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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 Agenc y 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.



### ii DRAFT

— DO NOT CITE OR QUOTE DISCLAIMER

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

distributed solely for the purpose of pre

- dissemination peer review under applicable

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

represent and should not be construed to represent any Agency determination or policy.

Mention of trade names or commercial products does not constitute endorsement of

recommendation for use.

### Toxicological Review of benzo[a]pyrene

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



### iii DRAFT

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# CONTENTS

APPENDIX A. OTHER AGENCY AND INTERNATIONAL ASSESSMENTS

................................ ........ A

- 1

APPENDIX B. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE

REPONSE 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 DE RIVATION 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 APPROACH ES FOR CROSS

- SPECIES SCALING OF T HE

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

### Toxicological Review of benzo[a]pyrene

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



### iv DRAFT

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# TABLES AND FIGURES

Table A

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

1

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

- 2 5

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 ± SD a 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 B6C3F 1 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 lif e

................................ ................................ ................................ ........ 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 t o 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

### Toxicological Review of benzo[a]pyrene

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### v DRAFT

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................................ ................................ ................................ ................................ ............................ 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

– PN D14

................................ ... 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 ± SD a 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 ± SD a 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 mice C

21

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

### Toxicological Review of benzo[a]pyrene

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### vi DRAFT

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................................ ................................ ................................ ................................ .......... C

- 34

Table C

- 16. Derivation of HEDs for dose

- response modeling of B6C3F 1 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 d ata for rats from Kroese et al. (1981)

................................ ........ C

- 36

Table C

- 18. Summary of human equivalent overall oral slope factors, based on male and f emale 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

benz o[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

.... C

71

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]p yrene (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 ski n 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., 1 984); graph and model output.

................................ ............. C

- 89

Figure C

- 17. Fit of log

- logistic model to skin tumors in female CFLP mice exposed dermally to

b enzo[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

### Toxicological Review of benzo[a]pyrene

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### vii DRAFT

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# ABBREVIATIONS

3

- MC 3

- methylcholanthrene

8

- OHdG 8

- hydroxydeoxyguanosine

ADAF age

- dependent adjustment factor

Ah a ryl 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

- N 2

- BPDE 10β

- (deoxyguanosin

- N 2

- yl)

-

7β,8α,9α

- trihydroxy

7,8,9,10

- 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

ER OD 7

- ethoxyresorufin

- O

- deethylase

ETS environmental tobacco smoke

Fe 2 O 3 ferrous oxide

FOB functional observational battery

Ga 2 O 3 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

- adver se

- 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 chai n 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

### Toxicological Review of benzo[a]pyrene

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### viii DRAFT

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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

- tetradecano ylphorbol

- 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

### Toxicological Review of benzo[a]pyrene

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# APPENDIX A. OTHER AG ENCY AND 1



# INTERNATIONAL ASSESSMENTS 2

Table A

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

and international agencies 4

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) - 1 derived 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/m 3 ) - 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 - 5 μg/m 3 benzo[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/m 3 ) - 1 was derived based on Thyssen et al. (1981).

EU (2005) Target value of 1 ng/m 3 benzo[a]pyrene (averaged over one calendar year) as a

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

was derived.

### Toxicological Review of benzo[a]pyrene

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### A

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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

### Toxicological Review of benzo[a]pyrene

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### B

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# APPENDIX B. INFORMATION IN SUPPORT OF 1



# HAZARD IDENTIFICATION AND DOSE

- REPONSE 2



# ANALYSIS 3

TOXICOKINETICS 4

Overview 

### 5

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

### 6

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

### 7

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

### 8

metabolites in serum and excreta dem onstrate wide systemic tissue distribution. Benzo[a]pyrene 

### 9

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

### 10

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

### 11

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

### 12

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

### 13

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

### 14

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

### 15

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

### 16

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

### 17

benzo[a]pyrene is eliminated quickly with a biological half

- life of several hours. 

### 18

Absorption 

### 19

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

### 20

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

### 21

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

### 22

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

### 23

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

### 24

were correlated to urine concentrations of benzo[a]pyrene

- 9,10

- dihydrodiol, a specific metabolite 

### 25

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

### 26

benzo[a]pyrene extracted

- from personal air monitoring devices (a surrogate for ambient PAHs) of 

### 27

coke oven workers were correlated with r

- 7,t

- 8,9,c 10 tetrahydroxy

- 7,8,9,10

- 

### 28

tetrahydrobenzo[a]pyrene (trans

- anti

- benz o[a]pyrene

- tetrol, a specific metabolite of 

### 29

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

### 30

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

### 31

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

### 32

described in the excretion section below). These occupational studies cannot be used to quantify 

### 33

### 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 absorption through inhalation

- only exposure in humans because the persistence of 

### 1

benz o[a]pyrene

- contaminated particulate matter on surfaces and food may lead to exposures via 

### 2

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

### 3

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

### 4

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

### 5

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

### 6

provide supporting, quantitative evidence that absorption by the respiratory tract i s rapid (Bevan 

### 7

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

### 8

instillation of 1 μg 3H

- labeled benzo[a]pyrene/kg dissolved in triethylene glycol to Sprague

- Dawley 

### 9

rats, radioactivity rapidly appeared in the liv er (reaching a maximum of about 21% of the 

### 10

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

### 11

with elimination half

- times of 5 and 116 minutes (Weyand and Bevan, 1986). In bile

- cannulated 

### 12

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

### 13

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

### 14

respiratory tract and rapid entry into hepatobiliary circulation following in tratracheal instillation. 

### 15

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

### 16

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

### 17

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

- bound 

### 18

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

### 19

neat compound (Gerde et al., 2001). 

### 20

Studies conducted to assess levels of benzo[a]pyrene metabolites or b enzo[a]pyrene

- DNA 

### 21

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

### 22

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

### 23

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

### 24

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

### 25

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

### 26

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

### 27

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

### 28

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

### 29

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

- fat diets, 

### 30

as compared with fiber

- or protein

- rich diets (O’Neill et al., 1991; Kawamura et al., 1988). Aqueous 

### 31

vehicles, quercetin, chlorog enic acid, or carbon particles reduced biliary excretion of 

### 32

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

### 33

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

### 34

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

### 35

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

### 36

dermally to benzo[a]pyrene

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

### 37

dermally. One study of dermal absorption in human vo lunteers found absorption rate constants 

### 38

### 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 ranging from 0.036 to 0.135/hour over a 45 minute exposure, suggesting 20

– 56% of the dose 

### 1

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

### 2

between different anatomical s ites (forehead, shoulder, volar forearm, palmar side of the hand, 

### 3

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

### 4

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

### 5

(maintained in short

- term organ cultures) exposed for 24 hours ranged from 0.09 to 2.6% of the 

### 6

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

### 7

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

### 8

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

### 9

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

### 10

exposure is an importan t factor in skin penetration. Exposure of female Sprague

- Dawley rats and 

### 11

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

- 

### 12

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

### 13

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

### 14

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

### 15

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

### 16

1985). 

### 17

Distribution 

### 18

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

### 19

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

### 20

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

### 21

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

### 22

excreta of PAH

- exposed populations suggest that benzo[a]pyren e is widely distributed. 

### 23

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

### 24

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

### 25

in various species (Sprague

- Dawley rats, Gu nn rats, guinea pigs, and hamsters) results in wide 

### 26

distribution throughout the body and rapid uptake into well

- perfused tissues (i.e. lung, kidney, and 

### 27

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

### 28

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

### 29

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

### 30

Weyand and Bevan, 1986; Morse and Carlson, 1985; Saunders et al., 2002; N eubert and Tapken 

### 31

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

### 32

in increased radioactivity in lung

- associated lymph nodes, suggesting distribution of 

### 33

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

### 34

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

### 35

thioether and glucuronic acid

- conjugated metabolites in intestines indicated enterohepatic 

### 36

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

### 37

vehicle for delivery of inhalated benzo[a]pyrene impacts the distribution with aerosolized 

### 38

### 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 benzo[a]pyrene more readily absorbed directly in the respiratory tract than particle

- ad sorbed 

### 1

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

### 2

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

### 3

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

### 4

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

### 5

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

### 6

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

### 7

metabolism, such. Serum of benzo[a]pyrene

- treated mice incubated with splenocytes or salmon 

### 8

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

### 9

were systemically dist ributed and available for interaction with target tissues (Ginsberg and 

### 10

Atherholt, 1989). 

### 11

Metabolism 

### 12

The metabolic pathways of benzo[a]pyrene (Figure B

- 1) and variation in species, strains, 

### 13

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

### 14

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

### 15

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

### 16

detected urinary metabolite (7,8,9 ,10

- tetrol) was observed in patients treated with coal tar 

### 17

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

### 18

humans. 

### 19

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

### 20

intermediates, dih ydrodiols, phenols, quinones, and their various combinations that are likely to 

### 21

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

### 22

Phase II metabolism of benzo[a]pyrene metabolites protects cellular macromolecu les from binding 

### 23

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

### 24

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

### 25

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

### 26

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

### 27

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

### 28

from these reviews and supplemented with recent findings. 

### 29

Phase I metabolism 

### 30

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

### 31

metabolites including epoxides, dihydrodiols, phenols and quinones (Figure B

- 2). The first step of 

### 32

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

### 33

the 2,3

- , 4,5

- , 7,8

- , and 9,10

- isomers (Gelboin, 1980). Once formed, these epoxides may undergo 

### 34

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

### 35

trans

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

### 36

binding with glutathione (either spontaneously or catalyzed by cytosolic glutathione

- S

- transferases 

### 37

### 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 (IARC 1983)). The metabolism of benzo[ a]pyrene to phenols occurs for 5 phenol isomers (1

- , 3

- , 6

- , 

### 1

7, and 9

- OH benzo[a]pyrene) (Pelkonen et al. 1982). The hydration of benzo[a]pyrene epoxides to 

### 2

trans

- dihydrodiols occurs for all four major epoxide isomers (2,3

- , 4,5

- , 7,8

- , and 9,10

- ). The 

### 3

7 ,8

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

### 4

the potent DNA

- binding metabolite benzo[a]pyrene

- 7,8

- diol

- 9,10

- epoxide (BPDE). BPDE is formed 

### 5

from benzo[a]pyrene 7,8

- transdiol by multiple mechanisms incl uding catalysis by CYPs (Deutsch 

### 6

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

### 7

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

### 8

diolepoxides can react further by s pontaneously hydrolyzing to tetrols (Hall and Grover 1988). 

### 9

### 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 1

Source: Miller and Ramos (2001). 

### 2

Figure B

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

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

### 4

microsomes from r ats stereospecifically oxidize the 7,8

- bond of benzo[a]pyrene to yield almost 

### 5

exclusively the (+)

- benzo[a]pyrene

- (7,8)

- oxide (see Figure B

- 2). Each enantiomer of the 7,8

- oxide 

### 6

is stereospecifically converted by epoxide hydrolase (EH) to a different dihydr odiol and further 

### 7

metabolism of the (

– )

- benzo[a]pyrene

- 7,8

- dihydrodiol enantiomer by rat CYP enzymes 

### 8

preferentially yields (+)

- benzo[a]pyrene

- 7R,8S

- diol

- 9S,10R

- epoxide [(+)

- anti

- benzo[a]pyrene

- 7,8

- 

### 9

diol

- 9,10

- epoxide (BPDE)], which is believed to be the mos t potent carcinogen among the four 

### 10

stereoisomers (Figure B

- 2). Formation of these stereoisomers does not occur at equimolar ratios, 

### 11

and the ratios differ between biological systems. For example a study in rabbit livers demonstrated 

### 12

### 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 that purified microsom es oxidized the (

- )

- benzo[a]pyrene

- 7,8

- dihydrodiol to isomeric diol epoxides 

### 1

in a ratio ranging from 1.8:1 to 11:1 in favor of the (+)

- anti

- BPDE isomer (Deutsch et al., 1979). 

### 2

3

4

Source: Grover (1986). 

### 5

Figure B

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

Several studies have attempted to determine which CYP isozyme is predominantly 

### 7

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

- benzo[a]pyrene 

### 8

to mice that have an Ah receptor (AhR) knock

- out (AhR

– /

– ) had significan tly decreased formation 

### 9

of (+)

- anti

- BPDE

- DNA adducts compared to WT and 1B1

– /

– mice (Kleiner et al. 2004). Gavage 

### 10

administration of benzo[a]pyrene in AhR knock

- out mice found the AhR

– /

– mice (with lower levels 

### 11

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

### 12

AhR+/+ or +/

– mice (Sagredo et al., 2006). Similarly, CYP1A1 (

– /

– ) knock

- out mice administered 

### 13

benzo[a]pyrene in feed for 18 days had higher steady

- state blood levels of benzo[a]pyrene and 

### 14

benzo[a]pyrene

– DNA ad ducts (Uno et al. 2006). DNA post

- labeling studies of mice administered by 

### 15

gavage demonstrated higher benzo[a]pyrene

- DNA adduct levels in CYP1A1(

– /

– ) than CYP1A1(+/+) 

### 16

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

### 17

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

### 18

between the CYP enzymes and biological activation or detoxification. 

### 19

Another important factor in evaluating variability in the metabolic activation of 

### 20

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

### 21

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

### 22

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

### 23

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

### 1

producing about three times the amount of (±)

- anti

- BPDE isomers as compared to the 

### 2

stereoisomers, (±)

- syn

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

### 3

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

### 4

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

### 5

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

### 6

transdihydrodiol

- benzo[a]pyrene or 6

- OH benzo[a]pyrene into quinones, primarily the 1,6

- , 3,6

- , 

### 7

7,8

- and 6,12

- isomers. Transdihydrodiol

- benzo[a]pyrene such as (+/

- )

- anti

- BPDE can be 

### 8

converted in a redox cycling reaction into benzo[a]pyrene

- 7,8

- quinone (BPQ) catalyzed by 

### 9

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

### 10

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

### 11

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

### 12

6

- Hydroxybenzo[a]pyrene can be oxidized into 6

- oxo

- benzo[a]pyrene semi

- quinone radical and 

### 13

further metabolized into 1,6

- , 3,6

- , or 6,12

- quinones spontan eously, or catalytically by 

### 14

prostaglandin endoperoxide synthetase (Eling, et al 1983). 

### 15

Phase II metabolism 

### 16

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

### 17

conjugation and detoxification enzyme systems that display preferential activity for specific 

### 18

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

### 19

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

### 20

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

### 21

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

### 22

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

### 23

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

### 24

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

### 25

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

### 26

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

### 27

intratracheal instillation to male Sprague

- Dawley rats were 49% glucuronides (quinol 

### 28

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

### 29

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

### 30

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

### 31

human GSTs expressed in mammalian cell lines have demonstrated the ability of GST t o metabolize 

### 32

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

### 33

benzo[a]pyrene

- derived diol epoxides and (±)anti

- BPDE with variation in activity across GST 

### 34

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

### 35

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

### 36

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

### 37

molecular epidemiol ogy studies. However, recent studies on the impact of polymorphism on 

### 38

### 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 adduct levels in PAH

- exposed human populations did not show a clear relationship between the 

### 1

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

### 2

(H emminki et al., 1997) or blood protein adducts (Pastorelli et al., 1998). 

### 3

Conjugation with UDP

- glucuronide catalyzed by UGT enzymes is another important 

### 4

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

### 5

alle lic variants, are expressed and have glucuronidation activity toward benzo[a]pyrene

- derived 

### 6

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

### 7

but not lung or liver (Zheng et al., 2002; Fang and Lazarus 20 04). UGT activity also shows 

### 8

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

### 9

covalent binding to protein with a 143

- fold interindividual variability and a statistically significant 

### 10

inverse correlation be tween glucuronidation and protein binding (Hu and Wells, 2004). 

### 11

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

### 12

rat or mouse liver, cytosolic sulfotransferase (in the presence of 3'

- phosphoadenosine 5'

- 

### 13

phosphosul fate) catalyzes formation of sulfates of three benzo[a]pyrene metabolites: 

### 14

benzo[a]pyrene

- 7,8,9,10

- tetrahydro

- 7

- ol, benzo[a]pyrene

- 7,8

- dihydrodiol, and benzo[a]pyrene



### 15

7,8,9,10

- tetrol. The benzo[a]pyrene

- 7,8,9,10

- tetrahydro

- 7

- ol

- sulfate is able to form pot entially 

### 16

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

- 

### 17

hydroxybenzo[a]pyrene conjugation to sulfate produces benzo[a]pyrene

- 3

- yl

- hydrogen sulfate, a 

### 18

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

### 19

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

### 20

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

### 21

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

### 22

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

### 23

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

### 24

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

### 25

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

- quinone adducts 

### 26

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

### 27

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

### 28

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

### 29

Tissue

- specific Metabolism 

### 30

Benzo[a]pyrene metabolism has been demo nstrated in vivo in laboratory animals for 

### 31

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

### 32

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

### 33

formation of dihyd rodiols, phenols, quinones, and tetrols in the nasal mucus and lung (Petridou



### 34

Fischer et al. 1988; Weyand and Bevan 1986, 1987a, 1988; Dahl et al. 1985; Wolff et al. 1989b). In 

### 35

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

### 36

benzo[a]pyrene, 16% conjugates or polyhydroxylated compounds, 10.7% 4,5

- , 7,8

- , and 9,10

- 

### 37

dihydrodiols, 9.3% 1,6

- , 3,6

- , 6,12

- quinone, and 6.9% 3

- and 9

- hydroxybenzo[a]pyrene (Weyand 

### 38

### 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 and Bevan 1986). In hamsters, approximately 50% o f the benzo[a]pyrene instilled was 

### 1

metabolized in the nose (nasal tissues had the highest metabolic acitivity per

- gram of the 

### 2

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

### 3

1985). 

### 4

In vitro studies of hum an and laboratory cells and cell lines provide further quantitative and 

### 5

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

### 6

liver and other tissues. Tracheobronchial tissues in culture of several species (i ncluding humans, 

### 7

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

### 8

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

### 9

degree of interindividual variability (a 33

- fold difference in human bronchus, a 5

- fold variation in 

### 10

human trachea, and a 3

- fold difference in bovine bronchus), but minimal variation among 

### 11

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

### 12

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

### 13

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

### 14

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

### 15

amount of unconjugated 7,8

- dihydrodiol (Monteith et al. 1987). 

### 16

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

### 17

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

### 18

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

### 19

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

### 20

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

### 21

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

### 22

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

### 23

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

### 24

human skin samples treated with 2 μg/cm 2 [ 14 C]

- benzo[a]pyrene in acetone and incubated for 

### 25

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

- soluble 

### 26

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

### 27

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

### 28

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

### 29

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

### 30

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

### 31

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

### 32

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

### 33

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

### 34

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

### 35

(Chakradeo et al. 1993). 

### 36

### 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 Elimination 

### 1

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

### 2

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

### 3

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

### 4

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

### 5

hamsters, guinea pigs, monkeys and dogs (Petridou

- Fischer et al., 1988; Wolff et al., 1989; Sun et al., 

### 6

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

### 7

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

### 8

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

### 9

after a single intratracheal instillation of benzo[a]pyrene (2 μg/kg) to male Sprague

- Dawley rats, 

### 10

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

### 11

monoglucuronides, 6.2% sulfate conj ugates, and 14.4% unconjugated metabolites (Bevan and 

### 12

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

### 13

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

### 14

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

### 15

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

### 16

elimination rate or to other factors such as the absorption rate. For [ 3 H ]

- benzo[a]pyrene 

### 17

administered to Sprague

- Dawley rats in hydrophilic triethylene glycol, 70.5% of the dose was 

### 18

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

### 19

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

### 20

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

### 21

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

### 22

BPDE adducts with guanine detected in urine of male Wistar rats was dose

- dependent. 48 hours 

### 23

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

### 24

dose was excreted in the urine as an adduct with guanine (Autrup and Seremet, 19 86). Overall, the 

### 25

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

### 26

qualitatively but to limited to estimate quantitative rates of elimination. 

### 27

Physiologically based pharmacokinetic models 28

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

### 29

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

### 30

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

### 31

d ata or in vitro experiments. 

### 32

Bevan and Weyand (1988) performed compartmental pharmacokinetic analysis of 

### 33

distribution of radioactivity in male Sprague

- Dawley rats, following the intratracheal instillation of 

### 34

benzo[a]pyrene to normal and bile duct

- cannulate d animals (Weyand and Bevan, 1987, 1986). 

### 35

However, implicit simulation approaches were used, as opposed to physiologically

- based 

### 36

### 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 approaches. The model calculated linear rate constants among compartments, and assumed the 

### 1

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

### 2

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

### 3

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

### 4

from Wiersma and Roth (1983a, b) and was evaluate d against tissue concentration data from 

### 5

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

### 6

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

### 7

was estimated usin g kinetic data from control rats and rats pretreated with 3

- MC to induce 

### 8

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

### 9

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

### 10

l iver. An adequate fit was obtained for some compartments; however tissue

- level data for 

### 11

calibration and validation of this model were limited. 

### 12

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

### 13

model development. R ats were injected with varying doses of [ 14 C]

- benzo[a]pyrene to 15 mg/kg 

### 14

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

### 15

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

### 16

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

### 17

modeled as diffusion

- limited, while the other tissues were flow

- limited. The model predicted the 

### 18

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

### 19

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

### 20

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

### 21

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

### 22

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

### 23

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

### 24

model. No metab olites were included in the model. This model was re

- calibrated by Crowell et al. 

### 25

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

### 26

However, it still did not incorporate metabolites, and some tissues conti nued to exhibit poor model 

### 27

fits. 

### 28

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

### 29

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

### 30

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

### 31

van Eijkeren, 1997). Most compartments were perfusion

- limited, and tissues modeled included 

### 32

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

### 33

How ever, there was no separate compartment for the lung. The liver compartment featured the 

### 34

AhR

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

### 35

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

### 36

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

### 37

model was experimentally calibrated in rats with the data for EROD and formation of DNA adducts 

### 38

### 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 in the liver after i.v. administration of a single dose and per os (p.o.) administration of a single or 

### 1

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

### 2

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

### 3

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

### 4

for the decay of the benzo[a]pyrene

- CYP450 complex, half

- life of the CYP450 protein, fraction and 

### 5

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

### 6

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

### 7

mechanism of AhR

- dependent induction of CYP450 dominated the simulated benzo[a]pyrene

- DNA 

### 8

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

### 9

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

### 10

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

### 11

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

### 12

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

### 13

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

### 14

mutagenic and carcinogenic metabolites c ould not be explicitly modeled in human liver because no 

### 15

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

### 16

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

### 17

benzo[a]pyre ne

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

### 18

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

### 19

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

### 20

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

### 21

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

- DNA 

### 22

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

### 23

DNA and other macromolecules. Alternatively, stable toxic metabolites (e.g., trans

- anti

- tetrol



### 24

benzo[a]pyrene) may be used as an internal dose surrogate. While the metabolic pattern of 

### 25

benzo[a]pyrene has bee n relatively well characterized qualitatively in animals, the quantitative 

### 26

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

### 27

not yet well defined. 

### 28

Recommendations for the use of PBPK models in toxicity value derivation 

### 29

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

### 30

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

### 31

uncertainties with respect to the inter

- species scaling of the metabolic parameters between rats 

### 32

and humans, these models were not used for cross

- species extrapolation. Furthermore, no 

### 33

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

### 34

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

### 35

carbonaceous particles. This precluded the model’s use for cross

- route extrapolation to the 

### 36

inhalation pathway. 

### 37

### 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 HUMAN STUDIES 1

Non

- Cancer Endpoints 

### 2

Cardiovascular Endpoints 

### 3

Burystn et al. (2005) repor ted the association of death from cardiovascular disease with 

### 4

B[a]P exposure in a cohort of 12,367 male European asphalt workers (Table B

- 1) . These workers 

### 5

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

### 6

Avera ge duration of follow

- up was 17 ± 9 years (mean ± SD), encompassing 193,889 person

- years 

### 7

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

### 8

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

### 9

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

### 10

quantitatively using measurement

- driven mixed effects exposure models, using data collected from 

### 11

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

### 12

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

### 13

this analysis was limited to diseases of the circulatory system (ICD codes 390

– 459), specifically 

### 14

ischem ic heart disease (IHD: ICD codes 410

– 414). Diesel exhaust exposure was also assessed in 

### 15

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

### 16

from cardiovascular disease. 0.25% of the cohort was lost to foll ow

- up, and 0.38% emigrated 

### 17

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

### 18

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

### 19

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

### 20

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

### 21

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

### 22

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

### 23

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

### 24

From analysis of smoking incidence in a sub

- cohort, the following sm oking distribution was 

### 25

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

### 26

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

### 27

Exposed subjects were stratified into quintiles base d upon IHD mortality, with 83

– 86 

### 28

deaths per exposure category, composing approximately 2/3 of the 660 cardiovascular disease



### 29

related deaths. Both cumulative and average exposure indices for B[a]P were positively associated 

### 30

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

### 31

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

### 32

all cardiovascular diseases (combined), although a dose

- response relationship was evident only fo r 

### 33

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

### 34

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

### 35

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

### 36

the contribution of smoking to cardiovascular disease etiology was greater than the original 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 17 DRAFT

— DO NOT CITE OR QUOTE assumptions. Furthermore, the RR for the high vs. low exposure quintile is 1.24 even if the 

### 1

distribution of non

- smokers/for mer smokers/current smokers shifts to 0/30/70%, using the 

### 2

original assumptions of cigarette smoke casual potency. 

### 3

Table B

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

cardiovascular diseases in a European cohort of asphalt paving workers 5

Effect measu red Cumulative exposure (ng/m 3

– years) P for

trend 0

– 189 a 189

– 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/m 3 ) P for

trend 0

– 68 a 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

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

### 7

chronic non

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

### 8

smelter workers (Table B

- 2 ) . Inclusion criteria required at least 3 years of continuous employment 

### 9

in either the smelter facility or power

- generating station from 1954

– 1997, with worker history 

### 10

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

### 11

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

### 12

standardized mortality ratios, which were adjusted for age, sex and time period. Ninety

- five % 

### 13

confidence intervals were calculated for the SMRs assu ming a Poisson distribution. Internal 

### 14

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

### 15

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

### 16

could not be differentiated from other IHD on death certificates issued previously. HRs were 

### 17

### Toxicological Review of benzo[a]pyrene

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



### B

- 18 DRAFT

— DO NOT CITE OR QUOTE calculated using Cox regression models, with age as a metamarker of time, also including smoking 

### 1

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

### 2

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

- smokers and 

### 3

25% never

- smokers. Quantitative exposure to coal tar pitch volatiles were estimated by B[a]P 

### 4

measurements, calculated by a job classification and time

- based e xposure matrix, as described in a 

### 5

previous report; annual arithmetic mean values were calculated for exposures from 1977

– 2000, 

### 6

while pre

- 1977 levels were backwards

- extrapolated from 1977 values, incorporating major 

### 7

technological changes in time periods a s appropriate. 

### 8

Cumulative exposure metrics were highly skewed. Cumulative B[a]P with a 5

- year lag (past 

### 9

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

### 10

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

### 11

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

### 12

5

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

– 

### 13

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

### 14

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

- term 

### 15

exposure. 

### 16

When exposed workers were pooled and compared externally to non

- ex posed referents, the 

### 17

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

### 18

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

### 19

stratified into quintiles based upon IHD mor tality, with approximately 56 deaths per exposure 

### 20

category. 5

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

### 21

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

### 22

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

### 23

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

### 24

significance. No association was observed between B[a]P exposure and non

- AM I IHD. While there 

### 25

was little difference in the exposure

- response association among 0, 2 and 5

- year lagged data, 10

- 

### 26

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

### 27

incorporation of work status and time

- since

- hire to account for the healthy worker effect, as 

### 28

evidenced by the SMR of 0.87 (95% CI: 0.82, 0.92) for all chronic non

- malignant diseases combined 

### 29

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

### 30

calculated tha t the risk of death from IHD to be 1.002 (95% CI: 1.000, 1.005) per μg/m 3 from 

### 31

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

### 32

that the association is nonlinear, suggesting that this value may be an underest imate. Restricting 

### 33

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

### 34

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

### 35

the highest exposure group was 2.39 (95% CI: 0.95, 6.05). Exposure

- response relationships were 

### 36

similarly examined in male smelter workers for chronic obstructive pulmonary disease (COPD) and 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 19 DRAFT

— DO NOT CITE OR QUOTE cerebrovascular disease, but neither was significantly associated with cumulative B[a]P exposure in 

### 1

either internal or external comparisons. 

### 2

Table B

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

cardiovascular diseases in a Canadian cohort of male aluminum smelter 4

workers 5

Effect measured Categorical cumulative e xposure with a 5

- year lag (μg/m 3

– year) P for

trend a Continuous b

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 follow

up

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 follow

up

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 ).

Reproductive and Developmental Endpoints 

### 6

Wu et al. (2010) conducted a study of benzo[a]pyrene

- DNA adduct levels in relation to risk 

### 7

of fetal death in Tianjin, China. This case

- control study included women who experienced a missed 

### 8

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

### 9

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

### 10

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

### 11

women who smoked, women with chronic d isease and pregnancy complications, and women with 

### 12

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

### 13

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

### 14

cont rols participated. Data pertaining to demographic characteristics, reproductive history, and 

### 15

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

### 16

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

### 17

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

### 18

benzo[a]pyrene

- BPDE adducts was assessed in the blood and tissue samples using HPLC. There 

### 19

### Toxicological Review of benzo[a]pyrene

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



### B

- 20 DRAFT

— DO NOT CITE OR QUOTE was no correlation between blood and abo rted tissue levels of benzo[a]pyrene adducts (r =

- 0.12 

### 1

for the 102 blood

- tissue pairs, r =

- 0.02 for the 51 case pai r s and r =

- 0.21 for the 51 control pairs). 

### 2

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

### 3

sampl es in terms of the interview

- based measures collected or in terms of the DNA

- adduct levels in 

### 4

aborted tissue.) Benzo[a]pyrene

- adduct levels were similar but slightly lower in the aborted tissue 

### 5

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

### 6

the blood samples, however, benzo[a]pyrene

- adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ± 

### 7

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

### 8

adduct me asure, the OR was 1.35 (95% CI 1.11

– 1.64) per adduct/10 8 nucleotide. These results 

### 9

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

### 10

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

– 12.99) 

### 11

in the high compared with low benzo[a]pyrene

- adduct group. There was no relation between 

### 12

benzo[a]pyrene

- adduct levels in the aborted tissue and missed abortion in the logistic regression 

### 13

analyses using either the continuous (adjus ted OR 0.97, 95% CI 0.93

– 1.02) or dichotomous 

### 14

exposure measure (adjusted OR 0.76, 95% CI 0.37

– 1.54). Associations between missed abortion 

### 15

and several interview

- based measures of potential PAH exposure were also seen: adjusted OR 3.07 

### 16

(95% CI 1.31

– 7.16) f or traffic congestion near residence, 3.52 (95% CI 1.44

– 8.57) for commuting 

### 17

by walking, 3.78 (95% CI 1.11

– 12.87) for routinely cooked during pregnancy, and 3.21 (95% CI 

### 18

0.98

– 10.48) for industrial site or stack near residence, but there was no association w ith other 

### 19

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

### 20

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

### 21

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

### 22

blood levels of benzo[a]pyrene (BPDE)

- DNA adducts were highest in study participants who lived 

### 23

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

### 24

the WTC. Neither cord blood adduct level nor ETS alone was positively correlat ed with adverse 

### 25

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

### 26

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

### 27

to ETS in utero, a doubling of cord blood benzo[a]pyr ene

- DNA adducts was associated with an 8% 

### 28

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

### 29

Perera et al. (2005b) compared various exposures

— ETS, nutrition, pesticides, material 

### 30

hardship

— with birth outcomes (length, he ad circumference, cognitive development). ETS 

### 31

exposure and intake of PAH

- rich foods by pregnant women were determined by questionnaire. 

### 32

Levels of benzo[a]pyrene diol epoxide (BPDE)

- DNA adducts were determined in umbilical cord 

### 33

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

- American 

### 34

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

### 35

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

### 36

c orrelated with each other. However, the interaction between benzo[a]pyrene

- DNA adducts and 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 21 DRAFT

— DO NOT CITE OR QUOTE ETS exposure was significantly associated with reduced birth weights (

- 6.8%; p = 0.03) and 

### 1

reduced head circumference (

- 2.9%; p = 0.04). 

### 2

Tang et al. (2006 ) measured benzo[a]pyrene diol epoxide (BPDE)

- DNA adducts in maternal 

### 3

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

### 4

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

- fired 

### 5

p ower plant; however, airborne PAH concentrations were not measured. Dietary PAH intake was 

### 6

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

### 7

sex, and maternal height and weight were considered as covaria tes. DNA adduct levels were 

### 8

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

### 9

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

### 10

and pregnancy weight total weigh t gain; complications of pregnancy and delivery; and medications 

### 11

used during pregnancy. 

### 12

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

### 13

weight at 18 months (β =

- 0.048, p = 0.03), 24 months (β =

- 0.041, p = 0.027), and 30 months of age 

### 14

(β =

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

### 15

adduct levels (β =

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

### 16

blood adduct levels nor with fetal and child g rowth. Among female infants, cord blood adduct levels 

### 17

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

### 18

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

### 19

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

### 20

associations were also inverse but were not statistically significant. 

### 21

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

### 22

accumula ted from epidemiological studies of time to pregnancy, ovulatory disorders, semen 

### 23

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

### 24

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

### 25

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

### 26

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

### 27

measures of benzo[a]pyrene and reproductive outcom es. 

### 28

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

### 29

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

### 30

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

### 31

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

### 32

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

### 33

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

### 34

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

### 35

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

### 36

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

### 37

(60.0, 14.3, and 21.0% below detection limit in nonsmoking, sidestream smoke, and active smoking 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 22 DRAFT

— DO NOT CITE OR QUOTE groups, respectively). A similar pattern was seen with follicular fluid benzo[ a]pyrene (30.0, 14.3, 

### 1

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

### 2

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

### 3

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

### 4

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

### 5

nonsmoking (0.03 ± 0.01 ng/mL) groups ( p = 0.04). The between

- group differences in serum 

### 6

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

### 7

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

### 8

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

### 9

smoked per day was strongly correlated with follicular fluid benzo[a]pyrene levels (r

= 0.7, p < 

### 10

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

### 11

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

### 12

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

### 13

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

### 14

A small case

- control study conducted between August 2005 and February 2006 in Lu cknow 

### 15

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

### 16

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

### 17

of gestation) and 31 term delivery controls. Demo graphic data smoking history, reproductive 

### 18

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

### 19

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

### 20

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

### 21

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

### 22

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

### 23

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

### 24

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

### 25

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

### 26

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

### 27

ppb in controls for benzo[a]pyrene compared with 25

– 30 ppb for anthracene, 

### 28

benzo(k)fluoranthene, benzo(b)fluoranthene, and dibe nzo(a,h)anthracene, 59 ppb for 

### 29

acenaphthylene, and 200

– 380 ppm for naphthalene, phenanthrene, fluoranthene, and pyrene; 

### 30

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

### 31

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

### 32

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

### 33

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

### 34

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

### 35

Wu et al. (2010) conducted a study of benzo[a]pyrene

- DNA adduct levels in relation to risk 

### 36

of fetal death in Tianjin, China. This case

- control study included women who experienced a miss ed 

### 37

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

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 23 DRAFT

— DO NOT CITE OR QUOTE required surgical intervention). Cases were matched by age and gravidity to controls (women 

### 1

undergoing induced abortion due to an unplanned or unwanted p regnancy). The study excluded 

### 2

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

### 3

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

### 4

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

### 5

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

### 6

factors relating to potential PAH exposure were collected using a structured interview, and s amples 

### 7

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

### 8

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

### 9

benzo[a]pyrene

- BPDE adducts was assessed in the blood and tissu e samples using HPLC. There 

### 10

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

- 0.12 

### 11

for the 102 blood

- tissue pairs, r =

- 0.02 for the 51 case pai r s and r =

- 0.21 for the 51 control pairs). 

### 12

(The authors noted that t here was little difference between women with and without blood 

### 13

samples in terms of the interview

- based measures collected or in terms of the DNA

- adduct levels in 

### 14

aborted tissue.) Benzo[a]pyrene

- adduct levels were similar but slightly lower in the aborted tissue 

### 15

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

### 16

the blood samples, however, benzo[a]pyrene

- adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ± 

### 17

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

### 18

adduct measure, the OR was 1.35 (95% CI 1.11

– 1.64) per adduct/10 8 nucleotide. These results 

### 19

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

### 20

Cat egorizing exposure at the median value resulted in an adjusted OR of 4.27 (95% CI 1.41

– 12.99) 

### 21

in the high compared with low benzo[a]pyrene

- adduct group. There was no relation between 

### 22

benzo[a]pyrene

- adduct levels in the aborted tissue and missed abortion i n the logistic regression 

### 23

analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93

– 1.02) or dichotomous 

### 24

exposure measure (adjusted OR 0.76, 95% CI 0.37

– 1.54). Associations between missed abortion 

### 25

and several interview

- based measures of potentia l PAH exposure were also seen: adjusted OR 3.07 

### 26

(95% CI 1.31

– 7.16) for traffic congestion near residence, 3.52 (95% CI 1.44

– 8.57) for commuting 

### 27

by walking, 3.78 (95% CI 1.11

– 12.87) for routinely cooked during pregnancy, and 3.21 (95% CI 

### 28

0.98

– 10.48) for in dustrial site or stack near residence, but there was no association with other 

### 29

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

### 30

Neurotoxicity 

### 31

Niu et al. (2010) studied 176 Chinese coke

- oven workers with elevated B[a]P exposure and 

### 32

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

### 33

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

### 34

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

### 35

a ssess emotional state, learning, memory and hand

- eye coordination. The authors self

- designed a 

### 36

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

### 37

drinking habits, personal habits, personal and family medical his tory, as well as any current 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 24 DRAFT

— DO NOT CITE OR QUOTE symptoms and medications used in the pervious several weeks. Workers were excluded from the 

### 1

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

### 2

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

### 3

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

### 4

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

### 5

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

### 6

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

### 7

warehouse, and dual automatic air sampling pumps collected samples at per sonal breathing zone 

### 8

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

### 9

relative exposure was compared to post

- shift urine levels of a B[a]P metabolite, 1

- hydroxypyrene 

### 10

(1

- OH

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

### 11

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

### 12

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

### 13

(AchE) levels measured in lysed RBCs using activity kits. 

### 14

B[a]P mean concentrations were 19.56 ± 13.2, 185.96 ± 38.6 and 1623.56 ± 435.8 ng/m 3 at 

### 15

the bottom, side and top of the coke oven, respectively, all of which were higher than the mean at 

### 16

the referents’ warehouse (10.26 ± 7.6 ng/m 3 ). The authors did not report stratified analysis by 

### 17

different levels of B[a]P exposure, and reported only comparisons between the referents and all 

### 18

exposed workers combined (Table B

- 3), or between workers grouped by urinary B[a]P metabolite 

### 19

1

- OH

- Py levels (Table B

- 4) . There were no significant differences in age, education, smoking or 

### 20

alcohol use between the coke oven and warehouse workers. Urinary 1

- OH

- Py levels were 32% 

### 21

higher in coke oven workers compared to the referent group, correspondi ng to the higher levels of 

### 22

B[a]P detected in all coke oven workstation compared to the supply warehouse. Performance in 

### 23

two neurobehavioral function tests, digit span and forward digit span, were significantly decreased 

### 24

in the exposed oven workers versus control group; when stratified by urinary metabolite level, 

### 25

scores significantly decreased with increasing 1

- OH

- Py levels. Of the neurotransmitters assessed, 

### 26

norepinephrine, dopamine, Asp and GABA were significantly decreased in exposed versus control 

### 27

wor kers; norepinephrine and Asp were also significantly and inversely related with 1

- OH

- Py levels. 

### 28

Dopamine levels appeared to decrease with increased urinary metabolite levels, although the 

### 29

relationship was not statistically significant. GABA levels were h ighly variable, and appeared to 

### 30

increase with increasing 1

- OH

- Py levels, although this relationship was statistically significant. 

### 31

Acetylcholine levels were 4

- fold higher in coke oven workers compared to referents, and AchE 

### 32

actiivty 30% lower; both Ach an d AchE were significantly associated with urinary B[a]P metabolite 

### 33

levels, although Ach increased and AchE activity decreased with increasing 1

- OH

- Py. The authors 

### 34

reported results of correlation analysis, indicating that digit span scores correlated negat ively with 

### 35

Ach and positively with AchE (coefficients of

- 0.230,

- 0.276 and 0.120, 0.170, respectively), 

### 36

although no indication of statistical significance was given. No other associations were reported. 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 25 DRAFT

— DO NOT CITE OR QUOTE Table B

- 3 . Exposure

- related effects in Chinese cok e oven workers or 1

warehouse controls exposed to benzo[a] pyrene in the workplace 2

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 3 9

. 71 ± 7.51

23/25

77%

27% 37. 86 ± 6.51

110/66

64%

39% 0. 098

0. 068

0.0 93

0. 140

Urine B[a]P metabolite ( μmol/mol Cr; mean ± SD )

1

- OH

- Py 2.7 7 ± 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 ( μ g/ml)

Glu ( μ g/ml)

GABA ( μ g/ml)

Ach ( μ g/ml)

AchE activity (U/mg prot ein ) 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 ).

### Toxicological Review of benzo[a]pyrene

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



### B

- 26 DRAFT

— DO NOT CITE OR QUOTE Table B

- 4 . Exposure

- related effects in Chinese coke oven workers or 1

warehouse controls exposed to benzo[a] pyrene in the workplace, 2

stratified by urinary metabolite levels 3

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 ( μ g/ml)

Glu ( μ g/ml)

GABA ( μ g/ml)

Ach ( μ g/ml)

AchE activity (U/mg

prot ein ) 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 ).

Immunotoxicity 

### 4

Zhang et al. (2012) studied 129 Chinese coke

- oven workers with elevated B[a]P exposure 

### 5

and compared them against 37 referents (workers in a supply warehouse), matched by 

### 6

socioeconomic status, lifestyle and health. Area B[a]P levels were quantified in the various work 

### 7

areas, and the primary endpoint was the level of early and late apoptosis in PBMCs isolated from 

### 8

each worker sub

- group the morning following an overnight fast. The authors self

- designed a study 

### 9

questionnaire to gather information on worker ed ucation, vocational history, smoking and drinking 

### 10

habits, personal habits, personal and family medical history, as well as any current symptoms and 

### 11

medications used in the pervious several weeks. “Smoking” was defined as ≥ 10 cigarettes/day 

### 12

during the pas t year, with “smoking index” defined as cigarettes/day x years smoking. Similarly, 

### 13

“drinking” was defined as wine/beer/spirits consumed ≥ 3 times/week for the past 6 months, and 

### 14

“drinking index” defined as grams of alcohol consumed/day x years drinking. Exposed workers 

### 15

were categorized by physical worksite location and expected differences in B[a]P exposure: 34 

### 16

oven bottom workers, 48 oven side workers, and 47 oven top workers. Workplace environmental 

### 17

sampling stations were established at each of the phy sical work locations, including the referent’s 

### 18

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

### 19

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

### 20

### Toxicological Review of benzo[a]pyrene

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



### B

- 27 DRAFT

— DO NOT CITE OR QUOTE relative exposure was c ompared to post

- shift urine levels of a B[a]P metabolite, 1

- hydroxypyrene 

### 1

(1

- OH

- Py). Collected and purified PBMCs were incubated with Annexin

- V and PI prior to analysis 

### 2

by flow cytometry; early apoptotic cells were considered to be Annexin V+/PI

- , while l ate apoptotic 

### 3

cells were considered Annexin V+/PI+. 

### 4

All apoptosis data was displayed graphically, and in all groupings early:late apoptotic 

### 5

PBMCs occurred at an approximate 2:1 frequency. PBMC apoptosis was similar in each of the three 

### 6

coke oven worker g roups, which were all statistically significantly higher than referents 

### 7

(approximately 2

- fold) for both early and late apoptosis. While self

- reported smoking incidence 

### 8

varied significantly among the 4 worker groups, stratification by smoking years or smok ing index 

### 9

did not reveal any significant association with PBMC apoptosis. Multiple linear stepwise regression 

### 10

analysis suggested that urine 1

- OH

- Py levels and years of coke oven operation were positively 

### 11

associated with increased early and late PMBC apopt osis (Table B

- 5) , and that years of ethanol 

### 12

consumption was negatively associated with only early apoptosis. These associations were tested 

### 13

by stratifying workers into three groups by urinary 1

- OH

- Py levels or coke oven operation years, 

### 14

and in both cases, the groups with the highest urinary metabolite levels or longest oven operating 

### 15

experience had statistically significantly higher levels of both early and late apoptotic PBMCs, vs. 

### 16

the lowest or shortest duration groups, respectively. Likewise, when sort ed into groups based 

### 17

upon years of ethanol consumption, the highest ethanol “years of consumption” group had 

### 18

statistically significantly lower early apoptosis rates when compared to the lowest ethanol 

### 19

consuming group. 

### 20

Table B

- 5

. Background information on Chinese coke oven workers or 21

warehouse controls exposed to benzo[a] pyrene in the workplace 22

Effect measured Exposure Group (ng/m 3 ; mean ± SD)

P value 10.2 ± 7.6 19.5 ± 13.2 185.9 ± 38.6 1623.5 ± 435.8

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

### Toxicological Review of benzo[a]pyrene

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



### B

- 28 DRAFT

— DO NOT CITE OR QUOTE Cancer

- related Endpoints 

### 1

Benzo[a]pyrene

- Induced Cytogenetic Damage 

### 2

Many studies measure cytogenetic damage as biomarkers of early biological effects which 

### 3

also reflect exposure to genotoxic chemicals. Standard cytogenetic end points include 

### 4

chromosomal aberration (CA), sister chromatid exchange (SCE), micronucleus (MN) f ormation, 

### 5

hypoxanthine guanine phosphoribosyl transferase (hprt) mutation frequency, and glycophorin A 

### 6

mutation frequency (Gyorffy et al., 2008). These biomarkers are often incorporated in multi



### 7

endpoint studies with other biomarkers of exposure. Because they indicate related but different 

### 8

endpoints, there is often a lack of correlation between the different categories of biomarkers. 

### 9

Merlo et al. (1997) evaluated DNA adduct formation (measured by [ 32 P]

- postlabelling) and 

### 10

MN in WBCs of 94 traffic policemen versus 52 residents from the metropolitan area of Genoa, Italy. 

### 11

All study subjects wore personal air samplers for 5 hours of one work shift, and levels of 

### 12

benzo[a]pyrene and other PAHs were measured. Policemen were exposed to 4.55 ng 

### 13

benzo[a]pyrene/m 3 a ir, compared with urban residents who were exposed to 0.15 ng/m 3 . DNA 

### 14

adduct levels in policemen were 35% higher than in urban residents (p

= 0.007), but MN in urban 

### 15

residents were 20% higher than in policemen (p = 0.02). Linear regressions of DNA adduct s and 

### 16

MN incidence, respectively, versus benzo[a]pyrene exposure levels did not reveal significant 

### 17

correlations. 

### 18

Perera and coworkers assessed DNA damage in Finnish iron foundry workers in two 

### 19

separate studies and using three methodologies. Based on resul ts from personal sampling and 

### 20

stationary monitoring in both studies, three levels of benzo[a]pyrene air concentrations were 

### 21

defined: low (<5 ng/m 3 benzo[a]pyrene), medium (5

– 12 ng/m 3 ), and high (>12 ng/m 3 ) (Perera et 

### 22

al., 1994, 1993). In the first study, involving 48 workers, several biomarkers were analyzed for 

### 23

dose

- response and interindividual variability (Perera et al., 1993). PAH

- DNA adducts were 

### 24

determined in WBCs using an immunoassay as described in Section 4.1.2.2.1 and enzyme

- linked 

### 25

immunosorbent assay with fluorescence detection. Mutations at the hprt locus were also measured 

### 26

in WBC DNA. The latter assay is based on the fact that each cell contains only one copy of the hprt 

### 27

gene, which is located on the X

- chromosome. While male cells have only one X

- chromosome, 

### 28

female cells inactivate one of the two X

- chromosomes at random. The gene is highly sensitive to 

### 29

mutations such that in the event of a crucial mutation in the gene, enzyme activity disappears 

### 30

completely from the cell. In addition, mutat ions at the glycophorin A gene locus were measured in 

### 31

red blood cells (RBCs). The glycophorin A mutation frequency was not correlated with either 

### 32

benzo[a]pyrene exposure or PAH

- DNA adduct formation. However, both PAH

- DNA adduct levels 

### 33

and hprt mutation f requency increased with increasing benzo[a]pyrene exposure. In addition, 

### 34

there was a highly significant correlation between incidence of hprt mutations and PAH

- DNA 

### 35

adduct levels (p = 0.004). 

### 36

In a second study, Perera et al. (1994) surveyed 64 iron foundry workers with assessments 

### 37

conducted in 2 successive years; 24 of the workers provided blood samples in both years. Exposure 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 29 DRAFT

— DO NOT CITE OR QUOTE to benzo[a]pyrene, collected by personal and area sampling in the first year of the study, ranged 

### 1

from <5 to 60 ng/m 3 and was estim ated to have decreased by 40% in the second year. The levels of 

### 2

PAH

- DNA adducts were roughly 50% lower in the 2nd year, presumably reflecting decreased 

### 3

exposure. The longer

- lived hprt mutations were not as strongly influenced by the decreasing 

### 4

exposure t o benzo[a]pyrene. Study subjects who did not have detectable levels of DNA adducts 

### 5

were excluded from the study. As in the previous study, a strong correlation between DNA adduct 

### 6

levels and incidence of hprt mutations was observed (Perera et al., 1993). 

