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EPA/635/R

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### www.epa.gov/iris



## Toxicological R eview of B enzo [ a ] pyrene

( CASRN 50

- 32

- 8 )

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

June 2012

NOTICE

This document is an Interagency Science Consultation draft. This information is distributed solely for the purpose of pre

- dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. It is being circulated for review of its technical accuracy and science policy implications.

National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency

Washington, DCToxicological 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 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 represen t and should not be

construed to represent any Agency determination or policy. Mention of trade names or commercial

products does not constitute endorsement of recommendation for use.Toxicological Review of Benzo[a]pyrene

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

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# CONTENTS

AUTHORS | CONTRIBUTORS | REVIEWERS

................................ ................................ ................................ . viii

PREFACE

................................ ................................ ................................ ................................ ........................ xi

PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS

................................ ................................ ............................ xiii

EXECUTIVE SUMMARY

................................ ................................ ................................ ............................. xxvii

LITERATURE SEARCH STRATEGY|STUDY SELECTION

................................ ................................ .............. xxxiii

1. HAZARD IDENTIFICATION

................................ ................................ ................................ .................... 1

- 1

1.1. Synthesis of Evidence

................................ ................................ ................................ .................... 1

- 1

1.1.1. Developmental Toxicity

................................ ................................ ................................ ..... 1

- 1

1. 1.2. Reproductive Toxicity

................................ ................................ ................................ ...... 1

- 16

1.1.3. Immunotoxicity

................................ ................................ ................................ ............... 1

- 30

1.1.4. Other Toxicological Effects

................................ ................................ .............................. 1

- 37

1.1.5. Carcinogenicity

................................ ................................ ................................ ................ 1

- 46

1.2. Summary and Evaluation

................................ ................................ ................................ ............ 1

- 69

1.2.1. Weight of Evidence for Effects Other Than Cancer

................................ ......................... 1

- 69

1.2.2. Weight of Evidence for Carcinogenicity

................................ ................................ .......... 1

- 70

2. DOSE

- RESPONSE ANALYSIS

................................ ................................ ................................ ................. 2

- 1

2.1. Oral Reference Dose for Effects Other Than Cancer

................................ ................................ .... 2

- 1

2.1.1. Identification of Studies and Effects for Dose

- Response Analysis

................................ .... 2

- 1

2.1.2. Methods of Analysis

................................ ................................ ................................ .......... 2

- 5

2.1.3. Derivation of Candidate Reference Doses

................................ ................................ ......... 2

- 8

2.1.4. Derivation of Organ/System

- specific Reference Doses

................................ ................... 2

- 10

2.1.5. Selection of the Proposed Overall Reference Dose

................................ ........................ 2

- 11

2.1.6. Confidence Statement

................................ ................................ ................................ ..... 2

- 12

2.1.7. Previous Reference Dose

................................ ................................ ................................ . 2

- 12

2.2. Inhalation Reference Concentration for Effects Other Than Cancer

................................ .......... 2

- 13

2.2.1. Identification of Studies and Effects for Dose

- Re sponse Analysis

................................ .. 2

- 13

2.2.2. Methods of Analysis

................................ ................................ ................................ ........ 2

- 14

2.2.3. Derivation of Candidate Reference Concentrations

................................ ....................... 2

- 16

2.2.4. Derivation of Organ/System

- specific Reference Concentrations

................................ .... 2

- 18Toxicological Review of Benzo[a]pyrene

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

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................................ ......... 2

- 19

2.2.6. Confidence Statement

................................ ................................ ................................ ..... 2

- 19

2.2.7. Previous Reference Concentration

................................ ................................ .................. 2

- 19

2.2.8. Uncertainties in the Derivation of the RfD and RfC

................................ ........................ 2

- 20

2.3. Oral Slope Factor for Cancer

................................ ................................ ................................ ....... 2

- 21

2.3.1. Analysis of Carcinogenicity Data

................................ ................................ ..................... 2

