 60 ng/m3 and was estimated to have decreased by 40% in the second year. The levels of 

3 PAH-DNA adducts were roughly 50% lower in the 2nd year, presumably reflecting decreased 

4 exposure. The longer-lived hprt mutations were not as strongly influenced by the decreasing 

5 exposure to benzo[a]pyrene. Study subjects who did not have detectable levels of DNA adducts 

6 were excluded from the study. As in the previous study, a strong correlation between DNA adduct 

7 levels and incidence of hprt mutations was observed (Perera et al., 1993).

8 Kalina et al. (1998) studied several cytogenetic markers in 64 coke oven workers and 

9 34 controls employed at other locations within the same plant. Airborne benzo[a]pyrene and seven 

10 other carcinogenic PAHs were collected by personal air samplers, which showed ambient 

benzo[a]pyrene concentrations ranging widely from 0.002 to 50 μg/m3 11 in coke oven workers and 

from 0.002 to 0.063 μg/m3 12 in controls. CAs, SCEs, high-frequency cells (HFCs), and SCE 

13 heterogeneity index were all significantly increased with benzo[a]pyrene exposure. Except for 

14 increases in HFCs, no effect of smoking was observed. Consistent with studies of PAH-DNA adduct 

15 formation, reduced cytogenetic response at high exposure levels produced a nonlinear dose16 response relationship. The authors also evaluated the potential influence of polymorphisms in 

17 enzymes involved in the metabolism of benzo[a]pyrene. Glutathione S-transferase M1 (GSTM-1) 

18 and N-acetyl transferase-2 polymorphisms were studied and no evidence of the two gene 

19 polymorphisms having any influence on the incidence of cytogenetic damage was found.

20 Motykiewicz et al. (1998) conducted a similar study of genotoxicity associated with 

21 benzo[a]pyrene exposure in 67 female residents of a highly polluted industrial urban area of Upper 

22 Silesia, Poland, and compared the results to those obtained from 72 female residents of another 

23 urban but less polluted area in the same province of Poland. Urinary mutagenicity and 1-

24 hydroxypyrene levels, PAH-DNA adducts in oral mucosa cells (detected by immunoperoxidase 

25 staining), SCEs, HFCs, CAs, bleomycin sensitivity, and GSTM-1 and CYP1A1 polymorphisms in blood 

26 lymphocytes were investigated. High volume air samplers and gas chromatography were used to 

quantify ambient benzo[a]pyrene levels, which were 3.7 ng/m3 27 in the polluted area and 0.6 ng/m3

in the control area during the summer. During winter, levels rose to 43.4 and 7.2 ng/m3 28 in the two 

29 areas, respectively. The cytogenetic biomarkers (CA and SCE/HFC), urinary mutagenicity, and 

30 urinary 1-hydroxypyrene excretion were significantly increased in females from the polluted area, 

31 and differences appeared to be more pronounced during winter time. PAH-DNA adduct levels were 

32 significantly increased in the study population, when compared to the controls, only in the winter 

33 season. No difference in sensitivity to bleomycin-induced lymphocyte chromatid breaks was seen 

34 between the two populations. As with the study by Kalina et al. (1998), genetic polymorphisms 

35 assumed to affect the metabolic transformation of benzo[a]pyrene were not associated with any 

36 difference in the incidence of DNA damage.

37 In a study of Thai school boys in urban (Bangkok) and rural areas, bulky (including but not 

38 limited to BPDE-type) DNA adduct levels were measured in lymphocytes along with DNA SSBs, 
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1 using the comet assay, and DNA repair capacity (Tuntawiroon et al., 2007). Ambient air and 

2 personal breathing zone measurements indicated that Bangkok school children experienced 

3 significantly higher exposures to benzo[a]pyrene and total PAHs. A significantly higher level of 

4 SSBs (tail length 1.93 ± 0.09 versus 1.28 ± 0.12 μm, +51%; p < 0.001) was observed in Bangkok 

5 school children when compared with rural children, and this parameter was significantly 

6 associated with DNA adduct levels. A significantly reduced DNA repair capacity (0.45 ± 0.01 versus 

7 0.26 ± 0.01 γ-radiation-induced deletions per metaphase, -42%; p < 0.001) was also observed in the 

8 city school children, again significantly associated with DNA adduct levels. It was not evident why 

9 higher environmental PAH exposure would be associated with lowered DNA repair capacity. 

10 However, because the personal breathing zone PAH levels and DNA adduct levels were not 

11 associated with each other, it is conceivable that the city school children had a priori lower DNA 

12 repair capacities that contributed significantly to the high adduct levels. The authors considered 

13 genetic differences between the two study populations as a possible reason for this observation.

14 Epidemiologic Findings in Humans

15 The association between human cancer and contact with PAH-containing substances, such 

16 as soot, coal tar, and pitch, has been widely recognized since the early 1900s (Bostrom et al., 2002). 

17 Although numerous epidemiology studies establish an unequivocal association between PAH 

18 exposure and human cancer, defining the causative role for benzo[a]pyrene and other specific PAHs 

19 remains a challenge. In essentially all reported studies, either the benzo[a]pyrene exposure and/or 

20 internal dose are not known, or the benzo[a]pyrene carcinogenic effect cannot be distinguished 

21 from the effects of other PAH and non-PAH carcinogens. Nevertheless, three types of investigations 

22 provide support for the involvement of benzo[a]pyrene in some human cancers: molecular 

23 epidemiology studies; population- and hospital-based case-control studies; and occupational cohort 

24 studies. In some cohort studies, benzo[a]pyrene exposure concentrations were measured and thus 

25 provide a means to link exposure intensity with observed cancer rates. In case-control studies, by 

26 their nature, benzo[a]pyrene and total PAH doses can only be estimated.

27 Molecular Epidemiology and Case-Control Cancer Studies

28 Defective DNA repair capacity leading to genomic instability and, ultimately, increased 

29 cancer risk is well documented (Wu et al., 2007, 2005). Moreover, sensitivity to mutagen-induced 

30 DNA damage is highly heritable and thus represents an important factor that determines individual 

31 cancer susceptibility. Based on studies comparing monozygotic and dizygotic twins, the genetic 

32 contribution to BPDE mutagenic sensitivity was estimated to be 48.0% (Wu et al., 2007). BPDE has 

33 been used as an etiologically relevant mutagen in case-control studies to examine the association 

34 between elevated lung and bladder cancer risk and individual sensitivity to BPDE-induced DNA 

35 damage. Mutagen sensitivity is determined by quantifying chromatid breaks or DNA adducts in 

36 phytohemagglutinin-stimulated peripheral blood lymphocytes as an indirect measure of DNA 

37 repair capacity.
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1 In a hospital-based, case-control study involving 221 lung cancer cases and 229 healthy 

2 controls, DNA adducts were measured in stimulated peripheral blood lymphocytes after incubation 

3 with BPDE in vitro (Li et al., 2001). Lung cancer cases showed consistent statistically significant 

4 elevations in induced BPDE-DNA adducts in lymphoctes, compared with controls, regardless of 

5 subgroup by age, sex, ethnicity, smoking history, weight loss, or family history of cancer. The 

6 lymphocyte BPDE-induced DNA adduct levels, when grouped by quartile using the levels in controls 

7 as cutoff points, were significantly dose-related with lung cancer risk (odds ratios [ORs] 1.11, 1.62, 

8 and 3.23; trend test, p < 0.001). In a related hospital-based, case-control study involving 155 lung 

9 cancer patients and 153 healthy controls, stimulated peripheral blood lymphocytes were exposed 

10 to BPDE in vitro (Wu et al., 2005). DNA damage/repair was evaluated in lymphocytes using the 

11 comet assay, and impacts on cell cycle checkpoints were measured using a fluorescence-activated 

12 cell-sorting method. The lung cancer cases exhibited significantly higher levels of BPDE-induced 

13 DNA damage than the controls (p < 0.001), with lung cancer risk positively associated with 

14 increasing levels of lymphocyte DNA damage when grouped in quartiles (trend test, p < 0.001). In 

15 addition, lung cancer patients demonstrated significantly shorter cell cycle delays in response to 

16 BPDE exposure to lymphocytes, which correlated with increased DNA damage.

17 Sensitivity to BPDE-induced DNA damage in bladder cancer patients supports the results 

18 observed in lung cancer cases. In a hospital-based, case-control study involving 203 bladder cancer 

19 patients and 198 healthy controls, BPDE-induced DNA damage was specifically evaluated at the 

20 chromosome 9p21 locus in stimulated peripheral blood lymphocytes (Gu et al., 2008). Deletions of 

21 9p21, which includes critical components of cell cycle control pathways, are associated with a 

22 variety of cancers. After adjusting for age, sex, ethnicity, and smoking status, individuals with high 

23 BPDE-induced damage at 9p21 were significantly associated with increased bladder cancer risk 

24 (OR 5.28; 95% confidence interval [CI] 3.26–8.59). Categorization of patients into tertiles for BPDE 

25 sensitivity relative to controls demonstrated a dose-related association between BPDE-induced 

26 9p21 damage and bladder cancer risk. Collectively, the results of molecular epidemiology studies 

27 with lung and bladder cancer patients indicate that individuals with a defective ability to repair 

28 BPDE-DNA adducts are at increased risk for cancer and, moreover, that specific genes linked to 

29 tumorigenesis pathways may be molecular targets for benzo[a]pyrene and other carcinogens.

30 Due to the importance of the diet as a benzo[a]pyrene exposure source, several population31 and hospital-based, case-control studies have investigated the implied association between dietary 

32 intake of benzo[a]pyrene and risk for several tumor types. In a study involving 193 pancreatic 

33 cancer cases and 674 controls (Anderson et al., 2005), another involving 626 pancreatic cancer 

34 cases and 530 controls (Li et al., 2007), and a third involving 146 colorectal adenoma cases and 228 

35 controls (Sinha et al., 2005), dietary intake of benzo[a]pyrene was estimated using food frequency 

36 questionnaires. In all studies, the primary focus was on estimated intake of benzo[a]pyrene (and 

37 other carcinogens) derived from cooked meat. Overall, cases when compared with controls had 

38 higher intakes of benzo[a]pyrene and other food carcinogens, leading to the conclusion that 
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1 benzo[a]pyrene plays a role in the etiology of these tumors in humans. In a supportive follow-up 

2 case-control study of colorectal adenomas, levels of leukocyte PAH-DNA adducts were significantly 

3 higher in cases when compared with controls (p = 0.02), using a method that recognizes BPDE and 

4 several other PAHs bound to DNA (Gunter et al., 2007).

5 Cohort Cancer Studies

6 Epidemiologic studies of workers in PAH-related occupations indicate increased human 

7 cancer risks associated with iron and steel production, roofing, carbon black production, and 

8 exposure to diesel exhaust (Bosetti et al., 2007). Exposure to benzo[a]pyrene is only one of 

9 numerous contributors to the cancer risk from complex PAH-containing mixtures that occur in the 

10 workplace. Although some occupational cohort studies report measured or estimated inhalation 

11 exposure concentrations for benzo[a]pyrene, none report biomarkers of internal benzo[a]pyrene 

12 dose in study subjects (reviewed in Bosetti et al., 2007; Armstrong et al., 2004). Several of these 

13 cohort studies (summarized below) demonstrate a positive exposure-response relationship with 

14 cumulative PAH exposure using benzo[a]pyrene—or a proxy such as benzene-soluble matter (BSM) 

15 that can be converted to benzo[a]pyrene—as an indicator substance. These studies provide insight 

16 and support for the causative role of benzo[a]pyrene in human cancer.

17

18 Cancer incidence in aluminum and electrode production plants 

19 Exposure to benzo[a]pyrene and BSM in aluminum smelter workers is strongly associated 

20 with bladder cancer and weakly associated with lung cancer (Boffetta et al., 1997; Tremblay et al., 

21 1995; Armstrong et al., 1994; Gibbs, 1985; Theriault et al., 1984). In an analysis of pooled data from 

22 nine cohorts of aluminum production workers, 688 respiratory tract cancer cases were observed 

23 versus 674.1 expected (pooled RR 1.03; CI 0.96–1.11) (Bosetti et al., 2007). A total of 196 bladder 

24 cancer cases were observed in eight of the cohorts, compared with 155.7 expected (pooled relative 

25 risk [RR] 1.29; CI 1.12–1.49). Based on estimated airborne benzo[a]pyrene exposures from a metaanalysis of eight cohort studies, the predicted lung cancer RR per 100 μg/m3 26 -years of cumulative 

27 benzo[a]pyrene exposure was 1.16 (95% CI 1.05–1.28) (Armstrong et al., 2004).

28 Spinelli et al. (2006) reported a 14-year update to a previously published historical cohort 

29 study (Spinelli et al., 1991) of Canadian aluminum reduction plant workers. The results confirmed 

30 and extended the findings from the earlier epidemiology study. The study surveyed a total of 6,423 

31 workers with ≥3 years of employment at an aluminum reduction plant in British Columbia, Canada, 

32 between the years 1954 and 1997, and evaluated all types of cancers. The focus was on cumulative 

33 exposure to coal tar pitch volatiles, measured as BSM and as benzo[a]pyrene. Benzo[a]pyrene 

34 exposure categories were determined from the range of predicted exposures over time from 

35 statistical exposure models. There were 662 cancer cases, of which approximately 98% had 

36 confirmed diagnoses. The overall cancer mortality rate (standardized mortality ratio 0.97; CI 0.87–

37 1.08) and cancer incidence rate (standardized incidence ratio [SIR] 1.00; CI 0.92–1.08) were not 

38 different from that of the British Columbia general population. However, this study identified 
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B-33 DRAFT—DO NOT CITE OR QUOTE

1 significantly increased incidence rates for cancers of the bladder (SIR 1.80; CI 1.45–2.21) and the 

2 stomach (SIR 1.46; CI 1.01–2.04). The lung cancer incidence rate was only slightly higher than 

3 expected (SIR 1.10; CI 0.93–1.30). Significant dose-response associations with cumulative 

4 benzo[a]pyrene exposure were seen for bladder cancer (p trend < 0.001), stomach cancer (p trend 

5 < 0.05), lung cancer (p trend < 0.001), non-Hodgkin lymphoma (p trend < 0.001), and kidney cancer 

6 (p trend < 0.01), although the overall incidence rates for the latter three cancer types were not 

7 significantly elevated versus the general population. Similar cancer risk results were obtained 

8 using BSM as the exposure measure; the cumulative benzo[a]pyrene and BSM exposures were 

9 highly correlated (r = 0.94).

10 In several occupational cohort studies of workers in Norwegian aluminum production 

11 plants, personal and stationary airborne PAH measurements were performed.

12 In a study covering 11,103 workers and 272,554 person × years of PAH exposure, cancer 

13 incidence was evaluated in six Norwegian aluminum smelters (Romundstad et al., 2000a, b). 

14 Reported estimates of PAH exposure concentrations reached a maximum of 3,400 μg/m3 PAH 

15 (680 μg/m3 benzo[a]pyrene). The overall number of cancers observed in this study did not differ 

16 significantly from control values (SIR 1.03; CI 1.0–1.1). The data from this study showed 

17 significantly increased incidences for cancer of the bladder (SIR 1.3; CI 1.1–1.5) and elevated, but 

18 not significant, SIRs for larynx (SIR 1.3; CI 0.8–1.9), thyroid (SIR 1.4; CI 0.7–2.5), and multiple 

19 myeloma (SIR 1.4; CI 0.9–1.9). Incidence rates for bladder, lung, pancreas, and kidney cancer (the 

20 latter three with SIRs close to unity) were subjected to a cumulative exposure-response analysis. 

21 The incidence rate for bladder cancer showed a trend with increasing cumulative exposure and 

22 with increasing lag times (up to 30 years) at the highest exposure level. The incidence of both lung 

23 and bladder cancers was greatly increased in smokers. The authors reported that using local 

24 county rates rather than national cancer incidence rates as controls increased the SIR for lung 

25 cancer (SIR 1.4; CI 1.2–1.6) to a statistically significant level.

26

27 Cancer incidence in coke oven, coal gasification, and iron and steel foundry workers 

28 An increased risk of death from lung and bladder cancer is reported in some studies 

29 involving coke oven, coal gasification, and iron and steel foundry workers (Bostrom et al., 2002; 

30 Boffetta et al., 1997). An especially consistent risk of lung cancer across occupations is noted when 

31 cumulative exposure is taken into consideration (e.g., RR of 1.16 per 100 unity-years for aluminum 

32 smelter workers, 1.17 for coke oven workers, and 1.15 for coal gasification workers). In an analysis 

33 of pooled data from 10 cohorts of coke production workers, 762 lung cancer cases were observed 

34 versus 512.1 expected (pooled RR 1.58; CI 1.47–1.69) (Bosetti et al., 2007). Significant variations in 

35 risk estimates among the studies were reported, particularly in the large cohorts (RRs of 1.1, 1.2, 

36 2.0, and 2.6). There was no evidence for increased bladder cancer risk in the coke production 

37 workers. Based on estimated airborne benzo[a]pyrene exposures from a meta-analysis of 10 
Toxicological Review of benzo[a]pyrene

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B-34 DRAFT—DO NOT CITE OR QUOTE

cohort studies, the predicted lung cancer RR per 100 μg/m3 1 -years of cumulative benzo[a]pyrene 

2 exposure was 1.17 (95% CI 1.12–1.22) (Armstrong et al., 2004).

3 A meta-analysis of data from five cohorts of gasification workers reported 251 deaths from 

4 respiratory tract cancer, compared with 104.7 expected (pooled RR 2.58; 95% CI 2.28–2.92) 

5 (Bosetti et al., 2007). Pooled data from three of the cohorts indicated 18 deaths from urinary tract 

6 cancers, versus 6.0 expected (pooled RR 3.27; 95% CI 2.06–5.19). Based on estimated airborne 

7 benzo[a]pyrene exposures from a meta-analysis of four gas worker cohort studies, the predicted 

lung cancer RR per 100 μg/m3 8 -years of cumulative benzo[a]pyrene exposure was 1.15 (95% CI 

9 1.11–1.20) (Armstrong et al., 2004).

10 Increased risks were reported in iron and steel foundry workers for cancers of the 

11 respiratory tract, bladder, and kidney. In an analysis of pooled data from 10 cohorts, 

12 1,004 respiratory tract cancer cases were observed versus 726.0 expected (pooled RR 1.40; 

13 CI 1.31–1.49) (Bosetti et al., 2007). A total of 99 bladder cancer cases were observed in seven of the 

14 cohorts, compared with 83.0 expected (pooled RR 1.29; CI 1.06–1.57). For kidney cancer, 40 cases 

15 were observed compared with 31.0 expected based on four studies (pooled RR 1.30; 95% CI 0.95–

16 1.77).

17 Xu et al. (1996) conducted a nested case-control study, surveying the cancer incidence 

18 among 196,993 active or retired workers from the Anshan Chinese iron and steel production 

19 complex. A large number of historical benzo[a]pyrene measurements (1956–1995) were available. 

20 The study included 610 cases of lung cancer and 292 cases of stomach cancer, with 959 age- and 

21 gender-matched controls from the workforce. After adjusting for nonoccupational risk factors such 

22 as smoking and diet, significantly elevated risks for lung cancer and stomach cancer were identified 

23 for subjects employed for ≥15 years, with ORs varying among job categories. For either type of 

24 cancer, highest risks were seen among coke oven workers: lung cancer, OR = 3.4 (CI 1.4–8.5); 

25 stomach cancer, OR = 5.4 (CI 1.8–16.0).

26 There were significant trends for long-term, cumulative benzo[a]pyrene exposure versus 

27 lung cancer (p = 0.004) or stomach cancer (p = 0.016) incidence. For cumulative total 

28 benzo[a]pyrene exposures of <0.84, 0.85–1.96, 1.97–3.2, and ≥3.2, respectively, the ORs for lung 

29 cancer were 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), respectively. For 

30 cumulative total benzo[a]pyrene exposures of <0.84, 0.85–1.96, 1.97–3.2, and ≥3.2, the ORs for 

31 stomach cancer 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), 

32 respectively. However, the investigators noted that additional workplace air contaminants were 

33 measured, which might have influenced the outcome. Of these, asbestos, silica, quartz, and iron 

34 oxide-containing dusts may have been confounders. For lung cancers, cumulative exposures to 

35 total dust and silica dust both showed significant dose-response trends (p = 0.001 and 0.007, 

36 respectively), while for stomach cancer, only cumulative total dust exposure showed a marginally 

37 significant trend (p = 0.061). For cumulative total dust exposures of <69, 69–279, 280–882, and 

≥883 mg/m3 38 , the ORs for lung cancer were 1.4 (CI 1.2–1.9), 1.2 (CI 1.0–2.19), 1.4 (CI 1.0–2.0), and 
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1 1.9 (CI 1.3–2.5), respectively. For cumulative silica dust exposures of <3.7, 3.7–10.39, 10.4–27.71, 

and ≥27.72 mg/m3 2 , 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), 

3 and 1.8 (CI 1.2–2.5), respectively. For cumulative total dust exposures of <69, 69–279, 280–882, 

and ≥883 mg/m3 4 , 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 

5 1.6 (CI 1.1–2.5), respectively.

6 Exposure-response data from studies of coke oven workers in the United States have often 

7 been used to derive quantitative risk estimates for PAH mixtures, and for benzo[a]pyrene as an 

8 indicator substance (Bostrom et al., 2002). However, there are numerous studies of coke oven 

9 worker cohorts that do not provide estimates of benzo[a]pyrene exposure. An overview of the 

10 results of these and other studies can be obtained from the review of Boffetta et al. (1997).

11

12 Cancer incidence in asphalt workers and roofers 

13 These groups encompass different types of work (asphalt paving versus roofing) and also 

14 different types of historical exposure that have changed from using PAH-rich coal tar pitch to the 

15 use of bitumen or asphalt, both of which are rather low in PAHs due to their source (crude oil 

16 refinery) and a special purification process. Increased risks for lung cancer were reported in large 

17 cohorts of asphalt workers and roofers; evidence for increased bladder cancer risk is weak 

18 (Burstyn et al., 2007; Partanen and Boffetta, 1994; Chiazze et al., 1991; Hansen, 1991, 1989; 

19 Hammond et al., 1976). In an analysis of pooled data from two cohorts of asphalt workers, 822 lung 

20 cancer cases were observed versus 730.7 expected (pooled RR 1.14; 95% CI 1.07–1.22) (Bosetti et 

21 al., 2007). In two cohorts of roofers, analysis of pooled data indicated that 138 lung cancer cases 

22 were observed, compared with 91.9 expected (pooled RR 1.51; 95% CI 1.28–1.78) (Bosetti et al., 

23 2007).

24
Toxicological Review of benzo[a]pyrene

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1 ANIMAL BIOASSAYS

2 Oral Bioassays

3 Subchronic Studies

4 De Jong et al. (1999) treated male Wistar rats (eight/dose group) with benzo[a]pyrene 

5 (98.6% purity) dissolved in soybean oil by gavage 5 days/week for 35 days at doses of 0, 3, 10, 30, 

6 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 

7 exposure period, rats were necropsied, organ weights were determined, and major organs and 

8 tissues were prepared for histological examination (adrenals, brain, bone marrow, colon, caecum, 

9 jejunum, heart, kidney, liver, lung, lymph nodes, esophagus, pituitary, spleen, stomach, testis, and 

10 thymus). Blood was collected for examination of hematological endpoints, but there was no 

11 indication that serum biochemical parameters were analyzed. Immune parameters included 

12 determinations of serum immunoglobulin (Ig) levels (IgG, IgM, IgE, and IgA), relative spleen cell 

13 distribution, and spontaneous cytotoxicity of spleen cell populations determined in a natural-killer 

14 (NK) cell assay.

15 Body weight gain was decreased beginning at week 2 at the high dose of 90 mg/kg-day; 

16 there was no effect at lower doses (De Jong et al., 1999). Hematology revealed a dose-related 

17 decrease in RBC count, hemoglobin, and hematocrit at ≥10 mg/kg-day (Table B-6). A minimal but 

18 significant increase in mean cell volume and a decrease in mean cell hemoglobin concentration 

19 were noted at 90 mg/kg-day, and may indicate dose-related toxicity for the RBCs and/or RBC 

20 precursors in the bone marrow. A decrease in WBCs, attributed to a decrease in the number of 

21 lymphocytes (approximately 50%) and eosinophils (approximately 90%), was observed at 

22 90 mg/kg-day; however, there was no effect on the number of neutrophils or monocytes. A 

23 decrease in the cell number in the bone marrow observed in the 90 mg/kg-day dose group was 

24 consistent with the observed decrease in the RBC and WBC counts at this dose level. In the 

25 90 mg/kg-day dose group, brain, heart, kidney, and lymph node weights were decreased and liver 

26 weight was increased (Table B-6). Decreases in heart weight at 3 mg/kg-day and in kidney weight 

27 at 3 and 30 mg/kg-day were also observed, but these changes did not show dose-dependent 

28 responses. Dose-related decreases in thymus weight were statistically significant at ≥10 mg/kg29 day (Table B-6).

30 Table B-6. Exposure-related effects in male Wistar rats exposed to 

31 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)

WBCs (109/L)

RBCs (109/L)

14.96 ± 1.9

8.7 ± 0.2

13.84 ± 3.0

8.6 ± 0.2

13.69 ± 1.8 a

8.3 ± 0.2

13.58 ± 2.9 a

7.8 ± 0.4

8.53 ± 1.1a

7.1 ± 0.4a
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Effect

Dose (mg/kg-d)

0 3 10 30 90

Hemoglobin (mmol/L)

Hematocrit (L/L)

10.5 ± 0.2

0.5 ± 0.01

10.4 ± 0.3

0.5 ± 0.01

9.8 ± 0.2a

0.47 ± 0.01a

9.5 ± 0.4a

0.46 ± 0.02a

8.6 ± 0.6a

0.43 ± 0.02a

Serum Ig 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 of 

3 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%), 

7 30 (18%), and 90 mg/kg-day (41%); significant decreases in absolute number of cells harvested in 

8 the spleen (31%), in the number of B cells in the spleen (61%), and NK cell activity in the spleen 
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1 (E:T ratio was 40.9 ± 28.4% that of the controls) at 90 mg/kg-day; and a decrease in serum IgM 

2 (33%) and IgA (61%) in rats treated with 30 and 90 mg/kg-day, respectively. The decrease in the 

3 spleen cell count was attributed by the study authors to the decreased B cells and suggested a 

4 possible selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The study 

5 authors considered the decrease in IgA and IgM to be due to impaired production of antibodies, 

6 suggesting a role of thymus toxicity in the decreased (T-cell dependent) antibody production. In 

7 addition to the effects on the thymus and spleen, histopathologic examination revealed treatment8 related lesions only in the liver and forestomach at the two highest dose levels, but the incidence 

9 data for these lesions were not reported by De Jong et al. (1999). Increased incidence for 

10 forestomach basal cell hyperplasia (p < 0.05 by Fisher’s exact test) was reported at 30 and 

11 90 mg/kg-day, and increased incidence for oval cell hyperplasia in the liver was reported at 

12 90 mg/kg-day (p < 0.01, Fisher’s exact test). The results indicate that 3 mg/kg-day was a no13 observed-adverse-effect level (NOAEL) for effects on hematological parameters (decreased RBC 

14 count, hemoglobin, and hematocrit) and immune parameters (decreased thymus weight and 

15 percent of B cells in the spleen) noted in Wistar rats at 10 mg/kg-day (the lowest-observed16 adverse-effect level [LOAEL]) and above. Lesions of the liver (oval cell hyperplasia) and 

17 forestomach (basal cell hyperplasia) occurred at doses ≥30 mg/kg-day.

18 Knuckles et al. (2001) exposed male and female F344 rats (20/sex/dose group) to 

19 benzo[a]pyrene (98% purity) at doses of 0, 5, 50, or 100 mg/kg-day in the diet for 90 days. Food 

20 consumption and body weight were monitored, and the concentration of benzo[a]pyrene in the 

21 food was adjusted every 3–4 days to maintain the target dose. The authors indicated that the actual 

22 intake of benzo[a]pyrene by the rats was within 10% of the calculated intake, and the nominal 

23 doses were not corrected to actual doses. Hematology and serum chemistry parameters were 

24 evaluated. Urinalysis was also performed. Animals were examined for gross pathology, and 

25 histopathology was performed on selected organs (stomach, liver, kidney, testes, and ovaries). 

26 Statistically significant decreases in RBC counts and hematocrit level (decreases as much as 10 and 

27 12%, respectively) were observed in males at doses ≥50 mg/kg-day and in females at 100 mg/kg28 day. A maximum 12% decrease (statistically significant) in hemoglobin level was noted in both 

29 sexes at 100 mg/kg-day. Blood chemistry analysis showed a significant increase in blood urea 

30 nitrogen (BUN) only in high-dose (100 mg/kg-day) males. Histopathology examination revealed an 

31 apparent increase in the incidence of abnormal tubular casts in the kidney in males at 5 mg/kg-day 

32 (40%), 50 mg/kg-day (80%), and 100 mg/kg-day (100%), compared to 10% in the controls. Only 

33 10% of the females showed significant kidney tubular changes at the two high-dose levels 

34 compared to zero animals in the female control group. The casts were described as molds of distal 

35 nephron lumen and were considered by the study authors to be indicative of renal dysfunction. 

36 From this study, male F344 rats appeared to be affected more severely by benzo[a]pyrene 

37 treatment than the female rats. However, the statistical significance of the kidney lesions are 

38 unclear. Several reporting gaps and inconsistencies regarding the reporting of kidney 
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1 abnormalities in Knuckles et al. (2001) make interpretation of the results difficult. Results of 

2 histopathological kidney abnormalities (characterized primarily as kidney casts) were presented 

3 graphically and the data were not presented numerically in this report. No indication was given in 

4 the graph that any groups were statistically different than controls, although visual examination of 

5 the magnitude of response and error bars appears to indicate a fourfold increase in kidney casts in 

6 males compared to the control group (40 compared to 10%). The figure legend reported the data 

7 as “percentage incidence of abnormal kidney tissues” and reported values as mean ± SD. However, 

8 the text under the materials and methods section stated that Fisher’s exact test was used for 

9 histopathological data, which would involve the pairwise comparison of incidence and not means. 

10 There are additional internal inconsistencies in the data presented. The data appeared to indicate 

11 that incidences for males were as follows: control, 10%; 5 mg/kg-day, 40%; 50 mg/kg-day, 80%; 

12 and 100 mg/kg-day, 100%; however, these incidences are inconsistent with the size of the study 

13 groups, which were reported as 6–8 animals per group. The study authors were contacted, but did 

14 not respond to EPA’s request for clarification of study design and/or results. Due to issues of data 

15 reporting, a LOAEL could not be established for the increased incidence of kidney lesions. Based on 

16 the statistically significant hematological effects including decreases in RBC counts, hematocrit, and 

17 BUN, the NOAEL in males was 5 mg/kg-day and the LOAEL was 50 mg/kg-day, based on in F344 

18 rats. No exposure-related histological lesions were identified in the stomach, liver, testes, or 

19 ovaries in this study.

20 In a range-finding study, Wistar (specific pathogen-free [SPF] Riv:TOX) rats (10/sex/dose 

21 group) were administered benzo[a]pyrene (97.7% purity) dissolved in soybean oil by gavage at 

22 dose levels of 0, 1.5, 5, 15, or 50 mg/kg body weight-day, 5 days/week for 5 weeks (Kroese et al., 

23 2001). Behavior, clinical symptoms, body weight, and food and water consumption were 

24 monitored. None of the animals died during the treatment period. Animals were sacrificed 

25 24 hours after the last dose. Urine and blood were collected for standard urinalysis and 

26 hematology and clinical chemistry evaluation. Liver enzyme induction was monitored based on 

27 EROD activity in plasma. Animals were subjected to macroscopic examination, and organ weights 

28 were recorded. The esophagus, stomach, duodenum, liver, kidneys, spleen, thymus, lung, and 

29 mammary gland (females only) from the highest-dose and control animals were evaluated for 

30 histopathology. Intermediate-dose groups were examined if abnormalities were observed in the 

31 higher-dose groups.

32 A significant, but not dose-dependent, increase in food consumption in males at ≥1.5 mg/kg33 day and a decrease in food consumption in females at ≥5 mg/kg-day was observed (Kroese et al., 

34 2001). Water consumption was statistically significantly altered in males only: a decrease at 1.5, 5, 

35 and 15 mg/kg-day and an increase at 50 mg/kg-day. Organ weights of lung, spleen, kidneys, 

36 adrenals, and ovaries were not affected by treatment. There was a dose-related, statistically 

37 significant decrease in thymus weight in males at 15 and 20 mg/kg-day (decreased by 28 and 33%, 

38 respectively) and a significant decrease in thymus weight in females at 50 mg/kg-day (decreased by 
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1 17%) (Table B-7). In both sexes, liver weight was statistically significantly increased only at 

2 50 mg/kg-day by about 18% (Table B-7).

3 Table B-7. Exposure-related effects in Wistar rats exposed to benzo[a]-

4 pyrene by gavage 5 days/week for 5 weeks

Organ

Dose (mg/kg-d)

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).

5

6 Hematological evaluation revealed only statistically nonsignificant, small, dose-related 

7 decreases in hemoglobin in both sexes and RBC counts in males. Clinical chemistry analysis 

8 showed a small, but statistically significant, increase in creatinine levels in males only at 1.5 mg/kg9 day, but this effect was not dose-dependent. A dose-dependent induction of liver microsomal EROD 

10 activity was observed, with a 5-fold induction at 1.5 mg/kg-day compared to controls, reaching 36-

11 fold in males at 50 mg/kg-day; the fold induction in females at the top dose was less than in males. 

12 At necropsy, significant, dose-dependent macroscopic findings were not observed.

13 Histopathology examination revealed a statistically significant increase in basal cell 

14 hyperplasia in the forestomach of females at doses ≥15 mg/kg-day (Kroese et al., 2001). The 

15 induction of liver microsomal EROD was not accompanied by any adverse histopathologic findings 

16 in the liver at the highest dose, 50 mg/kg-day, so the livers from intermediate-dose groups were, 

17 therefore, not examined. An increased incidence of brown pigmentation of red pulp (hemosiderin) 

18 in the thymus was observed in treated animals of both sexes. However, this tissue was not 

19 examined in intermediate-dose groups. This range-finding, 5-week study identified a NOAEL of 

20 5 mg/kg-day and a LOAEL of 15 mg/kg-day, based on decreased thymus weight and forestomach 

21 hyperplasia in Wistar rats.

22 Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (10/sex/dose group) to benzo[a]pyrene 

23 (98.6% purity, dissolved in soybean oil) by gavage at 0, 3, 10, or 30 mg/kg body weight-day, 
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1 5 days/week for 90 days. The rats were examined daily for behavior and clinical symptoms and by 

2 palpation. Food and water consumption, body weights, morbidity, and mortality were monitored. 

3 At the end of the exposure period, rats were subjected to macroscopic examination and organ 

4 weights were recorded. Blood was collected for hematology and serum chemistry evaluation, and 

5 urine was collected for urinalysis. All gross abnormalities, particularly masses and lesions 

6 suspected of being tumors, were evaluated. The liver, stomach, esophagus, thymus, lung, spleen, 

7 and mesenteric lymph node were examined histopathologically. In addition, cell proliferation in 

8 forestomach epithelium was measured as the prevalence of S-phase epithelial cells displaying 

9 bromodeoxyuridine (BrdU) incorporation.

10 There were no obvious effects on behavior of the animals, and no difference was observed 

11 in survival or food consumption between exposed animals and controls (Kroese et al., 2001). 

12 Higher water consumption and slightly lower body weights than the controls were observed in 

13 males but not females at the high dose of 30 mg/kg-day. Hematological investigations showed only 

14 nonsignificant, small dose-related decreases in RBC count and hemoglobin level in both sexes. 

15 Clinical chemistry evaluation did not show any treatment-related group differences or dose16 response relationships for alanine aminotransferase (ALT), serum aspartate transaminase (AST), 

17 lactate dehydrogenase (LDH), or creatinine, but a small dose-related decrease in -glutamyl 

18 transferase (GGT) activity was observed in males only. Urinalysis revealed an increase in urine 

19 volume in males at 30 mg/kg-day, which was not dose related. At the highest dose, both sexes 

20 showed increased levels of urinary creatinine and a dose-related increase in urinary protein. 

21 However, no further investigation was conducted to determine the underlying mechanisms for 

22 these changes. At necropsy, reddish to brown/gray discoloration of the mandibular lymph nodes 

23 was consistently noted in most rats; occasional discoloration was also observed in other regional 

24 lymph nodes (axillary). Statistically significant increases in liver weight were observed at 10 and 

25 30 mg/kg-day in males (15 and 29%) and at 30 mg/kg-day in females (17%). A decrease in thymus 

26 weight was seen in both sexes at 30 mg/kg-day (17 and 33% decrease in females and males, 

27 respectively, compared with controls) (Table B-8). At 10 mg/kg-day, thymus weight in males was 

28 decreased by 15%, but the decrease did not reach statistical significance.
Toxicological Review of benzo[a]pyrene

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Table B-8. Means ± SDa 1 for liver and thymus weights in Wistar rats 

2 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).

3

4 Histopathologic examination revealed what was characterized by Kroese et al. (2001) as 

5 basal cell disturbance in the epithelium of the forestomach in males (p < 0.05) and females 

6 (p < 0.01) at 30 mg/kg-day. The basal cell disturbance was characterized by increased number of 

7 basal cells, mitotic figures, and remnants of necrotic cells; occasional early nodule development; 

8 infiltration by inflammatory cells (mainly histiocytes); and capillary hyperemia, often in 

9 combination with the previous changes (Kroese et al., 2001). Incidences for these lesions (also 

10 described as “slight basal cell hyperplasia”) in the 0, 3, 10, and 30-mg/kg-day groups were 0/10, 

11 2/10, 3/10, and 7/10, respectively, in female rats and 2/10, 0/10, 6/10, and 7/10, respectively, in 

12 male rats. Nodular hyperplasia was noted in one animal of each sex at 30 mg/kg-day. A significant 

13 (p < 0.05) increase in proliferation of forestomach epithelial cells was detected at doses ≥10 mg/kg14 day by morphometric of analysis of nuclei with BrdU incorporation. The mean numbers of BrdU15 staining nuclei per unit surface area of the underlying lamina muscularis mucosa were increased by 

16 about two- and three–fourfold at 10 and 30 mg/kg-day, respectively, compared with controls. A 

17 reduction of thymus weight and increase in the incidence of thymus atrophy (the report described 

18 the atrophy as slight, but did not specify the full severity scale used in the pathology examination) 

19 was observed in males only at 30 mg/kg-day (p < 0.01 compared with controls). Respective 

20 incidences for thymus atrophy for the control through high-dose groups were 0/10, 0/10, 0/10, 

21 and 3/10 for females and 0/10, 2/10, 1/10, and 6/10 for males. No significant differences were 

22 observed in the lungs of control and treated animals. In the esophagus, degeneration and 

23 regeneration of muscle fibers and focal inflammation of the muscular wall were judged to be a 

24 result of the gavage dosing rather than of benzo[a]pyrene treatment.
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B-43 DRAFT—DO NOT CITE OR QUOTE

1 The target organs of benzo[a]pyrene toxicity in this 90-day dietary study of Wistar rats 

2 were the forestomach, thymus, and liver. The LOAEL for forestomach hyperplasia, decreased 

3 thymus weight, and thymus atrophy was 30 mg/kg-day and the NOAEL was 10 mg/kg-day.

4 Chronic Studies and Cancer Bioassays

5 Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (52/sex/dose group) to benzo[a]pyrene 

6 (98.6% purity) in soybean oil by gavage at nominal doses of 0, 3, 10, or 30 mg/kg-day, 5 days/week, 

7 for 104 weeks. Mean achieved dose levels were 0, 2.9, 9.6, and 29 mg/kg-day. Additional rats 

8 (6/sex/group) were sacrificed after 4 and 5 months of exposure for analysis of DNA adduct 

9 formation in blood and major organs and tissues. The rats were 6 weeks old at the start of 

10 exposure. The rats were examined daily for behavior and clinical symptoms and by palpation. 

11 Food and water consumption, body weights, morbidity, and mortality were monitored during the 

12 study. Complete necropsy was performed on all animals that died during the course of the study, 

13 were found moribund, or at terminal sacrifice (organ weight measurement was not mentioned in 

14 the report by Kroese et al., 2001). The organs and tissues collected and prepared for microscopic 

15 examination included: brain, pituitary, heart, thyroid, salivary glands, lungs, stomach, oesophagus, 

16 duodenum, jejunum, ileum, caecum, colon, rectum, thymus, kidneys, urinary bladder, spleen, lymph 

17 nodes, liver pancreas, adrenals, sciatic nerve, nasal cavity, femur, skin including mammary tissue, 

18 ovaries/uterus, and testis/accessory sex glands. Some of these tissues were examined only when 

19 gross abnormalities were detected. All gross abnormalities, particularly masses and lesions that 

20 appeared to be tumors, were also examined.

21 At 104 weeks, survival in the control group was 65% (males) and 50% (females), whereas 

22 mortality in the 30 mg/kg-day dose group was 100% after about week 70. At 80 weeks, survival 

23 percentages were about 90, 85 and 75% in female rats in the 0, 3, and 10 mg/kg-day groups, 

24 respectively; in males, respective survival percentages were ~95, 90, and 85% at 80 weeks. 

25 Survival of 50% of animals occurred at 104, 104, ~90, and 60 weeks for control through high-dose 

26 females; for males, the respective times associated with 65% survival were 104, 104, 104, and ~60 

27 weeks. The high mortality rate in high-dose rats was attributed to liver or forestomach tumor 

28 development, not to noncancer systemic effects. After 20 weeks, body weight was decreased 

29 (compared with controls by >10%) in 30-mg/kg-day males, but not in females. This decrease was 

30 accompanied by a decrease in food consumption. Body weights and food consumption were not 

31 adversely affected in the other dose groups compared to controls. In males, there was a dose32 dependent increase in water consumption starting at week 13, but benzo[a]pyrene treatment had 

33 no significant effects on water consumption in females.

34 Tumors were detected at significantly elevated incidences at several tissue sites in female 

35 and male rats at doses ≥10 and ≥3 mg/kg-day, respectively (Table 4-5; Kroese et al., 2001). The 

36 tissue sites with the highest incidences of tumors were the liver (hepatocellular adenoma and 

37 carcinoma) and forestomach (squamous cell papilloma and carcinoma) in both sexes (Table B-9). 