### 7

Kalina et al. (1998) studied several cytogenetic markers in 64 coke oven workers and 

### 8

34 controls employed at other locations within the same plant. Airborne benzo[a]pyrene and seven 

### 9

other carcinogenic PAHs were collected by personal air samplers, which sh owed ambient 

### 10

benzo[a]pyrene concentrations ranging widely from 0.002 to 50 μg/m 3 in coke oven workers and 

### 11

from 0.002 to 0.063 μg/m 3 in controls. CAs, SCEs, high

- frequency cells (HFCs), and SCE 

### 12

heterogeneity index were all significantly increased with benz o[a]pyrene exposure. Except for 

### 13

increases in HFCs, no effect of smoking was observed. Consistent with studies of PAH

- DNA adduct 

### 14

formation, reduced cytogenetic response at high exposure levels produced a nonlinear dose



### 15

response relationship. The authors also evaluated the potential influence of polymorphisms in 

### 16

enzymes involved in the metabolism of benzo[a]pyrene. Glutathione S

- transferase M1 (GSTM

- 1) 

### 17

and N

- acetyl transferase

- 2 polymorphisms were studied and no evidence of the two gene 

### 18

polymorphisms havi ng any influence on the incidence of cytogenetic damage was found. 

### 19

Motykiewicz et al. (1998) conducted a similar study of genotoxicity associated with 

### 20

benzo[a]pyrene exposure in 67 female residents of a highly polluted industrial urban area of Upper 

### 21

Silesi a, Poland, and compared the results to those obtained from 72 female residents of another 

### 22

urban but less polluted area in the same province of Poland. Urinary mutagenicity and 1

- 

### 23

hydroxypyrene levels, PAH

- DNA adducts in oral mucosa cells (detected by immun operoxidase 

### 24

staining), SCEs, HFCs, CAs, bleomycin sensitivity, and GSTM

- 1 and CYP1A1 polymorphisms in blood 

### 25

lymphocytes were investigated. High volume air samplers and gas chromatography were used to 

### 26

quantify ambient benzo[a]pyrene levels, which were 3.7 ng/m 3 in the polluted area and 0.6 ng/m 3 

### 27

in the control area during the summer. During winter, levels rose to 43.4 and 7.2 ng/m 3 in the two 

### 28

areas, respectively. The cytogenetic biomarkers (CA and SCE/HFC), urinary mutagenicity, and 

### 29

urinary 1

- hydroxypyren e excretion were significantly increased in females from the polluted area, 

### 30

and differences appeared to be more pronounced during winter time. PAH

- DNA adduct levels were 

### 31

significantly increased in the study population, when compared to the controls, only in the winter 

### 32

season. No difference in sensitivity to bleomycin

- induced lymphocyte chromatid breaks was seen 

### 33

between the two populations. As with the study by Kalina et al. (1998), genetic polymorphisms 

### 34

assumed to affect the metabolic transformation of b enzo[a]pyrene were not associated with any 

### 35

difference in the incidence of DNA damage. 

### 36

In a study of Thai school boys in urban (Bangkok) and rural areas, bulky (including but not 

### 37

limited to BPDE

- type) DNA adduct levels were measured in lymphocytes along wit h DNA SSBs, 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 30 DRAFT

— DO NOT CITE OR QUOTE using the comet assay, and DNA repair capacity (Tuntawiroon et al., 2007). Ambient air and 

### 1

personal breathing zone measurements indicated that Bangkok school children experienced 

### 2

significantly higher exposures to benzo[a]pyrene and total PAHs. A significantly higher level of 

### 3

SSBs (tail length 1.93 ± 0.09 versus 1.28 ± 0.12 μm, +51%; p < 0.001) was observed in Bangkok 

### 4

school children when compared with rural children, and this parameter was significantly 

### 5

associated with DNA adduct levels. A si gnificantly reduced DNA repair capacity (0.45 ± 0.01 versus 

### 6

0.26 ± 0.01 γ

- radiation

- induced deletions per metaphase,

- 42%; p < 0.001) was also observed in the 

### 7

city school children, again significantly associated with DNA adduct levels. It was not evident why 

### 8

higher environmental PAH exposure would be associated with lowered DNA repair capacity. 

### 9

However, because the personal breathing zone PAH levels and DNA adduct levels were not 

### 10

associated with each other, it is conceivable that the city school children had a priori lower DNA 

### 11

repair capacities that contributed significantly to the high adduct levels. The authors considered 

### 12

genetic differences between the two study populations as a possible reason for this observation. 

### 13

Epidemiologic Findings in Humans 

### 14

The association between human cancer and contact with PAH

- containing substances, such 

### 15

as soot, coal tar, and pitch, has been widely recognized since the early 1900s (Bostrom et al., 2002). 

### 16

Although numerous epidemiology studies establish an unequivocal assoc iation between PAH 

### 17

exposure and human cancer, defining the causative role for benzo[a]pyrene and other specific PAHs 

### 18

remains a challenge. In essentially all reported studies, either the benzo[a]pyrene exposure and/or 

### 19

internal dose are not known, or the be nzo[a]pyrene carcinogenic effect cannot be distinguished 

### 20

from the effects of other PAH and non

- PAH carcinogens. Nevertheless, three types of investigations 

### 21

provide support for the involvement of benzo[a]pyrene in some human cancers: molecular 

### 22

epidemiolog y studies; population

- and hospital

- based case

- control studies; and occupational cohort 

### 23

studies. In some cohort studies, benzo[a]pyrene exposure concentrations were measured and thus 

### 24

provide a means to link exposure intensity with observed cancer rates. In case

- control studies, by 

### 25

their nature, benzo[a]pyrene and total PAH doses can only be estimated. 

### 26

Molecular Epidemiology and Case

- Control Cancer Studies 

### 27

Defective DNA repair capacity leading to genomic instability and, ultimately, increased 

### 28

cancer risk i s well documented (Wu et al., 2007, 2005). Moreover, sensitivity to mutagen

- induced 

### 29

DNA damage is highly heritable and thus represents an important factor that determines individual 

### 30

cancer susceptibility. Based on studies comparing monozygotic and dizygo tic twins, the genetic 

### 31

contribution to BPDE mutagenic sensitivity was estimated to be 48.0% (Wu et al., 2007). BPDE has 

### 32

been used as an etiologically relevant mutagen in case

- control studies to examine the association 

### 33

between elevated lung and bladder can cer risk and individual sensitivity to BPDE

- induced DNA 

### 34

damage. Mutagen sensitivity is determined by quantifying chromatid breaks or DNA adducts in 

### 35

phytohemagglutinin

- stimulated peripheral blood lymphocytes as an indirect measure of DNA 

### 36

repair capacity. 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 31 DRAFT

— DO NOT CITE OR QUOTE I n a hospital

- based, case

- control study involving 221 lung cancer cases and 229 healthy 

### 1

controls, DNA adducts were measured in stimulated peripheral blood lymphocytes after incubation 

### 2

with BPDE in vitro (Li et al., 2001). Lung cancer cases showed consisten t statistically significant 

### 3

elevations in induced BPDE

- DNA adducts in lymphoctes, compared with controls, regardless of 

### 4

subgroup by age, sex, ethnicity, smoking history, weight loss, or family history of cancer. The 

### 5

lymphocyte BPDE

- induced DNA adduct leve ls, when grouped by quartile using the levels in controls 

### 6

as cutoff points, were significantly dose

- related with lung cancer risk (odds ratios [ORs] 1.11, 1.62, 

### 7

and 3.23; trend test, p < 0.001). In a related hospital

- based, case

- control study involving 15 5 lung 

### 8

cancer patients and 153 healthy controls, stimulated peripheral blood lymphocytes were exposed 

### 9

to BPDE in vitro (Wu et al., 2005). DNA damage/repair was evaluated in lymphocytes using the 

### 10

comet assay, and impacts on cell cycle checkpoints were meas ured using a fluorescence

- activated 

### 11

cell

- sorting method. The lung cancer cases exhibited significantly higher levels of BPDE

- induced 

### 12

DNA damage than the controls (p < 0.001), with lung cancer risk positively associated with 

### 13

increasing levels of lymphocyte DNA damage when grouped in quartiles (trend test, p < 0.001). In 

### 14

addition, lung cancer patients demonstrated significantly shorter cell cycle delays in response to 

### 15

BPDE exposure to lymphocytes, which correlated with increased DNA damage. 

### 16

Sensitivity to B PDE

- induced DNA damage in bladder cancer patients supports the results 

### 17

observed in lung cancer cases. In a hospital

- based, case

- control study involving 203 bladder cancer 

### 18

patients and 198 healthy controls, BPDE

- induced DNA damage was specifically evaluate d at the 

### 19

chromosome 9p21 locus in stimulated peripheral blood lymphocytes (Gu et al., 2008). Deletions of 

### 20

9p21, which includes critical components of cell cycle control pathways, are associated with a 

### 21

variety of cancers. After adjusting for age, sex, eth nicity, and smoking status, individuals with high 

### 22

BPDE

- induced damage at 9p21 were significantly associated with increased bladder cancer risk 

### 23

(OR 5.28; 95% confidence interval [CI] 3.26

– 8.59). Categorization of patients into tertiles for BPDE 

### 24

sensitivity relative to controls demonstrated a dose

- related association between BPDE

- induced 

### 25

9p21 damage and bladder cancer risk. Collectively, the results of molecular epidemiology studies 

### 26

with lung and bladder cancer patients indicate that individuals with a defe ctive ability to repair 

### 27

BPDE

- DNA adducts are at increased risk for cancer and, moreover, that specific genes linked to 

### 28

tumorigenesis pathways may be molecular targets for benzo[a]pyrene and other carcinogens. 

### 29

Due to the importance of the diet as a benzo[a]pyrene exposure source, several population



### 30

and hospital

- based, case

- control studies have investigated the implied association between dietary 

### 31

intake of benzo[a]pyrene and risk for several tumor types. In a study involving 193 pancreatic 

### 32

cancer ca ses and 674 controls (Anderson et al., 2005), another involving 626 pancreatic cancer 

### 33

cases and 530 controls (Li et al., 2007), and a third involving 146 colorectal adenoma cases and 228 

### 34

controls (Sinha et al., 2005), dietary intake of benzo[a]pyrene was e stimated using food frequency 

### 35

questionnaires. In all studies, the primary focus was on estimated intake of benzo[a]pyrene (and 

### 36

other carcinogens) derived from cooked meat. Overall, cases when compared with controls had 

### 37

higher intakes of benzo[a]pyrene an d other food carcinogens, leading to the conclusion that 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 32 DRAFT

— DO NOT CITE OR QUOTE benzo[a]pyrene plays a role in the etiology of these tumors in humans. In a supportive follow

- up 

### 1

case

- control study of colorectal adenomas, levels of leukocyte PAH

- DNA adducts were significantly 

### 2

hig her in cases when compared with controls (p = 0.02), using a method that recognizes BPDE and 

### 3

several other PAHs bound to DNA (Gunter et al., 2007). 

### 4

Cohort Cancer Studies 

### 5

Epidemiologic studies of workers in PAH

- related occupations indicate increased human 

### 6

c ancer risks associated with iron and steel production, roofing, carbon black production, and 

### 7

exposure to diesel exhaust (Bosetti et al., 2007). Exposure to benzo[a]pyrene is only one of 

### 8

numerous contributors to the cancer risk from complex PAH

- containing mixtures that occur in the 

### 9

workplace. Although some occupational cohort studies report measured or estimated inhalation 

### 10

exposure concentrations for benzo[a]pyrene, none report biomarkers of internal benzo[a]pyrene 

### 11

dose in study subjects (reviewed in Boset ti et al., 2007; Armstrong et al., 2004). Several of these 

### 12

cohort studies (summarized below) demonstrate a positive exposure

- response relationship with 

### 13

cumulative PAH exposure using benzo[a]pyrene

— or a proxy such as benzene

- soluble matter (BSM) 

### 14

that can b e converted to benzo[a]pyrene

— as an indicator substance. These studies provide insight 

### 15

and support for the causative role of benzo[a]pyrene in human cancer. 

### 16

17

Cancer incidence in aluminum and electrode production plants 

### 18

Exposure to benzo[a]pyrene and BSM in aluminum smelter workers is strongly associated 

### 19

with bladder cancer and weakly associated with lung cancer (Boffetta et al., 1997; Tremblay et al., 

### 20

1995; Armstrong et al., 1994; Gibbs, 1985; Theriault et al., 1984). In an analysis of pooled data from 

### 21

nine cohorts of aluminum production workers, 688 respiratory tract cancer cases were observed 

### 22

versus 674.1 expected (pooled RR 1.03; CI 0.96

– 1.11) (Bosetti et al., 2007). A total of 196 bladder 

### 23

cancer cases were observed in eight of the cohorts, compared with 155.7 expected (pooled relative 

### 24

risk [RR] 1.29; CI 1.12

– 1.49). Based on estimated airborne benzo[a]pyrene exposures from a meta



### 25

analysis of eight cohort studies, the predicted lung cancer RR per 100 μg/m 3

- years of cumulative 

### 26

benzo[a]pyrene exposure w as 1.16 (95% CI 1.05

– 1.28) (Armstrong et al., 2004). 

### 27

Spinelli et al. (2006) reported a 14

- year update to a previously published historical cohort 

### 28

study (Spinelli et al., 1991) of Canadian aluminum reduction plant workers. The results confirmed 

### 29

and extende d the findings from the earlier epidemiology study. The study surveyed a total of 6,423 

### 30

workers with ≥3 years of employment at an aluminum reduction plant in British Columbia, Canada, 

### 31

between the years 1954 and 1997, and evaluated all types of cancers. T he focus was on cumulative 

### 32

exposure to coal tar pitch volatiles, measured as BSM and as benzo[a]pyrene. Benzo[a]pyrene 

### 33

exposure categories were determined from the range of predicted exposures over time from 

### 34

statistical exposure models. There were 662 ca ncer cases, of which approximately 98% had 

### 35

confirmed diagnoses. The overall cancer mortality rate (standardized mortality ratio 0.97; CI 0.87

– 

### 36

1.08) and cancer incidence rate (standardized incidence ratio [SIR] 1.00; CI 0.92

– 1.08) were not 

### 37

different from t hat of the British Columbia general population. However, this study identified 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 33 DRAFT

— DO NOT CITE OR QUOTE significantly increased incidence rates for cancers of the bladder (SIR 1.80; CI 1.45

– 2.21) and the 

### 1

stomach (SIR 1.46; CI 1.01

– 2.04). The lung cancer incidence rate was only s lightly higher than 

### 2

expected (SIR 1.10; CI 0.93

– 1.30). Significant dose

- response associations with cumulative 

### 3

benzo[a]pyrene exposure were seen for bladder cancer (p trend < 0.001), stomach cancer (p trend 

### 4

< 0.05), lung cancer (p trend < 0.001), non

- Hodgk in lymphoma (p trend < 0.001), and kidney cancer 

### 5

(p trend < 0.01), although the overall incidence rates for the latter three cancer types were not 

### 6

significantly elevated versus the general population. Similar cancer risk results were obtained 

### 7

using BSM as the exposure measure; the cumulative benzo[a]pyrene and BSM exposures were 

### 8

highly correlated (r = 0.94). 

### 9

In several occupational cohort studies of workers in Norwegian aluminum production 

### 10

plants, personal and stationary airborne PAH measurements were perf ormed. 

### 11

In a study covering 11,103 workers and 272,554 person × years of PAH exposure, cancer 

### 12

incidence was evaluated in six Norwegian aluminum smelters (Romundstad et al., 2000a, b). 

### 13

Reported estimates of PAH exposure concentrations reached a maximum of 3 ,400 μg/m 3 PAH 

### 14

(680 μg/m 3 benzo[a]pyrene). The overall number of cancers observed in this study did not differ 

### 15

significantly from control values (SIR 1.03; CI 1.0

– 1.1). The data from this study showed 

### 16

significantly increased incidences for cancer of the bladder (SIR 1.3; CI 1.1

– 1.5) and elevated, but 

### 17

not significant, SIRs for larynx (SIR 1.3; CI 0.8

– 1.9), thyroid (SIR 1.4; CI 0.7

– 2.5), and multiple 

### 18

myeloma (SIR 1.4; CI 0.9

– 1.9). Incidence rates for bladder, lung, pancreas, and kidney cancer (the 

### 19

latter t hree with SIRs close to unity) were subjected to a cumulative exposure

- response analysis. 

### 20

The incidence rate for bladder cancer showed a trend with increasing cumulative exposure and 

### 21

with increasing lag times (up to 30 years) at the highest exposure level . The incidence of both lung 

### 22

and bladder cancers was greatly increased in smokers. The authors reported that using local 

### 23

county rates rather than national cancer incidence rates as controls increased the SIR for lung 

### 24

cancer (SIR 1.4; CI 1.2

– 1.6) to a sta tistically significant level. 

### 25

26

Cancer incidence in coke oven, coal gasification, and iron and steel foundry workers 

### 27

An increased risk of death from lung and bladder cancer is reported in some studies 

### 28

involving coke oven, coal gasification, and iron and steel foundry workers (Bostrom et al., 2002; 

### 29

Boffetta et al., 1997). An especially consistent risk of lung cancer across occupations is noted when 

### 30

cumulative exposure is taken into consideration (e.g., RR of 1.16 per 100 unity

- years for aluminum 

### 31

smelter w orkers, 1.17 for coke oven workers, and 1.15 for coal gasification workers). In an analysis 

### 32

of pooled data from 10 cohorts of coke production workers, 762 lung cancer cases were observed 

### 33

versus 512.1 expected (pooled RR 1.58; CI 1.47

– 1.69) (Bosetti et al. , 2007). Significant variations in 

### 34

risk estimates among the studies were reported, particularly in the large cohorts (RRs of 1.1, 1.2, 

### 35

2.0, and 2.6). There was no evidence for increased bladder cancer risk in the coke production 

### 36

workers. Based on estima ted airborne benzo[a]pyrene exposures from a meta

- analysis of 10 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 34 DRAFT

— DO NOT CITE OR QUOTE cohort studies, the predicted lung cancer RR per 100 μg/m 3

- years of cumulative benzo[a]pyrene 

### 1

exposure was 1.17 (95% CI 1.12

– 1.22) (Armstrong et al., 2004). 

### 2

A meta

- analysis of data from five cohorts of gasification workers reported 251 deaths from 

### 3

respiratory tract cancer, compared with 104.7 expected (pooled RR 2.58; 95% CI 2.28

– 2.92) 

### 4

(Bosetti et al., 2007). Pooled data from three of the cohorts indicated 18 deaths from urinary tract 

### 5

cancers , versus 6.0 expected (pooled RR 3.27; 95% CI 2.06

– 5.19). Based on estimated airborne 

### 6

benzo[a]pyrene exposures from a meta

- analysis of four gas worker cohort studies, the predicted 

### 7

lung cancer RR per 100 μg/m 3

- years of cumulative benzo[a]pyrene exposure w as 1.15 (95% CI 

### 8

1.11

– 1.20) (Armstrong et al., 2004). 

### 9

Increased risks were reported in iron and steel foundry workers for cancers of the 

### 10

respiratory tract, bladder, and kidney. In an analysis of pooled data from 10 cohorts, 

### 11

1,004 respiratory tract cancer c ases were observed versus 726.0 expected (pooled RR 1.40; 

### 12

CI 1.31

– 1.49) (Bosetti et al., 2007). A total of 99 bladder cancer cases were observed in seven of the 

### 13

cohorts, compared with 83.0 expected (pooled RR 1.29; CI 1.06

– 1.57). For kidney cancer, 40 ca ses 

### 14

were observed compared with 31.0 expected based on four studies (pooled RR 1.30; 95% CI 0.95

– 

### 15

1.77). 

### 16

Xu et al. (1996) conducted a nested case

- control study, surveying the cancer incidence 

### 17

among 196,993 active or retired workers from the Anshan Chinese i ron and steel production 

### 18

complex. A large number of historical benzo[a]pyrene measurements (1956

– 1995) were available. 

### 19

The study included 610 cases of lung cancer and 292 cases of stomach cancer, with 959 age

- and 

### 20

gender

- matched controls from the workfor ce. After adjusting for nonoccupational risk factors such 

### 21

as smoking and diet, significantly elevated risks for lung cancer and stomach cancer were identified 

### 22

for subjects employed for ≥15 years, with ORs varying among job categories. For either type of 

### 23

cancer, highest risks were seen among coke oven workers: lung cancer, OR

= 3.4 (CI 1.4

– 8.5); 

### 24

stomach cancer, OR = 5.4 (CI 1.8

– 16.0). 

### 25

There were significant trends for long

- term, cumulative benzo[a]pyrene exposure versus 

### 26

lung cancer (p

= 0.004) or stomach cancer (p = 0.016) incidence. For cumulative total 

### 27

benzo[a]pyrene exposures of <0.84, 0.85

– 1.96, 1.97

– 3.2, and ≥3.2, respectively, the ORs for lung 

### 28

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 

### 29

cum ulative total benzo[a]pyrene exposures of <0.84, 0.85

– 1.96, 1.97

– 3.2, and ≥3.2, the ORs for 

### 30

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), 

### 31

respectively. However, the investigators noted that additional workpla ce air contaminants were 

### 32

measured, which might have influenced the outcome. Of these, asbestos, silica, quartz, and iron 

### 33

oxide

- containing dusts may have been confounders. For lung cancers, cumulative exposures to 

### 34

total dust and silica dust both showed si gnificant dose

- response trends (p = 0.001 and 0.007, 

### 35

respectively), while for stomach cancer, only cumulative total dust exposure showed a marginally 

### 36

significant trend (p = 0.061). For cumulative total dust exposures of <69, 69

– 279, 280

– 882, and 

### 37

≥883 mg/m 3 , 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 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 35 DRAFT

— DO NOT CITE OR QUOTE 1.9 (CI 1.3

– 2.5), respectively. For cumulative silica dust exposures of <3.7, 3.7

– 10.39, 10.4

– 27.71, 

### 1

and ≥27.72 mg/m 3 , the ORs for lung cancer were 1.7 (CI 1.2

– 2.4) , 1.5 (CI 1.0

– 2.1), 1.5 (CI 1.0

– 2.1), 

### 2

and 1.8 (CI 1.2

– 2.5), respectively. For cumulative total dust exposures of <69, 69

– 279, 280

– 882, 

### 3

and ≥883 mg/m 3 , ORs for stomach cancer were 1.3 (CI 0.8

– 2.1), 14 (CI 0.9

– 2.2), 12 (CI 0.8

– 1.9), and 

### 4

1.6 (CI 1.1

– 2.5), re spectively. 

### 5

Exposure

- response data from studies of coke oven workers in the United States have often 

### 6

been used to derive quantitative risk estimates for PAH mixtures, and for benzo[a]pyrene as an 

### 7

indicator substance (Bostrom et al., 2002). However, there are numerous studies of coke oven 

### 8

worker cohorts that do not provide estimates of benzo[a]pyrene exposure. An overview of the 

### 9

results of these and other studies can be obtained from the review of Boffetta et al. (1997). 

### 10

11

Cancer incidence in asphalt worker s and roofers 

### 12

These groups encompass different types of work (asphalt paving versus roofing) and also 

### 13

different types of historical exposure that have changed from using PAH

- rich coal tar pitch to the 

### 14

use of bitumen or asphalt, both of which are rather low in PAHs due to their source (crude oil 

### 15

refinery) and a special purification process. Increased risks for lung cancer were reported in large 

### 16

cohorts of asphalt workers and roofers; evidence for increased bladder cancer risk is weak 

### 17

(Burstyn et al., 200 7; Partanen and Boffetta, 1994; Chiazze et al., 1991; Hansen, 1991, 1989; 

### 18

Hammond et al., 1976). In an analysis of pooled data from two cohorts of asphalt workers, 822 lung 

### 19

cancer cases were observed versus 730.7 expected (pooled RR 1.14; 95% CI 1.07

– 1.22 ) (Bosetti et 

### 20

al., 2007). In two cohorts of roofers, analysis of pooled data indicated that 138 lung cancer cases 

### 21

were observed, compared with 91.9 expected (pooled RR 1.51; 95% CI 1.28

– 1.78) (Bosetti et al., 

### 22

2007). 

### 23

24

### Toxicological Review of benzo[a]pyrene

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



### B

- 36 DRAFT

— DO NOT CITE OR QUOTE ANIMAL BIOASSAYS 1

Oral Bioassays 

### 2

Subch ronic Studies 

### 3

De Jong et al. (1999) treated male Wistar rats (eight/dose group) with benzo[a]pyrene 

### 4

(98.6% purity) dissolved in soybean oil by gavage 5 days/week for 35 days at doses of 0, 3, 10, 30, 

### 5

or 90 mg/kg

- day (adjusted doses: 0, 2.14, 7.14, 21.4, a nd 64.3 mg/kg

- day). At the end of the 

### 6

exposure period, rats were necropsied, organ weights were determined, and major organs and 

### 7

tissues were prepared for histological examination (adrenals, brain, bone marrow, colon, caecum, 

### 8

jejunum, heart, kidney, liver , lung, lymph nodes, esophagus, pituitary, spleen, stomach, testis, and 

### 9

thymus). Blood was collected for examination of hematological endpoints, but there was no 

### 10

indication that serum biochemical parameters were analyzed. Immune parameters included 

### 11

deter minations of serum immunoglobulin (Ig) levels (IgG, IgM, IgE, and IgA), relative spleen cell 

### 12

distribution, and spontaneous cytotoxicity of spleen cell populations determined in a natural

- killer 

### 13

(NK) cell assay. 

### 14

Body weight gain was decreased beginning at week 2 at the high dose of 90 mg/kg

- day; 

### 15

there was no effect at lower doses (De Jong et al., 1999). Hematology revealed a dose

- related 

### 16

decrease in RBC count, hemoglobin, and hematocrit at ≥10 mg/kg

- day ( Table B

- 6 ). A minimal but 

### 17

significant increase in m ean cell volume and a decrease in mean cell hemoglobin concentration 

### 18

were noted at 90 mg/kg

- day, and may indicate dose

- related toxicity for the RBCs and/or RBC 

### 19

precursors in the bone marrow. A decrease in WBCs, attributed to a decrease in the number of 

### 20

ly mphocytes (approximately 50%) and eosinophils (approximately 90%), was observed at 

### 21

90 mg/kg

- day; however, there was no effect on the number of neutrophils or monocytes. A 

### 22

decrease in the cell number in the bone marrow observed in the 90 mg/kg

- day dose gro up was 

### 23

consistent with the observed decrease in the RBC and WBC counts at this dose level. In the 

### 24

90 mg/kg

- day dose group, brain, heart, kidney, and lymph node weights were decreas ed and liver 

### 25

weight was increased ( Table B

- 6 ). Decreases in heart weight at 3 mg/kg

- day and in kidney weight 

### 26

at 3 and 30 mg/kg

- day were also observed, but these changes did not show dose

- dependent 

### 27

responses. Dose

- related decreases in thymus weight were statistically significant at ≥10 mg/kg



### 28

day ( Table B

- 6 ). 

### 29

Table B

- 6 . Exposur e

- related effects in male Wistar rats exposed to 30

benzo[a]pyrene by gavage 5 days/week for 5 weeks 31

Effect Dose (mg/kg

- d)

0 3 10 30 90

Hematologic effects

(mean ± SD; n = 7

– 8)

WBCs (10 9 /L)

RBCs (10 9 /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 ± 1.1 a

7.1 ± 0.4 a

### Toxicological Review of benzo[a]pyrene

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



### B

- 37 DRAFT

— DO NOT CITE OR QUOTE 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 ± 0.2 a

0.47 ± 0.01 a 9.5 ± 0.4 a

0.46 ± 0.02 a 8.6 ± 0.6 a

0.43 ± 0.02 a

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 ± 16 a

106 ± 19

72 ± 22

145 ± 176 81 ± 26

99 ± 29

39 ± 19 a

75 ± 55

Cellularity (mean ± SD; n = 7

– 8)

Spleen (cell number × 10 7 )

Bone marrow (G/L) 59 ± 15

31 ± 7 71 ± 14

36 ± 5 59 ± 13

31 ± 8 63 ± 10

27 ± 8 41 ± 10 a

19 ± 4 a

Spleen cell distribution (%)

B cells

T cells

Th cells

Ts cells 39± 4

40 ± 9

23 ± 7

24 ± 5 36 ± 2

48 ± 12

26 ± 7

26 ± 6 34 ± 3 a

40 ± 9

24 ± 5

24 ± 7 32 ± 4 a

36 ± 2

22 ± 4

19 ± 2 23 ± 4 a

44 ± 6

26 ± 4

27 ± 5

Body (g) and organ (mg) weights

(means; n = 7

– 8)

Body weight

Brain

Heart

Kidney

Liver

Thymus

Spleen

Mandibular lymph nodes

Mesenteric lymph nodes

Popliteal lymph nodes 305

1,858

1,030

1,986

10,565

517 ± 47

551

152

165

19 282 a

1,864

934 a

1,761 a

9,567

472 ± 90

590

123

148

18 300

1,859

1,000

1,899

11,250

438 ± 64 a

538

160

130 a

19 293

1,784

967

1,790 a

11,118

388 ± 71 a

596

141

158

17 250 a

1,743 a

863 a

1,626 a

12,107 a

198 ± 65 a

505

89 a

107 a

10 a

Thymus cortex surface area

(% of total surface area of thymus;

mean ± SD; n = 6

– 8) 77.9 ± 3.8 74.4 ± 2.2 79.2 ± 5.9 75.8 ± 4.0 68.9 ± 5.2 a

a Significantly ( p < 0.05) different from control mean. For body weight and organ weight means, SDs

were only reported for thymus weights.

Source: De Jong et al. (1999).



### 1

Statistically significant reductions were also observed in the relative cortex surface area of 

### 2

the thymus and thymic medullar weight at 90 mg/kg

- day, but there was no difference in cell 

### 3

proliferation between treated and control animals using the proliferating cell nuclear antigen 

### 4

(PCNA) technique. Changes in the following immune parameters were noted: dose

- related and 

### 5

statistically significant decrease in the relative number of B cells in the spleen at 10 (13%), 

### 6

30 (18%), and 90 mg/kg

- day (41%); significant decreases in absolute number of cells harvested in 

### 7

the spleen (31%), in the number of B cells in the spleen (61%), and NK cell activity in the spleen 

### 8

### Toxicological Review of benzo[a]pyrene

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



### B

- 38 DRAFT

— DO NOT CITE OR QUOTE (E:T ratio was 40.9 ± 28.4% that of the controls) at 90 mg/kg

- day; and a decrease in serum IgM 

### 1

(33%) and IgA (61%) in rats treated with 30 and 90 mg/kg

- day, respectively. The decrease in the 

### 2

spleen cel l count was attributed by the study authors to the decreased B cells and suggested a 

### 3

possible selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The study 

### 4

authors considered the decrease in IgA and IgM to be due to impaired prod uction of antibodies, 

### 5

suggesting a role of thymus toxicity in the decreased (T

- cell dependent) antibody production. In 

### 6

addition to the effects on the thymus and spleen, histopathologic examination revealed treatment



### 7

related lesions only in the liver and f orestomach at the two highest dose levels, but the incidence 

### 8

data for these lesions were not reported by De Jong et al. (1999). Increased incidence for 

### 9

forestomach basal cell hyperplasia (p < 0.05 by Fisher’s exact test) was reported at 30 and 

### 10

90 mg/kg

- da y, and increased incidence for oval cell hyperplasia in the liver was reported at 

### 11

90 mg/kg

- day (p < 0.01, Fisher’s exact test). The results indicate that 3 mg/kg

- day was a no



### 12

observed

- adverse

- effect level (NOAEL) for effects on hematological parameters (d ecreased RBC 

### 13

count, hemoglobin, and hematocrit) and immune parameters (decreased thymus weight and 

### 14

percent of B cells in the spleen) noted in Wistar rats at 10 mg/kg

- day (the lowest

- observed



### 15

adverse

- effect level [LOAEL]) and above. Lesions of the liver (o val cell hyperplasia) and 

### 16

forestomach (basal cell hyperplasia) occurred at doses ≥30 mg/kg

- day. 

### 17

Knuckles et al. (2001) exposed male and female F344 rats (20/sex/dose group) to 

### 18

benzo[a]pyrene (98% purity) at doses of 0, 5, 50, or 100 mg/kg

- day in the diet f or 90 days. Food 

### 19

consumption and body weight were monitored, and the concentration of benzo[a]pyrene in the 

### 20

food was adjusted every 3

– 4 days to maintain the target dose. The authors indicated that the actual 

### 21

intake of benzo[a]pyrene by the rats was withi n 10% of the calculated intake, and the nominal 

### 22

doses were not corrected to actual doses. Hematology and serum chemistry parameters were 

### 23

evaluated. Urinalysis was also performed. Animals were examined for gross pathology, and 

### 24

histopathology was performe d on selected organs (stomach, liver, kidney, testes, and ovaries). 

### 25

Statistically significant decreases in RBC counts and hematocrit level (decreases as much as 10 and 

### 26

12%, respectively) were observed in males at doses ≥50 mg/kg

- day and in females at 100 mg/kg



### 27

day. A maximum 12% decrease (statistically significant) in hemoglobin level was noted in both 

### 28

sexes at 100 mg/kg

- day. Blood chemistry analysis showed a significant increase in blood urea 

### 29

nitrogen (BUN) only in high

- dose (100 mg/kg

- day) males. Hist opathology examination revealed an 

### 30

apparent increase in the incidence of abnormal tubular casts in the kidney in males at 5 mg/kg

- day 

### 31

(40%), 50 mg/kg

- day (80%), and 100 mg/kg

- day (100%), compared to 10% in the controls. Only 

### 32

10% of the females showed sign ificant kidney tubular changes at the two high

- dose levels 

### 33

compared to zero animals in the female control group. The casts were described as molds of distal 

### 34

nephron lumen and were considered by the study authors to be indicative of renal dysfunction. 

### 35

Fro m this study, male F344 rats appeared to be affected more severely by benzo[a]pyrene 

### 36

treatment than the female rats. However, the statistical significance of the kidney lesions are 

### 37

unclear. Several reporting gaps and inconsistencies regarding the reporti ng of kidney 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 39 DRAFT

— DO NOT CITE OR QUOTE abnormalities in Knuckles et al. (2001) make interpretation of the results difficult. Results of 

### 1

histopathological kidney abnormalities (characterized primarily as kidney casts) were presented 

### 2

graphically and the data were not presented numer ically in this report. No indication was given in 

### 3

the graph that any groups were statistically different than controls, although visual examination of 

### 4

the magnitude of response and error bars appears to indicate a fourfold increase in kidney casts in 

### 5

male s compared to the control group (40 compared to 10%). The figure legend reported the data 

### 6

as “percentage incidence of abnormal kidney tissues” and reported values as mean ± SD. However, 

### 7

the text under the materials and methods section stated that Fisher’ s exact test was used for 

### 8

histopathological data, which would involve the pairwise comparison of incidence and not means. 

### 9

There are additional internal inconsistencies in the data presented. The data appeared to indicate 

### 10

that incidences for males were as follows: control, 10%; 5 mg/kg

- day, 40%; 50 mg/kg

- day, 80%; 

### 11

and 100 mg/kg

- day, 100%; however, these incidences are inconsistent with the size of the study 

### 12

groups, which were reported as 6

– 8 animals per group. The study authors were contacted, but did 

### 13

no t respond to EPA’s request for clarification of study design and/or results. Due to issues of data 

### 14

reporting, a LOAEL could not be established for the increased incidence of kidney lesions. Based on 

### 15

the statistically significant hematological effects inc luding decreases in RBC counts, hematocrit, and 

### 16

BUN, the NOAEL in males was 5 mg/kg

- day and the LOAEL was 50 mg/kg

- day, based on in F344 

### 17

rats. No exposure

- related histological lesions were identified in the stomach, liver, testes, or 

### 18

ovaries in this study

. 

### 19

In a range

- finding study, Wistar (specific pathogen

- free [SPF] Riv:TOX) rats (10/sex/dose 

### 20

group) were administered benzo[a]pyrene (97.7% purity) dissolved in soybean oil by gavage at 

### 21

dose levels of 0, 1.5, 5, 15, or 50 mg/kg body weight

- day, 5 days/week for 5 weeks (Kroese et al., 

### 22

2001). Behavior, clinical symptoms, body weight, and food and water consumption were 

### 23

monitored. None of the animals died during the treatment period. Animals were sacrificed 

### 24

24 hours after the last dose. Urine and blood were collected for standard urinalysis and 

### 25

hematology and clinical chemistry evaluation. Liver enzyme induction was monitored based on 

### 26

EROD activity in plasma. Animals were subjected to macroscopic examination, and organ weights 

### 27

were recorded. The esophagus , stomach, duodenum, liver, kidneys, spleen, thymus, lung, and 

### 28

mammary gland (females only) from the highest

- dose and control animals were evaluated for 

### 29

histopathology. Intermediate

- dose groups were examined if abnormalities were observed in the 

### 30

higher

- do se groups. 

### 31

A significant, but not dose

- dependent, increase in food consumption in males at ≥1.5 mg/kg



### 32

day and a decrease in food consumption in females at ≥5 mg/kg

- day was observed (Kroese et al., 

### 33

2001). Water consumption was statistically significantly a ltered in males only: a decrease at 1.5, 5, 

### 34

and 15 mg/kg

- day and an increase at 50 mg/kg

- day. Organ weights of lung, spleen, kidneys, 

### 35

adrenals, and ovaries were not affected by treatment. There was a dose

- related, statistically 

### 36

significant decrease in t hymus weight in males at 15 and 20 mg/kg

- day (decreased by 28 and 33%, 

### 37

respectively) and a significant decrease in thymus weight in females at 50 mg/kg

- day (decreased by 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 40 DRAFT

— DO NOT CITE OR QUOTE 17%) ( Table B

- 7 ). In both sexes, liver weight was statistically significantly increas ed only at 

### 1

50 mg/kg

- day by about 18% ( Table B

- 7 ). 

### 2

Table B

- 7 . Exposure

- related effects in Wistar rats exposed to benzo[a]

- 3

pyrene by gavage 5 days/week for 5 weeks 4

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 ± 0.18 a

5.03 ± 0.15 a

Thymus weight (mg; mean ± SD)

Males

Females 471 ± 19

326 ± 12 434 ± 20

367 ± 23 418 ± 26

351 ± 25 342 ± 20 a

317 ± 30 317 ± 21 a

271 ± 16 a

Basal cell hyperplasia of the

forestomach (incidence with slight

severity)

Males

Females 1/10

0/10 1/10

1/10 4/10

1/10 3/10

3/10 a 7/10

7/10 a

a Significantly ( p < 0.05) different from control mean; n = 10/sex/group.

Source: Kroese et al. (2001).



### 5

Hematological evaluation revealed only statistically nonsignificant, small, dose

- related 

### 6

decreases in hemoglobin in both sexes and RBC counts in males. Clinical chemistry analysis 

### 7

showed a small, but statistically significant, increase in creatinine levels in males only at 1.5 mg/kg



### 8

day, but this effect was not dose

- dependent. A dose

- dependent induction of liver microsomal EROD 

### 9

activity was observed, with a 5

- fold induction at 1.5 mg/kg

- day compared to controls, reaching 36

- 

### 10

fold in males a t 50 mg/kg

- day; the fold induction in females at the top dose was less than in males. 

### 11

At necropsy, significant, dose

- dependent macroscopic findings were not observed. 

### 12

Histopathology examination revealed a statistically significant increase in basal cell 

### 13

h yperplasia in the forestomach of females at doses ≥15 mg/kg

- day (Kroese et al., 2001). The 

### 14

induction of liver microsomal EROD was not accompanied by any adverse histopathologic findings 

### 15

in the liver at the highest dose, 50 mg/kg

- day, so the livers from in termediate

- dose groups were, 

### 16

therefore, not examined. An increased incidence of brown pigmentation of red pulp (hemosiderin) 

### 17

in the thymus was observed in treated animals of both sexes. However, this tissue was not 

### 18

examined in intermediate

- dose groups. This range

- finding, 5

- week study identified a NOAEL of 

### 19

5 mg/kg

- day and a LOAEL of 15 mg/kg

- day, based on decreased thymus weight and forestomach 

### 20

hyperplasia in Wistar rats. 

### 21

Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (10/sex/dose group) to benzo[a]p yrene 

### 22

(98.6% purity, dissolved in soybean oil) by gavage at 0, 3, 10, or 30 mg/kg body weight

- day, 

### 23

### Toxicological Review of benzo[a]pyrene

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



### B

- 41 DRAFT

— DO NOT CITE OR QUOTE 5 days/week for 90 days. The rats were examined daily for behavior and clinical symptoms and by 

### 1

palpation. Food and water consumption, body weights, morbid ity, and mortality were monitored. 

### 2

At the end of the exposure period, rats were subjected to macroscopic examination and organ 

### 3

weights were recorded. Blood was collected for hematology and serum chemistry evaluation, and 

### 4

urine was collected for urinalysi s. All gross abnormalities, particularly masses and lesions 

### 5

suspected of being tumors, were evaluated. The liver, stomach, esophagus, thymus, lung, spleen, 

### 6

and mesenteric lymph node were examined histopathologically. In addition, cell proliferation in 

### 7

f orestomach epithelium was measured as the prevalence of S

- phase epithelial cells displaying 

### 8

bromodeoxyuridine (BrdU) incorporation. 

### 9

There were no obvious effects on behavior of the animals, and no difference was observed 

### 10

in survival or food consumption between exposed animals and controls (Kroese et al., 2001). 

### 11

Higher water consumption and slightly lower body weights than the controls were observed in 

### 12

males but not females at the high dose of 30 mg/kg

- day. Hematological investigations showed only 

### 13

nonsignificant, small dose

- related decreases in RBC count and hemoglobin level in both sexes. 

### 14

Clinical chemistry evaluation did not show an y treatment

- related group differences or dose



### 15

response relationships for alanine aminotransferase (ALT), serum aspartate transaminase (AST), 

### 16

lactate dehydrogenase (LDH), or creatinine, but a small dose

- related decrease in

- glutamyl 

### 17

transferase (GGT) activ ity was observed in males only. Urinalysis revealed an increase in urine 

### 18

volume in males at 30 mg/kg

- day, which was not dose related. At the highest dose, both sexes 

### 19

showed increased levels of urinary creatinine and a dose

- related increase in urinary pro tein. 

### 20

However, no further investigation was conducted to determine the underlying mechanisms for 

### 21

these changes. At necropsy, reddish to brown/gray discoloration of the mandibular lymph nodes 

### 22

was consistently noted in most rats; occasional discoloration w as also observed in other regional 

### 23

lymph nodes (axillary). Statistically significant increases in liver weight were observed at 10 and 

### 24

30 mg/kg

- day in males (15 and 29%) and at 30 mg/kg

- day in females (17%). A decrease in thymus 

### 25

weight was seen in both s exes at 30 mg/kg

- day (17 and 33% decrease in females and males, 

### 26

respectively, compared with controls) ( Table B

- 8 ). At 10 mg/kg

- day, thymus weight in males was 

### 27

decreased by 15%, but the decrease did not reach statistical significance. 

### 28

### Toxicological Review of benzo[a]pyrene

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



### B

- 42 DRAFT

— DO NOT CITE OR QUOTE Table B

- 8 . Means ± S D a 

### for liver and thymus weights in Wistar rats 1

exposed to benzo[a]pyrene by gavage 5 days/week for 90 days 2

Organ Dose (mg/kg

- d)

0 3 10 30

Liver weight (g)

Males

Females 7.49 ± 0.97

5.54 ± 0.70 8.00 ± 0.85

5.42 ± 0.76 8.62 ± 1.30 b

5.76 ± 0.71 9.67 ± 1.17 b

6.48 ± 0.78 b

Thymus weight (mg)

Males

Females 380 ± 60

320 ± 60 380 ± 110

310 ± 50 330 ± 60

300 ± 40 270 ± 40 b

230 ± 30 b

a Reported as SE, but judged to be SD (and confirmed by study authors).

b Significantly ( p < 0.05) different from control mean; student t

- test (unpaired, two

- tailed); n =

10/sex/group.

Source: Kroese et al. (2001).



### 3

Histopathologic examination revealed what was characterized by Kroese et al. (2001) as 

### 4

basal cell disturbance in the epithelium of the forestomach in males ( p < 0.05) and females 

### 5

( p < 0.01) at 30 mg/kg

- day. The basal cell disturbance was characterized by increased number of 

### 6

basal cells, mitotic figures, and remnants of necrotic cells; occasional early nodule development; 

### 7

infiltrat ion by inflammatory cells (mainly histiocytes); and capillary hyperemia, often in 

### 8

combination with the previous changes (Kroese et al., 2001). Incidences for these lesions (also 

### 9

described as “slight basal cell hyperplasia”) in the 0, 3, 10, and 30

- mg/kg

- d ay groups were 0/10, 

### 10

2/10, 3/10, and 7/10, respectively, in female rats and 2/10, 0/10, 6/10, and 7/10, respectively, in 

### 11

male rats. Nodular hyperplasia was noted in one animal of each sex at 30 mg/kg

- day. A significant 

### 12

( p < 0.05) increase in proliferatio n of forestomach epithelial cells was detected at doses ≥10 mg/kg



### 13

day by morphometric of analysis of nuclei with BrdU incorporation. The mean numbers of BrdU



### 14

staining nuclei per unit surface area of the underlying lamina muscularis mucosa were increased b y 

### 15

about two

- and three

– fourfold at 10 and 30 mg/kg

- day, respectively, compared with controls. A 

### 16

reduction of thymus weight and increase in the incidence of thymus atrophy (the report described 

### 17

the atrophy as slight, but did not specify the full severity s cale used in the pathology examination) 

### 18

was observed in males only at 30 mg/kg

- day ( p < 0.01 compared with controls). Respective 

### 19

incidences for thymus atrophy for the control through high

- dose groups were 0/10, 0/10, 0/10, 

### 20

and 3/10 for females and 0/10, 2 /10, 1/10, and 6/10 for males. No significant differences were 

### 21

observed in the lungs of control and treated animals. In the esophagus, degeneration and 

### 22

regeneration of muscle fibers and focal inflammation of the muscular wall were judged to be a 

### 23

result o f the gavage dosing rather than of benzo[a]pyrene treatment. 

### 24

### Toxicological Review of benzo[a]pyrene

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



### B

- 43 DRAFT

— DO NOT CITE OR QUOTE The target organs of benzo[a]pyrene toxicity in this 90

- day dietary study of Wistar rats 

### 1

were the forestomach, thymus, and liver. The LOAEL for forestomach hyperplasia, decreased 

### 2

thymus weight, and thymus atrophy was 30 mg/kg

- day and the NOAEL was 10 mg/kg

- day. 

### 3

Chronic Studies and Cancer Bioassays 

### 4

Kroese et al. (2001) exposed Wistar (Riv:TOX) rats (52/sex/dose group) to benzo[a]pyrene 

### 5

(98.6% purity) in soybean oil by gavage at nominal doses of 0, 3, 10, or 30 mg/kg

- day, 5 days/week, 

### 6

for 104 weeks. Mean achieved dose levels were 0, 2.9, 9.6, and 29 mg/kg

- day. Additional rats 

### 7

(6/sex/group) were sacrificed after 4 and 5 months of exposure for analysis of DNA adduct 

### 8

formation in blood and major orga ns and tissues. The rats were 6 weeks old at the start of 

### 9

exposure. The rats were examined daily for behavior and clinical symptoms and by palpation. 

### 10

Food and water consumption, body weights, morbidity, and mortality were monitored during the 

### 11

study. Co mplete necropsy was performed on all animals that died during the course of the study, 

### 12

were found moribund, or at terminal sacrifice (organ weight measurement was not mentioned in 

### 13

the report by Kroese et al., 2001). The organs and tissues collected and pr epared for microscopic 

### 14

examination included: brain, pituitary, heart, thyroid, salivary glands, lungs, stomach, oesophagus, 

### 15

duodenum, jejunum, ileum, caecum, colon, rectum, thymus, kidneys, urinary bladder, spleen, lymph 

### 16

nodes, liver pancreas, adrenals, s ciatic nerve, nasal cavity, femur, skin including mammary tissue, 

### 17

ovaries/uterus, and testis/accessory sex glands. Some of these tissues were examined only when 

### 18

gross abnormalities were detected. All gross abnormalities, particularly masses and lesions t hat 

### 19

appeared to be tumors, were also examined. 

### 20

At 104 weeks, survival in the control group was 65% (males) and 50% (females), whereas 

### 21

mortality in the 30 mg/kg

- day dose group was 100% after about week 70. At 80 weeks, survival 

### 22

percentages were about 90, 8 5 and 75% in female rats in the 0, 3, and 10 mg/kg

- day groups, 

### 23

respectively; in males, respective survival percentages were ~95, 90, and 85% at 80 weeks. 

### 24

Survival of 50% of animals occurred at 104, 104, ~90, and 60 weeks for control through high

- dose 

### 25

fema les; for males, the respective times associated with 65% survival were 104, 104, 104, and ~60 

### 26

weeks. The high mortality rate in high

- dose rats was attributed to liver or forestomach tumor 

### 27

development, not to noncancer systemic effects. After 20 weeks, bo dy weight was decreased 

### 28

(compared with controls by >10%) in 30

- mg/kg

- day males, but not in females. This decrease was 

### 29

accompanied by a decrease in food consumption. Body weights and food consumption were not 

### 30

adversely affected in the other dose groups co mpared to controls. In males, there was a dose



### 31

dependent increase in water consumption starting at week 13, but benzo[a]pyrene treatment had 

### 32

no significant effects on water consumption in females. 

### 33

Tumors were detected at significantly elevated incidences at several tissue sites in female 

### 34

and male rats at doses ≥10 and ≥3 mg/kg

- day, respectively (Table 4

- 5; Kroese et al., 2001). The 

### 35

tissue sites with the highest incidences of tumors were the liver (hepatocellular adenoma and 

### 36

carcinoma) and forestomach (squ amous cell papilloma and carcinoma) in both sexes ( Table B

- 9 ). 

### 37

The first liver tumors were detected in week 35 in high

- dose male rats. Liver tumors were 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 44 DRAFT

— DO NOT CITE OR QUOTE described as complex, with a considerable proportion (59/150 tumors) metastasizing to the lungs. 

### 1

At the highest dose level, 95% of rats with liver tumors had ma lignant carcinomas (95/100; Table 

### 2

B

- 9 ). Forestomach tumors were associated with the basal cell proliferation observed (without 

### 3

diffuse hyperplasia) in the forestomach of rats in the preliminary range

- finding and 90

- day 

### 4

exposure studies described previousl y in Section 4.2.1. At the highest dose level, 59% of rats with 

### 5

forestomach tumors had malignant carcinomas (60/102; Table B

- 9 ). Other tissue sites with 

### 6

distinctly elevated incidences of tumors in the 30 mg/kg

- day dose group included the oral cavity 

### 7

(pap illoma and squamous cell carcinoma [SCC]) in both sexes, and the jejunum (adenocarcinoma), 

### 8

kidney (cortical adenoma), and skin (basal cell adenoma and carcinoma) in male rats ( Table B

- 9 ). 