- 21

2.3.2. Dose Response Analysis

– Adjustments and Extrapolations Methods

........................... 2

- 23

2.3.3. Deriv ation of the Oral Slope Factor

................................ ................................ ................. 2

- 24

2.3.4. Uncertainties in the Derivation of the Oral Slope Factor

................................ ................ 2

- 26

2.3.5. Previous Oral Slope Factor

................................ ................................ .............................. 2

- 28

2.4. Inhalation Unit Risk for Cancer

................................ ................................ ................................ ... 2

- 28

2.4.1. Analysis of Carcinogenicity Data

................................ ................................ ..................... 2

- 28

2.4.2. Dose Response Analysis

— Adjustments and Extrapolations Methods

............................ 2

- 29

2.4.3. Derivation of the Inhalation Unit Risk

................................ ................................ ............. 2

- 30

2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk

................................ ............ 2

- 31

2.4.5. Previous Inhalation Unit Risk

................................ ................................ ........................... 2

- 33

2.5. Dermal Slope Factor for Cancer

................................ ................................ ................................ .. 2

- 33

2.5.1. Analysis of Carcinogenicity Data

................................ ................................ ..................... 2

- 33

2.5.2. Dose Response Analysis

– Adjustments and Extrapolations Methods

........................... 2

- 34

2.5.3. Derivation of the Dermal Slope Factor

................................ ................................ ............ 2

- 35

2.5.4. Dermal Slope Factor Cross Species Scaling

................................ ................................ ..... 2

- 37

2.5.5. Uncertainties in the Derivation of the Dermal Slope Factor

................................ ........... 2

- 38

2.5.6. Previous Dermal Slope Factor

................................ ................................ ......................... 2

- 40

2.6. Application of Age

- dependent Adjustment Factors (ADAFs)

................................ ..................... 2

- 40

REFERENCES

................................ ................................ ................................ ................................ ............... R

- 1Toxicological Review of Benzo[a]pyrene

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

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# TABLES

Table ES

- 1. Summary of proposed reference dose (RfD) derivation

................................ ....................... xxviii
Table ES

- 2. Summary of proposed reference concentration (RfC) derivation

................................ ....... xxix

Table LS

- 1. Details of the literature search strategy employed

................................ ................................ .. xxxiv
Table 1

- 1. Evidence pertaining to the developmental effects of benzo[a]pyrene in humans

............. 1

- 4

Table 1

- 2. Evidence pertaining to the developmental effects of benzo[a]pyrene in animals

.............. 1

- 5

Table 1

- 3. Evidence pertaining to the neurodevelopmental effects of benzo[a]pyrene in humans

.... 1

- 11

Table 1

- 4. Evidence pertaining to the neurodevelopmental effects of benzo[a]pyrene in animals 1

- 12

Table 1

- 5. Evidence pertaining to the male reproductive toxicity of benzo[a]pyrene in animals

... 1

- 19

Ta ble 1

- 6. Evidence pertaining to the female reproductive effects of benzo[a]pyrene in humans 1

- 25

Table 1

- 7. Evidence pertaining to the female reproductive effects of benzo[a]pyrene in animals

. 1

- 26

Table 1

- 8. Evidence pertaining to the immune effects of benzo[a]pyrene in animals

........................ 1

- 33

Table 1

- 9. Evidence pertaining to the forestomach effects of benzo[a]pyrene in animals

................. 1

- 38

Table 1

- 10. Evidence pertaining to other systemic effects of benzo[a]pyrene in animals

................. 1

- 44

Table 1

- 11. Cancer sites for PAH

- related agents reviewed by IARC

................................ ............................ 1

- 48

Table 1

- 12. Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation to lung cancer risk: Tier 1 studies

................................ ................................ ................................ .................... 1

- 48

Table 1

- 13. Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation to lung cancer risk: Tier 2 studies

................................ ................................ ................................ .................... 1

- 50

Table 1

- 14. Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation to

bladder cancer risk

................................ ................................ ................................ ................................ ........... 1

- 52

Table 1

- 15. Tumors observed in chronic oral animal bioassays.