38 The first liver tumors were detected in week 35 in high-dose male rats. Liver tumors were 
Toxicological Review of benzo[a]pyrene

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B-44 DRAFT—DO NOT CITE OR QUOTE

1 described as complex, with a considerable proportion (59/150 tumors) metastasizing to the lungs. 

2 At the highest dose level, 95% of rats with liver tumors had malignant carcinomas (95/100; Table 

3 B-9). Forestomach tumors were associated with the basal cell proliferation observed (without 

4 diffuse hyperplasia) in the forestomach of rats in the preliminary range-finding and 90-day 

5 exposure studies described previously in Section 4.2.1. At the highest dose level, 59% of rats with 

6 forestomach tumors had malignant carcinomas (60/102; Table B-9). Other tissue sites with 

7 distinctly elevated incidences of tumors in the 30 mg/kg-day dose group included the oral cavity 

8 (papilloma and squamous cell carcinoma [SCC]) in both sexes, and the jejunum (adenocarcinoma), 

9 kidney (cortical adenoma), and skin (basal cell adenoma and carcinoma) in male rats (Table B-9). 

10 In addition, auditory canal tumors (carcinoma or squamous cell papilloma originating from pilo11 sebaceous units including the Zymbal’s gland) were also detected in both sexes at 30 mg/kg-day, 

12 but auditory canal tissue was not histologically examined in the lower dose groups and the controls 

13 (Table B-9). Gross examination revealed auditory canal tumors only in the high-dose group.

14 Table B-9. Incidences of exposure-related neoplasms in Wistar rats 

15 treated by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks

Dose (mg/kg-d)

0 3 10 30a

Site Femalesb

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

Malesb
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Dose (mg/kg-d)

0 3 10 30a

Oral cavity

 Papilloma

 SCC

 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

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

This group had significantly decreased survival.

b

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.

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 rats 

3 sacrificed during the 2-year study, because the focus was to identify and quantitate tumor 
Toxicological Review of benzo[a]pyrene

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1 occurrence. However, incidences were reported for nonneoplastic lesions in tissues or organs in 

2 which tumors were detected (i.e., oral cavity, oesophagus, forestomach, jejunum, liver, kidney, skin, 

3 mammary, and auditory canal). The reported nonneoplastic lesions associated with exposure were 

4 the forestomach basal cell hyperplasia and clear cell foci of cellular alteration in the liver. 

5 Incidences for forestomach basal cell hyperplasia in the control through high-dose groups were 

6 1/52, 8/51, 13/51, and 2/52 for females and 2/50, 8/52, 8/52, and 0/52 in males. Incidences for 

7 hepatic clear cell foci of cellular alteration were 22/52, 33/52, 4/52, and 2/52 for females and 

8 8/52, 22/52, 1/52, and 1/52 for males. These results indicate that the lowest dose group, 3 mg/kg9 day, was a LOAEL for increased incidence of forestomach hyperplasia and hepatic histological 

10 changes in male and female Wistar rats exposed by gavage to benzo[a]pyrene for up to 104 weeks 

11 (see Table 4-5). The lack of an increase in incidence of these nonneoplastic lesions in the 

12 forestomach and liver at the intermediate and high doses (compared with controls) were 

13 associated with increased incidences of forestomach and liver tumors at these dose levels. The 

14 authors of this study note that non-neoplastic effects were not quantified in organs with tumors. 

15 As an adjunct study to the 2-year gavage study with Wistar rats, Kroese et al. (2001) 

16 sacrificed additional rats (6/sex/group) after 4 and 5 months of exposure (0, 1, 3, 10, or 30 mg/kg17 day) for analysis of DNA adduct formation in WBCs and major organs and tissues. Additional rats 

18 (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 [ 19 32P]-postlabeling technique, five benzo[a]pyrene-DNA 

20 adducts were identified in all of the examined tissues at 4 months (WBCs, liver, kidney, heart, lung, 

21 skin, forestomach, glandular stomach, brain). Only one of these adducts (adduct 2) was identified 

22 based on co-chromatography with a standard. This adduct, identified as 10β-(deoxyguanosin-N2-

yl)-7β,8α,9α-trihydroxy-7,8,9,10 tetrahydro-benzo[a]pyrene (dG-N2 23 -BPDE), was the predominant 

24 adduct in all organs of female rats exposed to 10 mg/kg-day, except the liver and kidney, in which 

25 another adduct (unidentified adduct 4) was predominant. Levels of total adducts (number of 

26 benzo[a]pyrene-DNA adducts per 1010 nucleotides) in examined tissues (from the single 10 mg/kg27 day female rat) showed the following order: liver > heart > kidney > lung > skin > forestomach ≈ 

28 WBCs > brain. Mean values for female levels of total benzo[a]pyrene-DNA adducts (number per 

29 1010 nucleotides) in four organs showed the same order, regardless of exposure group: liver > lung 

30 > forestomach ≈ WBCs; comparable data for males were not reported). Mean total benzo[a]pyrene31 DNA adduct levels in livers increased in both sexes from about 100 adducts per 1010 nucleotides at 

32 0.1 mg/kg-day to about 70,000 adducts per 1010 nucleotides at 30 mg/kg-day. In summary, these 

33 results suggest that total benzo[a]pyrene-DNA adduct levels in tissues at 4 months were not 

34 independently associated with the carcinogenic responses noted after 2 years of exposure to 

35 benzo[a]pyrene. The liver showed the highest total DNA adduct levels and a carcinogenic response, 

36 but total DNA adduct levels in heart, kidney, and lung (in which no carcinogenic responses were 

37 detected) were higher than levels in forestomach and skin (in which carcinogenic responses were 

38 detected).
Toxicological Review of benzo[a]pyrene

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1 Groups of Sprague-Dawley rats (32/sex/dose) were fed diets delivering a daily dose of 

2 0.15 mg benzo[a]pyrene/kg body weight every ninth day or 5 times/week (Brune et al., 1981). 

3 Other groups (32/ sex/dose) were given gavage doses of 0.15 mg benzo[a]pyrene (in aqueous 1.5% 

4 caffeine solution)/kg every ninth day, every third day, or 5 times/week. The study included an 

5 untreated control group (to compare with the dietary exposed groups) and a gavage vehicle control 

6 group (each with 32 rats/sex). Rats were treated until moribundity or death occurred, with 

7 average annual doses are reported in Table 4-6 (mg/kg-year, calculated by Brune et al. [1981]). 

8 The following tissues were prepared for histopathological examination: tongue, larynx, lung, heart, 

9 trachea, esophagus, stomach, small intestine, colon, rectum, spleen, liver, urinary bladder, kidney, 

10 adrenal gland, and any tissues showing tumors or other gross changes. Survival was similar among 

11 the groups, with the exception that the highest gavage-exposure group showed a decreased median 

12 time of survival (Table B-10). Increased incidences of portal-of-entry tumors (forestomach, 

13 esophagus, and larynx) were observed in all of the gavage-exposed groups and in the highest 

14 dietary exposure group (Table B-10). Following dietary administration, all observed tumors were 

15 papillomas. Following gavage administration, two malignant forestomach tumors were found (one 

16 each in the mid- and high-dose groups) and the remaining tumors were benign. The data in Table 

17 4-6 show that the carcinogenic response to benzo[a]pyrene was stronger with the gavage protocol 

18 compared with dietary exposure, and that no distinct difference in response was apparent between 

19 the sexes. Tumors at distant sites (mammary gland, kidney, pancreas, lung, urinary bladder, testes, 

20 hematopoietic, and soft tissue) were not considered treatment-related as they were also observed 

21 at similar rates in the control group (data not provided). The study report did not address 

22 noncancer systemic effects.

23 Table B-10. Incidences of alimentary tract tumors in Sprague-Dawley 

24 rats chronically exposed to benzo[a]pyrene in the diet or by gavage in 

25 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%)d13/64 (20.3%) 112

18 0.049 26/64 (40.1%)e26/64 (40.6%) 113

39 0.107 14/64 (21.9%)e14/64 (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%)d10/64 (15.6%) 131
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a

Average annual dose divided by 365 days.

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 2 test that accounted for group 

differences in survival time.

e

Significantly (p < 0.05) different from control using a modified 2test that accounted for group 

differences in survival time.

Source: Brune et al. (1981).

1

2 In the other modern cancer bioassay with benzo[a]pyrene, female B6C3F1 mice (48/dose 

3 group) were administered benzo[a]pyrene (98.5% purity) at concentrations of 0 (acetone vehicle), 

4 5, 25, or 100 ppm in the diet for 2 years (Beland and Culp, 1998; Culp et al., 1998). This study was 

5 designed to compare the carcinogenicity of coal tar mixtures with that of benzo[a]pyrene and 

6 included groups of mice fed diets containing one of several concentrations of two coal tar mixtures. 

7 Benzo[a]pyrene was dissolved in acetone before mixing with the feed. Control mice received only 

8 acetone-treated feed. Female mice were chosen because they have a lower background incidence of 

9 lung tumors than male B6C3F1 mice. Culp et al. (1998) reported that the average daily intakes of 

10 benzo[a]pyrene in the 25- and 100-ppm groups were 104 and 430 μg/day, but did not report 

11 intakes for the 5-ppm group. Based on the assumption that daily benzo[a]pyrene intake at 5 ppm 

12 was one-fifth of the 25-ppm intake (about 21 μg/day), average daily doses for the three 

13 benzo[a]pyrene groups are estimated at 0.7, 3.3, and 16.5 mg/kg-day. Estimated doses were 

14 calculated using time-weighted average (TWA) body weights of 0.032 kg for the control, 5- and 25-

15 ppm groups and 0.026 kg for the 100-ppm group (estimated from graphically presented data). 

16 Food consumption, body weights, morbidity, and mortality were monitored at intervals, and lung, 

17 kidneys, and liver were weighed at sacrifice. Necropsy was performed on all mice that died during 

18 the experiment or survived to the end of the study period. Limited histopathologic examinations 

19 (liver, lung, small intestine, stomach, tongue, esophagus) were performed on all control and high20 dose mice and on all mice that died during the experimental period, regardless of treatment group. 

21 In addition, all gross lesions found in mice of the low- and mid-dose groups were examined 

22 histopathologically.

23 None of the mice administered 100 ppm benzo[a]pyrene survived to the end of the study, 

24 and morbidity/mortality was 100% by week 78. Decreased survival was also observed at 25 ppm 

25 with only 27% survival at 104 weeks, compared with 56 and 60%, in the 5-ppm and control groups, 

26 respectively. In the mid- and high-dose group, 60% of mice were alive at about 90 and 60 weeks, 

27 respectively. Early deaths in exposed mice were attributed to tumor formation rather than other 

28 causes of systemic toxicity. Food consumption was not statistically different in 
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1 benzo[a]pyrene-exposed and control mice. Body weights of mice fed 100 ppm were similar to 

2 those of the other treated and control groups up to week 46, and after approximately 52 weeks, 

3 body weights were reduced in 100-ppm mice compared with controls. Body weights for the 5- and 

4 25-ppm groups were similar to controls throughout the treatment period. Compared with the 

5 control group, no differences in liver, kidney, or lung weights were evident in any of the treated 

6 groups (other organ weights were not measured).

7 Papillomas and/or carcinomas of the forestomach, esophagus, tongue, and larynx at 

8 elevated incidences occurred in groups of mice exposed to 25 or 100 ppm, but no exposure-related 

9 tumors occurred in the liver or lung (Table B-11; Beland and Culp, 1998; Culp et al., 1998). The 

10 forestomach was the most sensitive tissue, and demonstrated the highest tumor incidence among 

11 the examined tissues and was the only tissue with an elevated incidence of tumors at 25 ppm 

12 (Table B-11). In addition, most of the forestomach tumors in the exposed groups were carcinomas, 

13 as 1, 31, and 45 mice had forestomach carcinomas in the 5-, 25-, and 100-ppm groups respectively. 

14 Nonneoplastic lesions were also found in the forestomach at significantly (p < 0.05) elevated 

15 incidences: hyperplasia at ≥5 ppm and hyperkeratosis at ≥25 ppm (Table B-11). The esophagus 

16 was the only other examined tissue showing elevated incidence of a nonneoplastic lesion (basal cell 

17 hyperplasia, see Table B-11). Tumors (papillomas and carcinomas) were also significantly elevated 

18 in the esophagus and tongue at 100 ppm (Table B-11). Esophogeal carcinomas were detected in 1 

19 mouse at 25 ppm and in 11 mice at 100 ppm. Tongue carcinomas were detected in seven 100-ppm 

20 mice; the remaining tongue tumors were papillomas. Although incidences of tumors of the larynx 

21 were not significantly elevated in any of the exposed groups, a significant dose-related trend was 

22 apparent (Table B-11).

23 Table B-11. Incidence of nonneoplastic and neoplastic lesions in female 

24 B6C3F1 mice fed benzo[a]pyrene in the diet for up to 2 years

Tissue and lesion

Incidence (%)

Benzo[a]pyrene concentration (ppm) in diet

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) 1/48b

(2)

3/47

(6)

36/46a

(78)

46/47a

(98)

Forestomach (hyperplasia) 13/48b

(27)

23/47

(49)

33/46a

(72)

37/47a

(79)

Forestomach (hyperkeratosis) 13/48b22/47 33/46a38/47a
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(27) (47) (72) (81)

Esophagus (papilloma and/or carcinoma) 0/48b

(0)

0/48

(0)

2/45

(0)

27/46a

(59)

Esophagus (basal cell hyperplasia) 1/48b

(2)

0/48

(0)

5/45

(11)

30/46a

(65)

Tongue (papilloma and/or carcinoma) 0/49b

(0)

0/48

(0)

2/46

(4)

23/48a

(48)

Larynx (papilloma and/or carcinoma) 0/35b

(0)

0/35

(0)

3/34

(9)

5/38

(13)

a

Significantly different from control incidence (p < 0.05); using a modified Bonferonni procedure for 

multiple comparisons to the same control.

b

Significant (p < 0.05) dose-related trend calculated for incidences of these lesions.

Sources: Beland and Culp (1998); Culp et al. (1998). 

1

2 Neal and Rigdon (1967) fed benzo[a]pyrene (purity not reported) at concentrations of 0, 1, 

3 10, 20, 30, 40, 45, 50, 100, and 250 ppm to male and female CFW-Swiss mice in the diet. 

4 Corresponding doses (in mg/kg-day) were calculated1 as 0, 0.2, 1.8, 3.6, 5.3, 7.1, 8, 8.9, 17.8, and 

5 44.4 mg/kg-day. The age of the mice ranged from 17 to 180 days old and the treatment time was 

6 from 1 to 197 days; the size of the treated groups ranged from 9 to 73. There were 289 mice 

7 (number of mice/sex not stated) in the control group. No forestomach tumors were reported at 0, 

8 0.2, or 1.8 mg/kg-day. The incidence of forestomach tumors at 20, 30, 40, 45, 50, 100, and 250 ppm 

9 dose groups (3.6, 5.3, 7.1, 8, 8.9, 17.8, and 44.4 mg/kg-day) were 1/23, 0/37, 1/40, 4/40, 23/34, 

10 19/23, and 66/73, respectively.

11 Other Oral Exposure Cancer Bioassays in Mice

12 Numerous other oral exposure cancer bioassays in mice have limitations that restrict their 

13 usefulness for characterizing dose-response relationships between chronic-duration oral exposure 

14 to benzo[a]pyrene and noncancer effects or cancer, but collectively, they provide strong evidence 

15 that oral exposure to benzo[a]pyrene can cause portal-of-entry site tumors (see Table B-12 for 

16 references).

 

1

Calculation: mg/kg-day = (ppm in feed × 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 kg (females) were used (U.S. EPA, 1988) and resulting 

doses were averaged between males and females.
Toxicological Review of benzo[a]pyrene

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

B-51 DRAFT—DO NOT CITE OR QUOTE

1 Table B-12. Other oral exposure cancer bioassays in mice

Species/strain Exposure Results Comments Reference

Rat/SpragueDawleyGroups of SpragueDawley 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, esophagus, 

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. 

Nonstandard 

treatment 

protocol involved 

animals being 

treated for ≤5 

days/week; 

relatively high 

control incidence 

compared to 

other gavage 

studies.

Brune et al., 

1981

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/kgd.

Incidence with 

forestomach tumors:

Low 11/20 (18 wks)

Mid 13/19 (20 wks)

High 12/12 (12 wks)

Less-than-lifetime 

exposure 

duration; only 

stomachs were 

examined for 

tumors; tumors 

found only in 

forestomach.

Wattenberg

, 1972

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 

duration; 

glandular 

stomach, lung, 

and livers from 

control and 

exposed mice 

showed no 

tumors. 

Triolo et al., 

1977

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 

duration; only 

stomachs were 

examined for 

tumors; tumors 

found only in 

forestomach; no 

nonexposed 

controls were 

mentioned.

Wattenberg

, 1974
Toxicological Review of benzo[a]pyrene

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

B-52 DRAFT—DO NOT CITE OR QUOTE

Species/strain Exposure Results Comments Reference

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 

was 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 

duration; only 

stomach were 

examined for 

tumors; tumors 

found only in 

forestomach.

El-Bayoumy, 

1985

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 

duration; the 

following details 

were not 

reported: 

inclusion of 

controls, 

methods for 

detecting tumors, 

and body weight 

data. 

Biancifiori 

et al., 1967

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.

Berenblum 

and Haran, 

1955

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

 1/17

200 8/17

 NE

Less-than-lifetime 

exposure 

duration; GI tract 

examined for 

tumors with hand 

lens; body weight 

data not 

reported.

Field and 

Roe, 1965

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; daily 

dose levels and 

methods of 

detecting tumors 

were not clearly 

reported.

Chouroulink

ov et al., 

1967
Toxicological Review of benzo[a]pyrene

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

B-53 DRAFT—DO NOT CITE OR QUOTE

Species/strain Exposure Results Comments Reference

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 

duration; no 

vehicle control 

group; animals 

ranged from 3 

wks to 6 mo old 

at the start of 

dosing; only 

alimentary tract 

was examined for 

tumors (see also 

Rigdon and Neal, 

1969, 1967, 

1966).

Neal and 

Rigdon, 

1967

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 glycol-400 

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 

exposure; 

exposure-related 

tumors only 

found in 

forestomach.

Roe et al., 

1970

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 

exposure; only 

stomachs were 

examined for 

tumors; tumors 

found only in 

forestomach;

nonexposed 

controls were not 

mentioned.

Benjamin et 

al., 1988
Toxicological Review of benzo[a]pyrene

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

B-54 DRAFT—DO NOT CITE OR QUOTE

Species/strain Exposure Results Comments Reference

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.

Fedorenko 

and 

Yansheva, 

1967; as 

cited in U.S. 

EPA, 1991a

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; only 

lungs examined 

for tumors; no 

nonexposed 

controls were 

mentioned.

Wattenberg

, 1974

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 were 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; A/J 

strain of mice 

particularly 

sensitive to 

chemically 

induced cancer; 

only lungs and 

stomachs were 

examined for 

tumors.

Weyand et 

al., 1995 

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 

exposure; only 

lungs and GI tract 

were examined 

for tumors.

Robinson et 

al., 1987

NE = not evaluated

1
Toxicological Review of benzo[a]pyrene

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

B-55 DRAFT—DO NOT CITE OR QUOTE

1 Inhalation Studies

2 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/m3 4 for 2 hours/day, 5 days/week for 4 weeks (corresponding to a TWA of 

0.45 mg/m3 5 ). Rats were 10–11 weeks old at the beginning of the experiment. Benzo[a]pyrene 

6 (>98% pure) aerosols were formed by heating and then condensing the vaporized benzo[a]pyrene. 

7 The particle MMAD was 0.21 μm. Subgroups of these animals (six/sex/dose) were exposed for 

8 4 days or 6 months after the end of the 4-week exposure to radiolabeled aluminosilicate particles. 

9 Lung injury was assessed by analyzing clearance of radiolabeled aluminosilicate particles and via 

10 histopathologic evaluations. Body and lung weights, measured in subgroups from 1 day to 12 

11 months after the exposure did not differ between controls and treated animals. Radiolabeled 

12 particle clearance did not differ 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/m3 13 for a short14 term exposure.

15 Chronic Studies and Cancer Bioassays

16 Thyssen et al. (1981) conducted an inhalation study in which male Syrian golden hamsters 

17 were exposed to benzo[a]pyrene for their natural lifetime. Groups of 20–30 animals (8 weeks old) 

were exposed by nose-only inhalation to NaCl aerosols (controls; 240 μg NaCl/m3 18 ) or 

19 benzo[a]pyrene condensed onto NaCl aerosols at three nominal concentrations of 2, 10, or 50 mg 

benzo[a]pyrene/m3 20 for 3–4.5 hours/day, 5 days/week for 1–41 weeks, followed by 3 hours/day, 

21 7 days/week for the remainder of study (until hamsters died 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/m3 22 . 

23 More than 99% of the particles were between 0.2 and 0.5 μm in diameter, and over 80% had 

24 diameters between 0.2 and 0.3 μm. The particle analysis of the aerosols was not reported to 

25 modern standards (MMAD and geometric SD were not reported). Each group initially consisted of 

26 24 hamsters; final group sizes were larger as animals dying during the first 12 months of the study 

27 were replaced.

28 Survival was similar in the control, low-dose, and mid-dose groups, but was significantly 

29 decreased in the high-dose group. Average survival times in the control, low-, mid-, and high-dose 

30 groups were 96.4 ± 27.6, 95.2 ± 29.1, 96.4 ± 27.8, and 59.5 ± 15.2 weeks, respectively. After the 60th

31 week, body weights decreased and mortality increased steeply in the highest dose group. 

32 Histologic examination of organs (a complete list of organs examined histologically was not 

33 reported by Thyssen et al. [1981]) revealed a dose-related increase in tumors in the upper 

34 respiratory tract, including the nasal cavity, pharynx, larynx, and trachea, and in the digestive tract 

35 in the mid- and high-dose groups (Table B-13). A statistical analysis was not included in the 

36 Thyssen et al. (1981) report. No lung tumors were observed. Squamous cell tumors in the 

37 esophagus and forestomach were also observed in the high-dose group, presumably as a 
Toxicological Review of benzo[a]pyrene

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

B-56 DRAFT—DO NOT CITE OR QUOTE

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 B-13).

4 Table B-13. Incidence of respiratory and upper digestive tract tumors 

5 in male hamsters treated for life with benzo[a]pyrene by inhalation

Tumor site

Reported benzo[a]pyrene concentration (mg/m3)

0

a

2

b

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)

a

Effective number of animals in control group: n = 27.

b

Effective number of animals in 2 mg/m3dose group: n = 27.

cMean ± SD.

Source: Thyssen et al. (1981).

6

7 Under contract to the U.S. EPA, Clement Associates (1990) obtained the individual animal 

8 data (including individual animal pathology reports, time-to-death data, and exposure chamber 

9 monitoring data) collected by Thyssen et al. (1981). Re-analysis of the original data revealed 

10 several errors and omissions in the published report. The actual exposure protocol was as follows: 

11 4.5 hours/day, 5 days/week on weeks 1–12; 3 hours/day, 5 days/week on weeks 13–29; 3.7 

12 hours/day, 5 days/week on week 30; 3 hours/day, 5 days/week on weeks 31–41; and 3 hours/day, 

13 7 days/week for the reminder of the experiment. In addition, actual exposure concentrations 

14 varied widely from week to week. Because different animals were started at different times, each 

15 individual animal had an exposure history somewhat different than others in the same exposure 

16 group. In order to deal with this problem, Clement Associates (1990) used the original individual 

17 animal data to calculate average continuous lifetime exposures for each individual hamster. Group 

18 averages of individual average continuous lifetime exposure concentrations were 0, 0.25, 1.01, and 

4.29 mg/m3 19 for the control through high-exposure groups.

20 For this assessment, the individual animal pathology reports prepared by Thyssen et al. 

21 (1981) and obtained by Clement Associates (1990) were examined to independently assess the 

22 numbers of hamsters with tumors in the larynx, pharynx, and nose in each group. Table B-14

23 presents the number of animals with tumors in the larynx and pharynx and the numbers of animals 
Toxicological Review of benzo[a]pyrene

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B-57 DRAFT—DO NOT CITE OR QUOTE

1 in each exposure group. Numbers of animals with either laryngeal or pharyngeal tumors are also 

2 noted in Table B-14, since these two types of tumors arise in close anatomical proximity from 

3 similar cell types. Examination of the individual animal pathology reports also showed that all of 

4 the nasal, forestomach, esophageal, and tracheal tumors occurred in animals that also had either 

5 laryngeal or pharyngeal tumors, except for two animals in the mid-dose group that displayed nasal 

6 tumors (one malignant and one benign) without displaying tumors in the pharynx or larynx.

7 Table B-14. Number of animals with pharynx and larynx tumors in 

8 male hamsters exposed by inhalation to benzo[a]pyrene for life

Average 

continuous 

benzo[a]pyrene 

concentrationa

(mg/m3)

Number of 

hamsters in 

groupb

LarynxbPharynxb

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

a

As calculated by Clement Associates (1990) from air monitoring data collected by Thyssen and 

colleagues.

b

As counted from information in Table E-1 in Appendix E, which was obtained from examination of 

individual animal pathology reports prepared by Thyssen and colleagues and obtained by Clement 

Associates.

c

As counted from information in Table E-1 in Appendix E. 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.

9

10 Several studies have investigated the carcinogenicity of benzo[a]pyrene in hamsters 

11 exposed by intratracheal instillation. Single-dose studies verified that benzo[a]pyrene is 

12 tumorigenic, but do not provide data useful for characterizing dose-response relationships because 

13 of their design (Kobayashi, 1975; Reznik-Schuller and Mohr, 1974; Henry et al., 1973; Mohr, 1971; 

14 Saffiotti et al., 1968; Gross et al., 1965; Herrold and Dunham, 1962). One multiple-dose study, 

15 which utilized very low doses (0.005, 0.02, and 0.04 mg, once every 2 weeks), failed to find any 

16 tumorigenic response (Kunstler, 1983). Tumorigenic responses (mostly in the respiratory tract) 

17 were found at higher dosage levels (0.25–2 mg benzo[a]pyrene once per week for 30–52 weeks) in 

18 four multiple-dose studies (Feron and Kruysse, 1978; Ketkar et al., 1978; Feron et al., 1973; Saffiotti 

19 et al., 1972). These studies identify the respiratory tract as a cancer target with exposure to 

20 benzo[a]pyrene by intratracheal instillation and provide supporting evidence for the 

21 carcinogenicity of benzo[a]pyrene at portal-of-entry sites.

22
Toxicological Review of benzo[a]pyrene

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B-58 DRAFT—DO NOT CITE OR QUOTE

1 Dermal studies

2 Skin-Tumor Initiation-Promotion Assays

3 Results from numerous studies indicate that acute dermal exposure to benzo[a]pyrene 

4 induces skin tumors in mice when followed by repeated exposure to a potent tumor promoter 

5 (Weyand et al., 1992; Cavalieri et al., 1991, 1981; Rice et al., 1985; El-Bayoumy et al., 1982; LaVoie 

6 et al., 1982; Raveh et al., 1982; Slaga et al., 1980, 1978; Wood et al., 1980; Hoffmann et al., 1972). 

7 The typical exposure protocol in these studies involved the application of a single dose of 

8 benzo[a]pyrene (typically ≥20 nmol per mouse) to dorsal skin of mice followed by repeated 

9 exposure to a potent tumor promoter, such as 12-O-tetradecanoylphorbol-13-acetate (TPA).

10 Carcinogenicity Bioassays

11 Repeated application of BaP to skin (in the absence of exogenous promoters) has been 

12 variously demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs (IARC, 2010, 

13 1983, 1973; WHO, 1998; ATSDR, 1995). Mice have been most extensively studied, presumably 

14 because of early evidence that they may be more sensitive than other animal species, but 

15 comprehensive comparison of species differences in sensitivity to lifetime dermal exposure are not 

16 available. Early studies of complete dermal carcinogenicity in other species (rats, hamsters, guinea 

17 pigs, and rabbits) have several limitations which make them not useful for dose-response analysis 

18 (see IARC, 1973 for descriptions of studies by Nakano et al., 1937, Shubik et al., 1960; Oberling et 

19 al., 1937; Schürch and Winterstein, 1935; Wynder et al., 1957). The limitations in these studies

20 include inadequate reporting of the amount of BaP applied, use of the carcinogen benzene as a 

21 vehicle, and less than lifetime exposure duration. 

22 This section discusses complete carcinogenicity bioassays in mice that provide the best 

23 available dose-response data for skin tumors caused by repeated dermal exposure to BaP (Sivak et 

24 al., 1997; Higginbotham et al., 1993; Albert et al., 1991; Habs et al., 1984, 1980; Grimmer et al., 

25 1984, 1983; Schmähl et al., 1977; Schmidt et al., 1973; Roe et al., 1970; Poel, 1960, 1959). Early 

26 studies of BaP complete carcinogenicity in mouse skin (Wynder and Hoffman 1959; Wynder et al, 

27 1957) are not further described herein, because the investigators applied solutions of BaP at 

28 varying concentrations on the skin, but did not report volumes applied. As such, applied doses in 

29 these studies cannot be determined. Other complete carcinogenicity mouse skin tumor bioassays 

30 with BaP are available, but these are not described further in this review, because: (1) they only 

31 included one BaP dose level (e.g., Emmett et al., 1981) or only dose levels inducing 90–100% 

32 incidence of mice with tumors (e.g., Wilson and Holland, 1988; Warshawsky and Barkley, 1987) and 

33 thus provide no information about the shape of the dose-response relationship; (2) they used a 1-

34 time/week (e.g., Nesnow et al., 1983) or 1-time every 2 weeks (e.g., Levin et al., 1977) exposure 

35 protocol, which is less useful for extrapolating to daily human exposure; or (3) they used a vehicle 

36 demonstrated to interact with or enhance benzo[a]pyrene carcinogenicity (Bingham and Falk, 

37 1969).
Toxicological Review of benzo[a]pyrene

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B-59 DRAFT—DO NOT CITE OR QUOTE

1 Poel (1959) applied benzo[a]pyrene in toluene to shaved interscapular skin of groups of 

2 13–56 male C57L mice at doses of 0, 0.15, 0.38, 0.75, 3.8, 19, 94, 188, 376, or 752 μg, 3 times/week 

3 for up to 103 weeks or until the appearance of a tumor by gross examination (3 times weekly). 

4 Some organs (not further specified) and interscapular skin in sacrificed mice were examined 

5 histologically. With increasing dose level, the incidence of mice with skin tumors increased and the 

6 time of tumor appearance decreased (see Table B-15). Doses >3.8 μg were associated with 100% 

7 mortality after increasingly shorter exposure periods, none greater than 44 weeks. Poel (1959) did 

8 not mention the appearance of exposure-related tumors in tissues other than interscapular skin.

9 Table B-15. Skin tumor incidence and time of appearance in male C57L 

10 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/week for up to 103 weeks or until time of 

appearance of a grossly detected skin tumor.

b

Carcinomas were histologically confirmed.

c

Ranges reflect differing information in Tables 4 and 6 of Poel (1959).

Source: Poel (1959).

11

12 Poel (1960) applied benzo[a]pyrene in a toluene vehicle to shaved interscapular skin of 

13 groups of 14–25 male SWR, C3HeB, or A/He mice 3 times/week at doses of 0, 0.15, 0.38, 0.75, 3.8, 

14 19.0, 94.0, or 470 μg benzo[a]pyrene per application, until mice died or a skin tumor was observed. 

15 Time ranges for tumor observations were provided, but not times of death for mice without tumors, 

16 so it was not possible to evaluate differential mortality among all dose groups or the length of 

17 exposure for mice without tumors. With increasing dose level, the incidence of mice with skin 

18 tumors increased and the time of tumor appearance decreased (Table B-16). The lowest dose level 

19 did not induce an increased incidence of mice with skin tumors in any strain, but strain differences 

20 in susceptibility were evident at higher dose levels. SWR and C3HeB mice showed skin tumors at 
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1 doses ≥0.38 μg benzo[a]pyrene, whereas AH/e mice showed tumors at doses ≥19 μg 

2 benzo[a]pyrene (Table B-16). Except for metastases of the skin tumors to lymph nodes and lung, 

3 Poel (1960) did not mention the appearance of exposure-related tumors in tissues other than 

4 interscapular skin.

5 Table B-16. Skin tumor incidence and time of appearance in male SWR, 

6 C3HeB, and A/He mice dermally exposed to benzo[a]pyrene for life or 

7 until a skin tumor was detected

Dose (μg)a

SWR mice C3HeB mice A/He mice

Tumor 

incidenceb

Time of first 

tumor 

appearance

(wks)

Tumor 

incidenceb

Time of first 

tumor 

appearance

(wks)

Tumor 

incidenceb

Time of fist 

tumor 

appearance

(wks)

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 3 times/week for life or until a skin tumor was detected. Mice were 10–

14 weeks old at initial exposure.

b

Incidence of mice exposed ≥10 weeks with a skin tumor.

Source: Poel (1960).

8

9 Roe et al. (1970) treated groups of 50 female Swiss mice with 0 (acetone vehicle), 0.1, 0.3, 1, 

10 3, or 9 μg benzo[a]pyrene applied to the shaved dorsal skin 3 times/week for up to 93 weeks; all 

11 surviving mice were killed and examined for tumors during the following 3 weeks. The dorsal skin 

12 of an additional control group was shaved periodically but was not treated with the vehicle. Mice 

13 were examined every 2 weeks for the development of skin tumors at the site of application. 

14 Histologic examinations included: (1) all skin tumors thought to be possibly malignant; (2) lesions 

15 of other tissues thought to be neoplastic; and (3) limited nonneoplastic lesions in other tissues. As 

16 shown in Table B-17, markedly elevated incidences of mice with skin tumors were only found in the 

17 two highest dose groups (3 or 9 μg), compared with no skin tumors in the control groups. 

18 Malignant skin tumors (defined as tumors with invasion or penetration of the panniculus carnosus 

19 muscle) were detected in 4/41 and 31/40 mice in the 3- and 9-μg groups, respectively, surviving to 

20 at least 300 days. Malignant lymphomas were detected in all groups, but the numbers of cases were 

21 not elevated compared with expected numbers after adjustment for survival differences. Lung 
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1 tumors were likewise detected in control and exposed groups at incidences that were not 

2 statistically different.

3 Table B-17. Tumor incidence in female Swiss mice dermally exposed to 

4 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 

incidencec200 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/week for up to 93 weeks to shaved dorsal skin.

bNumerator: number of mice detected with a skin tumor. Denominator: number of mice surviving to 

300 days for all groups except the highest dose group. For the highest dose group (in which skin tumors 

were first detected between 200 and 300 days), the number of mice surviving to 200 days was used as 

the denominator.

cNumerator: number of mice detected with specified tumor. Denominator: number of mice surviving 

to 300 days unless a tumor was detected earlier, in which case, the number dying before 300 days 

without a tumor was subtracted from the number of animals reported to have been examined.

Source: Roe et al. (1970).

5

6 Schmidt et al. (1973) dermally administered benzo[a]pyrene in acetone to female NMRI 

7 mice (100/group) and female Swiss mice. Benzo[a]pyrene was applied to the shaved dorsal skin 

8 twice weekly with doses of 0, 0.05, 0.2, 0.8, or 2 μg until spontaneous death occurred or until an 

9 advanced carcinoma was observed. Skin carcinomas were identified by the presence of crater10 shaped ulcerations, infiltrative growth, and the beginning of physical wasting (i.e., cachexia). 

11 Necropsy was performed for all animals, and histopathological examination of the dermal site of 

12 application and any other tissues with gross abnormalities was conducted. Skin tumors were 

13 observed at the two highest doses in both strains of female mice (see Table B-18), with induction 

14 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 the 

15 Swiss mice, respectively. The authors indicated that the latency period for tumor formation was 

16 highly variable and significant differences among exposure groups could not be identified, but no 

17 further timing information was available, including overall survival. Carcinoma was the primary 

18 tumor type seen after lifetime application of benzo[a]pyrene to mouse skin.
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1 Table B-18. Skin tumor incidence in female NMRI and Swiss mice 

2 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/week to shaved skin of the back.

Source: Schmidt et al. (1973).

3

4 Schmähl et al. (1977) applied benzo[a]pyrene 2 times/week to the shaved dorsal skin of 

5 female NMRI mice (100/group) at doses of 0, 1, 1.7, or 3 μg in 20 μL acetone. The authors reported 

6 that animals were observed until natural death or until they developed a carcinoma at the site of 

7 application. The effective numbers of animals at risk was about 80% of the nominal group sizes, 

8 which the authors attributed to autolyis; no information was provided concerning when tumors 

9 appeared in the relevant groups, how long treatment lasted in each group, or any times of death. 

10 Necropsy was performed on all mice and the skin of the back, as well as any organs that exhibited 

11 macroscopic changes, were examined histopathologically. The incidence of all types of skin tumors 

12 was increased in a dose-related manner compared to controls (see Table B-19). Carcinoma was the 

13 primary tumor type observed following chronic dermal exposure to benzo[a]pyrene, and skin 

14 papillomas occurred infrequently. Dermal sarcoma was not observed.

15 Table B-19. Skin tumor incidence in female NMRI mice dermally 

16 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%)
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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/week 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 NMRI 

3 mice (40/group) at doses of 0, 1.7, 2.8, or 4.6 μg in 20 μL acetone twice weekly, from 10 weeks of 

4 age until natural death or gross observation of infiltrative tumor growth. Latency of tumors, either 

5 as time of first appearance or as average time of appearance of tumors, was not reported. Necropsy 

6 was performed on all animals, and the dorsal skin, as well as any organs showing gross alterations 

7 at autopsy, was prepared for histopathological examination. Age-standardized mortality rates, 

8 using the total population of the experiment as the standard population, were used to adjust tumor 

9 incidence findings in the study. Benzo[a]pyrene application was associated with a statistically 

10 significant increase in the incidence of skin tumors at each dose level (see Table B-20).

11 Table B-20. Skin tumor incidence in female NMRI mice dermally 

12 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/week 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. (1984, 1983) applied benzo[a]pyrene (in 0.1 mL of a 1:3 solution of 

15 acetone:dimethyl sulfoxide [DMSO]) to the interscapular skin of female CFLP mice (65–80/group) 2 

16 times/week for 104 weeks. Doses were 0, 3.9, 7.7, and 15.4 μg in the 1983 experiment, and 0, 3.4, 

17 6.7, and 13.5 μg in the 1984 experiment. Mice were observed until spontaneous death, unless an 

18 advanced tumor was observed or if animals were found moribund. Survival information was not 

19 provided; incidences reflect the number of animals placed on study. Necropsy was performed on 

20 all mice. Histopathological examination of the skin and any other organ showing gross 

21 abnormalities was performed. Chronic dermal exposure to benzo[a]pyrene produced a dose22 related increase in skin tumor incidence and a decrease in tumor latency (see Table B-21). 
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1 Carcinoma was the primary tumor type observed and a dose-response relationship was evident for 

2 carcinoma formation and incidence of all types of skin tumors.

3 Table B-21. Skin tumor incidence and time of appearance in female 

4 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.

bMean ± SD.

cMedian with 95% CI.

Sources: Grimmer et al. (1984, 1983)

5

6 Habs et al. (1984) applied benzo[a]pyrene (in 0.01 mL acetone) to the shaved interscapular 

7 skin of female NMRI mice at doses of 0, 2, or 4 μg, 2 times/week for life. Animals were observed 

8 twice daily until spontaneous death, unless an invasive tumor was observed. All animals were 

9 necropsied and histopathological examination was performed on the dorsal skin and any other 

10 organ with gross abnormalities. Chronic dermal exposure to benzo[a]pyrene did not affect body 

11 weight gain, but appeared to reduce survival at the highest dose with mean survival times of 691, 

12 648, and 528 days for the 0, 2, and 4 μg/day groups, respectively. The total length of exposure for 

13 each group was not reported, but can be inferred from the survival data. Latency also was not 

14 reported. Benzo[a]pyrene application resulted in a dose-related increase the incidence of total skin 

15 tumors and skin carcinomas (see Table B-22). Hematopoietic tumors (at 6/20, 3/20, and 3/20) and 

16 lung adenomas (at 2/20, 1/20, and 0/20) were observed in the controls and in the benzo[a]pyrene 

17 treatment groups, but did not appear to be treatment related according to the study authors.
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1 Table B-22. Skin tumor incidence in female NMRI mice dermally 

2 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% CI)

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/week to shaved interscapular skin.

Source: Habs et al. (1984).

3

4 Groups of 23–27 female Ah-receptor-responsive Swiss mice were treated on a shaved area 

5 of dorsal skin with 0, 1, 4, or 8 nmol (0, 0.25, 1, or 2 μg/treatment) benzo[a]pyrene (>99% pure) in 

6 acetone 2 times weekly for 40 weeks (Higginbotham et al., 1993). Surviving animals were 

7 sacrificed 8 weeks later. Complete necropsies were performed, and tissues from the treated area, 

8 lung, liver, kidney, spleen, urinary bladder, ovary, and uterus were harvested for histopathologic 

9 examination. Histopathologic examination was performed on tissues from the treated area, lungs, 

10 liver, kidneys, spleen, urinary bladder, uterus, and ovaries, as well as any other grossly abnormal 

11 tissue. Lung adenomas occurred in each group (1/27, 2/24, 1/23, 1/23), and other tumors were 

12 noted in isolated mice (i.e., malignant lymphoma [spleen] in one low-dose and one mid-dose mouse; 

13 malignant lymphoma with middle organ involvement in one high-dose mouse; and hemangioma 

14 [liver] in one mid-dose mouse) and were not considered dose related. In addition, benzo[a]pyrene 

15 showed no skin tumors under the conditions of this bioassay.

16 Sivak et al. (1997) designed a study to compare the carcinogenicity of condensed asphalt 

17 fumes (including benzo[a]pyrene and other PAHs) with several doses of benzo[a]pyrene alone. For 

18 the purposes of this assessment, the exposure groups exposed to PAH mixtures are not discussed. 