### 9

In addition, auditory canal tumors (carcinoma or squamous cell papi lloma originating from pilo



### 10

sebaceous units including the Zymbal’s gland) were also detected in both sexes at 30 mg/kg

- day, 

### 11

but auditory canal tissue was not histologically examined in the lower dose groups and the controls 

### 12

( Table B

- 9 ). Gross examination revealed auditory canal tumors only in the high

- dose group. 

### 13

Table B

- 9 . Incidences of exposure

- related neoplasms in Wistar rats 14

treated by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks 15

Dose (mg/kg

- d)

0 3 10 30 a

Site Females b

Oral cavity

Papilloma

SCC

Basal cell adenoma

Sebaceous cell carcinoma 0/19

1/19

0/19

0/19 0/21

0/21

0/21

0/21 0/9

0/9

1/9

0/9 9/31 c

9/31 c

4/31

1/31

Oesophagus

Sarcoma undifferentiated

Rhabdomyosarcoma

Fibrosarcoma 0/52

0/52

0/52 0/52

1/52

0/52 2/52

4/52

3/52 0/52

0/52

0/52

Forestomach

Squamous cell papilloma

SCC 1/52

0/52 3/51

3/51 20/51 c

10/51 c 25/52 c

25/52 c

Liver

Hepatocellular adenoma

Hepatocellular carcinoma

Cholangiocarcinoma

Anaplastic carcinoma 0/52

0/52

0/52

0/52 2/52

0/52

0/52

0/52 7/52 c

32/52 c

1/52

1/52 1/52

50/52 c

0/52

0/52

Auditory canal

Benign tumor

Squamous cell papilloma

Carcinoma 0/0

0/0

0/0 0/0

0/1

0/1 0/0

0/0

0/0 1/20

1/20

13/20 c

Males b

### Toxicological Review of benzo[a]pyrene

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



### B

- 45 DRAFT

— DO NOT CITE OR QUOTE

Dose (mg/kg

- d)

0 3 10 30 a

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 10/38 c

11/38 c

2/38

2/38

Forestomach

Squamous cell papilloma

SCC 0/52

0/52 7/52 c

1/52 18/52 c

25/52 c 17/52 c

35/52 c

Jejunum

Adenocarcinoma 0/51 0/50 1/51 8/49 c

Liver

Hepatocellular adenoma

Hepatocellular carcinoma

Cholangiocarcinoma 0/52

0/52

0/52 3/52

1/52

0/52 15/52 c

23/52 c

0/52 4/52

45/52 c

1/52

Kidney

Cortical adenoma

Adenocarcinoma

Urothelial carcinoma 0/52

0/52

0/52 0/52

0/52

0/52 7/52 c

2/52

0/52 8/52 c

0/52

3/52

Auditory canal

Benign

Squamous cell papilloma

Carcinoma

Sebaceous cell adenoma 0/1

0/1

0/1

0/1 0/0

0/0

0/0

0/0 1/7

0/7

2/7

0/7 0/33

4/33

19/33 c

1/33

Skin and mammary

Basal cell adenoma

Basal cell carcinoma

SCC

Keratoacanthoma

Trichoepithelioma

Fibrosarcoma

Fibrous histiocytoma (malignant) 2/52

1/52

0/52

1/52

0/52

0/52

0/52 0/52

1/52

1/52

0/52

1/52

3/52

0/52 1/52

0/52

1/52

1/52

2/52

5/52

1/52 10/51 c

4/51

5/51

4/51

8/51 c

0/51

1/52

a 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 ma croscopic 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

Kroese et al. (2001) did not systematically investigate nonneoplastic lesions detected in rats 

### 2

sacrificed during the 2

- year study, because the focus was to identify and quantitate tumor 

### 3

### Toxicological Review of benzo[a]pyrene

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



### B

- 46 DRAFT

— DO NOT CITE OR QUOTE occurrence. However, incidences were reported for nonneoplastic lesions in tissues or org ans in 

### 1

which tumors were detected (i.e., oral cavity, oesophagus, forestomach, jejunum, liver, kidney, skin, 

### 2

mammary, and auditory canal). The reported nonneoplastic lesions associated with exposure were 

### 3

the forestomach basal cell hyperplasia and clear ce ll foci of cellular alteration in the liver. 

### 4

Incidences for forestomach basal cell hyperplasia in the control through high

- dose groups were 

### 5

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 

### 6

hepatic clear cell foci of cellular alteration were 22/52, 33/52, 4/52, and 2/52 for females and 

### 7

8/52, 22/52, 1/52, and 1/52 for males. These results indicate that the lowest dose group, 3 mg/kg



### 8

day, was a LOAEL for increased incidence of forestomach hyperplasia and hepati c histological 

### 9

changes in male and female Wistar rats exposed by gavage to benzo[a]pyrene for up to 104 weeks 

### 10

(see Table 4

- 5). The lack of an increase in incidence of these nonneoplastic lesions in the 

### 11

forestomach and liver at the intermediate and high dos es (compared with controls) were 

### 12

associated with increased incidences of forestomach and liver tumors at these dose levels. The 

### 13

authors of this study note that non

- neoplastic effects were not quantified in organs with tumors. 

### 14

As an adjunct study to the 2

- year gavage study with Wistar rats, Kroese et al. (2001) 

### 15

sacrificed additional rats (6/sex/group) after 4 and 5 months of exposure (0, 1, 3, 10, or 30 mg/kg



### 16

day) for analysis of DNA adduct formation in WBCs and major organs and tissues. Additional rats 

### 17

( 6/sex/time period) were exposed to 0.1 mg/kg

- day benzo[a]pyrene for 4 and 5 months for 

### 18

analysis of DNA adduct formation. Using the [ 32 P]

- postlabeling technique, five benzo[a]pyrene

- DNA 

### 19

adducts were identified in all of the examined tissues at 4 months (WB Cs, liver, kidney, heart, lung, 

### 20

skin, forestomach, glandular stomach, brain). Only one of these adducts (adduct 2) was identified 

### 21

based on co

- chromatography with a standard. This adduct, identified as 10β

- (deoxyguanosin

- N2

- 

### 22

yl)

- 7β,8α,9α

- trihydroxy

- 7,8,9,1 0 tetrahydro

- benzo[a]pyrene (dG

- N 2

- BPDE), was the predominant 

### 23

adduct in all organs of female rats exposed to 10 mg/kg

- day, except the liver and kidney, in which 

### 24

another adduct (unidentified adduct 4) was predominant. Levels of total adducts (number of 

### 25

ben zo[a]pyrene

- DNA adducts per 10 10 nucleotides) in examined tissues (from the single 10 mg/kg



### 26

day female rat) showed the following order: liver > heart > kidney > lung > skin > forestomach ≈ 

### 27

WBCs > brain. Mean values for female levels of total benzo[a]pyre ne

- DNA adducts (number per 

### 28

10 10 nucleotides) in four organs showed the same order, regardless of exposure group: liver > lung 

### 29

> forestomach ≈ WBCs; comparable data for males were not reported). Mean total benzo[a]pyrene



### 30

DNA adduct levels in livers increa sed in both sexes from about 100 adducts per 10 10 nucleotides at 

### 31

0.1 mg/kg

- day to about 70,000 adducts per 10 10 nucleotides at 30 mg/kg

- day. In summary, these 

### 32

results suggest that total benzo[a]pyrene

- DNA adduct levels in tissues at 4 months were not 

### 33

inde pendently associated with the carcinogenic responses noted after 2 years of exposure to 

### 34

benzo[a]pyrene. The liver showed the highest total DNA adduct levels and a carcinogenic response, 

### 35

but total DNA adduct levels in heart, kidney, and lung (in which no c arcinogenic responses were 

### 36

detected) were higher than levels in forestomach and skin (in which carcinogenic responses were 

### 37

detected). 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 47 DRAFT

— DO NOT CITE OR QUOTE Groups of Sprague

- Dawley rats (32/sex/dose) were fed diets delivering a daily dose of 

### 1

0.15 mg benzo[a]pyrene/kg body weigh t every ninth day or 5 times/week (Brune et al., 1981). 

### 2

Other groups (32/ sex/dose) were given gavage doses of 0.15 mg benzo[a]pyrene (in aqueous 1.5% 

### 3

caffeine solution)/kg every ninth day, every third day, or 5 times/week. The study included an 

### 4

untreate d control group (to compare with the dietary exposed groups) and a gavage vehicle control 

### 5

group (each with 32 rats/sex). Rats were treated until moribundity or death occurred, with 

### 6

average annual doses are reported in Table 4

- 6 (mg/kg

- year, calculated by Brune et al. [1981]). 

### 7

The following tissues were prepared for histopathological examination: tongue, larynx, lung, heart, 

### 8

trachea, esophagus, stomach, small intestine, colon, rectum, spleen, liver, urinary bladder, kidney, 

### 9

adrenal gland, and any tissues showing tumors or other gross changes. Survival was similar among 

### 10

the groups, with the exception that the highest gavage

- exposure group showed a decreased median 

### 11

time of survival ( Table B

- 10 ). Increased incidences of portal

- of

- entry tumors (forestomach, 

### 12

esophagus, and larynx) were observed in all of the gavage

- exposed groups and in the highest 

### 13

dietary exposure group ( Table B

- 10 ). Following dietary administration, all observed tumors were 

### 14

papillomas. Following gavage administration, two malignant forestomach tumors were found (one 

### 15

each in the mid

- and high

- dose groups) and the remaining tumors were benign. The data in Table 

### 16

4

- 6 show that the carcinogenic response to benzo[a]pyrene was stronger with the gavage protocol 

### 17

compared with dietary exposur e, and that no distinct difference in response was apparent between 

### 18

the sexes. Tumors at distant sites (mammary gland, kidney, pancreas, lung, urinary bladder, testes, 

### 19

hematopoietic, and soft tissue) were not considered treatment

- related as they were also observed 

### 20

at similar rates in the control group (data not provided). The study report did not address 

### 21

noncancer systemic effects. 

### 22

Table B

- 10 . Incidences of alimentary tract tumors in Sprague

- Dawley 23

rats chronically exposed to benzo[a]pyrene in the diet o r by gavage in 24

caffeine solution 25

Average annual

dose (mg/kg

- yr) Estimated average

daily dose a

(mg/kg

- d) Forestomach tumors b Total alimentary tract

tumors c (larynx,

esophagus,

forestomach) Median

survival time

(wks)

Benzo[a]pyrene by gavage in 1.5% caffeine solution

0 0 3/64 (4.7%) 6/64 (9.4%) 102

6 0.016 12/64 (18.8%) d 13/64 (20.3%) 112

18 0.049 26/64 (40.1%) e 26/64 (40.6%) 113

39 0.107 14/64 (21.9%) e 14/64 (21.9%) 87

Benzo[a]pyrene in diet

0 0 2/64 (3.1%) 3/64 (4.7%) 129

6 0.016 1/64 (1.6%) 3/64 (4.7%) 128

39 0.107 9/64 (14.1%) d 10/64 (15.6%) 131

### Toxicological Review of benzo[a]pyrene

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



### B

- 48 DRAFT

— DO NOT CITE OR QUOTE a Average annual dose divided by 365 days.

b No sex

- specific forestomach tumor incidence data were reported by Brune et al. (1981).

c Sex

- specific incidences for total alimentary tract tumors were rep orted 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 2 test that accounted for group

differences in survival time.

Source: Brune et al. (1981).



### 1

In the other modern cancer bioassay with benzo[a]pyrene, female B6C3F 1 mice (48/dose 

### 2

group) were administered ben zo[a]pyrene (98.5% purity) at concentrations of 0 (acetone vehicle), 

### 3

5, 25, or 100 ppm in the diet for 2 years (Beland and Culp, 1998; Culp et al., 1998). This study was 

### 4

designed to compare the carcinogenicity of coal tar mixtures with that of benzo[a]pyr ene and 

### 5

included groups of mice fed diets containing one of several concentrations of two coal tar mixtures. 

### 6

Benzo[a]pyrene was dissolved in acetone before mixing with the feed. Control mice received only 

### 7

acetone

- treated feed. Female mice were chosen be cause they have a lower background incidence of 

### 8

lung tumors than male B6C3F 1 mice. Culp et al. (1998) reported that the average daily intakes of 

### 9

benzo[a]pyrene in the 25

- and 100

- ppm groups were 104 and 430 μg/day, but did not report 

### 10

intakes for the 5

- ppm group. Based on the assumption that daily benzo[a]pyrene intake at 5 ppm 

### 11

was one

- fifth of the 25

- ppm intake (about 21 μg/day), average daily doses for the three 

### 12

benzo[a]pyrene groups are estimated at 0.7, 3.3, and 16.5 mg/kg

- day. Estimated doses were 

### 13

ca lculated using time

- weighted average (TWA) body weights of 0.032 kg for the control, 5

- and 25

- 

### 14

ppm groups and 0.026 kg for the 100

- ppm group (estimated from graphically presented data). 

### 15

Food consumption, body weights, morbidity, and mortality were monitor ed at intervals, and lung, 

### 16

kidneys, and liver were weighed at sacrifice. Necropsy was performed on all mice that died during 

### 17

the experiment or survived to the end of the study period. Limited histopathologic examinations 

### 18

(liver, lung, small intestine, st omach, tongue, esophagus) were performed on all control and high



### 19

dose mice and on all mice that died during the experimental period, regardless of treatment group. 

### 20

In addition, all gross lesions found in mice of the low

- and mid

- dose groups were examined 

### 21

histopathologically. 

### 22

None of the mice administered 100 ppm benzo[a]pyrene survived to the end of the study, 

### 23

and morbidity/ mortality was 100% by week 78. Decreased survival was also observed at 25 ppm 

### 24

with only 27% survival at 104 weeks, compared with 56 and 60%, in the 5

- ppm and control groups, 

### 25

respectively. In the mid

- and high

- dose group, 60% of mice were alive at about 90 and 60 weeks, 

### 26

respectively. Early deaths in exposed mice were attributed to tumor formation rather than other 

### 27

causes of systemic t oxicity. Food consumption was not statistically different in 

### 28

### Toxicological Review of benzo[a]pyrene

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



### B

- 49 DRAFT

— DO NOT CITE OR QUOTE benzo[a]pyrene

- exposed and control mice. Body weights of mice fed 100 ppm were similar to 

### 1

those of the other treated and control groups up to week 46, and after approximately 52 weeks, 

### 2

body wei ghts were reduced in 100

- ppm mice compared with controls. Body weights for the 5

- and 

### 3

25

- ppm groups were similar to controls throughout the treatment period. Compared with the 

### 4

control group, no differences in liver, kidney, or lung weights were evident i n any of the treated 

### 5

groups (other organ weights were not measured). 

### 6

Papillomas and/or carcinomas of the forestomach, esophagus, tongue, and larynx at 

### 7

elevated incidences occurred in groups of mice exposed to 25 or 100 ppm, but no exposure

- related 

### 8

tumors o ccurred in the liver or lung ( Table B

- 11 ; Beland and Culp, 1998; Culp et al., 1998). The 

### 9

forestomach was the most sensitive tissue, and demonstrated the highest tumor incidence among 

### 10

the examined tissues and was the only tissue with an elevated incidence of tumors at 25 ppm 

### 11

( Table B

- 11 ). In addition, most of the forestomach tumors in the exposed groups were carcinomas, 

### 12

as 1, 31, and 45 mice had forestomach carcinomas in the 5

- , 25

- , and 100

- ppm groups respectively. 

### 13

Nonneoplastic lesions were also found i n the forestomach at significantly ( p < 0.05) elevated 

### 14

incidences: hyperplasia at ≥5 ppm and hyperkeratosis at ≥25 ppm ( Table B

- 11 ). The esophagus 

### 15

was the only other examined tissue showing elevated incidence of a nonneoplastic lesion (basal cell 

### 16

hyperpl asia, see Table B

- 11 ). Tumors (papillomas and carcinomas) were also significantly elevated 

### 17

in the esophagus and tongue at 100 ppm ( Table B

- 11 ). Esophogeal carcinomas were detected in 1 

### 18

mouse at 25 ppm and in 11 mice at 100 ppm. Tongue carcinomas were de tected in seven 100

- ppm 

### 19

mice; the remaining tongue tumors were papillomas. Although incidences of tumors of the larynx 

### 20

were not significantly elevated in any of the exposed groups, a significant dose

- related trend was 

### 21

apparent ( Table B

- 11 ). 

### 22

Table B

- 11 . Incidence of nonneoplastic and neoplastic lesions in female 23

B6C3F 1 

### mice fed benzo[a]pyrene in the diet for up to 2 years 24

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) 1/48 b

(2) 3/47

(6) 36/46 a

(78) 46/47 a

(98)

Forestomach (hyperplasia) 13/48 b

(27) 23/47

(49) 33/46 a

(72) 37/47 a

(79)

Forestomach (hyperkeratosis) 13/48 b 22/47 33/46 a 38/47 a

### Toxicological Review of benzo[a]pyrene

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



### B

- 50 DRAFT

— DO NOT CITE OR QUOTE (27) (47) (72) (81)

Esophagus (papilloma and/or carcinoma) 0/48 b

(0) 0/48

(0) 2/45

(0) 27/46 a

(59)

Esophagus (basal cell hyperplasia) 1/48 b

(2) 0/48

(0) 5/45

(11) 30/46 a

(65)

Tongue (papilloma and/or carcinoma) 0/49 b

(0) 0/48

(0) 2/46

(4) 23/48 a

(48)

Larynx (papilloma and/or carcinoma) 0/35 b

(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

Neal and Rigdon (1967) fed benzo[a]pyrene (purity not reported) at concentrations of 0, 1, 

### 2

10, 20, 30, 40, 45, 50, 100, and 250 ppm to male and female CFW

- Swiss mice in the diet. 

### 3

Corresponding doses (in mg/kg

- day) were calculated 1 as 0, 0.2, 1.8, 3.6, 5.3, 7.1, 8, 8.9, 1 7.8, and 

### 4

44.4 mg/kg

- day. The age of the mice ranged from 17 to 180 days old and the treatment time was 

### 5

from 1 to 197 days; the size of the treated groups ranged from 9 to 73. There were 289 mice 

### 6

(number of mice/sex not stated) in the control group. No f orestomach tumors were reported at 0, 

### 7

0.2, or 1.8 mg/kg

- day. The incidence of forestomach tumors at 20, 30, 40, 45, 50, 100, and 250 ppm 

### 8

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, 

### 9

19/23, and 66/73, re spectively. 

### 10

Other Oral Exposure Cancer Bioassays in Mice 

### 11

Numerous other oral exposure cancer bioassays in mice have limitations that restrict their 

### 12

usefulness for characterizing dose

- response relationships between chronic

- duration oral exposure 

### 13

to benzo[a] pyrene and noncancer effects or cancer, but collectively, they provide strong evidence 

### 14

that oral exposure to benzo[a]pyrene can cause portal

- of

- entry site tumors (see Table B

- 12 for 

### 15

references). 

### 16

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) a nd 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 Table B

- 12. Other oral exposure cancer bioassays in mice 1

Spec ies/strain Exposure Results Comments Reference

Rat/Sprague

Dawley Groups of Sprague

Dawley rats

(32/sex/dose) were fed

diets delivering a daily

dose of 0.15 mg

benzo[a]pyrene/kg body

weight every 9 th 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 9 th day,

every 3 rd 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/kg

d. Incidence with

forestomach tumors:

Low 11/20 (18 wks)

Mid 13/19 (20 wks)

High 12/12 (12 wks) Less

- than

- lifetime

exposure

duration; only

stomachs were

examined for

tumo rs; 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

th e 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 Spec ies/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,

metho ds 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 w ere 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

durat ion; 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 Spec ies/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, 5 0,

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 exami ned 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

fo und 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 Spec ies/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, an d 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:

L ung 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 Inhalation Studies 

### 1

Short

- term and Subchronic Studies 

### 2

Wolff et al. (1989) exposed groups of 40 male and 40 female F344/Crl rats, via nose only, to 

### 3

7.5 mg benzo[a]pyrene/m 3 for 2 hours/day, 5 days/week for 4 weeks (corresponding to a TWA of 

### 4

0.45 mg/m 3 ). Rats were 10

– 11 weeks old at the beginning of the experiment. Benzo[a]pyrene 

### 5

(>98% pure) aerosols were formed by heating and then condensing the vaporized benzo[a]pyrene. 

### 6

The particle MMAD was 0.21 μm. Subgroups of these animals (six/sex/dose) were exposed for 

### 7

4 days or 6 mo nths after the end of the 4

- week exposure to radiolabeled aluminosilicate particles. 

### 8

Lung injury was assessed by analyzing clearance of radiolabeled aluminosilicate particles and via 

### 9

histopathologic evaluations. Body and lung weights, measured in subgrou ps from 1 day to 12 

### 10

months after the exposure did not differ between controls and treated animals. Radiolabeled 

### 11

particle clearance did not differ between the control and treated groups, and there were no 

### 12

significant lung lesions. This study identified a NOAEL for lung effects of 0.45 mg/m 3 for a short



### 13

term exposure. 

### 14

Chronic Studies and Cancer Bioassays 

### 15

Thyssen et al. (1981) conducted an inhalation study in which male Syrian golden hamsters 

### 16

were exposed to benzo[a]pyrene for their natural lifetime. Groups of 20

– 30 animals (8 weeks old) 

### 17

were exposed by nose

- only inhalation to NaCl aerosols (controls; 240 μg NaCl/m 3 ) or 

### 18

benzo[a]pyrene condensed onto NaCl aerosols at three nominal concentrations of 2, 10, or 50 mg 

### 19

benzo[a]pyrene/m 3 for 3

– 4.5 hours/day, 5 days/week for 1

– 41 weeks, followed by 3 hours/day, 

### 20

7 days/week for the remainder of study (until hamsters died or became moribund). Thyssen et al. 

### 21

(1981) reported average measured benzo[a]pyrene concentrations to be 0, 2.2, 9.5, or 46.5 mg/m 3

. 

### 22

More than 99% of the particles were between 0.2 and 0.5 μm in diameter, and over 80% had 

### 23

diameters between 0.2 and 0.3 μm. The particle analysis of the aerosols was not reported to 

### 24

modern standards (MMAD and geometric SD were not reported). Each group initially consisted of 

### 25

24 hamsters; final group sizes were larger as animals dying during the first 12 months of the study 

### 26

were replaced. 

### 27

Survival was similar in the control, low

- dose, and mid

- dose groups, but was significantly 

### 28

decreased in the high

- dose group. Average survival times in the control, low

- , mid

- , and high

- dose 

### 29

groups were 96.4 ± 27.6, 95.2 ± 29.1, 96.4 ± 27.8, and 59.5 ± 15.2 weeks, respectively. After the 60 th 

### 30

week, body weights decreased and mortality increased steeply in the highest dose group. 

### 31

Histologic examination of organs (a complete list of organs examined histologically was not 

### 32

reported by Thyssen et al. [1981]) revealed a dose

- related increase in tumors in the upper 

### 33

respiratory tract, including the nasal cavity, phary nx, larynx, and trachea, and in the digestive tract 

### 34

in the mid

- and high

- dose groups ( Table B

- 13 ). A statistical analysis was not included in the 

### 35

Thyssen et al. (1981) report. No lung tumors were observed. Squamous cell tumors in the 

### 36

esophagus and fores tomach were also observed in the high

- dose group, presumably as a 

### 37

### 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 consequence of mucociliary particle clearance. Tumors were detected in other sites, but none of 

### 1

these appeared to be related to exposure. The results indicated that the pharynx and larynx, 

### 2

including the epiglottis, were the main cancer targets ( Table B

- 13 ). 

### 3

Table B

- 13 . Incidence of respiratory and upper digestive tract tumors 4

in male hamsters treated for life with benzo[a]pyrene by inhalation 5

Tumor site Reported benzo[a]pyrene concentratio n (mg/m 3 )

0 a 2 b 10 50

Tumor incidence (latency in wks c )

Nasal cavity 0 0 3/26 (116 ± 1.5) 1/25 (79)

Larynx 0 0 8/26 (107.1 ± 15.5) 13/25 (67.6 ± 12.1)

Trachea 0 0 1/26 (115) 3/25 (63.3 ± 33.3)

Lung 0 0 0 0

Pharynx 0 0 6/26 (97.2 ± 16.9) 14/25 (67.5 ± 12.2)

Esophagus 0 0 0 2/25 (70, 79)

Forestomach 0 0 1/26 (119) 1/25 (72)

a Effective number of animals in control group: n = 27.

b Effective number of animals in 2 mg/m 3 dose group: n = 27.

c Mean ± SD.

Source: Thyssen et al. (1981).



### 6

Under contract to the U.S. EPA, Clement Associates (1990) obtained the individual animal 

### 7

data (including individual animal pathology reports, time

- to

- death data, and exposure chamber 

### 8

monitoring data) collected by Thyssen et al. (1981). Re

- analysis of the origin al data revealed 

### 9

several errors and omissions in the published report. The actual exposure protocol was as follows: 

### 10

4.5 hours/day, 5 days/week on weeks 1

– 12; 3 hours/day, 5 days/week on weeks 13

– 29; 3.7 

### 11

hours/day, 5 days/week on week 30; 3 hours/day, 5 d ays/week on weeks 31

– 41; and 3 hours/day, 

### 12

7 days/week for the reminder of the experiment. In addition, actual exposure concentrations 

### 13

varied widely from week to week. Because different animals were started at different times, each 

### 14

individual animal had a n exposure history somewhat different than others in the same exposure 

### 15

group. In order to deal with this problem, Clement Associates (1990) used the origi n al individual 

### 16

animal data to calculate average continuous lifetime exposures for each individual hamster. Group 

### 17

averages of individual average continuous lifetime exposure concentrations were 0, 0.25, 1.01, and 

### 18

4.29 mg/m 3 for the control through high

- exposure groups. 

### 19

For this assessment, the individual animal pathology reports prepared by Thyssen et al. 

### 20

(1981) and obtained by Clement Associates (1990) were examined to independently assess the 

### 21

numbers of hamsters with tumors in the larynx, pharynx, and nose in each group. Table B

- 14 

### 22

presents the number of animals with tumors in the larynx and pharynx and the numbers of animals 

### 23

### Toxicological Review of benzo[a]pyrene

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



### B

- 57 DRAFT

— DO NOT CITE OR QUOTE in each exposure group. Numbers of animals with either laryngeal or pharyngeal tumors are also 

### 1

noted in Table B

- 14 , since these two types of tumors arise in close anatomical proximity from 

### 2

similar cell types. Examination of the individual animal pathology reports also showed that all of 

### 3

the nasal, forestomach, esophageal, and tracheal tumors occurred in animals that also had either 

### 4

laryngeal or pharyngeal tumors, except for two animals in the mid

- dose group that displayed nasal 

### 5

tumors (one malignant and one benign) without displaying tumors in the pharynx or larynx. 

### 6

Table B

- 1 4 . Number of animals with pharynx and larynx tumors in 7

male hamsters exposed by inhalation to benzo[a]pyrene for life 8

Average

continuous

benzo[a]pyrene

conc entration a

(mg/m 3 ) Number of

hamsters in

group b Larynx b Pharynx b Larynx or pharynx,

combined c

Malignant All Malignant All Malignant All

Control 27 0 0 0 0 0 0

0.25 27 0 0 0 0 0 0

1.01 26 8 11 7 9 11 16

4.29 34 9 12 17 18 17 18

a As calculated by Clement Associates (1990) from air monitoring data collected by Thyssen and

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 a nd 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 grou p that displayed nasal tumors (one malignant and one benign) without displaying

tumors in the pharynx or larynx.



### 9

Several studies have investigated the carcinogenicity of benzo[a]pyrene in hamsters 

### 10

exposed by intratracheal instillation. Single

- dose studies verified that benzo[a]pyrene is 

### 11

tumorigenic, but do not provide data useful for characterizing dose

- response relationships because 

### 12

of their design (Kobayashi, 1975; Reznik

- Schuller and Mohr, 1974; Henry et al., 1973; Mohr, 1971; 

### 13

Saffiotti et al., 1 968; Gross et al., 1965; Herrold and Dunham, 1962). One multiple

- dose study, 

### 14

which utilized very low doses (0.005, 0.02, and 0.04 mg, once every 2 weeks), failed to find any 

### 15

tumorigenic response (Kunstler, 1983). Tumorigenic responses (mostly in the resp iratory tract) 

### 16

were found at higher dosage levels (0.25

– 2 mg benzo[a]pyrene once per week for 30

– 52 weeks) in 

### 17

four multiple

- dose studies (Feron and Kruysse, 1978; Ketkar et al., 1978; Feron et al., 1973; Saffiotti 

### 18

et al., 1972). These studies identify the respiratory tract as a cancer target with exposure to 

### 19

benzo[a]pyrene by intratracheal instillation and provide supporting evidence for the 

### 20

carcinogenicity of benzo[a]pyrene at portal

- of

- entry sites. 

### 21

22

### Toxicological Review of benzo[a]pyrene

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



### B

- 58 DRAFT

— DO NOT CITE OR QUOTE Dermal studies 

### 1

Skin

- Tumor Initiation

- Promotion Assays 

### 2

R esults from numerous studies indicate that acute dermal exposure to benzo[a]pyrene 

### 3

induces skin tumors in mice when followed by repeated exposure to a potent tumor promoter 

### 4

(Weyand et al., 1992; Cavalieri et al., 1991, 1981; Rice et al., 1985; El

- Bayoumy e t al., 1982; LaVoie 

### 5

et al., 1982; Raveh et al., 1982; Slaga et al., 1980, 1978; Wood et al., 1980; Hoffmann et al., 1972). 

### 6

The typical exposure protocol in these studies involved the application of a single dose of 

### 7

benzo[a]pyrene (typically ≥20 nmol per m ouse) to dorsal skin of mice followed by repeated 

### 8

exposure to a potent tumor promoter, such as 12

- O

- tetradecanoylphorbol

- 13

- acetate (TPA). 

### 9

Carcinogenicity Bioassays 

### 10

Repeated application of BaP to skin (in the absence of exogenous promoters) has been 

### 11

variou sly demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs (IARC, 2010, 

### 12

1983, 1973; WHO, 1998; ATSDR, 1995). Mice have been most extensively studied, presumably 

### 13

because of early evidence that they may be more sensitive than other anima l species, but 

### 14

comprehensive comparison of species differences in sensitivity to lifetime dermal exposure are not 

### 15

available. Early studies of complete dermal carcinogenicity in other species (rats, hamsters, guinea 

### 16

pigs, and rabbits) have several limitati ons which make them not useful for dose

- response analysis 

### 17

(see IARC, 1973 for descriptions of studies by Nakano et al., 1937, Shubik et al., 1960; Oberling et 

### 18

al., 1937; Schürch and Winterstein, 1935; Wynder et al., 1957). The limitations in these studies 

### 19

include inadequate reporting of the amount of BaP applied, use of the carcinogen benzene as a 

### 20

vehicle, and less than lifetime exposure duration. 

### 21

This section discusses complete carcinogenicity bioassays in mice that provide the best 

### 22

available dose

- resp onse data for skin tumors caused by repeated dermal exposure to BaP (Sivak et 

### 23

al., 1997; Higginbotham et al., 1993; Albert et al., 1991; Habs et al., 1984, 1980; Grimmer et al., 

### 24

1984, 1983; Schmähl et al., 1977; Schmidt et al., 1973; Roe et al., 1970; Poel , 1960, 1959). Early 

### 25

studies of BaP complete carcinogenicity in mouse skin (Wynder and Hoffman 1959; Wynder et al, 

### 26

1957) are not further described herein, because the investigators applied solutions of BaP at 

### 27

varying concentrations on the skin, but did no t report volumes applied. As such, applied doses in 

### 28

these studies cannot be determined. Other complete carcinogenicity mouse skin tumor bioassays 

### 29

with BaP are available, but these are not described further in this review, because: (1) they only 

### 30

included one BaP dose level (e.g., Emmett et al., 1981) or only dose levels inducing 90

– 100% 

### 31

incidence of mice with tumors (e.g., Wilson and Holland, 1988; Warshawsky and Barkley, 1987) and 

### 32

thus provide no information about the shape of the dose

- response relations hip; (2) they used a 1

- 

### 33

time/week (e.g., Nesnow et al., 1983) or 1

- time every 2 weeks (e.g., Levin et al., 1977) exposure 

### 34

protocol, which is less useful for extrapolating to daily human exposure; or (3) they used a vehicle 

### 35

demonstrated to interact with or e nhance benzo[a]pyrene carcinogenicity (Bingham and Falk, 

### 36

1969). 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 59 DRAFT

— DO NOT CITE OR QUOTE Poel (1959) applied benzo[a]pyrene in toluene to shaved interscapular skin of groups of 

### 1

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 

### 2

for up to 103 weeks or until the appearance of a tumor by gross examination (3 times weekly). 

### 3

Some organs (not further specified) and interscapular skin in sacrificed mice were examined 

### 4

histologically. With increasing dose level, the incidence of mice with skin tumors increased and the 

### 5

time of tumor appearance decreased (see Table B

- 15 ). Doses >3.8 μg were associated with 100% 

### 6

mortality after increasingly shorter exposure periods, none greater than 44 weeks. Poel (1959) did 

### 7

not mention the appearance of exposure

- related tumors in tissues other than interscapular skin. 

### 8

Table B

- 1 5 . Skin tumor incidence and time of appearance in male C57L 9

mice dermally exposed to benzo[a]pyrene for up to 103 weeks 10

Dose (μg) a Incidence of mice with

gross skin tumors Time of first tumor

appearance (wks) Incidence of mice

with epidermoid

carcinoma b Length of exposure

period (wks)

0 (Toluene) 0/33 (0%)

− 0/33 (0%) 92

0.15 5/55 (9%) 42

– 44 c 0/55 (0%) 98

0.38 11/55 (20%) 24 2/55 (4%) 103

0.75 7/56 (13%) 36 4/56 (7%) 94

3.8 41/49 (84%) 21

– 25 32/49 (65%) 82

19 38/38 (100%) 11

– 21 37/38 (97%) 25

– 44 c

94 35/35 (100%) 8

– 19 35/35 (100%) 22

– 43

188 12/14 (86%) 9

– 18 10/14 (71%) 20

– 35

376 14/14 (100%) 4

– 15 12/14 (86%) 19

– 35

752 13/13 (100%) 5

– 13 13/13 (100%) 19

– 30

a Indicated doses were applied to interscapular skin 3 times/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

Poel (1960) applied benzo[a]pyrene in a toluene vehicle to shaved interscapular skin of 

### 12

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, 

### 13

19.0, 94.0, or 470 μg benzo[a]pyrene per application, until mice died or a skin tumor was observed. 

### 14

Time ranges for tumor observations were provided, but not times of death for mice without tumors, 

### 15

so it was not possible to evaluate differential mortality among all dose groups or the length of 

### 16

exposure for m ice without tumors. With increasing dose level, the incidence of mice with skin 

### 17

tumors increased and the time of tumor appearance decreased ( Table B

- 16 ). The lowest dose level 

### 18

did not induce an increased incidence of mice with skin tumors in any strain, but strain differences 

### 19

in susceptibility were evident at higher dose levels. SWR and C3HeB mice showed skin tumors at 

### 20

### Toxicological Review of benzo[a]pyrene

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



### B

- 60 DRAFT

— DO NOT CITE OR QUOTE doses ≥0.38 μg benzo[a]pyrene, whereas AH/e mice showed tumors at doses ≥19 μg 

### 1

benzo[a]pyrene ( Table B

- 16 ). Except for metastases of the skin tumors to lymph nodes and lung, 

### 2

Poel (1960) did not mention the appearance of exposure

- related tumors in tissues other than 

### 3

interscapular skin. 

### 4

Table B

- 1 6 . Skin tumor incidence and time of appearance in male SWR, 5

C3HeB, and A/He mice dermally expose d to benzo[a]pyrene for life or 6

until a skin tumor was detected 7

Dose (μg) a SWR mice C3HeB mice A/He mice

Tumor

incidence b Time of first

tumor

appearance

(wks) Tumor

incidence b Time of first

tumor

appearance

(wks) Tumor

incidence b 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

Roe et al. (1970) treated groups of 50 female Swiss mice with 0 (acetone vehicle), 0.1, 0.3, 1, 

### 9

3, or 9 μg benzo[a]pyrene applied to the shaved dorsal skin 3 times/week for up to 93 weeks; all 

### 10

surviving mice were killed and examined for tumors during the f ollowing 3 weeks. The dorsal skin 

### 11

of an additional control group was shaved periodically but was not treated with the vehicle. Mice 

### 12

were examined every 2 weeks for the development of skin tumors at the site of application. 

### 13

Histologic examinations includ ed: (1) all skin tumors thought to be possibly malignant; (2) lesions 

### 14

of other tissues thought to be neoplastic; and (3) limited nonneoplastic lesions in other tissues. As 

### 15

shown in Table B

- 17 , markedly elevated incidences of mice with skin tumors were on ly found in the 

### 16

two highest dose groups (3 or 9 μg), compared with no skin tumors in the control groups. 

### 17

Malignant skin tumors (defined as tumors with invasion or penetration of the panniculus carnosus 

### 18

muscle) were detected in 4/41 and 31/40 mice in the 3

- and 9

- μg groups, respectively, surviving to 

### 19

at least 300 days. Malignant lymphomas were detected in all groups, but the numbers of cases were 

### 20

not elevated compared with expected numbers after adjustment for survival differences. Lung 

### 21

### Toxicological Review of benzo[a]pyrene

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



### B

- 61 DRAFT

— DO NOT CITE OR QUOTE tumors were likewi se detected in control and exposed groups at incidences that were not 

### 1

statistically different. 

### 2

Table B

- 1 7 . Tumor incidence in female Swiss mice dermally exposed to 3

benzo[a]pyrene for up to 93 weeks 4

Dose (μg) a Cumulative number of mice with skin

tumor/surv ivors Skin tumor

incidence b Malignant

lymphoma

incidence c Lung tumor

incidence c 200 d 300 d 400 d 500 d 600 d 700 d

No treatment 0/48 0/43 0/40 0/31 0/21 0/0 0/43 (0%) 19/44 (43%) 12/41 (29%)

Acetone 0/49 0/47 0/45 0/37 0/23 0/0 0/47 (0%) 12/47 (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%)

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. 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.

c Numerator: nu mber 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

Schmidt et al. (1973) dermally administered benzo[a]pyrene in acetone to female NMRI 

### 6

mice (100/group) and female Swiss mice. Benzo[a]pyrene was applied to the shaved dorsal skin 

### 7

twice weekly with doses of 0, 0 .05, 0.2, 0.8, or 2 μg until spontaneous death occurred or until an 

### 8

advanced carcinoma was observed. Skin carcinomas were identified by the presence of crater



### 9

shaped ulcerations, infiltrative growth, and the beginning of physical wasting (i.e., cachexia). 

### 10

Necropsy was performed for all animals, and histopathological examination of the dermal site of 

### 11

application and any other tissues with gross abnormalities was conducted. Skin tumors were 

### 12

observed at the two highest doses in both strains of female mice ( see Table B

- 18 ), with induction 

### 13

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 

### 14

Swiss mice, respectively. The authors indicated that the latency period for tumor formation was 

### 15

highly variable and significant differences among exposure groups could not be identified, but no 

### 16

further timing information was available, including overall survival. Carcinoma was the primary 

### 17

tumor type seen after lifetime application of benzo[a]pyrene to mouse skin. 

### 18

### Toxicological Review of benzo[a]pyrene

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



### B

- 62 DRAFT

— DO NOT CITE OR QUOTE Table B

- 1 8 . Ski n tumor incidence in female NMRI and Swiss mice 1

dermally exposed to benzo[a]pyrene 2

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%)

a Mice were exposed until natural death or until they developed a carcinoma at the site of application.

b Indicated doses were applied 2 times/week to shaved skin of the back.

Source: Schmidt et al. (1973).



### 3

Schmähl et al. (1977) applied benzo[a]pyrene 2 times/week to the shaved dorsal skin of 

### 4

female NMRI mice (100/group) at doses of 0, 1, 1.7, or 3 μg in 20 μL acetone. The authors reported 

### 5

that animals were observed u ntil natural death or until they developed a carcinoma at the site of 

### 6

application. The effective numbers of animals at risk was about 80% of the nominal group sizes, 

### 7

which the authors attributed to autolyis; no information was provided concerning when tum ors 

### 8

appeared in the relevant groups, how long treatment lasted in each group, or any times of death. 

### 9

Necropsy was performed on all mice and the skin of the back, as well as any organs that exhibited 

### 10

macroscopic changes, were examined histopathologically. The incidence of all types of skin tumors 

### 11

was increased in a dose

- related manner compared to controls (see Table B

- 19 ). Carcinoma was the 

### 12

primary tumor type observed following chronic dermal exposure to benzo[a]pyrene, and skin 

### 13

papillomas occurred infreq uently. Dermal sarcoma was not observed. 

### 14

Table B

- 1 9 . Skin tumor incidence in female NMRI mice dermally 15

exposed to benzo[a]pyrene 16

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%)

### Toxicological Review of benzo[a]pyrene

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



### B

- 63 DRAFT

— DO NOT CITE OR QUOTE 1.7 25/88 (28%) 0/88 (0%) 25/88 (28%)

3 45/81 (56%) 2/81 (3%) 43/81 (53%)

a Mice were exposed until natural death or until they developed a carcinoma at the site of application.

b Indicated doses were applied 2 times/week to shaved skin of the back.

Source: Schmähl et al. (1977).



### 1

Habs et al. (1980) applied benzo[a]pyrene to the shaved interscapular skin of female NMRI 

### 2

mice (40/group) at doses of 0, 1.7, 2.8, or 4.6 μg in 20 μL acetone twice weekly, from 10 we eks of 

### 3

age until natural death or gross observation of infiltrative tumor growth. Latency of tumors, either 

### 4

as time of first appearance or as average time of appearance of tumors, was not reported. Necropsy 

### 5

was performed on all animals, and the dorsal sk in, as well as any organs showing gross alterations 

### 6

at autopsy, was prepared for histopathological examination. Age

- standardized mortality rates, 

### 7

using the total population of the experiment as the standard population, were used to adjust tumor 

### 8

incidence findings in the study. Benzo[a]pyrene application was associated with a statistically 

### 9

significant increase in the incidence of skin tumors at each dose level (see Table B

- 20 ). 

### 10

Table B

- 20 . Skin tumor incidence in female NMRI mice dermally 11

exposed to benzo [a]pyrene 12

Dose (μg) a,b Skin tumor incidence Age

- standardized tumor incidence c

0 (acetone) 0/35 (0%) 0%

1.7 8/34 (24%) 24.8%

2.8 24/35 (68%) 89.3%

4.6 22/36 (61%) 91.7%

a Mice were exposed until natural death or until they developed a carcinoma at the site of application.

b Indicated doses were applied 2 times/week to shaved skin of the back.

c Mortality data of the total study population were used to derive the age

- standardized tumor incidence.

Source: Habs et al. (1980).



### 13

Grimmer et al. (1984, 1983) applied benzo[a]pyrene (in 0.1 mL of a 1:3 solution of 

### 14

acetone:dimethyl sulfoxide [DMSO]) to the interscapular skin of female CFLP mice (65

– 80/group) 2 

### 15

times/week for 104 weeks. Doses were 0, 3.9, 7.7, and 15.4 μg in the 1983 e xperiment, and 0, 3.4, 

### 16

6.7, and 13.5 μg in the 1984 experiment. Mice were observed until spontaneous death, unless an 

### 17

advanced tumor was observed or if animals were found moribund. Survival information was not 

### 18

provided; incidences reflect the number of a nimals placed on study. Necropsy was performed on 

### 19

all mice. Histopathological examination of the skin and any other organ showing gross 

### 20

abnormalities was performed. Chronic dermal exposure to benzo[a]pyrene produced a dose



### 21

related increase in skin tumor incidence and a decrease in tumor latency (see Table B

- 21 ). 

### 22

### Toxicological Review of benzo[a]pyrene

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



### B

- 64 DRAFT

— DO NOT CITE OR QUOTE Carcinoma was the primary tumor type observed and a dose

- response relationship was evident for 

### 1

carcinoma formation and incidence of all types of skin tumors. 

### 2

Table B

- 2 1. Skin tumor incidence an d time of appearance in female 3

CFLP mice dermally exposed to benzo[a]pyrene for 104 weeks 4

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 ± 16.78 b

7.7 39/64 (61%) 5/64 (8%) 34/64 (53%) 60.9 ± 13.90

15.4 56/64 (88%) 2/64 (3%) 54/64 (84%) 44.1 ± 7.66

Grimmer et al. (1984)

0 (1:3 Solution of

acetone:DMSO) 0/65 (0%) 0/65 (0%) 0/65 (0%)

–

3.4 43/64 (67%) 6/64 (9%) 37/64 (58%) 61 (53

– 65) c

6.7 53/65 (82%) 8/65 (12%) 45/65 (69%) 47 (43

– 50)

13.5 57/65 (88%) 4/65 (6%) 53/65 (82%) 35 (32

– 36)

a 

### Indicated doses were applied twice/week to shaved skin of the back.

b 

### Mean ± SD.

c 

### Median with 95% CI.

Sources: Grimmer et al. (1984, 1983)

5

Habs et al. (1984) applied benzo[a]pyrene (in 0.01 mL acetone) to the shaved interscapular 

### 6

skin of female NMRI mice at doses of 0, 2, or 4 μg, 2 times/week for life. Animals were observed 

### 7

twice daily until spontaneous death, unless an invasive tumor was o bserved. All animals were 

### 8

necropsied and histopathological examination was performed on the dorsal skin and any other 

### 9

organ with gross abnormalities. Chronic dermal exposure to benzo[a]pyrene did not affect body 

### 10

weight gain, but appeared to reduce surviv al at the highest dose with mean survival times of 691, 

### 11

648, and 528 days for the 0, 2, and 4 μg/day groups, respectively. The total length of exposure for 

### 12

each group was not reported, but can be inferred from the survival data. Latency also was not 

### 13

repo rted. Benzo[a]pyrene application resulted in a dose

- related increase the incidence of total skin 

### 14

tumors and skin carcinomas (see Table B

- 22 ). Hematopoietic tumors (at 6/20, 3/20, and 3/20) and 

### 15

lung adenomas (at 2/20, 1/20, and 0/20) were observed in the controls and in the benzo[a]pyrene 

### 16

treatment groups, but did not appear to be treatment related according to the study authors. 

### 17

### Toxicological Review of benzo[a]pyrene

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



### B

- 65 DRAFT

— DO NOT CITE OR QUOTE Table B

- 22 . Skin tumor incidence in female NMRI mice dermally 1

exposed to benzo[a]pyrene for life 2

Dose (μg) a,b Skin tumor

incide nce (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)

a Mice 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

Groups of 23

– 27 female Ah

- receptor

- respons ive Swiss mice were treated on a shaved area 

### 4

of dorsal skin with 0, 1, 4, or 8 nmol (0, 0.25, 1, or 2 μg/treatment) benzo[a]pyrene (>99% pure) in 

### 5

acetone 2 times weekly for 40 weeks (Higginbotham et al., 1993). Surviving animals were 

### 6

sacrificed 8 weeks la ter. Complete necropsies were performed, and tissues from the treated area, 

### 7

lung, liver, kidney, spleen, urinary bladder, ovary, and uterus were harvested for histopathologic 

### 8

examination. Histopathologic examination was performed on tissues from the trea ted area, lungs, 

### 9

liver, kidneys, spleen, urinary bladder, uterus, and ovaries, as well as any other grossly abnormal 

### 10

tissue. Lung adenomas occurred in each group (1/27, 2/24, 1/23, 1/23), and other tumors were 

### 11

noted in isolated mice (i.e., malignant lymph oma [spleen] in one low

- dose and one mid

- dose mouse; 

### 12

malignant lymphoma with middle organ involvement in one high

- dose mouse; and hemangioma 

### 13

[liver] in one mid

- dose mouse) and were not considered dose related. In addition, benzo[a]pyrene 

### 14

showed no skin tu mors under the conditions of this bioassay. 

### 15

Sivak et al. (1997) designed a study to compare the carcinogenicity of condensed asphalt 

### 16

fumes (including benzo[a]pyrene and other PAHs) with several doses of benzo[a]pyrene alone. For 

### 17

the purposes of this asses sment, the exposure groups exposed to PAH mixtures are not discussed. 

### 18

Groups of 30 male C3H/HeJ mice were treated dermally twice/week to 0, 0.0001, 0.001, or 0.01% 

### 19

(0, 0.05, 0.5, or 5 μg) benzo[a]pyrene in a 50 μL volume of cyclohexanone/ acetone (1:1) fo r 104 

### 20

weeks beginning at 8 weeks of age. Mice dying durin g the exposure period or sacrificed at the 24 

### 21

month termination were necropsied; mice with skin tumors that persisted for 4 consecutive weeks 

### 22

with diameters > 3 cm were sacrificed before the study termination and also necropsied. Skin 

### 23

samples and any grossly observed lesions were subjected to histopathological examination. 

### 24

Carcinomas and sarcomas were referred to as carcinomas, whereas papillomas, keratoacanthomas, 

### 25

and fibromas were referred to as papillomas. The incidences of mice with skin tumors and mean 

### 26

survival times for each group are shown in Table B

- 23 . All high

- dose mice died before the final 

### 27

sacrifice, and 80% showed scabs and sores at the site of application. The time of first tumor 

### 28

ap pearance was not reported for the tumor

- inducing groups, but from a plot of the tumor incidence 

### 29

### Toxicological Review of benzo[a]pyrene

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



### B

- 66 DRAFT

— DO NOT CITE OR QUOTE in the high

- dose group versus treatment days, an estimate of ~320 days (~43 weeks) is obtained 

### 1

for this group. The extent of deaths prior to 1 year in each gro up was not provided, so that the 

### 2

reported incidence may underestimate the tumor rate of animals exposed long enough to develop 

### 3

tumors. However, the crude skin tumor rates show an increasing trend in incidence. 