................................ ................................ .. 1

- 54

Table 1

- 16. Tumors observed in chronic inhalation animal bioassays.

................................ ...................... 1

- 56

Table 1

- 17. Tumor observations in dermal animal bioassays.

................................ ................................ ....... 1

- 57

Table 1

- 18. Experimental support for the postulated key events for the hypothesized mu tagenic

MOA

................................ ................................ ................................ ................................ ................................ ......... 1

- 65

Table 1

- 19. Supporting evidence for the carcinogenic to humans cancer descriptor for benzo[a]pyrene

................................ ................................ ................................ ................................ .................. 1

- 74

Table 2

- 1. Summary of Derivation of Points of Departure

................................ ................................ ................ 2

- 7

Table 2

- 2. Effects and corresponding derivation of candidate RfDs

................................ ............................. 2

- 8

Table 2

- 3. Organ/system

- specific RfDs and proposed RfD for benzo[a]pyrene

................................ ..... 2

- 11

Table 2

- 4. Summary of Derivation of Points of Departure

................................ ................................ .............. 2

- 16

Table 2

- 5. Effects and corresponding derivation of candidate RfCs

................................ ........................... 2

- 17

Table 2

- 6. Organ/system

- specific RfCs and proposed RfC for benzo[a]pyrene

................................ ...... 2

- 19

Table 2

- 7. Summary of the Oral Slope Factor Derivations

................................ ................................ .............. 2

- 25

Table 2

- 8. Summary of uncertainties in the benzo[a]pyrene oral cancer slope factor (OSF)

.......... 2

- 27

Table 2

- 9. Summary of the Inhalation Unit Risk Derivation

................................ ................................ ........... 2

- 31

Table 2

- 10. Summary of uncertainties in the benzo[a]pyrene cancer inhalation unit risk (IUR)

... 2

- 32

Table 2

- 11. Summary of Dermal Slope Facto r Derivations

................................ ................................ ............. 2

- 36

Table 2

- 12. Summary of uncertainties in the benzo[a]pyrene cancer dermal slope factor (DSF)

.. 2

- 39

Table 2

- 13. Application of ADAFs for the estimation of benzo[a]pyrene cancer risk following lifetime (70

- year) oral exposure

................................ ................................ ................................ ................. 2

- 41

Table 2

- 14. Application of ADAFs for the estimation of benzo[a]pyrene cancer risk following lifetime (70

- year) inhalation ex posure

................................ ................................ ................................ .... 2

- 42

Table 2

- 15. Application of ADAFs for the estimation of benzo[a]pyrene cancer risk following lifetime (70

- year) dermal exposure

................................ ................................ ................................ ........... 2

- 42Toxicological Review of Benzo[a]pyrene

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

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# FIGURES

Figure LS

- 1. Study selection strategy.

................................ ................................ ................................ ..................... xxxv Figure 1

- 1. Exposure

- response array for developmental effects following oral exposure

................. 1

- 7 Figure 1

- 2. Exposure

- response array for neurodevelopmental effects following oral exposure

... 1

- 14 Figure 1

- 3. Exposure

- response array for male reproductive effects following oral exposure

........ 1

- 22 Figure 1

- 4. Exposure

- response array for female reproductive effects following oral exposure

.... 1

- 27 Figure 1

- 5. Exposure

- response array for immune effects following oral exposure

............................. 1

- 35 Figure 1

- 6. Exposure

- response array for forestomach hyperplasia following oral exposure

......... 1

- 39 Figure 1

- 7. Proposed metabolic activation pathways and key events in the carcinogenic mode of action for benzo[a]pyrene

................................ ................................ ................................ .............................. 1