19 Groups of 30 male C3H/HeJ mice were treated dermally twice/week to 0, 0.0001, 0.001, or 0.01% 

20 (0, 0.05, 0.5, or 5 μg) benzo[a]pyrene in a 50 μL volume of cyclohexanone/acetone (1:1) for 104 

21 weeks beginning at 8 weeks of age. Mice dying during the exposure period or sacrificed at the 24

22 month termination were necropsied; mice with skin tumors that persisted for 4 consecutive weeks 

23 with diameters > 3 cm were sacrificed before the study termination and also necropsied. Skin 

24 samples and any grossly observed lesions were subjected to histopathological examination. 

25 Carcinomas and sarcomas were referred to as carcinomas, whereas papillomas, keratoacanthomas, 

26 and fibromas were referred to as papillomas. The incidences of mice with skin tumors and mean 

27 survival times for each group are shown in Table B-23. All high-dose mice died before the final 

28 sacrifice, and 80% showed scabs and sores at the site of application. The time of first tumor 

29 appearance was not reported for the tumor-inducing groups, but from a plot of the tumor incidence 
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1 in the high-dose group versus treatment days, an estimate of ~320 days (~43 weeks) is obtained 

2 for this group. The extent of deaths prior to 1 year in each group was not provided, so that the 

3 reported incidence may underestimate the tumor rate of animals exposed long enough to develop 

4 tumors. However, the crude skin tumor rates show an increasing trend in incidence.

5 Table B-23. Skin tumor incidence in male C3H/HeJ mice dermally 

6 exposed to benzo[a]pyrene for 24 months

Dose (μg)a

Skin tumor incidence 

(all types)b

Number of mice that 

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.

bNumber of skin tumor-bearing mice. In the high-dose group, 1 papilloma and 28 carcinomas were 

detected. In the 0.5 μg group, 2 papillomas and 3 carcinomas were detected. 

Source: Sivak et al. (1997).

7

8 To examine dose-response relationships and the time course of benzo[a]pyrene-induced 

9 skin damage, DNA adduct formation, and tumor formation, groups of 43–85 female Harlan mice 

10 were treated dermally with 0, 16, 32, or 64 μg of benzo[a]pyrene in 50 μL of acetone once per week 

11 for 29 weeks (Albert et al., 1991). Interscapular skin of each mouse was clipped 3 days before the 

12 first application and every 2 weeks thereafter. Additional groups of mice were treated for 9 weeks 

13 with 0, 8, 16, 32, or 64 μg radiolabeled benzo[a]pyrene to determine BPDE-DNA adduct formation 

14 in the epidermis at several time points (1, 2, 4, and 9 weeks). Tumor formation was monitored only 

15 in the skin.

16 No tumors were present in vehicle-treated or untreated control mice. In exposed groups, 

17 incidences of mice with skin tumors were not reported, but time-course data for cumulative 

18 number of tumors per mouse, corrected for deaths from nontumor causes, were reported. Tumors 

19 began appearing after 12–14 weeks of exposure for the mid- and high-dose groups and at 18 weeks 

20 for the low-dose group. At study termination (35 weeks after start of exposure), the mean number 

21 of tumors per mouse was approximately one per mouse in the low- and mid-dose groups and eight 

22 per mouse in the high-dose group; indicating that most, if not all, mice in each exposure group 

23 developed skin tumors and that the tumorigenic response was greatest in the highest dose group. 

24 The majority of tumors were initially benign, with an average time of 8 weeks for progression from 

25 benign papillomas to malignant carcinomas. Epidermal damage occurred in a dose-related manner 
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1 (more severe in the high-dose group than in the low- and mid-dose groups) and included 

statistically significant increases (compared with controls) in: [ 2 3H]-thymidine labeling and mitotic 

3 indices; incidence of pyknotic and dark cells (signs of apoptosis); and epidermal thickness. Only a 

4 minor expansion of the epidermal cell population was observed. In the high-dose group, indices of 

5 epidermal damage increased to a plateau by 2 weeks of exposure. The early time course of 

6 epidermal damage indices was not described in the low- or mid-dose groups, since data for these 

7 endpoints were only collected at 20, 24, and 30 weeks of exposure. An increased level of BPDE8 DNA adducts, compared with controls, was apparent in all exposed groups after 4 weeks of 

9 exposure in the following order: 64 > 32 > 16 > 8 μg/week. The time-course data indicate that 

10 benzo[a]pyrene-induced increases in epidermal damage indices and BPDE-DNA adducts preceded 

11 the appearance of skin tumors.

12 Reproductive and Developmental Toxicity Studies

13 Oral

14 In a study evaluating the combined effects of DBP and benzo[a]pyrene on the male 

15 reproductive tract, Chen et al. (2011) administered benzo[a]pyrene alone in corn oil via daily 

16 gavage at 5 mg/kg-day to 30 male Sprague-Dawley rats (28-30 days old); a group of 30 rats 

17 received only vehicle. Body weight was measured weekly. Groups of 10 rats per group were 

18 sacrificed after 4, 8, and 12 weeks of exposure. At sacrifice, blood was collected for analysis of 

19 serum testosterone levels by radioimmunoassay. The testes and epidiymides were weighed, and 

20 the right testis and epididymis were examined microscopically. The left epididymis was used for 

21 evaluation of sperm parameters (sperm count and morphology). Oxidative stress, as measured by 

22 superoxide dismutase, glutathione peroxidase, and catalase activity and malondioaldehyde levels, 

23 was evaluated in the left testis of each rat. Exposure to benzo[a]pyrene did not affect body weight, 

24 and no signs of toxicity were seen. Testes and epididymides weights of exposed rats were similar 

25 to controls at all time points. Sperm counts and percent abnormal sperm were also similar to 

26 controls at 4 and 8 weeks of exposure, but were significantly (p<0.05) different from controls after 

27 12 weeks of exposure to benzo[a]pyrene (29% decrease in sperm count and 54% increase in 

28 percent abnormal sperm). Serum testosterone levels were significantly increased relative to 

29 controls after 4 weeks (>two-fold higher) and 8 weeks (~1.5-fold higher) of benzo[a]pyrene 

30 exposure, but were comparable to controls after 12 weeks. Histopathology evaluation of the testes 

31 revealed irregular and disordered arrangement of germ cells in the seminiferous tubules of treated 

32 rats; the authors did not report incidence or severity of these changes. Among measures of 

33 testicular oxidative stress, only catalase activity was significantly affected by benzo[a]pyrene 

34 exposure, showing an increase of ~50% after 12 weeks of exposure. These data suggest a LOAEL of 

35 5 mg/kg-day (the only dose tested) for decreased sperm count, increased percentage of abnormal 

36 sperm, altered testosterone levels, and histopathology changes in the testes following 13 weeks of 

37 exposure.
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1 Chung et al. (2011) evaluated the effects of low-dose benzo[a]pyrene exposure on 

2 spermatogenesis, and the role of altered steroidogenesis on the sperm effects. Groups of 20-25 

3 male Sprague-Dawley rats (8 wks old) were given daily gavage doses of 0, 0.001, 0.01, or 0.1 

4 mg/kg-day benzo[a]pyrene in DMSO for 90 consecutive days. At the end of exposure, the animals 

5 were sacrificed for removal of the pituitary, testes, and epididymides, and collection of serum and 

6 testicular interstitial fluid. Subgroups of each exposure group were used for various analyses. 

7 Serum levels of testosterone and LH were measured, as was testosterone concentration in the 

8 interstitial fluid (ELISA assays). Body and testes weights were recorded. Sections of the testis 

9 were analyzed for apoptotic germ cells using TUNEL assay. Evaluation of the epididymis included 

10 histopathology as well as measurement of caput and caudal epididymal tubule diameters. In 

11 addition, sperm were isolated from the cauda epididymis for analysis of sperm number and 

12 motility, acrosomal integrity, and immunocytochemistry for ADAM3 (a disintegrin and 

13 metallopeptidse domain 3; a sperm surface protein associated with fertilization).

14 Leydig cells were isolated from the right testis of animals from each dose group and 

15 cultured with or without human chorionic gondatotropin (hCG) or dibutyl cyclic adenosine 

16 monophosphate (dbcAMP) to evaluate testosterone production (Chung et al., 2011). Cultured 

17 Leydig cells were also subjected to western blot and immunocytochemistry analyses to evaluate 

18 changes in the expression of genes involved in steroidogenesis (StAR[steroidogenic acute 

19 regulatory protein], p450scc [p450 side-chain cleavage], and 3β-HSD[3β-hydroxysteroid 

20 dehydrogenase isomerase]). Finally, pituitary gland extracts were evaluated for LH protein 

21 content using immunohistochemistry. Data were reported graphically and analyzed by ANOVA 

22 followed by Duncan’s post hoc test, using a p-value cutoff of 0.05 for significant difference.

23 At termination of exposure, body weights of treated animals were similar to controls, as 

24 were absolute testes weights (Chung et al., 2011). Testosterone concentrations in both serum and 

25 testicular interstitial fluid were significantly reduced at the high dose of benzo[a]pyrene (0.1 

26 mg/kg-day); based on visual inspection of the data, the mean serum concentration in this group 

27 was ~20% of the control and the mean intersitital fluid concentration was ~60% of the control 

28 (n=9 animals/dose for these evaluations). In addition, baseline production of testosterone by 

29 cultured Leydig cells was significantly decreased (~50% based on data shown graphically) at 0.1 

30 mg/kg-day. Both hCG- and dbcAMP-stimulated testosterone production measurements were lower 

31 (~60% lower than controls) in Leydig cells from rats exposed to either 0.01 or 0.1 mg/kg-day. 

32 Serum LH was significantly increased at both 0.01 and 0.1 mg/kg-day (~65-75% higher than 

33 controls based on visual inspection of graphs); concordant increases in the intensity of LH 

34 immunoreactivity were evident in pituitary extracts from exposed rats. 

35 Dose-related increases in the number of apoptotic germ cells, primarily spermatogonia, 

36 were demonstrated both via TUNEL assay and caspase-3 staining; the number per tubule was 

37 significantly increased over control at all doses (Chung et al., 2011). Numbers of sperm were lower 

38 in the treatment groups, but did not differ significantly from the control group. However, sperm 
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1 motility was significantly reduced in exposed groups compare with control. The authors did not 

2 report sperm motility for all dose groups, but showed only the significant decrease in the 0.01 

3 mg/kg-day mid-dose group (~30% lower than controls based on visual inspection of graph). 

4 Acrosomal integrity (measured by LysoTracker staining) was diminished in sperm heads from 

5 exposed rats; likewise, the expression of ADAM3 protein was downregulated by exposure to 

6 benzo[a]pyrene; the authors reported a significant decrease in the 0.01 mg/kg-day group but did 

7 not provide details of the analysis of other exposure groups. Histopathology examination of the 

8 caput and cauda epididymides revealed dose-related decreases in both cauda and caput tubule 

9 diameters that were statistically significantly lower than controls at all doses (~10-30% smaller 

10 mean diameter than control based on measurements of 175 tubules collected from 5 samples in 

11 each group; data reported graphically). 

12 Statistically significant effects observed at the lowest dose (0.001 mg/kg-day) of 

13 benzo[a]pyrene in this study included decreased caput and cauda epididymal tubule diameters 

14 (~10-15% lower than controls) and increased numbers of apoptotic germ cells (~twofold higher 

15 than controls) by TUNEL assay (Chung et al., 2011). The authors reported that “sperm motility was 

16 significantly reduced in the benzo[a]pyrene-exposed groups in comparison to that of the control” 

17 but provided quantitative data only for the middle dose group, which exhibited a ~30% decrease in 

18 percent motile sperm. No statistically significant decrease in sperm count was reported at any 

19 dose. The middle dose (0.01 mg/kg-day) is considered to be a LOAEL, based on reduced sperm 

20 motility. 

21 Gao et al. (2011) examined effects of benzo[a]pyrene exposure via on cervical cell 

22 morphology. Female ICR mice (18-22 g) were exposed to doses of 0, 2.5, 5, or 10 mg/kg twice per 

23 week for 14 weeks, either by oral gavage or by intraperitoneal injection (for this review, only oral 

24 results are reported). After adjustment for equivalent continuous dosing (2/7 days/week), the 

25 equivalent daily doses are estimated to be 0.7, 1.4, 2.9 mg/kg-day. Both vehicle (sesame oil) and 

26 untreated control groups were maintained. Body weights were determined weekly. Groups of 26 

27 mice per dose per exposure route were sacrificed at the end of exposure for evaluation of cervical 

28 weight and histopathology. Additional groups of 10 mice were exposed for 14 weeks and used for 

29 determination of lipid peroxidation (malondialdehyde and glutathione-S-transferase levels) and 

30 CYP1A1 activity (EROD) in both liver and cervix, as well as creatine kinase activity, AST activity, and 

31 IL-6 levels in cervix and serum.

32 Mortality was observed in all exposure groups with the exception of the low dose oral 

33 exposure group; the authors did not indicate the timing or causes of death (Gao et al., 2011). There 

34 were no control deaths. Mortality incidences in the oral exposure groups (low to high dose) were 

35 0/26 (untreated control), 0/26 (vehicle control), 0/26, 1/36, and 2/26. Benzo[a]pyrene treatment 

36 resulted in dose-dependent decreases in body weight gain. In the high dose group of both 

37 treatments, body weight began to decline after ~7 weeks of exposure. Based on visual examination 

38 of data presented graphically, mean terminal body weights in the low, mid-, and high-dose oral 
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1 exposure groups were ~10, 15, and 30% lower (respectively) than the vehicle control mean. The 

2 untreated control mean body weight for the oral exposure group was similar to the vehicle control 

3 mean body weight. Cervical weight as a function of body weight was not affected by oral 

4 benzo[a]pyrene exposure. Microscopic examination of the cervix revealed increased incidences of 

5 epithelial hyperplasia and inflammatory cells in the cervix of all groups of exposed mice, and 

6 atypical hyperplasia of the cervix in mice exposed to 1.4 or 2.9 mg/kg benzo[a]pyrene. Statistical 

7 analysis of the findings was conducted, but was poorly reported in the publication. Table B-24

8 shows the incidences in the oral exposure groups, along with the results of Fisher’s exact tests 

9 performed for this review.

10 Table B-24. Mortality and cervical histopathology incidences in female 

11 ICR mice exposed to benzo[a]pyrene via gavage for 14 weeks

Endpoint

Dose (mg/kg-d)

Untreated 

control

Vehicle 

control 0.7 1.4 2.9

Mortality 0/26 0/26 0/26 1/26 2/26

Cervical epithelial hyperplasia 0/26 0/26 4/26 6/25a7/24a

Atypical hyperplasia of cervix 0/26 0/26 0/26 2/25 4/24a

Inflammatory cells in cervix 2/26 3/26 10/26a12/25a18/24a

a

Significantly different from vehicle control by Fisher’s exact test performed for this review (one-sided p

< 0.05).

Source: Gao et al. (2011).

12

13 Levels of malondialdehyde in both the cervix and liver were significantly higher than 

14 controls in all dose groups of animals treated by either oral (1.5 to 2-fold higher in the cervix and 

15 ~3-fold to 7-fold higher in the liver after oral exposure p<0.05) or intraperitoneal exposure. 

16 Concomitant decreases in GST activity (~15% to 50% lower than controls in the cervix and ~30% 

17 to 60% lower in the liver after oral exposure; p<0.05) were also observed at all doses and in both 

18 organs and both treatments. EROD activity was increased in the cervix (~4- to ~12-fold) and liver 

19 (~12- to ~35-fold) of all exposure groups. Measurement of CK and AST activity in the cervix and 

20 serum also showed significant increases at all doses and after both exposures (~1.5- to 2-fold in the 

21 cervix, and ~20% to 50% higher than controls in the liver after oral exposure). Finally, levels of the 

22 inflammatory cytokine IL-6 were significantly (p<0.05) increased in the cervix of all treated mice, 

23 and were markedly increased (from more than two-fold higher than untreated or vehicle controls 

24 at the low dose, to ~six-fold higher at the high dose) in the serum of treated mice. 

25 Based on the observations of decreased body weight and increased cervical epithelial 

26 inflammation and hyperplasia, a LOAEL of 0.7 mg/kg-day (the lowest dose tested) is identified for 

27 this study. 
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1 Mohamed et al. (2010) investigated multi-generational effects in male mice following 

2 exposure of 6-week old-C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day 

3 benzo[a]pyrene for 6 weeks by gavage. Following final treatment, male mice were allowed to 

4 stabilize for 1 week prior to being mated with two untreated female mice to produce an 

5 F1 generation. Male mice were sacrificed 1 week after mating. F1 males were also mated with 

6 untreated female mice as were F2 males. The mice of the F1, F2, and F3 generations were not 

7 exposed to benzo[a]pyrene. The F0, F1, F2, and F3 mice were all sacrificed at the same age 

8 (14 weeks) and endpoints including testis histology, sperm count, sperm motility, and in vitro 

9 sperm penetration (of hamster oocytes) were evaluated. These endpoints were analyzed 

10 statistically using analysis of variance (ANOVA) and Tukey’s honest significance test and results 

11 were reported graphically as means ± SD.

12 Testicular atrophy was observed in the benzo[a]pyrene treatment groups, but was not 

13 statistically different than controls. Statistically significant reductions were observed in epididymal 

14 sperm counts of F0 and F1 generations treated with the high or low dose of benzo[a]pyrene. For F0 

15 and F1 generations, epididymal sperm counts were reduced approximately 50 and 70%, 

16 respectively, in the low- and high-dose groups. Additionally, sperm motility was statistically 

17 significantly decreased at the high dose in the F0 and F1 generations. Sperm parameters of the F3 

18 generation were not statistically different from controls. An in vitro sperm penetration assay 

19 revealed statistically significantly reduced fertilization in F0 and F1 generations of the low- and 

20 high-dose groups. However, the value of this in vitro test is limited as it bypasses essential 

21 components of the intact animal system (U.S. EPA, 1996). Based on decreased epididymal sperm 

22 counts of F0 and F1 generations, a LOAEL of 1 mg/kg-day was established from this study (no 

23 NOAEL was identified).

24 Arafa et al. (2009) exposed groups of 12 male Swiss albino rats to benzo[a]pyrene in olive 

25 oil (0 or 50 mg/kg-day via gavage) for 10 consecutive days, either alone or after similar treatment 

26 with 200 mg/kg-day of the flavonoid hesperidin, which has been shown to exert anti-inflammatory, 

27 antioxidant, and anticarcinogenic activity. One day after the final dose, the animals were sacrificed 

28 for removal of the cauda epididymides and testes. Epididymal sperm count and motility were 

29 assessed, as was daily sperm production in the testes. The study authors also investigated the 

30 testicular activity of LDH, SOD, and GST, as well as GSH, malondialdehyde, and protein content. The 

31 testes were examined under light microscope.

32 Relative testes weights (normalized to body weight) of benzo[a]pyrene exposed-animals 

33 were significantly decreased compared with controls (35% lower, p < 0.05) (Arafa et al., 2009). In 

34 addition, exposure to benzo[a]pyrene alone resulted in significantly decreased sperm count, 

35 numbers of motile sperm, and daily sperm production (~40% decrease from control in each 

36 parameter, p < 0.05). Effects on sperm count and production were abolished by hesperidin 

37 pretreatment, but the number of motile sperm remained significantly depressed (compared with 

38 the control group) in the group exposed to both benzo[a]pyrene and hesperidin. Measures of 
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1 antioxidant enzymes and lipid peroxidation showed statistically significant induction of oxidative 

2 stress in the testes of benzo[a]pyrene-exposed rats. With the exception of the decrease in testicular 

3 GSH content (which was partially mitigated), pretreatment with hesperidin eliminated the effects of 

4 benzo[a]pyrene on lipid peroxidation and antioxidant enzymes. 

5 Xu et al. (2010) treated female Sprague-Dawley rats (6/group) to 0 (corn oil only), 5, or 10 

6 mg/kg-day benzo[a]pyrene by gavage every other day for a duration of 60 days. This resulted in 

7 TWA doses of 0, 2.5, and 5 mg/kg-day over the study period of 60 days. Endpoints examined 

8 included ovary weight, estrous cycle, 17B-estradiol blood level, and ovarian follicle populations 

9 (including primordial, primary, secondary, atretic, and corpora leutea). Animals were observed 

10 daily for any clinical signs of toxicity and following sacrifice, gross pathological examinations were 

11 made and any findings were recorded. All animals survived to necropsy. A difference in clinical 

12 signs was not observed for the treated groups and body weights were not statistically different in 

13 treated animals (although they appear to be depressed 6% at the high dose). Absolute ovary 

14 weight was statistically significantly reduced in both the low- and high-dose groups (11 and 15%, 

15 respectively) (see Table B-25). Animals treated with the high dose were noted to have a 

16 statistically significantly prolonged duration of the estrous cycle and nonestrus phase compared to 

17 controls. Animals in the high-dose group also had statistically significantly depressed levels of 

18 estradiol (by approximately 25%) and decreased numbers of primordial follicles (by approximately 

19 20%). This study also indicated a strong apoptotic response of ovarian granulosa cells as visualized

20 through TUNEL labeling; however, the strongest response was seen at the low dose; decreased 

21 apoptosis was also observed at the high dose. Based on decreased ovary weight following a 60-day 

22 oral exposure to benzo[a]pyrene, a LOAEL of 2.5 mg/kg-day was established from this study (no 

23 NOAEL was identified).

24 Table B-25. Means ± SD for ovary weight in female Sprague-Dawley 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).

25

26 Zheng et al. (2010) treated male Sprague-Dawley rats to 0 (corn oil only), 1, or 5 mg/kg-day 

27 benzo[a]pyrene by daily gavage for a duration of 30 (8/group) or 90 days (8/group). At necropsy, 

28 the left testis of each animal was collected and weighed. Testes testosterone concentrations were 

29 determined by radioimmunassay and results were expressed as ng/g testis and reported 

30 graphically. Testicular testosterone was statistically significantly decreased in the high-dose group 
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1 approximately 15% following 90 days of exposure. The low-dose group also appeared to have a 

2 similar average depression of testosterone levels; however, the change did not reach statistical 

3 significance. Testosterone levels measured in animals sacrificed following 30 days of 

4 benzo[a]pyrene exposure were not statistically different than controls. Based on decreased 

5 testicular testosterone levels following a 90-day oral exposure to benzo[a]pyrene, a LOAEL of 5 

6 mg/kg-day and a NOAEL of 1 mg/kg-day were identified.

7 McCallister et al. (2008) administered 0 or 300 μg/kg benzo[a]pyrene by gavage in peanut 

8 oil to pregnant Long Evans rats (n = 5 or 6) on GDs 14–17. At this exposure level, no significant 

9 changes were see in number of pups per litter, pup growth, or liver to body weight ratios in control 

10 compared to benzo[a]pyrene exposed offspring. Treatment-related differences in brain to body 

11 weight ratios were observed only on PNDs 15 and 30. Decreases in cerebrocortical mRNA 

12 expression of the glutamatergic N-methyl-D-aspartate (NMDA) receptor subunit was significantly 

13 reduced (50%) in treated offspring compared to controls. In addition, in utero exposed offspring 

14 exhibited decreased evoked cortical neuronal activity in the barrel field cortex when tested at PNDs 

15 90–120.

16 Rigdon and Neal (1965) administered diets containing 1,000 ppm benzo[a]pyrene to 

17 pregnant mice (nine/group) on GDs 10–21 or 5–21. The pups were reported as appearing 

18 generally normal at birth, but cannibalism was elevated in the exposed groups. These results are in 

19 contrast with an earlier study (Rigdon and Rennels, 1964) in which rats (strain not specified) were 

20 fed diets containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and 

21 during gestation. In the earlier study, five of eight treated females mated with untreated males 

22 became pregnant, but only one delivered live young. The treated dam that delivered had two live 

23 and two stillborn pups; one dead pup was grossly malformed. In the remaining treated females, 

24 vaginal bleeding was observed on GDs 23 or 24. In the inverse experimental design, three of six 

25 controls mated to benzo[a]pyrene-treated males became pregnant and delivered live young. 

26 Visceral and skeletal examinations of the pups were not conducted. These studies were limited by 

27 the small numbers of animals, minimal evaluation of the pups, lack of details on days of treatment 

28 (food consumption, weight gain), and the occurrence of cannibalism.

29 Reproductive effects of in utero exposure via oral route

30 MacKenzie and Angevine (1981) conducted a two-generation reproductive and 

31 developmental toxicity study for benzo[a]pyrene in CD-1 mice. Benzo[a]pyrene was administered 

32 by gavage in 0.2 mL of corn oil to groups of 30 or 60 pregnant (the F0 generation) mice at doses of 

33 0, 10, 40, or 160 mg/kg-day on GDs 7–16 only. Therefore, unlike the standard two-generation 

34 study, F1 animals were exposed only in utero. F1 offspring were evaluated for postnatal 

35 development and reproductive function as follows. F1 pups (four/sex when possible) were allowed 

36 to remain with their mothers until weaning on PND 20. Crossover mating studies were then 

37 conducted. Beginning at 7 weeks of age, each F1 male mouse (n = 20–45/group) was allowed to 

38 mate with two untreated virgin females for 5-day periods for 25 days (for a total exposure of 10 
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1 untreated females/F1 male), after which time the males were separated from the females. 

2 Fourteen days after separation from the males (i.e., on days 14–19 of gestation), the females were 

3 sacrificed and the numbers of implants, fetuses, and resorptions were recorded. The F2 fetuses 

4 were then examined for gross abnormalities. Similarly, each F1 female mouse (n = 20–55/group), 

5 beginning at 6 weeks of age, was paired with an untreated male for a period of 6 months. Males 

6 were replaced if the females failed to produce a litter during the first 30-day period. All F2 young 

7 were examined for gross abnormalities on day 1 of life and their weights were recorded on day 4 of 

8 age. This F2 group was sacrificed on day 20 postpartum, while the F1 female was left with a male 

9 until the conclusion of the study. At 6 weeks of age, gonads of groups of 10 male and 10 female F1 

10 mice exposed to 0, 10, or 40 mg/kg-day benzo[a]pyrene in utero were subjected to gross pathology 

11 and histologic examinations.

12 No maternal toxicity was observed. The number of F0 females with viable litters at 

13 parturition at the highest dose was statistically significantly reduced by about 35% (Table B-26), 

14 but progeny were normal by gross observation. Parturition rates of the low- and mid-dose groups 

15 were unaffected by treatment, and litter sizes of all treated groups were similar to the control group 

16 throughout lactation. However, body weights of the F1 pups in the mid- and high-dose groups were 

17 statistically significantly decreased on PND 20, by 7 and 13%, respectively, and in all treated pups 

18 on PND 42, 6, 6, and 10% for the low, mid, and high dose, respectively (Table B-26). The number of 

19 F1 pups surviving to PNDs 20 and 42 was significantly reduced at the high dose (p < 0.01), by 8 and 

20 16%, respectively. When F1 males were bred to untreated females and F1 females were mated 

21 with untreated males, a marked dose-related decrease in fertility of >30% was observed in both 

22 sexes, starting at the lowest exposure. There were no treatment-associated gross abnormalities or 

23 differences in body weights in the F2 offspring.

24 Table B-26. Reproductive effects in male and female CD-1 F1 mice 

25 exposed in utero to benzo[a]pyrene

Effect

Dose (mg/kg-d)a

0 10 40 160

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
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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.

c

Beginning at 7 weeks of age, each F1 male mouse (20–45/group) was exposed to 10 untreated females 

over a period of 25 days. Index = (females pregnant/females exposed to males) × 100.

d

Beginning at 6 weeks of age, each F1 female mouse (20–55/group) was cohabitated with an untreated 

male for a period of 6 months.

SEM = standard error of the mean

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 the 

5 weight of testes from the control animals (no F2 offspring were produced in the high-dose group). 

6 This was confirmed by histopathologic observation of atrophic seminiferous tubules in the 

7 40 mg/kg-day group that were smaller than those of controls and were empty except for a basal 

8 layer of cells. The number of interstitial cells in the testes was also increased in this group. Males 

9 from the 10 mg/kg-day group showed limited testicular damage; although all exhibited evidence of 

10 tubular injury, each animal had some seminiferous tubules that displayed active spermatogenesis. 

11 Ovarian tissue was absent or reduced in F1 females such that organ weights were not possible to 

12 obtain. Examination of available tissue in these females revealed hypoplastic ovaries with few 

13 follicles and corpora lutea (10 mg/kg-day) or with no evidence of folliculogenesis (40 mg/kg-day). 

14 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 (<5%) 

16 at PND 42 of F1 offspring of dams treated with 10, 40, or 160 mg/kg-day benzo[a]pyrene, marked 

17 decreases in the reproductive capacity (as measured by fertility index) of both male and female F1 

18 offspring exposed at all three treatment levels of benzo[a]pyrene (by approximately 30% in males 

19 and females), decreased litter size (by about 20%) in offspring of F1 dams, and the dramatic 

20 decrease in size and alteration in anatomy of the gonads of both male and female F1 mice exposed 

21 to 10 and 40 mg/kg-day benzo[a]pyrene in utero. A NOAEL was not identified.

22 In another reproductive and developmental toxicity study, benzo[a]pyrene was 

23 administered by gavage in corn oil to nine female NMRI mice at a dose of 10 mg/kg-day on GDs 7–

24 16; a group of nine controls received corn oil (Kristensen et al., 1995). Body weights were 

25 monitored. F0 females were kept with their offspring until after weaning (21 days after delivery). 

26 At 6 weeks of age, one F1 female from each litter (n = 9) was caged with an untreated male. The 

27 F2 offspring were inspected for gross deformities at birth, weight and sex were recorded 2 days 

28 after birth, and the pups were sacrificed. The F1 females were sacrificed after 6 months of 

29 continuous breeding. The effects of benzo[a]pyrene treatment on fertility, ovary weights, follicles, 

30 and corpora lutea were evaluated. F0 females showed no signs of general toxicity, and there was no 
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1 effect on their fertility. F1 females had statistically significantly lower median numbers of offspring, 

2 number of litters, and litter sizes and a statistically significantly greater median number of days 

3 between litters as compared with the controls (Table B-27). At necropsy, the F1 females from 

4 treated F0 females had statistically significantly reduced ovary weights; histologic examination of 

5 the ovaries revealed decreased numbers of small, medium, or large follicles and corpora lutea 

6 (Table B-27). Only one dose group was used in this study, with decreased F1 female fertility 

7 observed following in utero exposure at the LOAEL of 10 mg/kg-day; no NOAEL was identified.

8 Table B-27. Effect of prenatal exposure to benzo[a]pyrene on indices of 

9 reproductive performance in F1 female NMRI mice

Endpoint (median with range in parentheses) Control

a

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-day by gavage 

in corn oil on GDs 7–16. One F1 female from each litter was continuously bred with an untreated male 

for 6 months.

b

Significantly (p < 0.05) different from control group by Wilcoxon rank sum test or Kruskall-Wallis twotailed test.

Source: Kristensen et al. (1995).

10

11 Chen et al., (2012) treated male and female neonatal Sprague-Dawley rats (10/sex/group) 

12 with benzo[a]pyrene (unspecified purity) dissolved in peanut oil by gavage daily from post-natal 

13 day (PND) 5 – 11, at doses of 0.02, 0.2 or 2 mg/kg in 3 mL vehicle/kg b.w., determined individually 

14 based upon daily measurements. This time period was described as representing the brain growth 

15 spurt in rodents, analogous to brain developmental occurring from the third trimester to 2 years of 

16 age in human infants. Breeding was performed by pairs of nine week old rats, with delivery 

17 designated as PND0. Litters were culled to 8 pups/dam (4/ea male and female, when possible) and 

18 randomly redistributed at PND1 among the nursing dams; dams themselves were rotated every 2-3 

19 days to control for caretaking differences, and cage-side observations of maternal behavior were 

20 made daily. One male and female from each litter were assigned per treatment group, and the 

21 following physical maturation landmarks were assessed daily in all treatment groups until weaning 

22 at PND21: incisor eruption, eye opening, development of fur, testis decent and vaginal opening.
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1 Neonatal sensory and motor developmental tests were administered to pups during the 

2 preweaning period at PNDs 12, 14, 16 and 18, and were behavioral tests administered to rats as 

3 adolescents (PND 35, 36) or as adults (PND 70, 71): each rat was only tested during one 

4 developmental period. All dosing was performed from 1300 – 1600 hrs, and behavioral testing was 

5 during the “dark” period from 1900 – 2300 hrs, although tests were performed in a lighted 

6 environment. Pups were observed individually and weighed daily, the order of testing litters was 

7 randomized each day, and all observations were recored by investigators blinded to group 

8 treatment. 

9 Sensory and motor developmental tests including the surface righting reflex test, negative 

10 geotaxis test, and cliff aversion test were performed only once, while the forelimb grip strength test 

11 was assessed during three 60 second trials on PND12. Rat movements during the open-field test 

12 were recorded by camera, and two blinded investigators scored movement and rearing separately 

13 during a 5 min. evaluation period. Blinded investigators directly observed video monitoring of rat 

14 movements during the elevated plus maze, and after a 5 min. free exploration period, recored 

15 number of entries into the closed and open arms, the time spent in the open arms, and latency to 

16 the first arm entry. Assessment of the Morris water maze was slightly different, in that the rats 

17 were habituated to the testing pool by a 60 second swim without a platform on the day prior to 

18 testing. The rats were then tested during a 60 second swim with a hidden platform present at a 

19 constant position each day for four days; on the fifth day, the rats were evaluated during a 60 

20 second probe swim without a platform. The number of times each animal crossed the original 

21 platform location and the duration of time spent in the platform quadrant were recorded during 

22 this final evaluation. One pup/sex/litter were assigned for behavioral testing to each of four tracks: 

23 Track 1, surface righting reflex test, cliff aversion test, and open-field test (PND 12 – 18); Track 2, 

24 negative geotaxis test, forelimb grip strength test, and open-field test (PND 12 – 20); Track 3, 

25 elevated plus maze, Morris water maze, and open-field test (PND 34 – 36); Track 4, elevated plus 

26 maze, Morris water maze, and open-field test (PND 69 – 71). All results were presented in 

27 graphical form only.

28 No significant effects on pup body weight were observed during the 7-day treatment period 

29 (PND 5 – 11). Three-way ANOVA (time x B[a]P treatment x sex) indicated that effects of B[a]P were 

30 not sex-dependent throughout the 71 day experiment, so both sexes were pooled together. From 

31 this pooled analysis, pups in the 2 mg/kg treatment group gained significantly less weight at both 

32 PND36 and PND71. There were no differences among treatment groups in incisor eruption, eye 

33 opening, development of fur, testis decent or vaginal opening.

34 For all measurements of neonatal sensory and motor development, results from both sexes 

35 were analyzed together since B[a]P was reported to have no significant interaction with sex by 3-

36 way ANOVA. No significant differences were observed in either the cliff aversion or forelimb grip 

37 strength tests. In the surface righting reflex test, latency was increased in the 0.2 mg/kg group at 

38 PND12, in the 0.02 and 2 mg/kg groups at PND14, in only the high dose 2 mg/kg group at PND16, 
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1 and was not significantly different in any group at PND18. At PND12 there was a dose-related 

2 increase in negative geotaxis latency associated with 0.02, 2 and 2 mg/kg B[a]P, which was also 

3 present in the 2 mg/kg group at PND14, but returned to control levels at PND16 and PND18. In the 

4 open field test, there were no significant differences in either locomotion or rearing activity at 

5 PND18 or 20. At PND34, the 2 mg/kg group exhibited significantly increased movement, but 

6 increases in rearing were not significant. At PND69, increased locomotion was observed in both the 

7 0.2 and 2 mg/kg groups, while rearing was significantly increased in only the 2 mg/kg treatment 

8 group.

9 The elevated plus maze performance was only evaluated in adolescent and adult rats. 

10 Unlike the previous tests, 3-way ANOVA revealed a statistically significant interaction between 

11 neonatal B[a]P treatment and sex, so male and female performance was analyzed independently. 

12 No significant differences in PND35 males were observed, and the only significant observation in 

13 PND35 females was increased time spent in the open maze arms by 2 mg/kg treatment group. 

14 Significantly decreased latency time to first open arm entry was observed in PND70 males and 

15 females in both 0.2 and 2 mg/kg treatment groups; these groups also spent significantly more time 

16 in open maze arms, along with the 0.02 mg/kg female group. PND70 2 mg/kg males, along with 0.2 

17 and 2 mg/kg females, entered more frequently into open arms and less frequently into closed arms 

18 than vehicle controls. In the Morris water maze, escape latency (time to reach the platform during 

19 each of the four testing days) was consistently increased in the 2 mg/kg treatment group of both 

20 sexes, in both adolescent and adult animals. These increases were statistically significant in both 

21 males and females treated with 2 mg/kg B[a]P at both PND39 and PND74, and were also 

22 significantly elevated in 0.2 mg/kg animals of both sexes at PND74. Likewise, performance during 

23 the fifth test day, in the absence of the escape platform, was significantly adversely affected by both 

24 metrics (decreased time spent in the target quadrant and decreased number of attempts to cross 

25 the platform location) in 2 mg/kg rats of both sexes at both PND40 and PND75. PND75 females 

26 treated with 0.2 mg/kg B[a]P also showed significant decreases in both performance metrics, while 

27 PND75 0.2 mg/kg males only demonstrated significant differences in “time spent in target 

28 quadrant”. Swim speed was also assessed, but there were no differences among any treatment 

29 group at either age evaluated.

30 Jules et al., (2009) treated pregnant Long Evans Hooded (LEH) rats with benzo[a]pyrene 

31 (unspecified purity) dissolved in 0.875 mL peanut oil by gavage daily from GD14 – GD17, at doses of 

32 150, 300, 600 and 1,200 μg B[a]P /kg b.w., with animals weighed daily. Cage-side observations 

33 were performed until pup weaning, and litter size evaluated for each treatment group. Pups from 4 

34 – 5 individual litters were analyzed for each endpoint, which was independently repeated for a total 

35 of 3 replicates. Delivery was designated PND0, and pups were harvested from PND0 – 15 for B[a]P 

36 metabolite identification, or for other endpoints as young adults at PND53. Systolic/diastolic blood 

37 pressure and heart rate was recorded by a volume pressure recording sensor and occlusion tail-cuff 

38 applied to conscious, non-anesthetized animals. Animals were preconditioned to the restraint 
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1 device and tail-cuff by daily acclimatization sessions during PND46 – 50, to minimize stress effects 

2 during data collection. Cardiac function values were averaged from 15 readings each collected over 

3 a 1 minute interval every other minute for 30 minutes on PND53. Whole blood was collected from 

4 the heart and aorta prior to surgical resection and tissue processing. Plasma and heart tissue B[a]P 

5 metabolite content was quantified by reverse-phase HPLC with UV and fluorescence detection, 

6 while heart and aortic tissue was subjected to SDS-PAGE for qualitative protein analysis, and RNA 

7 extraction. Quantitative RT-PCR was performed for levels of angiotensin II (AngII), neuronal NOS 

8 (nNOS), endothelial NOS (eNOS) and 7,8-Dihydrobiopterin oxidoreductase (BH4/BH2 

9 oxidoreductase). Total RNA was also used to probe a cDNA microarray, and targets with ≥ 2-fold 

10 changes in expression were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and 

11 Gene Ontology (GO) biological process pathway analysis. 

12 No significant differences in litter size or pup weight gain from PND0 – 15 were reported in 

13 any treatment group, and no convulsions, tremors or abnormal movements were reproducibly 

14 observed. Most analytical data was reported graphically, as mean ± SEM of three replicates of 3 – 5 

15 offspring measured/group. Plasma and heart tissue total B[a]P metabolite levels were maximal at 

16 PND0 (the first time point sampled) and progressively decreased from PND0 – 13. Compared to the 

17 low-dose group (150 μg/kg), plasma metabolite levels were significantly elevated in the 600 and 

18 1,200 μg/kg B[a]P groups through PND13, while heart metabolite levels were significantly 

19 increased through PND11. Metabolites in mid-dose group, 300 μg/kg, trended between the 150 

20 and 600 μg/kg group levels from PND0 – 7, while not achieving statistically significant differences 

21 in pair-wise comparisons. Three principle groups of B[a]P metabolites were identified. More than 

22 70% of the total heart metabolite burden was composed of diol metabolites through PND13, while 

23 the more reactive hydroxyl metabolites increased in relative composition from PND9 – 13, and the 

24 dione population remained constant at ≤ 5%. 

25 Cardiovascular function was evaluated in pups exposed in utero to 600 or 1,200 μg/kg B[a]P 

26 vs. controls. A dose-related and statistically significant increase in both systolic (20, 50%) and 

27 diastolic pressure (30, 80%) was observed in mid and high-dose pups, respectively. Heart rate was 

28 also significantly altered; a 10% increased heart rate was reported in the 600 μg/kg B[a]P group, 

29 while the average heart rate of the 1,200 μg/kg B[a]P groups decreased 8%. Cardiac tissue eNOS 

30 protein levels fluctuated as a result of B[a]P treatment; in both the 600 and 1,200 μg/kg groups, 

31 eNOS expression by semi-quantitative SDS-PAGE was significantly decreased at PND0 and PND5, 

32 while it was significantly elevated above controls at PND10 and PND15. While eNOS expression in 

33 the 600 μg/kg B[a]P group had returned to control levels by PND53, eNOS expression was 

34 significantly higher (approximately 2-fold) in the 1,200 μg/kg group. Compared to vehicle-treated 

35 controls, cardiac message levels of nNOS and eNOS were not significantly affected by B[a]P 

36 treatment at PND0, and while nNOS mRNA levels were 2-fold higher at PND53 in the 600 μg/kg 

37 group, and eNOS mRNA was 3-fold higher in the 1,200 μg/kg group, consistent with the increased 

38 eNOS protein levels detected at PND53. Message levels of BH4/BH2 oxidoreductase were 
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1 suppressed in both B[a]P treatment groups at PND0, and while mRNA expression remained 

2 suppressed at PND53 in the 600 μg/kg group, BH4/BH2 message returned to control levels in the 

3 1,200 μg/kg group. Angiotensin II mRNA levels were 1.8-fold higher in both B[a]P groups at PND0, 

4 and while expression increased to 5-fold more than controls at PND53 in the 600 μg/kg group, 

5 AngII expression remained closer to 1.5-fold greater in the high-dose group. The following 

6 pathways were identified as being enriched by 1,200 μg/kg B[a]P treatment in utero using KEGG 

7 analysis, and correcting for multiple comparisons using the false-discovery rate method: PPARγ, 

8 renin–angiotensin system (AngII, adiponectin C1Q and collagen domain, adrenergicβ3R, tachykinin 

9 R1), hematopoietic cell lineage, CYP450 metabolism (CYP2a2, CYP7a1 and CYP2b12), retinol 

10 metabolism, cell adhesion molecules-CAMs, primary bile acid biosynthesis and tight junctions.