### 4

Table B

- 23 . Skin tumor incidence in male C3H /HeJ mice dermally 5

exposed to benzo[a]pyrene for 24 months 6

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.

b 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.

Source: Sivak et al. (1997).



### 7

To examine dose

- response relationships and the time course of benzo[a]pyrene

- induced 

### 8

skin damage, DNA adduct formation, and tumor formation, groups of 43

– 85 female Harlan mice 

### 9

were treated dermally with 0, 16, 32, or 64 μg of benzo[a]pyrene in 50 μL of acetone once per week 

### 10

for 29 weeks (Albert et al., 1991). Interscapular skin of each mouse was clipped 3 days before the 

### 11

first application and every 2 weeks thereafter. Additional groups of mice were treated for 9 weeks 

### 12

with 0, 8, 16, 32, or 64 μg radiolabeled benzo[a]pyrene to determine BPDE

- DNA adduct formation 

### 13

in the epidermis at several time points (1, 2, 4, and 9 weeks). Tumor formation was monitored only 

### 14

in the skin. 

### 15

No tumors were present in vehicle

- treated or untreated control mice. In exposed groups, 

### 16

incidences of mice with skin tumors were no t reported, but time

- course data for cumulative 

### 17

number of tumors per mouse, corrected for deaths from nontumor causes, were reported. Tumors 

### 18

began appearing after 12

– 14 weeks of exposure for the mid

- and high

- dose groups and at 18 weeks 

### 19

for the low

- dose g roup. At study termination (35 weeks after start of exposure), the mean number 

### 20

of tumors per mouse was approximately one per mouse in the low

- and mid

- dose groups and eight 

### 21

per mouse in the high

- dose group; indicating that most, if not all, mice in each e xposure group 

### 22

developed skin tumors and that the tumorigenic response was greatest in the highest dose group. 

### 23

The majority of tumors were initially benign, with an average time of 8 weeks for progression from 

### 24

benign papillomas to malignant carcinomas. Ep idermal damage occurred in a dose

- related manner 

### 25

### Toxicological Review of benzo[a]pyrene

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



### B

- 67 DRAFT

— DO NOT CITE OR QUOTE (more severe in the high

- dose group than in the low

- and mid

- dose groups) and included 

### 1

statistically significant increases (compared with controls) in: [ 3 H]

- thymidine labeling and mitotic 

### 2

indices; incidence of pyknotic and dark cells (signs of apoptosis); and epidermal thickness. Only a 

### 3

minor expansion of the epidermal cell population was observed. In the high

- dose group, indices of 

### 4

epidermal damage increased to a plateau by 2 weeks of exposure. The early time course of 

### 5

epidermal damage indices was not described in the low

- or mid

- dose groups, since data for these 

### 6

endpoints were only collected at 20, 24, and 30 weeks of exposure. An increased level of BPDE



### 7

DNA adducts, compared with controls, was apparent in all exposed groups after 4 weeks of 

### 8

exposure in the following order: 64 > 32 > 16 > 8 μg/week. The time

- course data indicate that 

### 9

benzo[a]pyrene

- induced increases in epidermal damage indices and BPDE

- DNA adducts preceded 

### 10

the appearance of skin tumors

. 

### 11

Reproductive and Developmental Toxicity Studies 

### 12

Oral 

### 13

In a study evaluating the combined effects of DBP and benzo[a]pyrene on the male 

### 14

reproductive tract, Chen et al. (2011) administered benzo[a]pyrene alone in corn oil via daily 

### 15

gavage at 5 mg/kg

- day to 30 male Sprague

- Dawley rats (28

- 30 days old); a group of 30 rats 

### 16

received only vehicle. Body weight was measured weekly. Groups of 10 rats per group were 

### 17

sacrificed after 4, 8, and 12 weeks of exposure. At sacrifice, blood was collected for analysis of 

### 18

serum testosterone levels by radioimmunoassay. The testes and epidiymides were weighed, and 

### 19

the right testis and epididymis were examined microscopically. The left epididymis was used for 

### 20

evaluation of sperm parameters (sperm count and morphology). Oxida tive stress, as measured by 

### 21

superoxide dismutase, glutathione peroxidase, and catalase activity and malondioaldehyde levels, 

### 22

was evaluated in the left testis of each rat. Exposure to benzo[a]pyrene did not affect body weight, 

### 23

and no signs of toxicity were seen. Testes and epididymides weights of exposed rats were similar 

### 24

to controls at all time points. Sperm counts and percent abnormal sperm were also similar to 

### 25

controls at 4 and 8 weeks of exposure, but were significantly (p<0.05) different from control s after 

### 26

12 weeks of exposure to benzo[a]pyrene (29% decrease in sperm count and 54% increase in 

### 27

percent abnormal sperm). Serum testosterone levels were significantly increased relative to 

### 28

controls after 4 weeks (>two

- fold higher) and 8 weeks (~1.5

- fold hi gher) of benzo[a]pyrene 

### 29

exposure, but were comparable to controls after 12 weeks. Histopathology evaluation of the testes 

### 30

revealed irregular and disordered arrangement of germ cells in the seminiferous tubules of treated 

### 31

rats; the authors did not report i ncidence or severity of these changes. Among measures of 

### 32

testicular oxidative stress, only catalase activity was significantly affected by benzo[a]pyrene 

### 33

exposure, showing an increase of ~50% after 12 weeks of exposure. These data suggest a LOAEL of 

### 34

5 mg /kg

- day (the only dose tested) for decreased sperm count, increased percentage of abnormal 

### 35

sperm, altered testosterone levels, and histopathology changes in the testes following 13 weeks of 

### 36

exposure. 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 68 DRAFT

— DO NOT CITE OR QUOTE Chung et al. (2011) evaluated the effects of low

- dose be nzo[a]pyrene exposure on 

### 1

spermatogenesis, and the role of altered steroidogenesis on the sperm effects. Groups of 20

- 25 

### 2

male Sprague

- Dawley rats (8 wks old) were given daily gavage doses of 0, 0.001, 0.01, or 0.1 

### 3

mg/kg

- day benzo[a]pyrene in DMSO for 90 co nsecutive days. At the end of exposure, the animals 

### 4

were sacrificed for removal of the pituitary, testes, and epididymides, and collection of serum and 

### 5

testicular interstitial fluid. Subgroups of each exposure group were used for various analyses. 

### 6

Serum levels of testosterone and LH were measured, as was testosterone concentration in the 

### 7

interstitial fluid (ELISA assays). Body and testes weights were recorded. Sections of the testis 

### 8

were analyzed for apoptotic germ cells using TUNEL assay. Evaluation of the epididymis included 

### 9

histopathology as well as measurement of caput and caudal epididymal tubule diameters. In 

### 10

addition, sperm were isolated from the cauda epididymis for analysis of sperm number and 

### 11

motility, acrosomal integrity, and immunocytoch emistry for ADAM3 (a disintegrin and 

### 12

metallopeptidse domain 3; a sperm surface protein associated with fertilization). 

### 13

Leydig cells were isolated from the right testis of animals from each dose group and 

### 14

cultured with or without human chorionic gondatotrop in (hCG) or dibutyl cyclic adenosine 

### 15

monophosphate (dbcAMP) to evaluate testosterone production (Chung et al., 2011). Cultured 

### 16

Leydig cells were also subjected to western blot and immunocytochemistry analyses to evaluate 

### 17

changes in the expression of genes involved in steroidogenesis (StAR[steroidogenic acute 

### 18

regulatory protein], p450scc [p450 side

- chain cleavage], and 3β

- HSD[3β

- hydroxysteroid 

### 19

dehydrogenase isomerase]). Finally, pituitary gland extracts were evaluated for LH protein 

### 20

content using immunohi stochemistry. Data were reported graphically and analyzed by ANOVA 

### 21

followed by Duncan’s post hoc test, using a p

- value cutoff of 0.05 for significant difference. 

### 22

At termination of exposure, body weights of treated animals were similar to controls, as 

### 23

were absolute testes weights (Chung et al., 2011). Testosterone concentrations in both serum and 

### 24

testicular interstitial fluid were significantly reduced at the high dose of benzo[a]pyrene (0.1 

### 25

mg/kg

- day); based on visual inspection of the data, the mean serum concentration in this group 

### 26

was ~20% of the control and the mean intersitital fluid concentration was ~60% of the control 

### 27

(n=9 animals/dose for these evaluations). In addition, baseline production of testosterone by 

### 28

cultured Leydig cells was significantl y decreased (~50% based on data shown graphically) at 0.1 

### 29

mg/kg

- day. Both hCG

- and dbcAMP

- stimulated testosterone production measurements were lower 

### 30

(~60% lower than controls) in Leydig cells from rats exposed to either 0.01 or 0.1 mg/kg

- day. 

### 31

Serum LH wa s significantly increased at both 0.01 and 0.1 mg/kg

- day (~65

- 75% higher than 

### 32

controls based on visual inspection of graphs); concordant increases in the intensity of LH 

### 33

immunoreactivity were evident in pituitary extracts from exposed rats. 

### 34

Dose

- related increases in the number of apoptotic germ cells, primarily spermatogonia, 

### 35

were demonstrated both via TUNEL assay and caspase

- 3 staining; the number per tubule was 

### 36

significantly increased over control at all doses (Chung et al., 2011). Numbers of sperm we re lower 

### 37

in the treatment groups, but did not differ significantly from the control group. However, sperm 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 69 DRAFT

— DO NOT CITE OR QUOTE motility was significantly reduced in exposed groups compare with control. The authors did not 

### 1

report sperm motility for all dose groups, but showed only the significant decrease in the 0.01 

### 2

mg/kg

- day mid

- dose group (~30% lower than controls based on visual inspection of graph). 

### 3

Acrosomal integrity (measured by LysoTracker staining) was diminished in sperm heads from 

### 4

exposed rats; likewise, the expre ssion of ADAM3 protein was downregulated by exposure to 

### 5

benzo[a]pyrene; the authors reported a significant decrease in the 0.01 mg/kg

- day group but did 

### 6

not provide details of the analysis of other exposure groups. Histopathology examination of the 

### 7

caput a nd cauda epididymides revealed dose

- related decreases in both cauda and caput tubule 

### 8

diameters that were statistically significantly lower than controls at all doses (~10

- 30% smaller 

### 9

mean diameter than control based on measurements of 175 tubules collected from 5 samples in 

### 10

each group; data reported graphically). 

### 11

Statistically significant effects observed at the lowest dose (0.001 mg/kg

- day) of 

### 12

benzo[a]pyrene in this study included decreased caput and cauda epididymal tubule diameters 

### 13

(~10

- 15% lower than controls) and increased numbers of apoptotic germ cells (~twofold higher 

### 14

than controls) by TUNEL assay (Chung et al., 2011). The authors reported that “sperm motility was 

### 15

significantly reduced in the benzo[a]pyrene

- exposed groups in comparison to that of the control” 

### 16

but provided quantitative data only for the middle dose group, which exhibited a ~30% decrease in 

### 17

percent motile sperm. No statistically significant decrease in sperm count was reported at any 

### 18

dose. The middle dose (0.01 mg/kg

- day) is conside red to be a LOAEL, based on reduced sperm 

### 19

motility. 

### 20

Gao et al. (2011) examined effects of benzo[a]pyrene exposure via on cervical cell 

### 21

morphology. Female ICR mice (18

- 22 g) were exposed to doses of 0, 2.5, 5, or 10 mg/kg twice per 

### 22

week for 14 weeks, eit her by oral gavage or by intraperitoneal injection (for this review, only oral 

### 23

results are reported). After adjustment for equivalent continuous dosing (2/7 days/week), the 

### 24

equivalent daily doses are estimated to be 0.7, 1.4, 2.9 mg/kg

- day. Both vehicle (sesame oil) and 

### 25

untreated control groups were maintained. Body weights were determined weekly. Groups of 26 

### 26

mice per dose per exposure route were sacrificed at the end of exposure for evaluation of cervical 

### 27

weight and histopathology. Additional groups of 10 mice were exposed for 14 weeks and used for 

### 28

determination of lipid peroxidation (malondialdehyde and glutathione

- S

- transferase levels) and 

### 29

CYP1A1 activity (EROD) in both liver and cervix, as well as creatine kinase activity, AST activity, and 

### 30

IL

- 6 le vels in cervix and serum. 

### 31

Mortality was observed in all exposure groups with the exception of the low dose oral 

### 32

exposure group; the authors did not indicate the timing or causes of death (Gao et al., 2011). There 

### 33

were no control deaths. Mortality inciden ces in the oral exposure groups (low to high dose) were 

### 34

0/26 (untreated control), 0/26 (vehicle control), 0/26, 1/36, and 2/26. Benzo[a]pyrene treatment 

### 35

resulted in dose

- dependent decreases in body weight gain. In the high dose group of both 

### 36

treatments, body weight began to decline after ~7 weeks of exposure. Based on visual examination 

### 37

of data presented graphically, mean terminal body weights in the low, mid

- , and high

- dose oral 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 70 DRAFT

— DO NOT CITE OR QUOTE exposure groups were ~10, 15, and 30% lower (respectively) than the vehicl e control mean. The 

### 1

untreated control mean body weight for the oral exposure group was similar to the vehicle control 

### 2

mean body weight. Cervical weight as a function of body weight was not affected by oral 

### 3

benzo[a]pyrene exposure. Microscopic examinatio n of the cervix revealed increased incidences of 

### 4

epithelial hyperplasia and inflammatory cells in the cervix of all groups of exposed mice, and 

### 5

atypical hyperplasia of the cervix in mice exposed to 1.4 or 2.9 mg/kg benzo[a]pyrene. Statistical 

### 6

analysis of the findings was conducted, but was poorly reported in the publication. Table B

- 24 

### 7

shows the incidences in the oral exposure groups, along with the results of Fisher’s exact tests 

### 8

performed for this review. 

### 9

Table B

- 2 4 . Mortality and cervical histopatholo gy incidences in female 10

ICR mice exposed to benzo[a]pyrene via gavage for 14 weeks 11

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 6/25 a 7/24 a

Atypical hyperplasia of cervix 0/26 0/26 0/26 2/25 4/24 a

Inflammatory cells in cervix 2/26 3/26 10/26 a 12/25 a 18/24 a

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

Levels of malondialdehyde in both the cervix and liver were significantly higher than 

### 13

controls in all dose groups of animals treated by either oral (1.5 to 2

- fold higher in the cervix and 

### 14

~3

- fold to 7

- fold higher in the liver after oral exposure p<0.05) or intraperitoneal exposure. 

### 15

Concomitant decreases in GST activity (~15% to 50% lower than controls in the cervix and ~30% 

### 16

to 60% lower in the liver after oral exposure; p<0.05) were also observed at all doses and in both 

### 17

organs and both treatments. EROD a ctivity was increased in the cervix (~4

- to ~12

- fold) and liver 

### 18

(~12

- to ~35

- fold) of all exposure groups. Measurement of CK and AST activity in the cervix and 

### 19

serum also showed significant increases at all doses and after both exposures (~1.5

- to 2

- fold in the 

### 20

cervix, and ~20% to 50% higher than controls in the liver after oral exposure). Finally, levels of the 

### 21

inflammatory cytokine IL

- 6 were significantly (p<0.05) increased in the cervix of all treated mice, 

### 22

and were markedly increased (from more than t wo

- fold higher than untreated or vehicle controls 

### 23

at the low dose, to ~six

- fold higher at the high dose) in the serum of treated mice. 

### 24

Based on the observations of decreased body weight and increased cervical epithelial 

### 25

inflammation and hyperplasia, a LOA EL of 0.7 mg/kg

- day (the lowest dose tested) is identified for 

### 26

this study. 

### 27

### Toxicological Review of benzo[a]pyrene

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



### B

- 71 DRAFT

— DO NOT CITE OR QUOTE Mohamed et al. (2010) investigated multi

- generational effects in male mice following 

### 1

exposure of 6

- week old

- C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg

- day 

### 2

benzo[a]pyr ene for 6 weeks by gavage. Following final treatment, male mice were allowed to 

### 3

stabilize for 1 week prior to being mated with two untreated female mice to produce an 

### 4

F1 generation. Male mice were sacrificed 1 week after mating. F1 males were also mated with 

### 5

untreated female mice as were F2 males. The mice of the F1, F2, and F3 generations were not 

### 6

exposed to benzo[a]pyrene. The F0, F1, F2, and F3 mice were all sacrificed at the same age 

### 7

(14 weeks) and endpoints including testis histology, sperm count, sperm motility, and in vitro 

### 8

sperm penetration (of hamster oocytes) were evaluated. These endpoints were analyzed 

### 9

statistically using analysis of variance (ANOVA) and Tukey’s honest significance test and results 

### 10

were reported graphically as means ± SD. 

### 11

T esticular atrophy was observed in the benzo[a]pyrene treatment groups, but was not 

### 12

statistically different than controls. Statistically significant reductions were observed in epididymal 

### 13

sperm counts of F0 and F1 generations treated with the high or low d ose of benzo[a]pyrene. For F0 

### 14

and F1 generations, epididymal sperm counts were reduced approximately 50 and 70%, 

### 15

respectively, in the low

- and high

- dose groups. Additionally, sperm motility was statistically 

### 16

significantly decreased at the high dose in th e F0 and F1 generations. Sperm parameters of the F3 

### 17

generation were not statistically different from controls. An in vitro sperm penetration assay 

### 18

revealed statistically significantly reduced fertilization in F0 and F1 generations of the low

- and 

### 19

high

- do se groups. However, the value of this in vitro test is limited as it bypasses essential 

### 20

components of the intact animal system (U.S. EPA, 1996). Based on decreased epididymal sperm 

### 21

counts of F0 and F1 generations, a LOAEL of 1 mg/kg

- day was established f rom this study (no 

### 22

NOAEL was identified). 

### 23

Arafa et al. (2009) exposed groups of 12 male Swiss albino rats to benzo[a]pyrene in olive 

### 24

oil (0 or 50 mg/kg

- day via gavage) for 10 consecutive days, either alone or after similar treatment 

### 25

with 200 mg/kg

- day of the flavonoid hesperidin, which has been shown to exert anti

- inflammatory, 

### 26

antioxidant, and anticarcinogenic activity. One day after the final dose, the animals were sacrificed 

### 27

for removal of the cauda epididymides and testes. Epididymal sperm count and motility were 

### 28

assessed, as was daily sperm production in the testes. The study authors also investigated the 

### 29

testicular activity of LDH, SOD, and GST, as well as GSH, malondialdehyde, and protein content. The 

### 30

testes were examined under light microscope. 

### 31

Relative testes weights (normalized to body weight) of benzo[a]pyrene exposed

- animals 

### 32

were significantly decreased compared with controls (35% lower, p < 0.05) (Arafa et al., 2009). In 

### 33

addition, exposure to benzo[a]pyrene alone resulted in significantly decreased sperm count, 

### 34

numbers of motile sperm, and daily sperm production (~40% decrease from control in each 

### 35

parameter, p < 0.05). Effects on sperm cou nt and production were abolished by hesperidin 

### 36

pretreatment, but the number of motile sperm remained significantly depressed (compared with 

### 37

the control group) in the group exposed to both benzo[a]pyrene and hesperidin. Measures of 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 72 DRAFT

— DO NOT CITE OR QUOTE antioxidant enzymes and lipid peroxidation showed statistically significant induction of oxidative 

### 1

stress in the testes of benzo[a]pyrene

- exposed rats. With the exception of the decrease in testicular 

### 2

GSH content (which was partially mitigated), pretreatment with hesperidin elim inated the effects of 

### 3

benzo[a]pyrene on lipid peroxidation and antioxidant enzymes. 

### 4

Xu et al. (2010) treated female Sprague

- Dawley rats (6/group) to 0 (corn oil only), 5, or 10 

### 5

mg/kg

- day benzo[a]pyrene by gavage every other day for a duration of 60 days. This resulted in 

### 6

TWA doses of 0, 2.5, and 5 mg/kg

- day over the study period of 60 days. Endpoints examined 

### 7

included ovary weight, estrous cycle, 17B

- estradiol blood level, and ovarian follicle populations 

### 8

(including primordial, primary, secondary, atret ic, and corpora leutea). Animals were observed 

### 9

daily for any clinical signs of toxicity and following sacrifice, gross pathological examinations were 

### 10

made and any findings were recorded. All animals survived to necropsy. A difference in clinical 

### 11

signs w as not observed for the treated groups and body weights were not statistically different in 

### 12

treated animals (although they appear to be depressed 6% at the high dose). Absolute ovary 

### 13

weight was statistically significantly reduced in both the low

- and high

- dose groups (11 and 15%, 

### 14

respectively) (see Table B

- 25 ). Animals treated with the high dose were noted to have a 

### 15

statistically significantly prolonged duration of the estrous cycle and nonestrus phase compared to 

### 16

controls. Animals in the high

- dose group also had statistically significantly depressed levels of 

### 17

estradiol (by approximately 25%) and decreased numbers of primordial follicles (by approximately 

### 18

20%). This study also indicated a strong apoptotic response of ovarian granulosa cells as visualized 

### 19

through TUNEL labeling; however, the strongest response was seen at the low dose; decreased 

### 20

apoptosis was also observed at the high dose. Based on decreased ovary weight following a 60

- day 

### 21

oral exposure to benzo[a]pyrene, a LOAEL of 2.5 mg/kg

- day was est ablished from this study (no 

### 22

NOAEL was identified). 

### 23

Table B

- 2 5 . Means ± SD for ovary weight in female Sprague

- Dawley rats 24

Dose (mg/kg

- d) a

0 2.5 5

Ovary weight (g) 0.160 ± 0.0146 0.143 ± 0.0098 b 0.136 ± 0.0098 b

Body weight (g) 261.67 ± 12.0 249.17 ± 11.2 247.25 ± 11.2

a TWA doses over the 60

- day study period.

b Statistically different from controls ( p < 0.05) using one

- way ANOVA.

Source: Xu et al. (2010).



### 25

Zheng et al. (2010) treated male Sprague

- Dawley rats to 0 (corn oil only), 1, or 5 mg/kg

- day 

### 26

benzo[a]pyrene by daily gavage for a duration of 30 (8/group) or 90 days (8/group). At necropsy, 

### 27

the left testis of each animal was collected and weighed. Testes testosterone concentrations were 

### 28

determined by radioimmunassay and results were expressed as ng/g testis and reported 

### 29

graphically. Testicular testosterone was statistically significantly decreased in the high

- dose group 

### 30

### Toxicological Review of benzo[a]pyrene

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



### B

- 73 DRAFT

— DO NOT CITE OR QUOTE approximately 15% following 90 days of exposure. The low

- dose group also appeared to have a 

### 1

similar average depression of test osterone levels; however, the change did not reach statistical 

### 2

significance. Testosterone levels measured in animals sacrificed following 30 days of 

### 3

benzo[a]pyrene exposure were not statistically different than controls. Based on decreased 

### 4

testicular tes tosterone levels following a 90

- day oral exposure to benzo[a]pyrene, a LOAEL of 5 

### 5

mg/kg

- day and a NOAEL of 1 mg/kg

- day were identified. 

### 6

McCallister et al. (2008) administered 0 or 300 μg/kg benzo[a]pyrene by gavage in peanut 

### 7

oil to pregnant Long Evans rats (n = 5 or 6) on GDs 14

– 17. At this exposure level, no significant 

### 8

changes were see in number of pups per litter, pup growth, or liver to body weight ratios in control 

### 9

compared to benzo[a]pyrene exposed offspring. Treatment

- related differences in brain t o body 

### 10

weight ratios were observed only on PNDs 15 and 30. Decreases in cerebrocortical mRNA 

### 11

expression of the glutamatergic N

- methyl

- D

- aspartate (NMDA) receptor subunit was significantly 

### 12

reduced (50%) in treated offspring compared to controls. In additi on, in utero exposed offspring 

### 13

exhibited decreased evoked cortical neuronal activity in the barrel field cortex when tested at PNDs 

### 14

90

– 120. 

### 15

Rigdon and Neal (1965) administered diets containing 1,000 ppm benzo[a]pyrene to 

### 16

pregnant mice (nine/group) on GDs 10

– 21 or 5

– 21. The pups were reported as appearing 

### 17

generally normal at birth, but cannibalism was elevated in the exposed groups. These results are in 

### 18

contrast with an earlier study (Rigdon and Rennels, 1964) in which rats (strain not specified) were 

### 19

fed diets containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and 

### 20

during gestation. In the earlier study, five of eight treated females mated with untreated males 

### 21

became pregnant, but only one delivered live young. The treated d am that delivered had two live 

### 22

and two stillborn pups; one dead pup was grossly malformed. In the remaining treated females, 

### 23

vaginal bleeding was observed on GDs 23 or 24. In the inverse experimental design, three of six 

### 24

controls mated to benzo[a]pyrene

- treated males became pregnant and delivered live young. 

### 25

Visceral and skeletal examinations of the pups were not conducted. These studies were limited by 

### 26

the small numbers of animals, minimal evaluation of the pups, lack of details on days of treatment 

### 27

(f ood consumption, weight gain), and the occurrence of cannibalism. 

### 28

Reproductive effects of in utero exposure via oral route 

### 29

MacKenzie and Angevine (1981) conducted a two

- generation reproductive and 

### 30

developmental toxicity study for benzo[a]pyrene in CD

- 1 mic e. Benzo[a]pyrene was administered 

### 31

by gavage in 0.2 mL of corn oil to groups of 30 or 60 pregnant (the F0 generation) mice at doses of 

### 32

0, 10, 40, or 160 mg/kg

- day on GDs 7

– 16 only. Therefore, unlike the standard two

- generation 

### 33

study, F1 animals were expo sed only in utero. F1 offspring were evaluated for postnatal 

### 34

development and reproductive function as follows. F1 pups (four/sex when possible) were allowed 

### 35

to remain with their mothers until weaning on PND 20. Crossover mating studies were then 

### 36

conduct ed. Beginning at 7 weeks of age, each F1 male mouse (n = 20

– 45/group) was allowed to 

### 37

mate with two untreated virgin females for 5

- day periods for 25 days (for a total exposure of 10 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 74 DRAFT

— DO NOT CITE OR QUOTE untreated females/F1 male), after which time the males were separated fro m the females. 

### 1

Fourteen days after separation from the males (i.e., on days 14

– 19 of gestation), the females were 

### 2

sacrificed and the numbers of implants, fetuses, and resorptions were recorded. The F2 fetuses 

### 3

were then examined for gross abnormalities. Similarly, each F1 female mouse (n = 20

– 55/group), 

### 4

beginning at 6 weeks of age, was paired with an untreated male for a period of 6 months. Males 

### 5

were replaced if the females failed to produce a litter during the first 30

- day period. All F2 young 

### 6

were ex amined for gross abnormalities on day 1 of life and their weights were recorded on day 4 of 

### 7

age. This F2 group was sacrificed on day 20 postpartum, while the F1 female was left with a male 

### 8

until the conclusion of the study. At 6 weeks of age, gonads of g roups of 10 male and 10 female F1 

### 9

mice exposed to 0, 10, or 40 mg/kg

- day benzo[a]pyrene in utero were subjected to gross pathology 

### 10

and histologic examinations. 

### 11

No maternal toxicity was observed. The number of F0 females with viable litters at 

### 12

parturition at the highest dose was statistically significantly reduced by about 35% ( Table B

- 26 ), 

### 13

but progeny were normal by gross observation. Parturition rates of the low

- and mid

- dose groups 

### 14

were unaffected by treatment, and litter sizes of all treated groups wer e similar to the control group 

### 15

throughout lactation. However, body weights of the F1 pups in the mid

- and high

- dose groups were 

### 16

statistically significantly decreased on PND 20, by 7 and 13%, respectively, and in all treated pups 

### 17

on PND 42, 6, 6, and 10% f or the low, mid, and high dose, respectively (Table B

- 26 ). The number of 

### 18

F1 pups surviving to PNDs 20 and 42 was significantly reduced at the high dose ( p < 0.01), by 8 and 

### 19

16%, respectively. When F1 males were bred to untreated females and F1 females we re mated 

### 20

with untreated males, a marked dose

- related decrease in fertility of >30% was observed in both 

### 21

sexes, starting at the lowest exposure. There were no treatment

- associated gross abnormalities or 

### 22

differences in body weights in the F2 offspring. 

### 23

Tabl e B

- 2 6 . Reproductive effects in male and female CD

- 1 F1 mice 24

exposed in utero to benzo[a]pyrene 25

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 index c

F1 female fertility index d 46/60 (77%)

11.2 ± 0.1

29.9 ± 0.2

80.4

100.0 21/30 (70%)

11.6 ± 0.1

28.2 ± 0.3 b

52.0 b

65.7 b 44/60 (73%)

10.4 ± 0.1 b

28.0 ± 0.2 b

4.7 b

0.0 b 13/30 (43%) b

9.7 ± 0.2 b

26.8 ± 0.4 b

0.0 b

0.0 b

### Toxicological Review of benzo[a]pyrene

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



### B

- 75 DRAFT

— DO NOT CITE OR QUOTE 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

Exposure to benzo[a]pyrene caused a marked dose

- related decrease in the size of the 

### 2

gonads. In F1 males, testes weights were statistically significantly reduced. Testes from animals 

### 3

exposed in utero to 10 and 40 mg/kg

- day weighed approximately 60 and 18%, respectively, of the 

### 4

weight of testes from the control animals (no F2 offspring were produced in the high

- dose group). 

### 5

This was confirmed by histopathologic observation of atrophic seminiferous tubules in the 

### 6

40 mg/kg

- day group t hat were smaller than those of controls and were empty except for a basal 

### 7

layer of cells. The number of interstitial cells in the testes was also increased in this group. Males 

### 8

from the 10 mg/kg

- day group showed limited testicular damage; although all ex hibited evidence of 

### 9

tubular injury, each animal had some seminiferous tubules that displayed active spermatogenesis. 

### 10

Ovarian tissue was absent or reduced in F1 females such that organ weights were not possible to 

### 11

obtain. Examination of available tissue i n these females revealed hypoplastic ovaries with few 

### 12

follicles and corpora lutea (10 mg/kg

- day) or with no evidence of folliculogenesis (40 mg/kg

- day). 

### 13

Ovarian tissue was not examined in highest

- dose females. 

### 14

The LOAEL in this study was 10 mg/kg

- day, bas ed on decreases in mean pup weight (<5%) 

### 15

at PND 42 of F1 offspring of dams treated with 10, 40, or 160 mg/kg

- day benzo[a]pyrene, marked 

### 16

decreases in the reproductive capacity (as measured by fertility index) of both male and female F1 

### 17

offspring exposed at all three treatment levels of benzo[a]pyrene (by approximately 30% in males 

### 18

and females), decreased litter size (by about 20%) in offspring of F1 dams, and the dramatic 

### 19

decrease in size and alteration in anatomy of the gonads of both male and female F1 mic e exposed 

### 20

to 10 and 40 mg/kg

- day benzo[a]pyrene in utero. A NOAEL was not identified. 

### 21

In another reproductive and developmental toxicity study, benzo[a]pyrene was 

### 22

administered by gavage in corn oil to nine female NMRI mice at a dose of 10 mg/kg

- day on GDs 7

– 

### 23

16; a group of nine controls received corn oil (Kristensen et al., 1995). Body weights were 

### 24

monitored. F0 females were kept with their offspring until after weaning (21 days after delivery). 

### 25

At 6 weeks of age, one F1 female from each litter (n = 9) w as caged with an untreated male. The 

### 26

F2 offspring were inspected for gross deformities at birth, weight and sex were recorded 2 days 

### 27

after birth, and the pups were sacrificed. The F1 females were sacrificed after 6 months of 

### 28

continuous breeding. The eff ects of benzo[a]pyrene treatment on fertility, ovary weights, follicles, 

### 29

and corpora lutea were evaluated. F0 females showed no signs of general toxicity, and there was no 

### 30

### Toxicological Review of benzo[a]pyrene

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



### B

- 76 DRAFT

— DO NOT CITE OR QUOTE effect on their fertility. F1 females had statistically significantly lower median numbers of offspring, 

### 1

number of litters, and litter sizes and a statistically significantly greater median number of days 

### 2

between litters as compared with the controls ( Table B

- 27 ). At necropsy, the F1 females from 

### 3

treated F0 females had statistically si gnificantly reduced ovary weights; histologic examination of 

### 4

the ovaries revealed decreased numbers of small, medium, or large follicles and corpora lutea 

### 5

( Table B

- 27 ). Only one dose group was used in this study, with decreased F1 female fertility 

### 6

observe d following in utero exposure at the LOAEL of 10 mg/kg

- day; no NOAEL was identified. 

### 7

Table B

- 2 7 . Effect of prenatal exposure to benzo[a]pyrene on indices of 8

reproductive performance in F1 female NMRI mice 9

Endpoint (median with range in parentheses) Contro l a Benzo[a]pyrene

exposed a (10 mg/kg

- d)

Number of F2 offspring

Number of F2 litters

F2 litter size (number of pups per litter)

Number of d between F2 litters 92 (26

– 121)

8 (3

– 8)

11.5 (6

– 15)

20.5 (20

– 21) 22 b (0

– 86)

3 b (0

– 8)

8 b (3

– 11)

21 b (20

– 23)

F1 ovary weight (mg)

Number of small follicles

Number of medium follicles

Number of large follicles

Number of corpora lutea 13 (13

– 20)

44 (1

– 137)

9 (5

– 25)

14 (6

– 23)

16 (6

– 35) 9 b (7

– 13)

0 b (0

– 68)

0 b (0

– 57)

0 b (0

– 19)

0 b (0

– 14)

a Groups of nine female NMRI F0 mice were 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 tes t or Kruskall

- Wallis two

tailed test.

Source: Kristensen et al. (1995).



### 10

Chen et al., (2012) treated male and female neonatal Sprague

- Dawley rats (10/sex/group) 

### 11

with benzo[a]pyrene (unspecified purity) dissolved in peanut oil by gavage daily from post

- natal 

### 12

day (PND) 5

– 11, at doses of 0.02, 0.2 or 2 mg/kg in 3 mL vehicle/kg b.w., determined individually 

### 13

based upon daily measurements. This time period was described as representing the brain growth 

### 14

spurt in rodents, analogous to brain developmenta l occurring from the third trimester to 2 years of 

### 15

age in human infants. Breeding was performed by pairs of nine week old rats, with delivery 

### 16

designated as PND0. Litters were culled to 8 pups/dam (4/ea male and female, when possible) and 

### 17

randomly redistr ibuted at PND1 among the nursing dams; dams themselves were rotated every 2

- 3 

### 18

days to control for caretaking differences, and cage

- side observations of maternal behavior were 

### 19

made daily. One male and female from each litter were assigned per treatment gro up, and the 

### 20

following physical maturation landmarks were assessed daily in all treatment groups until weaning 

### 21

at PND21: incisor eruption, eye opening, development of fur, testis decent and vaginal opening. 

### 22

### Toxicological Review of benzo[a]pyrene

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



### B

- 77 DRAFT

— DO NOT CITE OR QUOTE Neonatal sensory and motor developmental tests wer e administered to pups during the 

### 1

preweaning period at PNDs 12, 14, 16 and 18, and were behavioral tests administered to rats as 

### 2

adolescents (PND 35, 36) or as adults (PND 70, 71): each rat was only tested during one 

### 3

developmental period. All dosing was p erformed from 1300

– 1600 hrs, and behavioral testing was 

### 4

during the “dark” period from 1900

– 2300 hrs, although tests were performed in a lighted 

### 5

environment. Pups were observed individually and weighed daily, the order of testing litters was 

### 6

randomized each day, and all observations were recored by investigators blinded to group 

### 7

treatment. 

### 8

Sensory and motor developmental tests including the surface righting reflex test, negative 

### 9

geotaxis test, and cliff aversion test were performed only once, while th e forelimb grip strength test 

### 10

was assessed during three 60 second trials on PND12. Rat movements during the open

- field test 

### 11

were recorded by camera, and two blinded investigators scored movement and rearing separately 

### 12

during a 5 min. evaluation period. B linded investigators directly observed video monitoring of rat 

### 13

movements during the elevated plus maze, and after a 5 min. free exploration period, recored 

### 14

number of entries into the closed and open arms, the time spent in the open arms, and latency to 

### 15

the first arm entry. Assessment of the Morris water maze was slightly different, in that the rats 

### 16

were habituated to the testing pool by a 60 second swim without a platform on the day prior to 

### 17

testing. The rats were then tested during a 60 second swim with a hidden platform present at a 

### 18

constant position each day for four days; on the fifth day, the rats were evaluated during a 60 

### 19

second probe swim without a platform. The number of times each animal crossed the original 

### 20

platform location and the duration of time spent in the platform quadrant were recorded during 

### 21

this final evaluation. One pup/sex/litter were assigned for behavioral testing to each of four tracks: 

### 22

Track 1, surface righting reflex test, cliff aversion test, and open

- field test (PND 12

– 18); Track 2, 

### 23

negative geotaxis test, forelimb grip strength test, and open

- field test (PND 12

– 20); Track 3, 

### 24

elevated plus maze, Morris water maze, and open

- field test (PND 34

– 36); Track 4, elevated plus 

### 25

maze, Morris water maze, and open

- field test (PND 69

– 71). All results were presented in 

### 26

graphical form only. 

### 27

No significant effects on pup body weight were observed during the 7

- day treatment period 

### 28

(PND 5

– 11). Three

- way ANOVA (time x B[a]P treatment x sex) indicated that effects of B[a]P were 

### 29

not sex

- dependent throughout the 71 day experiment, so both sexes were pooled together. From 

### 30

this pooled analysis, pups in the 2 mg/kg treatment group gained significantly less weight at both 

### 31

PND36 and PND71. There were no differences among treatment groups in incisor eruption, eye 

### 32

opening, development of fur, testis decent or vaginal opening. 

### 33

For all measurements of neonatal sensory and motor development, results from both sexes 

### 34

were analyzed together since B[a]P was reported to have no significant interaction with sex by 3

- 

### 35

way ANOVA. No significant differences were observed in either the cliff aversion or forelimb grip 

### 36

strength tests. In the surface righting reflex test, latency was increased in the 0.2 mg/kg group at 

### 37

PND12, in the 0.02 and 2 mg/kg groups at PND14, in only the high dose 2 mg/kg group at PND16, 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 78 DRAFT

— DO NOT CITE OR QUOTE and was not significantly different in any group at PND18. At PND12 there was a dose

- related 

### 1

increase in negative geotaxis latency associated with 0.02, 2 and 2 mg/kg B[a]P, which was also 

### 2

present in th e 2 mg/kg group at PND14, but returned to control levels at PND16 and PND18. In the 

### 3

open field test, there were no significant differences in either locomotion or rearing activity at 

### 4

PND18 or 20. At PND34, the 2 mg/kg group exhibited significantly increa sed movement, but 

### 5

increases in rearing were not significant. At PND69, increased locomotion was observed in both the 

### 6

0.2 and 2 mg/kg groups, while rearing was significantly increased in only the 2 mg/kg treatment 

### 7

group. 

### 8

The elevated plus maze performance was only evaluated in adolescent and adult rats. 

### 9

Unlike the previous tests, 3

- way ANOVA revealed a statistically significant interaction between 

### 10

neonatal B[a]P treatment and sex, so male and female performance was analyzed independently. 

### 11

No significant d ifferences in PND35 males were observed, and the only significant observation in 

### 12

PND35 females was increased time spent in the open maze arms by 2 mg/kg treatment group. 

### 13

Significantly decreased latency time to first open arm entry was observed in PND70 ma les and 

### 14

females in both 0.2 and 2 mg/kg treatment groups; these groups also spent significantly more time 

### 15

in open maze arms, along with the 0.02 mg/kg female group. PND70 2 mg/kg males, along with 0.2 

### 16

and 2 mg/kg females, entered more frequently into open arms and less frequently into closed arms 

### 17

than vehicle controls. In the Morris water maze, escape latency (time to reach the platform during 

### 18

each of the four testing days) was consistently increased in the 2 mg/kg treatment group of both 

### 19

sexes, in both a dolescent and adult animals. These increases were statistically significant in both 

### 20

males and females treated with 2 mg/kg B[a]P at both PND39 and PND74, and were also 

### 21

significantly elevated in 0.2 mg/kg animals of both sexes at PND74. Likewise, performa nce during 

### 22

the fifth test day, in the absence of the escape platform, was significantly adversely affected by both 

### 23

metrics (decreased time spent in the target quadrant and decreased number of attempts to cross 

### 24

the platform location) in 2 mg/kg rats of both sexes at both PND40 and PND75. PND75 females 

### 25

treated with 0.2 mg/kg B[a]P also showed significant decreases in both performance metrics, while 

### 26

PND75 0.2 mg/kg males only demonstrated significant differences in “time spent in target 

### 27

quadrant”. Swim speed was also assessed, but there were no differences among any treatment 

### 28

group at either age evaluated. 

### 29

Jules et al., (2009) treated pregnant Long Evans Hooded (LEH) rats with benzo[a]pyrene 

### 30

(unspecified purity) dissolved in 0.875 mL peanut oil by gavage dail y from GD14

– GD17, at doses of 

### 31

150, 300, 600 and 1,200 μg B[a]P /kg b.w., with animals weighed daily. Cage

- side observations 

### 32

were performed until pup weaning, and litter size evaluated for each treatment group. Pups from 4 

### 33

– 5 individual litters were an alyzed for each endpoint, which was independently repeated for a total 

### 34

of 3 replicates. Delivery was designated PND0, and pups were harvested from PND0

– 15 for B[a]P 

### 35

metabolite identification, or for other endpoints as young adults at PND53. Systolic/di astolic blood 

### 36

pressure and heart rate was recorded by a volume pressure recording sensor and occlusion tail

- cuff 

### 37

applied to conscious, non

- anesthetized animals. Animals were preconditioned to the restraint 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 79 DRAFT

— DO NOT CITE OR QUOTE device and tail

- cuff by daily acclimatization ses sions during PND46

– 50, to minimize stress effects 

### 1

during data collection. Cardiac function values were averaged from 15 readings each collected over 

### 2

a 1 minute interval every other minute for 30 minutes on PND53. Whole blood was collected from 

### 3

the hear t and aorta prior to surgical resection and tissue processing. Plasma and heart tissue B[a]P 

### 4

metabolite content was quantified by reverse

- phase HPLC with UV and fluorescence detection, 

### 5

while heart and aortic tissue was subjected to SDS

- PAGE for qualitativ e protein analysis, and RNA 

### 6

extraction. Quantitative RT

- PCR was performed for levels of angiotensin II (AngII), neuronal NOS 

### 7

(nNOS), endothelial NOS (eNOS) and 7,8

- Dihydrobiopterin oxidoreductase (BH4/BH2 

### 8

oxidoreductase). Total RNA was also used to probe a cDNA microarray, and targets with ≥ 2

- fold 

### 9

changes in expression were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and 

### 10

Gene Ontology (GO) biological process pathway analysis. 

### 11

No significant differences in litter size or pup weight gain f rom PND0

– 15 were reported in 

### 12

any treatment group, and no convulsions, tremors or abnormal movements were reproducibly 

### 13

observed. Most analytical data was reported graphically, as mean ± SEM of three replicates of 3

– 5 

### 14

offspring measured/group. Plasma a nd heart tissue total B[a]P metabolite levels were maximal at 

### 15

PND0 (the first time point sampled) and progressively decreased from PND0

– 13. Compared to the 

### 16

low

- dose group (150 μg/kg), plasma metabolite levels were significantly elevated in the 600 and 

### 17

1 ,200 μg/kg B[a]P groups through PND13, while heart metabolite levels were significantly 

### 18

increased through PND11. Metabolites in mid

- dose group, 300 μg/kg, trended between the 150 

### 19

and 600 μg/kg group levels from PND0

– 7, while not achieving statistically significant differences 

### 20

in pair

- wise comparisons. Three principle groups of B[a]P metabolites were identified. More than 

### 21

70% of the total heart metabolite burden was composed of diol metabolites through PND13, while 

### 22

the more reactive hydroxyl metabolites increased in relative composition from PND9

– 13, and the 

### 23

dione population remained constant at ≤ 5%. 

### 24

Cardiovascular function was evaluated in pups exposed in utero to 600 or 1,200 μg/kg B[a]P 

### 25

vs. controls. A dose

- related and statistically significant increase in both systolic (20, 50%) and 

### 26

diastolic pressure (30, 80%) was observed in mid and high

- dose pups, respectively. Heart rate was 

### 27

also significantly altered; a 10% increased heart rate was reported in the 600 μg/kg B[a]P group, 

### 28

while the average h eart rate of the 1,200 μg/kg B[a]P groups decreased 8%. Cardiac tissue eNOS 

### 29

protein levels fluctuated as a result of B[a]P treatment; in both the 600 and 1,200 μg/kg groups, 

### 30

eNOS expression by semi

- quantitative SDS

- PAGE was significantly decreased at PND0 and PND5, 

### 31

while it was significantly elevated above controls at PND10 and PND15. While eNOS expression in 

### 32

the 600 μg/kg B[a]P group had returned to control levels by PND53, eNOS expression was 

### 33

significantly higher (approximately 2

- fold) in the 1,200 μg/k g group. Compared to vehicle

- treated 

### 34

controls, cardiac message levels of nNOS and eNOS were not significantly affected by B[a]P 

### 35

treatment at PND0, and while nNOS mRNA levels were 2

- fold higher at PND53 in the 600 μg/kg 

### 36

group, and eNOS mRNA was 3

- fold high er in the 1,200 μg/kg group, consistent with the increased 

### 37

eNOS protein levels detected at PND53. Message levels of BH4/BH2 oxidoreductase were 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 80 DRAFT

— DO NOT CITE OR QUOTE suppressed in both B[a]P treatment groups at PND0, and while mRNA expression remained 

### 1

suppressed at PND53 in th e 600 μg/kg group, BH4/BH2 message returned to control levels in the 

### 2

1,200 μg/kg group. Angiotensin II mRNA levels were 1.8

- fold higher in both B[a]P groups at PND0, 

### 3

and while expression increased to 5

- fold more than controls at PND53 in the 600 μg/kg gro up, 

### 4

AngII expression remained closer to 1.5

- fold greater in the high

- dose group. The following 

### 5

pathways were identified as being enriched by 1,200 μg/kg B[a]P treatment in utero using KEGG 

### 6

analysis, and correcting for multiple comparisons using the false

- discovery rate method: PPARγ, 

### 7

renin

– angiotensin system (AngII, adiponectin C1Q and collagen domain, adrenergicβ3R, tachykinin 

### 8

R1), hematopoietic cell lineage, CYP450 metabolism (CYP2a2, CYP7a1 and CYP2b12), retinol 

### 9

metabolism, cell adhesion molecules

- CAMs, primary bile acid biosynthesis and tight junctions. 

### 10

Table B

- 28 . Exposure

- related effects in Long Evans Hooded rats 11

exposed to benzo[a] pyrene by gavage daily in utero from GD14

– GD17 12

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

Bouayed et al., (2009) treated nursing female Swiss Albino OF1 mice (5/dose group) with 

### 14

benzo[a]pyrene (unspecified purity) dissolved in avocado oil by gavage daily while nursing pups 

### 15

from PND1

– 14 at 0, 2 or 20mg/kg

- day in 10 mL/kg b.w., individually det ermined each day. Prior 

### 16

to benzo[a]pyrene treatment, Swiss Albino litters were culled to 10 pups (5/sex when possible), 

### 17

and nurturing females assigned to litters that were stratified randomly to achieve equivalent mean 

### 18

pup litter body weights across the d esignated treatment groups. As the effects of B[a]P on maternal 

### 19

nurturing behavior was unknown, dam behavior was visually monitored daily until weaning. 

### 20

Furthermore, maternal nurturing performance from PND0

– 21 was assessed by two methods: a 

### 21

nest

- buildi ng test administered q.2.d., where nest quality/complexity was scored 15 minutes after 

### 22

cotton material was supplied; and pup retrieval, in which latency to return the displaced pup to the 

### 23

nest was measured twice and averaged, was evaluated q.d. At the ind icated times 2 mice/sex/litter 

### 24

were randomly selected, weighed, and brains resected for later mRNA expression analysis (n = 

### 25

20/group). 

### 26

Pup neuromotor maturation and behavior was assessed during pre

- weaning by four 

### 27

standard methods (administered between 1 000

– 1300 on testing days, and in temporal order as 

### 28

indicated): 1) righting reflex test , maximum duration 120 seconds, administered on PNDs 3, 5, 7 and 

### 29

### Toxicological Review of benzo[a]pyrene

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



### B

- 81 DRAFT

— DO NOT CITE OR QUOTE 9; 2) negative geotaxis test , maximum duration 120 seconds, administered on PNDs 5, 7, 9 and 11; 

### 1

3) for elimb grip test , duration until failure, administered on PNDs 9 and 11; and 4) open field test , 6 

### 2

minute evaluation of locomotor activity and rearing following a 1 minute habituation period, 

### 3

administered on PND15. Adolescent function was evaluated by thre e methods: water escape pole 

### 4

climbing (WESPOC) test , administered at PND20, in which the time to find the pole, time to climb the 

### 5

pole, and the time to reach the safety platform were reported; elevated plus maze , administered at 

### 6

PND32 for 5 minutes, in whi ch the latency time to first open arm entry, number of entries into open 

### 7

arms, total number of entries, percent of time spent in open arms, and percent of entries into open 

### 8

arms was determined; and Y

- maze spontaneous alternation test , administered at PND40 for 5 

### 9

minutes, in which the % spontaneous alternation was calculated by: [(the number of successful 

### 10

overlapping triplets)/(total number of arm entries

– 2) x 100%]. 

### 11

B enzo[a]pyrene treatment did not significantly affect the body weight of nursing mothers 

### 12

d uring the 2 week treatment period. Since three

- way ANOVA indicated that changes in pup weight 

### 13

as a result of B[a]P treatment were not sex

- dependent, data from male and female pups were 

### 14

combined. B[a]P treatment of nursing mothers was associated with a 8

- 9% weight gain in pups 

### 15

nursing from the 2 mg/kg group, and a 10

- 12% weight gain in pups from the 20 mg/kg group at 

### 16

PND12

– 20. While not significantly different from PND26

– 40, pup weight in the 20 mg/kg group 

### 17

was continuously higher than either the 2 mg/ kg group or vehicle

- treated controls. There were no 

### 18

significant differences in pup brain weight or eye opening observed. Likewise, B[a]P treatment of 

### 19

nursing mothers did not affect nest

- building interest or quality, and while not significantly 

### 20

impacting pup retrieval time, the retrieval latency period was observed to increase with increasing 

### 21

treatment duration in both B[a]P groups vs. controls. 