- 59 Figure 2

- 1. Candidate RfDs with corresponding POD and composite UF

................................ .................. 2

- 10 Figure 2

- 2. Candidate RfCs with corresponding POD and composite UF

................................ ................... 2

- 18Toxicological Review of Benzo[a]pyrene

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

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# ABBREVIATIONS

ADAF age

- dependent adjustment factor

AhR aryl hydrocarbon receptor

AIC Akaike’s information criterion

AKR aldo

- keto reductase

ALT alanine aminotransferase

AST aspartate aminotransferase

ATSDR Agency for Toxic Substances and

Disease Registry B[a]P benzo[a]pyrene

BMC benchmark concentration

BMD benchmark dose

BMCL benc hmark concentration lower

confidence limit

BMDL benchmark dose lower confidence limit

BMDS Benchmark Dose Software

BMR benchmark response

BPDE benzo[a]pyrene

- 7,8

- diol

- 9,10

- epoxide

BUN blood urea nitrogen

BW body weight

CA chromosomal aberration

CASRN Chemical Abstracts Service Registry

Number

CERCLA Comprehensive Environmental

Response, Compensation, and Liability

Act

CI confidence interval

CYP450 cytochrome P450

DAF dosimetric adjustment factor

DHH dihydrodiol dehydrogenase

DNA deoxyribonu cleic acid

DSF dermal slope factor

EPA Environmental Protection Agency

ETS environmental tobacco smoke

FSH follicle stimulating hormone

F TOT total fractional deposition

GD gestation day

GGT γ

- glutamyl transferase

HEC human equivalent concentration

HED human equivalent dose

HERO Health and Environmental Research

Online

HSC hematopoietic stem cells i.p. intraperitoneal

IRIS Integrated Risk Information System

i.t. intratracheal

IUR inhalation unit risk

IVF in vitro fertilization

LH luteinizing hormone

LOAEL lowest

- observed

- adverse

- effect level

MMAD mass median aerodynamic diameter MN micronuclei

MOA mode of action

MPPP Multi

- Path Particle Deposition

NCEA National Center for Environmental

Assessment

NMDA N

- methyl

- D

- aspartate

NOAEL no

- obser ved

- adverse

- effect level

NPL National Priorities List

NTP National Toxicology Program

OR odds ratio

ORD Office of Research and Development

OSF oral slope factor

PAH polycyclic aromatic hydrocarbon

PBPK physiologically based pharmacokinetic

PCNA pro liferating cell nuclear antigen

PND post

- natal day

POD point of departure

POD [ADJ] duration

- adjusted POD

RBC red blood cells

RDDR ER regional deposited dose ratio for

extrarespiratory effects

RfC inhalation reference concentration

RfD oral reference dose

RNA ribonucleic acid

ROS reactive oxygen species

RR relative risk

SCC squamous cell carcinoma

SCE sister chromatid exchange

s.c. subcutaneous

SC SA sperm chromatic structure assay

SD standard deviation

SE standard error

SIR sta ndardized incidence ratio

SMR standardized mortality ratio

SRBC sheep red blood cells

SSB single strand break

TWA time

- weighted average

UCL upper confidence limit

UF uncertainty factor

UF A interspecies uncertainty factor

UF H intraspecies uncertainty factor

UF L LOAEL

- to

- NOAEL uncertainty factor

UF S subchronic

- to

- chronic uncertainty

factor

UF D database deficiencies uncertainty factor

U.S. United States of America

WBC white blood cells

WHO

- NCTB World Health Organization

Neurobehavioral Core Test BatteryToxicological Review of Benzo[a]pyrene

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

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# AUTHORS | CONTRIBUTORS | REVIEWER S

Assessment Team

Christine Cai

, MS Glinda Cooper , Ph.D. Louis D’Amico, Ph.D. Jason Fritz, Ph.D.