11 Table B-28. Exposure-related effects in Long Evans Hooded rats 

12 exposed to benzo[a]pyrene by gavage daily in utero from GD14 – GD17

Effect measured

Dose (mg/kg-d)

0 0.600 1.20

Heart rate (bmp; mean ± SEM) 504.6 ± 15.7 554.6 ± 26.2* 466.3 ± 16.9*

Blood pressure measured by tail cuff (mmHg; mean ± SEM)

Systolic pressure

Diastolic pressure

131.6 ± 1.2

85.0 ± 4.2

151.6 ± 45*

113.0 ± 3.3*

200.4 ± 2.4*

155.6 ± 3.2*

*Significantly (p < 0.05) different from control mean; n = 4-5/replicate, 3 replicates performed.

Source: Jules et al. (2012).

13

14 Bouayed et al., (2009) treated nursing female Swiss Albino OF1 mice (5/dose group) with 

15 benzo[a]pyrene (unspecified purity) dissolved in avocado oil by gavage daily while nursing pups 

16 from PND1 – 14 at 0, 2 or 20mg/kg-day in 10 mL/kg b.w., individually determined each day. Prior 

17 to benzo[a]pyrene treatment, Swiss Albino litters were culled to 10 pups (5/sex when possible), 

18 and nurturing females assigned to litters that were stratified randomly to achieve equivalent mean 

19 pup litter body weights across the designated treatment groups. As the effects of B[a]P on maternal 

20 nurturing behavior was unknown, dam behavior was visually monitored daily until weaning. 

21 Furthermore, maternal nurturing performance from PND0 – 21 was assessed by two methods: a 

22 nest-building test administered q.2.d., where nest quality/complexity was scored 15 minutes after 

23 cotton material was supplied; and pup retrieval, in which latency to return the displaced pup to the 

24 nest was measured twice and averaged, was evaluated q.d. At the indicated times 2 mice/sex/litter 

25 were randomly selected, weighed, and brains resected for later mRNA expression analysis (n = 

26 20/group). 

27 Pup neuromotor maturation and behavior was assessed during pre-weaning by four 

28 standard methods (administered between 1000 – 1300 on testing days, and in temporal order as 

29 indicated): 1) righting reflex test, maximum duration 120 seconds, administered on PNDs 3, 5, 7 and 
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1 9; 2) negative geotaxis test, maximum duration 120 seconds, administered on PNDs 5, 7, 9 and 11; 

2 3) forelimb grip test, duration until failure, administered on PNDs 9 and 11; and 4) open field test, 6 

3 minute evaluation of locomotor activity and rearing following a 1 minute habituation period, 

4 administered on PND15. Adolescent function was evaluated by three methods: water escape pole 

5 climbing (WESPOC) test, administered at PND20, in which the time to find the pole, time to climb the 

6 pole, and the time to reach the safety platform were reported; elevated plus maze, administered at 

7 PND32 for 5 minutes, in which the latency time to first open arm entry, number of entries into open 

8 arms, total number of entries, percent of time spent in open arms, and percent of entries into open 

9 arms was determined; and Y-maze spontaneous alternation test, administered at PND40 for 5 

10 minutes, in which the % spontaneous alternation was calculated by: [(the number of successful 

11 overlapping triplets)/(total number of arm entries – 2) x 100%].

12 Benzo[a]pyrene treatment did not significantly affect the body weight of nursing mothers 

13 during the 2 week treatment period. Since three-way ANOVA indicated that changes in pup weight 

14 as a result of B[a]P treatment were not sex-dependent, data from male and female pups were 

15 combined. B[a]P treatment of nursing mothers was associated with a 8-9% weight gain in pups 

16 nursing from the 2 mg/kg group, and a 10-12% weight gain in pups from the 20 mg/kg group at 

17 PND12 – 20. While not significantly different from PND26 – 40, pup weight in the 20 mg/kg group 

18 was continuously higher than either the 2 mg/kg group or vehicle-treated controls. There were no 

19 significant differences in pup brain weight or eye opening observed. Likewise, B[a]P treatment of 

20 nursing mothers did not affect nest-building interest or quality, and while not significantly 

21 impacting pup retrieval time, the retrieval latency period was observed to increase with increasing 

22 treatment duration in both B[a]P groups vs. controls.

23 Behavioral test data was reported graphically, as mean ± SEM of n = 20/group. For the pre24 weaning neuromotor developmental tests, B[a]P treatment was found to not depend on sex, and so 

25 data from male and female pups was combined. Pups nursing from mothers administered 2 or 20 

26 mg/kg-day B[a]P had significantly elevated righting reflex times at PND3 – 5, which decreased to 

27 control times at PND7 – 9. Only pups from the 20 mg/kg treatment group demonstrated 

28 significantly increased negative geotaxis latency, which was 2-fold greater than controls at PNDs 5, 

29 7 and 9, but returned to control levels at PND11. Interestingly, mice in the 20 mg/kg group had 

30 increased forelimb grip strength, which was significantly greater than control mice at PND9 and 11, 

31 corresponding to increased body weight in the B[a]P-treated mice vs. controls. Mice in the 2 mg/kg 

32 group also performed better than controls at PND9, but were equivalent at PND11. No treatment or 

33 sex-related effects were reported on locomotion or rearing activity during the open field test. Sex34 dependency on test performance became evident during the analysis of the WESPOC test data: 

35 female pups were not significantly affected using any metric, while males in the 20 mg/kg group 

36 demonstrated a statistically significantly longer pole-grasping latency (3-fold), and took 13-times 

37 longer to escape the pole and board the safety platform, vs. vehicle controls. While performance of 

38 male pups from the 2 mg/kg group was not statistically significantly worse than vehicle controls by 
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1 pair-wise comparison, latency for both pole-grasping and escape in this treatment group 

2 contributed to a significant trend for treatment dose and these effects. In the evaluation of the 

3 elevated plus maze, treatment effects did not appear to depend upon sex, so both male and female 

4 performance was analyzed together. Mice in both B[a]P treatment groups demonstrated 

5 significantly decreased latency time to first entering an open arm (30 – 50%), as well as entered 

6 open arms 2-times more frequently and spent twice as much time there vs. vehicle controls. While 

7 mice in the 2 mg/kg treatment group entered into closed arms 20% less frequently than controls, 

8 mice in the 20 mg/kg group were not significantly different. Likewise, mice nursing from mothers 

9 treated with 2 mg/kg B[a]P performed 15% more spontaneous alternations in the Y-maze 

10 spontaneous alternation test compared to controls, while mice in the high-dose group were not 

11 significantly different. The brains of pups nursing from the 20 mg/kg group expressed 

12 approximately 50% lower levels of 5-hydroxytryptamine (serotonin) 1A (5HT1A), and mu 1-opioid 

13 (MOR1) mRNA, and a trend was observed in the low-dose group as well. No significant changes in 

14 alpha-1D adrenergic (ADRA1D) or gamma-aminobutyric acid A (GABAA) mRNA levels were 

15 detected. 

16 Table B-29. Exposure-related effects in Swiss Albino OF1 mice exposed 

17 as pups to benzo[a]pyrene in breast milk from dams treated by gavage 

18 daily from PND1 – PND14

Effect measured

Dose (mg/kg-d)

0 2 20

Pup body weight (g; mean ± SEM, n = 20)

PND0

PND4

PND8

PND12

PND20

PND26

PND32

PND40

1.70 ± 0.02

3.01 ± 0.08

5.08 ± 0.1

6.57 ± 0.12

12.51 ± 0.24

17.71 ± 0.49

24.47 ± 0.55

30.55 ± 0.94

1.73 ± 0.02

3.08 ± 0.06

5.26 ± 0.09

7.16 ± 0.06***

13.55 ± 0.25**

18.60 ± 0.36

25.59 ± 0.57

30.90 ± 0.93

1.74 ± 0.02

3.16 ± 0.04

5.30 ± 0.08

7.39 ± 0.05***

13.79 ± 0.14***

18.35 ± 0.34

25.38 ± 0.54

31.78 ± 0.97

** p < 0.01, *** p < 0.001 significantly different from control mean

Source: Bouayed et al. (2009).

19 Reproductive effects in adults and repeated oral exposure

20 Rigdon and Neal (1965) conducted a series of experiments to assess the reproductive 

21 effects of orally administered benzo[a]pyrene to Ah-responsive white Swiss mice. Female animals 

22 (number not stated) were administered benzo[a]pyrene at 250, 500, or 1,000 ppm in the feed 

23 before or during a 5-day mating period. Based on the initial body weight, the doses can be 

24 estimated as 32, 56, and 122 mg/kg-day, respectively. No effect on fertility was observed at any 

25 treatment dose, even when animals were fed 1,000 ppm benzo[a]pyrene for 20 days prior to 
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1 mating, but interpretation of this finding was marred by large variability in numbers of pregnant 

2 females and litter sizes for both treated and control mice. In separate experiments, the fertility of 

3 five male mice/group was not affected by exposure to 1,000 ppm in food for up to 30 days prior to 

4 mating with untreated females. Histologic examinations showed that male mice fed 500 ppm 

5 benzo[a]pyrene for 30 days had spermatozoa present in their testes; further details were not 

6 provided. The only treatment-related effect was a lack of weight gain related to feed unpalatability. 

7 While this study suggests that premating exposure of male or female mice to doses up to 

8 122 mg/kg-day for 20 days may not affect fertility, the sample sizes were too small and study 

9 designs were too inconsistent to provide reliable NOAELs and LOAELs for 

10 reproductive/developmental toxicity.

11 In an earlier study (Rigdon and Rennels, 1964), rats (strain not specified) were fed diets 

12 containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and during 

13 gestation. In this study, five of eight treated females mated with untreated males became pregnant, 

14 but only one delivered live young. The treated dam that delivered had two live and two stillborn 

15 pups; one dead pup was grossly malformed. In the remaining treated females, vaginal bleeding was 

16 observed on GDs 23 or 24. In the inverse experimental design, three of six controls mated to 

17 benzo[a]pyrene-treated males became pregnant and delivered live young. Visceral and skeletal 

18 examinations of the pups were not conducted. These studies are insufficiently reported and of 

19 insufficient design (e.g., inadequate numbers of animals for statistical analysis) to provide reliable 

20 NOAELs or LOAELs for reproductive effects from repeated oral exposure to benzo[a]pyrene.

21 Inhalation

22 Reproductive toxicity and in utero exposure via inhalation

23 Archibong et al. (2002) evaluated the effect of exposure to inhaled benzo[a]pyrene on fetal 

24 survival and luteal maintenance in timed-pregnant F344 rats. Prior to exposure on GD 8, 

25 laparotomy was performed to determine the number of implantation sites, and confirmed pregnant 

26 rats were divided into three groups, consisting of rats that had four to six, seven to nine, or more 

27 than nine conceptuses in utero. Rats in these groups were then assigned randomly to the treatment 

28 groups or control groups to ensure a similar distribution of litter sizes. Animals (10/group) were 

29 exposed to benzo[a]pyrene:carbon black aerosols at concentrations of 25, 75, or 100 μg/m3 via 

30 nose-only inhalation, 4 hours/day on GDs 11–20. Control animals were either sham-exposed to 

31 carbon black or remained entirely unexposed. Results of particle size analysis of generated 

32 aerosols were reported by several other reports from this laboratory (Inyang et al., 2003; Ramesh 

33 et al., 2001a; Hood et al., 2000). Aerosols showed a trimodal distribution with averages of 95% 

34 cumulative mass with diameters <15.85 μm; 89% <10 μm; 55% <2.5 μm; and 38% <1 μm (Inyang 

35 et al., 2003). Ramesh et al. (2001a) reported that the (MMAD ± geometric SD) for the 55% mass 

36 fraction with diameters <2.5 μm was 1.7 ± 0.085. Progesterone, estradiol-17β, and prolactin 

37 concentrations were determined in plasma collected on GDs 15 and 17. Fetal survival was 
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1 calculated as the total number of pups divided by the number of all implantation sites determined 

2 on GD 8. Individual pup weights and crown-rump length per litter per treatment were determined 

3 on PND 4 (PND 0 = day of parturition).

4 Archibong et al. (2002) reported that exposure of rats to benzo[a]pyrene caused 

5 biologically and statistically significant (p ≤ 0.05) reductions in fetal survival compared with the 

6 two control groups; fetal survival rates were 78.3, 38.0, and 33.8% per litter at 25, 75, and 

100 μg/m3 7 , respectively, and 96.7% with carbon black or 98.8% per litter in untreated controls (see 

8 Table 4-24). 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/m3 9 , compared with 

10 sham-exposed and unexposed control groups. No effects on hormone levels were observed on 

11 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 12 (14 and 16% decreases at 75 and 100 

μg/m3 13 , respectively, p < 0.05). Exposure to benzo[a]pyrene did not affect crown-rump length (see 

14 Table B-30).

15 Table B-30. Pregnancy outcomes in female F344 rats treated with 

16 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 ± 0.5b4.2 ± 0.1b3.0 ± 0.2b

Survival (litter %) 98.9 ± 1.1 96.7 ± 1.7 78.3 ± 4.1b38.0 ± 2.1b33.8 ± 1.3b

Pup weight (g/litter) 10.6 ± 0.1 8.8 ± 0.1 10.5 ± 0.2 9.1 ± 0.2b8.9 ± 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

a

Values 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).

17

Benzo[a]pyrene exposure at 75 μg/m3 18 caused a statistically significant decrease in plasma 

19 progesterone, estradiol, and prolactin on GD 17; these levels were not determined in the rats 

exposed to 100 μg/m3 20 (Archibong et al., 2002). Plasma prolactin is an indirect measure of the 

21 activity of decidual luteotropin, a prolactin-like hormone whose activity is necessary for luteal 

22 maintenance during pregnancy in rats. Control levels of prolactin increased from GD 15 to 17, but 

this increase did not occur in the rats exposed to 75 μg/m3 23 . Although the progesterone 

24 concentration at 75 μg/m3 was significantly lower than in controls on GD 17, the authors thought 

25 that the circulating levels should have been sufficient to maintain pregnancy; thus, the increased 
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1 loss of fetuses was thought to be caused by the lower prolactin levels rather than progesterone 

2 deficiency. The reduced circulating levels of progesterone and estradiol-17β among 

3 benzo[a]pyrene-treated rats were thought to be a result of limited decidual luteotropic support for 

4 the corpora lutea. The authors proposed the following mechanism for the effects of benzo[a]pyrene 

5 on fertility: benzo[a]pyrene or its metabolites decreased prolactin and decidual luteotropin levels, 

6 compromising the luteotropic support for the corpora lutea and thereby decreasing the plasma 

7 levels of progesterone and estradiol-17β. The low estradiol-17β may decrease uterine levels of 

8 progesterone receptors, thereby resulting in fetal mortality. Based on biologically and statistically 

significant decreases in pups/litter and percent fetal survival/per litter, the LOAEL was 25 μg/m3 9 ; 

10 no NOAEL was identified.

11 Neurotoxicity and in utero exposure via inhalation

12 To evaluate the effects of benzo[a]pyrene on the developing CNS, Wormley et al. (2004) 

13 exposed timed-pregnant F344 rats (10/group) to benzo[a]pyrene:carbon black aerosols by noseonly inhalation on GDs 11–21 for 4 hours/day at a concentration of 100 μg/m3 14 . Results of particle 

15 size analysis of genenerated aerosols were reported by other reports from this laboratory (Ramesh 

16 et al., 2001a; Hood et al., 2000). Particle size analysis of a 100-μg/m3 aerosol showed a trimodal 

17 distribution with averages of 95% cumulative mass with diameters <15.85 μm; 90% <10 μm; 

18 67.5% <2.5 μm; and 66.2% <1 μm; the MMAD ± geometric SD for the latter fraction was 0.4 ± 0.02 

19 μm (Hood et al., 2000). Dams were maintained to term and pups were weaned on PND 30. 

20 Benzo[a]pyrene reduced the number of live pups to one-third of control values without affecting 

21 the number of implantation sites. During PNDs 60–70, electrical stimulation and evoked field 

22 potential responses were recorded via electrodes implanted into the brains of the animals. Direct 

23 stimulation of perforant paths in the entorhinal region revealed a diminution in long-term 

24 potentiation of population spikes across the perforant path-granular cell synapses in the dentate 

25 gyrus of the hippocampus of F1 generation benzo[a]pyrene-exposed animals; responses in exposed 

26 offspring were about 25% weaker than in control offspring. Additionally, NMDA receptor subunit 1 

27 protein (important for synaptic functioning) was down-regulated in the hippocampus of 

28 benzo[a]pyrene exposed F1 pups. The authors interpreted their results as suggesting that 

29 gestational exposure to benzo[a]pyrene inhalation attenuates the capacity for long-term 

30 potentiation (a cellular correlate of learning and memory) in the F1 generation.

31 In another study by this same group of investigators, Wu et al. (2003) evaluated the 

32 generation of benzo[a]pyrene metabolites in F1 generation pups, as well as the developmental 

33 profile for AhR and mRNA. In this study, confirmed pregnant F344 rats were exposed to 

34 benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 μg/m3 via nose-only inhalation, 

35 4 hours/day, for 10 days (GDs 11–21). Control animals were exposed to carbon black (sham) to 

36 control for inert carrier effects or they remained untreated. Each benzo[a]pyrene concentration 

37 had its own set of controls (carbon black and untreated). Two randomly selected pups were 

38 sacrificed on each of PND 0, 3, 5, 10, 15, 20, and 30. Body, brain, and liver weights were recorded. 
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1 Benzo[a]pyrene metabolites were analyzed in the cerebral cortex, hippocampus, liver, and plasma. 

2 A dose-related increase in plasma and cortex benzo[a]pyrene metabolite concentrations in pups 

3 was observed. Dihydrodiols (4,5-; 7,8-; 9,10-) dominated the metabolite distribution profile up to 

4 PND 15 and the hydroxy (3-OH-benzo[a]pyrene; 9-OH-benzo[a]pyrene) metabolites after PND 15 

at 100 μg/m3 5 (the only exposure concentration reported). Results indicated a dose-related 

6 decrease 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/m3 7 , was 

8 statistically significantly increased compared with controls.

9 Adult male reproductive effects and repeated inhalation exposure

10 Inyang et al. (2003) evaluated the effect of subacute exposure to inhaled benzo[a]pyrene on 

11 testicular steroidogenesis and epididymal function in rats. Male F344 rats (10/group), 13 weeks of 

12 age, were exposed to benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 μg/m3 via nose-only 

13 inhalation, 4 hours/day for 10 days. Control animals were either exposed to carbon black (sham) to 

14 control for exposure to the inert carrier, or they remained untreated. Each benzo[a]pyrene 

15 concentration had its own set of controls (carbon black and untreated). Aerosols showed a 

16 trimodal distribution with averages of 95% cumulative mass <15.85 μm; 89% <10 μm; 55% <2.5 

17 μm; and 38% <1 μm (Inyang et al., 2003); an earlier report from this laboratory indicated that the 

18 55% mass fraction had a MMAD ± geometric SD of 1.7 ± 0.085 (Ramesh et al., 2001a). Blood 

19 samples were collected at 0, 24, 48, and 72 hours after cessation of exposure to assess the effect of 

20 benzo[a]pyrene on systemic concentrations of testosterone and luteinizing hormone (LH), 

21 hormones that regulate testosterone synthesis. Reproductive endpoints such as testis weight and 

22 motility and density of stored (epididymal) sperm were evaluated.

23 Regardless of the exposure concentration, inhaled benzo[a]pyrene did not affect testis 

24 weight or the density of stored sperm compared with controls. However, inhaled benzo[a]pyrene 

25 caused a concentration-dependent reduction in the progressive motility of stored sperm. 

Progressive motility was similar at 75 and 100 μg/m3 26 , but these values were significantly lower (p < 

27 0.05) than in any other group. The reduction in sperm motility postcessation of exposure was 

28 thought to be the result of benzo[a]pyrene limiting epididymal function. Benzo[a]pyrene exposure 

to 75 μg/m3 29 caused a decrease in circulating concentrations of testosterone compared with controls 

30 from the time of cessation of exposure (time 0) to 48 hours posttermination of exposure (p < 0.05). 

31 However, the decrease was followed by a compensatory increase in testosterone concentration at 

72 hours postcessation of exposure. Exposure to 75 μg/m3 32 caused a nonsignificant increase in 

33 plasma LH concentrations at the end of exposure compared with controls, which increased further 

34 and turned significant (p < 0.05) for the remaining time of the study period. The decreased plasma 

35 concentration of testosterone, accompanied by an increased plasma LH level, was thought to 

36 indicate that benzo[a]pyrene did not have a direct effect on LH. The authors also noted that the 

37 decreased circulating testosterone may have been secondary to induction of liver CYP450 enzymes 

38 by benzo[a]pyrene. The authors concluded that subacute exposure to benzo[a]pyrene contributed 
Toxicological Review of benzo[a]pyrene

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

B-87 DRAFT—DO NOT CITE OR QUOTE

1 to impaired testicular endocrine function that ultimately impaired epididymal function. Based on 

this study, the NOAEL was 25 μg/m3 and the LOAEL was 75 μg/m3 2 , based on a statistically 

3 significant reduction in the progressive motility of stored sperm and impairment of testicular 

function with 10 days of exposure at 75 μg/m3 4 .

5 In a follow-up study with longer exposure duration, adult male F344 rats (10 per group) 

6 were exposed to benzo[a]pyrene:carbon black aerosols at 75 μg/m3 via nose-only inhalation, 

7 4 hours/day for 60 days (Archibong et al., 2008; Ramesh et al., 2008). Rats in the control group 

8 were subjected to the nose-only restraint, but were not exposed to the carbon black carrier. Blood 

9 samples were collected at 0, 24, 48, and 72 hours after exposure terminated, and the animals 

10 sacrificed for tissue analyses following the last blood sampling. Data were analyzed statistically for 

11 benzo[a]pyrene effects on weekly body weights, total plasma testosterone and LH concentrations, 

12 testis weights, density of stored spermatozoa, sperm morphological forms and motility, 

13 benzo[a]pyrene metabolite concentrations and AHH activity, and morphometric assessments of 

14 testicular histologies. Relative to controls, the results indicated 34% reduced testis weight (p < 

15 0.025), reduced daily sperm production (p < 0.025) and reduced intratesticular testosterone 

16 concentrations (p < 0.025). Plasma testosterone concentrations were reduced to about one-third of 

17 the level in controls on the last day of exposure (day 60) and at 24, 48, and 72 hours later (p < 0.05). 

18 However, plasma LH concentrations in benzo[a]pyrene exposed rats were elevated throughout the 

19 blood sampling time periods compared with controls (p < 0.05). In testis, lung, and liver, AHH 

20 activity, and benzo[a]pyrene-7,8-dihydrodiol (precursor to the DNA-reactive BPDE) and 

21 benzo[a]pyrene-3,6-dione metabolites were significantly (p < 0.05) elevated relative to controls. 

22 Progressive motility and mean density of stored spermatozoa were significantly reduced (p < 0.05). 

23 Weekly body weight gains were not affected by benzo[a]pyrene exposure. These results indicate 

24 that 60-day exposure of adult male rats to benzo[a]pyrene:carbon black aerosols at 75 μg/m3

25 produced decreased testis weight; decreased intratesticular and plasma testosterone 

26 concentrations; and decreased sperm production, motility, and density.
Toxicological Review of benzo[a]pyrene

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

B-88 DRAFT—DO NOT CITE OR QUOTE

1 OTHER PERTINENT TOXICITY INFORMATION

2 Table B-31. In vitro genotoxicity studies of benzo[a]pyrene in non3 mammalian cells

Result

Reference

+

S9

–

S9

Endpoint/test system: prokaryotic cells

Forward mutation

Salmonella typhimurium TM677 + – Rastetter et al., 1982

S. typhimurium TM677 + ND Babson et al., 1986

Reverse mutation

S. typhimurium TA98, TA1538 + ND Ames et al., 1975

S. typhimurium TA98, TA100, TA1538 + ND McCann et al., 1975

S. typhimurium TA1538, TA98 + – Wood et al., 1976

S. typhimurium TA98, TA100, TA1537 + – Epler et al., 1977

S. typhimurium TA98, TA100 + –

Obermeier and Frohberg, 

1977

S. typhimurium TA98 + – Pitts et al., 1978

S. typhimurium TA98, TA100 + ND LaVoie et al., 1979

S. typhimurium TA98, TA100 + – Simmon, 1979a

S. typhimurium TA98 + ND Hermann, 1981

S. typhimurium TA98, TA100 + ND Alfheim and Randahl, 1984

S. typhimurium TA98, TA100, TA1538 ND – Glatt et al., 1985

S. typhimurium TA97, TA98, TA100 + – Sakai et al., 1985

S. typhimurium TA97, TA98, TA100, TA1537 + – Glatt et al., 1987

S. typhimurium TA97, TA98, TA100 + ND Marino, 1987

S. typhimurium TA98 + – Alzieu et al., 1987

S. typhimurium TA98, TA100 + – Prasanna et al., 1987

S. typhimurium TA98 + ND Ampy et al., 1988

S. typhimurium TA98, TA100 + ND Bos et al., 1988

S. typhimurium TA98 + ND Lee and Lin, 1988

S. typhimurium TA98 + ND Antignac et al., 1990

S. typhimurium TA98 – ND Gao et al., 1991

S. typhimurium TA98 + ND Balansky et al., 1994

S. typhimurium TA100 + ND Norpoth et al., 1984

S. typhimurium TA100 + – Carver et al., 1986

S. typhimurium TA100 + ND Pahlman and Pelkonen, 1987
Toxicological Review of benzo[a]pyrene

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

B-89 DRAFT—DO NOT CITE OR QUOTE

Result

Reference

+

S9

–

S9

S. typhimurium TA100 + ND Tang and Friedman, 1977

S. typhimurium TA100 + ND Bruce and Heddle, 1979

S. typhimurium TA100 + ND Phillipson and Ioannides, 1989

S. typhimurium TA100 – ND Balansky et al., 1994

S. typhimurium TA1537, TA1538 + – Ames et al., 1973

S. typhimurium TA1537, TA1538 + – Glatt et al., 1975

S. typhimurium TA1537 + ND Oesch et al., 1976

S. typhimurium TA1538 + ND Egert and Greim, 1976

S. typhimurium TA1538 + – Rosenkranz and Poirier, 1979

S. typhimurium TA1535 – – Ames et al., 1973

S. typhimurium TA 1535 – – Glatt et al., 1975

S. typhimurium TA 1535 – ND McCann et al., 1975

S. typhimurium TA1535 – – Epler et al., 1977

DNA damage

E. coli/pol A + – Rosenkranz and Poirier, 1979

E. coli/differential killing test + – Tweats, 1981

E. coli WP2-WP100/rec-assay + ND Mamber et al., 1983

E. coli/SOS chromotest Pq37 + –

Mersch-Sundermann et al., 

1992

Endpoint/test system: nonmammalian eukaryotes

Mitotic recombination

S. cerevisiae D4-RDII ND – Siebert et al., 1981

S. cerevisiae D3 – – Simmon, 1979b

+ = positive; – = negative; ND = not determined

1 Table B-32. In vitro genotoxicity studies of benzo[a]pyrene in 

2 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
Toxicological Review of benzo[a]pyrene

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B-90 DRAFT—DO NOT CITE OR QUOTE

Assay/test system

Result

+S9 – S9 Reference

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

Human HSC172 lung fibroblasts + – Gupta and Goldstein, 1981

Human Q3-wp normal lung keratinocytes + ND Allen-Hofmann and 

Rheinwald, 1984

Human SCC-13Y lung keratinocytes ND + Allen-Hofmann and 

Rheinwald, 1984

Mouse lacZ transgenic MutaTMMouse 

primary hepatocytes

ND + Chen et al., 2010

Mouse L5178Y/HGPRT + – Clive et al., 1979

Mouse lymphoma (L5178Y/TK+/-) cells + – Clive et al., 1979

Mouse lymphoma (L5178Y/TK+/-) cells + ND Amacher and Turner, 1980; 

Amacher et al., 1980

Mouse lymphoma (L5178Y/TK+/-) cells + – Amacher and Paillet, 1983

Mouse lymphoma (L5178Y/TK+/-) cells + ND Arce et al., 1987

Chinese hamster ovary (CHO) cells (aprt) + ND Yang et al., 1999

CHO 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

Human peripheral blood lymphocytes ND + Wiencke et al., 1990

Human peripheral blood lymphocytes ND + Li et al., 2001

Human peripheral blood lymphocytes ND + Wu et al., 2005

Human peripheral blood lymphocytes ND + Gu et al., 2008

Human fibroblast (MRC5CV1) cell line + ND Hanelt et al., 1997

Human hepatoma (HepG2) cell line ND + Tarantini et al., 2009

Hamster tracheal cells ND + Roggeband et al., 1994

Chinese hamster V79 lung epithelial cells + ND Arce et al., 1987
Toxicological Review of benzo[a]pyrene

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

B-91 DRAFT—DO NOT CITE OR QUOTE

Assay/test system

Result

+S9 – S9 Reference

Virus transformed SHE 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., 1994

Unscheduled DNA synthesis

HeLa cells + ND Martin et al., 1978

Human fibroblasts + ND Agrelo and Amos, 1981

Human fibroblasts + – Robinson and Mitchell, 

1981

Human HepG2 ND + Valentin-Severin et al., 2004

Hamster primary embryo cells ND + Casto et al., 1976

Hamster tracheal cells ND + Roggeband et al., 1994

Rat hepatocytes ND + Michalopoulos et al., 1978

Rat tracheal cells ND – Roggeband et al., 1994

DNA repair

Human mammary epithelial cells ND + Leadon et al., 1988

Human skin fibroblasts ND + Milo et al., 1978

Baby hamster kidney (BHK21/c13) cells ND + Feldman et al., 1978

secondary mouse embryo fibroblasts 

(C57BL/6) and human lymphocytes

ND + Shinohara and Cerutti, 1977

Rat/F344 hepatocytes ND + Williams et al., 1982

Cytogenetic damage

CAs

Human blood cells ND + Salama et al., 2001

Human WI38 fibroblasts + – Weinstein et al., 1977

Chinese hamster lung cells + – 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

MN

Human AHH-1 lymphoblastoid cells ND + Crofton-Sleigh et al., 1993

Human HepG2 liver cells ND + Wu et al., 2003

Human lymphoblastoid (TK) cells ND + Fowler et al., 2010

Human MCL-5 lymphoblastoid cells ND + Crofton-Sleigh et al., 1993

Human peripheral blood lymphocytes + ND Lo Jacono et al., 1992

Chinese hamster V79 cells ND + Whitwell et al., 2010
Toxicological Review of benzo[a]pyrene

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

B-92 DRAFT—DO NOT CITE OR QUOTE

Assay/test system

Result

+S9 – S9 Reference

Chinese hamster V79-MZ cells ND + Matsuoka et al., 1999

DNA strand breaks

Human sperm + + Sipinen et al., 2010

Human peripheral blood lymphocytes + + Rodriguez-Romero et al., 

2012

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, 1988; 

Cosma et al., 1988

Rat lymphocytes ND + Gao et al., 1991

SCEs

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, 

1977

Human peripheral blood lymphocytes ND + Schönwald et al., 1977

Human peripheral blood lymphocytes ND + Wiencke et al., 1990

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., 1981

Chinese hamster V79 lung epithelial cells + ND Arce et al., 1987

Chinese hamster V79 lung epithelial cells ND + Kulka et al., 1993a

CHO cells + – de Raat, 1979

CHO cells + – Husgafvel-Pursiainen et al., 

1986

CHO cells ND + Wolff and Takehisa, 1977
Toxicological Review of benzo[a]pyrene

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

B-93 DRAFT—DO NOT CITE OR QUOTE

Assay/test system

Result

+S9 – S9 Reference

CHO cells ND + Pal et al., 1978

Chinese hamster lung cells ND + Shimizu et al., 1984

Rabbit peripheral blood lymphocytes ND + Takehisa and Wolff, 1978

Rat 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., 1993b

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 ND + van Agen et al., 1997

Human breast epithelial (MCF-10F, MCF-7, 

T24) cell lines

ND + Calaf and Russo, 1993

Baby hamster kidney (BHK21/c13) cells + ND Greb et al., 1980

Golden hamster embryo cells + ND Mager et al., 1977

Syrian hamster embryo (SHE) cells ND + DiPaolo et al., 1971, 1969

SHE cells ND + Dunkel et al., 1981

SHE cells ND + LeBoeuf et al., 1990

SHE cells/focus assay ND + Casto et al., 1977

Fetal Syrian hamster lung (FSHL) cells ND + Emura et al., 1987, 1980

Virus infected rat embryo RLV/RE and RAT 

cells; mouse embryo AKR/Me cells; Syrian 

hamster embryo cells

ND + Heidelberger et al., 1983

Virus transformed SHE and mouse 

C3H10T1/2 cells

ND + Arce et al., 1987

Mouse C3H/10T1/2 embryo fibroblasts ND + Nesnow et al., 2002, 1997

Mouse 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

ND + Heidelberger et al., 1983

Mouse BALB/c-3T3 cells ND + Dunkel et al., 1981

Mouse BALB/c-3T3 cells ND + Matthews, 1993

Mouse BALB/c-3T3 clone A31-1-1 ND + Little and Vetroys, 1988
Toxicological Review of benzo[a]pyrene

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

B-94 DRAFT—DO NOT CITE OR QUOTE

Assay/test system

Result

+S9 – S9 Reference

Rat embryo cells/SA7 virus transformation ND + DiPaolo and Casto, 1976

Rat/Fischer, embryo cells (leukemia virus 

transformed)

ND + Dunkel et al., 1981

Rat/Fischer, embryo cells/OuaR ND + Mishra et al., 1978

+ = positive; – = negative; ND = not determined; SHE = Syrian hamster embryo; TK = thymidine kinase

1
Toxicological Review of benzo[a]pyrene

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

B-1 DRAFT—DO NOT CITE OR QUOTE

1

2 Table B-33. In vivo genotoxicity studies of benzo[a]pyrene

Endpoint Test system Test conditions Results Dose Comment Reference

Mutation 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, base-pair 

substitutions, frameshift, and 

deletion mutations observed. 

Smokers and nonsmokers had 

GC→TA transversions (13 and 

6%, respectively) and GC→AT 

transitions (24 and 35%, 

respectively) in hprt gene 

consistent with in vitro 

mutagenicity of 

benzo[a]pyrene

Hackman 

et al., 2000

Mutation, 

germline

Mouse, T-stock, (SEC × 

C57BL)F1, (C3H × 

101)F1, or (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 

d posttreatment with 12-wk-old 

females from different stocks; 

sacrificed on d 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 

experiment; fertilized eggs collected 

from 9 to 11 hrs after mating and firstcleavage metaphase chromosomes 

prepared 20 hrs after mating.

+ 500 mg/kg The percent of dominant lethal 

mutations were in the order of 

T-stock = (C3H × 101)F1 > 

(SEC × C57BL)F1 > 

(C3H × C57BL)F1

Generoso 

et al., 1979
Toxicological Review of benzo[a]pyrene

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

B-2 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

Mutation, 

germline 

Mouse, male stocks: 

(101 × C3H)F1; female 

stocks (A): 

(101 × C3H)F1, (B): 

(C3H × 101)F1, (C): 

(C3H × C57BL)F1, 

(D):(SEC × C57BL)F1, 

(E):T-stock females; 

dominant lethal 

mutations

In dominant lethal assay, 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 d postdosing, or with (#3b) 

with stocks B, C, D, and E females 3.5–

6.5 d postdosing. Control group mated 

at time corresponding to 1.5–4.5 d 

posttreatment in the test groups.

+ 500 mg/kg Dominant lethal effects were 

observed in early to middle 

(4.5–5.5 and 6.5–7.5 d 

posttreatment, respectively) 

spermatozoa and in 

preleptotene spermatocytes 

(32.5–33.5 and 34.5–35.5 d 

post-treatment). 

Generoso 

et al., 1982

Mutation, 

germline 

Mouse, male stocks: 

(101 × C3H)F1; female 

stocks (A): (101 × 

C3H)F1, (B): (C3H × 

101)F1, (C): (C3H × 

C57BL)F1, (D): (SEC × 

C57BL)F1, (E): T-stock 

females; heritable 

translocations

For heritable translocation assay, 

males were mated with stocks B and D 

females 3.5–7.7 d post-benzo[a]pyrene 

treatment and male progeny screened 

for translocation heterozygosity.

– 500 mg/kg No significant differences were 

observed between treated and 

control progeny.

Generoso 

et al., 1982

Mutations 

and BPDEDNA 

adducts, 

germline

Mouse, C57BL/6, cII

transgenic (Big Blue®)

Benzo[a]pyrene administered i.p. in 

corn oil on d 0, 1, and 2; sacrificed at d 

4, 16, 30, 44, or 119. Caput and cauda 

epididymal spermatozoa analyzed for 

cII mutation frequency, and DNA 

adducts analyzed in testis by LCMS/MS SRM with 15N-deoxyguanosine 

labeling.

+ 50 mg/kg Exposed spermatocytes 

acquired persistent BPDE-DNA 

adducts; exposed 

spermatogonia gave rise to 

spermatocytes with mutations 

consistent with a 

benzo[a]pyrene spectrum 

(GC>TA transversions).

Olsen et 

al., 2010
Toxicological Review of benzo[a]pyrene

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

B-3 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

Mutations 

and BPDEDNA 

adducts, 

germline

Mouse, C57BL/6 males, 

wild type and Xpc-/-

with pUR288 lacZ

reporter gene

Benzo[a]pyrene given via gavage in 

sunflower oil 3 times/wk for 1, 4, or 6 

wks (Xpc-/-) or 6 wks (Wt). Spleen, 

testis, and sperm cells analyzed for 

lacZ mutation frequency, and DNA 

adducts analyzed in testis by 32Ppostlabeling.

+ 13 mg/kg Statistically significant 

increases in lacZ mutation 

frequencies in Xpc-/- spleen at 

4 and 6 wks (dose dependent) 

and in Wt spleen and sperm at 

6 wks; DNA adducts were 

statistically significant in testis 

in all exposed groups.

Verhofstad 

et al., 2011

Mutations 

and BPDEDNA 

adducts 

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 d posttreatment; 

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 BPDE-dG adduct levels peaked 

between 5 and 7 days 

posttreatment, followed by 

gradual decline; rate of 

removal highest in lung, liver, 

and spleen and lowest in 

kidney and brain; mutant 

frequencies peaked between 7 

and 14 days in lung, spleen, 

liver, and kidney; brain was not 

significant at any time point.

Boerrigter, 

1999

Mutation Mouse, C57BL female × 

T-strain male; somatic 

mutation assay

Mice mated for a 5-d period; 10.25 d 

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 and recessive spots.

+ 100 or 

500 mg/kg 

Induced coat color mosaics 

represent genetic changes 

(e.g., point mutations) in 

somatic cells. White nearmidline ventral spots and 

recessive spots represent 

melanocyte cell killing and 

mutagenicity, respectively. 

Benzo[a]pyrene caused high 

incidence of recessive spots 

but did not correlate with 

white near-midline ventral 

spots.

Russell, 

1977
Toxicological Review of benzo[a]pyrene

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

B-4 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

Mutation Mouse, lacZ transgenic 

(MutaTMMouse)

Benzo[a]pyrene given via gavage in 

olive oil daily for 28 consecutive d; 

sacrificed 3 d after last dosing; 4 

organs analyzed for lacZ mutation 

frequency.

+ 25, 50, and 75 

mg/kg-day

Highest lacZ mutation 

frequency observed in small 

intestine, followed by bone 

marrow, glandular stomach, 

and liver

Lemieux et 

al., 2011

Mutation Mouse, lacZ transgenic 

(MutaTMMouse)

Benzo[a]pyrene given orally in corn oil 

for 5 consecutive d; sacrificed 14 d 

after last dosing; 11 organs analyzed 

for lacZ mutation frequency.

+ 125 mg/kg-day Highest mutation frequency 

observed in colon followed by 

ileum > forestomach > bone 

marrow = spleen > glandular 

stomach > liver = lung > 

kidney = heart

Hakura et 

al., 1998

Mutation Mouse, C57BL/6J Dlb-1

congenic; Dlb-1 locus 

assay

Animals dosed: (1) i.p. with vehicle or 

benzo[a]pyrene two, four, or six doses 

at 96-hr intervals; or (2) single dose of 

benzo[a]pyrene given i.p. or p.o. alone 

or 96 hrs following a single i.p. dosing 

with 10 μg/kg TCDD.

+ 40 mg/kg Benzo[a]pyrene caused a dosedependent increase in mutant 

frequency; i.p. route showed 

higher mutant frequency than 

p.o. route; induction of 

mutations were associated 

with Ah-responsiveness.

Brooks et 

al., 1999

Mutation Mouse, C57BL/6 (lacZ

negative and XPA+/+

and XPA-/-); hprt 

mutations in T 

lymphocytes

Gavage in corn oil 3 times/wk for 0, 1, 

5, 9, or 13 wks; sacrificed 7 wks after 

last treatment.

+ 13 mg/kg Mutation sensitivity:

XPA-/-> XPA+/+.

Bol et al., 

1998

Mutation Mouse, Cockayne 

syndrome-deficient

(Csb-/-); heterozygous

(Csb+/-) and WT 

controls (Csb+/+); hprt 

mutation frequency 

assay

Csb-/-/lacZ+/-and Csb+/-/lacZ+/- mice 

were dosed i.p. with benzo[a]pyrene 3 

times/wk for 5, 9, or 13 wks; for hprt

mutation frequency analysis mice were 

sacrificed 3 wks after last treatment; 

splenocytes collected; for lacZ

mutation frequency analysis, mice 

were sacrificed 3 d after last treatment 

and liver, lung, and spleen collected.

+ 13 mg/kg lacZ mutation frequency 

detected in all tissues but no 

differences between WT and 

Csb-/- mice; hprt mutations 

significantly higher in Csb-/-

mice than control mice. BPDEdGuo adducts in hprt gene are 

preferentially removed in WT 

mice than Csb-/- mice.