### 22

Behavioral test data was reported graphically, as mean ± SEM of n = 20/group. For the pre



### 23

weaning neuromotor de velopmental tests, B[a]P treatment was found to not depend on sex, and so 

### 24

data from male and female pups was combined. Pups nursing from mothers administered 2 or 20 

### 25

mg/kg

- day B[a]P had significantly elevated righting reflex times at PND3

– 5, which decre ased to 

### 26

control times at PND7

– 9. Only pups from the 20 mg/kg treatment group demonstrated 

### 27

significantly increased negative geotaxis latency, which was 2

- fold greater than controls at PNDs 5, 

### 28

7 and 9, but returned to control levels at PND11. Interesting ly, mice in the 20 mg/kg group had 

### 29

increased forelimb grip strength, which was significantly greater than control mice at PND9 and 11, 

### 30

corresponding to increased body weight in the B[a]P

- treated mice vs. controls. Mice in the 2 mg/kg 

### 31

group also performed better than controls at PND9, but were equivalent at PND11. No treatment or 

### 32

sex

- related effects were reported on locomotion or rearing activity during the open field test. Sex



### 33

dependency on test performance became evident during the analysis of the WESPO C test data: 

### 34

female pups were not significantly affected using any metric, while males in the 20 mg/kg group 

### 35

demonstrated a statistically significantly longer pole

- grasping latency (3

- fold), and took 13

- times 

### 36

longer to escape the pole and board the safety platform, vs. vehicle controls. While performance of 

### 37

male pups from the 2 mg/kg group was not statistically significantly worse than vehicle controls by 

### 38

### Toxicological Review of benzo[a]pyrene

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



### B

- 82 DRAFT

— DO NOT CITE OR QUOTE pair

- wise comparison, latency for both pole

- grasping and escape in this treatment group 

### 1

contributed to a significant trend for treatment dose and these effects. In the evaluation of the 

### 2

elevated plus maze, treatment effects did not appear to depend upon sex, so both male and female 

### 3

performance was analyzed together. Mice in both B[a]P treatment groups dem onstrated 

### 4

significantly decreased latency time to first entering an open arm (30

– 50%), as well as entered 

### 5

open arms 2

- times more frequently and spent twice as much time there vs. vehicle controls. While 

### 6

mice in the 2 mg/kg treatment group entered into c losed arms 20% less frequently than controls, 

### 7

mice in the 20 mg/kg group were not significantly different. Likewise, mice nursing from mothers 

### 8

treated with 2 mg/kg B[a]P performed 15% more spontaneous alternations in the Y

- maze 

### 9

spontaneous alternation tes t compared to controls, while mice in the high

- dose group were not 

### 10

significantly different. The brains of pups nursing from the 20 mg/kg group expressed 

### 11

approximately 50% lower levels of 5

- hydroxytryptamine (serotonin) 1A (5HT1A), and mu 1

- opioid 

### 12

(MOR1) m RNA, and a trend was observed in the low

- dose group as well. No significant changes in 

### 13

alpha

- 1D adrenergic (ADRA1D) or gamma

- aminobutyric acid A (GABAA) mRNA levels were 

### 14

detected. 

### 15

Table B

- 2 9 . Exposure

- related effects in Swiss Albino OF1 mice exposed 16

as pups to benzo[a] pyrene in breast milk from dams treated by gavage 17

daily from PND1

– PND14 18

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).

Reproductive effects in adults and repeated oral exposure 

### 19

Rigdon and Neal (1965) conducted a series of experiments to assess the reproductive 

### 20

effects of orally administered benzo[a]pyrene to Ah

- responsive white Swiss mice. Female animals 

### 21

(number not stated) were administered benzo[a]pyrene at 250, 500, or 1,000 ppm in the feed 

### 22

before or during a 5

- day mating period. Based on t he initial body weight, the doses can be 

### 23

estimated as 32, 56, and 122 mg/kg

- day, respectively. No effect on fertility was observed at any 

### 24

treatment dose, even when animals were fed 1,000 ppm benzo[a]pyrene for 20 days prior to 

### 25

### Toxicological Review of benzo[a]pyrene

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



### B

- 83 DRAFT

— DO NOT CITE OR QUOTE mating, but interpretation o f this finding was marred by large variability in numbers of pregnant 

### 1

females and litter sizes for both treated and control mice. In separate experi ments, the fertility of 

### 2

five male mice/group was not affected by exposure to 1,000 ppm in food for up to 3 0 days prior to 

### 3

mating with untreated females. Histologic examinations showed that male mice fed 500 ppm 

### 4

benzo[a]pyrene for 30 days had spermatozoa present in their testes; further details were not 

### 5

provided. The only treatment

- related effect was a lack o f weight gain related to feed unpalatability. 

### 6

While this study suggests that premating exposure of male or female mice to doses up to 

### 7

122 mg/kg

- day for 20 days may not affect fertility, the sample sizes were too small and study 

### 8

designs were too inconsiste nt to provide reliable NOAELs and LOAELs for 

### 9

reproductive/developmental toxicity. 

### 10

In an earlier study (Rigdon and Rennels, 1964), rats (strain not specified) were fed diets 

### 11

containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and during 

### 12

gestation. In this study, five of eight treated females mated with untreated males became pregnant, 

### 13

but only one delivered live young. The treated dam that delivered had two live and two stillborn 

### 14

pups; one dead pup was grossly malformed. In the remaining treated females, vaginal bleeding was 

### 15

observed on GDs 23 or 24. In the inverse experimental design, three of six controls mated to 

### 16

benzo[a]pyrene

- treated males became pregnant and delivered live young. Visceral and skeletal 

### 17

examinations of the pups were not conducted. These studies are insufficiently reported and of 

### 18

insufficient design (e.g., inadequate numbers of animals for statistical analysis) to provide reliable 

### 19

NOAELs or LOAELs for reproductive effects from repeated oral exposure to b enzo[a]pyrene. 

### 20

Inhalation 

### 21

Reproductive toxicity and in utero exposure via inhalation 

### 22

Archibong et al. (2002) evaluated the effect of exposure to inhaled benzo[a]pyrene on fetal 

### 23

survival and luteal maintenance in timed

- pregnant F344 rats. Prior to exposure on GD 8, 

### 24

laparotomy was performed to determine the number of implantation sites, and confirmed pregnant 

### 25

rats were divided into three groups, consisting of rats that had four to six, seven to nine, or more 

### 26

than nine conceptuses in utero. Rats in these gro ups were then assigned randomly to the treatment 

### 27

groups or control groups to ensure a similar distribution of litter sizes. Animals (10/group) were 

### 28

exposed to benzo[a]pyrene:carbon black aerosols at concentrations of 25, 75, or 100 μg/m 3 via 

### 29

nose

- only inh alation, 4 hours/day on GDs 11

– 20. Control animals were either sham

- exposed to 

### 30

carbon black or remained entirely unexposed. Results of particle size analysis of generated 

### 31

aerosols were reported by several other reports from this laboratory (Inyang et al. , 2003; Ramesh 

### 32

et al., 2001a; Hood et al., 2000). Aerosols showed a trimodal distribution with averages of 95% 

### 33

cumulative mass with diameters <15.85 μm; 89% <10 μm; 55% <2.5 μm; and 38% <1 μm (Inyang 

### 34

et al., 2003). Ramesh et al. (2001a) reported that the (MMAD ± geometric SD) for the 55% mass 

### 35

fraction with diameters <2.5 μm was 1.7 ± 0.085. Progesterone, estradiol

- 17β, and prolactin 

### 36

concentrations were determined in plasma collected on GDs 15 and 17. Fetal survival was 

### 37

### Toxicological Review of benzo[a]pyrene

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



### B

- 84 DRAFT

— DO NOT CITE OR QUOTE calculated as the total number of pups divided by the number of all implantation sites determined 

### 1

on GD 8. Individual pup weights and crown

- rump length per litter per treatment were determined 

### 2

on PND 4 (PND 0 = day of parturition). 

### 3

Archibong et al. (2002) reported that exposure of rats to benzo[a]pyrene caused 

### 4

biologically and statistically significant ( p ≤ 0.05) reductions in fetal survival compared with the 

### 5

two control groups; fetal survival rates were 78.3, 38.0, and 33.8% per litter at 25, 75, and 

### 6

100 μg/m 3 , respectively, and 96.7% wit h carbon black or 98.8% per litter in untreated controls (see 

### 7

Table 4

- 24). Consequently, the number of pups per litter was also decreased in a concentration



### 8

dependent manner. The decrease was ~50% at 75 μg/m 3 and ~65% at 100 μg/m 3 , compared with 

### 9

sham

- exp osed and unexposed control groups. No effects on hormone levels were observed on 

### 10

GDs 15 or 17 at the low

- dose. Biologically significant decreases in mean pup weights (expressed as 

### 11

g per litter) of >5% were observed at doses ≥75 μg/m 3 (14 and 16% decrease s at 75 and 100 

### 12

μg/m 3 , respectively, p < 0.05). Exposure to benzo[a]pyrene did not affect crown

- rump length (see 

### 13

Table B

- 30 ). 

### 14

Table B

- 30 . Pregnancy outcomes in female F344 rats treated with 15

benzo[a]pyrene on GDs 11

– 21 by inhalation 16

Parameter a Administere d concentration of benzo[a]pyrene (μg/m 3 )

0 (unexposed

control) 0

(carbon black) 25 75 100

Implantation sites 8.6 ± 0.2 8.8 ± 0.1 8.8 ± 0.5 9.0 ± 0.2 8.8 ± 0.1

Pups per litter 8.5 ± 0.2 8.7 ± 0.2 7.4 ± 0.5 b 4.2 ± 0.1 b 3.0 ± 0.2 b

Survival (litter %) 98.9 ± 1.1 96.7 ± 1.7 78.3 ± 4.1 b 38.0 ± 2.1 b 33.8 ± 1.3 b

Pup weight (g/litter) 10.6 ± 0.1 8.8 ± 0.1 10.5 ± 0.2 9.1 ± 0.2 b 8.9 ± 0.1 b

Crown

- rump length

(mm/litter) 29.4 ± 0.6 29.3 ± 0.5 28.0 ± 0.6 27.3 ± 0.7 27.9 ± 0.7

a Values presented as means ± SEM.

b Significantly different from controls at p < 0.05 by one

- tailed post

- hoc t

- testing following ANOVA.

Source: Archibong et al. (2002).



### 17

Benzo[a]pyrene exposure at 75 μg/m 3 caused a statistically significant decrease in plasma 

### 18

progesterone, estradiol, and prolactin on GD 17; these levels were not determined in the rats 

### 19

exposed to 100 μg/m 3 (Archibong et al., 2002). Plasma prolactin is an indirect measure of the 

### 20

activity of decidual luteotropin, a prolactin

- like hormone whose activity is necessary for luteal 

### 21

maintenance during pregnancy in rats. Control levels of prolactin increased from GD 15 to 17, but 

### 22

this increase did not occur in the rats exposed to 75 μg/m 3 . Although the progesterone 

### 23

concentration at 75 μg/m 3 was significantly lower than in controls on GD 17, the authors thought 

### 24

that the circulating levels should have been sufficient to maintain pregnancy; thus, the increased 

### 25

### Toxicological Review of benzo[a]pyrene

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— DO NOT CITE OR QUOTE loss of fetuses was thought to be caused by the lower prolactin levels rather than progesterone 

### 1

deficiency. The reduced circulating levels of progesterone and estradiol

- 17β among 

### 2

benzo[a]pyrene

- treated rats were thought to be a result of limited decidual luteotropic support for 

### 3

the corpora lutea. The authors proposed the following mechanism for the effects of benzo[a]pyrene 

### 4

on fertility: benzo[a]pyrene or its metabolites decreased prolactin and decidual luteotropin levels, 

### 5

compromising the luteotropic support for the corpora lutea and thereby dec reasing the plasma 

### 6

levels of progesterone and estradiol

- 17β. The low estradiol

- 17β may decrease uterine levels of 

### 7

progesterone receptors, thereby resulting in fetal mortality. Based on biologically and statistically 

### 8

significant decreases in pups/litter a nd percent fetal survival/per litter, the LOAEL was 25 μg/m 3 ; 

### 9

no NOAEL was identified. 

### 10

Neurotoxicity and in utero exposure via inhalation 

### 11

To evaluate the effects of benzo[a]pyrene on the developing CNS, Wormley et al. (2004) 

### 12

exposed t imed

- pregnant F344 rat s (10/group) to benzo[a]pyrene:carbon black aerosols by nose



### 13

only inhalation on GDs 11

– 21 for 4 hours/day at a concentration of 100 μg/m 3 . Results of particle 

### 14

size analysis of genenerated aerosols were reported by other reports from this laboratory (Rames h 

### 15

et al., 2001a; Hood et al., 2000). Particle size analysis of a 100

- μg/m 3 aerosol showed a trimodal 

### 16

distribution with averages of 95% cumulative mass with diameters <15.85 μm; 90% <10 μm; 

### 17

67.5% <2.5 μm; and 66.2% <1 μm; the MMAD ± geometric SD for the la tter fraction was 0.4 ± 0.02 

### 18

μm (Hood et al., 2000). Dams were maintained to term and pups were weaned on PND 30. 

### 19

Benzo[a]pyrene reduced the number of live pups to one

- third of control values without affecting 

### 20

the number of implantation sites. During PN Ds 60

– 70, electrical stimulation and evoked field 

### 21

potential responses were recorded via electrodes implanted into the brains of the animals. Direct 

### 22

stimulation of perforant paths in the entorhinal region revealed a diminution in long

- term 

### 23

potentiation of population spikes across the perforant path

- granular cell synapses in the dentate 

### 24

gyrus of the hippocampus of F1 generation benzo[a]pyrene

- exposed animals; responses in exposed 

### 25

offspring were about 25% weaker than in control offspring. Additionally, NMDA receptor subunit 1 

### 26

protein (important for synaptic functioning) was down

- regulated in the hippocampus of 

### 27

benzo[a]pyrene exposed F1 pups. The authors interpreted their results as suggesting that 

### 28

gestational exposure to benzo[a]pyrene inhalation attenuates the capacity for long

- term 

### 29

potentiation (a cellular correlate of learning and memory) in the F1 generation. 

### 30

In another study by this same group of investigators, Wu et al. (2003) evaluated the 

### 31

generation of benzo[a]pyrene metabolites in F1 generation pups, as well as the developmental 

### 32

profile for AhR and mRNA. In this study, confirmed pregnant F344 rats were exposed to 

### 33

benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 μg/m 3 via nose

- only inhalation, 

### 34

4 hours/day, for 10 days (GDs 11

– 21). Control anima ls were exposed to carbon black (sham) to 

### 35

control for inert carrier effects or they remained untreated. Each benzo[a]pyrene concentration 

### 36

had its own set of controls (carbon black and untreated). Two randomly selected pups were 

### 37

sacrificed on each of PND 0, 3, 5, 10, 15, 20, and 30. Body, brain, and liver weights were recorded. 

### 38

### Toxicological Review of benzo[a]pyrene

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— DO NOT CITE OR QUOTE Benzo[a]pyrene metabolites were analyzed in the cerebral cortex, hippocampus, liver, and plasma. 

### 1

A dose

- related increase in plasma and cortex benzo[a]pyrene metabolite concentrat ions in pups 

### 2

was observed. Dihydrodiols (4,5

- ; 7,8

- ; 9,10

- ) dominated the metabolite distribution profile up to 

### 3

PND 15 and the hydroxy (3

- OH

- benzo[a]pyrene; 9

- OH

- benzo[a] pyrene) metabolites after PND 15 

### 4

at 100 μg/m 3 (the only exposure concentration repor ted). Results indicated a dose

- related 

### 5

decrease in the ratio of the total number of pups born per litter to the total number of implantation 

### 6

sites per litter. The number of resorptions at 75 and 100 μg/m 3 , but not at 25 μg/m 3 , was 

### 7

statistically significa ntly increased compared with controls. 

### 8

Adult male reproductive effects and repeated inhalation exposure 

### 9

Inyang et al. (2003) evaluated the effect of subacute exposure to inhaled benzo[a]pyrene on 

### 10

testicular steroidogenesis and epididymal function in rats. Male F344 rats (10/group), 13 weeks of 

### 11

age, were exposed to benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 μg/m 3 via nose

- only 

### 12

inhalation, 4 hours/day for 10 days. Control animals were either exposed to carbon black (sham) to 

### 13

control for exposure to the inert carrier, or they remained untreated. Each benzo[a]pyrene 

### 14

concentration had its own set of controls (carbon black and untreated). Aerosols showed a 

### 15

trimodal distribution with averages of 95% cumulative mass <15.85 μm; 89% <10 μm; 55% <2.5 

### 16

μm ; and 38% <1 μm (Inyang et al., 2003); an earlier report from this laboratory indicated that the 

### 17

55% mass fraction had a MMAD ± geometric SD of 1.7 ± 0.085 (Ramesh et al., 2001a). Blood 

### 18

samples were collected at 0, 24, 48, and 72 hours after cessation of exposure to assess the effect of 

### 19

benzo[a]pyrene on systemic concentrations of testosterone and luteinizing hormone (LH), 

### 20

hormones that regulate testosterone synthesis. Reproductive endpoints such as testis weight and 

### 21

motility and density of stored (epidid ymal) sperm were evaluated. 

### 22

Regardless of the exposure concentration, inhaled benzo[a]pyrene did not affect testis 

### 23

weight or the density of stored sperm compared with controls. However, inhaled benzo[a]pyrene 

### 24

caused a concentration

- dependent reduction in the progressive motility of stored sperm. 

### 25

Progressive motility was similar at 75 and 100 μg/m 3 , but these values were significantly lower ( p < 

### 26

0.05) than in any other group. The reduction in sperm motility postcessation of exposure was 

### 27

thought to be the result of benzo[a]pyrene limiting epididymal function. Benzo[a]pyrene exposure 

### 28

to 75 μg/m 3 caused a decrease in circulating concentrations of testosterone compared with controls 

### 29

from the time of cessation of exposure (time 0) to 48 hours posttermination o f exposure ( p < 0.05). 

### 30

However, the decrease was followed by a compensatory increase in testosterone concentration at 

### 31

72 hours postcessation of exposure. Exposure to 75 μg/m 3 caused a nonsignificant increase in 

### 32

plasma LH concentrations at the end of expo sure compared with controls, which increased further 

### 33

and turned significant ( p < 0.05) for the remaining time of the study period. The decreased plasma 

### 34

concentration of testosterone, accompanied by an increased plasma LH level, was thought to 

### 35

indicate tha t benzo[a]pyrene did not have a direct effect on LH. The authors also noted that the 

### 36

decreased circulating testosterone may have been secondary to induction of liver CYP450 enzymes 

### 37

by benzo[a]pyrene. The authors concluded that subacute exposure to benzo[ a]pyrene contributed 

### 38

### Toxicological Review of benzo[a]pyrene

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### B

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— DO NOT CITE OR QUOTE to impaired testicular endocrine function that ultimately impaired epididymal function. Based on 

### 1

this study, the NOAEL was 25 μg/m 3 and the LOAEL was 75 μg/m 3 , based on a statistically 

### 2

significant reduction in the progressive motility of stored sperm and impairment of testicular 

### 3

function with 10 days of exposure at 75 μg/m 3

. 

### 4

In a follow

- up study with longer exposure duration, adult male F344 rats (10 per group) 

### 5

were exposed to benzo[a]pyrene:carbon black aerosols at 75 μg/m 3 via nose

- o nly inhalation, 

### 6

4 hours/day for 60 days (Archibong et al., 2008; Ramesh et al., 2008). Rats in the control group 

### 7

were subjected to the nose

- only restraint, but were not exposed to the carbon black carrier. Blood 

### 8

samples were collected at 0, 24, 48, and 7 2 hours after exposure terminated, and the animals 

### 9

sacrificed for tissue analyses following the last blood sampling. Data were analyzed statistically for 

### 10

benzo[a]pyrene effects on weekly body weights, total plasma testosterone and LH concentrations, 

### 11

testi s weights, density of stored spermatozoa, sperm morphological forms and motility, 

### 12

benzo[a]pyrene metabolite concentrations and AHH activity, and morphometric assessments of 

### 13

testicular histologies. Relative to controls, the results indicated 34% reduced te stis weight ( p < 

### 14

0.025), reduced daily sperm production ( p < 0.025) and reduced intratesticular testosterone 

### 15

concentrations ( p < 0.025). Plasma testosterone concentrations were reduced to about one

- third of 

### 16

the level in controls on the last day of exposur e (day 60) and at 24, 48, and 72 hours later ( p < 0.05). 

### 17

However, plasma LH concentrations in benzo[a]pyrene exposed rats were elevated throughout the 

### 18

blood sampling time periods compared with controls ( p < 0.05). In testis, lung, and liver, AHH 

### 19

activity, and benzo[a]pyrene

- 7,8

- di hydrodiol (precursor to the DNA

- reactive BPDE) and 

### 20

benzo[a]pyrene

- 3,6

- dione metabolites were significantly ( p < 0.05) elevated relative to controls. 

### 21

Progressive motility and mean density of stored spermatozoa were significantly reduced ( p < 0.05). 

### 22

Weekly body weight gains were not affected by benzo[a]pyrene exposure. These results indicate 

### 23

that 60

- day exposure of adult male rats to benzo[a]pyrene: carbon black aerosols at 75 μg/ m 3 

### 24

produced decreased testis weight; decreased intratesticular and plasma testosterone 

### 25

concentrations; and decreased sperm production, motility, and density. 

### 26

### Toxicological Review of benzo[a]pyrene

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### B

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— DO NOT CITE OR QUOTE OTHER PERTINENT TOXICITY INFORMATION 1

Table B

- 31 . In vitro genotoxicity studies of benzo[a]pyrene in non

2

mammal ian cells 3

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

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### B

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— 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



### Table B

- 32 . In vitro genotoxicity studies of benzo[a]pyrene in 1

mammalian cells 2

Assay

/test system Result

Reference +S9

– S9

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

Reference +S9

– S9

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 Muta TM Mouse

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/Oua R 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

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### B

- 91 DRAFT

— DO NOT CITE OR QUOTE Assay /test system Result

Reference +S9

– S9

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

Reference +S9

– S9

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

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### B

- 93 DRAFT

— DO NOT CITE OR QUOTE Assay /test system Result

Reference +S9

– S9

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

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### B

- 94 DRAFT

— DO NOT CITE OR QUOTE Assay /test system Result

Reference +S9

– S9

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/Oua R ND + Mishra et al., 1978

+ = positive;

– = negative; ND = not determined; SHE = Syrian hamster embryo; TK

= thymidine kinase



### 1

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### B

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Table B

- 33 . In vivo genotoxicity studies of benzo[a]pyrene 2

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 wit h 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

e xperiment; fertilized eggs collected

from 9 to 11 hrs after mating and first

cleavage 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

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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 BPDE

DNA

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 LC

MS/MS SRM with 15 N

- 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

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### B

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Mutations

and BPDE

DNA

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 32 P

postlabeling. + 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 BPDE

DNA

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 b y [ 32 P]

- 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 near

midline 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 vent ral

spots. Russell,

1977

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### B

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Mutation Mouse, lacZ transgenic

(Muta TM Mouse) 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

(Muta TM Mouse) 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 dose

dependent 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 h prt

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; h prt mutations

significantly higher in Csb

- /

-

mice than control mice. BPDE

dGuo adducts in hprt gene are

preferentially removed in WT

mice than Csb

-

/

- mice. Wijnhoven

et al., 2000

### Toxicological Review of benzo[a]pyrene

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### B

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Mutation Mouse, B6C3F 1 ,

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

(Muta TM 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 dose

and 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

h prt 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

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### B

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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 β

glucuronidase. + 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

BPDE

DNA

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 95 th

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

BPDE

DNA

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

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### B

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BPDE

DNA

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 anti

BPDE

- 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 PAH

rich meals (>52 versus <52

times/year), and long time

periods spent outdo ors (>4

versus <4 hours/day) were risk

factors as all increased BPDE

DNA adduct levels significantly. Pavanello

et al., 2006

BPDE

DNA

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

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### B

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BPDE

DNA

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/m 3 of

benzo[a]pyrene, respectively. + <0.15, 0.15

– 4,

or >4 μg/m 3 of

benzo[a]pyren

e PAH exposure, CYP1A1 status

and smoking significantly

affected DNA adduct levels,

i.e., CYP1A1 ( *1/*2 or *2A/*2 a)

> CYP1A1 * 1/*1 ; occupational >

environmental exposure;

smokers > nonsmokers;

adducts increased with dose

and duration of smoking. Rojas et

al., 2000

BPDE

DNA

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/m 3 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 nons mokers; no

correlation with air

benzo[a]pyrene levels and

adduct levels. Mensing et

al., 2005

BPDE

DNA

adducts Mouse, lacZ transgenic

(Muta TM Mouse) 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

32 P

- 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

BPDE

DNA

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

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### B

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BPDE

DNA

adducts Mouse, C57BL/6J

Cyp1a1 (+/

- ) and

Cyp1a1 (

- /

- ) Single i.p. injection; sacrificed 24 hrs

posttreatment; liver DNA analyzed by

[ 32 P]

- postlabeling assay. + 500 mg/kg BPDE

- DNA adduct levels

fourfold higher in Cyp1a1 (

- /

- )

mice than Cyp1a1 (+/

- ) mice. Uno et al.,

2001

BPDE

DNA

adducts Mouse, B6C3F 1 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 [ 32 P]

- 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

BPDE

DNA

adducts Mouse, BALB/c Single i.p. injection; sacrificed 12 hrs

postinjection; liver and forestomach

collected; DNA binding of [ 3 H]

- 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

BPDE

DNA

adducts Mouse, BALB/cAnN

(BALB), CBA/JN (CBA);

[ 32 P]

- 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

BPDE

DNA

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

[ 32 P]

- postlabeling assay. + 4 × 1.2 μmol/

animal Five adducts spots detected. Reddy et

al., 1984

### Toxicological Review of benzo[a]pyrene

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### B

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BPDE

DNA

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

BPDE

DNA

adducts Rat, CD, peripheral

blood lymphocytes,

lungs, and liver Single i.p. injection; sacrificed 3 d

posttreatment; DNA analyzed by

Nuclease P1

- endhanced [ 32 P]

- postlabeling assay. + 2.5 mg/animal BPDE

- dG as major adducts and

several minor adducts

detected in all tissues. Ross et al.,

1991

BPDE

DNA

adducts Rat, Sprague

- Dawley,

liver Single i.p. injection followed by

sacrifice at 4 hrs posttreatment; liver

DNA isolated and analyzed by [ 32 P]

- postlabeling assay. + 100 mg/kg Two adduct spots detected. Reddy et

al., 1984

BPDE

DNA

adducts Rat, Lewis, lung and

liver Animals received a single oral dose of

benzo[a]pyrene in tricaprylin;

sacrificed 1, 2, 4, 11, and 21 d

postdosing; analyzed liver and lung

DNA for BP

- DNA adducts by [ 32 P]

- postlabeling assay and urine for

8

- oxodG adducts by HPLC

electrochemical 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

BPDE

DNA

adducts Rat, F344;

[ 32 P]

- 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

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### B

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BPDE

DNA

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 [ 32 P]

- 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

b one 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

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### B

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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

(Muta TM Mouse) 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, dose

dependent increases in % PCEs

and NCEs at all doses. Lemieux et

al., 2011

MN Mouse, B6C3F 1

(hybrid) i.p. injection; several doses given to

calculate LD

50

. + 232 mg/kg

(LD 50/7 );

259 mg/kg

(LD 50/4 ) Study conducted to determine

the toxicity of benzo[a]pyrene

(LD

50 ). 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. Dose

dependent 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

collec ted; controls received normal

saline instillation; all cells analyzed by

comet assay. + Experiment

#1: 3 mg of

benzo[a]pyren

e; Experiment

#2: dose

response

study with

0.75, 1.5, or 3

mg

benzo[a]pyren

e All time points showed

significant increase in SSBs

(Experiment #1); a dose

response 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 liv er (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 time

dependent 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 [ 3 H]

- 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

[ 3 H]thymidine; cultured; epidermal

UDS measured. + 0, 0.25, 0.5,

and 1% (w/v)

in acetone UDS index showed a dose

dependent 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;

[ 3 H]

- 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 vehicle

treated controls. Roszinsky

Kocher 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 (AHH

inducible) < DBA/2 (AHH

noninducible). 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 (AHH

inducible) < DBA/2 (AHH

noninducible). 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

### Toxicological Review of benzo[a]pyrene

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



### B

- 17 DRAFT

— DO NOT CITE OR QUOTE Endpoint Test system Test conditions Results Dose Comment Reference

Mutation Drosophila

melanogaster, sex

linked recessive lethal

test Basc males exposed to benzo[a]pyrene

were mated with virgin females of

Berlin K or mei

- 9 L1 strains. ± 10 mM Data inconclusive due to low

fertility rates of mei

- 9 L1

females. Vogel et

al., 1983

Mutation D. melanogaster , sex

linked 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 , sex

linked 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 dose

response observed. Fahmy and

Fahmy,

1980

### Toxicological Review of benzo[a]pyrene

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



### B

- 18 DRAFT

— DO NOT CITE OR QUOTE Endpoint Test system Test conditions Results Dose Comment Reference

Cell trans

formation Hamster, LVG:LAK

strain (virus free);

transplacental host

mediated assay Pregnant animals dosed i.p. with

benzo[a]pyrene on GD 10; sacrificed

on GD 13, fetal cell cultures prepared,

10 × 10 6 cells/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

### 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 Tumor Promotion and Progression 

### 1

Cytotoxicity and inflammatory response 

### 2

The cytotoxicity of benzo[a]pyrene metabolites may contribute to tumor promotion via 

### 3

inflammatory responses leading to cell proliferation (Burdick et al., 2003). Benzo[a]pyrene is 

### 4

metabolized to o

- quinones, which are cytotoxic, and can generate ROS (Bolton et al., 2000; Penning, 

### 5

1999). Benzo[a]pyrene o

- quinones reduce the viability and survival of rat and human hepatoma 

### 6

cells (Flowers

- Geary et al., 1996, 1993). Cytotoxicity was also induced by benzo[a]pyrene and 

### 7

BPDE in a human prostate carcinoma cell line (Nwagbara et al., 2007). Inflammatory responses to 

### 8

cytotoxicity may contribu te to the tumor promotion process. For example, benzo[a]pyrene 

### 9

quinones (1,6

- , 3,6

- , and 6,12

- benzo[a]pyrene

- quinone) generated ROS and increased cell 

### 10

proliferation by enhancing the epidermal growth factor receptor pathway in cultured breast 

### 11

epithelial ce lls (Burdick et al., 2003). 

### 12

Several studies have demonstrated that exposure to benzo[a]pyrene increases the 

### 13

production of inflammatory cytokines, which may contribute to cancer progression. Garçon et al. 

### 14

(2001a, b) exposed Sprague

- Dawley rats by inhalati on to benzo[a]pyrene with or without ferrous 

### 15

oxide (Fe 2 O 3 ) particles. They found that benzo[a]pyrene alone or in combination with Fe 2 O 3 

### 16

particles elicited mRNA and protein synthesis of the inflammatory cytokine, IL

- 1. Tamaki et al. 

### 17

(2004) also demonstrated a benzo[a]pyrene

- induced increase in IL

- 1 expression in a human 

### 18

fibroblast

- like synoviocyte cell line (MH7A). Benzo[a]pyrene increas es the expression of the mRNA 

### 19

for CCL1, an inflammatory chemokine, in human macrophages (N’Diaye et al., 2006). The 

### 20

benzo[a]pyrene

- induced increase in CCL1 mRNA was inhibited by the potent AhR antagonist, 

### 21

3 ′

- methoxy

- 4 ′

- nitroflavone. 

### 22

AhR

- mediated effects 

### 23

The promotional effects of benzo[a]pyrene may also be related to AhR affinity and the 

### 24

upregulation of genes related to biotransformation (i.e., induction of CYP1A1), growth, and 

### 25

differentiation (Bostrom et al., 2002). Figure B

- 3 illustrates the function o f the AhR and depicts the 

### 26

genes regulated by this receptor as belonging to two major functional groups (i.e., induction of 

### 27

metabolism or regulation cell differentiation and proliferation). PAHs bind to the cytosolic AhR in 

### 28

complex with heat shock protein 90 (Hsp90). The ligand

- bound receptor is then transported to 

### 29

nucleus in complex with the Ah receptor nuclear translocator. The AhR complex interacts with the 

### 30

Ah responsive elements of the DNA to increase the transcription of proteins associated with 

### 31

indu ction of metabolism and regulation of cell differentiation and proliferation. 

### 32

### 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 1

AHRE DNA = Ah

- responsive elements of DNA; ARNT = Ah receptor nuclear translocator; Hsp90 = heat 

### 2

shock protein 90 

### 3

Sourc e: Okey et al. (1994). 

### 4

5

Figure B

- 3 . Interaction of PAHs with the AhR. 

### 6

7

Binding to the AhR induces enzymes that increase the formation of reactive metabolites, 

### 8

resulting in DNA binding and, eventually, tumor initiation. In addition, with persistent exposure, 

### 9

the ligand

- activated AhR triggers epithelial hyperplasia, which provides the second step leading 

### 10

from tumor initiation to promotion and progression (Nebert et al., 1993). Ma and Lu (2007) 

### 11

reviewed several studies of benzo[a]pyrene toxicity and t umorigenicity in mouse strains with high 

### 12

and low affinity AhRs. Disparities were observed in the tumor pattern and toxicity of 

### 13

Ah

- responsive (+/+ and +/

- ) and Ah

- nonresponsive (

- /

- ) mice. Ah

- responsive mice were more 

### 14

susceptible to toxicity and tumorigen icity in proximal target tissues such as the liver, lung, and skin. 

### 15

For example, Shimizu et al. (2000) reported that AhR knock

- out mice (

- /

- ), treated with 

### 16

benzo[a]pyrene by s.c. injection or dermal painting, did not develop skin cancers at the treatment 

### 17

site, while AhR

- responsive (+/+) or heterozygous (+/

- ) mice developed tumors within 18

– 25 weeks 

### 18

after treatment. Benzo[a]pyrene treatment increased CYP1A1 expression in the skin and liver of 

### 19

AhR

- positive mice (+/

- or +/+), but CYP1A1 expression was not al tered by benzo[a]pyrene 

### 20

### 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 treatment in AhR knock

- out mice (

- /

- ). Talaska et al. (2006) also showed that benzo[a]pyrene 

### 1

adduct levels in skin were reduced by 50% in CYP1A2 knock

- out mice and by 90% in AhR knock



### 2

out mice compared with WT C57Bl6/J mice followi ng a single dermal application of 33 mg/kg 

### 3

benzo[a]pyrene for 24 hours. Ma and Lu (2007) further noted that Ah

- non responsive mice were at 

### 4

greater risk of toxicity and tumorigenicity in remote organs, distant from the site of exposure (i.e., 

### 5

bone marrow). As an example, Uno et al. (2006) showed that benzo[a]pyrene (125 mg/kg

- day, p.o. 

### 6

for 18 days) caused marked wasting, immunosuppression, and bone marrow hypocellularity in 

### 7

CYP 1A1 knock

- out mice, but not in WT mice. 

### 8

Some studies have demonstrated the form ation of DNA adducts in the liver of AhR knock



### 9

out mice following i.p. or oral exposure to benzo[a]pyrene (Sagredo et al., 2006; Uno et al., 2006; 

### 10

Kondraganti et al., 2003). These findings suggest that there may be alternative (i.e., non

- AhR 

### 11

mediated) mec hanisms of benzo[a]pyrene activation in the mouse liver. Sagredo et al. (2006) 

### 12

studied the relationship between the AhR genotype and CYP metabolism in different organs of the 

### 13

mouse . AhR +/+

, +/

– , and

– /

– mice were treated once with 100 mg/kg benzo[a]pyren e by gavage. 

### 14

CYP1A1, CYP1B1, and AhR expression was evaluated in the lung, liver, spleen, kidney, heart, and 

### 15

blood, via real

- time or reverse transcriptase polymerase chain reaction, 24 hours after treatment. 

### 16

CYP1A1 RNA was increased in the lung and live r and CYP1B1 RNA was increased in the lung 

### 17

following benzo[a]pyrene treatment in AhR +/+ and +/

– mice (generally higher in heterozygotes). 

### 18

Benzo[a]pyrene treatment did not induce CYP1A1 or CYP1B1 enzymes in AhR

– /

– mice. The 

### 19

expression of CYP1A1 RNA, as st andardized to β

- actin expression, was generally about 40 times 

### 20

that of CYP1B1. The concentration of benzo[a]pyrene metabolites and the levels of DNA and 

### 21

protein adducts were increased in mice lacking the AhR, suggesting that there may be an 

### 22

AhR

- independen t pathway for benzo[a]pyrene metabolism and activation. The high levels of 

### 23

benzo[a]pyrene DNA adducts in organs other than the liver of AhR

– /

– mice may be the result of slow 

### 24

detoxification of benzo[a]pyrene in the liver, allowing high concentrations of th e parent compound 

### 25

to reach distant tissues. 

### 26

Uno et al. (2006) also demonstrated a paradoxical increase in liver DNA adducts in AhR 

### 27

knock

- out mice following oral exposure to benzo[a]pyrene. WT C57BL/6 mice and several knock



### 28

out mouse strains (CYP1A2

– /

– an d CYP1B1

– /

– single knock

- out, CYP1A1/1B1

– /

– and CYP1A2/1B1

– /

– 

### 29

double knock

- out) were studied. Benzo[a]pyrene was administered in the feed at 1.25, 12.5, or 125 

### 30

mg/kg for 18 days (this dose is well tolerated by WT C57BL/6 mice for 1 year, but lethal within 30 

### 31

days to the CYP1A1

– /

– mice). Steady

- state blood levels of benzo[a]pyrene, reached within 5 days of 

### 32

treatment, were ~25 times higher in CYP 1A1

– /

– and ~75 times higher in CYP 1A1/1B1

– /

– than in WT 

### 33

mice, while clearance was similar to WT mice in the other knock

- out mouse strains. DNA adduct 

### 34

levels, measured by [ 32 P]

- postlabeling in liver, spleen, and bone marrow, were highest in the 

### 35

CYP1A1

– /

– mice at the two higher doses, and in the CYP1A1/1B1

– /

– mice at the mid dose only. 

### 36

Adduct patterns, as revealed by 2

- dimensional chromatography, differed substantially between 

### 37

organs in the various knock

- out types. 

### 38

### 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 Dertinger et al. (2001, 2000) demonstrated that AhR signaling may play a role in 

### 1

cytogenetic damage caused by benzo[a]pyrene. The in vivo formation of MN in peripheral blood 

### 2

reticulocytes of C57Bl/6J mice induced by a single i.p. injection of benzo[a]pyrene (150 mg/kg) was 

### 3

eliminated by prior treatment with the potent AhR antagonist 3'

- methoxy

- 4'

- nitroflavone. This 

### 4

antagonist also protected AhR null allel e mice from benzo[a]pyrene

- induced increases in MN 

### 5

formation, suggesting that 3'

- methoxy

- 4'

- nitroflavone may also act through a mechanism 

### 6

independent of the AhR (Dertinger et al., 2000). 

### 7

Several in vitro studies have suggested that the AhR plays a role in the disruption of cell 

### 8

cycle control, possibly leading to cell proliferation and tumor promotion following exposure to 

### 9

benzo[a]pyrene (Andrysik et al., 2007; Chung et al., 2007; Chen et al., 2003). Chung et al. (2007) 

### 10

showed that benzo[a]pyrene

- induced c ytotoxicity and apoptosis in mouse hepatoma (Hepa1c1c7) 

### 11

cells occurred through a p53 and caspase

- dependent process requiring the AhR. An accumulation 

### 12

of cells in the S

- phase of the cell cycle (i.e., DNA synthesis and replication) was also observed, 

### 13

sugges ting that this process may be related to cell proliferation. Chen et al. (2003) also 

### 14

demonstrated the importance of the AhR in benzo[a]pyrene

- 7,8

- dihydrodiol

- and BPDE

- induced 

### 15

apoptosis in human HepG2 cells. Both the dihydrodiol and BPDE affected Bcl2 (a member of a 

### 16

family of apoptosis suppressors) and activated caspase and p38 mitogen

- activated protein (MAP) 

### 17

kinases, both enzymes that promote apoptosis. When the experiments were conducted in a cell line 

### 18

that does not contain Ah receptor nuclear transloc ator (see Figure 4

- 1), the dihydrodiol was not 

### 19

able to initiate apoptotic event sequences, indicating that activation to BPDE by CYP1A1 was 

### 20

required. BPDE did not induce apoptosis

- related events in a p38

- defective cell line, illustrating the 

### 21

importance of MAP kinases in this process. In rat liver epithelial cells (WB

- F344 cells), in vitro 

### 22

exposure to benzo[a]pyrene resulted in apoptosis, a decrease in cell number, an increase in the 

### 23

percentage of cells in S

- phase (comparable to a proliferating population of WB

- F334 cells), and 

### 24

increased expression of cell cycle proteins (e.g., cyclin A) (Andrysik et al., 2007). Benzo[a]pyrene



### 25

induced apoptosis was attenuated in cells transfected with a dominant

- negative mutation of the 

### 26

AhR. 

### 27

Inhibition of gap junctional in tercellular communication (GJIC) 

### 28

Gap junctions are channels between cells that allow substances of a molecular weight up to 

### 29

roughly 1 kDa to pass from one cell to the other. This process of metabolic cooperation is crucial 

### 30

for differentiation, proliferation, apoptosis, and cell death and consequently for the two epigenetic 

### 31

steps of tumor formation, promotion, and progression. Chronic exposure to many toxicants results 

### 32

in down

- regulation of gap junctions. For tumor promoters, such as TPA or TCD D, inhibition of 

### 33

intercellular communication is correlated with their promoting potency (Sharovskaya et al., 2006; 

### 34

Yamasaki, 1990). 

### 35

Blaha et al. (2002) surveyed the potency of 35 PAHs, including benzo[a]pyrene, to inhibit 

### 36

GJIC. The scrape loading/dye tra nsfer assay was employed using a rat liver epithelial cell line that 

### 37

was incubated in vitro for 15, 30, or 60 minutes with 50 μM benzo[a]pyrene. After incubation, cells 

### 38

### 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 were washed, and then a line was scraped through the cells with a surgical blade. Cel ls were 

### 1

exposed to the fluorescent dye lucifer yellow for 4 minutes and then fixed with formalin. Spread of 

### 2

the dye from the scrape line into cells remote from the scrape was estimated under a fluorescence 

### 3

microscope. Benzo[a]pyrene reduced spread of the dye after 30 minutes of exposure 

### 4

(approximately 50% of control). Recovery of GJIC was observed 60 minutes after exposure. 

### 5

Sharovskaya et al. (2006) studied the effects of carcinogenic and noncarcinogenic PAHs on 

### 6

GJIC in HepG2 cells. Individual carcinog enic PAHs inhibited GJIC in a temporary fashion (70

– 100% 

### 7

within 24 hours), but removal of the PAH from culture reversed the effect. Noncarcinogenic PAHs 

### 8

had very little effect on GJIC. Benzo[a]pyrene at 20 μM inhibited GJIC completely within 24 hours, 

### 9

wh ile its noncarcinogenic homolog, benzo[e]pyrene, produced <20% inhibition. The effect was not 

### 10

AhR

- dependent, because benzo[a]pyrene inhibited GJIC in HepG2 cells to the same extent as in 

### 11

hepatoma G27 cells, which express neither CYP1A1 nor AhR. The autho rs concluded that the 

### 12

effects of benzo[a]pyrene and benzo[e]pyrene on GJIC were direct (i.e., not caused by metabolites). 

### 13

### Toxicological Review of benzo[a]pyrene

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



### C

- 1 DRAFT

— DO NOT CITE OR QUOTE 

# APPENDIX C. DOSE

- RESPONSE MODELING FOR 1



# THE DERIVATION OF REFERENCE VALUES FOR 2



# EFFECTS OTHER THAN CANCER AND THE 3



# DERIVATION OF C ANCER RISK ESTIMATES 4

This appendix provides technical detail on dose

- response evaluation and determination of 

### 5

points of departure (POD) for relevant toxicological endpoints. Except where other software is 

### 6

noted, all endpoints were modeled using the U.S. E PA’s Benchmark Dose Software (BMDS ; U.S. EPA, 

### 7

2012; version 2. 0 or later ). The preambles for the cancer and non

- cancer parts below describe the 

### 8

common practices used in evaluating the model fit and selecting the appropriate model for 

### 9

determining the POD, as outlined in the draft Benchmark Dose Technical Guidance Document (U.S. 

### 10

EPA, 20 00 ). 

### 11

DOSE

- RESPONSE MODELING FOR DERVIATION OF RFD 12

Evaluation of Model Fit 

### 13

For each dichotomous endpoint, BMDS dichotomous models were fitted to the data using 

### 14

the maximum li kelihood method. Each model was tested for goodness

- of

- fit using a chi

- square 

### 15

goodness

- of

- fit test (χ 2 p

- value < 0.10 indicates lack of fit). Other factors were also used to assess 

### 16

model fit, such as scaled residuals, visual fit, and adequacy of fit in t he low

- dose region and in the 

### 17

vicinity of the BMR. 

### 18

For each continuous endpoint, BMDS continuous models were fitted to the data using the 

### 19

maximum likelihood method. Model fit was assessed by a series of tests as follows. For each model, 

### 20

first the homog eneity of the variances was tested using a likelihood ratio test (BMDS Test 2). If Test 

### 21

2 was not rejected (χ 2 p

- value ≥ 0.10), the model was fitted to the data assuming constant variance. 

### 22

If Test 2 was rejected (χ 2 p

- value < 0.10), the variance was modeled as a power function of the 

### 23

mean, and the variance model was tested for adequacy of fit using a likelihood ratio test (BMDS 

### 24

Test 3). For fitting models using either constant variance or modeled variance, models for the mean 

### 25

response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 4, with χ 2 p

- value < 

### 26

0.10 indicating inadequate fit). Other factors were also used to assess the model fit, such as scaled 

### 27

residuals, visual fit, and adequacy of fit in the low

- dose region and in t he vicinity of the BMR. 

### 28

Model Selection 

### 29

For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as 

### 30

estimated by the profile likelihood method) and AIC value were used to select a best

- fit model from 

### 31

among the models exhibiting adequate fit. If the BMDL estimates were “sufficiently close,” that is, 

### 32

### Toxicological Review of benzo[a]pyrene

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



### C

- 2 DRAFT

— DO NOT CITE OR QUOTE differed by at most threefold, the model selected was the one that yielded the lowest AIC value. If 

### 1

the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the POD. 

### 2

Decreased thymus weight, males (Kroese et al., 2001) 

### 3

Table C

- 1 . Means ± SD a 

### for thymus weight in male Wistar rats exposed 4

to benzo[a]pyrene by gavage 5 days/week for 90 days 5

Organ Dose (mg/kg

- d)

0 3 10 30

Thymus weight

(mg) , m ales 380 ± 60 380 ± 110 330 ± 60 270 ± 40 b

a Reported as SE, but judged to be SD (and confirmed by study authors).

b Significantly ( p < 0.05) different from control mean; student t

- test (unpaired, two

- tailed); n =

10/sex/group.



### Table C

- 2 . Model predictions for decreased thymus weight in male 6

Wistar rats

— 90 days 7

Model Variance

p

- value a Goodness

- of

fit

p

- value AIC BMD 1SD (mg/kg

- d) BMDL 1SD

(mg/kg

- d)

Constant variance

Linear 0.01 0.74 384.84 12.97 8.97

Nonconstant variance

Hill c Insufficient degrees of freedom

Linear, Polynomial (2

- degree), Power c 0.30 0.23 380.71 16.40 11.30



### 8

### Toxicological Review of benzo[a]pyrene

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



### C

- 3 DRAFT

— DO NOT CITE OR QUOTE 1

Figure C

- 1 . Fit of linear model (nonconstant variance) to data on 2

decreased thymus weight in male Wistar rats

— 90 days. 3

BMDs and BMDLs indicated are associated with a change of 1 SD from the 4

control, and are in units of mg/kg

- day. 5

==================================================================== 

### 6

Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

### 7

Input Data File: 

### 8

C: \ USEPA \ IRIS \ benzo[a]pyrene \ RfD \ Kroese2001 \ 90day \ thymusweight \ male \ durationadjusted \ 2Linkrolin.( 

### 9

d) 

### 10

Gnuplot Plotting File: 

### 11

C: \ USEPA \ IRIS \ benzo[a]pyrene \ RfD \ Kroese2001 \ 90day \ thymusweight \ male \ durationadjusted \ 2Linkrolin.p 

### 12

lt 

### 13

==================================================================== 

### 14

15

BMDS Model Run 

### 16

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 17

18

The form of the response function is: 

### 19

20

Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 

### 21

22

23

Dependent variable = mean 

### 24

Independent variable = dose 

### 25

The polynomial coeff icients are restricted to be negative 

### 26

The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho) 

### 27

28

Total number of dose groups = 4 

### 29

### Toxicological Review of benzo[a]pyrene

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



### C

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— DO NOT CITE OR QUOTE Total number of records with missing values = 0 

### 1

Maximum number of iterations = 250 

### 2

Relative Fun ction Convergence has been set to: 1e

- 008 

### 3

Parameter Convergence has been set to: 1e

- 008 

### 4

5

6

7

Default Initial Parameter Values 

### 8

lalpha = 8.56121 

### 9

rho = 0 

### 10

beta_0 = 380.763 

### 11

beta_1 =

- 5.3285 

### 12

13

14

Asymptotic Correlation Matrix of Parameter Estimates 

### 15

16

lalpha rho beta_0 beta_1 

### 17

18

lalpha 1

- 1 0.048

- 0.061 

### 19

20

rho

- 1 1

- 0.048 0.061 

### 21

22

beta_0 0.048

- 0.048 1

- 0.84 

### 23

24

beta_1

- 0.061 0.061

- 0.84 1 

### 25

26

27

28

Parameter Estimates 

### 29

30

95.0% Wald Confidence Interval 

### 31

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 32

lalpha

- 18.8293 9.75429

- 37.9473 0.288754 

### 33

rho 4.66515 1.67581 1.38062 7.94967 

### 34

beta_0 378.954 16.5291 346.558 411.351 

### 35

beta_1

- 5.14219 1.00497

- 7.11189

- 3.17249 

### 36

37

38

39

Table of Data and Estimated Values of Interest 

### 40

41

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

### 42

------ --- -------- -------- ----------- ----------- ---------- 

### 43

44

0 10 380 379 60 84.3 0.0392 

### 45

2.1 10 380 368 110 78.8 0.475 

### 46

7.1 10 330 342 60 66.6

- 0.591 

### 47

21.4 10 270 269 40 37.9 0.0908 

### 48

49

50

51

Model Descriptions for likelihoods calculated 

### 52

53

54

Model A1: Yij = Mu(i) + e(ij) 

### 55

Var{e(ij)} = Sigma^2 

### 56

57

Model A2: Yij = Mu(i) + e(ij) 

### 58

Var{e(ij)} = Sigma(i)^2 

### 59

60

Model A3: Yij = Mu(i) + e(ij) 

### 61

Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 

### 62

Model A3 uses any fixed variance parameters that 

### 63

were specified by the user 

### 64

65

Model R: Yi = Mu + e(i) 

### 66

Var{e(i)} = Sigma^2 

### 67

68

69

Likelihoods of Interest 

### 70

71

### Toxicological Review of benzo[a]pyrene

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



### C

- 5 DRAFT

— DO NOT CITE OR QUOTE Model Log(likelihood) # Param's AIC 

### 1

A1

- 189.116991 5 388.233982 

### 2

A2

- 183.673279 8 383.346558 

### 3

A3

- 184.883626 6 381.767253 

### 4

fitted

- 186.353541 4 380.707081 

### 5

R

- 196.353362 2 396.706723 

### 6

7

8

Explanation of Tests 

### 9

10

Test 1: Do responses and/or variances differ among Dose levels? 