Martin Gehlhaus , MHS Catherine Gibbons, Ph.D. Karen Hogan

, M S Kathleen Newhouse, MS (Chemical Manager) Margaret Pratt , Ph.D. Keith Salazar, Ph.D. John Schaum (retired) Suryanarayana Vulimiri , DVM Gene Ching

- Hung Hsu , Ph.D. (formerly with EPA) U.S. EPA

O ffice of R esearch and D evelopment

National Center for Environmental Assessment Washington, DC

John Cowden , Ph.D. John Stanek , Ph.D. U.S. EPA

O ffice of R esearch and D evelopment

National Center for Environmental Assessment Research Triangle Park, NC

Chris Brinkerhoff, Ph.D. Oak Ridge Insti tute for Science and Education F ellow

Scott Glaberman , Ph.D. American Association for the Advancement of

Science F ellow

1 Scientific Support Team

Lynn Flowers, Ph.D., DABT John Fox Martin Gehlhaus, M HS Connie Kang

- Sickel , Ph.D. Jamie B. Strong, Ph.D.

Paul White U.S. EPA

O ffice of R esearch and D evelopment

National Center for Environmental Assessment Washington, DC

Reeder Sams, Ph.D. John Cowden , Ph.D. U.S. EPA

O ffice of R esearch and D evelopment

National Center for Environmental Assessment Research Triangle Park, NC

2

Production Team

Taukecha Cunningham Maureen Johnson

Terri Konoza

Vicki Soto U.S. EPA

O ffice of R esearch and D evelopment

National Center for Environmental Assessment Washington, DC

3Toxicological Review of Benzo[a]pyrene

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

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Heather Carlson

- Lynch , S.M.

Peter McClure , Ph.D. Megan Riccardi Kelly Salinas Joe Santodonato Julie Stickney , Ph.D., DABT SRC, Inc., Chemical, Biological, and Environmental Center, Syracuse, NY

George Holdsworth , Ph.D.

Lutz W. Weber , Ph.D., DABT Oak Ridge Institute for Science and Education, Oak Ridge, TN

Janusz Z. Byczkowski

, Fairborn, OH , Ph.D., D.Sc., DABT

1

Executive Direction

Rebecca Clark , MPH Vincent Cogliano, Ph.D. Lynn Flowers, Ph.D., DABT Samantha Jones, Ph.D. Jamie Strong, Ph.D. U.S. EPA

O ffice of R esearch and D evelopment

National Center for Environmental Assessment Washington, DC

2

Internal Review Team

Stephen Nesnow , Ph.D. U.S. EPA

National Health and Environmental Effects Research Laboratory Research Triangle Park, NC

Rita Schoeny , Ph.D. U.S. EPA

Office of Water Washington, DC

3Toxicological Review of Benzo[a]pyrene

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

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— DO NOT CITE OR QUOTE Reviewers

This assessment was provided for review to scientists in EPA’s Program and Region al Offices. 1 Comments were submitted by : 2

Office of Children ’ s He alth Protection, Washington DC

Office of Policy, Washington, DC Office of Solid Waste and Emergency Response, Washington DC Office of Water, Washington DC Region 2, New York, NY Region 3, Philadelphia, PA Region 8, Denver, CO

This assessment was provided for review to other federal agencies and Executive O ffices of the 3
President. C omments were submitted by : 4

Agency for Toxic Substances and Disease Registry, Centers for Disease Control, Department of

Health and Human Services Department of Defense National Aeronautics and Space Administration National Institute for Occupational Safety and Health, Centers for Disease Control, Department of

Health and Human Services

Offi ce of Management and Budget, Executive Office of the President White House Council on Environmental Quality, Executive Office of the President

5Toxicological Review of Benzo[a]pyrene

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

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# PREFACE

This Toxicological Review, prepared under the auspices of EPA’s Integrated Risk 1
Information System (IRIS) program, critically reviews the publicly available studies on 2

benzo[a]pyrene in order to identify potential adverse health effects and to characterize exposure