Wijnhoven 

et al., 2000
Toxicological Review of benzo[a]pyrene

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

B-5 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

Mutation Mouse, B6C3F1, 

forestomach H-ras, 

K-ras, and p53

mutations

Benzo[a]pyrene given in feed in a 2-yr 

chronic feeding study.

+ 5, 25, or 

100 ppm

68% K-ras (codons 12,13), 10% 

H-ras (codon 13), 10% p53

mutations; all G→T 

transversions

Culp et al., 

2000

Mutation 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 d 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 significant doseand time-dependent increase 

in mutation frequency; liver 

and lung showed no 

mutations; mutation frequency 

for single- or multiple-dose 

regimens was similar.

Dean et al., 

1998

Mutation 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 × 2 mg)

Davidson 

and 

Dawson, 

1976

Mutation Mouse, 129/Ola (WT); 

hprt mutations in 

splenic T lymphocytes

Single i.p. injection followed by 

sacrifice 7 wks posttreatment.

+ 0, 50, 100, 

200, or 400

mg/kg

Dose-dependent increase in 

hprt mutation frequency.

Bol et al., 

1998

Mutation Mouse, A/J, male Single i.p. injection followed by 

sacrifice 28 days posttreatment.

+ 0, 0.05, 0.5, 5, 

or 50 mg/kg

Dose-dependent increase in 

lung tissue K-ras codon 12 

G→T mutation frequency.

Meng et 

al., 2010

Mutation 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 initiation promoted 

twice weekly with 5 nmol TPA for 14 

wks. One month after stopping TPA 

application, papillomas were collected 

and DNA from 10 individual papillomas 

were analyzed for Ha-ras mutations by 

PCR and direct sequencing.

+ 600 

nmol/mouse

About 90% of papillomas 

contained Ha-ras mutations, all 

of them being transversions at 

codons 12 (20% GGA→GTA), 

13 (50% GGC→GTC), and 61 

(20% CAA→CTA).

Colapietro 

et al., 1993
Toxicological Review of benzo[a]pyrene

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

B-6 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

Mutation Rat, Wistar Single dose by gavage; urine and feces 

collected 0–24, 24–48, and 48–72 hrs 

posttreatment; urine and extracts of 

feces tested in S. typhimurium TA100 

strain with or without S9 mix and βglucuronidase.

+ 0, 1, 5, 10, or 

100 mg/kg

Fecal extracts and urine 

showed mutagenicity ≥1 and 

10 mg/kg body weight 

benzo[a]pyrene, respectively. 

Highest mutagenic activity 

observed for 0–24 hrs 

posttreatment for feces and 

24–48 hrs posttreatment for 

urine with β-glucuronidase ±

S9 mix.

Willems et

al., 1991

BPDEDNA 

adducts

Human, WBCs 96 people occupationally or medically 

exposed to PAH mixtures (psoriatic 

patients, coke oven workers, chimney 

sweeps, and aluminum anode plant 

workers); adducts measured by 

HPLC/fluorescence analysis.

+ Percentages of subjects with 

adduct levels > the 95th

percentile control value were 

47% (7/15), 21% (4/19) and 3% 

(1/34) in coke oven workers, 

chimney sweeps, and controls, 

respectively.

Pavanello 

et al., 1999

BPDEDNA 

adducts

Human, WBCs 67 highly exposed coke oven workers 

were tested for genetic factors that 

can modulate individual responses to 

carcinogenic PAHs; adducts measured 

by HPLC/fluorescence analysis.

+ Levels of BPDE-DNA adducts 

were significantly associated 

with workplace PAH exposure 

(as correlated with urinary 

excretion of 1-pyrenol), lack of 

GSTM1 activity, and low NER 

capacity.

Pavanello 

et al., 2005
Toxicological Review of benzo[a]pyrene

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

B-7 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

BPDEDNA 

adducts

Human, peripheral 

lymphocytes

585 Caucasian municipal workers (52% 

males, 20–62 years old) from 

northeast Italy environmentally 

exposed to PAH mixtures were 

screened for adducts measured by 

HPLC/fluorescence analysis.

+ Forty-two percent of the 

participants had elevated antiBPDE-DNA adduct levels, 

defined as >0.5 adducts/108 

nucleotides (mean, 1.28 ± 2.80 

adducts/108 nucleotides). 

Comparison of adduct levels 

with questionnaire responses 

indicated that smoking, 

frequent consumption of PAHrich meals (>52 versus <52 

times/year), and long time 

periods spent outdoors (>4 

versus <4 hours/day) were risk 

factors as all increased BPDEDNA adduct levels significantly. 

Pavanello 

et al., 2006

BPDEDNA 

adducts

Human, maternal and 

umbilical cord blood

Maternal and umbilical cord blood 

obtained following normal delivery 

from 329 nonsmoking pregnant 

women exposed to emissions from 

fires during the 4 weeks following the 

collapse of the World Trade Center 

(WTC) building in New York City on 

09/11/2001. 

+ BPDE-DNA adduct levels in 

cord and maternal blood were 

highest in study participants 

who lived within 1 mile of the 

WTC, with inverse correlation 

between cord blood levels and 

distance from WTC. 

Perera et 

al., 2005a
Toxicological Review of benzo[a]pyrene

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

B-8 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

BPDEDNA 

adducts

Human, WBCs Workers were exposed for 6–8 hrs/d 

for at least 4–6 mo before blood 

collection; leukocyte DNA isolated and 

digested, and benzo[a]pyrene tetrols 

analyzed by HPLC with fluorescent 

detection. Low, medium, and high 

exposure groups correspond to <0.15, 

0.15–4, and >4 mg/m3of 

benzo[a]pyrene, respectively.

+ <0.15, 0.15– 4, 

or >4 μg/m3of 

benzo[a]pyren

e

PAH exposure, CYP1A1 status 

and smoking significantly 

affected DNA adduct levels, 

i.e., CYP1A1(*1/*2 or *2A/*2a) 

> CYP1A1*1/*1; occupational > 

environmental exposure; 

smokers > nonsmokers; 

adducts increased with dose 

and duration of smoking.

Rojas et 

al., 2000

BPDEDNA 

adducts

Human, WBCs Coke oven workers were exposed to 

PAHs and benzo[a]pyrene-WBC DNA 

analyzed by HPLC-fluorescence 

detection for BPDE-DNA adducts.

± 0.14 μg/m3 Median detectable BPDE-DNA 

adducts in workers vs. controls 

not significant due to low 

number of subjects (9 workers, 

26 controls); 4/9 workers had 

adducts substantially higher 

than all controls. No significant 

difference between smokers 

and nonsmokers; no 

correlation with air 

benzo[a]pyrene levels and 

adduct levels.

Mensing et 

al., 2005

BPDEDNA 

adducts

Mouse, lacZ transgenic 

(MutaTMMouse)

Benzo[a]pyrene given via gavage in 

olive oil daily for 28 consecutive d; 

sacrificed 3 d after last dosing; 4 

organs analyzed for DNA adducts using 

32P-postlabeling with nuclease P1 

digestion enrichment.

+ 25, 50, and 75 

mg/kg-day

Highest adduct levels observed 

in liver, followed by glandular 

stomach, small intestine, and 

bone marrow 

Lemieux et 

al., 2011

BPDEDNA 

adducts

Mouse, (Ahr+/+, Ahr+/-, 

Ahr-/-)

Gavage; sacrificed 24 hrs 

posttreatment.

+ 100 mg/kg No induction of CYP in Ahr-/-, 

but all alleles positive for 

adduct formation.

Sagredo et 

al., 2006
Toxicological Review of benzo[a]pyrene

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

B-9 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

BPDEDNA 

adducts

Mouse, C57BL/6J 

Cyp1a1(+/-) and 

Cyp1a1 (-/-)

Single i.p. injection; sacrificed 24 hrs 

posttreatment; liver DNA analyzed by 

[

32P]-postlabeling assay.

+ 500 mg/kg BPDE-DNA adduct levels 

fourfold higher in Cyp1a1(-/-) 

mice than Cyp1a1(+/-) mice.

Uno et al., 

2001

BPDEDNA 

adducts

Mouse, B6C3F1 Benzo[a]pyrene fed in diet for 4 wks 

(100 ppm) or for 1, 2, 8, 16, and 32 wks 

(5 ppm); sacrificed and liver, lungs, 

forestomach, and small intestine 

collected; DNA analyzed by [32P]-

postlabeling assay.

+ 5 ppm (32 

wks) and 100 

ppm (4 wks)

Linear dose-response in 4-wk 

study; the 5 ppm groups 

showed a plateau after 4 wks 

of feeding.

Culp et al., 

2000

BPDEDNA 

adducts

Mouse, BALB/c Single i.p. injection; sacrificed 12 hrs 

postinjection; liver and forestomach 

collected; DNA binding of [3H]-

benzo[a]pyrene analyzed by 

scintillation counting.

+ 140 μCi/100 g 

body weight

Liver DNA had threefold higher 

binding of benzo[a]pyrene than 

that of forestomach.

Gangar et 

al., 2006

BPDEDNA 

adducts

Mouse, 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 in both

strains dosed with 

benzo[a]pyrene alone. TCDD 

pretreatment had a greater 

suppressive effect on adduct 

formation in BALB relative to 

CBA mice at low dose but 

resulted in no significant 

difference in adduct levels at 

high dose.

Wu et al., 

2008

BPDEDNA 

adducts

Mouse, 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 

[

32P]-postlabeling assay.

+ 4 × 1.2 μmol/ 

animal

Five adducts spots detected. Reddy et 

al., 1984
Toxicological Review of benzo[a]pyrene

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

B-10 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

BPDEDNA 

adducts

Mouse, Swiss, 

epidermal and dermal 

skin

Single topical application on shaved 

backs; sacrificed 1, 3, and 7 d 

posttreatment; 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 for 

benzo[a]pyrene adducts; 

epidermis > dermis; adducts 

persisted up to 7 d with a 

gradual decline in levels.

Oueslati et 

al., 1992

BPDEDNA 

adducts

Rat, CD, peripheral 

blood lymphocytes, 

lungs, and liver

Single i.p. injection; sacrificed 3 d 

posttreatment; DNA analyzed by 

Nuclease P1-endhanced [32P]-

postlabeling assay.

+ 2.5 mg/animal BPDE-dG as major adducts and 

several minor adducts 

detected in all tissues.

Ross et al., 

1991

BPDEDNA 

adducts

Rat, Sprague-Dawley, 

liver

Single i.p. injection followed by 

sacrifice at 4 hrs posttreatment; liver 

DNA isolated and analyzed by [ 32P]-

postlabeling assay.

+ 100 mg/kg Two adduct spots detected. Reddy et 

al., 1984

BPDEDNA 

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 d 

postdosing; analyzed liver and lung 

DNA for BP-DNA adducts by [32P]-

postlabeling assay and urine for 

8-oxodG adducts by HPLCelectrochemical detection.

+ 10 mg/kg BPDE-dG levels peaked 2 d 

after exposure in both tissues, 

higher in lungs than liver at all 

time points, decline faster in 

liver than lung; Increased 8-

oxodG levels in urine and 

decreased levels in liver and 

lung.

Briedé et 

al., 2004

BPDEDNA 

adducts

Rat, F344; 

[

32P]-postlabeling assay

Benzo[a]pyrene given in the diet for 

30, 60, or 90 d; animals sacrificed and 

liver and lung isolated and DNA 

extracted and analyzed for adducts.

+ 0, 5, 50, or 

100 mg/kg

Adduct levels linear at low and 

intermediate doses, nonlinear 

at high dose.

Ramesh 

and 

Knuckels, 

2006
Toxicological Review of benzo[a]pyrene

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

B-11 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

BPDEDNA 

adducts

Rat, Wistar; liver and 

peripheral blood 

lymphocyte adducts

Single dose by gavage; sacrificed 24 hrs 

post-dosing; peripheral blood 

lymphocytes and liver DNA analyzed 

by [32P]-postlabeling for BP-DNA 

adducts.

+ 0, 10, or 

100 mg/kg 

At 100 mg/kg dose, total 

adduct levels in peripheral 

blood lymphocytes were 

twofold higher than the levels 

in liver; adduct profiles differed 

between peripheral blood 

lymphocytes and liver.

Willems et 

al., 1991

CAs Mouse, C57 (high AHH 

inducible) and DBA 

(low AHH inducible) 

strains; 11-d-old 

embryos; adult bone 

marrows

Study used four matings (female × 

male): C57 × C57; DBA × DBA; C57 × 

DBA; and DBA × C57; pregnant mice 

treated orally on GD 11 with 

benzo[a]pyrene; sacrificed 15 hrs 

posttreatment; material liver, bone 

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.

+ 150 mg/kg Levels of CAs: hybrid embryos 

> homozygous DBA embryos > 

homozygous C57 embryos; 

tissue AHH activity: C57 

mothers and their embryos > 

DBA females and their 

homozygous embryos. No 

quantitative correlation 

between BP-induced CAs and 

AHH inducibility. No 

differences in bone marrow 

mitotic index of males of 

different strains between 

control and treatment groups.

Adler et 

al., 1989

CAs Mouse, 1C3F1 hybrid 

(101/E1 × C31 × 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 CAs in 

benzo[a]pyrene-treated 

animals compared to controls.

Adler and 

Ingwersen, 

1989

CAs Rat, Wistar; peripheral 

blood lymphocytes 

Single dose by gavage; sacrificed 6, 24, 

and 48 hrs posttreatment; blood from 

abdominal aorta collected, whole 

blood cultures set up, CAs scored in 

100 first-division peripheral blood 

lymphocytes per animal.

– 0, 10, 100, or 

200 mg/kg 

No difference between control 

and treatment groups at any 

dose or at any sampling time 

observed.

Willems et 

al., 1991
Toxicological Review of benzo[a]pyrene

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

B-12 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

CAs Hamster; bone 

marrow

Single, i.p. injection of benzo[a]pyrene 

dissolved in tricapryline; animals

sacrificed 24 hrs post-exposure.

+ 25, 50, or 

100 mg/kg 

Benzo[a]pyrene induced CAs at 

50 mg/kg body weight only, 

with negative responses at the 

low and high dose.

Bayer, 

1978

MN Mouse, lacZ transgenic 

(MutaTMMouse)

Benzo[a]pyrene given via gavage in 

olive oil daily for 28 consecutive d; 

blood samples were collected 48 h 

after last dose; % of PCEs and NCEs 

reported.

+ 25, 50, and 75 

mg/kg-day

Statistically significant, dosedependent increases in % PCEs 

and NCEs at all doses. 

Lemieux et 

al., 2011

MN Mouse, 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 of benzo[a]pyrene 

(LD50).

Salamone 

et al., 1981

MN 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, 

1,000, or 

2,000 mg/kg 

Significant increase at all 

doses; no dose-response; 

double dosing at 500 mg/kg 

dose gave best response.

Shimada et 

al., 1990

MN Mouse, CD-1 and 

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, 

or 500 mg/kg 

Maximum response seen at 48 

hrs posttreatment.

Shimada et 

al., 1992

MN 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 All groups significantly higher 

than controls for MN; fetal 

liver more sensitive than any 

other group.

Harper et 

al., 1989

MN Mouse, Swiss albino; 

bone marrow

Given orally in corn oil; sacrificed 24 

hrs post-exposure.

+ 75 mg/kg Koratkar et 

al., 1993
Toxicological Review of benzo[a]pyrene

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

B-13 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

MN Mouse, Swiss; bone 

marrow polychromatic 

erythrocytes

Given by gavage and sacrificed 36 hrs 

posttreatment.

+ 75 mg/kg Rao and 

Nandan, 

1990

MN Mouse, CD-1 and 

MS/Ae strains

i.p. and p.o. administration. + 62.5, 125, 250, 

or 500 mg/kg

Good dose response by both 

routes, strains; i.p. better than 

p.o.; MS/Ae strain more 

sensitive than CD-1 strain.

Awogi and 

Sato, 1989

MN 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 May-Grunwald-Giemsa 

technique and scored for MN 

polychromatic erythrocytes.

+ 0, 25, 50, or 

60 mg/kg 

Positive at all doses, time 

points and sexes tested. Dosedependent increase in MN 

observed in both sexes; males 

responded better than 

females; highest positive 

response observed at 72 hrs 

postinjection.

Balansky et 

al., 1994

MN Mouse, HRA/Skh 

hairless, keratinocytes

Single topical application. + 0.5, 5, 50, 100, 

or 

500 mg/mous

e

He and 

Baker, 

1991

MN Mouse, HOS:HR-1, 

hairless; skin 

micronuclei

Topical application once daily for 3 d; 

sacrificed 24 hrs after last treatment.

+ 0.4, 1, 2, or 

4 mg

Nishikawa 

et al., 2005

MN Mouse, HR-1 hairless, 

skin (benzo[a]pyrene 

with slight radiation)

+ Exposure to sunlight simulator 

to evaluate photogenotoxicity 

and chemical exposure.

Hara et al., 

2007

MN 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, or 

1,000 mg/kg 

Maximum response seen at 72 

hrs posttreatment.

Shimada et 

al., 1992

MN Rat, Sprague-Dawley, 

pulmonary alveolar 

macrophages

Intratracheal instillation, once/day for 

3 d.

+ 25 mg/kg De Flora et 

al., 1991
Toxicological Review of benzo[a]pyrene

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

B-14 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

MN Rat, Sprague-Dawley, 

bone marrow cells

Intratracheal instillation, once/day for 

3 d.

– 25 mg/kg De Flora et 

al., 1991

MN Hamster; bone marrow Single, i.p. injection of benzo[a]pyrene 

dissolved in tricaprylin; animals 

sacrificed 30 hours post-exposure.

– 100, 300, or 

500 mg/kg 

Bayer, 

1978

MN Fish (carp, rainbow 

trout, clams); blood 

and hemolymph

+ 0.05, 0.25, 0.5, 

or1 ppm

Kim and 

Hyun, 2006

DNA 

strand 

breaks

Rat, Sprague-Dawley; 

comet assay

Instilled intratracheally with: (1) single 

dose of benzo[a]pyrene in aqueous 

suspension; sacrificed at 3, 24, and 48 

hrs posttreatment; alveolar 

macrophages, lung cells, and 

lymphocytes, hepatocytes collected or 

(2) dose-response study and sacrificed 

at 24 hrs posttreatment; lungs 

collected; controls received normal 

saline instillation; all cells analyzed by 

comet assay.

+ Experiment 

#1: 3 mg of 

benzo[a]pyren

e; Experiment 

#2: doseresponse 

study with 

0.75, 1.5, or 3 

mg 

benzo[a]pyren

e

All time points showed 

significant increase in SSBs 

(Experiment #1); a doseresponse in SSBs was observed 

(Experiment #2).

Garry et 

al., 2003a, 

b

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 d, 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 posttreatment; 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-response for 

strand breaks observed; carp 

and trout liver showed highest 

response at 48 hrs and clam 

digestive gland showed timedependent increase at highest 

concentration.

Kim and 

Hyun, 2006

DNA 

strand 

breaks

Rat, Brown Norway UDS determined after 5 and 18 hrs of a 

single intragastric dosing.

– 62.5 mg/kg Negative at both time points. Mullaart et 

al., 1989
Toxicological Review of benzo[a]pyrene

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

B-15 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

UDS Rat, F344 Single i.p. injection of benzo[a]pyrene 

or DMSO; sacrificed at 2 or 12 hrs 

post-exposure; liver isolated, 

hepatocyte cultures were set up and 

incubated with 10 mCi/mL [3H]-

thymidine for 4 hrs; washed and 

autoradiography performed.

– 100 mg/kg Benzo[a]pyrene was negative 

at both time points.

Mirsalis et 

al., 1982

UDS 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 

posttreatment, skin isolated 

[

3H]thymidine; cultured; epidermal 

UDS measured.

+ 0, 0.25, 0.5, 

and 1% (w/v) 

in acetone

UDS index showed a dosedependent increase up to 0.5% 

benzo[a]pyrene dose and then 

plateaued.

Mori et al., 

1999

UDS Rat, Brown Norway; 

liver

Single intragastric injection; sacrificed 

at 5 and 18 hrs post-injection.

– 62.5 mg/kg Benzo[a]pyrene was negative 

at both time points.

Mullaart et 

al., 1989

UDS Mouse, (C3Hf × 101)F1 

hybrid, germ cells

i.p. injection of benzo[a]pyrene; 

[

3H]-thymidine injection later.

– 0.3 mL Concentration not specified. Sega, 1979

UDS Mouse, early 

spermatid

i.p. injection. – 250–500 

mg/kg 

Reviewed by Sotomayor and 

Sega (2000).

Sega, 1982

SCEs Hamster; 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 Significant increase in 

metaphase SCEs in 

benzo[a]pyrene-treated 

animals compared to vehicletreated controls.

RoszinskyKocher et 

al., 1979
Toxicological Review of benzo[a]pyrene

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

B-16 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

SCEs Hamster Animals implanted s.c. with 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 body weight, 

sacrificed 2 hrs later; bone marrow 

from femur prepared for SCE assay.

+ 50 or 

100 mg/kg 

SCEs increased with low dose 

of phorone significantly.

Bayer et 

al., 1981

SCEs Hamster; fetal liver i.p. injection to pregnant animals on 

GDs 11, 13, or 15; fetal liver SCEs were 

analyzed.

+ 50 and 

125 mg/kg 

Produced doubling of SCE 

frequency.

Pereira et 

al., 1982

SCEs Hamster; bone marrow NA + 2.5, 25, 40, 50, 

75, or 

100 mg/kg 

Frequency of SCEs increased 

≥40 mg/kg body weight

Bayer, 

1978

SCEs Mouse, DBA/2 and 

C57BL/6, bone marrow 

cells

Two intragastric injections given; mice 

implanted with BrdU tablets, sacrificed 

on d 5, SCEs estimated.

+ 10 or 

100 mg/kg 

SCEs and BP-DNA adducts in 

the order of C57Bl/6 (AHHinducible) < DBA/2 (AHHnoninducible).

Wielgosz 

et al., 1991

SCEs Mouse, DBA/2 and 

C57BL/6, splenic 

lymphocytes

Two intragastric injections given; mice 

killed on 5th day and cells cultured for 

48 hrs with BrdU.

+ 10 or 

100 mg/kg 

SCEs and BP-DNA adducts in 

the order of C57Bl/6 (AHHinducible) < DBA/2 (AHHnoninducible).

Wielgosz 

et al., 1991

SCEs Rat, Wistar; peripheral 

blood lymphocytes 

Single dose by gavage; sacrificed 6, 24, 

and 48 hrs posttreatment; blood from 

abdominal aorta collected, whole 

blood cultures set up, SCEs scored in 

50 second-division metaphases in 

peripheral blood lymphocytes per 

animal.

+ 0, 10, 100, or 

200 mg/kg

Linear dose-response at any 

sampling time; however, 

significant at the highest dose 

only; no interaction between 

dose and sampling time.

Willems et 

al., 1991
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B-17 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

Mutation Drosophila 

melanogaster, sexlinked recessive lethal 

test

Basc males exposed to benzo[a]pyrene 

were mated with virgin females of 

Berlin K or mei-9

L1strains.

± 10 mM Data inconclusive due to low 

fertility rates of mei-9

L1 

females.

Vogel et 

al., 1983

Mutation D. melanogaster, sexlinked recessive lethal 

test

Adult Berlin males treated orally with 

benzo[a]pyrene.

+ 5 or 7.5 mM Low mutagenic activity. Vogel et 

al., 1983

Mutation D. melanogaster, 

Berlin-K and Oregon-K 

strains; sex-linked 

recessive lethal test

Benzo[a]pyrene dissolved in special fat 

and injected into the abdomen of flies.

– 2 or 5 mM Negative at both doses. Zijlstra and 

Vogel, 

1984

Mutation D. melanogaster, sexlinked recessive lethal 

test

Male Berlin K larvae treated with 

benzo[a]pyrene for 9–11 d.

+ 0.1–4 mM Threefold enhancement in 

lethals in treated versus 

controls.

Vogel et 

al., 1983

Mutation D. melanogaster, 

Canton-S (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 d; four broods 

obtained; a group of 100 daughters of 

each male were mated again; scored 

for percent lethal.

– 250 or 500 

ppm

Authors report incomplete 

dissolution of benzo[a]pyrene 

in DMSO as a possible cause of 

negative result.

Valencia 

and 

Houtchens, 

1981

Mutation D. melanogaster; 

somatic mutation, eye 

color mosaicism

Fifty 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 

mosaic eye sectors.

+ 1, 2, or 3 mM Benzo[a]pyrene was effective 

as a mutagen; no doseresponse observed.

Fahmy and 

Fahmy, 

1980
Toxicological Review of benzo[a]pyrene

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B-18 DRAFT—DO NOT CITE OR QUOTE

Endpoint Test system Test conditions Results Dose Comment Reference

Cell transformationHamster, 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 × 106cells/plate; 5 d post-culture 

trypsinized; subcultured every 4–6 d 

thereafter and scored for plating 

efficiency and transformation.

+ 3 mg/100 g 

body weight

Quarles et 

al., 1979

CSB = Cockayne syndrome; FM6 = First Multiple No. 6 is an X chromosome with a complex of inversions (to suppress cross-over) and visible 

markers such as yellow body and white and narrow eyes; Li = liver; Lu = lung; Sk = skin; UDS = unscheduled DNA synthesis; XPA = xeroderma 

pigmentosum group A

1

2
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1 Tumor Promotion and Progression

2 Cytotoxicity and inflammatory response

3 The cytotoxicity of benzo[a]pyrene metabolites may contribute to tumor promotion via 

4 inflammatory responses leading to cell proliferation (Burdick et al., 2003). Benzo[a]pyrene is 

5 metabolized to o-quinones, which are cytotoxic, and can generate ROS (Bolton et al., 2000; Penning, 

6 1999). Benzo[a]pyrene o-quinones reduce the viability and survival of rat and human hepatoma 

7 cells (Flowers-Geary et al., 1996, 1993). Cytotoxicity was also induced by benzo[a]pyrene and 

8 BPDE in a human prostate carcinoma cell line (Nwagbara et al., 2007). Inflammatory responses to 

9 cytotoxicity may contribute to the tumor promotion process. For example, benzo[a]pyrene 

10 quinones (1,6-, 3,6-, and 6,12-benzo[a]pyrene-quinone) generated ROS and increased cell 

11 proliferation by enhancing the epidermal growth factor receptor pathway in cultured breast 

12 epithelial cells (Burdick et al., 2003).

13 Several studies have demonstrated that exposure to benzo[a]pyrene increases the 

14 production of inflammatory cytokines, which may contribute to cancer progression. Garçon et al. 

15 (2001a, b) exposed Sprague-Dawley rats by inhalation to benzo[a]pyrene with or without ferrous 

16 oxide (Fe2O3) particles. They found that benzo[a]pyrene alone or in combination with Fe2O3

17 particles elicited mRNA and protein synthesis of the inflammatory cytokine, IL-1. Tamaki et al. 

18 (2004) also demonstrated a benzo[a]pyrene-induced increase in IL-1 expression in a human 

19 fibroblast-like synoviocyte cell line (MH7A). Benzo[a]pyrene increases the expression of the mRNA 

20 for CCL1, an inflammatory chemokine, in human macrophages (N’Diaye et al., 2006). The 

21 benzo[a]pyrene-induced increase in CCL1 mRNA was inhibited by the potent AhR antagonist, 

22 3′-methoxy-4′-nitroflavone.

23 AhR-mediated effects

24 The promotional effects of benzo[a]pyrene may also be related to AhR affinity and the 

25 upregulation of genes related to biotransformation (i.e., induction of CYP1A1), growth, and 

26 differentiation (Bostrom et al., 2002). Figure B-3 illustrates the function of the AhR and depicts the 

27 genes regulated by this receptor as belonging to two major functional groups (i.e., induction of 

28 metabolism or regulation cell differentiation and proliferation). PAHs bind to the cytosolic AhR in 

29 complex with heat shock protein 90 (Hsp90). The ligand-bound receptor is then transported to 

30 nucleus in complex with the Ah receptor nuclear translocator. The AhR complex interacts with the 

31 Ah responsive elements of the DNA to increase the transcription of proteins associated with 

32 induction of metabolism and regulation of cell differentiation and proliferation.
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B-2 DRAFT—DO NOT CITE OR QUOTE

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; ARNT = Ah receptor nuclear translocator; Hsp90 = heat 

3 shock protein 90

4 Source: Okey et al. (1994).

5

6 Figure B-3. 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 high 

13 and low affinity AhRs. Disparities were observed in the tumor pattern and toxicity of 

14 Ah-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 skin. 

16 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 18–25 weeks 

19 after treatment. Benzo[a]pyrene treatment increased CYP1A1 expression in the skin and liver of 

20 AhR-positive mice (+/- or +/+), but CYP1A1 expression was not altered by benzo[a]pyrene 
Toxicological Review of benzo[a]pyrene

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1 treatment in AhR knock-out mice (-/-). Talaska et al. (2006) also showed that benzo[a]pyrene 

2 adduct levels in skin were reduced by 50% in CYP1A2 knock-out mice and by 90% in AhR knock3 out mice compared with WT C57Bl6/J mice following a single dermal application of 33 mg/kg 

4 benzo[a]pyrene for 24 hours. Ma and Lu (2007) further noted that Ah-nonresponsive mice were at 

5 greater risk of toxicity and tumorigenicity in remote organs, distant from the site of exposure (i.e., 

6 bone marrow). As an example, Uno et al. (2006) showed that benzo[a]pyrene (125 mg/kg-day, p.o. 

7 for 18 days) caused marked wasting, immunosuppression, and bone marrow hypocellularity in 

8 CYP1A1 knock-out mice, but not in WT mice.

9 Some studies have demonstrated the formation of DNA adducts in the liver of AhR knock10 out mice following i.p. or oral exposure to benzo[a]pyrene (Sagredo et al., 2006; Uno et al., 2006; 

11 Kondraganti et al., 2003). These findings suggest that there may be alternative (i.e., non-AhR 

12 mediated) mechanisms of benzo[a]pyrene activation in the mouse liver. Sagredo et al. (2006) 

13 studied the relationship between the AhR genotype and CYP metabolism in different organs of the 

mouse. AhR+/+, 

+/–, and 14 –/– mice were treated once with 100 mg/kg benzo[a]pyrene by gavage. 

15 CYP1A1, CYP1B1, and AhR expression was evaluated in the lung, liver, spleen, kidney, heart, and 

16 blood, via real-time or reverse transcriptase polymerase chain reaction, 24 hours after treatment. 

17 CYP1A1 RNA was increased in the lung and liver and CYP1B1 RNA was increased in the lung 

following benzo[a]pyrene treatment in AhR+/+ and 18 +/– mice (generally higher in heterozygotes). 

19 Benzo[a]pyrene treatment did not induce CYP1A1 or CYP1B1 enzymes in AhR–/– mice. The 

20 expression of CYP1A1 RNA, as standardized to β-actin expression, was generally about 40 times 

21 that of CYP1B1. The concentration of benzo[a]pyrene metabolites and the levels of DNA and 

22 protein adducts were increased in mice lacking the AhR, suggesting that there may be an 

23 AhR-independent pathway for benzo[a]pyrene metabolism and activation. The high levels of 

24 benzo[a]pyrene DNA adducts in organs other than the liver of AhR–/– mice may be the result of slow 

25 detoxification of benzo[a]pyrene in the liver, allowing high concentrations of the parent compound 

26 to reach distant tissues.

27 Uno et al. (2006) also demonstrated a paradoxical increase in liver DNA adducts in AhR 

28 knock-out mice following oral exposure to benzo[a]pyrene. WT C57BL/6 mice and several knockout mouse strains (CYP1A2–/– and CYP1B1–/– 29 single knock-out, CYP1A1/1B1–/– and CYP1A2/1B1–/–

30 double knock-out) were studied. Benzo[a]pyrene was administered in the feed at 1.25, 12.5, or 125 

31 mg/kg for 18 days (this dose is well tolerated by WT C57BL/6 mice for 1 year, but lethal within 30 

32 days to the CYP1A1–/– 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–/– 33 than in WT 

34 mice, while clearance was similar to WT mice in the other knock-out mouse strains. DNA adduct 

levels, measured by [ 35 32P]-postlabeling in liver, spleen, and bone marrow, were highest in the 

36 CYP1A1–/– mice at the two higher doses, and in the CYP1A1/1B1–/– mice at the mid dose only. 

37 Adduct patterns, as revealed by 2-dimensional chromatography, differed substantially between 

38 organs in the various knock-out types.
Toxicological Review of benzo[a]pyrene

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B-4 DRAFT—DO NOT CITE OR QUOTE

1 Dertinger et al. (2001, 2000) demonstrated that AhR signaling may play a role in 

2 cytogenetic damage caused by benzo[a]pyrene. The in vivo formation of MN in peripheral blood 

3 reticulocytes of C57Bl/6J mice induced by a single i.p. injection of benzo[a]pyrene (150 mg/kg) was 

4 eliminated by prior treatment with the potent AhR antagonist 3'-methoxy-4'-nitroflavone. This 

5 antagonist also protected AhR null allele mice from benzo[a]pyrene-induced increases in MN 

6 formation, suggesting that 3'-methoxy-4'-nitroflavone may also act through a mechanism 

7 independent of the AhR (Dertinger et al., 2000).

8 Several in vitro studies have suggested that the AhR plays a role in the disruption of cell 

9 cycle control, possibly leading to cell proliferation and tumor promotion following exposure to 

10 benzo[a]pyrene (Andrysik et al., 2007; Chung et al., 2007; Chen et al., 2003). Chung et al. (2007) 

11 showed that benzo[a]pyrene-induced cytotoxicity and apoptosis in mouse hepatoma (Hepa1c1c7) 

12 cells occurred through a p53 and caspase-dependent process requiring the AhR. An accumulation 

13 of cells in the S-phase of the cell cycle (i.e., DNA synthesis and replication) was also observed, 

14 suggesting that this process may be related to cell proliferation. Chen et al. (2003) also 

15 demonstrated the importance of the AhR in benzo[a]pyrene-7,8-dihydrodiol- and BPDE-induced 

16 apoptosis in human HepG2 cells. Both the dihydrodiol and BPDE affected Bcl2 (a member of a 

17 family of apoptosis suppressors) and activated caspase and p38 mitogen-activated protein (MAP) 

18 kinases, both enzymes that promote apoptosis. When the experiments were conducted in a cell line 

19 that does not contain Ah receptor nuclear translocator (see Figure 4-1), the dihydrodiol was not 

20 able to initiate apoptotic event sequences, indicating that activation to BPDE by CYP1A1 was 

21 required. BPDE did not induce apoptosis-related events in a p38-defective cell line, illustrating the 

22 importance of MAP kinases in this process. In rat liver epithelial cells (WB-F344 cells), in vitro 

23 exposure to benzo[a]pyrene resulted in apoptosis, a decrease in cell number, an increase in the 

24 percentage of cells in S-phase (comparable to a proliferating population of WB-F334 cells), and 

25 increased expression of cell cycle proteins (e.g., cyclin A) (Andrysik et al., 2007). Benzo[a]pyrene26 induced apoptosis was attenuated in cells transfected with a dominant-negative mutation of the 

27 AhR.

28 Inhibition of gap junctional intercellular communication (GJIC)

29 Gap junctions are channels between cells that allow substances of a molecular weight up to 

30 roughly 1 kDa to pass from one cell to the other. This process of metabolic cooperation is crucial 

31 for differentiation, proliferation, apoptosis, and cell death and consequently for the two epigenetic 

32 steps of tumor formation, promotion, and progression. Chronic exposure to many toxicants results 

33 in down-regulation of gap junctions. For tumor promoters, such as TPA or TCDD, inhibition of 

34 intercellular communication is correlated with their promoting potency (Sharovskaya et al., 2006; 

35 Yamasaki, 1990).

36 Blaha et al. (2002) surveyed the potency of 35 PAHs, including benzo[a]pyrene, to inhibit 

37 GJIC. The scrape loading/dye transfer assay was employed using a rat liver epithelial cell line that 

38 was incubated in vitro for 15, 30, or 60 minutes with 50 μM benzo[a]pyrene. After incubation, cells 
Toxicological Review of benzo[a]pyrene

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B-5 DRAFT—DO NOT CITE OR QUOTE

1 were washed, and then a line was scraped through the cells with a surgical blade. Cells were 

2 exposed to the fluorescent dye lucifer yellow for 4 minutes and then fixed with formalin. Spread of 

3 the dye from the scrape line into cells remote from the scrape was estimated under a fluorescence 

4 microscope. Benzo[a]pyrene reduced spread of the dye after 30 minutes of exposure 

5 (approximately 50% of control). Recovery of GJIC was observed 60 minutes after exposure.

6 Sharovskaya et al. (2006) studied the effects of carcinogenic and noncarcinogenic PAHs on 

7 GJIC in HepG2 cells. Individual carcinogenic PAHs inhibited GJIC in a temporary fashion (70–100% 

8 within 24 hours), but removal of the PAH from culture reversed the effect. Noncarcinogenic PAHs 

9 had very little effect on GJIC. Benzo[a]pyrene at 20 μM inhibited GJIC completely within 24 hours, 

10 while its noncarcinogenic homolog, benzo[e]pyrene, produced <20% inhibition. The effect was not 

11 AhR-dependent, because benzo[a]pyrene inhibited GJIC in HepG2 cells to the same extent as in 

12 hepatoma G27 cells, which express neither CYP1A1 nor AhR. The authors concluded that the 

13 effects of benzo[a]pyrene and benzo[e]pyrene on GJIC were direct (i.e., not caused by metabolites).
Toxicological Review of benzo[a]pyrene

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C-1 DRAFT—DO NOT CITE OR QUOTE

1 APPENDIX C. DOSE-RESPONSE MODELING FOR 

2 THE DERIVATION OF REFERENCE VALUES FOR 

3 EFFECTS OTHER THAN CANCER AND THE 

4 DERIVATION OF CANCER RISK ESTIMATES

5 This appendix provides technical detail on dose-response evaluation and determination of 

6 points of departure (POD) for relevant toxicological endpoints. Except where other software is 

7 noted, all endpoints were modeled using the U.S. EPA’s Benchmark Dose Software (BMDS; U.S. EPA, 

8 2012; version 2.0 or later). The preambles for the cancer and non-cancer parts below describe the 

9 common practices used in evaluating the model fit and selecting the appropriate model for 

10 determining the POD, as outlined in the draft Benchmark Dose Technical Guidance Document (U.S. 

11 EPA, 2000). 

12 DOSE-RESPONSE MODELING FOR DERVIATION OF RFD

13 Evaluation of Model Fit

14 For each dichotomous endpoint, BMDS dichotomous models were fitted to the data using 

15 the maximum likelihood method. Each model was tested for goodness-of-fit using a chi-square 

16 goodness-of-fit test (χ2 p-value < 0.10 indicates lack of fit). Other factors were also used to assess 

17 model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the 

18 vicinity of the BMR. 

19 For each continuous endpoint, BMDS continuous models were fitted to the data using the 

20 maximum likelihood method. Model fit was assessed by a series of tests as follows. For each model, 

21 first the homogeneity of the variances was tested using a likelihood ratio test (BMDS Test 2). If Test 

22 2 was not rejected (χ2 p-value ≥ 0.10), the model was fitted to the data assuming constant variance. 

23 If Test 2 was rejected (χ2 p-value < 0.10), the variance was modeled as a power function of the 

24 mean, and the variance model was tested for adequacy of fit using a likelihood ratio test (BMDS 

25 Test 3). For fitting models using either constant variance or modeled variance, models for the mean 

26 response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 4, with χ2 p-value < 

27 0.10 indicating inadequate fit). Other factors were also used to assess the model fit, such as scaled 

28 residuals, visual fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.

29 Model Selection

30 For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as 

31 estimated by the profile likelihood method) and AIC value were used to select a best-fit model from 

32 among the models exhibiting adequate fit. If the BMDL estimates were “sufficiently close,” that is, 
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C-2 DRAFT—DO NOT CITE OR QUOTE

1 differed by at most threefold, the model selected was the one that yielded the lowest AIC value. If 

2 the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the POD.

3 Decreased thymus weight, males (Kroese et al., 2001)

Table C-1. Means ± SDa 4 for thymus weight in male Wistar rats exposed 

5 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.

6 Table C-2. Model predictions for decreased thymus weight in male 

7 Wistar rats—90 days

Model

Variance

p-valuea

Goodness-offit 

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

HillcInsufficient degrees of freedom

Linear, Polynomial (2-

degree), Powerc

0.30 0.23 380.71 16.40 11.30

8
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C-3 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-1. Fit of linear model (nonconstant variance) to data on 

3 decreased thymus weight in male Wistar rats—90 days.

4 BMDs and BMDLs indicated are associated with a change of 1 SD from the 

5 control, and are in units of mg/kg-day.

6 ==================================================================== 

7 Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

8 Input Data File: 

9 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\male\durationadjusted\2Linkrolin.(

10 d) 

11 Gnuplot Plotting File: 

12 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\male\durationadjusted\2Linkrolin.p

13 lt

14 ==================================================================== 

15

16 BMDS Model Run 

17 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

18

19 The form of the response function is: 

20

21 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ...

22

23

24 Dependent variable = mean

25 Independent variable = dose

26 The polynomial coefficients are restricted to be negative

27 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

28

29 Total number of dose groups = 4

 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
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C-4 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 lalpha = 8.56121

10 rho = 0

11 beta_0 = 380.763

12 beta_1 = -5.3285

13

14

15 Asymptotic Correlation Matrix of Parameter Estimates

16

17 lalpha rho beta_0 beta_1

18

19 lalpha 1 -1 0.048 -0.061

20

21 rho -1 1 -0.048 0.061

22

23 beta_0 0.048 -0.048 1 -0.84

24

25 beta_1 -0.061 0.061 -0.84 1

26

27

28

29 Parameter Estimates

30

31 95.0% Wald Confidence Interval

32 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

33 lalpha -18.8293 9.75429 -37.9473 0.288754

34 rho 4.66515 1.67581 1.38062 7.94967

35 beta_0 378.954 16.5291 346.558 411.351

36 beta_1 -5.14219 1.00497 -7.11189 -3.17249

37

38

39

40 Table of Data and Estimated Values of Interest

41

42 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.