### 11

(A2 vs. R) 

### 12

Test 2: Are Variances Homogeneous? (A1 vs A2) 

### 13

Test 3: Are variances adequately modeled? (A2 vs. A3) 

### 14

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

### 15

(Note: W hen rho=0 the results of Test 3 and Test 2 will be the same.) 

### 16

17

Tests of Interest 

### 18

19

Test

- 2*log(Likelihood Ratio) Test df p

- value 

### 20

21

Test 1 25.3602 6 0.0002928 

### 22

Test 2 10.8874 3 0.01235 

### 23

Test 3 2.42069 2 0.2981 

### 24

Test 4 2.93983 2 0.2299 

### 25

26

The p

- value for Test 1 is less than .05. There appears to be a 

### 27

difference betwee n response and/or variances among the dose levels 

### 28

It seems appropriate to model the data 

### 29

30

The p

- value for Test 2 is less than .1. A non

- homogeneous variance 

### 31

model appears to be appropriate 

### 32

33

The p

- value for Test 3 is greater than .1. The modeled variance appears 

### 34

to be appropriate here 

### 35

36

The p

- value for Test 4 is greater than .1. The model chosen seems 

### 37

to adequately describe the data 

### 38

39

40

Benchmark Dose Computation 

### 41

42

Specified effect = 1 

### 43

44

Risk Type = Estimated standard d eviations from the control mean 

### 45

46

Confidence level = 0.95 

### 47

48

BMD = 16.4008 

### 49

50

51

BMDL = 11.2965 

### 52

53

### Toxicological Review of benzo[a]pyrene

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



### C

- 6 DRAFT

— DO NOT CITE OR QUOTE Decreased thymus weight, females (Kroese et al., 2001) 

### 1

Table C

- 3 . Means ± SD a 

### for thymus weight in female Wistar rats 2

exposed to benzo[a]pyrene by gavage 5 days/week for 90 days 3

Organ Dose (mg/kg

- d)

0 3 10 30

Thymus weight

(mg)

- Females 320 ± 60 310 ± 50 300 ± 40 230 ± 30 b

a Reported as SE, but judged to be SD (and confirmed by study authors).

b Significantly ( p < 0.05) different from control mean; student t

- test (unpaired, two

- tailed); n =

10/sex/group.



### Table C

- 4 . Model predictions for decreased thymus weight in female 4

Wistar rats

— 90 days 5

Model (constant variance) Variance

p

- value a Mean

p

- value a AIC BMD 1SD (mg/kg

- d) BMDL 1SD

(mg/kg

- d)

Hill b NA

Linear c 0.17 0.81 349.12 10.52 7.64

Polynomial (2

- degree) c,d 0.17 0.77 350.80 13.29 7.77

Power b NA

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Coefficients restricted to be negative.

d Lowest degree polynomial with an adequate fit is 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

BMDs and BMDLs indicated are associated with a change of 1 SD from the 2

control, and are in units of mg/kg

- day. 3

Figure C

- 2 . Fit of linear model (constant variance) to data on decreased 4

thymus weight in female Wistar rats

— 90 days. 5

==================================================================== 

### 6

Polynomial Model. (Version: 2.13; Date: 04/08/2008) 

### 7

Input Data File: 

### 8

C: \ USEPA \ IRIS \ benzo[a]pyrene \ RfD \ Kroese2001 \ 90day \ thymusweight \ female \ durationadjusted \ 2Linkrolin 

### 9

.(d) 

### 10

Gnuplot Plotting File: 

### 11

C: \ USEPA \ IRIS \ benzo[a]pyrene \ RfD \ Kroese2001 \ 90day \ thymusweight \ female \ durationadjusted \ 2Linkrolin 

### 12

.plt 

### 13

Thu Oct 15 16:27:44 2009 

### 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

rho is set to 0 

### 27

The polynomial coefficients are restricted to be negative 

### 28

### 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 A constant variance model is fit 

### 1

2

Total number of dose groups = 4 

### 3

Total number of records with missing values = 0 

### 4

Maximum number of iterations = 250 

### 5

Relative Function Convergence h as been set to: 1e

- 008 

### 6

Parameter Convergence has been set to: 1e

- 008 

### 7

8

9

10

Default Initial Parameter Values 

### 11

alpha = 1 

### 12

rho = 0 Specified 

### 13

beta_0 = 322.144 

### 14

beta_1 =

- 4.2018 

### 15

16

17

Asymptotic Correlation Matrix of Parameter Estimates 

### 18

19

( *** The model parameter(s)

- rho 

### 20

have been estimated at a boundary point, or have been specified by the user, 

### 21

and do not appear in the correlation matrix ) 

### 22

23

alpha beta_0 beta_1 

### 24

25

alpha 1 2.4e

- 008

- 2.3e

- 0 08 

### 26

27

beta_0 2.4e

- 008 1

- 0.68 

### 28

29

beta_1

- 2.3e

- 008

- 0.68 1 

### 30

31

32

33

Parameter Estimates 

### 34

35

95.0% Wald Confidence Interval 

### 36

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 37

alpha 1954.92 437.134 1098.16 2811.69 

### 38

beta_0 322.144 9.48287 303.558 340.73 

### 39

beta_1

- 4.2018 0.837537

- 5.84334

- 2.56026 

### 40

41

42

43

Table of Data and Estimated Values of Interest 

### 44

45

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

### 46

------ --- -------- -------- ----------- ----------- ---------- 

### 47

48

0 10 320 322 60 44.2

- 0.153 

### 49

2.1 10 310 313 50 44.2

- 0.237 

### 50

7.1 10 300 292 40 44.2 0.55 

### 51

21.4 10 230 232 30 44.2

- 0.159 

### 52

53

54

55

Model Descriptions for likelihoods calculated 

### 56

57

58

Model A1: Yij = Mu(i) + e(ij) 

### 59

Var{e(ij)} = Sigma^2 

### 60

61

Model A2: Yij = Mu(i) + e(ij) 

### 62

Var{e(ij)} = Sigma(i)^2 

### 63

64

Model A3: Yij = Mu(i) + e(ij) 

### 65

Var{e(ij)} = Sigma^2 

### 66

Model A3 uses any fixed variance parameters that 

### 67

were specified by the user 

### 68

69

Model R: Yi = Mu + e(i) 

### 70

Var{e(i)} = Sigma^2 

### 71

### 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

Likelihoods of Interest 

### 3

4

Model Log(likelihood) # Param's AIC 

### 5

A1

- 171.357252 5 352.714504 

### 6

A2

- 168.857234 8 353.714467 

### 7

A3

- 171.357252 5 352.714504 

### 8

fitted

- 171.562118 3 349.124237 

### 9

R

- 181.324151 2 366.648303 

### 10

11

12

Explanation of Tests 

### 13

14

Test 1: Do responses and/or variances differ among Dose levels? 

### 15

(A2 vs. R) 

### 16

Test 2: Are Variances Homogeneous? (A1 vs A2) 

### 17

Test 3: Are variances adequately modeled? (A2 vs. A3) 

### 18

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

### 19

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

### 20

21

Tests of Interest 

### 22

23

Test

- 2*log(Likelihood Ratio) Test df p

- value 

### 24

25

Test 1 24.9338 6 0.0003512 

### 26

Test 2 5.00004 3 0.1718 

### 27

Test 3 5.00004 3 0.1718 

### 28

Test 4 0.409733 2 0.8148 

### 29

30

The p

- value for Test 1 is less than .05. There appears t o be a 

### 31

difference between response and/or variances among the dose levels 

### 32

It seems appropriate to model the data 

### 33

34

The p

- value for Test 2 is greater than .1. A homogeneous variance 

### 35

model appears to be appropriate here 

### 36

37

38

The p

- value for Test 3 is greater than .1. The modeled variance appears 

### 39

to be appropriate here 

### 40

41

The p

- value for Test 4 is greater than .1. The model chosen seems 

### 42

to adequately describe the data 

### 43

44

45

Benchmark Dose Computation 

### 46

47

Specified eff ect = 1 

### 48

49

Risk Type = Estimated standard deviations from the control mean 

### 50

51

Confidence level = 0.95 

### 52

53

BMD = 10.5228 

### 54

55

56

BMDL = 7.64037 

### 57

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 Decreased ovary weight

— female rats, 60 days (Xu et al., 2010) 

### 1

Table C

- 5 . Means ± SDs for ovary weight in female Sprague

- Dawley rats 2

Organ Dose (mg/kg

- d) a

0 2.5 5

Ovary weight (mg) 0.160 ± 0.0146 0.143 ± 0.0098 b 0.136 ± 0.0098 b

a TWA doses over the 60

- day study period.

b Statistically different ( p < 0.05) from controls using one

- way ANOVA.



### Table C

- 6 . Model predictions for decreased ovary weight in female 3

Sprague

- Dawley rats 4

Model Goodness

- of

- fit

p

- value AIC BMD 1SD (mg/kg

- d) BMDL 1SD

(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

Figure C

- 3 . Fit of linear/polynomial (1°) model to data on decreased 2

ovary weight

. 3

==================================================================== 

### 4

Polynomial Model. (Version: 2.16; Date: 05/26/2010) 

### 5

Input Data File: 

### 6

C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_1SD.(d) 

### 7

Gnuplot Plotting File: 

### 8

C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeigh t/Xu2010_AbsOvaryWeight_Linear_1SD.plt 

### 9

Tue Dec 14 13:51:32 2010 

### 10

==================================================================== 

### 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 12

13

The form of the response function is: 

### 14

15

Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 

### 16

17

18

Dependent variable = Mean 

### 19

Independent variable = Dose 

### 20

rho is set to 0 

### 21

Signs of the polynomial coefficients are not restricted 

### 22

A constant variance model is fit 

### 23

24

Total number of dose groups = 3 

### 25

Total number of records with missing values = 0 

### 26

Maximum number of iterations = 250 

### 27

Relative Function Convergence has been set to: 1e

- 008 

### 28

Parameter Convergence has been set to: 1e

- 008 

### 29

30

### 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

Defau lt Initial Parameter Values 

### 3

alpha = 0.000136 

### 4

rho = 0 Specified 

### 5

beta_0 = 0.158333 

### 6

beta_1 =

- 0.0048 

### 7

8

9

Asymptotic Co rrelation Matrix of Parameter Estimates 

### 10

11

( *** The model parameter(s)

- rho 

### 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

alpha beta_0 beta_1 

### 16

17

alpha 1 4e

- 010

- 4.5e

- 010 

### 18

19

beta_0 4e

- 010 1

- 0.77 

### 20

21

beta_1

- 4.5e

- 010

- 0.77 1 

### 22

23

24

25

Parameter Estimat es 

### 26

27

95.0% Wald Confidence Interval 

### 28

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 29

alpha 0.000118889 3.96296e

- 005 4.12162e

- 005 0.000196562 

### 30

beta_0 0.158333 0.00406354 0.150369 0.166298 

### 31

beta_1

- 0.0048 0.00125904

- 0.00726768

- 0.00233232 

### 32

33

34

35

Table of Data and Estimated Values of Interest 

### 36

37

D ose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

### 38

------ --- -------- -------- ----------- ----------- ---------- 

### 39

40

0 6 0.16 0.158 0.0147 0.0109 0.374 

### 41

2.5 6 0.14 3 0.146 0.0098 0.0109

- 0.749 

### 42

5 6 0.136 0.134 0.0098 0.0109 0.374 

### 43

44

45

46

Model Descriptions for likelihoods calculated 

### 47

48

49

Model A1: Yij = Mu(i) + e(ij) 

### 50

Var{e(ij)} = Sigma^2 

### 51

52

Model A2: Yij = Mu(i) + e(ij) 

### 53

Var{e(ij)} = Sigma(i)^2 

### 54

55

Model A3: Yij = Mu(i) + e(ij) 

### 56

Var{e(ij)} = Sigma^2 

### 57

Model A3 uses any fixed variance parameters that 

### 58

were specified by the user 

### 59

60

Model R: Yi = M u + e(i) 

### 61

Var{e(i)} = Sigma^2 

### 62

63

64

Likelihoods of Interest 

### 65

66

Model Log(likelihood) # Param's AIC 

### 67

A1 72.766595 4

- 137.533190 

### 68

A2 73.468565 6

- 134.937129 

### 69

A3 72.766595 4

- 137.533190 

### 70

fitted 72.335891 3

- 138.671782 

### 71

### 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 R 67.008505 2

- 130.017010 

### 1

2

3

Explanation of Tests 

### 4

5

Test 1: Do responses and/or variances differ among Dose levels? 

### 6

(A2 vs. R) 

### 7

Test 2: Are Variances Homogeneous? (A1 vs A2) 

### 8

Test 3: Are variances adequately modeled? (A2 vs. A3) 

### 9

Test 4: Does the Mo del for the Mean Fit? (A3 vs. fitted) 

### 10

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

### 11

12

Tests of Interest 

### 13

14

Test

- 2*log(Likelihood Ratio) Test df p

- value 

### 15

16

Test 1 12.9201 4 0.01167 

### 17

Test 2 1.40394 2 0.4956 

### 18

Test 3 1.40394 2 0.4956 

### 19

Test 4 0.861408 1 0.3533 

### 20

21

The p

- value for Test 1 is less than .05. There appears to be a 

### 22

difference between response and/or variances among the dose levels 

### 23

It seems appropriate to model the data 

### 24

25

The p

- value for Test 2 is greater than .1. A homogeneous variance 

### 26

model appears to be appropriate here 

### 27

28

29

The p

- value for Test 3 is greater than .1. The modeled variance appears 

### 30

to be appropriate here 

### 31

32

The p

- value for Test 4 is greater than .1. The model chosen seems 

### 33

to adequately describe the data 

### 34

35

36

Benchmark Dose Computation 

### 37

38

Specified eff ect = 1 

### 39

40

Risk Type = Estimated standard deviations from the control mean 

### 41

42

Confidence level = 0.95 

### 43

44

BMD = 2.27159 

### 45

46

47

BMDL = 1.49968 

### 48

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 Morris water maze results

— male and female Sprague

- Daw ley rats, Chen et al. (2012) 

### 1

Data from Morris water maze was presented graphically in Chen et al., 2012, but dose group 

### 2

means and standard deviations were provided upon request by the study authors which enabled 

### 3

modeling of this endpoint. In addition, th e data for male and female rats were combined for dose



### 4

response analysis because there was no substantive difference between males and females for each 

### 5

dose group (supported by statistical testing using two

- way ANOVA, and allowing for interactions), 

### 6

and be cause there was no rationale or information available suggesting there would be sex



### 7

mediated differences for these neurologic tests. 

### 8

Table C

- 7 . Means ± SDs for Escape Latency and Time Spent in Target 9

Quadrant 10

Test Dose (mg/kg

- d)

0 0.02 0.2 2.0

Escape l atency

(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



### Table C

- 8 . Model predictions for increase in Morris water maze test for 11

escape latency, male and female rats 12

Model a Goodness

- of

- fit

p

- value AIC BMD 1SD (mg/kg

- d) BMDL 1SD

(mg/kg

- d)

Hill b 0.515 386.3 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 var iances (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 Figure C

- 4 . Fit of Hill model to data on Morris water maze test escape 1

latency. 2

==================================================================== 

### 3

Hill Model. (Version: 2.16; Date: 04/06/2011) 

### 4

Input Data File: C: \ Documents and Settings \ jfox \ My Documents \ _CURRENTWORK \ _CAST 

### 5

plus \ BaP \ BMDS \ hil_Chen.FM.latency_Hil

- ModelVari ance

- BMR1Std

- Restrict.(d) 

### 6

Gnuplot Plotting File: C: \ Documents and Settings \ jfox \ My Documents \ _CURRENTWORK \ _CAST 

### 7

plus \ BaP \ BMDS \ hil_Chen.FM.latency_Hil

- ModelVariance

- BMR1Std

- Restrict.plt 

### 8

Tue Apr 24 14:41:26 2012 

### 9

================================ ==================================== 

### 10

11

BMDS Model Run 

### 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 13

14

The form of the response function is: 

### 15

16

Y[dose] = intercept + v*dose^n/(k^n + dose^n) 

### 17

18

19

Dependent variable = Mean 

### 20

Independent variable = Dose 

### 21

Power parameter restricted to be greater than 1 

### 22

The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i))) 

### 23

24

Total number of dose groups = 4 

### 25

Total number of records with missing values = 0 

### 26

Maximu m number of iterations = 250 

### 27

Relative Function Convergence has been set to: 1e

- 008 

### 28

Parameter Convergence has been set to: 1e

- 008 

### 29

30

31

32

Default Initial Parameter Values 

### 33

lalpha = 3.87128 

### 34

rho = 0 

### 35

intercept = 9.888 

### 36

v = 23.6385 

### 37

n = 0.187055 

### 38

k = 3.47082 

### 39

40

41

### 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 Asymptotic Correlation Mat rix of Parameter Estimates 

### 1

2

( *** The model parameter(s)

- n 

### 3

have been estimated at a boundary point, or have been specified by the user, 

### 4

and do not appear in the correlation matrix ) 

### 5

6

lalpha rho intercept v k 

### 7

8

lalpha 1

- 0.99

- 0.27 0.062

- 0.11 

### 9

10

rho

- 0.99 1 0.24

- 0.063 0.12 

### 11

12

intercept

- 0.27 0.24 1 0.017 0.47 

### 13

14

v 0.062

- 0.063 0.017 1 0.73 

### 15

16

k

- 0.11 0.12 0.47 0.73 1 

### 17

18

19

20

Parameter Estim ates 

### 21

22

95.0% Wald Confidence Interval 

### 23

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 24

lalpha 0.88775 0.974841

- 1.0229 2.7984 

### 25

rho 0.998033 0.338845 0.33391 1.66216 

### 26

intercept 10.6545 0.914127 8.86283 12.4461 

### 27

v 28.7081 3.94381 20.9783 36.4378 

### 28

n 1 NA 

### 29

k 0.494812 0.213359 0.0766351 0.912988 

### 30

31

NA

- Indicates that this parameter has hit a bound 

### 32

implied by some inequalit y constraint and thus 

### 33

has no standard error. 

### 34

35

36

37

Table of Data and Estimated Values of Interest 

### 38

39

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

### 40

------ --- -------- -------- ----------- ----------- ---------- 

### 41

42

0 20 9.89 10.7 5.76 5.08

- 0.675 

### 43

0.02 20 12.5 11.8 5.1 5.33 0.641 

### 44

0.2 20 19.1 18.9 5.85 6.76 0.0952 

### 45

2 20 33.5 33.7 9.93 9.01

- 0.0706 

### 46

47

48

49

Model Descriptions for likelihoods calculated 

### 50

51

52

Model A1: Yij = Mu(i) + e(ij) 

### 53

Var{e(ij)} = Sigma^2 

### 54

55

Model A2: Yij = Mu(i) + e(ij) 

### 56

Var{e(ij)} = Sigma(i )^2 

### 57

58

Model A3: Yij = Mu(i) + e(ij) 

### 59

Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 

### 60

Model A3 uses any fixed variance parameters that 

### 61

were specified by the user 

### 62

63

Model R: Yi = Mu + e(i) 

### 64

Var{e(i)} = Sigma^2 

### 65

66

67

Likelihoods of Interest 

### 68

69

Model Log(likelihood) # Param's AIC 

### 70

A1

- 192.799518 5 395.599036 

### 71

### 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 A2

- 186.795503 8 389.591006 

### 1

A3

- 187.957975 6 387.915949 

### 2

fitted

- 188.169983 5 386.339965 

### 3

R

- 234.549118 2 473.098237 

### 4

5

6

Explanation of Tests 

### 7

8

Test 1: Do responses and/or variances differ among Dose levels? 

### 9

(A2 vs. R) 

### 10

Test 2: Are Variances Homogeneous? (A1 vs A2) 

### 11

Test 3: Are variances adequately modeled? (A2 vs. A3) 

### 12

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

### 13

(Note: When rho=0 the results of Test 3 an d Test 2 will be the same.) 

### 14

15

Tests of Interest 

### 16

17

Test

- 2*log(Likelihood Ratio) Test df p

- value 

### 18

19

Test 1 95.5072 6 <.0001 

### 20

Test 2 12.008 3 0.007356 

### 21

Test 3 2.32494 2 0.3127 

### 22

Test 4 0.424016 1 0.5149 

### 23

24

The p

- value for Test 1 is less than .05. There appears to be a 

### 25

difference between response and/or variances among the dose levels 

### 26

It seems appropriate to model the data 

### 27

28

The p

- value for Test 2 is less than .1. A non

- homogeneous variance 

### 29

model appears to be appropriate 

### 30

31

The p

- value for Test 3 is greater than .1. The modeled variance appears 

### 32

to be appropriate here 

### 33

34

The p

- value for Test 4 is greater than .1. The model chosen seems 

### 35

to adequately describe the data 

### 36

37

38

Benchmark Dose Computation 

### 39

40

Specified effect = 1 

### 41

42

Risk Type = Estimated standard deviations from the control mean 

### 43

44

Confidence level = 0.95 

### 45

46

BMD = 0.106284 

### 47

48

BMDL = 0.0609511 

### 49

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 Table C

- 9 . Model predictions for decrease in Morris water maze test for 1

time spent in target quadrant, male and female rats 2

Model a Goodness

- of

- fit

p

- value AIC BMD 1SD (mg/kg

- d) BMDL 1SD

(mg/kg

- d)

Exponential 4 0.576 395.4 0.065 0.043

Exponential 5 NA b 397.1 0.084 0.044

Hill NA b 397.1 0.071 0.038

Linear, Powe r, 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

Figure C

- 5 . Fit of Exponential 4 model to data on Morris water maze 5

time spent in target quadrant. 6

==================================================================== 

### 7

Exponential Model. (Version: 1.7; Date: 12/10/2009) 

### 8

Input Data File: C: \ Documents and Settings \

... \ exp_Chen.FM.target_Exp

- ModelVariance



### 9

BMR1Std

- Down.(d) 

### 10

==================================================================== 

### 11

12

BMDS Model Run 

### 13

~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 14

15

The form of the response function by Model: 

### 16

Model 2: Y[dose] = a * exp{sign * b * dose} 

### 17

Model 3: Y[dose] = a * exp{sign * (b * dose)^d} 

### 18

Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}] 

### 19

Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}] 

### 20

21

Note: Y[dose] is the median response for exposure = dose; 

### 22

sign = +1 for increasing trend in data; 

### 23

### 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 sign =

- 1 for decreasing trend. 

### 1

2

Model 2 is nested within Models 3 and 4. 

### 3

Model 3 is nested within Model 5. 

### 4

Model 4 is nested within Model 5. 

### 5

6

7

Dependent variable = Mean 

### 8

Independent variable = Dose 

### 9

Data are assumed to be distributed: norm ally 

### 10

Variance Model: exp(lnalpha +rho *ln(Y[dose])) 

### 11

The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho) 

### 12

13

Total number of dose groups = 4 

### 14

Total number of records with missing values = 0 

### 15

Maximum number of iterations = 25 0 

### 16

Relative Function Convergence has been set to: 1e

- 008 

### 17

Parameter Convergence has been set to: 1e

- 008 

### 18

19

MLE solution provided: Exact 

### 20

21

22

Initial Parameter Values 

### 23

24

Variable Model 4 

### 25

-------- -------- 

### 26

lnalpha 0.666712 

### 27

rho 1.04799 

### 28

a 35.3094 

### 29

b 1.97191 

### 30

c 0.300675 

### 31

d 1 

### 32

33

34

35

Parameter Estimates 

### 36

37

Variable Model 4 

### 38

-------- ------- 

### 39

lnalpha 0.601192 

### 40

rho 1.05452 

### 41

a 34.3199 

### 42

b 7.26795 

### 43

c 0.325841 

### 44

d 1 

### 45

46

NC = No Convergence 

### 47

48

49

Table of Stats From Input Data 

### 50

51

Dose N Obs Mean Obs Std Dev 

### 52

----- --- ---------- ------------- 

### 53

0 20 33.63 8.924 

### 54

0.02 20 31.94 8.633 

### 55

0.2 20 16.56 5.744 

### 56

2 20 11.15 5.117 

### 57

58

59

Estimated Values of Interest 

### 60

61

Dose Est Mean Est Std Scaled Residual 

### 62

------ ---------- --------- ---------------- 

### 63

0 34.32 8.713

- 0.3551 

### 64

0.02 31.19 8.285 0.4069 

### 65

0.2 16.59 5.939

- 0.02044 

### 66

2 11.18 4.824

- 0.03277 

### 67

68

69

70

Other models for which likelihoo ds are calculated: 

### 71

72

Model A1: Yij = Mu(i) + e(ij) 

### 73

Var{e(ij)} = Sigma^2 

### 74

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 Model A2: Yij = Mu(i) + e(ij) 

### 1

Var{e(ij)} = Sigma(i)^2 

### 2

3

Model A3: Yij = Mu(i) + e(ij) 

### 4

Var{e(ij)} = exp(lalpha + log(mean(i)) * rho) 

### 5

6

Model R: Yij = Mu + e(i) 

### 7

Var{e(ij)} = Sigma^2 

### 8

9

10

Likelihoods of Interest 

### 11

12

Model Log(likelihood) DF AIC 

### 13

------- ----------------- ---- ------------ 

### 14

A1

- 197.0118 5 404.0235 

### 15

A2

- 192.448 8 400.896 

### 16

A3

- 192 .5331 6 397.0662 

### 17

R

- 238.8696 2 481.7393 

### 18

4

- 192.6894 5 395.3787 

### 19

20

21

Additive constant for all log

- likelihoods =

- 73.52. This constant added t o the 

### 22

above values gives the log

- likelihood including the term that does not 

### 23

depend on the model parameters. 

### 24

25

26

Explanation of Tests 

### 27

28

Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R) 

### 29

T est 2: Are Variances Homogeneous? (A2 vs. A1) 

### 30

Test 3: Are variances adequately modeled? (A2 vs. A3) 

### 31

32

Test 6a: Does Model 4 fit the data? (A3 vs 4) 

### 33

34

35

Tests of Interest 

### 36

37

Test

- 2*log(Likelihood Ratio) D. F. p

- value 

### 38

-------- ------------------------ ------ -------------- 

### 39

Test 1 92.84 6 < 0.0001 

### 40

Test 2 9.127 3 0.02764 

### 41

Test 3 0.1701 2 0.9185 

### 42

Test 6a 0.3126 1 0.5761 

### 43

44

45

The p

- value for Test 1 is less than .05. There appears to be a 

### 46

difference between response and/or variances among the dose 

### 47

levels, it seems appropriate to model the data. 

### 48

49

The p

- value for Test 2 is less than .1. A non

- homogeneous 

### 50

variance model appears to be appropriate. 

### 51

52

The p

- value for Test 3 is greater than .1. The modeled 

### 53

variance appears to be appropriate here. 

### 54

55

The p

- value for Test 6a is greater than .1. Model 4 seems 

### 56

to adequately describe the data. 

### 57

58

59

Benchmark Dose Computations: 

### 60

61

Specified Effect = 1.000000 

### 62

63

Risk Type = Estimated standard deviations from control 

### 64

65

Confidence Level = 0.950000 

### 66

67

BMD = 0.0650194 

### 68

69

BMDL = 0.0432761 

### 70

### 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 Cervical epithelial hyperplasia

– female ICR mice (Gao et al., 2011) 

### 1

Table C

- 1 0 . Incidence of cervical epithelial hyperplasia 2

Observation Dose (mg/kg

- day) a

0 0.71 1.4 2.9

Cervical epithelial hyperplasia 0/26 4/26 6/25 7/24

a doses converted to mg/kg

- day after adjustment for equivalent continuous dosing (2/7 

### 3

days/week) 

### 4

Table C

- 1 1

. Model predictions for increased incidence of epithelial 5

hyperplasia in female ICR mice 6

Model a Goodness

- of

- fit

p

- value AIC BMD 1SD (mg/kg

- d) BMDL 1SD

(mg/kg

- d)

G amma 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. 6 8 6

M ultistage 0.6874 82.2821 0.65 9 0.452



### 7

==================================================================== 

### 8

Logistic Model. (Version: 2.13; Date: 10/28/2009) 

### 9

Input Data File: C: \ Users \ hclynch \ Documents \ _Active Projects \ _FA498 IRIS \ xBaP \ IASC Aug 

### 10

2011 \ bmd modeling \ lnl_gao 2011 inflamm cells_Opt.(d) 

### 11

Gnuplot Plotting File: C: \ Users \ hclynch \ Documents \ _Ac tive Projects \ _FA498 

### 12

IRIS \ xBaP \ IASC Aug 2011 \ bmd modeling \ lnl_gao 2011 inflamm cells_Opt.plt 

### 13

==================================================================== 

### 14

15

BMDS_Model_Run 

### 16

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 17

18

The form of the probability function is: 

### 19

20

P[response] = background+(1

- background)/[1+EXP(

- intercept

- slope*Log(dose))] 

### 21

22

23

Dependent variable = Col3 

### 24

Independent variable = Col1 

### 25

Slope parameter is restricted as slope >= 1 

### 26

27

Total number of observations = 4 

### 28

Total number of records with missing values = 0 

### 29

Maximum number of iterations = 250 

### 30

Relative Function Convergence has been set to: 1e

- 008 

### 31

Parameter Convergence has been set to: 1e

- 008 

### 32

33

34

35

User has chosen the log transformed model 

### 36

37

38

Default Initial Parameter Values 

### 39

background = 0 

### 40

intercept =

- 1.60901 

### 41

slope = 1 

### 42

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

Asymptotic Correlation M atrix of Parameter Estimates 

### 2

3

( *** The model parameter(s)

- background

- slope 

### 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

intercept 

### 8

9

intercept 1 

### 10

11

12

13

Parameter Estimates 

### 14

15

95.0% Wald Confidence Interval 

### 16

Variable Estimate Std. Err. Lower Co nf. Limit Upper Conf. Limit 

### 17

background 0 * * * 

### 18

intercept

- 1.6502 * * * 

### 19

slope 1 * * * 

### 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

- 39.4267 4 

### 29

Fitted mod el

- 39.8502 1 0.847034 3 0.8382 

### 30

Reduced model

- 45.7739 1 12.6945 3 0.005346 

### 31

32

AIC: 81.7004 

### 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 26 0.000 

### 40

0.7100 0.1200 3.119 4.000 26 0.532 

### 41

1.4000 0.2119 5.297 6.000 25 0.344 

### 42

2.9000 0.3577 8.584 7.000 24

- 0.675 

### 43

44

Chi^2 = 0.86 d.f. = 3 P

- value = 0.8360 

### 45

46

47

Benchmark Dose Computation 

### 48

49

Specified effect = 0.1 

### 50

51

Risk Type = Extra risk 

### 52

53

Confidence level = 0.95 

### 54

55

BMD = 0.578668 

### 56

57

BMDL = 0.368701 

### 58

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 INHALATION DOSIMETRY MODELING FOR RFC DERIVATION 1

2

3

Figure C

- 6 . Human fractional deposition. 4

Species = humanlimited 

### 5

FRC = 3300.0 

### 6

Head volume = 50.0 

### 7

Density = 1.0 

### 8

Number of particles calculated = single 

### 9

Diameter = 1.7000000000000002 μm MMAD 

### 10

Inhalability = yes 

### 11

GSD = 1.0 

### 12

Breathing interval: One single breath 

### 13

Concentration = 4.2 

### 14

Breathing Frequency = 16.0 

### 15

Tidal Volume = 860.0 

### 16

Inspiratory Fraction = 0.5 

### 17

Pause Fraction = 0.0 

### 18

Breathing Route = nasal 

### 19

20

Region: Entire Lung 

### 21

Region: Entire Lung 

### 22

Region Deposition F raction 

### 23

-- -- 

### 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 Head 0.449 

### 1

TB 0.045 

### 2

P 0.127 

### 3

Total 0.621 

### 4

5

6

7

Figure C

- 7 . Rat fractional deposition. 8

Species = rat 

### 9

FRC = 4.0 

### 10

Head volume = 0.42 

### 11

Density = 1.0 

### 12

Number of particles calculated = single 

### 13

Diameter = 1.7000000000000002 μm MMAD 

### 14

Inhalability = yes 

### 15

GSD = 1.0 

### 16

Breathing interval: One single breath 

### 17

Concentration = 4.2 

### 18

Breathing Frequency = 102.0 

### 19

Tidal Volume = 1.8 

### 20

Inspiratory Fraction = 0.5 

### 21

Pause Fraction = 0.0 

### 22

Breathing Route = nasal 

### 23

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 Region: Entire Lung 

### 1

Region: Entire Lung 

### 2

Region Deposition Fraction 

### 3

-- -- 

### 4

Head 0.072 

### 5

TB 0.041 

### 6

P 0.068 

### 7

Total 0.181 

### 8

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 DOSE

- RESPONSE MODELING FOR CANCER RISK VALUES 1

The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend that the 

### 2

method used to characterize and quantify cancer risk from a chemical is determined by what is 

### 3

known about the mode of action of the carcinogen and the shape of the cancer dose

- response curve. 

### 4

No biologically based mod els for BaP carcinogenicity following oral, inhalation, or dermal exposure 

### 5

were identified. 

### 6

Methods for the Oral Slope Factor and Inhalation Unit Risk 

### 7

Due to the occurrence of multiple tumor types, earlier occurrence with increasing exposure, 

### 8

and early t ermination of the high

- dose group in each of the oral and inhalation carcinogenicity 

### 9

studies (see Appendix B for study details) , methods that can reflect the influence of competing risks 

### 10

and intercurrent mortality on site

- specific tumor incidence rates are preferred. EPA has generally 

### 11

used a model that incorporates the time at which death

- with

- tumor occurred as well as the dose; 

### 12

the multistage

- Weibull model is multistage in dose and Weibull in time, and has the form: 

### 13

14

P(d, t) = 1

- exp[

- (q 0 + q 1 d + q 2 d

2 + ... + q k d k

) × (t ± t 0 ) c ]

, 

### 15

16

where P(d, t) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent 

### 17

exposure in this case) and age t (in bioassay weeks); parameters q i ≥ 0, for i = 0, 1, ..., k; t is the time 

### 18

at which the tumor was observed; and c is a parameter which characterizes the change in response 

### 19

with age. The parameter t 0 represents the time between when a potentially fatal tumor becomes 

### 20

observable and when it causes death, and is generally set to 0 either when all tumors are 

### 21

considered incidental or because of a lack of data to estimate the time reliably. The dose

- response 

### 22

analyses were conducted using the computer software program MultiStage

- Weibull (U.S. EP A, 

### 23

2010), which is based on Weibull models drawn from Krewski et al. (1983). Parameters were 

### 24

estimated using the method of maximum likelihood. From specific model fits using stages up to n

- 1, 

### 25

where n is the number of dose groups, the model fit with the l owest AIC was selected

. 

### 26

Two general characteristics of the observed tumor types were considered prior to 

### 27

modeling; allowance for different, although unidentified modes of action, and allowance for relative 

### 28

severity of tumor types. First, etiologically dif ferent tumor types were not combined across sites 

### 29

prior to modeling (that is, overall counts of tumor

- bearing animals were not tabulated) in order to 

### 30

allow for the possibility that different tumor types could have different dose

- response relationships 

### 31

due to different underlying mechanisms or factors, such as latency. Consequently, all of the tumor 

### 32

types were also modeled separately. 

### 33

Additionally, the multistage

- Weibull model can address relative severity of tumor types by 

### 34

distinguishing between tumors as being either fatal or incidental to the death of an animal in order 

### 35

to adjust partially for competing risks. I n contrast to fatal tumors, i ncidental tumors are those 

### 36

tumors thought not to have caused the death of an animal. Cause

- of

- death information for most 

### 37

### 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 early animal deaths was provided by the investigators of both of the bioassays. In the rat study, 

### 1

tumors of the forestomach or liver were the principal cause of death for most animals dying or 

### 2

sacrificed (due to moribundity) before the end of the st udy, while tumors of the forestomach were 

### 3

the most common cause of early deaths in the mouse study. 

### 4

PODs for estimating low

- dose risk were identified at doses at the lower end of the observed 

### 5

data, generally corresponding to 10% extra risk, where extra ris k is defined as [P(d)

- P(0)]/[1

- 

### 6

P(0)]. The lifetime oral cancer slope factor for humans is defined as the slope of the line from the 

### 7

lower 95% bound on the exposure at the POD to the control response (slope factor = 0.1/BMDL 10 ). 

### 8

This slope, a 95% uppe r confidence limit (UCL) represents a plausible upper bound on the true risk. 

### 9

Although the time

- to

- tumor modeling helps account for competing risks associated with 

### 10

decreased survival times and other tumors, considering the tumor sites individually still do es not 

### 11

convey the total amount of risk potentially arising from the sensitivity of multiple sites

— that is, the 

### 12

risk of developing any combination of the increased tumor types, not just the risk of developing all 

### 13

simultaneously. One approach suggested in t he Guidelines for Carcinogen Risk Assessment (U.S. EPA, 

### 14

2005a) would be to estimate cancer risk from tumor

- bearing animals. EPA traditionally used this 

### 15

approach until the National Resource Council (NRC) document Science and Judgment (NRC, 1994) 

### 16

made a cas e that this approach would tend to underestimate overall risk when tumor types occur in 

### 17

a statistically independent manner. In addition, application of one model to a composite data set 

### 18

does not accommodate biologically relevant information that may vary across sites or may only be 

### 19

available for a subset of sites. For instance, the time courses of the multiple tumor types evaluated 

### 20

varied, as is suggested by the variation in estimates of c , from 1.5 (e.g., male rat skin or mammary 

### 21

gland basal cell tumors) , indicating relatively little effect of age on tumor incidence, to 3.7 (e.g., male 

### 22

mouse alimentary tract tumors), indicating a more rapidly increasing response with increasing age 

### 23

(in addition to exposure level). The result of fitting a model with param eters that can reflect 

### 24

underlying mechanisms, such as z in the multistage

- Weibull model, would be difficult to interpret 

### 25

with composite data (i.e., counts of tumor

- bearing animals). A simpler model, such as the 

### 26

multistage model, could be used for the comp osite data but relevant biological information would 

### 27

then be ignored. 

### 28

Following the recommendations of the NRC (1994) regarding combining risk estimates, 

### 29

statistical methods that can accommodate the underlying distribution of slope factors are optimal, 

### 30

suc h as through maximum likelihood estimation or through bootstrapping or Bayesian analysis. 

### 31

However, these methods have not yet been extended to models such as the multistage

- Weibull 

### 32

model. A method involving the assumption that the variability in the slop e factors could be 

### 33

characterized by a normal distribution is detailed below (U.S. EPA, 2010). Using the results in 

### 34

female rats to illustrate, the overall risk estimate involved the following steps: 

### 35

36

(1) It was assumed that the tumor groupings modeled above were statistically independent

— 

### 37

that is, that the occurrence of a liver tumor was not dependent upon whether there was a 

### 38

### 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 forestomach tumor. This assumption cannot currently be verified, and if not correct, could 

### 1

lead to an overestimate of risk from s umming across tumor sites. However, NRC (1994) 

### 2

argued that a general assumption of statistical independence of tumor

- type occurrences 

### 3

within animals was not likely to introduce substantial error in assessing carcinogenic 

### 4

potency from rodent bioassay data. 

### 5

6

(2) The models previously fitted to estimate the BMDs and BMDLs were used to extrapolate to a 

### 7

lower level of risk (R), in order to reach the region of each estimated dose

- response 

### 8

function where the slope was reasonably constant and upper bound estimatio n was still 

### 9

numerically stable. For these data, a 10

- 3 risk was generally the lowest risk necessary. The 

### 10

oral slope factor for each site was then estimated by R/BMDL R , as for the estimates for each 

### 11

tumor site above. 

### 12

13

(3) The maximum likelihood estimates (MLE) of unit potency (that is, risk per unit of exposure) 

### 14

estimated by R/BMD R , were summed across the alimentary tract, liver, and 

### 15

jejunum/duodenum in female rats. 

### 16

17

(4) An estimate of the 95% (one

- sided) upper bound on the summed oral slope factor was 

### 18

cal culated by assuming a normal distribution for the individual risk estimates, and deriving 

### 19

the variance of the risk estimate for each tumor site from its 95% UCL according to the 

### 20

formula: 

### 21

22

95% UCL = MLE + 1.645 × SD, 

### 23

rearranged to: 

### 24

s.d. = (UCL

– MLE) / 1.6 45, 

### 25

26

where 1.645 is the t

- statistic corresponding to a one

- sided 95% CI and >120 degrees of freedom, 

### 27

and the SD is the square root of the variance of the MLE. The variances (variance = SD 2 ) for each 

### 28

site

- specific estimate were summed across tumor sites to obtain the variance of the sum of the 

### 29

MLEs. The 95% UCL on the sum of MLEs was calculated from the expression above for the UCL, 

### 30

using the variance of the sum of the MLE to obtain the relevant SD (SD

= variance 1/2 ). 

### 31

The results of this analysis are provi ded in Table C

- 17

. 

### 32

### 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 Table C

- 1 2

. Tumor incidence data, with time to death with tumor; male 1

rats exposed by gavage to benzo [a]pyrene

— Kroese et al. (2001) 2

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

Incidental a Fatal a Incidental 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

Incidental a Fatal a Incidental 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 Table C

- 1 3 . Tumor incidence data, with time to death with tumor; 1

female rats exposed by gavage to benzo[a]pyrene

— Kroese et al. (2001) 2

Dose

(mg/kg

- d) Week of

death Total

examined Numbers of animals with

Oral cavity or forestomach

tumors Liver tumors Duodenum or

jejunum tumors

Incidental a Fatal a Incidental Fatal Incidental

0 64

69

75

104

106

107

108

109 1

1

1

1

4

7

7

30 0

0

0

0

0

0

0

1 0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0

3 8

47

52

60

65

76

77

83

85

86

88

93

94

97

107

108

109 1

1

1

1

1

1

1

2

1

1

1

2

1

1

6

9

21 0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

2

1 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

10 42

43

44

45

48

55

59

75

76

77

80

81

82

83

85

86

87

88

89

91

95

96

98

99

102 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

Incidental a Fatal a Incidental 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 Table C

- 14 . Tumor incidence, with time to death with tumor; female 1

mice exposed to benzo[a]pyrene via diet

— Beland and Culp (1998) 2

Dose group

(ppm in diet) Week of death Total examined Number of animals with alimentary tract squamous cell tumors

Fatal a Incidental

0 31

74

89

91

93

94

97

98

99

100

101

104

105 1

1

2

1

2

2

2

2

1

2

2

1

29 0

0

0

0

0

0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0

0

0

0

0

1

5 25

55

83

86

87

88

90

94

95

96

97

98

101

102

105 1

1

1

1

2

2

1

1

2

1

2

2

2

2

27 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

25 44

47

64

70

77

80

81

84

85

86

88

89

90

93

94

96

97

98

99

100

101

102

104

105 1

1

1

1

1

1

1

2

1

1

1

1

4

3

2

3

1

1

2

1

1

2

1

13 1

0

0

1

1

0

1

1

1

1

1

0

4

2

2

0

1

1

1

1

0

2

1

0 0

0

0

0

0

0

0

1

0

0

0

0

0

1

0

2

0

0

1

0

0

0

0

10

### 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

Fatal a Incidental

100 39

40

42

47

49

50

53

55

56

57

58

59

60

61

62

63

64

65

66

68

69

70

71

72

73

74

79 1

1

1

2

1

1

1

3

1

1

1

3

1

3

5

4

3

2

3

1

2

2

1

1

1

1

1 1

1

1

2

0

1

0

3

1

1

1

3

1

3

5

4

3

2

3

1

2

2

1

1

1

1

1 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

a “Incidental” denotes presence of tumors not known to have caused death of particular animals. “Fatal” denotes incidence of

tumors indicated by the study investigators to have caused death of particular animals.



### Table C

- 1 5 . Derivation of HEDs to use for BMD modeling of Wistar rat 1

tumor incidence data from Kroese et al. (2001) 2

Benzo[a]pyrene dose (mg/kg

- d) TWA body weight (kg) Interspecies scaling

factor a HED b (mg/kg

- d)

Male

3 0.349 0.27 0.54

10 0.349 0.27 1.81

30 0.288 0.25 5.17

Female

3 0.222 0.24 0.49

10 0.222 0.24 1.62

30 0.222 0.24 4.85

a Scaling factors were calculated using U.S. EPA (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 Table C

- 1 6 . Derivation of HEDs for dose

- response modeling of B6C3F 1 

### 1

female mouse tumor incidence data from Beland and Culp (1998) 2

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 Table C

- 1 7

. Summary of model selection and modeling results for best

1

fitting multistage

- Weibull models, using time

- to

- tumor data for rats 2

from Kroese et al. (1981) 3

Endpoints Model

stages AIC BMD 10 BMD L 10 − BMD U 10 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 Male Rat (Kroese et al., 2001): Squamous Cell Papilloma or Carcinoma in Oral Cavity or Forestomach 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: OralForstKroeseM3.(d) 

### 6

======================================================================= 

### 7

The f orm of the probability function is: 

### 8

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 9

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 10

11

The parameter betas are restricted to be positive 

### 12

13

Dependent variable = CONTEXT 

### 14

Independent variables = DOSE, TIME 

### 15

16

Total number of observations = 208 

### 17

Total number of records with missing values = 0 

### 18

Total number of parameters in model = 6 

### 19

Total number of specified parameters = 0 

### 20

Degree of polynomial = 3 

### 21

22

Maximum number of ite rations = 64 

### 23

Relative Function Convergence has been set to: 2.22045e

- 016 

### 24

Parameter Convergence has been set to: 1.49012e

- 008 

### 25

26

27

Default Initial Parameter Values 

### 28

c = 3.6 

### 29

t_0 = 39.1111 

### 30

beta_0 = 0 

### 31

beta_1 = 8.8911e

- 009 

### 32

beta_2 = 1.60475e

- 031 

### 33

beta_3 = 1.95818e

- 008 

### 34

35

36

Asymptotic Correlation Matrix of Para meter Estimates 

### 37

( *** The model parameter(s)

- beta_0

- beta_2 

### 38

have been estimated at a boundary point, or have been specified by the user, 

### 39

and do not appear in the correlation matrix ) 

### 40

41

c t_0 beta_1 beta_3 

### 42

43

c 1

- 0.53

- 0.93

- 0.99 

### 44

45

t_0

- 0.53 1 0.47 0.57 

### 46

47

beta_1

- 0.93 0.47 1 0.9 

### 48

49

beta_3

- 0.99 0.57 0.9 1 

### 50

51

52

Parameter Estimates 

### 53

95.0% Wald Confidence Interval 

### 54

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 55

c 3.74559 0.447309 2.86888 4.6223 

### 56

t_0 41.4581 2.14975 37.2447 45.6716 

### 57

beta_0 0 NA 

### 58

beta_1 4.37816e

- 009 1.07528e

- 008

- 1.6697e

- 008 2.54533e

- 008 

### 59

beta_2 0 NA 

### 60

beta_3 1.01904e

- 008 1.94164e

- 008

- 2.78651e

- 008 4.82458e

- 008 

### 61

62

NA

- Indi cates 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

- 108.512 6 229.024 

### 68

69

70

Data Summary 

### 71

CONTEXT 

### 72

### 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 C F I U Total Expected Response 

### 1

DOSE 

### 2

0 52 0 0 0 52 0.00 

### 3

0.54 44 0 8 0 52 6.77 

### 4

1.8 7 0 45 0 52 41.69 

### 5

5.2 0 9 43 0 52 49.97 

### 6

7

Minimum observation time for F tumor context = 44 

### 8

9

10

Benchmark Dose Computation 

### 11

Risk Response = Incidental 

### 12

Risk Type = Extra 

### 13

Confidence level = 0.9 

### 14

Time = 104 

### 15

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 Male Rat (Kroese et al., 2001): Hepatocellular Adenoma or Carcinoma 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: LiverKroeseM3.(d) 

### 6

======================================================================= 

### 7

8

The form of the probability function is: 

### 9

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 10

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 11

12

The parameter betas are restricted to be 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

24

Maximum number of iterations = 64 

### 25

Relative Function Convergence has been set to: 2.22045e

- 016 

### 26

Parameter Convergence has been set to: 1.49012e

- 008 

### 27

28

29

Default Initial Parameter Values 

### 30

c = 3.6 

### 31

t_0 = 34.6667 

### 32

beta_0 = 0 

### 33

beta_1 = 2.73535e

- 009 

### 34

beta_2 = 8.116e

- 028 

### 35

beta_3 = 1.43532e

- 008 

### 36

37

38

Asymptotic Correlation Matrix of Parameter Estimates 

### 39

( *** The model parameter(s)

- beta_0

- beta_ 2 

### 40

have been estimated at a boundary point, or have been specified by the user, 

### 41

and do not appear in the correlation matrix ) 

### 42

43

c t_0 beta_1 beta_3 

### 44

45

c 1

- 0.84

- 0.88

- 1 

### 46

47

t_0

- 0.84 1 0.71 0.86 

### 48

49

beta_1

- 0.88 0.71 1 0.86 

### 50

51

beta_3

- 1 0.86 0.86 1 

### 52

53

54

Parameter Estimates 

### 55

95.0% Wald Confidence Interval 

### 56

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 57

c 3.49582 0.629257 2.26249 4.72914 

### 58

t_0 40.2211 5.65421 29.1391 51.3032 

### 59

beta_0 0 NA 

### 60

beta_1 4.4390 6e

- 009 1.76051e

- 008

- 3.00664e

- 008 3.89445e

- 008 

### 61

beta_2 0 NA 

### 62

beta_3 2.35065e

- 008 6.47999e

- 008

- 1.03499e

- 007 1.50512e

- 007 

### 63

64

NA

- Indicates that this parameter has hit a bound implied by some inequality constraint 

### 65

and thus has no standard error. 

### 66

67

68

Log(likelihood) # Param AIC 

### 69

Fitted Model

- 138.544 6 289.088 

### 70

71

72

Data Summary 

### 73

### 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 CONTEXT 

### 1

C F I U Total Expected Response 

### 2

DOSE 

### 3

0 52 0 0 0 52 0.00 

### 4

0.54 48 0 4 0 52 3.38 

### 5

1.8 14 2 36 0 5 2 36.81 

### 6

5.2 3 17 32 0 52 49.55 

### 7

8

Minimum observation time for F tumor context = 52 

### 9

10

11

Benchmark Dose Computation 

### 12

Risk Response = Incidental 

### 13

Risk Type = Extra 

### 14

Confidence level = 0.9 

### 15

Time = 104 

### 16

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

### 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 Male Rat (Kroese et al., 2001): Duodenum or Jejunum Adenocarcinoma 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Da ta File: DuoJejKroeseM3.(d) 

### 6

======================================================================= 

### 7

8

The form of the probability function is: 

### 9

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 10

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 11

12

The parameter betas are restricted to be 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

25

User specifies the following parameters: 

### 26

t_0 = 0 

### 27

28

Maximum number of iterations = 64 

### 29

Relative Function Convergence has been set to: 2.22045e

- 016 

### 30

Parameter Con vergence has been set to: 1.49012e

- 008 

### 31

32

33

Default Initial Parameter Values 

### 34

c = 1.63636 

### 35

t_0 = 0 Specified 

### 36

beta_0 = 4.31119e

- 027 

### 37

beta_1 = 2.96347e

- 025 

### 38

beta_2 = 0 

### 39

beta_3 = 1.76198e

- 006 

### 40

41

42

Asymptotic Correlation Matrix of Parameter Estimates 

### 43

( *** The model parameter(s)

- t_0

- beta_0

- beta_1

- beta_2 

### 44

have been estimated at a boundary point, or have been specified by the user, 

### 45

and do not appear in the correlation matrix ) 

### 46

47

c beta_3 

### 48

49

c 1

- 1 

### 50

51

beta_3

- 1 1 

### 52

53

54

Parameter Estimates 

### 55

95.0% Wald Confidence Interval 

### 56

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 57

c 1.77722 2.03042

- 2.20233 5.75677 

### 58

beta_0 0 NA 

### 59

beta_1 0 NA 

### 60

beta_2 0 NA 

### 61

beta_3 9.82635e

- 007 8.29355e

- 006

- 1.52724e

- 005 1.72377e

- 005 

### 62

63

NA

- Indicates that this parameter has hit a bound implied by some inequality constraint 

### 64

and thus has no standard error. 

### 65

66

67

Log(likelihood) # Param AIC 

### 68

Fitted Model

- 28.4387 5 66.8773 

### 69

70

71

Data Summary 

### 72

CONTEXT 

### 73

C F I U Total Expected Response 

### 74

### 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 DOSE 

### 1

0 52 0 0 0 52 0.00 

### 2

0.54 52 0 0 0 52 0.03 

### 3

1.8 51 0 1 0 52 1.04 

### 4

5.2 43 0 9 0 52 8.96 

### 5

6

7

Benchmark Dose Computation 

### 8

Risk Response = Incidental 

### 9

Risk Type = Extra 

### 10

Specified effect = 0.1 

### 11

Confidence level = 0.9 

### 12

Time = 104 

### 13

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

### 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

Male Rat (Kroese et al., 2001): Skin or Mammary Gland Basal Cell Tumors 

### 2

3

======================================================================= 

### 4

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 5

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 6

Input Data File: SKinMamBasalKroeseM3.(d) 

### 7

== ===================================================================== 

### 8

9

The form of the probability function is: 

### 10

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 11

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 12

13

The parameter betas are restricted to be positive 

### 14

15

Dependent variable = CONTEXT 

### 16

Independent variables = DOSE, TIME 

### 17

18

Total number of observations = 208 

### 19

Total number of records with missing values = 0 

### 20

Total number of parameters in model = 6 

### 21

Total number of specified parameters = 1 

### 22

Degree of polynomial = 3 

### 23

24

25

26

User specifies the following parameters: 

### 27

t_0 = 0 

### 28

29

Maximum number of iterations = 64 

### 30

Relative Function Convergence has been set to: 2.22045e

- 016 

### 31

Parameter Con vergence has been set to: 1.49012e

- 008 

### 32

33

34

Default Initial Parameter Values 

### 35

c = 1.38462 

### 36

t_0 = 0 Specified 

### 37

beta_0 = 3.84298e

- 005 

### 38

beta_1 = 1.06194e

- 028 

### 39

beta_2 = 0 

### 40

beta_3 = 6.84718e

- 006 

### 41

42

43

Asymptotic Correlation Matrix of Parameter Estimates 

### 44

( *** The model parameter(s)

- t_0

- beta_1

- beta_2 

### 45

have been estimated at a boundary point, or have been specified by the user, 

### 46

and do not appear in the correlation matrix ) 

### 47

48

c beta_0 beta_3 

### 49

50

c 1

- 1

- 1 

### 51

52

beta_0

- 1 1 0.99 

### 53

54

beta_3

- 1 0.99 1 

### 55

56

57

Parameter Estimates 

### 58

95.0% Wald Confidence Interval 

### 59

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 60

c 1.47227 1.76686

- 1.9907 4.93525 

### 61

beta_0 2.54786e

- 005 0.000211261

- 0.000388585 0.000439542 

### 62

beta_1 0 NA 

### 63

beta_2 0 NA 

### 64

beta_3 4.81611e

- 006 3.49e

- 005

- 6.35866e

- 005 7.32188e

- 005 

### 65

66

NA

- Indicates that this parameter has hit a bound implied by some inequality constraint 

### 67

and thus has no standard error. 

### 68

69

70

Log(likelihood) # Param AIC 

### 71

### 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 Fitted Model

- 47.3623 5 104.725 

### 1

2

3

Data Summary 

### 4

CONTEXT 

### 5

C F I U Total Expected Response 

### 6

DOSE 

### 7

0 50 0 2 0 52 1.18 

### 8

0.54 51 0 1 0 52 1.22 

### 9

1.8 51 0 1 0 52 2.32 

### 10

5.2 39 0 13 0 52 12.54 

### 11

12

13

Benchmark Dose Computation 

### 14

Risk Response = Incidental 

### 15

Risk Type = Extra 

### 16

Confidence level = 0.9 

### 17

Time = 104 

### 18

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

### 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 Male Rat (Kroese et al., 2001): Skin or Mammary Gland Squamous Cell Tumors 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- int v, (c) by P. Spellucci 

### 5

Input Data File: SKinMamSCCKroeseM3.(d) 

### 6

======================================================================= 

### 7

8

The form of the probability function is: 

### 9

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 10

(beta_0+beta_1 *dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 11

12

The parameter betas are restricted to be 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

25

User specifies the following parameters: 

### 26

t_0 = 0 

### 27

28

Maximum number of iterations = 64 

### 29

Relative Function Convergence has been set to: 2.22045e

- 016 

### 30

Parameter Convergence has been set to: 1.49012e

- 008 

### 31

32

33

Default Initial Parameter Values 

### 34

c = 3 

### 35

t_0 = 0 Specified 

### 36

beta_0 = 0 

### 37

beta_1 = 1.25256e

- 008 

### 38

beta_2 = 1.25627e

- 030 

### 39

beta_3 = 3.34696e

- 009 

### 40

41

42

Asymptotic Correlation Matrix of Param eter Estimates 

### 43

( *** The model parameter(s)

- t_0

- beta_0

- beta_2 

### 44

have been estimated at a boundary point, or have been specified by the user, 

### 45

and do not appear in the correlation matrix ) 

### 46

47

c beta_1 beta_3 

### 48

49

c 1

- 0.99

- 1 

### 50

51

beta_1

- 0.99 1 0.99 

### 52

beta_3

- 1 0.99 1 

### 53

54

55

Parameter Estimates 

### 56

95.0% Wald Confidence Interval 

### 57

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 58

c 2.96213 2.591

- 2.11613 8.04039 

### 59

beta_0 0 NA 

### 60

beta_1 1.50104e

- 008 1.86972e

- 007

- 3.51447e

- 007 3.81468e

- 007 

### 61

beta_2 0 NA 

### 62

beta_3 3.9084e

- 009 4.15374e

- 008

- 7.75033e

- 008 8.53201e

- 008 

### 63

64

NA

- Indicates that this parameter has hit a bound implied by some inequality constraint 

### 65

and thus has no standard error. 

### 66

67

68

Log(likelihood) # P aram AIC 

### 69

Fitted Model

- 27.652 5 65.304 

### 70

71

72

Data Summary 

### 73

CONTEXT 

### 74

### 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 C F I U Total Expected Response 

### 1

DOSE 

### 2

0 52 0 0 0 52 0.00 

### 3

0.54 51 0 1 0 52 0.42 

### 4

1.8 51 0 1 0 52 2.12 

### 5

5.2 46 0 6 0 52 5.51 

### 6

7

8

Benchmark Dose Computation 

### 9

Risk Response = Incidental 

### 10

Risk Type = Extra 

### 11

Confidence level = 0.9 

### 12

Time = 104 

### 13

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 Male Rat (Kroese et al., 2001): Kidney Urothelial Carcinomas 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: KidneyUrothelialCarKroeseM3.(d) 

### 6

======================================================================= 

### 7

8

The form of the probability function is: 

### 9

P[response] = 1

- E XP{

- (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

25

User specifies the following parameters: 

### 26

t_0 = 0 

### 27

28

Maximum number of iterations = 64 

### 29

Relative Function Convergence has been set to: 2.22045e

- 016 

### 30

Parameter Convergence has been set to: 1.49012e

- 008 

### 31

32

33

Default Initial Parameter Values 

### 34

c = 1.63636 

### 35

t_0 = 0 Specified 

### 36

beta_0 = 3.78734e

- 027 

### 37

beta_1 = 1.59278e

- 027 

### 38

beta_2 = 2.718e

- 024 

### 39

beta_3 = 4.96063e

- 007 

### 40

41

42

Asy mptotic Correlation Matrix of Parameter Estimates 

### 43

( *** The model parameter(s)

- t_0

- beta_0

- beta_1

- beta_2 

### 44

have been estimated at a boundary point, or have been specified by the user, 

### 45

and do not appear in the correlation matrix ) 

### 46

47

c beta_3 

### 48

49

c 1

- 1 

### 50

51

beta_3

- 1 1 

### 52

53

54

Parameter Estimates 

### 55

95.0% Wald Confidence Interval 

### 56

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 57

c 1.74897 3.79403

- 5.68719 9.18512 

### 58

beta_0 0 NA 

### 59

beta_1 0 NA 

### 60

beta_2 0 NA 

### 61

beta_3 3.11107e

- 007 4.90313e

- 006

- 9.29885e

- 006 9.92107e

- 006 

### 62

63

NA

- Indicates that this parameter has hit a 

### 64

bound implied by some inequality constraint 

### 65

and thus has no standard error. 

### 66

67

68

Log(likelihood) # Param AIC 

### 69

Fitted Model

- 11.3978 5 32.7956 

### 70

71

72

Data Summary 

### 73

CONTEXT 

### 74

### 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 C F I U Total Expected Response 

### 1

DOSE 

### 2

0 52 0 0 0 52 0.00 

### 3

0.54 52 0 0 0 52 0.01 

### 4

1.8 52 0 0 0 52 0.29 

### 5

5.2 49 0 3 0 52 2.71 

### 6

7

8

Benchmark Dose Computation 

### 9

Risk Response = Incidental 

### 10

Risk Type = Extra 

### 11

Confidence level = 0.9 

### 12

Time = 104 

### 13

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

### 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 Female Rat (Kroese et al., 2001): Oral Cavity or Forestomach, Squamous Cell Papilloma or Carcinoma 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: Or alForstKroeseF3.(d) 

### 6

======================================================================= 

### 7

8

The form of the probability function is: 

### 9

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 10

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 11

12

The p arameter 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

24

Maximum number of iterations = 64 

### 25

Relative Function Convergence has been set to: 2.22045e

- 016 

### 26

Parameter Convergence has been set to: 1.49012e

- 008 

### 27

28

29

Default Initial Parameter Values 

### 30

c = 3.6 

### 31

t_0 = 45.1111 

### 32

beta_0 = 1.11645e

- 009 

### 33

beta_1 = 4.85388e

- 009 

### 34

beta_2 = 0 

### 35

beta_3 = 1.95655e

- 008 

### 36

37

Asymptotic Correlation Matrix of Parameter Estimates 

### 38

( *** The model parameter(s)

- beta_2 

### 39

have been estimated at a boundary point, or h ave been specified by the user, 

### 40

and do not appear in the correlation matrix ) 

### 41

42

c t_0 beta_0 beta_1 beta_3 

### 43

44

c 1

- 0.79

- 0.92

- 0.93

- 1 

### 45

46

t_ 0

- 0.79 1 0.73 0.72 0.8 

### 47

48

beta_0

- 0.92 0.73 1 0.79 0.92 

### 49

50

beta_1

- 0.93 0.72 0.79 1 0.91 

### 51

52

beta_3

- 1 0.8 0.92 0.91 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 3.52871 0.701117 2.15454 4.90287 

### 59

t_0 46.553 5.93306 34.9244 58.1816 

### 60

beta_0 1.53589e

- 009 5.40523e

- 009

- 9.05817e

- 009 1.21299e

- 008 

### 61

beta_1 7.57004e

- 009 2.9647e

- 008

- 5.05369e

- 008 6.5677e

- 008 

### 62

beta_2 0 NA 

### 63

beta_3 2.53126e

- 008 7.66404e

- 008

- 1.249e

- 007 1.75525e

- 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) # P aram AIC 

### 70

Fitted Model

- 94.5119 6 201.024 

### 71

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 Data Summary 

### 1

CONTEXT 

### 2

C F I U Total Expected Response 

### 3

DOSE 

### 4

0 51 0 1 0 52 1.14 

### 5

0.49 46 0 6 0 52 4.90 

### 6

1.6 22 0 30 0 52 31.81 

### 7

4.6 2 7 43 0 52 49.43 

### 8

9

Minimum observation time for F tumor context = 58 

### 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

0.538801

0.3281 35

0.717127 0.01

0.0981283

0.0345104

0.325

909 0.001

0.0100797

0.00344714

>

0.0806373



### 18

### 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 Female Rat (Kroese et al., 2001): Hepatocellular Adenoma or Carcinoma 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: LiverKroeseF3.(d) 

### 6

Fri Apr 16 09:08:03 2010 

### 7

======================================================================= 

### 8

9

Timer to Tumor Model, Liver Hepatocellular Tumors, Kroese et al, Female 

### 10

11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 12

13

The form of the probability f unction is: 

### 14

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 15

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 16

17

The parameter betas are restricted to be positive 

### 18

19

Dependent variable = CONTEXT 

### 20

Independent variables = DOSE, TIME 

### 21

22

Total number of observations = 208 

### 23

Total number of records with missing values = 0 

### 24

Total number of parameters in model = 6 

### 25

Total number of specified parameters = 0 

### 26

Degree of polynomial = 3 

### 27

28

29

Maximum number of iterations = 64 

### 30

Relative Function Conver gence 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 = 3.6 

### 36

t_0 = 31.7778 

### 37

beta_0 = 0 

### 38

beta_1 = 4.9104e

- 031 

### 39

beta_2 = 5.45766e

- 030 

### 40

beta_3 = 3.44704e

- 008 

### 41

42

43

Asymptotic Correlation Matrix of Parameter Estimates 

### 44

( *** The model parameter(s)

- beta_0

- beta_1

- beta_2 

### 45

have been estimated at a boundary point, or have been specified by the user, 

### 46

and do not appear in the correlation matrix ) 

### 47

48

c t_0 beta_3 

### 49

50

c 1

- 0.9

- 1 

### 51

52

t_0

- 0.9 1 0.92 

### 53

54

beta_3

- 1 0.92 1 

### 55

56

57

Parameter Estimates 

### 58

95.0% Wald Confidence Interval 

### 59

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 60

c 3.11076 0.549208 2.03434 4.18719 

### 61

t_0 38.6965 5.21028 28.4846 48.9085 

### 62

beta_0 0 NA 

### 63

beta_1 0 NA 

### 64

beta_2 0 NA 

### 65

beta_3 2.94354e

- 007 7.19418e

- 007

- 1.11568e

- 006 1.70439e

- 006 

### 66

67

NA

- Indicates that this parameter has hit a bound implied by some inequality constraint 

### 68

and thus has no standard error. 

### 69

70

71

Log(likelihood) # P aram AIC 

### 72

Fitted Model

- 228.17 6 468.34 

### 73

### 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

Data Summary 

### 3

CONTEXT 

### 4

C F I U Total Expected Response 

### 5

DOSE 

### 6

0 52 0 0 0 52 0.00 

### 7

0.49 51 0 1 0 52 3.02 

### 8

1.6 13 12 27 0 52 38.36 

### 9

4.6 1 38 13 0 52 51.36 

### 10

11

Minimum observation time for F tumor context = 44 

### 12

13

14

Benchmar k Dose Computation 

### 15

Risk Response = Incidental 

### 16

Risk Type = Extra 

### 17

Confidence level = 0.9 

### 18

Time = 104 

### 19

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

### 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 Female Rat (Kroese et al., 2001): Duodenum or Jejunum Adenocarcinoma 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: DuoJejKroeseF3.(d) 

### 6

======================================================================= 

### 7

The form of the probability function is: 

### 8

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 9

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 10

11

The parameter betas are restricted to be positive 

### 12

13

Dependent variable = CONTEXT 

### 14

Independent variables = DOSE, TIME 

### 15

16

Total number of observations = 208 

### 17

Total number of records with missing values = 0 

### 18

Total number of parameters in model = 6 

### 19

Total number of specified parameters = 1 

### 20

Degree of polynomial = 3 

### 21

22

23

User specifies the following parameters: 

### 24

t_0 = 0 

### 25

26

Maximum number of iterations = 64 

### 27

Relative Function Convergence has been set to: 2.22045e

- 016 

### 28

Parameter Convergence has been set to: 1.49012e

- 008 

### 29

30

31

Default Initial Parameter Values 

### 32

c = 2.25 

### 33

t_0 = 0 Specified 

### 34

beta_0 = 0 

### 35

beta_1 = 0 

### 36

beta_2 = 0 

### 37

beta_3 = 7.289e

- 008 

### 38

39

40

Asymptotic Correlation Matrix of Parameter Estimates 

### 41

( *** The model parameter(s)

- t_0

- beta_0

- beta_1

- beta_2 

### 42

have been estimated at a boundary point, or have been specified by the user, 

### 43

and do not appear in the correlation matrix ) 

### 44

45

c beta_3 

### 46

47

c 1

- 1 

### 48

49

beta_3

- 1 1 

### 50

51

52

Parameter Estimates 

### 53

95.0% Wald Confidence Interval 

### 54

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 55

c 2.32531 3.58729

- 4.70565 9.35626 

### 56

beta_0 0 NA 

### 57

beta_1 0 NA 

### 58

beta_2 0 NA 

### 59

beta_3 5.32209e

- 008 7.98487e

- 007

- 1.51178e

- 006 1.61823e

- 006 

### 60

61

NA

- Indicates that this parameter has hit a bound implied by some inequality constraint 

### 62

and thus has no standard error. 

### 63

64

65

Log(likelihood) # P aram AIC 

### 66

Fitted Model

- 13.8784 5 37.7569 

### 67

68

69

Data Summary 

### 70

CONTEXT 

### 71

C F I U Total Expected Response 

### 72

DOSE 

### 73

### 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 0 52 0 0 0 52 0.00 

### 1

0.49 52 0 0 0 52 0.01 

### 2

1.6 52 0 0 0 52 0.44 

### 3

4.6 48 0 4 0 52 3.57 

### 4

5

6

Benchmark Dose Computation 

### 7

Risk Res ponse = Incidental 

### 8

Risk Type = Extra 

### 9

Confidence level = 0.9 

### 10

Time = 104 

### 11

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

### 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 Table C

- 1 8 . Summary of human equivalent overall oral slope factors

, 1

based on male and female rat tumor incidence 2

Data set Tumor site BMD 001 BMDL 001 Risk value a at

SD SD 2 Properties

of total

variance BMD 001 BMDL 001

Males Oral cavity/forestomach 6.37 × 10 - 3 2.86 × 10 - 3 1.57 × 10 - 1 3.50 × 10 - 1 1.17 × 10 - 1 1.38 × 10 - 2 0.64

Liver 2.00 × 10 - 2 5.30 × 10 - 3 5.00 × 10 - 2 1.89 × 10 - 1 8.42 × 10 - 2 7.09 × 10 - 3 0.33

Duodenum/jejunum 6.42 × 10 - 1 4.21 × 10 - 2 1.56 × 10 - 3 2.38 × 10 - 2 1.35 × 10 - 2 1.82 × 10 - 4 0.01

Skin/mammary gland:

basal cell 6.06 × 10 - 1 4.24 × 10 - 2 1.65 × 10 - 3 2.36 × 10 - 2 1.33 × 10 - 2 1.78 × 10 - 4 0.01

Skin/mammary gland:

squam. cell 7.06 × 10 - 2 2.11 × 10 - 2 1.42 × 10 - 2 4.75 × 10 - 2 2.03 × 10 - 2 4.10 × 10 - 4 0.02

Kidney 9.84 × 10 - 1 7.48 × 10 - 2 1.02 × 10 - 3 1.34 × 10 - 2 7.51 × 10 - 3 5.64 × 10 - 5 0.00

Sum, risk values at BMD 001 : 2.25 × 10 - 1 Sum, SD 2

: 2.17 × 10 - 2

Overall SD b

: 1.47 × 10 - 1

Upper bound on sum of risk estimates c : 4.68 × 10 - 1

Females Oral cavity/forestomach 3.45 × 10 - 3 1.01 × 10 - 2 2.90 × 10 - 1 9.92 × 10 - 2 1.16 × 10 - 1 1.35 × 10 - 2 0.91

Liver 1.53 × 10 - 2 1.22 × 10 - 1 6.54 × 10 - 2 8.21 × 10 - 3 3.48 × 10 - 2 1.21 × 10 - 3 0.08

Duodenum/jejunum 5.85 × 10 - 2 7.27 × 10 - 1 1.71 × 10 - 2 1.38 × 10 - 3 9.56 × 10 - 3 9.13 × 10 - 5 0.01

Sum, risk values at BMD 001 : 1.09 × 10 - 1 Sum, SD 2

: 1.48 × 10 - 2

Overall SD: 1.22 × 10 - 1

Upper bound on sum of risk estimates c : 3.09E

- 01

a Risk value = 0.001/BMDL

001

. b Overall SD = (sum, SD 2 ) 0.5

.

c Upper bound on the overall risk estimate = sum of BMD 001 risk values + 1.645 × overall SD.

Source of data : Kroese et al. (2001).



### Table C

- 1 9

. Summary of model selection among multistage

- Weibull 3

models fit to alimentary tract tumor data for female mice 4

Model

stages AIC BMD 10 BMD L 10 − BMD U 10 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

Source of data: Beland and Culp ( 1998) 

### 6

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 Female Mice (Beland and Culp, 1998): Alimentary Tract Squamous Cell Tumors 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: C: \ msw10

- 09 \ benzo[a]pyrene_FemaleSquamF3i.(d) 

### 6

======================================================================= 

### 7

8

The form of the probability function is: 

### 9

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 10

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 11

12

The parameter betas are restricted to be positive 

### 13

14

Dependent variable = Class 

### 15

Independent variables = Dose, time 

### 16

17

Total number of observations = 191 

### 18

Total number of records wi th missing values = 0 

### 19

Total number of parameters in model = 6 

### 20

Total number of specified parameters = 0 

### 21

Degree of polynomial = 3 

### 22

23

24

Maximum number of iterations = 64 

### 25

Relative Function Convergence has been set to: 2.22045e

- 016 

### 26

Parameter Convergence has been set to: 1.49012e

- 008 

### 27

28

29

30

User Inputs Initial Parameter Values 

### 31

c = 2 

### 32

t_0 = 15 

### 33

beta_0 = 1.6e

- 014 

### 34

beta_1 = 0 

### 35

beta_2 = 5.5e

- 012 

### 36

beta_3 = 4.4e

- 012 

### 37

38

39

Asymptotic Correlation Matrix of Parameter Estimates 

### 40

c t_0 beta_0 beta_1 beta_2 beta_3 

### 41

42

c 1

- 0.78

- 0.97

- 0.42

- 0.99

- 0.99 

### 43

44

t_0

- 0.78 1 0.76 0.39 0.74 0.84 

### 45

46

beta_0

- 0.97 0.76 1 0.33 0.97 0.96 

### 47

48

beta_1

- 0.42 0.39 0.33 1 0.31 0.46 

### 49

50

beta_2

- 0.99 0.74 0.97 0.31 1 0.97 

### 51

52

beta_3

- 0.99 0.84 0.96 0.46 0.97 1 

### 53

54

55

Parameter Estimates 

### 56

95.0% Wald Confidence Interval 

### 57

Var iable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 58

c 6.92317 1.33874 4.29929 9.54705 

### 59

t_0 13.9429 4.96646 4.20881 2 3.677 

### 60

beta_0 2.46916e

- 016 1.47619e

- 015

- 2.64636e

- 015 3.14019e

- 015 

### 61

beta_1 0 1.30525e

- 014

- 2.55825e

- 014 2.55825e

- 014 

### 62

beta_2 5.85452e

- 014 3.75144e

- 013

- 6.76723e

- 013 7.93813e

- 013 

### 63

beta_3 9.76542e

- 014 5.62017e

- 013

- 1.00388e

- 012 1.19919e

- 012 

### 64

65

66

Log(likelihood) # Param AIC 

### 67

Fitted Model

- 306.265 6 624.53 

### 68

69

70

Data Summary 

### 71

Class 

### 72

C F I U Total Expected Response 

### 73

Dose 

### 74

### Toxicological Review of benzo[a]pyrene

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



### C

- 57 DRAFT

— DO NOT CITE OR QUOTE 0 47 0 1 0 48 0.93 

### 1

0.1 45 0 3 0 48 3.21 

### 2

0.48 8 23 15 1 47 30.82 

### 3

2.3 1 46 0 1 48 41.91 

### 4

5

Minimum observation time for F tumor context = 39 

### 6

7

8

Benchmark Dose Computation 

### 9

Risk Response = Incidental 

### 10

Risk Type = Ex tra 

### 11

Specified effect = 0.1 

### 12

Confidence level = 0.9 

### 13

14

Time = 104 

### 15

16

BMD = 0.126983 

### 17

BMDL = 0.0706103 

### 18

BMDU = 0.179419 

### 19

20

21

22

### Toxicological Review of benzo[a]pyrene

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



### C

- 58 DRAFT

— DO NOT CITE OR QUOTE DOSE

- RESPONSE MODELING FOR THE INHALATION UNIT RISK 1

As with the tumor data used for the oral slope factor (see Dose Response

- modeling for the 

### 2

Oral Slope Factor Section ), there was earlier occurrence of tumors with increasing exposure, and 

### 3

early termination of the high

- dose group (Thyssen et al., 1981; see Appendix B for study details). 

### 4

The computer software program MSW (U.S. EPA, 2010) was used as described in the analysis of the 

### 5

oral carcinogenicity data. 

### 6

Thyssen et al. (1981) did not determine cause of death for an y of the animals. Bounding 

### 7

estimates for the Thyssen et al. (1981) data were developed by treating the tumors alternately as 

### 8

either all incidental or all fatal. In either case, therefore, an estimate of t 0 (the time between a 

### 9

tumor first becoming observa ble and causing death) could not be estimated. The data analyzed are 

### 10

summarized in Table C

- 20 , the results are summarized in Table C

- 2 1 , and the modeling details 

### 11

follow. 

### 12

Table C

- 20 . Individual pathology and tumor occurrence data for male 13

Syrian hamsters exposed to benzo[a]pyrene via inhalation for lifetime

— 

### 14

Thyssen et al. (1981). 15

Nominal

exposure

concentration

(mg/m 3 ) 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

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



### C

- 59 DRAFT

— DO NOT CITE OR QUOTE Nominal

exposure

concentration

(mg/m 3 ) 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

### Toxicological Review of benzo[a]pyrene

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



### C

- 60 DRAFT

— DO NOT CITE OR QUOTE Nominal

exposure

concentration

(mg/m 3 ) 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.



### Table C

- 2 1

. Summary of model selection among multistage

- Weibull 1

models fit to tumor data for male hamsters 2

Tumor context Model

stages AIC BMD 10 BMD L 10 Model selection rationale

A ll 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

( BMDU 10 = 0 .350 )

A ll 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

( BMDU1 0 = 0.719 )

Data source: Thyssen et al. (1981) 

### 3

Output for squamous cell neoplasia following inhalation exposure to BaP : all tumors considered 

### 4

incidental to cause of death 

### 5

6

======================================================================= 

### 7

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 8

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 9

Input Data File: C: \ msw \ benzo[a]pyrene

- Thyssen_inc2st.(d) 

### 10

======================================================================= 

### 11

12

The form of the probability function is: 

### 13

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 14

(beta_0+beta_1*dose^1+beta_2*dose^2)} 

### 15

16

The parameter betas are restricted to be positive 

### 17

18

Dependent variable = Class 

### 19

Independent variables = Conc, Time 

### 20

21

Total number of observations = 96 

### 22

Total number of records with missing valu es = 0 

### 23

Total number of parameters in model = 5 

### 24

Total number of specified parameters = 1 

### 25

Degree of polynomial = 2 

### 26

27

28

29

User specifies the following parameters: 

### 30

t_0 = 0 

### 31

### Toxicological Review of benzo[a]pyrene

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



### C

- 61 DRAFT

— DO NOT CITE OR QUOTE 1

Maximum number of iterations = 32 

### 2

Relative Function Converge nce has been set to: 1e

- 008 

### 3

Parameter Convergence has been set to: 1e

- 008 

### 4

5

6

Default Initial Parameter Values 

### 7

c = 3.6 

### 8

t_0 = 0 Specified 

### 9

beta_0 = 1.18657e

- 031 

### 10

beta_1 = 1.49e

- 030 

### 11

beta_2 = 6.10362e

- 008 

### 12

13

14

Asymptotic Correlation Matrix of Parameter Estimates 

### 15

( *** The model parameter(s)

- t_0

- beta_0

- beta_1 

### 16

have been estimated at a boundary point, or have been specified by the user, 

### 17

and do not appear in the correlation matrix ) 

### 18

19

c beta_2 

### 20

21

c 1

- 1 

### 22

23

beta_2

- 1 1 

### 24

25

26

Parameter Estimates 

### 27

95.0% Wald Confidence Interval 

### 28

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 29

c 4.21938 0.840997 2.57105 5.8677 

### 30

beta_0 0 NA 

### 31

beta_1 0 NA 

### 32

beta_2 4.00402e

- 009 1.495e

- 008

- 2.52974e

- 008 3.33054e

- 008 

### 33

34

NA

- Indicates that this parameter has hit a 

### 35

bound implied by some inequality constraint 

### 36

and thus has no standard error. 

### 37

38

39

Log(likelihood) # Param AIC 

### 40

Fitted Model

- 19.967 4 47.9339 

### 41

42

43

Data Summary 

### 44

Class 

### 45

C F I U Total Expected Response 

### 46

Conc 

### 47

0 23 0 0 0 23 0.00 

### 48

0.25 24 0 0 0 24 1.92 

### 49

1 8 0 18 0 26 16.04 

### 50

4.3 5 0 18 0 23 18.22 

### 51

52

53

Benchmark Dose Computation 

### 54

Risk Resp onse = Incidental 

### 55

Risk Type = Extra 

### 56

Specified effect = 0.1 

### 57

Confidence level = 0.9 

### 58

59

Time = 104 

### 60

61

BMD = 0.284958 

### 62

BMDL = 0.197807 

### 63

BMDU = 0.350247 

### 64

65

66

### Toxicological Review of benzo[a]pyrene

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



### C

- 62 DRAFT

— DO NOT CITE OR QUOTE 1

### Toxicological Review of benzo[a]pyrene

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



### C

- 63 DRAFT

— DO NOT CITE OR QUOTE Output for respiratory tract tumors: all tumors considered to be cause of death 

### 1

2

======================================================================= 

### 3

Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) 

### 4

Solutions are obtained using donlp2

- intv, (c) by P. Spellucci 

### 5

Input Data File: C: \ msw \ benzo[a]pyrene

- Thyssen_allfatal_noU_3st.(d) 

### 6

======================================================================= 

### 7

8

The form of the probability function is: 

### 9

P[response] = 1

- EXP{

- (t

- t_0)^c * 

### 10

(beta_0+beta_1*dose^1+beta_2*dose^2+beta_3*dose^3)} 

### 11

12

The parameter betas are restricted to be positive 

### 13

14

Dependent variable = Class 

### 15

Independent variables = Conc, Time 

### 16

17

Total number of observati ons = 96 

### 18

Total number of records with missing values = 0 

### 19

Total number of parameters in model = 6 

### 20

Total number of specified parameters = 1 

### 21

Degree of polynomial = 3 

### 22

23

24

25

User specifies the following parameters: 

### 26

t_0 = 0 

### 27

28

Maximum number of iterations = 32 

### 29

Relative Function Convergence has been set to: 1e

- 008 

### 30

Parameter Convergence has been set to: 1e

- 008 

### 31

32

33

Default Initial Parameter Values 

### 34

c = 4.5 

### 35

t_0 = 0 Specified 

### 36

beta_0 = 0 

### 37

beta_1 = 1.37501e

- 010 

### 38

beta_2 = 2.84027e

- 010 

### 39

beta_3 = 1.44668e

- 037 

### 40

41

42

Asymptotic Correlation Matrix of Parameter Estimates 

### 43

( *** The model parameter(s)

- t_0

- beta_0

- beta_1

- beta_2 

### 44

have been estimated at a boundary point, or have been specified by the user, 

### 45

and do not appear in the correlation matrix ) 

### 46

47

c beta_3 

### 48

49

c 1

- 1 

### 50

51

beta_3

- 1 1 

### 52

53

54

Parameter Estimates 

### 55

95.0% Wald Confidence Interval 

### 56

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 57

c 8.95016 0.896607 7.19284 10.7075 

### 58

beta_0 0 NA 

### 59

beta_1 0 NA 

### 60

beta_2 0 NA 

### 61

beta_3 3.43452e

- 019 1.39727e

- 018

- 2.39515e

- 018 3.08205e

- 018 

### 62

63

N A

- Indicates that this parameter has hit a 

### 64

bound implied by some inequality constraint 

### 65

and thus has no standard error. 

### 66

67

68

Log(likelihood) # Param AIC 

### 69

Fitted Model

- 144.522 5 299.043 

### 70

71

72

Data Summary 

### 73

### Toxicological Review of benzo[a]pyrene

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



### C

- 64 DRAFT

— DO NOT CITE OR QUOTE Class 

### 1

C F I U Total 

### 2

Conc 

### 3

0 23 0 0 0 23 

### 4

0.25 24 0 0 0 24 

### 5

1 8 18 0 0 26 

### 6

4.3 5 18 0 0 23 

### 7

8

Minimum observation time for F tumor context = 40 

### 9

10

11

Benchmark Dose Computation 

### 12

Risk Response = Fatal 

### 13

Risk Type = Extra 

### 14

Specified effect = 0.1 

### 15

Confidence level = 0.9 

### 16

17

Time = 104 

### 18

19

BMD = 0.647659 

### 20

BMDL = 0.461415 

### 21

BMDU = 0.719325 

### 22

23

24

### Toxicological Review of benzo[a]pyrene

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



### C

- 65 DRAFT

— DO NOT CITE OR QUOTE DOSE

- RESPONSE MODELING FOR THE DERMAL SLOPE FACTOR 1

M odeling methods: 

### 2

For e ach endpoint, multistage models (BMDS; U.S. EPA, 2012 ; v 2.1 ) were fitted to the data 

### 3

using the maximum likelihood method. Each model was tested for goodness

- of

- fit using a chi



### 4

square goodness

- of

- fit test (χ 2 p

- value < 0.05 indicates lack of fit). Other factors were used to 

### 5

assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the low

- dose region and in 

### 6

the vicinity of the BMR. T he BMDL estimate (95% lower confidence limit on the BMD, as estim ated 

### 7

by the profile likelihood method) and AIC value were used to select a best

- fit model from among the 

### 8

models exhibiting adequate fit. The data modeled are summarized in Tables C

- 2 2 through C

- 2 5

. 

### 9

The modeling results are summarized in Table C

- 2 6 . The modeling details are provided with 

### 10

Figures C

- 8 through C

- 19

. 

### 11

Data adjustments prior to modeling : 

### 12

Roe et al. (1970) applied benzo[a]pyrene dermally for 93 weeks or until natural death; with 

### 13

the exception of the highest dose group, each group still had app roximately 20 animals at 86 weeks 

### 14

(see Table C

- 2 2 ) . The tumors were first observed in the lowest and highest dose groups during the 

### 15

interval of weeks 29

– 43. Mice that died before week 29 were likely not at risk of tumor 

### 16

development. However, because tum or incidence and mortality were reported in 100

- day 

### 17

intervals, mice that had not been on study long enough to develop tumors were not easily 

### 18

identifiable. Incidence denominators reflect the number of animals alive at week 29, and may thus 

### 19

tend to lead to underestimates of tumor risk if the number of animals at risk ha s been 

### 20

overestimated. 

### 21

Schmidt et al. (1973) did not report survival information; instead, the authors provided 

### 22

incidences based on the numbers of mice initially included in each dose group at the start of the 

### 23

study. Overall latency was reported for the two high

- dose groups in each series, but these data only 

### 24

describe the survival of mice with tumors (animals were removed from study when a tumor 

### 25

appeared). It is not clear how long exposures la sted overall in each dose group, or whether some 

### 26

mice may have died on study from other causes before tumors appeared. While it is possible that 

### 27

no mice died during the study, all of the other studies considered here demonstrate mortality. 

### 28

However, the d ata were modeled as reported, recognizing the possibility of underestimating risk 

### 29

associated with incidences reported and lack of duration of exposure. (See Table C

- 2 2 .) 

### 30

Schmähl et al. (1977) reported that reduced numbers of animals at risk (77−88 mice pe r 

### 31

dose group compared with the initial group sizes of 100) resulted from varying rates of autolysis. 

### 32

No other survival or latency information was provided, so all exposures were assumed to have 

### 33

lasted for 104 weeks and were modeled as reported. Given the results of the other studies, it seems 

### 34

possible that the numbers at risk in each group may be overestimated, which could lead to an 

### 35

underestimate of lifetime risk. (See Table C

- 2 2 .) 

### 36

Habs et al. (1980) reported age

- standardized skin tumor incidence rates, indicating earlier 

### 37

mortality in the two highest dose groups (2.8 and 4.6 μg/application). These rates were used to 

### 38

estimate the number at risk in the dose

- response modeling, by dividing the number of mice with 

### 39

### Toxicological Review of benzo[a]pyrene

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



### C

- 66 DRAFT

— DO NOT CITE OR QUOTE tumors by the age

- standardized rates. Exposu re lasted longer than 104 weeks in the two lower 

### 1

exposure groups, at about 120 and 112 weeks, and until about 88 weeks in the highest exposure 

### 2

group. Incidence in the two lower exposure groups may be higher than if the exposure had lasted 

### 3

just 104 weeks. There was mortality in the first 52 weeks of exposure, about 10−15% in the three 

### 4

exposure groups, but because there was no information concerning when tumors first appeared, it 

### 5

is not possible to determine how much the early mortality may have impacted th e number of mice 

### 6

at risk in each group. (See Table C

- 2 2 .) 

### 7

Habs et al. (1984) reported mean survival times (with 95% CIs) for each dose group. The 

### 8

CIs supported the judgment that the control and lower dose groups were treated for 104 weeks. 

### 9

The higher do se group (4 μg/application) was probably treated for <104 weeks, because the upper 

### 10

95% confidence limit for the mean survival was approximately 79 weeks. However, since it was 

### 11

not possible to estimate a more realistic duration for this group, an estimate of 104 weeks was 

### 12

used. (See Table C

- 2 2 .) 

### 13

The studies by Poel (1960, 1959) were conducted in male mice and used toluene as the 

### 14

vehicle. In addition to a control group, the 1959 study included nine dose groups of one mouse 

### 15

strain (C57L) and the 1960 study included seven dose groups of three other mouse strains. Both 

### 16

studies demonstrated high mortality and tumor incidence at higher exposure levels. All C57L mice 

### 17

in dose groups with >3.8 μg/application died by week 44 of the study (Poel, 1959). Therefore, 

### 18

these five dose groups were omitted prior to dose

- response modeling because of the relatively 

### 19

large uncertainty in extrapolating cancer risk as a result of lifetime exposure. Four dose groups in 

### 20

addition to control remained. Among these groups, mice surv ived and were exposed until weeks 

### 21

83

– 103. According to the lifespan ranges provided, at least one mouse in each dose group died 

### 22

before the first appearance of tumor, but insufficient information was available to determine how 

### 23

many; consequently, the incid ence denominators were not adjusted. The dose

- response data are 

### 24

summarized in Table C

- 2 3

. 

### 25

For the Poel (1960) studies, all tumors in the highest three dose groups for each of the three 

### 26

mouse strains had occurred by week 40. While these observations suppo rt concern for cancer risk, 

### 27

as noted above such results are relatively uncertain for estimating lifetime cancer risk. In addition, 

### 28

there was no information indicating duration of exposure for the mice without tumors; although 

### 29

exposure was for lifetime, it might have been as short as for the mice with tumors. Overall, these 

### 30

datasets did not provide sufficient information to estimate the extent of exposure associated with 

### 31

the observed tumor incidence. Consequently, the experiments reported by Poel (1960) w ere not 

### 32

used for dose

- response modeling. 

### 33

Grimmer et al. (1984, 1983), studied female CFLP mice, using acetone:DMSO (1:3) as the 

### 34

vehicle. Mean or median latency times were reported (as well as measures of variability), but no 

### 35

information concerning overall length of exposure or survival was included in the results. The total 

### 36

of tumor

- bearing mice and the reported percentages of mice with any skin tumors was reported 

### 37

and varied, at most, one animal from the number of animals initially placed on study. The 

### 38

decreasing latency and variability and increasing tumor incidence with increasing benzo[a]pyrene 

### 39

exposure suggests that exposure probably did not last for 104 weeks in at least the high

- dose 

### 40

group, but the available information did not provide duration of exposure. The data reported were 

### 41

### Toxicological Review of benzo[a]pyrene

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



### C

- 67 DRAFT

— DO NOT CITE OR QUOTE modeled under the assumption that at least some animals in each group were treated and survived 

### 1

until week 104. (See Table C

- 2 4 .) 

### 2

Sivak et al. (1997), exposed male C3H/HeJ mice dermally to benzo[a]pyrene in 

### 3

cyclohexanone/ acetone (1:1) for 24 months, and reported mean survival times for each group. All 

### 4

high

- dose mice died before the final sacrifice. From the information provided, it is apparent that 

### 5

the animals in the control and lower two dose groups survived until study termination at week 104. 

### 6

The study authors did not report how long treatment in the highest dose group lasted, but 

### 7

estimation of the figure from the publication suggest that exposure duration was 74 wee ks. (See 

### 8

Table C

- 2 5 ). 

### 9

### Toxicological Review of benzo[a]pyrene

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



### C

- 68 DRAFT

— DO NOT CITE OR QUOTE 1

Table C

- 2 2

. Skin tumor inc idence, benign or malignant in female Swiss 2

or NMRI mice dermally exposed to benzo[a]pyrene 3

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.,

1970 a,b Swiss 0 (acetone)

0.1

0.3

1

3

9 0

0.04

0.13

0.43

1.29

3.86 –

29

– 43

–

57

– 71

43

– 57

29

– 43 93

93

93

93

93

93 0.00

0.03

0.09

0.31

0.92

2.76 0/49 (0%)

1/45 (2%)

0/46 (0%)

1/48 (2%)

8/47 (20%)

34/46 (74%)

Schmidt et

al., 1973 c NMRI 0 (acetone)

0.05

0.2

0.8

2 0

0.01

0.06

0.23

0.57 –

–

–

53 e

76 e 104 d

104

104

104

104 0

0.01

0.06

0.23

0.57 0/100 (0%)

0/100 (0%)

0/100 (0%)

2/100 (2%)

30/100 (30%)

Swiss 0 (acetone)

0.05

0.2

0.8

2 0

0.01

0.06

0.23

0.57 –

–

–

58 e

61 e 104

104

104

104

104 0

0.01

0.06

0.23

0.57 0/80 (0%)

0/80 (0%)

0/80 (0%)

5/80 (6%)

45/80 (56%)

Schmähl et

al., 1977 c NMRI 0 (acetone)

1

1.7

3 0

0.29

0.49

0.86 –

NR

NR

NR 104

104

104

104 0

0.29

0.49

0.86 1/81 (1%)

11/77 (14%)

25/88 (28%)

45/81 (56%)

Habs et al.,

1980 c,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.,

1984 c NMRI 0 (acetone)

2

4 0

0.57

1.14 –

NR

NR 104

104

104 0

0.57

1.14 0/20 (0%)

9/20 (45%)

17/20 (85%)

a Doses were applied 3 times/week for up to 93 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. Hab s 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 tumor

bearing animals and denominators estimate d 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 Table C

- 2 3 . Skin tumor incidence, benign or malignant, in C57L male 1

mice dermally exposed to be nzo[a]pyrene 2

Study Mouse

strain Dose ( μg) a Average

daily dose

( μg/d) First

appearance of

tumor (wks) Length of

exposure

(wks) Lifetime

average daily

dose b 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 n ot 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.



### Table C

- 2 4 . Skin tumor incidence, benign or malignant, in female CFLP 3

mice dermally exposed to benzo[a]pyrene 4

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 ± 16.8 b

60.9 ± 13.9

44.1 ± 7.7 104

104

104

104 0

1.1

2.2

4.4 0/80 (0%)

22/65 (34%)

39/64 (61%)

56/64 (88%)

Grimmer et al.,

1984 0 (1:3 acetone:DMSO)

3.4

6.7

13.5 0

0.97

1.9

3.9 —

61 (53

– 65) c

47 (43

– 50)

35 (32

– 36) 104

104

104

104 0

0.97

1.9

3.9 0/80 (0%)

43/64 (67%)

53/65 (82%)

57/65 (88%)

a Indicated doses were applied twice/week to shaved skin of the back for up to 104 weeks.

b Mean ± SD.

c Median and 95% confidence limit.

d Assumed exposure period is indicated in italics.

e Incidence denominators were calculated from 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

- d ose 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 Table C

- 2 5 . Skin tumor incidence, benign or malignant, in male 1

C3H/HeJ mice dermally exposed to benzo[a]pyrene 2

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

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### C

- 71 DRAFT

— DO NOT CITE OR QUOTE Table C

- 2 6 . Summary of model selection and modeling results for best

1

fitting multistage models, for multiple data sets of skin tumors in mice 2

following dermal benzo[a]pyrene exposure 3

Data set Model Goodness

- of

- fit BMD 10 (μg/d) BMDL 10 (μg/d) Basis for Model Selection a Figure

number p

- value AIC

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

0.12 3 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

0.4 85

0.485 131.1

123.6

123.6 0. 318

0. 748

0.748 0.2 49

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., 1984 b

female CFLP Multistage 1 °

LogLogistic

Dich otomous

- 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 d ropped 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 L ogLogistic 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

Figure C

- 8 . Fit of multistage model to skin tumors in C57L mice exposed 2

dermally to benzo[a]pyrene (Poel, 1959); graph and model output. 3

==================================================================== 

### 4

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 5

Input Da ta File: 

### 6

C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Poel_1959_MultiCanc3_0.1.(d) 

### 7

Gnuplot Plotting File: 

### 8

C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Poel_1959_MultiCanc3_0.1.plt 

### 9

10

==================================================================== 

### 11

12

13

The form of the probability function is: 

### 14

15

P[response] = background + (1

- background)*[1

- EXP( 

### 16

- beta1*dose^1

- beta2*dose^2

- beta3*dose^3)] 

### 17

18

The parameter betas are restricted to be positive 

### 19

20

21

Dependent variable = NumAff 

### 22

Independent variable = LADD 

### 23

24

Total number of observations = 5 

### 25

Total number of records with missing values = 0 

### 26

Total number of parameters in model = 4 

### 27

Total number of specified parameters = 0 

### 28

Degree of polynomial = 3 

### 29

30

31

Maximum number of iterations = 250 

### 32

Relative Function Convergence has been set to: 1e

- 008 

### 33

Parameter Convergence has been set to: 1e

- 008 

### 34

35

36

37

Default Initial Parameter Values 

### 38

### 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 Background = 0.0449589 

### 1

Beta(1) = 0.49045 1 

### 2

Beta(2) = 0 

### 3

Beta(3) = 2.68146 

### 4

5

6

Asymptotic Correlation Matrix of Parameter Estimates 

### 7

8

( *** The model parameter(s)

- Beta(2) 

### 9

have been estimated at a boundary point, or have been specified by 

### 10

the user, 

### 11

and do not appear in the correlation matrix ) 

### 12

13

Background Beta(1) Beta(3) 

### 14

15

Background 1

- 0.87 0. 74 

### 16

17

Beta(1)

- 0.87 1

- 0.92 

### 18

19

Beta(3) 0.74

- 0.92 1 

### 20

21

22

23

Parameter Estimates 

### 24

25

95.0% Wald Confidence 

### 26

Interval 

### 27

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

### 28

Limit 

### 29

Background 0.0176699 * * * 

### 30

Beta(1) 0.79766 * * * 

### 31

Beta(2) 0 * * * 

### 32

Beta(3) 2.17146 * * * 

### 33

34

*

- Indicates that this value is not calculated. 