43 ------ --- -------- -------- ----------- ----------- ----------

44

45 0 10 380 379 60 84.3 0.0392

46 2.1 10 380 368 110 78.8 0.475

47 7.1 10 330 342 60 66.6 -0.591

48 21.4 10 270 269 40 37.9 0.0908

49

50

51

52 Model Descriptions for likelihoods calculated

53

54

55 Model A1: Yij = Mu(i) + e(ij)

56 Var{e(ij)} = Sigma^2

57

58 Model A2: Yij = Mu(i) + e(ij)

59 Var{e(ij)} = Sigma(i)^2

60

61 Model A3: Yij = Mu(i) + e(ij)

62 Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))

63 Model A3 uses any fixed variance parameters that

64 were specified by the user

65

66 Model R: Yi = Mu + e(i)

67 Var{e(i)} = Sigma^2

68

69

70 Likelihoods of Interest

71
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1 Model Log(likelihood) # Param's AIC

2 A1 -189.116991 5 388.233982

3 A2 -183.673279 8 383.346558

4 A3 -184.883626 6 381.767253

5 fitted -186.353541 4 380.707081

6 R -196.353362 2 396.706723

7

8

9 Explanation of Tests 

10

11 Test 1: Do responses and/or variances differ among Dose levels? 

12 (A2 vs. R)

13 Test 2: Are Variances Homogeneous? (A1 vs A2)

14 Test 3: Are variances adequately modeled? (A2 vs. A3)

15 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

16 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.)

17

18 Tests of Interest 

19

20 Test -2*log(Likelihood Ratio) Test df p-value 

21

22 Test 1 25.3602 6 0.0002928

23 Test 2 10.8874 3 0.01235

24 Test 3 2.42069 2 0.2981

25 Test 4 2.93983 2 0.2299

26

27 The p-value for Test 1 is less than .05. There appears to be a

28 difference between response and/or variances among the dose levels

29 It seems appropriate to model the data

30

31 The p-value for Test 2 is less than .1. A non-homogeneous variance 

32 model appears to be appropriate

33

34 The p-value for Test 3 is greater than .1. The modeled variance appears 

35 to be appropriate here

36

37 The p-value for Test 4 is greater than .1. The model chosen seems 

38 to adequately describe the data

39

40

41 Benchmark Dose Computation

42

43 Specified effect = 1

44

45 Risk Type = Estimated standard deviations from the control mean

46

47 Confidence level = 0.95

48

49 BMD = 16.4008

50

51

52 BMDL = 11.2965

53
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1 Decreased thymus weight, females (Kroese et al., 2001)

Table C-3. Means ± SDa 2 for thymus weight in female Wistar rats 

3 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.

4 Table C-4. Model predictions for decreased thymus weight in female 

5 Wistar rats—90 days

Model (constant variance)

Variance

p-valuea

Mean

p-valueaAIC

BMD1SD

(mg/kg-d)

BMDL1SD

(mg/kg-d)

Hillb NA

Linearc0.17 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

a

Values <0.10 fail to meet conventional goodness-of-fit criteria.

b

Power restricted to ≥1.

c

Coefficients restricted to be negative.

d

Lowest degree polynomial with an adequate fit is reported.

BMD/BMC = maximum likelihood estimate (MLE) of the dose/concentration associated with the 

selected BMR; NA = not applicable; model failed to generate

6
Toxicological Review of benzo[a]pyrene

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

C-7 DRAFT—DO NOT CITE OR QUOTE

1

2 BMDs and BMDLs indicated are associated with a change of 1 SD from the 

3 control, and are in units of mg/kg-day.

4 Figure C-2. Fit of linear model (constant variance) to data on decreased 

5 thymus weight in female Wistar rats—90 days.

6 ==================================================================== 

7 Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

8 Input Data File: 

9 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\female\durationadjusted\2Linkrolin

10 .(d) 

11 Gnuplot Plotting File: 

12 C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\female\durationadjusted\2Linkrolin

13 .plt

14 Thu Oct 15 16:27:44 2009

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 = mean

26 Independent variable = dose

27 rho is set to 0

28 The polynomial coefficients are restricted to be negative

 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
Toxicological Review of benzo[a]pyrene

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

C-8 DRAFT—DO NOT CITE OR QUOTE

1 A constant variance model is fit

2

3 Total number of dose groups = 4

4 Total number of records with missing values = 0

5 Maximum number of iterations = 250

6 Relative Function Convergence has been set to: 1e-008

7 Parameter Convergence has been set to: 1e-008

8

9

10

11 Default Initial Parameter Values 

12 alpha = 1

13 rho = 0 Specified

14 beta_0 = 322.144

15 beta_1 = -4.2018

16

17

18 Asymptotic Correlation Matrix of Parameter Estimates

19

20 ( *** The model parameter(s) -rho 

21 have been estimated at a boundary point, or have been specified by the user,

22 and do not appear in the correlation matrix )

23

24 alpha beta_0 beta_1

25

26 alpha 1 2.4e-008 -2.3e-008

27

28 beta_0 2.4e-008 1 -0.68

29

30 beta_1 -2.3e-008 -0.68 1

31

32

33

34 Parameter Estimates

35

36 95.0% Wald Confidence Interval

37 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

38 alpha 1954.92 437.134 1098.16 2811.69

39 beta_0 322.144 9.48287 303.558 340.73

40 beta_1 -4.2018 0.837537 -5.84334 -2.56026

41

42

43

44 Table of Data and Estimated Values of Interest

45

46 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.

47 ------ --- -------- -------- ----------- ----------- ----------

48

49 0 10 320 322 60 44.2 -0.153

50 2.1 10 310 313 50 44.2 -0.237

51 7.1 10 300 292 40 44.2 0.55

52 21.4 10 230 232 30 44.2 -0.159

53

54

55

56 Model Descriptions for likelihoods calculated

57

58

59 Model A1: Yij = Mu(i) + e(ij)

60 Var{e(ij)} = Sigma^2

61

62 Model A2: Yij = Mu(i) + e(ij)

63 Var{e(ij)} = Sigma(i)^2

64

65 Model A3: Yij = Mu(i) + e(ij)

66 Var{e(ij)} = Sigma^2

67 Model A3 uses any fixed variance parameters that

68 were specified by the user

69

70 Model R: Yi = Mu + e(i)

71 Var{e(i)} = Sigma^2
Toxicological Review of benzo[a]pyrene

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

C-9 DRAFT—DO NOT CITE OR QUOTE

1

2

3 Likelihoods of Interest

4

5 Model Log(likelihood) # Param's AIC

6 A1 -171.357252 5 352.714504

7 A2 -168.857234 8 353.714467

8 A3 -171.357252 5 352.714504

9 fitted -171.562118 3 349.124237

10 R -181.324151 2 366.648303

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 24.9338 6 0.0003512

27 Test 2 5.00004 3 0.1718

28 Test 3 5.00004 3 0.1718

29 Test 4 0.409733 2 0.8148

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 = 10.5228

55

56

57 BMDL = 7.64037

58
Toxicological Review of benzo[a]pyrene

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

C-10 DRAFT—DO NOT CITE OR QUOTE

1 Decreased ovary weight—female rats, 60 days (Xu et al., 2010)

2 Table C-5. Means ± SDs for ovary weight in female Sprague-Dawley rats

Organ

Dose (mg/kg-d)a

0 2.5 5

Ovary weight (mg) 0.160 ± 0.0146 0.143 ± 0.0098b0.136 ± 0.0098b

a

TWA doses over the 60-day study period.

b

Statistically different (p < 0.05) from controls using one-way ANOVA.

3 Table C-6. Model predictions for decreased ovary weight in female 

4 Sprague-Dawley rats

Model

Goodness-of-fit

p-value AIC

BMD1SD

(mg/kg-d)

BMDL1SD

(mg/kg-d)

Power NA

Linear, Polynomial (1°) 0.39 -138.67 2.27 1.49

NA = not applicable; model failed to generate

5
Toxicological Review of benzo[a]pyrene

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

C-11 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-3. Fit of linear/polynomial (1°) model to data on decreased 

3 ovary weight.

4 ==================================================================== 

5 Polynomial Model. (Version: 2.16; Date: 05/26/2010) 

6 Input Data File: 

7 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_1SD.(d) 

8 Gnuplot Plotting File: 

9 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_1SD.plt

10 Tue Dec 14 13:51:32 2010

11 ==================================================================== 

12 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

13

14 The form of the response function is: 

15

16 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ...

17

18

19 Dependent variable = Mean

20 Independent variable = Dose

21 rho is set to 0

22 Signs of the polynomial coefficients are not restricted

23 A constant variance model is fit

24

25 Total number of dose groups = 3

26 Total number of records with missing values = 0

27 Maximum number of iterations = 250

28 Relative Function Convergence has been set to: 1e-008

29 Parameter Convergence has been set to: 1e-008

30

 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
Toxicological Review of benzo[a]pyrene

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

C-12 DRAFT—DO NOT CITE OR QUOTE

1

2

3 Default Initial Parameter Values 

4 alpha = 0.000136

5 rho = 0 Specified

6 beta_0 = 0.158333

7 beta_1 = -0.0048

8

9

10 Asymptotic Correlation Matrix of Parameter Estimates

11

12 ( *** The model parameter(s) -rho 

13 have been estimated at a boundary point, or have been specified by the user,

14 and do not appear in the correlation matrix )

15

16 alpha beta_0 beta_1

17

18 alpha 1 4e-010 -4.5e-010

19

20 beta_0 4e-010 1 -0.77

21

22 beta_1 -4.5e-010 -0.77 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 alpha 0.000118889 3.96296e-005 4.12162e-005 0.000196562

31 beta_0 0.158333 0.00406354 0.150369 0.166298

32 beta_1 -0.0048 0.00125904 -0.00726768 -0.00233232

33

34

35

36 Table of Data and Estimated Values of Interest

37

38 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.

39 ------ --- -------- -------- ----------- ----------- ----------

40

41 0 6 0.16 0.158 0.0147 0.0109 0.374

42 2.5 6 0.143 0.146 0.0098 0.0109 -0.749

43 5 6 0.136 0.134 0.0098 0.0109 0.374

44

45

46

47 Model Descriptions for likelihoods calculated

48

49

50 Model A1: Yij = Mu(i) + e(ij)

51 Var{e(ij)} = Sigma^2

52

53 Model A2: Yij = Mu(i) + e(ij)

54 Var{e(ij)} = Sigma(i)^2

55

56 Model A3: Yij = Mu(i) + e(ij)

57 Var{e(ij)} = Sigma^2

58 Model A3 uses any fixed variance parameters that

59 were specified by the user

60

61 Model R: Yi = Mu + e(i)

62 Var{e(i)} = Sigma^2

63

64

65 Likelihoods of Interest

66

67 Model Log(likelihood) # Param's AIC

68 A1 72.766595 4 -137.533190

69 A2 73.468565 6 -134.937129

70 A3 72.766595 4 -137.533190

71 fitted 72.335891 3 -138.671782
Toxicological Review of benzo[a]pyrene

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

C-13 DRAFT—DO NOT CITE OR QUOTE

1 R 67.008505 2 -130.017010

2

3

4 Explanation of Tests 

5

6 Test 1: Do responses and/or variances differ among Dose levels? 

7 (A2 vs. R)

8 Test 2: Are Variances Homogeneous? (A1 vs A2)

9 Test 3: Are variances adequately modeled? (A2 vs. A3)

10 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

11 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.)

12

13 Tests of Interest 

14

15 Test -2*log(Likelihood Ratio) Test df p-value 

16

17 Test 1 12.9201 4 0.01167

18 Test 2 1.40394 2 0.4956

19 Test 3 1.40394 2 0.4956

20 Test 4 0.861408 1 0.3533

21

22 The p-value for Test 1 is less than .05. There appears to be a

23 difference between response and/or variances among the dose levels

24 It seems appropriate to model the data

25

26 The p-value for Test 2 is greater than .1. A homogeneous variance 

27 model appears to be appropriate here

28

29

30 The p-value for Test 3 is greater than .1. The modeled variance appears 

31 to be appropriate here

32

33 The p-value for Test 4 is greater than .1. The model chosen seems 

34 to adequately describe the data

35

36

37 Benchmark Dose Computation

38

39 Specified effect = 1

40

41 Risk Type = Estimated standard deviations from the control mean

42

43 Confidence level = 0.95

44

45 BMD = 2.27159

46

47

48 BMDL = 1.49968

49
Toxicological Review of benzo[a]pyrene

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

C-14 DRAFT—DO NOT CITE OR QUOTE

1 Morris water maze results—male and female Sprague-Dawley rats, Chen et al. (2012)

2 Data from Morris water maze was presented graphically in Chen et al., 2012, but dose group 

3 means and standard deviations were provided upon request by the study authors which enabled 

4 modeling of this endpoint. In addition, the data for male and female rats were combined for dose5 response analysis because there was no substantive difference between males and females for each 

6 dose group (supported by statistical testing using two-way ANOVA, and allowing for interactions), 

7 and because there was no rationale or information available suggesting there would be sex8 mediated differences for these neurologic tests.

9 Table C-7. Means ± SDs for Escape Latency and Time Spent in Target 

10 Quadrant

Test

Dose (mg/kg-d)

0 0.02 0.2 2.0

Escape latency 

(sec) 9.89 ± 5.76 12.5 ± 5.10 19.1 ± 5.85 33.5 ± 9.93

Time spent in 

target quadrant 

(sec)

33.6 ± 8.92 31.9 ± 8.63 16.6 ± 5.74 11.1 ± 5.12

11 Table C-8. Model predictions for increase in Morris water maze test for 

12 escape latency, male and female rats

Modela

Goodness-of-fit

p-value AIC

BMD1SD

(mg/kg-d)

BMDL1SD

(mg/kg-d)

Hillb0.515 386.3 0.106 0.061

Exponential 4, 5 0.466 386.4 0.115 0.071

Polynomial (2°) 0.423 386.6 0.123 0.083

Linear, Power 0.002 396.7 0.543 0.421

Exponential 2, 3 <0.001 400.3 0.815 0.687

a

Includes modeling of heterogeneous variances (BMDS Test 3, p = 0.313).

b

Power parameter n was estimated to be 1 (boundary of parameter space). 
Toxicological Review of benzo[a]pyrene

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

C-15 DRAFT—DO NOT CITE OR QUOTE

1 Figure C-4. Fit of Hill model to data on Morris water maze test escape 

2 latency.

3 ==================================================================== 

4 Hill Model. (Version: 2.16; Date: 04/06/2011) 

5 Input Data File: C:\Documents and Settings\jfox\My Documents\_CURRENTWORK\_CAST 

6 plus\BaP\BMDS\hil_Chen.FM.latency_Hil-ModelVariance-BMR1Std-Restrict.(d)

7 Gnuplot Plotting File: C:\Documents and Settings\jfox\My Documents\_CURRENTWORK\_CAST 

8 plus\BaP\BMDS\hil_Chen.FM.latency_Hil-ModelVariance-BMR1Std-Restrict.plt

9 Tue Apr 24 14:41:26 2012

10 ==================================================================== 

11

12 BMDS Model Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14

15 The form of the response function is: 

16

17 Y[dose] = intercept + v*dose^n/(k^n + dose^n)

18

19

20 Dependent variable = Mean

21 Independent variable = Dose

22 Power parameter restricted to be greater than 1

23 The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i)))

24

25 Total number of dose groups = 4

26 Total number of records with missing values = 0

27 Maximum number of iterations = 250

28 Relative Function Convergence has been set to: 1e-008

29 Parameter Convergence has been set to: 1e-008

30

31

32

33 Default Initial Parameter Values 

34 lalpha = 3.87128

35 rho = 0

36 intercept = 9.888

37 v = 23.6385

38 n = 0.187055

39 k = 3.47082

40

41

 5

 10

 15

 20

 25

 30

 35

 40

 0 0.5 1 1.5 2 Mean Response

dose

Hill Model with 0.95 Confidence Level

14:41 04/24 2012

BMDL BMD

 

Hill
Toxicological Review of benzo[a]pyrene

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

C-16 DRAFT—DO NOT CITE OR QUOTE

1 Asymptotic Correlation Matrix of Parameter Estimates

2

3 ( *** The model parameter(s) -n 

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 lalpha rho intercept v k

8

9 lalpha 1 -0.99 -0.27 0.062 -0.11

10

11 rho -0.99 1 0.24 -0.063 0.12

12

13 intercept -0.27 0.24 1 0.017 0.47

14

15 v 0.062 -0.063 0.017 1 0.73

16

17 k -0.11 0.12 0.47 0.73 1

18

19

20

21 Parameter Estimates

22

23 95.0% Wald Confidence Interval

24 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

25 lalpha 0.88775 0.974841 -1.0229 2.7984

26 rho 0.998033 0.338845 0.33391 1.66216

27 intercept 10.6545 0.914127 8.86283 12.4461

28 v 28.7081 3.94381 20.9783 36.4378

29 n 1 NA

30 k 0.494812 0.213359 0.0766351 0.912988

31

32 NA - Indicates that this parameter has hit a bound

33 implied by some inequality constraint and thus

34 has no standard error.

35

36

37

38 Table of Data and Estimated Values of Interest

39

40 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.

41 ------ --- -------- -------- ----------- ----------- ----------

42

43 0 20 9.89 10.7 5.76 5.08 -0.675

44 0.02 20 12.5 11.8 5.1 5.33 0.641

45 0.2 20 19.1 18.9 5.85 6.76 0.0952

46 2 20 33.5 33.7 9.93 9.01 -0.0706

47

48

49

50 Model Descriptions for likelihoods calculated

51

52

53 Model A1: Yij = Mu(i) + e(ij)

54 Var{e(ij)} = Sigma^2

55

56 Model A2: Yij = Mu(i) + e(ij)

57 Var{e(ij)} = Sigma(i)^2

58

59 Model A3: Yij = Mu(i) + e(ij)

60 Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))

61 Model A3 uses any fixed variance parameters that

62 were specified by the user

63

64 Model R: Yi = Mu + e(i)

65 Var{e(i)} = Sigma^2

66

67

68 Likelihoods of Interest

69

70 Model Log(likelihood) # Param's AIC

71 A1 -192.799518 5 395.599036
Toxicological Review of benzo[a]pyrene

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

C-17 DRAFT—DO NOT CITE OR QUOTE

1 A2 -186.795503 8 389.591006

2 A3 -187.957975 6 387.915949

3 fitted -188.169983 5 386.339965

4 R -234.549118 2 473.098237

5

6

7 Explanation of Tests 

8

9 Test 1: Do responses and/or variances differ among Dose levels? 

10 (A2 vs. R)

11 Test 2: Are Variances Homogeneous? (A1 vs A2)

12 Test 3: Are variances adequately modeled? (A2 vs. A3)

13 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

14 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.)

15

16 Tests of Interest 

17

18 Test -2*log(Likelihood Ratio) Test df p-value 

19

20 Test 1 95.5072 6 <.0001

21 Test 2 12.008 3 0.007356

22 Test 3 2.32494 2 0.3127

23 Test 4 0.424016 1 0.5149

24

25 The p-value for Test 1 is less than .05. There appears to be a

26 difference between response and/or variances among the dose levels

27 It seems appropriate to model the data

28

29 The p-value for Test 2 is less than .1. A non-homogeneous variance 

30 model appears to be appropriate

31

32 The p-value for Test 3 is greater than .1. The modeled variance appears 

33 to be appropriate here

34

35 The p-value for Test 4 is greater than .1. The model chosen seems 

36 to adequately describe the data

37

38

39 Benchmark Dose Computation

40

41 Specified effect = 1

42

43 Risk Type = Estimated standard deviations from the control mean 

44

45 Confidence level = 0.95

46

47 BMD = 0.106284

48

49 BMDL = 0.0609511

50
Toxicological Review of benzo[a]pyrene

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

C-18 DRAFT—DO NOT CITE OR QUOTE

1 Table C-9. Model predictions for decrease in Morris water maze test for 

2 time spent in target quadrant, male and female rats

Modela

Goodness-of-fit

p-value AIC

BMD1SD

(mg/kg-d)

BMDL1SD

(mg/kg-d)

Exponential 4 0.576 395.4 0.065 0.043

Exponential 5 NAb397.1 0.084 0.044

Hill NAb397.1 0.071 0.038

Linear, Power, Polynomial (1°, 2°, 3°) <0.001 433.1 1.23 0.98

a Includes modeling of heterogenous variances (BMDS Test 3, p = 0.919).

b NA: insufficient degrees of freedom to evaluate chi-square.

3

4

5 Figure C-5. Fit of Exponential 4 model to data on Morris water maze 

6 time spent in target quadrant.

7 ==================================================================== 

8 Exponential Model. (Version: 1.7; Date: 12/10/2009) 

9 Input Data File: C:\Documents and Settings\...\exp_Chen.FM.target_Exp-ModelVariance10 BMR1Std-Down.(d) 

11 ==================================================================== 

12

13 BMDS Model Run 

14 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

15

16 The form of the response function by Model: 

17 Model 2: Y[dose] = a * exp{sign * b * dose}

18 Model 3: Y[dose] = a * exp{sign * (b * dose)^d}

19 Model 4: Y[dose] = a * [c-(c-1) * exp{-b * dose}]

20 Model 5: Y[dose] = a * [c-(c-1) * exp{-(b * dose)^d}]

21

22 Note: Y[dose] is the median response for exposure = dose;

23 sign = +1 for increasing trend in data;

 10

 15

 20

 25

 30

 35

 40

 0 0.5 1 1.5 2 Mean Response

dose

Exponential Model 4 with 0.95 Confidence Level

14:35 04/24 2012

BMDL BMD

 

Exponential
Toxicological Review of benzo[a]pyrene

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

C-19 DRAFT—DO NOT CITE OR QUOTE

1 sign = -1 for decreasing trend.

2

3 Model 2 is nested within Models 3 and 4.

4 Model 3 is nested within Model 5.

5 Model 4 is nested within Model 5.

6

7

8 Dependent variable = Mean

9 Independent variable = Dose

10 Data are assumed to be distributed: normally

11 Variance Model: exp(lnalpha +rho *ln(Y[dose]))

12 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

13

14 Total number of dose groups = 4

15 Total number of records with missing values = 0

16 Maximum number of iterations = 250

17 Relative Function Convergence has been set to: 1e-008

18 Parameter Convergence has been set to: 1e-008

19

20 MLE solution provided: Exact

21

22

23 Initial Parameter Values

24

25 Variable Model 4

26 -------- --------

27 lnalpha 0.666712

28 rho 1.04799

29 a 35.3094

30 b 1.97191

31 c 0.300675

32 d 1

33

34

35

36 Parameter Estimates

37

38 Variable Model 4

39 -------- -------

40 lnalpha 0.601192

41 rho 1.05452

42 a 34.3199

43 b 7.26795

44 c 0.325841

45 d 1

46

47 NC = No Convergence

48

49

50 Table of Stats From Input Data

51

52 Dose N Obs Mean Obs Std Dev

53 ----- --- ---------- -------------

54 0 20 33.63 8.924

55 0.02 20 31.94 8.633

56 0.2 20 16.56 5.744

57 2 20 11.15 5.117

58

59

60 Estimated Values of Interest

61

62 Dose Est Mean Est Std Scaled Residual

63 ------ ---------- --------- ----------------

64 0 34.32 8.713 -0.3551

65 0.02 31.19 8.285 0.4069

66 0.2 16.59 5.939 -0.02044

67 2 11.18 4.824 -0.03277

68

69

70

71 Other models for which likelihoods are calculated:

72

73 Model A1: Yij = Mu(i) + e(ij)

74 Var{e(ij)} = Sigma^2

75
Toxicological Review of benzo[a]pyrene

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

C-20 DRAFT—DO NOT CITE OR QUOTE

1 Model A2: Yij = Mu(i) + e(ij)

2 Var{e(ij)} = Sigma(i)^2

3

4 Model A3: Yij = Mu(i) + e(ij)

5 Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

6

7 Model R: Yij = Mu + e(i)

8 Var{e(ij)} = Sigma^2

9

10

11 Likelihoods of Interest

12

13 Model Log(likelihood) DF AIC

14 ------- ----------------- ---- ------------

15 A1 -197.0118 5 404.0235

16 A2 -192.448 8 400.896

17 A3 -192.5331 6 397.0662

18 R -238.8696 2 481.7393

19 4 -192.6894 5 395.3787

20

21

22 Additive constant for all log-likelihoods = -73.52. This constant added to the

23 above values gives the log-likelihood including the term that does not

24 depend on the model parameters.

25

26

27 Explanation of Tests

28

29 Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R)

30 Test 2: Are Variances Homogeneous? (A2 vs. A1)

31 Test 3: Are variances adequately modeled? (A2 vs. A3)

32

33 Test 6a: Does Model 4 fit the data? (A3 vs 4)

34

35

36 Tests of Interest

37

38 Test -2*log(Likelihood Ratio) D. F. p-value

39 -------- ------------------------ ------ --------------

40 Test 1 92.84 6 < 0.0001

41 Test 2 9.127 3 0.02764

42 Test 3 0.1701 2 0.9185

43 Test 6a 0.3126 1 0.5761

44

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

48 levels, it seems appropriate to model the data.

49

50 The p-value for Test 2 is less than .1. A non-homogeneous

51 variance model appears to be appropriate.

52

53 The p-value for Test 3 is greater than .1. The modeled

54 variance appears to be appropriate here.

55

56 The p-value for Test 6a is greater than .1. Model 4 seems

57 to adequately describe the data.

58

59

60 Benchmark Dose Computations:

61

62 Specified Effect = 1.000000

63

64 Risk Type = Estimated standard deviations from control

65

66 Confidence Level = 0.950000

67

68 BMD = 0.0650194

69

70 BMDL = 0.0432761
Toxicological Review of benzo[a]pyrene

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

C-21 DRAFT—DO NOT CITE OR QUOTE

1 Cervical epithelial hyperplasia – female ICR mice (Gao et al., 2011)

2 Table C-10. Incidence of cervical epithelial hyperplasia

Observation Dose (mg/kg-day)a

0 0.71 1.4 2.9

Cervical epithelial hyperplasia 0/26 4/26 6/25 7/24

3 a doses converted to mg/kg-day after adjustment for equivalent continuous dosing (2/7 

4 days/week)

5 Table C-11. Model predictions for increased incidence of epithelial 

6 hyperplasia in female ICR mice

Modela

Goodness-of-fit

p-value AIC

BMD1SD

(mg/kg-d)

BMDL1SD

(mg/kg-d)

Gamma 0.6874 82.2821 0.659 0.452

Logistic 0.1422 88.4607 1.422 1.052

Log-logistic 0.8360 81.7004 0.578 0.369

Probit 0.1544 88.1151 1.326 0.979

Log-Probit 0.0775 88.2004 1.012 0.686

Multistage 0.6874 82.2821 0.659 0.452

7

8 ==================================================================== 

9 Logistic Model. (Version: 2.13; Date: 10/28/2009) 

10 Input Data File: C:\Users\hclynch\Documents\_Active Projects\_FA498 IRIS\xBaP\IASC Aug 

11 2011\bmd modeling\lnl_gao 2011 inflamm cells_Opt.(d)

12 Gnuplot Plotting File: C:\Users\hclynch\Documents\_Active Projects\_FA498 

13 IRIS\xBaP\IASC Aug 2011\bmd modeling\lnl_gao 2011 inflamm cells_Opt.plt

14 ==================================================================== 

15

16 BMDS_Model_Run 

17 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

18

19 The form of the probability function is: 

20

21 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

22

23

24 Dependent variable = Col3

25 Independent variable = Col1

26 Slope parameter is restricted as slope >= 1

27

28 Total number of observations = 4

29 Total number of records with missing values = 0

30 Maximum number of iterations = 250

31 Relative Function Convergence has been set to: 1e-008

32 Parameter Convergence has been set to: 1e-008

33

34

35

36 User has chosen the log transformed model

37

38

39 Default Initial Parameter Values 

40 background = 0

41 intercept = -1.60901

42 slope = 1

43
Toxicological Review of benzo[a]pyrene

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

C-22 DRAFT—DO NOT CITE OR QUOTE

1

2 Asymptotic Correlation Matrix of Parameter Estimates

3

4 ( *** The model parameter(s) -background -slope 

5 have been estimated at a boundary point, or have been specified by the user,

6 and do not appear in the correlation matrix )

7

8 intercept

9

10 intercept 1

11

12

13

14 Parameter Estimates

15

16 95.0% Wald Confidence Interval

17 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

18 background 0 * * *

19 intercept -1.6502 * * *

20 slope 1 * * *

21

22 * - Indicates that this value is not calculated.

23

24

25

26 Analysis of Deviance Table

27

28 Model Log(likelihood) # Param's Deviance Test d.f. P-value

29 Full model -39.4267 4

30 Fitted model -39.8502 1 0.847034 3 0.8382

31 Reduced model -45.7739 1 12.6945 3 0.005346

32

33 AIC: 81.7004

34

35

36 Goodness of Fit 

37 Scaled

38 Dose Est._Prob. Expected Observed Size Residual

39 ------------------------------------------------------------------------

40 0.0000 0.0000 0.000 0.000 26 0.000

41 0.7100 0.1200 3.119 4.000 26 0.532

42 1.4000 0.2119 5.297 6.000 25 0.344

43 2.9000 0.3577 8.584 7.000 24 -0.675

44

45 Chi^2 = 0.86 d.f. = 3 P-value = 0.8360

46

47

48 Benchmark Dose Computation

49

50 Specified effect = 0.1

51

52 Risk Type = Extra risk 

53

54 Confidence level = 0.95

55

56 BMD = 0.578668

57

58 BMDL = 0.368701

59

60
Toxicological Review of benzo[a]pyrene

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

C-23 DRAFT—DO NOT CITE OR QUOTE

1 INHALATION DOSIMETRY MODELING FOR RFC DERIVATION

2

3

4 Figure C-6. Human fractional deposition.

5 Species = humanlimited

6 FRC = 3300.0

7 Head volume = 50.0

8 Density = 1.0

9 Number of particles calculated = single

10 Diameter = 1.7000000000000002 μm MMAD

11 Inhalability = yes

12 GSD = 1.0

13 Breathing interval: One single breath

14 Concentration = 4.2

15 Breathing Frequency = 16.0

16 Tidal Volume = 860.0

17 Inspiratory Fraction = 0.5

18 Pause Fraction = 0.0

19 Breathing Route = nasal

20

21 Region: Entire Lung

22 Region: Entire Lung

23 Region Deposition Fraction

24 -- --
Toxicological Review of benzo[a]pyrene

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

C-24 DRAFT—DO NOT CITE OR QUOTE

1 Head 0.449

2 TB 0.045

3 P 0.127

4 Total 0.621

5

6

7

8 Figure C-7. Rat fractional deposition.

9 Species = rat

10 FRC = 4.0

11 Head volume = 0.42

12 Density = 1.0

13 Number of particles calculated = single

14 Diameter = 1.7000000000000002 μm MMAD

15 Inhalability = yes

16 GSD = 1.0

17 Breathing interval: One single breath

18 Concentration = 4.2

19 Breathing Frequency = 102.0

20 Tidal Volume = 1.8

21 Inspiratory Fraction = 0.5

22 Pause Fraction = 0.0

23 Breathing Route = nasal

24
Toxicological Review of benzo[a]pyrene

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

C-25 DRAFT—DO NOT CITE OR QUOTE

1 Region: Entire Lung

2 Region: Entire Lung

3 Region Deposition Fraction

4 -- --

5 Head 0.072

6 TB 0.041

7 P 0.068

8 Total 0.181

9
Toxicological Review of benzo[a]pyrene

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

C-26 DRAFT—DO NOT CITE OR QUOTE

1 DOSE-RESPONSE MODELING FOR CANCER RISK VALUES

2 The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend that the 

3 method used to characterize and quantify cancer risk from a chemical is determined by what is 

4 known about the mode of action of the carcinogen and the shape of the cancer dose-response curve. 

5 No biologically based models for BaP carcinogenicity following oral, inhalation, or dermal exposure 

6 were identified. 

7 Methods for the Oral Slope Factor and Inhalation Unit Risk

8 Due to the occurrence of multiple tumor types, earlier occurrence with increasing exposure, 

9 and early termination of the high-dose group in each of the oral and inhalation carcinogenicity 

10 studies (see Appendix B for study details), methods that can reflect the influence of competing risks 

11 and intercurrent mortality on site-specific tumor incidence rates are preferred. EPA has generally 

12 used a model that incorporates the time at which death-with-tumor occurred as well as the dose; 

13 the multistage-Weibull model is multistage in dose and Weibull in time, and has the form:

14

P(d, t) = 1 - exp[-(q0 + q1d + q2d2 + ... + qkdk) × (t ± t0)

c 15 ],

16

17 where P(d, t) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent 

18 exposure in this case) and age t (in bioassay weeks); parameters qi ≥ 0, for i = 0, 1, ..., k; t is the time 

19 at which the tumor was observed; and c is a parameter which characterizes the change in response 

20 with age. The parameter t0 represents the time between when a potentially fatal tumor becomes 

21 observable and when it causes death, and is generally set to 0 either when all tumors are 

22 considered incidental or because of a lack of data to estimate the time reliably. The dose-response 

23 analyses were conducted using the computer software program MultiStage-Weibull (U.S. EPA, 

24 2010), which is based on Weibull models drawn from Krewski et al. (1983). Parameters were 

25 estimated using the method of maximum likelihood. From specific model fits using stages up to n-1, 

26 where n is the number of dose groups, the model fit with the lowest AIC was selected.

27 Two general characteristics of the observed tumor types were considered prior to 

28 modeling; allowance for different, although unidentified modes of action, and allowance for relative 

29 severity of tumor types. First, etiologically different tumor types were not combined across sites 

30 prior to modeling (that is, overall counts of tumor-bearing animals were not tabulated) in order to 

31 allow for the possibility that different tumor types could have different dose-response relationships 

32 due to different underlying mechanisms or factors, such as latency. Consequently, all of the tumor 

33 types were also modeled separately.

34 Additionally, the multistage-Weibull model can address relative severity of tumor types by 

35 distinguishing between tumors as being either fatal or incidental to the death of an animal in order 

36 to adjust partially for competing risks. In contrast to fatal tumors, incidental tumors are those 

37 tumors thought not to have caused the death of an animal. Cause-of-death information for most 
Toxicological Review of benzo[a]pyrene

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

C-27 DRAFT—DO NOT CITE OR QUOTE

1 early animal deaths was provided by the investigators of both of the bioassays. In the rat study, 

2 tumors of the forestomach or liver were the principal cause of death for most animals dying or 

3 sacrificed (due to moribundity) before the end of the study, while tumors of the forestomach were 

4 the most common cause of early deaths in the mouse study.

5 PODs for estimating low-dose risk were identified at doses at the lower end of the observed 

6 data, generally corresponding to 10% extra risk, where extra risk is defined as [P(d) - P(0)]/[1 -

7 P(0)]. The lifetime oral cancer slope factor for humans is defined as the slope of the line from the 

8 lower 95% bound on the exposure at the POD to the control response (slope factor = 0.1/BMDL10). 

9 This slope, a 95% upper confidence limit (UCL) represents a plausible upper bound on the true risk.

10 Although the time-to-tumor modeling helps account for competing risks associated with 

11 decreased survival times and other tumors, considering the tumor sites individually still does not 

12 convey the total amount of risk potentially arising from the sensitivity of multiple sites—that is, the 

13 risk of developing any combination of the increased tumor types, not just the risk of developing all 

14 simultaneously. One approach suggested in the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 

15 2005a) would be to estimate cancer risk from tumor-bearing animals. EPA traditionally used this 

16 approach until the National Resource Council (NRC) document Science and Judgment (NRC, 1994) 

17 made a case that this approach would tend to underestimate overall risk when tumor types occur in 

18 a statistically independent manner. In addition, application of one model to a composite data set 

19 does not accommodate biologically relevant information that may vary across sites or may only be 

20 available for a subset of sites. For instance, the time courses of the multiple tumor types evaluated 

21 varied, as is suggested by the variation in estimates of c, from 1.5 (e.g., male rat skin or mammary 

22 gland basal cell tumors), indicating relatively little effect of age on tumor incidence, to 3.7 (e.g., male 

23 mouse alimentary tract tumors), indicating a more rapidly increasing response with increasing age 

24 (in addition to exposure level). The result of fitting a model with parameters that can reflect 

25 underlying mechanisms, such as z in the multistage-Weibull model, would be difficult to interpret 

26 with composite data (i.e., counts of tumor-bearing animals). A simpler model, such as the 

27 multistage model, could be used for the composite data but relevant biological information would 

28 then be ignored.

29 Following the recommendations of the NRC (1994) regarding combining risk estimates, 

30 statistical methods that can accommodate the underlying distribution of slope factors are optimal, 

31 such as through maximum likelihood estimation or through bootstrapping or Bayesian analysis. 

32 However, these methods have not yet been extended to models such as the multistage-Weibull 

33 model. A method involving the assumption that the variability in the slope factors could be 

34 characterized by a normal distribution is detailed below (U.S. EPA, 2010). Using the results in 

35 female rats to illustrate, the overall risk estimate involved the following steps:

36

37 (1) It was assumed that the tumor groupings modeled above were statistically independent—

38 that is, that the occurrence of a liver tumor was not dependent upon whether there was a 
Toxicological Review of benzo[a]pyrene

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

C-28 DRAFT—DO NOT CITE OR QUOTE

1 forestomach tumor. This assumption cannot currently be verified, and if not correct, could 

2 lead to an overestimate of risk from summing across tumor sites. However, NRC (1994) 

3 argued that a general assumption of statistical independence of tumor-type occurrences 

4 within animals was not likely to introduce substantial error in assessing carcinogenic 

5 potency from rodent bioassay data.

6

7 (2) The models previously fitted to estimate the BMDs and BMDLs were used to extrapolate to a 

8 lower level of risk (R), in order to reach the region of each estimated dose-response 

9 function where the slope was reasonably constant and upper bound estimation was still 

numerically stable. For these data, a 10-3 10 risk was generally the lowest risk necessary. The 

11 oral slope factor for each site was then estimated by R/BMDLR, as for the estimates for each 

12 tumor site above.

13

14 (3) The maximum likelihood estimates (MLE) of unit potency (that is, risk per unit of exposure) 

15 estimated by R/BMDR, were summed across the alimentary tract, liver, and 

16 jejunum/duodenum in female rats.

17

18 (4) An estimate of the 95% (one-sided) upper bound on the summed oral slope factor was 

19 calculated by assuming a normal distribution for the individual risk estimates, and deriving 

20 the variance of the risk estimate for each tumor site from its 95% UCL according to the 

21 formula:

22

23 95% UCL = MLE + 1.645 × SD,

24 rearranged to:

25 s.d. = (UCL – MLE) / 1.645,

26

27 where 1.645 is the t-statistic corresponding to a one-sided 95% CI and >120 degrees of freedom, 

and the SD is the square root of the variance of the MLE. The variances (variance = SD2 28 ) for each 

29 site-specific estimate were summed across tumor sites to obtain the variance of the sum of the 

30 MLEs. The 95% UCL on the sum of MLEs was calculated from the expression above for the UCL, 

using the variance of the sum of the MLE to obtain the relevant SD (SD = variance1/2 31 ).

32 The results of this analysis are provided in Table C-17.
Toxicological Review of benzo[a]pyrene

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

C-29 DRAFT—DO NOT CITE OR QUOTE

1 Table C-12. Tumor incidence data, with time to death with tumor; male 

2 rats exposed by gavage to benzo[a]pyrene—Kroese et al. (2001)

Dose 

(mg/kg-d)

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

IncidentalaFatalaIncidental Fatal Incidental Incidental Incidental Incidental

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
Toxicological Review of benzo[a]pyrene

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

C-30 DRAFT—DO NOT CITE OR QUOTE

Dose 

(mg/kg-d)

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

IncidentalaFatalaIncidental Fatal Incidental Incidental Incidental Incidental

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

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.
Toxicological Review of benzo[a]pyrene

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

C-31 DRAFT—DO NOT CITE OR QUOTE

1 Table C-13. Tumor incidence data, with time to death with tumor; 

2 female rats exposed by gavage to benzo[a]pyrene—Kroese et al. (2001)

Dose 

(mg/kg-d)

Week of 

death

Total 

examined

Numbers of animals with

Oral cavity or forestomach 

tumors Liver tumors

Duodenum or 

jejunum tumors

IncidentalaFatalaIncidental 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

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

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

0

0

0

0

0

0

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

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

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0
Toxicological Review of benzo[a]pyrene

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

C-32 DRAFT—DO NOT CITE OR QUOTE

Dose 

(mg/kg-d)

Week of 

death

Total 

examined

Numbers of animals with

Oral cavity or forestomach 

tumors Liver tumors

Duodenum or 

jejunum tumors

IncidentalaFatalaIncidental Fatal Incidental

104

105

106

107

108

109

1

2

1

5

7

4

1

1

1

5

7

2

0

0

0

0

0

0

1

1

0

5

7

2

0

1

1

0

0

0

0

0

0

0

0

0

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.
Toxicological Review of benzo[a]pyrene

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

C-33 DRAFT—DO NOT CITE OR QUOTE

1 Table C-14. Tumor incidence, with time to death with tumor; female 

2 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

FatalaIncidental

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
Toxicological Review of benzo[a]pyrene

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

C-34 DRAFT—DO NOT CITE OR QUOTE

Dose group

(ppm in diet) Week of death Total examined

Number of animals with alimentary tract squamous cell tumors

FatalaIncidental

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.

1 Table C-15. Derivation of HEDs to use for BMD modeling of Wistar rat 

2 tumor incidence data from Kroese et al. (2001)

Benzo[a]pyrene dose (mg/kg-d) TWA body weight (kg)

Interspecies scaling 

factoraHEDb(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.
Toxicological Review of benzo[a]pyrene

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

C-35 DRAFT—DO NOT CITE OR QUOTE

1 Table C-16. Derivation of HEDs for dose-response modeling of B6C3F1

2 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.

c

HED = administered dose × scaling factor.

3

4
Toxicological Review of benzo[a]pyrene

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

C-36 DRAFT—DO NOT CITE OR QUOTE

1 Table C-17. Summary of model selection and modeling results for best2 fitting multistage-Weibull models, using time-to-tumor data for rats

3 from Kroese et al. (1981)

Endpoints

Model

stages AIC BMD10 BMDL10 − BMDU10 Model selection rationale

Male rats Oral cavity and 

forestomach: 

squamous cell tumors

1

2

3

577.8

407.6

229.0

0.104

0.678

0.453 0.281 – 0.612 Lowest AIC, best fit to low dose data

Hepatocellular tumors 1

2

3

367.3

301.5

289.1

0.181

0.472

0.651 0.449 –0.772 Lowest AIC, best fit to low dose data

Duodenum and 

jejunum tumors

1

2

3

69.6

65.9

66.9

2.64

3.04

3.03 2.38 – 3.87 Best fit to data

Kidney: uroethelial 

carcinoma

1

2

3

31.9

31.7

32.8

9.16

5.71

4.65 2.50 – 9.01 Best fit to data

Skin and mammary 

gland: basal cell 

tumors

1

2

3

110.6

105.1

104.7

1.88

2.58

2.86 2.35 – 3.62 Lowest AIC, best fit to low dose data

Skin and mammary 

gland: squamous cell 

tumors

1

2

3

63.5

64.3

65.3

3.36

2.75

2.64

1.77 – 4.42 Lowest AIC, best fit to low dose data

Female rats Oral cavity and 

forestomach: 

squamous cell tumors

1

2

3

277.1

211.6

201.0

0.245

0.428

0.539 0.328 – 0.717 Lowest AIC, best fit to low dose data

Hepatocellular tumors 1

2

3

595.5

774.9

468.3

0.146

0.370

0.575 0.507 – 0.630 Lowest AIC, best fit to low dose data

Duodenum and 

jejunum tumors

1

2

3

37.9

37.0

37.8

6.00

4.33

3.43 1.95 – 5.70 Best fit to low dose data
Toxicological Review of benzo[a]pyrene

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

C-37 DRAFT—DO NOT CITE OR QUOTE

1 Male Rat (Kroese et al., 2001): Squamous Cell Papilloma or Carcinoma in Oral Cavity or Forestomach

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: OralForstKroeseM3.(d)

7 =======================================================================

8 The form of the probability function is: 

9 P[response] = 1-EXP{-(t - t_0)^c *

10 (beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)}

11

12 The parameter betas are restricted to be 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 = 0

21 Degree of polynomial = 3

22

23 Maximum number of iterations = 64

24 Relative Function Convergence has been set to: 2.22045e-016

25 Parameter Convergence has been set to: 1.49012e-008

26

27

28 Default Initial Parameter Values

29 c = 3.6

30 t_0 = 39.1111

31 beta_0 = 0

32 beta_1 = 8.8911e-009

33 beta_2 = 1.60475e-031

34 beta_3 = 1.95818e-008

35

36

37 Asymptotic Correlation Matrix of Parameter Estimates

38 ( *** The model parameter(s) -beta_0 -beta_2 

39 have been estimated at a boundary point, or have been specified by the user,

40 and do not appear in the correlation matrix )

41

42 c t_0 beta_1 beta_3

43

44 c 1 -0.53 -0.93 -0.99

45

46 t_0 -0.53 1 0.47 0.57

47

48 beta_1 -0.93 0.47 1 0.9

49

50 beta_3 -0.99 0.57 0.9 1

51

52

53 Parameter Estimates

54 95.0% Wald Confidence Interval

55 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

56 c 3.74559 0.447309 2.86888 4.6223

57 t_0 41.4581 2.14975 37.2447 45.6716

58 beta_0 0 NA

59 beta_1 4.37816e-009 1.07528e-008 -1.6697e-008 2.54533e-008

60 beta_2 0 NA

61 beta_3 1.01904e-008 1.94164e-008 -2.78651e-008 4.82458e-008

62

63 NA - Indicates that this parameter has hit a bound implied by some inequality constraint

64 and thus has no standard error.

65

66

67 Log(likelihood) # Param AIC

68 Fitted Model -108.512 6 229.024

69

70

71 Data Summary 

72 CONTEXT
Toxicological Review of benzo[a]pyrene

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

C-38 DRAFT—DO NOT CITE OR QUOTE

1 C F I U Total Expected Response

2 DOSE

3 0 52 0 0 0 52 0.00

4 0.54 44 0 8 0 52 6.77

5 1.8 7 0 45 0 52 41.69

6 5.2 0 9 43 0 52 49.97

7

8 Minimum observation time for F tumor context = 44

9

10

11 Benchmark Dose Computation

12 Risk Response = Incidental

13 Risk Type = Extra

14 Confidence level = 0.9

15 Time = 104

16

17

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

18

19

20
Toxicological Review of benzo[a]pyrene

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

C-39 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 
Toxicological Review of benzo[a]pyrene

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

C-40 DRAFT—DO NOT CITE OR QUOTE

1 CONTEXT

2 C F I U Total Expected Response

3 DOSE

4 0 52 0 0 0 52 0.00

5 0.54 48 0 4 0 52 3.38

6 1.8 14 2 36 0 52 36.81

7 5.2 3 17 32 0 52 49.55

8

9 Minimum observation time for F tumor context = 52

10

11

12 Benchmark Dose Computation

13 Risk Response = Incidental

14 Risk Type = Extra

15 Confidence level = 0.9

16 Time = 104

17

18

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

19

20

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)
Toxicological Review of benzo[a]pyrene

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

C-41 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
Toxicological Review of benzo[a]pyrene

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

C-42 DRAFT—DO NOT CITE OR QUOTE

1 DOSE

2 0 52 0 0 0 52 0.00

3 0.54 52 0 0 0 52 0.03

4 1.8 51 0 1 0 52 1.04

5 5.2 43 0 9 0 52 8.96

6

7

8 Benchmark Dose Computation

9 Risk Response = Incidental

10 Risk Type = Extra

11 Specified effect = 0.1

12 Confidence level = 0.9

13 Time = 104

14

15

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

16

17

18

19

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
Toxicological Review of benzo[a]pyrene

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

C-43 DRAFT—DO NOT CITE OR QUOTE

1

2 Male Rat (Kroese et al., 2001): Skin or Mammary Gland Basal Cell Tumors

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: SKinMamBasalKroeseM3.(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 = 1

23 Degree of polynomial = 3

24

25

26

27 User specifies the following parameters:

28 t_0 = 0

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 = 1.38462

37 t_0 = 0 Specified

38 beta_0 = 3.84298e-005

39 beta_1 = 1.06194e-028

40 beta_2 = 0

41 beta_3 = 6.84718e-006

42

43

44 Asymptotic Correlation Matrix of Parameter Estimates

45 ( *** The model parameter(s) -t_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 beta_0 beta_3

50

51 c 1 -1 -1

52

53 beta_0 -1 1 0.99

54

55 beta_3 -1 0.99 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 1.47227 1.76686 -1.9907 4.93525

62 beta_0 2.54786e-005 0.000211261 -0.000388585 0.000439542

63 beta_1 0 NA

64 beta_2 0 NA

65 beta_3 4.81611e-006 3.49e-005 -6.35866e-005 7.32188e-005

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
Toxicological Review of benzo[a]pyrene

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

C-44 DRAFT—DO NOT CITE OR QUOTE

1 Fitted Model -47.3623 5 104.725

2

3

4 Data Summary 

5 CONTEXT

6 C F I U Total Expected Response

7 DOSE

8 0 50 0 2 0 52 1.18

9 0.54 51 0 1 0 52 1.22

10 1.8 51 0 1 0 52 2.32

11 5.2 39 0 13 0 52 12.54

12

13

14 Benchmark Dose Computation

15 Risk Response = Incidental

16 Risk Type = Extra

17 Confidence level = 0.9

18 Time = 104

19

20

21

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

22

23

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
Toxicological Review of benzo[a]pyrene

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

C-45 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
Toxicological Review of benzo[a]pyrene

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

C-46 DRAFT—DO NOT CITE OR QUOTE

1 C F I U Total Expected Response

2 DOSE

3 0 52 0 0 0 52 0.00

4 0.54 51 0 1 0 52 0.42

5 1.8 51 0 1 0 52 2.12

6 5.2 46 0 6 0 52 5.51

7

8

9 Benchmark Dose Computation

10 Risk Response = Incidental

11 Risk Type = Extra

12 Confidence level = 0.9

13 Time = 104

14

15

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

16

17
Toxicological Review of benzo[a]pyrene

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

C-47 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
Toxicological Review of benzo[a]pyrene

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

C-48 DRAFT—DO NOT CITE OR QUOTE

1 C F I U Total Expected Response

2 DOSE

3 0 52 0 0 0 52 0.00

4 0.54 52 0 0 0 52 0.01

5 1.8 52 0 0 0 52 0.29

6 5.2 49 0 3 0 52 2.71

7

8

9 Benchmark Dose Computation

10 Risk Response = Incidental

11 Risk Type = Extra

12 Confidence level = 0.9

13 Time = 104

14

15

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

16

17

18

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
Toxicological Review of benzo[a]pyrene

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

C-49 DRAFT—DO NOT CITE OR QUOTE

1 Female Rat (Kroese et al., 2001): Oral Cavity or Forestomach, Squamous Cell Papilloma 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: OralForstKroeseF3.(d)

7 =======================================================================

8 

9 The form of the probability function is: 

10 P[response] = 1-EXP{-(t - t_0)^c *

11 (beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)}

12

13 The parameter betas are restricted to be 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 = 45.1111

33 beta_0 = 1.11645e-009

34 beta_1 = 4.85388e-009

35 beta_2 = 0

36 beta_3 = 1.95655e-008

37

38 Asymptotic Correlation Matrix of Parameter Estimates

39 ( *** The model parameter(s) -beta_2 

40 have been estimated at a boundary point, or have been specified by the user,

41 and do not appear in the correlation matrix )

42

43 c t_0 beta_0 beta_1 beta_3

44

45 c 1 -0.79 -0.92 -0.93 -1

46

47 t_0 -0.79 1 0.73 0.72 0.8

48

49 beta_0 -0.92 0.73 1 0.79 0.92

50

51 beta_1 -0.93 0.72 0.79 1 0.91

52

53 beta_3 -1 0.8 0.92 0.91 1

54

55

56 Parameter Estimates

57 95.0% Wald Confidence Interval

58 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

59 c 3.52871 0.701117 2.15454 4.90287

60 t_0 46.553 5.93306 34.9244 58.1816

61 beta_0 1.53589e-009 5.40523e-009 -9.05817e-009 1.21299e-008

62 beta_1 7.57004e-009 2.9647e-008 -5.05369e-008 6.5677e-008

63 beta_2 0 NA

64 beta_3 2.53126e-008 7.66404e-008 -1.249e-007 1.75525e-007

65

66 NA - Indicates that this parameter has hit a bound implied by some inequality constraint

67 and thus has no standard error.

68

69

70 Log(likelihood) # Param AIC

71 Fitted Model -94.5119 6 201.024

72

73
Toxicological Review of benzo[a]pyrene

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

C-50 DRAFT—DO NOT CITE OR QUOTE

1 Data Summary 

2 CONTEXT

3 C F I U Total Expected Response

4 DOSE

5 0 51 0 1 0 52 1.14

6 0.49 46 0 6 0 52 4.90

7 1.6 22 0 30 0 52 31.81

8 4.6 2 7 43 0 52 49.43

9

10 Minimum observation time for F tumor context = 58

11

12 Benchmark Dose Computation

13 Risk Response = Incidental

14 Risk Type = Extra

15 Confidence level = 0.9

16 Time = 104

17

Specified effect = 

BMD = 

BMDL = 

 BMDU = 

0.1

0.538801

0.328135

0.717127

0.01

0.0981283

0.0345104

0.325

909

0.001

0.0100797

0.00344714

> 

0.0806373

18

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)
Toxicological Review of benzo[a]pyrene

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

C-51 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
Toxicological Review of benzo[a]pyrene

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

C-52 DRAFT—DO NOT CITE OR QUOTE

1

2

3 Data Summary 

4 CONTEXT

5 C F I U Total Expected Response

6 DOSE

7 0 52 0 0 0 52 0.00

8 0.49 51 0 1 0 52 3.02

9 1.6 13 12 27 0 52 38.36

10 4.6 1 38 13 0 52 51.36

11

12 Minimum observation time for F tumor context = 44

13

14

15 Benchmark Dose Computation

16 Risk Response = Incidental

17 Risk Type = Extra

18 Confidence level = 0.9

19 Time = 104

20

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

21

22

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)
Toxicological Review of benzo[a]pyrene

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

C-53 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
Toxicological Review of benzo[a]pyrene

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

C-54 DRAFT—DO NOT CITE OR QUOTE

1 0 52 0 0 0 52 0.00

2 0.49 52 0 0 0 52 0.01

3 1.6 52 0 0 0 52 0.44

4 4.6 48 0 4 0 52 3.57

5

6

7 Benchmark Dose Computation

8 Risk Response = Incidental

9 Risk Type = Extra

10 Confidence level = 0.9

11 Time = 104

12

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

13

14

15

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
Toxicological Review of benzo[a]pyrene

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

C-55 DRAFT—DO NOT CITE OR QUOTE

1 Table C-18. Summary of human equivalent overall oral slope factors, 

2 based on male and female rat tumor incidence

Data set Tumor site BMD001 BMDL001

Risk valueaat

SD SD2

Properties 

of total 

BMD001 BMDL001 variance

Males Oral cavity/forestomach 6.37 × 10-32.86 × 10-31.57 × 10-13.50 × 10-11.17 × 10-11.38 × 10-20.64

Liver 2.00 × 10-25.30 × 10-35.00 × 10-21.89 × 10-18.42 × 10-27.09 × 10-30.33

Duodenum/jejunum 6.42 × 10-14.21 × 10-21.56 × 10-32.38 × 10-21.35 × 10-21.82 × 10-40.01

Skin/mammary gland: 

basal cell 6.06 × 10-1

4.24 × 10-21.65 × 10-32.36 × 10-21.33 × 10-21.78 × 10-40.01

Skin/mammary gland: 

squam. cell 7.06 × 10-2

2.11 × 10-21.42 × 10-24.75 × 10-22.03 × 10-24.10 × 10-40.02

Kidney 9.84 × 10-17.48 × 10-21.02 × 10-31.34 × 10-27.51 × 10-35.64 × 10-50.00

Sum, risk values at BMD001: 2.25 × 10-1Sum, SD2: 2.17 × 10-2

Overall SDb: 1.47 × 10-1

Upper bound on sum of risk estimatesc: 4.68 × 10-1

Females Oral cavity/forestomach 3.45 × 10-31.01 × 10-22.90 × 10-19.92 × 10-21.16 × 10-11.35 × 10-20.91

Liver 1.53 × 10-21.22 × 10-16.54 × 10-28.21 × 10-33.48 × 10-21.21 × 10-30.08

Duodenum/jejunum 5.85 × 10-27.27 × 10-11.71 × 10-21.38 × 10-39.56 × 10-39.13 × 10-50.01

Sum, risk values at BMD001: 1.09 × 10-1Sum, SD2: 1.48 × 10-2

Overall SD: 1.22 × 10-1

Upper bound on sum of risk estimatesc: 3.09E-01

a

Risk value = 0.001/BMDL001.

bOverall SD = (sum, SD2

)

0.5

.

cUpper bound on the overall risk estimate = sum of BMD001 risk values + 1.645 × overall SD.

Source of data: Kroese et al. (2001).

3 Table C-19. Summary of model selection among multistage-Weibull 

4 models fit to alimentary tract tumor data for female mice

Model

stages AIC BMD10 BMDL10 − BMDU10 Model selection rationale

1

2

3

688.5

629.2

624.5

0.104

0.102

0.127 0.071 −0.179 Lowest AIC, best fit to low dose data

5

6 Source of data: Beland and Culp (1998)

7
Toxicological Review of benzo[a]pyrene

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

C-56 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
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1 0 47 0 1 0 48 0.93

2 0.1 45 0 3 0 48 3.21

3 0.48 8 23 15 1 47 30.82

4 2.3 1 46 0 1 48 41.91

5

6 Minimum observation time for F tumor context = 39

7

8

9 Benchmark Dose Computation

10 Risk Response = Incidental

11 Risk Type = Extra

12 Specified effect = 0.1

13 Confidence level = 0.9

14

15 Time = 104

16

17 BMD = 0.126983

18 BMDL = 0.0706103

19 BMDU = 0.179419

20

21

22
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1 DOSE-RESPONSE MODELING FOR THE INHALATION UNIT RISK

2 As with the tumor data used for the oral slope factor (see Dose Response-modeling for the 

3 Oral Slope Factor Section), there was earlier occurrence of tumors with increasing exposure, and 

4 early termination of the high-dose group (Thyssen et al., 1981; see Appendix B for study details). 

5 The computer software program MSW (U.S. EPA, 2010) was used as described in the analysis of the 

6 oral carcinogenicity data. 

7 Thyssen et al. (1981) did not determine cause of death for any of the animals. Bounding 

8 estimates for the Thyssen et al. (1981) data were developed by treating the tumors alternately as 

9 either all incidental or all fatal. In either case, therefore, an estimate of t0 (the time between a 

10 tumor first becoming observable and causing death) could not be estimated. The data analyzed are 

11 summarized in Table C-20, the results are summarized in Table C-21, and the modeling details 

12 follow.

13 Table C-20. Individual pathology and tumor occurrence data for male 

14 Syrian hamsters exposed to benzo[a]pyrene via inhalation for lifetime—

15 Thyssen et al. (1981).

Nominal 

exposure 

concentration 

(mg/m3)

Time on 

study

Number 

examined

Papillomas, Polyps, Papillary polyps, Squamous cell carcinomas

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

1

1

1

1

1

1

1

0

a

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
Toxicological Review of benzo[a]pyrene

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Nominal 

exposure 

concentration 

(mg/m3)

Time on 

study

Number 

examined

Papillomas, Polyps, Papillary polyps, Squamous cell carcinomas

Larynx Pharynx Trachea Esophagus Forestomach Nasal cavity

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

2

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

a

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

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

c

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

1

1

2

1

1

1

2

1

2

1

1

1

1

1

1

1

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

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

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

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0
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Nominal 

exposure 

concentration 

(mg/m3)

Time on 

study

Number 

examined

Papillomas, Polyps, Papillary polyps, Squamous cell carcinomas

Larynx Pharynx Trachea Esophagus Forestomach Nasal cavity

60

63

64

66

68

70

71

72

73

79

1

1

1

1

1

1

1

1

2

4

0

0

0

1

0

1

1

1

2

3

1

0

1

1

1

1

1

1

2

4

0

0

0

0

0

0

1

0

0

1

0

0

0

0

0

1

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

a

Tissue was not examined for one animal of total examined.

b

Tissue was not examined for two animals of total examined.

c

An adenocarcinoma was observed in this tissue, but not included in the dose-response analysis because it was of a different 

cell type than the other tumors listed. It was judged to be an isolated finding not clearly associated with exposure.

1 Table C-21. Summary of model selection among multistage-Weibull 

2 models fit to tumor data for male hamsters

Tumor context

Model

stages AIC BMD10 BMDL10 Model selection rationale

All tumors considered incidental to 

cause of death

1

2

58.0

47.9

0.090

0.285

0.064

0.198 Lowest AIC, best fit to data 

(BMDU10 = 0.350)

All tumors considered to be cause 

of death

1

2

3

327.3

302.9

299.0

0.136

0.421

0.648

0.104

0.343

0.461 Lowest AIC; best fit to data

(BMDU10 = 0.719)

3 Data source: Thyssen et al. (1981)

4 Output for squamous cell neoplasia following inhalation exposure to BaP: all tumors considered 

5 incidental to cause of death

6

7 =======================================================================

8 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)

9 Solutions are obtained using donlp2-intv, (c) by P. Spellucci

10 Input Data File: C:\msw\benzo[a]pyrene-Thyssen_inc2st.(d)

11 =======================================================================

12

13 The form of the probability function is: 

14 P[response] = 1-EXP{-(t - t_0)^c *

15 (beta_0+beta_1*dose^1+beta_2*dose^2)}

16

17 The parameter betas are restricted to be positive

18

19 Dependent variable = Class

20 Independent variables = Conc, Time

21

22 Total number of observations = 96

23 Total number of records with missing values = 0

24 Total number of parameters in model = 5

25 Total number of specified parameters = 1

26 Degree of polynomial = 2

27

28

29

30 User specifies the following parameters:

31 t_0 = 0
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1

2 Maximum number of iterations = 32

3 Relative Function Convergence has been set to: 1e-008

4 Parameter Convergence has been set to: 1e-008

5

6

7 Default Initial Parameter Values

8 c = 3.6

9 t_0 = 0 Specified

10 beta_0 = 1.18657e-031

11 beta_1 = 1.49e-030

12 beta_2 = 6.10362e-008

13

14

15 Asymptotic Correlation Matrix of Parameter Estimates

16 ( *** The model parameter(s) -t_0 -beta_0 -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 c beta_2

21

22 c 1 -1

23

24 beta_2 -1 1

25

26

27 Parameter Estimates

28 95.0% Wald Confidence Interval

29 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

30 c 4.21938 0.840997 2.57105 5.8677

31 beta_0 0 NA

32 beta_1 0 NA

33 beta_2 4.00402e-009 1.495e-008 -2.52974e-008 3.33054e-008

34

35 NA - Indicates that this parameter has hit a

36 bound implied by some inequality constraint

37 and thus has no standard error.

38

39

40 Log(likelihood) # Param AIC

41 Fitted Model -19.967 4 47.9339

42

43

44 Data Summary 

45 Class

46 C F I U Total Expected Response

47 Conc

48 0 23 0 0 0 23 0.00

49 0.25 24 0 0 0 24 1.92

50 1 8 0 18 0 26 16.04

51 4.3 5 0 18 0 23 18.22

52

53

54 Benchmark Dose Computation

55 Risk Response = Incidental

56 Risk Type = Extra

57 Specified effect = 0.1

58 Confidence level = 0.9

59

60 Time = 104

61

62 BMD = 0.284958

63 BMDL = 0.197807

64 BMDU = 0.350247

65

66
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1

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
Toxicological Review of benzo[a]pyrene

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C-63 DRAFT—DO NOT CITE OR QUOTE

1 Output for respiratory 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 
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C-64 DRAFT—DO NOT CITE OR QUOTE

1 Class

2 C F I U Total 

3 Conc

4 0 23 0 0 0 23 

5 0.25 24 0 0 0 24 

6 1 8 18 0 0 26 

7 4.3 5 18 0 0 23 

8

9 Minimum observation time for F tumor context = 40

10

11

12 Benchmark Dose Computation

13 Risk Response = Fatal

14 Risk Type = Extra

15 Specified effect = 0.1

16 Confidence level = 0.9

17

18 Time = 104

19

20 BMD = 0.647659

21 BMDL = 0.461415

22 BMDU = 0.719325

23

24
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1 DOSE-RESPONSE MODELING FOR THE DERMAL SLOPE FACTOR

2 Modeling methods: 

3 For each endpoint, multistage models (BMDS; U.S. EPA, 2012; v 2.1) were fitted to the data 

4 using the maximum likelihood method. Each model was tested for goodness-of-fit using a chi5 square goodness-of-fit test (χ2 p-value < 0.05 indicates lack of fit). Other factors were used to 

6 assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in 

7 the vicinity of the BMR. The BMDL estimate (95% lower confidence limit on the BMD, as estimated 

8 by the profile likelihood method) and AIC value were used to select a best-fit model from among the 

9 models exhibiting adequate fit. The data modeled are summarized in Tables C-22 through C-25. 

10 The modeling results are summarized in Table C-26. The modeling details are provided with 

11 Figures C- 8 through C-19.

12 Data adjustments prior to modeling: 

13 Roe et al. (1970) applied benzo[a]pyrene dermally for 93 weeks or until natural death; with 

14 the exception of the highest dose group, each group still had approximately 20 animals at 86 weeks

15 (see Table C-22). The tumors were first observed in the lowest and highest dose groups during the 

16 interval of weeks 29–43. Mice that died before week 29 were likely not at risk of tumor 

17 development. However, because tumor incidence and mortality were reported in 100-day 

18 intervals, mice that had not been on study long enough to develop tumors were not easily 

19 identifiable. Incidence denominators reflect the number of animals alive at week 29, and may thus 

20 tend to lead to underestimates of tumor risk if the number of animals at risk has been 

21 overestimated.

22 Schmidt et al. (1973) did not report survival information; instead, the authors provided 

23 incidences based on the numbers of mice initially included in each dose group at the start of the 

24 study. Overall latency was reported for the two high-dose groups in each series, but these data only 

25 describe the survival of mice with tumors (animals were removed from study when a tumor 

26 appeared). It is not clear how long exposures lasted overall in each dose group, or whether some 

27 mice may have died on study from other causes before tumors appeared. While it is possible that 

28 no mice died during the study, all of the other studies considered here demonstrate mortality. 

29 However, the data were modeled as reported, recognizing the possibility of underestimating risk 

30 associated with incidences reported and lack of duration of exposure. (See Table C-22.)

31 Schmähl et al. (1977) reported that reduced numbers of animals at risk (77−88 mice per 

32 dose group compared with the initial group sizes of 100) resulted from varying rates of autolysis. 

33 No other survival or latency information was provided, so all exposures were assumed to have 

34 lasted for 104 weeks and were modeled as reported. Given the results of the other studies, it seems 

35 possible that the numbers at risk in each group may be overestimated, which could lead to an 

36 underestimate of lifetime risk. (See Table C-22.)

37 Habs et al. (1980) reported age-standardized skin tumor incidence rates, indicating earlier 

38 mortality in the two highest dose groups (2.8 and 4.6 μg/application). These rates were used to 

39 estimate the number at risk in the dose-response modeling, by dividing the number of mice with 
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C-66 DRAFT—DO NOT CITE OR QUOTE

1 tumors by the age-standardized rates. Exposure lasted longer than 104 weeks in the two lower 

2 exposure groups, at about 120 and 112 weeks, and until about 88 weeks in the highest exposure 

3 group. Incidence in the two lower exposure groups may be higher than if the exposure had lasted 

4 just 104 weeks. There was mortality in the first 52 weeks of exposure, about 10−15% in the three 

5 exposure groups, but because there was no information concerning when tumors first appeared, it 

6 is not possible to determine how much the early mortality may have impacted the number of mice 

7 at risk in each group. (See Table C-22.)

8 Habs et al. (1984) reported mean survival times (with 95% CIs) for each dose group. The 

9 CIs supported the judgment that the control and lower dose groups were treated for 104 weeks. 

10 The higher dose group (4 μg/application) was probably treated for <104 weeks, because the upper 

11 95% confidence limit for the mean survival was approximately 79 weeks. However, since it was 

12 not possible to estimate a more realistic duration for this group, an estimate of 104 weeks was 

13 used. (See Table C-22.)

14 The studies by Poel (1960, 1959) were conducted in male mice and used toluene as the 

15 vehicle. In addition to a control group, the 1959 study included nine dose groups of one mouse 

16 strain (C57L) and the 1960 study included seven dose groups of three other mouse strains. Both 

17 studies demonstrated high mortality and tumor incidence at higher exposure levels. All C57L mice 

18 in dose groups with >3.8 μg/application died by week 44 of the study (Poel, 1959). Therefore, 

19 these five dose groups were omitted prior to dose-response modeling because of the relatively 

20 large uncertainty in extrapolating cancer risk as a result of lifetime exposure. Four dose groups in 

21 addition to control remained. Among these groups, mice survived and were exposed until weeks 

22 83–103. According to the lifespan ranges provided, at least one mouse in each dose group died 

23 before the first appearance of tumor, but insufficient information was available to determine how 

24 many; consequently, the incidence denominators were not adjusted. The dose-response data are 

25 summarized in Table C-23.

26 For the Poel (1960) studies, all tumors in the highest three dose groups for each of the three 

27 mouse strains had occurred by week 40. While these observations support concern for cancer risk, 

28 as noted above such results are relatively uncertain for estimating lifetime cancer risk. In addition, 

29 there was no information indicating duration of exposure for the mice without tumors; although 

30 exposure was for lifetime, it might have been as short as for the mice with tumors. Overall, these 

31 datasets did not provide sufficient information to estimate the extent of exposure associated with 

32 the observed tumor incidence. Consequently, the experiments reported by Poel (1960) were not 

33 used for dose-response modeling.

34 Grimmer et al. (1984, 1983), studied female CFLP mice, using acetone:DMSO (1:3) as the 

35 vehicle. Mean or median latency times were reported (as well as measures of variability), but no 

36 information concerning overall length of exposure or survival was included in the results. The total 

37 of tumor-bearing mice and the reported percentages of mice with any skin tumors was reported 

38 and varied, at most, one animal from the number of animals initially placed on study. The 

39 decreasing latency and variability and increasing tumor incidence with increasing benzo[a]pyrene 

40 exposure suggests that exposure probably did not last for 104 weeks in at least the high-dose 

41 group, but the available information did not provide duration of exposure. The data reported were 
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C-67 DRAFT—DO NOT CITE OR QUOTE

1 modeled under the assumption that at least some animals in each group were treated and survived 

2 until week 104. (See Table C-24.)

3 Sivak et al. (1997), exposed male C3H/HeJ mice dermally to benzo[a]pyrene in 

4 cyclohexanone/acetone (1:1) for 24 months, and reported mean survival times for each group. All 

5 high-dose mice died before the final sacrifice. From the information provided, it is apparent that 

6 the animals in the control and lower two dose groups survived until study termination at week 104. 

7 The study authors did not report how long treatment in the highest dose group lasted, but 

8 estimation of the figure from the publication suggest that exposure duration was 74 weeks. (See 

9 Table C-25).
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C-68 DRAFT—DO NOT CITE OR QUOTE

1

2 Table C-22. Skin tumor incidence, benign or malignant in female Swiss 

3 or NMRI mice dermally exposed to benzo[a]pyrene

Study

Mouse 

strain Dose (μg)

Average 

daily 

dose 

(μg/d)

First 

appearance 

of tumor 

(wks)

Length of 

exposure 

(wks)

Lifetime 

average 

daily dose

(μg/d)

Skin tumor 

incidence (all 

types)

Roe et al., 

1970a,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., 1973c

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., 1977c

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., 

1980c,f

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%)

8/34 (24.8%)

24/27 (89.3%)

22/24 91.7%)

Habs et al., 

1984c

NMRI 0 (acetone)

2

4

0

0.57

1.14

–

NR

NR

104

104

104

0

0.57

1.14

0/20 (0%)

9/20 (45%)

17/20 (85%)

a

Doses were applied 3 times/week for up to 93 weeks to shaved dorsal skin.

b

Numerator: number of mice detected with a skin tumor. Tumors were thought to be malignant based on 

invasion or penetration of the panniculus carnosus muscle. Denominator: number of mice surviving to 29 weeks 

(200 days).

c

Doses 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. Schmidt et al. (1973): At 0.23 μg/d, all tumors were malignant in 

both strains; at 0.57 μg/d, tumors were predominately malignant: 28/30 for NMRI and 42/45 for Swiss. Schmähl 

et al., (1977): malignant/total tumors were 10/11, 25/25, and 43/45 for the 1-, 1.7-, and 3-μg/d groups. Habs et al. 

(1984): malignant/total tumors were 7/9 and 17/11 for the 2- and 4-μg/d groups.

d

Exposure periods not reported were assumed to be 104 weeks; indicated in italics.

e

Central tendency estimates; range or other variability measure not reported.

f

The percentages were reported by the authors as age-standardized incidences of animals with local tumors, 

derived using mortality data from the entire study population. The incidences reflect reported counts of tumorbearing animals and denominators estimated from the reported age-standardized rates. The authors did not 

report the percentages of local tumors which were carcinomas or papillomas.

NR = not reported
Toxicological Review of benzo[a]pyrene

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

C-69 DRAFT—DO NOT CITE OR QUOTE

1 Table C-23. Skin tumor incidence, benign or malignant, in C57L male 

2 mice dermally exposed to benzo[a]pyrene

Study

Mouse 

strain Dose (μg)a

Average 

daily dose 

(μg/d)

First 

appearance of 

tumor (wks)

Length of 

exposure 

(wks)

Lifetime 

average daily 

doseb

Skin tumor 

incidence (all 

types)c

Poel, 1959 C57L 0 (toluene)

0.15

0.38

0.75

3.8

0

0.06

0.16

0.32

1.63

–

42

24

36

21–25

92

98

103

94

82

0.00

0.05

0.16

0.24

0.80

0/33 (0%)

5/55 (9%)

11/55 (20%)

7/56 (13%)

41/49 (84%)

a

Doses were applied to interscapular skin 3 times/week for up to 103 weeks or until time of appearance of a 

grossly detected skin tumor. See Table B-15 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 2.5.2. of Toxicological Reivew for discussion of extrapolation to lifetime average daily doses.

c

Tumors were histologically confirmed as epidermoid carcinomas.

3 Table C-24. Skin tumor incidence, benign or malignant, in female CFLP 

4 mice dermally exposed to benzo[a]pyrene

Study Dose (μg)a

Average 

daily dose 

(μg/d)

Mean or 

median time 

of tumor 

appearance 

(wks)

Length of 

exposure 

(wks)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.

cMedian and 95% confidence limit.

d

Assumed exposure period is indicated in italics.

e

Incidence denominators were calculated from reported tumor-bearing animals and reported percentages. 

Grimmer et al. (1983): malignant/total tumors were 15/22, 34/39, and 54/56 for the low- through high-dose groups. 

Grimmer et al. (1984): malignant /total tumors were 37/43, 45/53, and 53/57 for the low- through high-dose 

groups.

5

6
Toxicological Review of benzo[a]pyrene

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

C-70 DRAFT—DO NOT CITE OR QUOTE

1 Table C-25. Skin tumor incidence, benign or malignant, in male 

2 C3H/HeJ mice dermally exposed to benzo[a]pyrene

Dose (μg)a

Average 

daily dose 

(μg/d)

First 

appearance 

of tumor 

(wks)

Length of 

exposure 

(wks)b

Lifetime 

average daily 

dose

(μg/d)

Skin tumor 

incidence (all 

types)c

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.

b

Assumed exposure period is indicated in italics.

c

Number of skin tumor-bearing mice. In the high-dose group, 1 papilloma and 28 carcinomas were detected. In 

the 0.5 μg group, 2 papillomas and 3 carcinomas were detected.

NR = not reported

Source: Sivak et al. (1997).

3

4
Toxicological Review of benzo[a]pyrene

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

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1 Table C-26. Summary of model selection and modeling results for best2 fitting multistage models, for multiple data sets of skin tumors in mice 

3 following dermal benzo[a]pyrene exposure

Data set Model

Goodness-of-fit BMD10

(μg/d)

BMDL10

(μg/d) Basis for Model Selectiona

Figure 

p-value AIC number

Poel, 1959

male C57L

Multistage 1°

Multistage 2°

Multistage 3°

Multistage 4°

0.011

0.027

0.053

0.068

191.5

188.6

186.9

186.2

0.070

0.134

0.127

0.123

0.057

0.078

0.078

0.077

No significant improvement in model fit 

with higher stage

C-9

Roe et al., 1970

female Swiss

Multistage 1°

Multistage 2°

Multistage 3°

0.110

0.485

0.485

131.1

123.6

123.6

0.318

0.748

0.748

0.249

0.480

0.480

No significant improvement in model fit 

with higher stages

C-10

Schmidt et al., 1973

female NMRI

Multistage 1°

Multistage 2°

Multistage 3°

0.008

0.609

0.999

162.7

147.4

143.9

0.256

0.329

0.381

0.194

0.287

0.326

No significant improvement in model fit 

with higher stages

C-11

Schmidt et al., 1973

female Swiss

Multistage 1°

Multistage 2°

Multistage 3°

Multistage 4°

<0.01

0.514

0.983

0.983

178.0

153.3

151.3

151.3

0.116

0.216

0.282

0.282

0.093

0.192

0.223

0.223

No significant improvement in model fit 

with higher stage

C-12

Schmähl et al., 1977

female NMRI

Multistage 1°

Multistage 2°

Multistage 3°

0.136

0.939

0.939

298.4

296.3

296.3

0.140

0.233

0.233

0.117

0.149

0.143

No significant improvement in model fit 

with higher stage

C-13

Habs et al., 1980

female NMRI

Multistage 1°

Multistage 2

Multistage 3°

0.0

0.009

0.207

96.5

84.4

76.7

0.063

0.198

0.294

0.050

0.143

0.215 Only model with adequate fit C-14

Habs et al., 1984

female NMRI

Multistage 1°

Multistage 2°

0.577

1.000

48.4

47.6

0.078

0.171

0.056

0.060

No significant improvement in model fit 

with higher stage

C-15

Grimmer et al., 1983

female CFLP

Multistage 1°

Multistage 2°

Multistage 3°

0.850

0.972

0.972

219.9

221.1

221.1

0.245

0.292

0.292

0.208

0.213

0.213

No significant improvement in model fit 

with higher stages

C-16

Grimmer et al., 1984b

female CFLP

Multistage 1°

LogLogistic

Dichotomous-Hill

LogProbit

Gamma, Weibull

Logistic

Probit

0.003

0.919

1.000

0.047

0.003

0.0

0.0

205.3

195.8

197.7

200.2

205.3

250.5

255.4

0.132

1.07

0.902

1.33

0.132

2.03

2.29

0.113

0.479

0.533

1.11

0.113

1.76

2.03

(Higher stages did not provide better fit)

Lowest AIC among adequately fitting 

models.

(Same as Multistage 1°)

C-17

C-18

Multistage 1°, high 

dose dropped

0.499 — 1.21 1.01 C-19

Sivak et al., 1997 

male CeH/HeJ

Multistage 1°

Multistage 2°

Multistage 3°

0.059

0.998

0.998

57.8

48.6

48.6

0.036

0.109

0.109

0.026

0.058

0.052

No significant improvement in model fit 

with higher stage

C-20

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

The POD for Grimmer et al. (1984) , using a BMR of 70% (near response at the lowest dose) , was based on the LogLogistic model. For 

comparison purposes, the multistage model was it fit to the Grimmer et al. (1984) data with the highest dose dropped (AIC not provided 

because it is not comparable to fits of the full dataset).

4
Toxicological Review of benzo[a]pyrene

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

C-72 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-8. Fit of multistage model to skin tumors in C57L mice exposed 

3 dermally to benzo[a]pyrene (Poel, 1959); graph and model output.

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_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

14 The form of the probability function is: 

15

16 P[response] = background + (1-background)*[1-EXP(

17 -beta1*dose^1-beta2*dose^2-beta3*dose^3)]

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 = 5

26 Total number of records with missing values = 0

27 Total number of parameters in model = 4

28 Total number of specified parameters = 0

29 Degree of polynomial = 3

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 

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
Toxicological Review of benzo[a]pyrene

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

C-73 DRAFT—DO NOT CITE OR QUOTE

1 Background = 0.0449589

2 Beta(1) = 0.490451

3 Beta(2) = 0

4 Beta(3) = 2.68146

5

6

7 Asymptotic Correlation Matrix of Parameter Estimates

8

9 ( *** The model parameter(s) -Beta(2) 

10 have been estimated at a boundary point, or have been specified by 

11 the user,

12 and do not appear in the correlation matrix )

13

14 Background Beta(1) Beta(3)

15

16 Background 1 -0.87 0.74

17

18 Beta(1) -0.87 1 -0.92

19

20 Beta(3) 0.74 -0.92 1

21

22

23

24 Parameter Estimates

25

26 95.0% Wald Confidence 

27 Interval

28 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

29 Limit

30 Background 0.0176699 * * *

31 Beta(1) 0.79766 * * *

32 Beta(2) 0 * * *

33 Beta(3) 2.17146 * * *

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 -87.1835 5

43 Fitted model -90.4265 3 6.48606 2 0.03905

44 Reduced model -141.614 1 108.86 4 <.0001

45

46 AIC: 186.853

47

48

49 Goodness of Fit 

50 Scaled

51 Dose Est._Prob. Expected Observed Size Residual

52 ------------------------------------------------------------------------

53 0.0000 0.0177 0.583 0.000 33 -0.770

54 0.0500 0.0563 3.098 5.000 55 1.112

55 0.1600 0.1430 7.866 11.000 55 1.207

56 0.2400 0.2128 11.917 7.000 56 -1.605

57 0.8000 0.8293 40.635 41.000 49 0.139

58

59 Chi^2 = 5.88 d.f. = 2 P-value = 0.0528

60

61

62 Benchmark Dose Computation

63

64 Specified effect = 0.1

65

66 Risk Type = Extra risk 

67
Toxicological Review of benzo[a]pyrene

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C-74 DRAFT—DO NOT CITE OR QUOTE

1 Confidence level = 0.95

2

3 BMD = 0.126567

4

5 BMDL = 0.0777875

6

7 BMDU = 0.272961

8

9 Taken together, (0.0777875, 0.272961) is a 90 % two-sided confidence

10 interval for the BMD

11

12 Multistage Cancer Slope Factor = 1.28555

13
Toxicological Review of benzo[a]pyrene

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

C-75 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-9. Fit of multistage model to skin tumors in female Swiss mice 

3 exposed dermally to benzo[a]pyrene (Roe et al., 1970); graph and 

4 model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Roe_1970_Setting.(d) 

8 Gnuplot Plotting File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Roe_1970_Setting.plt

9

10 ==================================================================== 

11 BMDS Model Run 

12 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

13

14 The form of the probability function is: 

15

16 P[response] = background + (1-background)*[1-EXP(

17 -beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^4-beta5*dose^5)]

18

19 The parameter betas are restricted to be positive

20

21 Dependent variable = tumors

22 Independent variable = LADD

23

24 Total number of observations = 6

25 Total number of records with missing values = 0

26 Total number of parameters in model = 6

27 Total number of specified parameters = 0

28 Degree of polynomial = 5

29

30 Maximum number of iterations = 250

31 Relative Function Convergence has been set to: 1e-008

32 Parameter Convergence has been set to: 1e-008

33

34

35

36 Default Initial Parameter Values 

37 Background = 0

0

0.2

0.4

0.6

0.8

0 0.5 1 1.5 2 2.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation

0

0.2

0.4

0.6

0.8

0 0.5 1 1.5

0

0.2

0.4

0.6

0.8

0 0.5 1 1.5 2 2.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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C-76 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 Parameter Estimates

24

25 95.0% Wald Confidence Interval

26 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

27 Background 0.00584893 * * *

28 Beta(1) 0.0379152 * * *

29 Beta(2) 0.166839 * * *

30 Beta(3) 0 * * *

31 Beta(4) 0 * * *

32 Beta(5) 0 * * *

33

34 * - Indicates that this value is not calculated.