### 35

36

37

38

Analysis of Deviance Table 

### 39

40

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 41

Full model

- 87.1835 5 

### 42

Fitted model

- 90.4265 3 6.48606 2 0.03905 

### 43

Reduc ed model

- 141.614 1 108.86 4 <.0001 

### 44

45

AIC: 186.853 

### 46

47

48

Goodness of Fit 

### 49

Scaled 

### 50

Dose Est._Prob. Expected Observed Size Residual 

### 51

------------------------------------------------------------------------ 

### 52

0.0000 0.0177 0.583 0.000 33

- 0.770 

### 53

0.0500 0.0563 3.098 5.000 55 1.112 

### 54

0.1600 0.1430 7.866 11.000 55 1.207 

### 55

0.2400 0.2128 11.917 7.000 56

- 1.605 

### 56

0.8000 0.8293 40.635 41.000 49 0.139 

### 57

58

Chi^2 = 5.88 d.f. = 2 P

- value = 0.0528 

### 59

60

61

Benchmark Dose Computation 

### 62

63

Specified effect = 0.1 

### 64

65

Risk Type = Extra risk 

### 66

67

### Toxicological Review of benzo[a]pyrene

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



### C

- 74 DRAFT

— DO NOT CITE OR QUOTE Confidence level = 0.95 

### 1

2

BMD = 0.126567 

### 3

4

BMDL = 0.0777875 

### 5

6

BMDU = 0.272961 

### 7

8

Taken together, (0.0777875, 0.272961) is a 90 % two

- sided confidence 

### 9

interval for the BMD 

### 10

11

Multistage Cancer Slope Factor = 1.28555 

### 12

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

Figure C

- 9 . Fit of multistage model to skin tumors in female Swiss mice 2

ex posed dermally to benzo[a]pyrene (Roe et al., 1970); graph and 3

model output. 4

==================================================================== 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

Input Data File: C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Roe_1970_Setting.(d) 

### 7

Gnuplot Plotting File: C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Roe_1970_Setting.plt 

### 8

9

==================================================================== 

### 10

BMDS Model Run 

### 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 12

13

The form of the probability function is: 

### 14

15

P[response] = background + (1

- background)*[1

- EXP( 

### 16

- beta1*dose^1

- beta2*dose^2

- beta3*dose^3

- beta4*dose^4

- beta5*dose^5)] 

### 17

18

The parameter betas are restricted to be positive 

### 19

20

Dependent variable = tumors 

### 21

Independent variable = LADD 

### 22

23

Total number of observations = 6 

### 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 = 5 

### 28

29

Maximum number of iterations = 250 

### 30

Relative Function Convergence has been set to: 1e

- 008 

### 31

Parameter Convergence has been set to: 1e

- 008 

### 32

33

34

35

Default Initial Parameter Values 

### 36

Background = 0 

### 37

### Toxicological Review of benzo[a]pyrene

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



### C

- 76 DRAFT

— DO NOT CITE OR QUOTE Beta(1) = 0.0962491 

### 1

Beta(2) = 0.141689 

### 2

Beta(3) = 0 

### 3

Beta(4) = 0 

### 4

Beta(5) = 0 

### 5

6

7

Asymptotic Correlation Matrix of Parameter Estimates 

### 8

9

( *** The model parameter(s)

- Beta(3)

- Beta(4)

- Beta(5) 

### 10

have been estimated at a boundary point, or have been specified by the user, 

### 11

and do not appear in the correlation matrix ) 

### 12

13

Background Beta(1) Beta(2) 

### 14

15

Background 1

- 0.57 0.45 

### 16

17

Beta(1)

- 0.57 1

- 0.94 

### 18

19

Beta(2) 0.45

- 0.94 1 

### 20

21

22

Parameter Estimates 

### 23

24

95.0% Wald Confidence Interval 

### 25

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 26

Background 0.00584 893 * * * 

### 27

Beta(1) 0.0379152 * * * 

### 28

Beta(2) 0.166839 * * * 

### 29

Beta(3) 0 * * * 

### 30

Beta(4) 0 * * * 

### 31

Beta(5) 0 * * * 

### 32

33

*

- Indicates that this value is not calculated. 

### 34

35

Analysis of Deviance Table 

### 36

37

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 38

Full model

- 56.1835 6 

### 39

Fitted model

- 57.5694 3 2.77176 3 0.4282 

### 40

Reduced model

- 118.948 1 125.529 5 <.0001 

### 41

42

AIC: 121.139 

### 43

44

Goodness of Fit 

### 45

Scaled 

### 46

Dose Est._Prob. Expected Observed Size Residual 

### 47

------------------------------------------------------------------------ 

### 48

0.0000 0.0058 0.275 0.000 47

- 0.526 

### 49

0.0300 0. 0071 0.321 1.000 45 1.204 

### 50

0.0900 0.0106 0.444 0.000 42

- 0.670 

### 51

0.3100 0.0331 1.423 1.000 43

- 0.361 

### 52

0.9200 0.1664 6.821 8.000 41 0.494 

### 53

2.7600 0.7488 34.444 34.000 46

- 0.151 

### 54

55

Chi^2 = 2.57 d.f. = 3 P

- value = 0.4626 

### 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 = 0.689131 

### 67

BMDL = 0.393806 

### 68

BMDU = 0.952365 

### 69

70

Taken together, (0.393806, 0.952365) is a 90 % two

- sided confidence 

### 71

interval for the BMD 

### 72

73

Multistage Cancer Slope Factor = 0. 253932 

### 74

### 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

Figure C

- 1 0 . Fit of multistage model to skin tumors in female NMRI 2

mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph 3

and model output. 4

==================================================================== 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

Input Data File: 

### 7

C: \ USEPA \ IRIS \ benzo[a]pyrene \ dermalslopefactor \ Schmidt1973femaleNMRI \ 2MulSchMS_.(d) 

### 8

Gnuplot Plotting File: 

### 9

C: \ USEPA \ IRIS \ benzo[a]pyrene \ dermalslopefactor \ Schmidt197 3femaleNMRI \ 2MulSchMS_.plt 

### 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)] 

### 18

19

The parameter betas are restricted to be positive 

### 20

21

Dependent variable = incidence 

### 22

Independent variable = dose 

### 23

24

Total number of observations = 5 

### 25

T otal number of records with missing values = 0 

### 26

Total number of parameters in model = 3 

### 27

Total number of specified parameters = 0 

### 28

Degree of polynomial = 2 

### 29

30

Maximum number of iterations = 250 

### 31

Relative Function Convergence has been set to: 2.22045e

- 016 

### 32

P arameter Convergence has been set to: 1.49012e

- 008 

### 33

34

**** We are sorry but Relative Function and Parameter Convergence   

### 35

** ** are currently unavailable in this model. Please keep checking   

### 36

### 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 ** ** the web sight for model updates which will event ually   

### 1

** ** incorporate these convergence criterion. Default values used.   

### 2

3

4

Default Initial Parameter Values 

### 5

Background = 0 

### 6

Beta(1) = 0 

### 7

Beta(2) = 1.11271 

### 8

9

10

Asymptotic Correlation Matrix of Parameter Estimates 

### 11

12

( ** * The model parameter(s)

- Background

- Beta(1) 

### 13

have been estimated at a boundary point, or have been spe cified by the user, 

### 14

and do not appear in the correlation matrix ) 

### 15

16

Beta(2) 

### 17

18

Beta(2) 1 

### 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) 0 * * * 

### 27

Beta(2) 0.970648 * * * 

### 28

29

*

- Indicates that this value is not calculated. 

### 30

31

32

33

Analysis of Dev iance Table 

### 34

35

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 36

Full model

- 70.8903 5 

### 37

Fitted model

- 72.6831 1 3.58562 4 0.465 

### 38

Reduced model

- 118.917 1 96.054 4 <.0001 

### 39

40

AIC: 147.366 

### 41

42

Goodness of Fit 

### 43

Scaled 

### 44

Dose Est._ Prob. Expected Observed Size Residual 

### 45

------------------------------------------------------------------------ 

### 46

0.0000 0.0000 0.000 0.000 100 0.000 

### 47

0.0100 0.0001 0.010 0.000 10 0

- 0.099 

### 48

0.0600 0.0035 0.349 0.000 100

- 0.592 

### 49

0.2300 0.0501 5.005 2.000 100

- 1.378 

### 50

0.5700 0.2705 27.048 30.000 100 0.665 

### 51

52

Chi^2 = 2.70 d.f. = 4 P

- value = 0.6091 

### 53

54

55

Benchmark Dose Computation 

### 56

57

Specified effect = 0.1 

### 58

59

Risk Type = Extra risk 

### 60

61

Confidence level = 0.95 

### 62

63

BMD = 0.329464 

### 64

65

BMDL = 0.286624 

### 66

67

BMDU = 0.384046 

### 68

69

Taken together, (0.286624, 0.384046) is a 90 % two

- sided confidence 

### 70

interval for the BMD 

### 71

72

Multistage Cancer Slope Factor = 0.348889 

### 73

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

Figure C

- 1 1 . Fit of multistage model to skin tumors in female Swiss 2

mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph 3

and model output. 4

==================================================================== 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

Input Data File: 

### 7

C: \ USEPA \ IRIS \ b enzo[a]pyrene \ dermalslopefactor \ Schmidt1973swissmice \ 3MulSchMS_.(d) 

### 8

Gnuplot Plotting File: 

### 9

C: \ USEPA \ IRIS \ benzo[a]pyrene \ dermalslopefactor \ Schmidt1973swissmice \ 3MulSchMS_.plt 

### 10

==================================================================== 

### 11

BMD S 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)] 

### 18

19

The parameter betas are restricted to be positive 

### 20

21

Dependent variable = incidence 

### 22

Independent variable = dose 

### 23

24

Total number of observations = 5 

### 25

Total number of records with missing values = 0 

### 26

Total number of parameters in model = 4 

### 27

Total number of sp ecified parameters = 0 

### 28

Degree of polynomial = 3 

### 29

30

Maximum number of iterations = 250 

### 31

Relative Function Convergence has been set to: 2.22045e

- 016 

### 32

Parameter Convergence has been set to: 1.49012e

- 008 

### 33

34

**** We are sorry but Relative Function and Parameter Convergence   

### 35

### 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 ** ** are currently unavailable in this model. Please keep checking   

### 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) = 0.338951 

### 9

Beta(3) = 3.8728 

### 10

11

12

Asymptotic Correlation Matrix of Parameter Estimates 

### 13

14

( ** * The model parameter(s)

- Background

- Beta(1) 

### 15

have been estimated at a boundary point, or have been specified by the 

### 16

user, 

### 17

and do not appear in the correlation matrix ) 

### 18

19

Beta(2) Beta(3) 

### 20

21

Beta(2) 1

- 0.99 

### 22

23

Beta(3)

- 0.99 1 

### 24

25

26

Parameter Estimates 

### 27

28

95.0% Wald Confidence Interval 

### 29

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

### 30

Limit 

### 31

Background 0 * * * 

### 32

Beta(1) 0 * * * 

### 33

Beta(2) 0.108125 * * * 

### 34

Beta(3) 4.31441 * * * 

### 35

36

*

- Indicates that this va lue is not calculated. 

### 37

38

Analysis of Deviance Table 

### 39

40

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 41

Full model

- 73.5285 5 

### 42

Fitted model

- 73.6628 2 0.268637 3 0.9658 

### 43

Reduced model

- 150.708 1 154.359 4 <.0001 

### 44

45

AIC: 151.326 

### 46

47

Goodness of Fit 

### 48

Scaled 

### 49

Dose Est._Prob. Expected Observed Size Residual 

### 50

------------------------------------------------------------------------ 

### 51

0.0000 0.0000 0.000 0.000 80 0.000 

### 52

0.0100 0.0000 0.001 0.000 80

- 0.035 

### 53

0.0600 0.0013 0.106 0.000 80

- 0.325 

### 54

0.2300 0.0566 4.524 5.000 80 0.230 

### 55

0.5700 0.5657 45.260 45.000 80

- 0.059 

### 56

57

Chi^2 = 0.16 d.f. = 3 P

- value = 0.9833 

### 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 = 0.282007 

### 68

BMDL = 0.2234 01 

### 69

BMDU = 0.309888 

### 70

71

Taken together, (0.223401, 0.309888) is a 90 % two

- sided confidence 

### 72

interval for the BMD 

### 73

74

Multistage Cancer Slope Factor = 0.447626 

### 75

### 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

Figure C

- 1 2 . Fit of multistage model to skin tumors in female NMRI 2

mice exposed dermally to benzo[a]pyrene (Schmähl et al., 1977); graph 3

and model output. 4

==================================================================== 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

Input Data File: 

### 7

C: \ USEPA \ IRIS \ b enzo[a]pyrene \ dermalslopefactor \ Schmahl1977femaleNMRI \ 2MulschMS_.(d) 

### 8

Gnuplot Plotting File: 

### 9

C: \ USEPA \ IRIS \ benzo[a]pyrene \ dermalslopefactor \ Schmahl1977femaleNMRI \ 2MulschMS_.plt 

### 10

==================================================================== 

### 11

B MDS 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)] 

### 18

19

The parameter betas are restricted to be positive 

### 20

21

22

Dependent variable = incidence 

### 23

Independent variable = dose 

### 24

25

Total number of observations = 4 

### 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

32

Maximum number of iterations = 250 

### 33

Relative Function Convergence has been set to: 2.22045e

- 016 

### 34

Parameter Convergence has been set to: 1.49012e

- 008 

### 35

### 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

**** We are sorry but Relative Function and Parameter Convergence   

### 2

** ** are currently unavailable in this model. Please keep checking   

### 3

** ** the web sight for model updates which will eventually   

### 4

** ** incorporate these convergence criterion. Default values used. **** 

### 5

6

7

Default Initial Parameter Values 

### 8

Background = 0.0115034 

### 9

Beta(1) = 0.284955 

### 10

Beta(2) = 0.750235 

### 11

12

13

Asymptotic Correlation Matrix of Pa rameter Estimates 

### 14

15

Background Beta(1) Beta(2) 

### 16

17

Background 1

- 0.67 0.47 

### 18

19

Beta(1)

- 0.67 1

- 0.94 

### 20

21

Beta(2) 0.47

- 0.94 1 

### 22

23

24

Parameter Estimates 

### 25

26

95.0% Wald Confidence Interval 

### 27

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

### 28

Limit 

### 29

Background 0.0123066 * * * 

### 30

Beta(1) 0.274413 * * * 

### 31

Beta(2) 0.764244 * * * 

### 32

33

*

- Indicates that this value i s 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

- 145.127 4 

### 40

Fitted model

- 145.13 3 0.00579898 1 0.9393 

### 41

Reduced model

- 184.158 1 78.0608 3 <.0001 

### 42

43

AIC: 296.261 

### 44

45

46

Goodness of Fit 

### 47

Scaled 

### 48

Dose Est._Prob. Expected Observed Size Residual 

### 49

------------------------------------------------------------------------ 

### 50

0.0000 0.0123 0.997 1.000 81 0.003 

### 51

0.2900 0.1446 11.137 11. 000 77

- 0.045 

### 52

0.4900 0.2813 24.756 25.000 88 0.058 

### 53

0.8600 0.5567 45.096 45.000 81

- 0.022 

### 54

55

Chi^2 = 0.01 d.f. = 1 P

- value = 0.9393 

### 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 = 0.232893 

### 67

BMDL = 0.148895 

### 68

BMDU = 0.320396 

### 69

70

Taken together, (0.148895, 0.320396) is a 90 % two

- sided confidence 

### 71

interval for the BMD 

### 72

73

Multistage Cancer Slope Factor = 0.671616 

### 74

### 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

Figure C

- 1 3 . Fit of multistage model to skin tumors in female NMRI 2

mice exposed dermally to benzo[a]pyrene (Habs et al., 1980); graph 3

and model output. 4

==== ================================================================ 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

Input Data File: M: \ _BMDS \ msc_BAP_HABS1980_MultiCanc3_0.1.(d) 

### 7

Gnuplot Plotting File: M: \ _BMDS \ msc_BAP_HABS1980_MultiCanc3_0.1.plt 

### 8

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)] 

### 18

19

The parameter betas are restricted to be positive 

### 20

21

22

Dependent variable = NumAff 

### 23

Independent variable = LADD 

### 24

25

Total number of observations = 4 

### 26

Total number of records with missing values = 0 

### 27

Total number of parameters in model = 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 

### 39

Background = 0 

### 40

Beta(1) = 0 

### 41

Beta(2) = 4.23649 

### 42

### 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 Beta(3) = 0 

### 1

2

3

Asymptotic Correlation Matrix of Parameter Estimates 

### 4

( *** The model parameter(s)

- Background

- Beta(1)

- Beta(2) 

### 5

have been estimated at a boundary point, or ha ve been specified by the 

### 6

user, 

### 7

and do not appear in the correlation matrix ) 

### 8

9

Beta(3) 

### 10

11

Beta(3) 1 

### 12

13

14

15

Parameter Estimates 

### 16

17

95.0% Wald Confidence Interval 

### 18

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

### 19

Limit 

### 20

Background 0 * * * 

### 21

Beta(1) 0 * * * 

### 22

Beta(2) 0 * * * 

### 23

Beta(3) 4.1289 * * * 

### 24

25

*

- Indicates that this value is not calculated. 

### 26

27

28

29

Analysis of Deviance Table 

### 30

31

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 32

Full model

- 34.8527 4 

### 33

Fitted model

- 37.3373 1 4.96903 3 0.1741 

### 34

Reduc ed model

- 82.5767 1 95.4478 3 <.0001 

### 35

36

AIC: 76.6745 

### 37

38

39

Goodness of Fit 

### 40

Scaled 

### 41

Dose Est._Prob. Expected Observed Size Residual 

### 42

------------------------------------------------------------------------ 

### 43

0.0000 0.0000 0.000 0.000 35 0.000 

### 44

0.4900 0.3848 13.082 8.000 34

- 1.791 

### 45

0.7400 0.8123 21.933 24.000 27 1.019 

### 46

0.8000 0.8792 21.102 22.000 24 0.563 

### 47

48

Chi^2 = 4.56 d.f. = 3 P

- value = 0.2067 

### 49

50

51

Benchmark Dose Computation 

### 52

53

Specified effect = 0.1 

### 54

55

Risk Type = Extra risk 

### 56

57

Confidence level = 0.95 

### 58

59

BMD = 0.294407 

### 60

61

BMDL = 0.215151 

### 62

63

BMDU = 0.320955 

### 64

65

Taken together, (0.215151, 0.320955) is a 90 % two

- sided co nfidence 

### 66

interval for the BMD 

### 67

68

Multistage Cancer Slope Factor = 0.46479 

### 69

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

Figure C

- 1 4 . Fit of multistage model to skin tumors in female NMRI 2

mice exposed dermally to benzo[a]pyrene (Habs et al., 1984); graph 3

and model output. 4

==================================================================== 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

Input Data File: C: \ Usepa \ BMDS21 \ mscDax_Setting.(d) 

### 7

Gnuplot Plotting File: C: \ Usepa \ BMDS21 \ mscDax_Setting.plt 

### 8

9

==================================================================== 

### 10

11

BMDS Model Run 

### 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 13

14

The form of the probabil ity 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 = tumors 

### 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

Pa rameter Convergence has been set to: 1e

- 008 

### 35

### 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

Default Initial Parameter Values 

### 4

Background = 0 

### 5

Beta(1) = 1.66414 

### 6

7

8

Asymptotic Correlation Matrix of Parameter Estimates 

### 9

10

( *** The model parameter(s)

- Background 

### 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

Beta(1) 

### 15

16

Beta(1) 1 

### 17

18

19

20

Parameter Estimates 

### 21

22

95.0% Wald Confidence Interval 

### 23

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 24

Background 0 * * * 

### 25

Beta(1) 1.35264 * * * 

### 26

27

*

- Indicates that this value is not c alculated. 

### 28

29

30

31

Analysis of Deviance Table 

### 32

33

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 34

Full model

- 22.217 3 

### 35

Fitted model

- 22.7878 1 1.14175 2 0.565 

### 36

Reduced model

- 41.0539 1 37.6739 2 <.0001 

### 37

38

AIC: 47.5757 

### 39

40

41

Goodness of Fit 

### 42

Scaled 

### 43

Dose Est._Prob. Expected Observed Size Residual 

### 44

------------------------------------------------------------------------ 

### 45

0.0000 0.0000 0.000 0.000 20 0.000 

### 46

0.5700 0.5375 10.749 9.000 20

- 0.784 

### 47

1.1400 0.7860 15.721 17.000 20 0.697 

### 48

49

Chi^2 = 1.10 d.f. = 2 P

- value = 0.5765 

### 50

51

52

Benchmark Dose Computation 

### 53

54

Spec ified effect = 0.1 

### 55

56

Risk Type = Extra risk 

### 57

58

Confidence level = 0.95 

### 59

60

BMD = 0.0778926 

### 61

62

BMDL = 0.0558466 

### 63

64

BMDU = 0.111853 

### 65

66

Taken together, (0.0558466, 0.111853) is a 90 % two

- sided confidence 

### 67

interval for the BMD 

### 68

69

Multistage Cancer Slope Factor = 1.79062 

### 70

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

Figure C

- 1 5 . Fit of multistage model to skin tumors in female CFLP mice 2

exposed dermally to benzo[a]pyrene (Grimmer et al., 1983); graph and 3

model output. 4

== ================================================================== 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

Input Data File: 

### 7

C: \ USEPA \ IRIS \ benzo[a]pyrene \ dermalslopefactor \ Grimmer1983CFLPmice \ 1MulGriMS_.(d) 

### 8

Gnuplot Plotting File: 

### 9

C: \ USEPA \ IRIS \ benzo[a]pyrene \ dermalslopefactor \ Grimmer1983CFLPmice \ 1MulGriMS_.plt 

### 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

Dependent variable = incidence 

### 23

Independent variable = dose 

### 24

25

Total number of observations = 4 

### 26

Total number of records with missing values = 0 

### 27

Total number of parameters in model = 2 

### 28

Total number of specified parameters = 0 

### 29

Degree of polynomial = 1 

### 30

31

32

Maximum number of iterations = 250 

### 33

Relative Function Convergence has been set to: 2.22045e

- 016 

### 34

Parameter Convergence has been set to: 1.49012e

- 008 

### 35

### 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

**** We are sorry but Relative Function and Parameter Convergence   

### 2

** ** are currently unavailable in this model. Please keep checking   

### 3

** ** the web sight for model updates which will eventually   

### 4

** ** incorporate these convergence criterion. Default values used.   

### 5

6

7

8

Default Initial Parameter Values 

### 9

Background = 0 

### 10

Beta(1) = 0.478645 

### 11

12

13

Asymptotic Correlation Matrix of Parameter Estimates 

### 14

15

( ** * The model parameter(s)

- Background 

### 16

have been estimated at a boundary point, or have been specified by the user, 

### 17

and do not appear in the correlation matrix ) 

### 18

19

Beta(1) 

### 20

21

Beta(1) 1 

### 22

23

24

25

Parameter Estimates 

### 26

27

95.0% Wald Confidence Interval 

### 28

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 29

Background 0 * * * 

### 30

Beta(1) 0 .430366 * * * 

### 31

32

*

- Indicates that this value is not calculated. 

### 33

34

35

Analysis of Deviance Table 

### 36

37

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 38

Full model

- 108.532 4 

### 39

Fitted model

- 108.943 1 0.823537 3 0.8438 

### 40

Reduced model

- 186.434 1 155.805 3 <.0001 

### 41

42

AIC: 219.887 

### 43

44

45

Goodness of Fit 

### 46

Scaled 

### 47

Dose Est._Prob. Expected Observed Size Residual 

### 48

-------------------------------------------------------- ---------------- 

### 49

0.0000 0.0000 0.000 0.000 80

- 0.000 

### 50

1.1100 0.3798 24.687 22.000 65

- 0.687 

### 51

2.2000 0.6120 39.169 39.000 64

- 0.043 

### 52

4.4000 0.8495 54.366 56.000 64 0.571 

### 53

54

Chi^2 = 0.80 d.f. = 3 P

- value = 0.8496 

### 55

56

57

Benchmark Dose Computation 

### 58

59

Specified effect = 0.1 

### 60

61

Risk Type = Extra risk 

### 62

63

Confidence level = 0.95 

### 64

65

BMD = 0.244816 

### 66

67

BMDL = 0.208269 

### 68

69

BMDU = 0.289606 

### 70

71

Taken together, (0.208269, 0.289606) is a 90 % two

- sided confidence 

### 72

interval for the BMD 

### 73

74

Multistage Cancer Slope Factor = 0.480148 

### 75

### 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

Figure C

- 16 . Fit of multistage model to skin tumors in female CFLP mice 2

exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and 3

model output. 4

==================================================================== 

### 5

Multistage Cancer Model. (Versi on: 1.7; Date: 05/16/2008) 

### 6

Input Data File: 

### 7

C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Grimmer1984_MultiCanc1_0.1.(d) 

### 8

Gnuplot Plotting File: 

### 9

C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Grimmer1984_MultiCanc1_0.1.plt 

### 10

Wed Apr 27 17:11:28 2011 

### 11

12

[add notes here] 

### 13

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 14

15

The form of the probability function is: 

### 16

17

P[response] = background + (1

- background)*[1

- EXP( 

### 18

- beta1*dose^1)] 

### 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 = 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 

### 36

37

38

39

Default Initial Parameter Values 

### 40

Background = 0.311241 

### 41

Beta(1) = 0.502556 

### 42

### 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

Asymptotic Correlation Matrix of Parameter Estimates 

### 3

4

( *** The model parameter(s)

- Background 

### 5

have been estimated at a boundary point, or have been specified by the 

### 6

user, 

### 7

and do not appear in the correlation matrix ) 

### 8

9

Beta(1) 

### 10

11

Beta(1) 1 

### 12

13

14

15

Parameter Estimates 

### 16

17

95.0% Wald Confidence Interval 

### 18

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

### 19

Limit 

### 20

Background 0 * * * 

### 21

Beta(1) 0.796546 * * * 

### 22

23

*

- Indicates that this value is not calculated. 

### 24

25

26

27

Analysis of Deviance Table 

### 28

29

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 30

Full model

- 95.8385 4 

### 31

Fitted model

- 101.643 1 11.61 3 0.008846 

### 32

Reduced model

- 175.237 1 158.797 3 <.0001 

### 33

34

AIC: 205.287 

### 35

36

37

Goodness of Fit 

### 38

Scaled 

### 39

Dose Est._Prob. Expected Observed Size Residual 

### 40

------------------------------------------------------------------------ 

### 41

0.0000 0.0000 0.000 0.000 65 0.000 

### 42

0.9700 0.5382 34.446 43.000 64 2.145 

### 43

1.9100 0.7816 50.804 53.000 65 0.659 

### 44

3.9000 0.9552 62.091 57.000 65

- 3.054 

### 45

46

Chi^2 = 14.36 d.f. = 3 P

- value = 0.0025 

### 47

48

49

Benchmark Dose Computation 

### 50

51

Specified effect = 0.1 

### 52

53

Risk Type = Extra risk 

### 54

55

Confidence level = 0.95 

### 56

57

BMD = 0.132272 

### 58

59

BMDL = 0.113427 

### 60

61

BMDU = 0.154848 

### 62

63

Taken together, (0.113427, 0.154848) is a 90 % two

- sided confidence 

### 64

interval for the BMD 

### 65

66

Multistage Cancer Slope Factor = 0.881621 

### 67

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

Figure C

- 17 . Fit of log

- logistic model to skin tumors in female CFLP 2

mice e xposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph 3

and model output. 4

==================================================================== 

### 5

Logistic Model. (Version: 2.12; Date: 05/16/2008) 

### 6

Input Data File: 

### 7

C: \ Usepa \ BMDS21 \ Data \ lnl_ benzo[a]pyrene_Grimmer1984_Grimmer1984_0.70u.(d) 

### 8

Gnuplot Plotting File: 

### 9

C: \ Usepa \ BMDS21 \ Data \ lnl_benzo[a]pyrene_Grimmer1984_Grimmer1984_0.70u.plt 

### 10

11

==================================================================== 

### 12

13

BMDS Model Run 

### 14

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 15

16

The form of the probability function is: 

### 17

18

P[response] = background+(1

- background)/[1+EXP(

- intercept

- slope*Log(dose))] 

### 19

20

21

Dependent variable = NumAff 

### 22

Independent variable = LADD 

### 23

Slope parameter is not restricted 

### 24

25

Total number of observations = 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 be en set to: 1e

- 008 

### 30

31

32

33

User has chosen the log transformed model 

### 34

35

36

Default Initial Parameter Values 

### 37

background = 0 

### 38

intercept = 0.799142 

### 39

slope = 0.8 94129 

### 40

41

42

### 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 Asymptotic Correlation Matrix of Parameter Estimates 

### 1

2

( *** The model parameter(s)

- background 

### 3

have been estimated at a boundary point, or have been specified 

### 4

by the user, 

### 5

and do not appea r in the correlation matrix ) 

### 6

7

intercept slope 

### 8

9

intercept 1

- 0.68 

### 10

11

slope

- 0.68 1 

### 12

13

14

15

Parameter Estimates 

### 16

17

95.0% Wald Confidence 

### 18

Interval 

### 19

Variable Estimate Std. Err. Lower Conf. Limit Upper 

### 20

Conf. Limit 

### 21

background 0 * * * 

### 22

intercept 0.783559 * * * 

### 23

slope 0.922655 * * * 

### 24

25

*

- Indicates that this value is not calculated. 

### 26

27

28

29

Analysis of Deviance Table 

### 30

31

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 32

Full model

- 95.8385 4 

### 33

Fitted model

- 95.9236 2 0.17031 2 0.9184 

### 34

Reduced model

- 175.237 1 158.797 3 <.0001 

### 35

36

AIC: 195.847 

### 37

38

39

Goodness of Fit 

### 40

Scaled 

### 41

Dose Est._Prob. Expected Observed Size Residual 

### 42

------------------------------------------------------------------------ 

### 43

0.0000 0.0000 0.000 0.000 65 0.000 

### 44

0.9700 0.6804 43.543 43.000 64

- 0.146 

### 45

1.9100 0.7991 51.941 53.000 65 0.328 

### 46

3.9000 0.8849 57.516 57.000 65

- 0.200 

### 47

48

Chi^2 = 0.17 d.f. = 2 P

- value = 0.9190 

### 49

50

51

Benchmark Dose Computation 

### 52

53

Specified effect = 0.7 

### 54

55

Risk Type = Extra risk 

### 56

57

Confidence level = 0.95 

### 58

59

BMD = 1.07152 

### 60

61

BMDL = 0.478669 

### 62

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 Figure C

- 18 . Fit of multistage model to skin tumors in female CFLP mice 1

exposed dermally to benzo[a]pyrene (Grimmer et al., 1984), highest 2

dose dropped; graph and model output. 3

==================================================================== 

### 4

Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) 

### 5

Input Data File: C:/Usepa/_BaP/msc_BaP_Grimmer1984_drophidose_MultiCanc1_0.7.(d) 

### 6

Gnuplot Plotting File: 

### 7

C:/Usepa/_BaP/msc_BaP_Grimmer1984_drophidose_MultiCanc1_0.7.plt 

### 8

==================================================================== 

### 9

10

[add_

notes_here] 

### 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 12

13

The form of the probability function is: 

### 14

15

P[response] = background + (1

- background)*[1

- EXP( 

### 16

- beta1*dose^1)] 

### 17

18

The parameter betas are restricted to be positive 

### 19

20

21

Dependent variable = NumAff 

### 22

Independent variable = LADD 

### 23

24

Total number of observations = 3 

### 25

Total number of records with missing values = 0 

### 26

Total number of parameters in model = 2 

### 27

Total number of specified parameters = 0 

### 28

Degree of polynomial = 1 

### 29

30

31

Maximum number of iterations = 250 

### 32

Relative Function Convergence has been set to: 1e

- 008 

### 33

Parameter Convergence has been set to: 1e

- 008 

### 34

35

36

37

Default Initial Parameter Values 

### 38

Background = 0.0806622 

### 39

Beta(1) = 0.88595 

### 40

41

42

Asymptotic Correlation Matrix of Parameter Estimates 

### 43

### 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

( *** The model parameter(s)

- Background 

### 2

have been estimated at a boundary point, or have been specified by the 

### 3

user, 

### 4

and do not appear in the correlation matrix ) 

### 5

6

Beta(1) 

### 7

8

Beta(1) 1 

### 9

10

11

12

Parameter Estimates 

### 13

14

95.0% Wald Confidence Interval 

### 15

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. 

### 16

Limit 

### 17

Background 0 * * * 

### 18

Beta(1) 0.997117 * * * 

### 19

20

*

- Indicates that this value is not calculated. 

### 21

22

23

24

Analysis of Deviance Table 

### 25

26

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 27

Full model

- 71.5928 3 

### 28

Fitted model

- 72.2756 1 1.36568 2 0.5052 

### 29

Reduced model

- 134.46 1 125.735 2 <.0001 

### 30

31

AIC: 146.551 

### 32

33

34

Goodness of Fit 

### 35

Scaled 

### 36

Dose Est._Prob. Expected Observed Size Residual 

### 37

----------------------------------------------------------------------- - 

### 38

0.0000 0.0000 0.000 0.000 65 0.000 

### 39

0.9700 0.6199 39.671 43.000 64 0.857 

### 40

1.9100 0.8511 55.322 53.000 65

- 0.809 

### 41

42

Chi^2 = 1.39 d.f. = 2 P

- value = 0.4992 

### 43

44

45

Benchmark Dose Computation 

### 46

47

Specified effect = 0.7 

### 48

49

Risk Type = Extra risk 

### 50

51

Confidence level = 0.95 

### 52

53

BMD = 1.20745 

### 54

55

BMDL = 1.00734 

### 56

57

BMDU = 1.45789 

### 58

59

Taken together, (1.00734, 1.45789) is a 90 % two

- sided confidence 

### 60

interval for the BMD 

### 61

62

Multistage Cancer Slope Factor = 0.6949 

### 63

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

Figure C

- 19 . Fit of multistage model to sk in tumors in male CeH/HeJ 2

mice exposed dermally to benzo[a]pyrene (Sivak et al., 1997); graph 3

and model output. 4

==================================================================== 

### 5

Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008) 

### 6

In put Data File: C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Sivak1993_MultiCanc2_0.1.(d) 

### 7

Gnuplot Plotting File: 

### 8

C: \ Usepa \ BMDS21 \ Data \ msc_benzo[a]pyrene_Sivak1993_MultiCanc2_0.1.plt 

### 9

10

==================================================================== 

### 11

12

[add notes here] 

### 13

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

### 14

15

The form of the probability function is: 

### 16

17

P[response] = background + (1

- background)*[1

- EXP( 

### 18

- beta1*dose^1

- beta2*dose^2)] 

### 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

Tot al number of records with missing values = 0 

### 28

Total number of parameters in model = 3 

### 29

Total number of specified parameters = 0 

### 30

Degree of polynomial = 2 

### 31

32

33

Maximum number of iterations = 250 

### 34

Relative Function Convergence has been set to: 1e

- 008 

### 35

Paramete r Convergence has been set to: 1e

- 008 

### 36

37

38

39

Default Initial Parameter Values 

### 40

Background = 0 

### 41

Beta(1) = 0.0936505 

### 42

### 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 Beta(2) = 8.67239 

### 1

2

3

Asymptotic Correlation Matrix of Parameter Estimates 

### 4

5

( *** The model parameter(s)

- Background

- Beta(1) 

### 6

have been estimated at a boundary point, or have been specified by the user, 

### 7

and do not a ppear in the correlation matrix ) 

### 8

9

Beta(2) 

### 10

11

Beta(2) 1 

### 12

13

14

15

Parameter Estimates 

### 16

17

95.0% Wald Confidence Interval 

### 18

Variable Est imate Std. Err. Lower Conf. Limit Upper Conf. Limit 

### 19

Background 0 * * * 

### 20

Beta(1) 0 * * * 

### 21

Beta(2) 8 .9375 * * * 

### 22

23

*

- Indicates that this value is not calculated. 

### 24

25

26

27

Analysis of Deviance Table 

### 28

29

Model Log(likelihood) # Param's Deviance Test d.f. P

- value 

### 30

Full model

- 23.2693 4 

### 31

Fitted model

- 23.3009 1 0.0631003 3 0.9959 

### 32

Reduced model

- 69.5898 1 92.641 3 <.0001 

### 33

34

AIC: 48.6018 

### 35

36

37

Goodness of Fit 

### 38

Scaled 

### 39

Dose Est._Prob. Expected Observed Size Residual 

### 40

------------------------------------------------------------------------ 

### 41

0.0000 0.0000 0.000 0.000 30 0.000 

### 42

0.0100 0.0009 0.027 0.000 30

- 0.164 

### 43

0.1400 0.1607 4.821 5.000 30 0.089 

### 44

0.5100 0.9022 27.065 27.0 00 30

- 0.040 

### 45

46

Chi^2 = 0.04 d.f. = 3 P

- value = 0.9982 

### 47

48

49

Benchmark Dose Computation 

### 50

51

Specified effect = 0.1 

### 52

53

Risk Type = Extra risk 

### 54

55

Confidence level = 0.95 

### 56

57

BMD = 0.108575 

### 58

59

BMDL = 0.058484 

### 60

61

BMDU = 0.129641 

### 62

63

Taken together, (0.058484, 0.129641) is a 90 % two

- sided confidence 

### 64

interval for the BMD 

### 65

66

Multistage Cancer Slope Factor = 1.70987 

### 67

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 ALTERNATIVE APPROACH ES FOR CROSS

- SPECIES S CALING OF THE DERMAL SLOPE 1

FACTOR 2

Several publications which develop a dermal slope factor for benzo[a]pyrene are available 

### 3

in the peer reviewed literature (Knafla et al., 2010; 2006; Hussain et al., 1998; LaGoy and Quirk 

### 4

1994; Sullivan et al., 1991). Wit h the exception of the 2010 Knafla et al. publication, none of these 

### 5

approaches applied quantitative adjustments to account for interspecies differences, though the 

### 6

proposed slope factors were developed to account for human risk. Knafla et al. (2010) qual itatively 

### 7

discuss processes which could affect the extrapolation between mice and humans including skin 

### 8

metabolic activity adduct formation, stratum corneum thickness, epidermal thickness, etc. 

### 9

Ultimately, the authors apply an adjustment based on the incr eased epidermal thickness of human 

### 10

skin on the arms and hands compared to mouse interscapular epidermal thickness. They 

### 11

hypothesize that the carcinogenic potential of benzo[a]pyrene may be related to the thickness of 

### 12

the epidermal layer. 

### 13

Because there i s no established methodology for cross

- species extrapolation of dermal 

### 14

toxicity, several alternative approaches were evaluated. Each approach begins with the POD of 

### 15

0.066 μg/day that was based on a 10% extra risk for skin tumors in male mice. Based on th e 

### 16

assumptions of each approach, a dermal slope factor for humans is calculated. The discussion of 

### 17

these approaches uses the following abbreviations: 

### 18

19

DSF = dermal slope factor 

### 20

POD M = point of departure (for 10% extra risk) from mouse bioassay, in μg/day 

### 21

BW M = mouse body weight = 0.035 kg (assumed) 

### 22

BW H = human body weight = 70 kg (assumed) 

### 23

SA H = total human surface area = 19,000 cm 2 (assumed) 

### 24

SA M = total mouse surface area = 100 cm 2 (assumed) 

### 25

26

Approach 1. No interspecies adjustment to daily applied dose (POD) in mouse model 

### 27

Under this approach, a given mass of benzo[a]pyrene, applied daily, would pose the same 

### 28

risk in an animal or in humans, regardless of whether it is applied to a small surface area or to a 

### 29

larger surface area at a proportionately lower concentration. 

### 30

31

DSF = 0.1/ POD M 

### 32

33

DSF= 0.1/0.0 6 8 μg/day = 1.5 (μg/day)

- 1 

### 34

35

Assumptions: The same mass of benzo[a]pyrene, applied daily, would have same potency in 

### 36

mice as in human skin regardless of treatment area. 

### 37

38

Approach 2. Cross

- species adjustment ba sed on whole body surface

- area scaling 

### 39

### 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 Under this approach, animals and humans are assumed to have equal lifetime cancer risk 

### 1

with equal average whole body exposures in loading units (μg/cm 2

- day). As long as doses are low 

### 2

enough that risk is proportional to the mass of applied compound, the daily dermal dose of 

### 3

benzo[a]pyrene can be normalized over the total surface area. 

### 4

5

POD (μg/cm 2

- day) = POD M/SA (μg/cm 2

- day) = POD M (μg/day) / SA M (cm 2 ) 

### 6

7

POD = (0.0 6 8 μg/day) / 100 cm 2 

### 8

= 0.0006 8 μg/cm 2

- day 

### 9

10

DSF = 0.1/(0.0006 8 μg/cm 2

- day) ≈ 1 47 (μg/cm 2

- day)

- 1 

### 11

12

Assumptions : Mouse and human slope factors are equipotent if total dermal dose is 

### 13

averaged over equal fractions of the entire surface area. T umor potency of benzo[a]pyrene is 

### 14

assumed to be related to overall dose and not dose per unit area. For example, a human exposed to 

### 15

0.01 μg/day on 10 cm 2 would be assumed to have the same potential to form a skin tumor as 

### 16

someone treated with 0.01 μg/day over 19,000 cm 2 (assumed human surface area). 

### 17

18

Approach 3. Cross

- species adjustment based on body weight 

### 19

Under this approach, a given mass of benzo[a]p yrene is normalized relative to the body 

### 20

weight of the animal or human. This approach has been used for oral doses for noncancer effects. 

### 21

22

POD M / BW M = 0.0 6 8 μg/0.035 kg

- day = 1.9 μg/kg

- day 

### 23

24

DSF = 0.1/ 1.9 μg/kg

- day = 0.0 51 (μg/kg

- day)

- 1 

### 25

26

Assumptions : The po tency of point of contact skin tumors is related to bodyweight and 

### 27

humans and mice would have an equal likelihood of developing skin tumors based on a dermal dose 

### 28

per kg basis. 

### 29

30

Issues : Skin cancer following benzo[a]pyrene exposure is a local effect and no t likely 

### 31

dependent on body weight. 

### 32

33

Approach 4. Cross

- species adjustment based on allometric scaling using body weight to the 

### 34

3/4 power 

### 35

Under this approach, rodents and humans exposed to the same daily dose of a carcinogen, 

### 36

adjusted for BW 3/4 , would be expected to have equal lifetime risks of cancer. That is, a lifetime dose 

### 37

expressed as μg/kg 3/4

- day would lead to an equal risk in rodents and humans. This scaling reflects 

### 38

the empirically observed phenomena of more rapid distribution, metabolism, and cl earance in 

### 39

smaller animals. The metabolism of benzo[a]pyrene to reactive intermediates is a critical step in 

### 40

the carcinogenicity of benzo[a]pyrene, and this metabolism occurs in the skin. 

### 41

### 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) = POD M (μg/day) × (BW H / BW M ) 3/4 

### 2

3

POD (μg/day) = 0.0 6 8 μg/day × (70 kg / 0.035 kg) 3/4 

### 4

= 2 0 .3 μg/day 

### 5

6

DSF = 0.1/( 20.3 μg/day) ≈ 0.00 49 (μg/day)

- 1 

### 7

8

Assumptions: Risk at low doses of benzo[a]pyrene is dependent on absolute dermal dose 

### 9

and not dose per unit of skin, meaning a higher exposure concentration of benzo[a]pyrene 

### 10

contacting a smaller area of exposed skin could carry the same risk of skin tumors as a lowe r 

### 11

exposure concentration of benzo[a]pyrene that contacts a larger area of skin. 

### 12

13

Issues: It is unclear if scaling of doses based on bodyweight ratios will correspond to 

### 14

differences in metabolic processes in the skin of mice and humans. 

### 15

16

Synthesis of the alternative approaches to cross

- species scaling 

### 17

A comparison of the above approaches is provided in Table C

- 2 7 below. T he lifetime risk 

### 18

from a nominal human dermal exposure to benzo[a]pyrene over a 5% area of exposed skin 

### 19

(approximately 950 cm 2 ), estimat ed at 1 x 10

- 4 μg/day*, is calculated for each of the approaches in 

### 20

order to judge whether the method yields risk estimates that are unrealistically high. 

### 21

22

Other potential interspecies adjustments 

### 23

The above discussion presents several mathematical appro aches that result from varying 

### 24

assumptions about what is the relevant dose metric for determining equivalence across species. 

### 25

Biological information (that is not presently comprehensive or detailed enough to develop robust 

### 26

models) that could be used in fu ture biologically based models for cross

- species extrapolation 

### 27

include: 

### 28

a. Quantitative information on interspecies differences in partitioning from exposure medium 

### 29

to the skin and absorption through the skin 

### 30

b. Thickness of the stratum corneum between anatomica l sites and between species 

### 31

c. Thickness of epidermal layer 

### 32

d. Skin permeability 

### 33

e. Metabolic activity of skin 

### 34

f. Formation of DNA adducts in skin 

### 35

### Toxicological Review of benzo[a]pyrene

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### C

- 100 DRAFT

— DO NOT CITE OR QUOTE Table C

- 2 7

. Alternative approaches to cross

- species scaling 1

Approach Assumptions Dose metric DSF Risk at nominal

exposure

(0.0001 μg/day)*

1. Mass

- per

day scaling Equal mass per day (μg /d), if applied to equal areas of skin (cm 2 ), will affect similar

numbers of cells across species. Cancer risk is proportional to the area (cm 2 ) exposed if

the lo ading rate (μg /cm 2

- 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 /cm 2

- day) and skin area exposed (cm 2 ) that have the same product when

mu ltiplied, will result in the same risk. μg/day 1.3 per

μg/day 1 x 10 - 4

2. Surface

area scaling Equal mass per day (μg /d), if applied to equal fractions of total skin surface (cm 2 ) 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 /cm 2

- d) gives

similar cancer risks across species. This approach assumes that risk is proportional to

dose expressed as mass per area per day. This appro ach implies that risk does not

increase with area exposed as long as dose per area remains constant. μg/cm 2

- day 128 per

μg/cm 2

- day 7 x 10 - 7

3. Body

weight

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 li fetime exposure

[e.g., 5%

- of

- the

- body lifetime exposure] at the same loading rate (μg /cm 2

- 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 i mplies 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

(BW 3/4 ) 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 exposur es. μg/day 0.0043 per μg

/day 4 x 10 - 7

* Nominal exposure calculated as a geometric mean of average daily doses ( μ g/day) calculated from a range of benzo[a]pyrene soil concentrations (1

- 1000 

### 2

ppb) reported from non

- contaminated rural/agricultural soils (ATSDR, 1995) and a range of standard exposure assumptions. 

### 3

### 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 

# APPENDIX D. SUMMARY OF EXTERNAL PEER 1



# REVIEW A ND PUBLIC COMMENTS AND EPA’S 2



# DISPOSITION 3

4

5

### Toxicological Review of benzo[a]pyrene

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### 1 DRAFT

— DO NOT CITE OR QUOTE 1



# REFERENCES FOR APPENDICES 2

Multiple references published in the same year by the same author(s) have been assigned a letter 

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have been retained for the appendices

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### Toxicological Review of benzo[a]pyrene

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### Toxicological Review of benzo[a]pyrene

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and formaldehyde in primary cultures of rat tracheal epithelial cells. Mutat Res 201(1):161

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cell line. Chem Biol Interact 53 :257

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Crofton

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### 43

cells, a proprietary human cell line expressing five human cytochromes P

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### 44

hydrolase. Mutagenesis 8(4):363

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### 45

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Crowell, SR ; Amin, SG ; Anderson, KA ; et al. (2011) Preliminary physiologically based pharmacokinetic models 

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for benzo [a] pyrene and dibenzo[def,p]chrysene in rod ents. Toxicol Appl Pharmacol. Dec 15;257(3):365

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### 50

benzo[a]pyrene in a 2

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### 51

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Culp, SJ; Warbritton, AR; Smith, BA; et al. (2000) DNA adduct measurements, cell proliferation and tumor 

### 53

mutation induction in relation to tum or formation in B6C3F1 mice fed coal tar or benzo[a]pyrene. 

### 54

Carcinogenesis 21(7):1433

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### 55

### Toxicological Review of benzo[a]pyrene

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



### 6 DRAFT

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Dahl, AR; Coslett, DS; Bond, JA; et al. (1985) Metabolism of benzo[a]pyrene on the nasal mucosa of Syrian 

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hamsters: comparison to metabolism by other extrahepatic tissues and possible role of nasally produced 

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metabolites in carcinogenesis. J Natl Cancer Inst 75(1):135

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### 4

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Danheiser, SL; Liber, HL; Thilly, WG. (1989) Long

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### 6

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Dean, JH; Luster, MI; Boorman, GA; et al. (1983) Selective immunosuppression resulting from exposure to the 

### 15

carcinogenic congener of benzopyrene in B6C3F1 mice. Clin Exp Immunol 52(1):199

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Dean, SW; Coates, A; Brooks, TM; et al. (1998) Benzo[a]pyrene site of contact mutagenicity in skin of Muta 

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mouse. Mutagenesis 13(5):515

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



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