35

36 Analysis of Deviance Table

37

38 Model Log(likelihood) # Param's Deviance Test d.f. P-value

39 Full model -56.1835 6

40 Fitted model -57.5694 3 2.77176 3 0.4282

41 Reduced model -118.948 1 125.529 5 <.0001

42

43 AIC: 121.139

44

45 Goodness of Fit 

46 Scaled

47 Dose Est._Prob. Expected Observed Size Residual

48 ------------------------------------------------------------------------

49 0.0000 0.0058 0.275 0.000 47 -0.526

50 0.0300 0.0071 0.321 1.000 45 1.204

51 0.0900 0.0106 0.444 0.000 42 -0.670

52 0.3100 0.0331 1.423 1.000 43 -0.361

53 0.9200 0.1664 6.821 8.000 41 0.494

54 2.7600 0.7488 34.444 34.000 46 -0.151

55

56 Chi^2 = 2.57 d.f. = 3 P-value = 0.4626

57

58

59 Benchmark Dose Computation

60

61 Specified effect = 0.1

62

63 Risk Type = Extra risk 

64

65 Confidence level = 0.95

66

67 BMD = 0.689131

68 BMDL = 0.393806

69 BMDU = 0.952365

70

71 Taken together, (0.393806, 0.952365) is a 90 % two-sided confidence

72 interval for the BMD

73

74 Multistage Cancer Slope Factor = 0.253932
Toxicological Review of benzo[a]pyrene

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

C-77 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-10. Fit of multistage model to skin tumors in female NMRI 

3 mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph 

4 and model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: 

8 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973femaleNMRI\2MulSchMS_.(d) 

9 Gnuplot Plotting File: 

10 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973femaleNMRI\2MulSchMS_.plt

11 ==================================================================== 

12 BMDS Model Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14

15 The form of the probability function is: 

16

17 P[response] = background + (1-background)*[1-EXP(

18 -beta1*dose^1-beta2*dose^2)]

19

20 The parameter betas are restricted to be positive

21

22 Dependent variable = incidence

23 Independent variable = dose

24

25 Total number of observations = 5

26 Total number of records with missing values = 0

27 Total number of parameters in model = 3

28 Total number of specified parameters = 0

29 Degree of polynomial = 2

30

31 Maximum number of iterations = 250

32 Relative Function Convergence has been set to: 2.22045e-016

33 Parameter Convergence has been set to: 1.49012e-008

34

35 **** We are sorry but Relative Function and Parameter Convergence  

36 ** ** are currently unavailable in this model. Please keep checking   0.4

0 0.1 0.2 0.3 0.4 0.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-78 DRAFT—DO NOT CITE OR QUOTE

1 ** ** the web sight for model updates which will eventually  

2 ** ** incorporate these convergence criterion. Default values used.  

3

4

5 Default Initial Parameter Values 

6 Background = 0

7 Beta(1) = 0

8 Beta(2) = 1.11271

9

10

11 Asymptotic Correlation Matrix of Parameter Estimates

12

13 ( ** * The model parameter(s) -Background -Beta(1) 

14 have been estimated at a boundary point, or have been specified by the user,

15 and do not appear in the correlation matrix )

16

17 Beta(2)

18

19 Beta(2) 1

20

21

22 Parameter Estimates

23

24 95.0% Wald Confidence Interval

25 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

26 Background 0 * * *

27 Beta(1) 0 * * *

28 Beta(2) 0.970648 * * *

29

30 * - Indicates that this value is not calculated.

31

32

33

34 Analysis of Deviance Table

35

36 Model Log(likelihood) # Param's Deviance Test d.f. P-value

37 Full model -70.8903 5

38 Fitted model -72.6831 1 3.58562 4 0.465

39 Reduced model -118.917 1 96.054 4 <.0001

40

41 AIC: 147.366

42

43 Goodness of Fit 

44 Scaled

45 Dose Est._Prob. Expected Observed Size Residual

46 ------------------------------------------------------------------------

47 0.0000 0.0000 0.000 0.000 100 0.000

48 0.0100 0.0001 0.010 0.000 100 -0.099

49 0.0600 0.0035 0.349 0.000 100 -0.592

50 0.2300 0.0501 5.005 2.000 100 -1.378

51 0.5700 0.2705 27.048 30.000 100 0.665

52

53 Chi^2 = 2.70 d.f. = 4 P-value = 0.6091

54

55

56 Benchmark Dose Computation

57

58 Specified effect = 0.1

59

60 Risk Type = Extra risk 

61

62 Confidence level = 0.95

63

64 BMD = 0.329464

65

66 BMDL = 0.286624

67

68 BMDU = 0.384046

69

70 Taken together, (0.286624, 0.384046) is a 90 % two-sided confidence

71 interval for the BMD

72

73 Multistage Cancer Slope Factor = 0.348889

74
Toxicological Review of benzo[a]pyrene

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

C-79 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-11. Fit of multistage model to skin tumors in female Swiss 

3 mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph 

4 and model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: 

8 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973swissmice\3MulSchMS_.(d) 

9 Gnuplot Plotting File: 

10 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973swissmice\3MulSchMS_.plt 

11 ==================================================================== 

12 BMDS Model Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14

15 The form of the probability function is: 

16

17 P[response] = background + (1-background)*[1-EXP(

18 -beta1*dose^1-beta2*dose^2-beta3*dose^3)]

19

20 The parameter betas are restricted to be positive

21

22 Dependent variable = incidence

23 Independent variable = dose

24

25 Total number of observations = 5

26 Total number of records with missing values = 0

27 Total number of parameters in model = 4

28 Total number of specified parameters = 0

29 Degree of polynomial = 3

30

31 Maximum number of iterations = 250

32 Relative Function Convergence has been set to: 2.22045e-016

33 Parameter Convergence has been set to: 1.49012e-008

34

35 **** We are sorry but Relative Function and Parameter Convergence  

0

0.1

0.2

0.3

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0.5

0.6

0.7

0 0.1 0.2 0.3 0.4 0.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation

0

0.1

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0.3

0.4

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0

0

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0 0.1 0.2 0.3 0.4 0.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-80 DRAFT—DO NOT CITE OR QUOTE

1 ** ** are currently unavailable in this model. Please keep checking  

2 ** ** the web sight for model updates which will eventually  

3 ** ** incorporate these convergence criterion. Default values used.  

4

5

6 Default Initial Parameter Values 

7 Background = 0

8 Beta(1) = 0

9 Beta(2) = 0.338951

10 Beta(3) = 3.8728

11

12

13 Asymptotic Correlation Matrix of Parameter Estimates

14

15 ( ** * The model parameter(s) -Background -Beta(1) 

16 have been estimated at a boundary point, or have been specified by the 

17 user,

18 and do not appear in the correlation matrix )

19

20 Beta(2) Beta(3)

21

22 Beta(2) 1 -0.99

23

24 Beta(3) -0.99 1

25

26

27 Parameter Estimates

28

29 95.0% Wald Confidence Interval

30 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

31 Limit

32 Background 0 * * *

33 Beta(1) 0 * * *

34 Beta(2) 0.108125 * * *

35 Beta(3) 4.31441 * * *

36

37 * - Indicates that this value is not calculated.

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 Goodness of Fit 

49 Scaled

50 Dose Est._Prob. Expected Observed Size Residual

51 ------------------------------------------------------------------------

52 0.0000 0.0000 0.000 0.000 80 0.000

53 0.0100 0.0000 0.001 0.000 80 -0.035

54 0.0600 0.0013 0.106 0.000 80 -0.325

55 0.2300 0.0566 4.524 5.000 80 0.230

56 0.5700 0.5657 45.260 45.000 80 -0.059

57

58 Chi^2 = 0.16 d.f. = 3 P-value = 0.9833

59

60 Benchmark Dose Computation

61

62 Specified effect = 0.1

63

64 Risk Type = Extra risk 

65

66 Confidence level = 0.95

67

68 BMD = 0.282007

69 BMDL = 0.223401

70 BMDU = 0.309888

71

72 Taken together, (0.223401, 0.309888) is a 90 % two-sided confidence

73 interval for the BMD

74

75 Multistage Cancer Slope Factor = 0.447626
Toxicological Review of benzo[a]pyrene

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

C-81 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-12. Fit of multistage model to skin tumors in female NMRI 

3 mice exposed dermally to benzo[a]pyrene (Schmähl et al., 1977); graph 

4 and model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: 

8 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahl1977femaleNMRI\2MulschMS_.(d) 

9 Gnuplot Plotting File: 

10 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahl1977femaleNMRI\2MulschMS_.plt

11 ==================================================================== 

12 BMDS Model Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14

15 The form of the probability function is: 

16

17 P[response] = background + (1-background)*[1-EXP(

18 -beta1*dose^1-beta2*dose^2)]

19

20 The parameter betas are restricted to be positive

21

22

23 Dependent variable = incidence

24 Independent variable = dose

25

26 Total number of observations = 4

27 Total number of records with missing values = 0

28 Total number of parameters in model = 3

29 Total number of specified parameters = 0

30 Degree of polynomial = 2

31

32

33 Maximum number of iterations = 250

34 Relative Function Convergence has been set to: 2.22045e-016

35 Parameter Convergence has been set to: 1.49012e-008

0

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dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation

0

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0

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dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-82 DRAFT—DO NOT CITE OR QUOTE

1

2 **** We are sorry but Relative Function and Parameter Convergence  

3 ** ** are currently unavailable in this model. Please keep checking  

4 ** ** the web sight for model updates which will eventually  

5 ** ** incorporate these convergence criterion. Default values used. ****

6

7

8 Default Initial Parameter Values 

9 Background = 0.0115034

10 Beta(1) = 0.284955

11 Beta(2) = 0.750235

12

13

14 Asymptotic Correlation Matrix of Parameter Estimates

15

16 Background Beta(1) Beta(2)

17

18 Background 1 -0.67 0.47

19

20 Beta(1) -0.67 1 -0.94

21

22 Beta(2) 0.47 -0.94 1

23

24

25 Parameter Estimates

26

27 95.0% Wald Confidence Interval

28 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

29 Limit

30 Background 0.0123066 * * *

31 Beta(1) 0.274413 * * *

32 Beta(2) 0.764244 * * *

33

34 * - Indicates that this value is not calculated.

35

36

37 Analysis of Deviance Table

38

39 Model Log(likelihood) # Param's Deviance Test d.f. P-value

40 Full model -145.127 4

41 Fitted model -145.13 3 0.00579898 1 0.9393

42 Reduced model -184.158 1 78.0608 3 <.0001

43

44 AIC: 296.261

45

46

47 Goodness of Fit 

48 Scaled

49 Dose Est._Prob. Expected Observed Size Residual

50 ------------------------------------------------------------------------

51 0.0000 0.0123 0.997 1.000 81 0.003

52 0.2900 0.1446 11.137 11.000 77 -0.045

53 0.4900 0.2813 24.756 25.000 88 0.058

54 0.8600 0.5567 45.096 45.000 81 -0.022

55

56 Chi^2 = 0.01 d.f. = 1 P-value = 0.9393

57

58

59 Benchmark Dose Computation

60

61 Specified effect = 0.1

62

63 Risk Type = Extra risk 

64

65 Confidence level = 0.95

66

67 BMD = 0.232893

68 BMDL = 0.148895

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
Toxicological Review of benzo[a]pyrene

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

C-83 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-13. Fit of multistage model to skin tumors in female NMRI 

3 mice exposed dermally to benzo[a]pyrene (Habs et al., 1980); graph 

4 and model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: M:\_BMDS\msc_BAP_HABS1980_MultiCanc3_0.1.(d) 

8 Gnuplot Plotting File: M:\_BMDS\msc_BAP_HABS1980_MultiCanc3_0.1.plt

9

10 ==================================================================== 

11

12 BMDS Model Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14

15 The form of the probability function is:

16

17 P[response] = background + (1-background)*[1-EXP(

18 -beta1*dose^1-beta2*dose^2-beta3*dose^3)]

19

20 The parameter betas are restricted to be positive

21

22

23 Dependent variable = NumAff

24 Independent variable = LADD

25

26 Total number of observations = 4

27 Total number of records with missing values = 0

28 Total number of parameters in model = 4

29 Total number of specified parameters = 0

30 Degree of polynomial = 3

31

32

33 Maximum number of iterations = 250

34 Relative Function Convergence has been set to: 1e-008

35 Parameter Convergence has been set to: 1e-008

36

37

38

39 Default Initial Parameter Values 

40 Background = 0

41 Beta(1) = 0

42 Beta(2) = 4.23649

0

0.2

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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
Toxicological Review of benzo[a]pyrene

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

C-84 DRAFT—DO NOT CITE OR QUOTE

1 Beta(3) = 0

2

3

4 Asymptotic Correlation Matrix of Parameter Estimates

5 ( *** The model parameter(s) -Background -Beta(1) -Beta(2) 

6 have been estimated at a boundary point, or have been specified by the 

7 user,

8 and do not appear in the correlation matrix )

9

10 Beta(3)

11

12 Beta(3) 1

13

14

15

16 Parameter Estimates

17

18 95.0% Wald Confidence Interval

19 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

20 Limit

21 Background 0 * * *

22 Beta(1) 0 * * *

23 Beta(2) 0 * * *

24 Beta(3) 4.1289 * * *

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 -34.8527 4

34 Fitted model -37.3373 1 4.96903 3 0.1741

35 Reduced model -82.5767 1 95.4478 3 <.0001

36

37 AIC: 76.6745

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 35 0.000

45 0.4900 0.3848 13.082 8.000 34 -1.791

46 0.7400 0.8123 21.933 24.000 27 1.019

47 0.8000 0.8792 21.102 22.000 24 0.563

48

49 Chi^2 = 4.56 d.f. = 3 P-value = 0.2067

50

51

52 Benchmark Dose Computation

53

54 Specified effect = 0.1

55

56 Risk Type = Extra risk 

57

58 Confidence level = 0.95

59

60 BMD = 0.294407

61

62 BMDL = 0.215151

63

64 BMDU = 0.320955

65

66 Taken together, (0.215151, 0.320955) is a 90 % two-sided confidence

67 interval for the BMD

68

69 Multistage Cancer Slope Factor = 0.46479

70
Toxicological Review of benzo[a]pyrene

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

C-85 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-14. Fit of multistage model to skin tumors in female NMRI 

3 mice exposed dermally to benzo[a]pyrene (Habs et al., 1984); graph 

4 and model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: C:\Usepa\BMDS21\mscDax_Setting.(d) 

8 Gnuplot Plotting File: C:\Usepa\BMDS21\mscDax_Setting.plt

9

10 ==================================================================== 

11

12 BMDS Model Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14

15 The form of the probability function is: 

16

17 P[response] = background + (1-background)*[1-EXP(

18 -beta1*dose^1)]

19

20 The parameter betas are restricted to be positive

21

22

23 Dependent variable = tumors

24 Independent variable = LADD

25

26 Total number of observations = 3

27 Total number of records with missing values = 0

28 Total number of parameters in model = 2

29 Total number of specified parameters = 0

30 Degree of polynomial = 1

31

32

33 Maximum number of iterations = 250

34 Relative Function Convergence has been set to: 1e-008

35 Parameter Convergence has been set to: 1e-008

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1 Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation

0

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0.6

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1

0 0.2 0.4

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1 Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-86 DRAFT—DO NOT CITE OR QUOTE

1

2

3

4 Default Initial Parameter Values 

5 Background = 0

6 Beta(1) = 1.66414

7

8

9 Asymptotic Correlation Matrix of Parameter Estimates

10

11 ( *** The model parameter(s) -Background 

12 have been estimated at a boundary point, or have been specified by the user,

13 and do not appear in the correlation matrix )

14

15 Beta(1)

16

17 Beta(1) 1

18

19

20

21 Parameter Estimates

22

23 95.0% Wald Confidence Interval

24 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

25 Background 0 * * *

26 Beta(1) 1.35264 * * *

27

28 * - Indicates that this value is not calculated.

29

30

31

32 Analysis of Deviance Table

33

34 Model Log(likelihood) # Param's Deviance Test d.f. P-value

35 Full model -22.217 3

36 Fitted model -22.7878 1 1.14175 2 0.565

37 Reduced model -41.0539 1 37.6739 2 <.0001

38

39 AIC: 47.5757

40

41

42 Goodness of Fit 

43 Scaled

44 Dose Est._Prob. Expected Observed Size Residual

45 ------------------------------------------------------------------------

46 0.0000 0.0000 0.000 0.000 20 0.000

47 0.5700 0.5375 10.749 9.000 20 -0.784

48 1.1400 0.7860 15.721 17.000 20 0.697

49

50 Chi^2 = 1.10 d.f. = 2 P-value = 0.5765

51

52

53 Benchmark Dose Computation

54

55 Specified effect = 0.1

56

57 Risk Type = Extra risk 

58

59 Confidence level = 0.95

60

61 BMD = 0.0778926

62

63 BMDL = 0.0558466

64

65 BMDU = 0.111853

66

67 Taken together, (0.0558466, 0.111853) is a 90 % two-sided confidence

68 interval for the BMD

69

70 Multistage Cancer Slope Factor = 1.79062

71
Toxicological Review of benzo[a]pyrene

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

C-87 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-15. Fit of multistage model to skin tumors in female CFLP mice 

3 exposed dermally to benzo[a]pyrene (Grimmer et al., 1983); graph and 

4 model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: 

8 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmer1983CFLPmice\1MulGriMS_.(d) 

9 Gnuplot Plotting File: 

10 C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmer1983CFLPmice\1MulGriMS_.plt

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)]

20

21 The parameter betas are restricted to be positive

22

23 Dependent variable = incidence

24 Independent variable = dose

25

26 Total number of observations = 4

27 Total number of records with missing values = 0

28 Total number of parameters in model = 2

29 Total number of specified parameters = 0

30 Degree of polynomial = 1

31

32

33 Maximum number of iterations = 250

34 Relative Function Convergence has been set to: 2.22045e-016

35 Parameter Convergence has been set to: 1.49012e-008

0

0.2

0.4

0.6

0.8

1

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation

0

0.2

0.4

0.6

0.8

1

0 0.5 1

0

0.2

0.4

0.6

0.8

1

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-88 DRAFT—DO NOT CITE OR QUOTE

1

2 **** We are sorry but Relative Function and Parameter Convergence  

3 ** ** are currently unavailable in this model. Please keep checking  

4 ** ** the web sight for model updates which will eventually  

5 ** ** incorporate these convergence criterion. Default values used.  

6

7

8

9 Default Initial Parameter Values 

10 Background = 0

11 Beta(1) = 0.478645

12

13

14 Asymptotic Correlation Matrix of Parameter Estimates

15

16 ( ** * The model parameter(s) -Background 

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(1)

21

22 Beta(1) 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) 0.430366 * * *

32

33 * - Indicates that this value is not calculated.

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
Toxicological Review of benzo[a]pyrene

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

C-89 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-16. Fit of multistage model to skin tumors in female CFLP mice 

3 exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and 

4 model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: 

8 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984_MultiCanc1_0.1.(d) 

9 Gnuplot Plotting File: 

10 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmer1984_MultiCanc1_0.1.plt

11 Wed Apr 27 17:11:28 2011

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)]

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 = 2

30 Total number of specified parameters = 0

31 Degree of polynomial = 1

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 = 0.311241

42 Beta(1) = 0.502556

0

0.2

0.4

0.6

0.8

1

0 0.5 1 1.5 2 2.5 3 3.5 4 Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-90 DRAFT—DO NOT CITE OR QUOTE

1

2

3 Asymptotic Correlation Matrix of Parameter Estimates

4

5 ( *** The model parameter(s) -Background 

6 have been estimated at a boundary point, or have been specified by the 

7 user,

8 and do not appear in the correlation matrix )

9

10 Beta(1)

11

12 Beta(1) 1

13

14

15

16 Parameter Estimates

17

18 95.0% Wald Confidence Interval

19 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

20 Limit

21 Background 0 * * *

22 Beta(1) 0.796546 * * *

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 -95.8385 4

32 Fitted model -101.643 1 11.61 3 0.008846

33 Reduced model -175.237 1 158.797 3 <.0001

34

35 AIC: 205.287

36

37

38 Goodness of Fit 

39 Scaled

40 Dose Est._Prob. Expected Observed Size Residual

41 ------------------------------------------------------------------------

42 0.0000 0.0000 0.000 0.000 65 0.000

43 0.9700 0.5382 34.446 43.000 64 2.145

44 1.9100 0.7816 50.804 53.000 65 0.659

45 3.9000 0.9552 62.091 57.000 65 -3.054

46

47 Chi^2 = 14.36 d.f. = 3 P-value = 0.0025

48

49

50 Benchmark Dose Computation

51

52 Specified effect = 0.1

53

54 Risk Type = Extra risk 

55

56 Confidence level = 0.95

57

58 BMD = 0.132272

59

60 BMDL = 0.113427

61

62 BMDU = 0.154848

63

64 Taken together, (0.113427, 0.154848) is a 90 % two-sided confidence

65 interval for the BMD

66

67 Multistage Cancer Slope Factor = 0.881621

68
Toxicological Review of benzo[a]pyrene

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

C-91 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-17. Fit of log-logistic model to skin tumors in female CFLP 

3 mice exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph 

4 and model output.

5 ==================================================================== 

6 Logistic Model. (Version: 2.12; Date: 05/16/2008) 

7 Input Data File: 

8 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmer1984_Grimmer1984_0.70u.(d) 

9 Gnuplot Plotting File: 

10 C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmer1984_Grimmer1984_0.70u.plt

11

12 ==================================================================== 

13

14 BMDS Model Run 

15 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

16

17 The form of the probability function is: 

18

19 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

20

21

22 Dependent variable = NumAff

23 Independent variable = LADD

24 Slope parameter is not restricted

25

26 Total number of observations = 4

27 Total number of records with missing values = 0

28 Maximum number of iterations = 250

29 Relative Function Convergence has been set to: 1e-008

30 Parameter Convergence has been set to: 1e-008

31

32

33

34 User has chosen the log transformed model

35

36

37 Default Initial Parameter Values 

38 background = 0

39 intercept = 0.799142

40 slope = 0.894129

41

42

0

0.2

0.4

0.6

0.8

1

0 0.5 1 1.5 2 2.5 3 3.5 4 Fraction Affected

dose

Log-Logistic Model with 0.95 Confidence Level

BMDL BMD

Log-Logistic
Toxicological Review of benzo[a]pyrene

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

C-92 DRAFT—DO NOT CITE OR QUOTE

1 Asymptotic Correlation Matrix of Parameter Estimates

2

3 ( *** The model parameter(s) -background 

4 have been estimated at a boundary point, or have been specified 

5 by the user,

6 and do not appear in the correlation matrix )

7

8 intercept slope

9

10 intercept 1 -0.68

11

12 slope -0.68 1

13

14

15

16 Parameter Estimates

17

18 95.0% Wald Confidence 

19 Interval

20 Variable Estimate Std. Err. Lower Conf. Limit Upper 

21 Conf. Limit

22 background 0 * * *

23 intercept 0.783559 * * *

24 slope 0.922655 * * *

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 -95.8385 4

34 Fitted model -95.9236 2 0.17031 2 0.9184

35 Reduced model -175.237 1 158.797 3 <.0001

36

37 AIC: 195.847

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 65 0.000

45 0.9700 0.6804 43.543 43.000 64 -0.146

46 1.9100 0.7991 51.941 53.000 65 0.328

47 3.9000 0.8849 57.516 57.000 65 -0.200

48

49 Chi^2 = 0.17 d.f. = 2 P-value = 0.9190

50

51

52 Benchmark Dose Computation

53

54 Specified effect = 0.7

55

56 Risk Type = Extra risk 

57

58 Confidence level = 0.95

59

60 BMD = 1.07152

61

62 BMDL = 0.478669

63
Toxicological Review of benzo[a]pyrene

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

C-93 DRAFT—DO NOT CITE OR QUOTE

1 Figure C-18. Fit of multistage model to skin tumors in female CFLP mice 

2 exposed dermally to benzo[a]pyrene (Grimmer et al., 1984), highest 

3 dose dropped; graph and model output.

4 ==================================================================== 

5 Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) 

6 Input Data File: C:/Usepa/_BaP/msc_BaP_Grimmer1984_drophidose_MultiCanc1_0.7.(d) 

7 Gnuplot Plotting File: 

8 C:/Usepa/_BaP/msc_BaP_Grimmer1984_drophidose_MultiCanc1_0.7.plt

9 ==================================================================== 

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 = 3

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 = 0.0806622

40 Beta(1) = 0.88595

41

42

43 Asymptotic Correlation Matrix of Parameter Estimates

0

0.2

0.4

0.6

0.8

0 0.5 1 1.5 2 Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-94 DRAFT—DO NOT CITE OR QUOTE

1

2 ( *** The model parameter(s) -Background 

3 have been estimated at a boundary point, or have been specified by the 

4 user,

5 and do not appear in the correlation matrix )

6

7 Beta(1)

8

9 Beta(1) 1

10

11

12

13 Parameter Estimates

14

15 95.0% Wald Confidence Interval

16 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

17 Limit

18 Background 0 * * *

19 Beta(1) 0.997117 * * *

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 -71.5928 3

29 Fitted model -72.2756 1 1.36568 2 0.5052

30 Reduced model -134.46 1 125.735 2 <.0001

31

32 AIC: 146.551

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 0.6199 39.671 43.000 64 0.857

41 1.9100 0.8511 55.322 53.000 65 -0.809

42

43 Chi^2 = 1.39 d.f. = 2 P-value = 0.4992

44

45

46 Benchmark Dose Computation

47

48 Specified effect = 0.7

49

50 Risk Type = Extra risk 

51

52 Confidence level = 0.95

53

54 BMD = 1.20745

55

56 BMDL = 1.00734

57

58 BMDU = 1.45789

59

60 Taken together, (1.00734, 1.45789) is a 90 % two-sided confidence

61 interval for the BMD

62

63 Multistage Cancer Slope Factor = 0.6949

64
Toxicological Review of benzo[a]pyrene

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

C-95 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure C-19. Fit of multistage model to skin tumors in male CeH/HeJ

3 mice exposed dermally to benzo[a]pyrene (Sivak et al., 1997); graph 

4 and model output.

5 ==================================================================== 

6 Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

7 Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivak1993_MultiCanc2_0.1.(d) 

8 Gnuplot Plotting File: 

9 C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivak1993_MultiCanc2_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)]

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 = 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

42 Beta(1) = 0.0936505

0

0.2

0.4

0.6

0.8

1

0 0.1 0.2 0.3 0.4 0.5

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

BMDL BMD

Multistage Cancer

Linear extrapolation
Toxicological Review of benzo[a]pyrene

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

C-96 DRAFT—DO NOT CITE OR QUOTE

1 Beta(2) = 8.67239

2

3

4 Asymptotic Correlation Matrix of Parameter Estimates

5

6 ( *** The model parameter(s) -Background -Beta(1) 

7 have been estimated at a boundary point, or have been specified by the user,

8 and do not appear in the correlation matrix )

9

10 Beta(2)

11

12 Beta(2) 1

13

14

15

16 Parameter Estimates

17

18 95.0% Wald Confidence Interval

19 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

20 Background 0 * * *

21 Beta(1) 0 * * *

22 Beta(2) 8.9375 * * *

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 -23.2693 4

32 Fitted model -23.3009 1 0.0631003 3 0.9959

33 Reduced model -69.5898 1 92.641 3 <.0001

34

35 AIC: 48.6018

36

37

38 Goodness of Fit 

39 Scaled

40 Dose Est._Prob. Expected Observed Size Residual

41 ------------------------------------------------------------------------

42 0.0000 0.0000 0.000 0.000 30 0.000

43 0.0100 0.0009 0.027 0.000 30 -0.164

44 0.1400 0.1607 4.821 5.000 30 0.089

45 0.5100 0.9022 27.065 27.000 30 -0.040

46

47 Chi^2 = 0.04 d.f. = 3 P-value = 0.9982

48

49

50 Benchmark Dose Computation

51

52 Specified effect = 0.1

53

54 Risk Type = Extra risk 

55

56 Confidence level = 0.95

57

58 BMD = 0.108575

59

60 BMDL = 0.058484

61

62 BMDU = 0.129641

63

64 Taken together, (0.058484, 0.129641) is a 90 % two-sided confidence

65 interval for the BMD

66

67 Multistage Cancer Slope Factor = 1.70987

68

69
Toxicological Review of benzo[a]pyrene

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

C-97 DRAFT—DO NOT CITE OR QUOTE

1 ALTERNATIVE APPROACHES FOR CROSS-SPECIES SCALING OF THE DERMAL SLOPE 

2 FACTOR

3 Several publications which develop a dermal slope factor for benzo[a]pyrene are available 

4 in the peer reviewed literature (Knafla et al., 2010; 2006; Hussain et al., 1998; LaGoy and Quirk 

5 1994; Sullivan et al., 1991). With the exception of the 2010 Knafla et al. publication, none of these 

6 approaches applied quantitative adjustments to account for interspecies differences, though the 

7 proposed slope factors were developed to account for human risk. Knafla et al. (2010) qualitatively 

8 discuss processes which could affect the extrapolation between mice and humans including skin 

9 metabolic activity adduct formation, stratum corneum thickness, epidermal thickness, etc. 

10 Ultimately, the authors apply an adjustment based on the increased epidermal thickness of human 

11 skin on the arms and hands compared to mouse interscapular epidermal thickness. They 

12 hypothesize that the carcinogenic potential of benzo[a]pyrene may be related to the thickness of 

13 the epidermal layer. 

14 Because there is no established methodology for cross-species extrapolation of dermal 

15 toxicity, several alternative approaches were evaluated. Each approach begins with the POD of 

16 0.066 μg/day that was based on a 10% extra risk for skin tumors in male mice. Based on the 

17 assumptions of each approach, a dermal slope factor for humans is calculated. The discussion of 

18 these approaches uses the following abbreviations: 

19

20 DSF = dermal slope factor

21 PODM = point of departure (for 10% extra risk) from mouse bioassay, in μg/day

22 BWM = mouse body weight = 0.035 kg (assumed)

23 BWH = human body weight = 70 kg (assumed)

SAH = total human surface area = 19,000 cm2 24 (assumed)

SAM = total mouse surface area = 100 cm2 25 (assumed)

26

27 Approach 1. No interspecies adjustment to daily applied dose (POD) in mouse model

28 Under this approach, a given mass of benzo[a]pyrene, applied daily, would pose the same 

29 risk in an animal or in humans, regardless of whether it is applied to a small surface area or to a 

30 larger surface area at a proportionately lower concentration.

31

32 DSF= 0.1/ PODM 

33

DSF= 0.1/0.068 μg/day = 1.5 (μg/day) 34 -1

35

36 Assumptions: The same mass of benzo[a]pyrene, applied daily, would have same potency in 

37 mice as in human skin regardless of treatment area.

38

39 Approach 2. Cross-species adjustment based on whole body surface-area scaling
Toxicological Review of benzo[a]pyrene

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

C-98 DRAFT—DO NOT CITE OR QUOTE

1 Under this approach, animals and humans are assumed to have equal lifetime cancer risk 

with equal average whole body exposures in loading units (μg/cm2 2 -day). As long as doses are low 

3 enough that risk is proportional to the mass of applied compound, the daily dermal dose of 

4 benzo[a]pyrene can be normalized over the total surface area. 

5

POD (μg/cm2-day) = PODM/SA (μg/cm2-day) = PODM (μg/day) / SAM (cm2 6 )

7

8 POD = (0.068 μg/day) / 100 cm2

 = 0.00068 μg/cm2 9 -day

10

DSF = 0.1/(0.00068 μg/cm2-day) ≈ 147 (μg/cm2-day) 11 -1

12

13 Assumptions: Mouse and human slope factors are equipotent if total dermal dose is 

14 averaged over equal fractions of the entire surface area. Tumor potency of benzo[a]pyrene is 

15 assumed to be related to overall dose and not dose per unit area. For example, a human exposed to 

16 0.01 μg/day on 10 cm2 would be assumed to have the same potential to form a skin tumor as 

someone treated with 0.01 μg/day over 19,000 cm2 17 (assumed human surface area). 

18

19 Approach 3. Cross-species adjustment based on body weight

20 Under this approach, a given mass of benzo[a]pyrene is normalized relative to the body 

21 weight of the animal or human. This approach has been used for oral doses for noncancer effects.

22

23 PODM/ BWM= 0.068 μg/0.035 kg-day = 1.9 μg/kg-day

24

DSF= 0.1/1.9 μg/kg-day = 0.051 (μg/kg-day) 25 -1

26

27 Assumptions: The potency of point of contact skin tumors is related to bodyweight and 

28 humans and mice would have an equal likelihood of developing skin tumors based on a dermal dose 

29 per kg basis.

30

31 Issues: Skin cancer following benzo[a]pyrene exposure is a local effect and not likely 

32 dependent on body weight.

33

34 Approach 4. Cross-species adjustment based on allometric scaling using body weight to the 

35 3/4 power

36 Under this approach, rodents and humans exposed to the same daily dose of a carcinogen, 

adjusted for BW3/4 37 , would be expected to have equal lifetime risks of cancer. That is, a lifetime dose 

expressed as μg/kg3/4 38 -day would lead to an equal risk in rodents and humans. This scaling reflects 

39 the empirically observed phenomena of more rapid distribution, metabolism, and clearance in 

40 smaller animals. The metabolism of benzo[a]pyrene to reactive intermediates is a critical step in 

41 the carcinogenicity of benzo[a]pyrene, and this metabolism occurs in the skin.
Toxicological Review of benzo[a]pyrene

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

C-99 DRAFT—DO NOT CITE OR QUOTE

1

POD(μg/day) = PODM (μg/day) × (BWH / BWM) 2 3/4

3

POD (μg/day) = 0.068 μg/day × (70 kg / 0.035 kg) 4 3/4

5 = 20.3 μg/day

6

DSF = 0.1/(20.3 μg/day) ≈ 0.0049 (μg/day)-1 7 

8

9 Assumptions: Risk at low doses of benzo[a]pyrene is dependent on absolute dermal dose 

10 and not dose per unit of skin, meaning a higher exposure concentration of benzo[a]pyrene 

11 contacting a smaller area of exposed skin could carry the same risk of skin tumors as a lower 

12 exposure concentration of benzo[a]pyrene that contacts a larger area of skin. 

13

14 Issues: It is unclear if scaling of doses based on bodyweight ratios will correspond to 

15 differences in metabolic processes in the skin of mice and humans. 

16

17 Synthesis of the alternative approaches to cross-species scaling

18 A comparison of the above approaches is provided in Table C-27 below. The lifetime risk 

19 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 20 -4 μg/day*, is calculated for each of the approaches in 

21 order to judge whether the method yields risk estimates that are unrealistically high. 

22

23 Other potential interspecies adjustments

24 The above discussion presents several mathematical approaches that result from varying 

25 assumptions about what is the relevant dose metric for determining equivalence across species. 

26 Biological information (that is not presently comprehensive or detailed enough to develop robust 

27 models) that could be used in future biologically based models for cross-species extrapolation 

28 include:

29 a. Quantitative information on interspecies differences in partitioning from exposure medium 

30 to the skin and absorption through the skin

31 b. Thickness of the stratum corneum between anatomical sites and between species

32 c. Thickness of epidermal layer 

33 d. Skin permeability

34 e. Metabolic activity of skin

35 f. Formation of DNA adducts in skin
Toxicological Review of benzo[a]pyrene

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

C-100 DRAFT—DO NOT CITE OR QUOTE

1 Table C-27. Alternative approaches to cross-species scaling

Approach Assumptions Dose metric DSF

Risk at nominal 

exposure 

(0.0001 μg/day)*

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 1.3 per 

μg/day

1 x 10-4

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, lifetime exposure normalized over the whole body 

[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 128 per 

μg/cm2-day 7 x 10-7

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 0.045 per 

μg/kg-day 6 x 10-8

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 0.0043 per μg 

/day

4 x 10-7

2 * Nominal exposure calculated as a geometric mean of average daily doses (μg/day) calculated from a range of benzo[a]pyrene soil concentrations (1- 1000 

3 ppb) reported from non-contaminated rural/agricultural soils (ATSDR, 1995) and a range of standard exposure assumptions. 
Toxicological Review of benzo[a]pyrene

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

D-1 DRAFT—DO NOT CITE OR QUOTE

1 APPENDIX D. SUMMARY OF EXTERNAL PEER 

2 REVIEW AND PUBLIC COMMENTS AND EPA’S 

3 DISPOSITION

4

5
Toxicological Review of benzo[a]pyrene

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

1 DRAFT—DO NOT CITE OR QUOTE

1

2 REFERENCES FOR APPENDICES

3 Multiple references published in the same year by the same author(s) have been assigned a letter 

4 (e.g., 1986a, 1986b) based on order of appearance in the text of the document. Those same letters 

5 have been retained for the appendices.

6

7 Achard, S; Perderiset, M; Jaurand, MC. (1987) Sister chromatid exchanges in rat pleural mesothelial cells 

8 treated with crocidolite, attapulgite, or benzo 3-4 pyrene. Br J Ind Med 44:281-283.

9

10 Adler, ID; Ingwersen, I. (1989) Evaluation of chromosomal aberrations in bone marrow of 1C3F1 mice. Mutat 

11 Res 224(3):343–345.

12

13 Adler, ID; Kliesch, U; Kiefer, F. (1989) Clastogenic effects of benzo[a]pyrene in postimplantation embryos with 

14 different genetic background. Teratog Carcinog Mutagen 9(6):383–392.

15

16 Agarwal, R; Medrano, EE; Khan, IU; et al. (1991) Metabolism of benzo[a]pyrene by human melanocytes in 

17 culture. Carcinogenesis 12(10):1963–1966.

18

19 Agrelo, C; Amos, H. (1981) DNA repair in human fibroblasts. In: De Serres, FJ.Ashby, J eds.: Elsevier/North20 Holland, 528–532.

21

22 Albert, RE; Miller, ML; Cody, T; et al. (1991) Benzo[a]pyrene-induced skin damage and tumor promotion in 

23 the mouse. Carcinogenesis 12(7):1273–1280.

24

25 Alexandrie, AK; Nyberg, F; Warholm, M; et al. (2004) Influence of CYP1A1, GSTM1, GSTT1, and NQO1 

26 genotypes and cumulative smoking dose on lung cancer risk in a Swedish population. Cancer Epidemiol 

27 Biomarkers Prev 13(6):908–914.

28

29 Alexandrov, K; Rojas, M; Goldberg, M; et al. (1990) A new sensitive fluorometric assay for the metabolism of 

30 (±)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by human hair follicles. Carcinogenesis 11:2157–2161.

31

32 Alfheim, I; Ramdahl, T. (1984) Contribution of wood combustion to indoor air pollution as measured by 

33 mutagenicity in Salmonella and polycyclic aromatic hydrocarbon concentration. Environ Mutagen 6(2):121–

34 130.

35

36 Alzieu, P; Cassand, P; Colin, C; et al. (1987) Effect of vitamins A, C and glutathione on the mutagenicity of 

37 benzo[a]pyrene mediated by S9 from vitamin A-deficient rats. Mutat Res 192(4):227–231.

38

39 Ames, BN; Durston, WE; Yamasaki, E; et al. (1973) Carcinogens are mutagens: a simple test system combining 

40 liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci USA 70(8):2281–2285.

41

42 Ames, BN; McCann, J; Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the 

43 Salmonella/mammalian-microsome mutagenicity test. Mutat Res 31(6):347–364.

44

45 Amacher, DE; Paillet, SC. (1983) The activation of procarcinogens to mutagens by cultured rat hepatocytes in 

46 the L5178Y/TK mutation assay. Mutat Res 113:77-88. 

47
Toxicological Review of benzo[a]pyrene

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

2 DRAFT—DO NOT CITE OR QUOTE

1 Amacher, DE; Paillet, SC; Turner, GN; et al. (1980) Point mutations at the thymidine kinase locus in L5178Y 

2 mouse lymphoma cells. II. Test validation and interpretation. Mutat Res 72:447-474. 

3

4 Amacher, DE; Turner, GN. (1980) Promutagen activation by rodent-liver postmitochondrial fractions in the 

5 L5178Y/TK cell mutation assay. Mutat Res 74:485-501.

6

7 Ampy, FR; Saxena, S; Verma, K. (1988) Mutagenicity of benzo(a)pyrene in uninduced tissues from BALB/c 

8 mice and Sprague-Dawley rats as an index of possible health risks using the Salmonella mutagenicity assay. 

9 Cytobios 56(225):81–87.

10

11 Andrysik, Z; Vondracek, J; Machala, M; et al. (2007) The aryl hydrocarbon receptor-dependent deregulation of 

12 cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells. Mutat Res 615(1-

13 2):87–97.

14

15 Antignac, E; Koch, B; Grolier, P; et al. (1990) Prochloraz as potent inhibitor of benzo[a]pyrene metabolism 

16 and mutagenic activity in rat liver fractions. Toxicol Lett. 54(2–3):309–315.

17

18 Arce, GT; Allen, JW; Doerr, CL; et al. (1987) Relationships between benzo(a)pyrene-DNA adduct levels and 

19 genotoxic effects in mammalian cells. Cancer Res 47(13):3388–3395.

20

21 Archibong, AE; Inyang, F; Ramesh, A; et al. (2002) Alteration of pregnancy related hormones and fetal 

22 survival in F-344 rats exposed by inhalation to benzo(a)pyrene. Reprod Toxicol 16(6):801–808.

23

24 Archibong, AE; Ramesh, A; Niaz, MS; et al. (2008) Effects of benzo(a)pyrene on intra-testicular function in F25 344 rats. Int J Environ Res Public Health 5(1):32–40.

26

27 ATSDR (Agency for Toxic Substances and Disease Registry). (1995) Toxicological profile for polycyclic 

28 aromatic hydrocarbons. Atlanta, GA: Agency for Toxic Substances and Disease Registry.

29

30 Autrup, H; Seremet, T. (1986) Excretion of benzo[a]pyrene-Gua adduct in the urine of benzo[a]pyrene-treated 

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55 Pharmacogenetics 13(6):349–355.
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Toxicological Review of benzo[a]pyrene

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Toxicological Review of benzo[a]pyrene

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Toxicological Review of benzo[a]pyrene

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53
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55
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55
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54
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54
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