3

response relationships. Benzo[a]pyrene is found i n the environment and in food

, it occurs in 4

conjunction with other structurally related chemical compounds known as polycyclic aromatic 5
hydrocarbons (PAHs). 1 Benzo[a]pyrene is universally present in these mixtures and is often used 6

as an indicator chemical to measure exposure to PAH mixtures. 7

Benzo[a]pyrene is listed as a hazardous substance under the Comprehensive 8
Environmental Response, Compensation, and Liability Act of 1980 (CERCLA)

, is foun d at 524 9

hazardous waste sites on the National Priorities List (NPL) and is ranked number 9 out of 275 10

chemicals on the Priority List of Hazardous Substances for CERCLA (ATSDR, 2011). 11
Benzo[a]pyrene is also listed as a drinking water contaminant under the Safe Drinking Water Act. 12

This assessment updates a previous IRIS assessment of benzo[a]pyrene that was developed 13

in 1987. The previous assessment included a cancer descriptor and oral slope factor. New 14

information has become available, and t his assessment reviews information on all health effects by 15

all exposure routes. O rgan/system

- specific r e f e r e n c e v a l u e s are calculated based on 16

developmental, reproductive and immune system toxicity data. These reference values may be 17

useful for cumulative risk asse ssments that consider the combined effect of multiple agents acting 18
on the same biological system. In addition, i n consideration of the Agency’s need to estimate the 19

potential for skin cancer from dermal exposure (US EPA 2004), especially in children expo sed to 20

contaminated soil, this assessment includes the IRIS Program’s first dermal slope factor. 21

This assessment was conducted in accordance with EPA guidance, which is cited and 22
summarized in the Preamble to IRIS Toxicological Reviews. The findings of this assessment and 23

related documents produced during its development are available on the IRIS website 24

( http://www.epa.gov/iris ). Appendices for chemical and physical properties, toxicokinetic 25

information, and summar ies of toxicity studies and other information are provided as Supplemental 26

Information to this assessment

. 27

For additional information about this assessment or for general questions regarding IRIS, 28
please contact EPA’s IRIS Hotline at 202

- 566

- 1676 (phone), 202

- 566

- 1749 (fax), or 29

hotline.iris@epa.gov

. 30

1 PAHs are a large class of chemical compounds formed during the incomplete combustion of organic matter. They consist of only carbon and hydrogen arranged in two or more fused rings.Toxicological Review of Benzo[a]pyrene

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

xii DRAFT

— DO NOT CITE OR QUOTE Chemical Properties and Uses 1

Benzo[a]pyrene is a five

- ring PAH . It is a pale yellow crystalline solid with a faint aromatic 2

odor. It is relatively insoluble in water a nd has low volatility. Benzo[a]pyrene is released to the air 3

from both natural and anthropogenic sources and removed from the atmosphere by photochemical 4
oxidation; reaction with nitrogen oxides, hydroxy and hydroperoxy radicals, ozone, sulfur oxides, 5

and peroxyacetyl nitrate; and dry deposition to land or water. In air, benzo[a]pyrene is 6

predominantly adsorbed to particulates but may also exist as a vapor at high temperatures (NLM, 7

2010). 8
There is no known commercial use for benzo[a]pyrene; it is only produced as a research 9

chemical. Benzo[a]pyrene is ubiquitous in the environment primarily as a result of incomplete 10

combustion emissions. It is released to the environment via both natural sources (such as forest 11
fires) and anthropogenic sources includi ng stoves/furnaces burning fossil fuels (especially wood 12
and coal), motor vehicle exhaust, cigarettes, and various industrial combustion processes (ATSDR, 13

1995). Benzo[a]pyrene is also found in soot and coal tars. Mahler et al. (2005) has reported that 14

u rban run

- off from asphalt

- paved car parks treated with coats of coal

- tar emulsion seal could 15
account for the majority of PAHs in many watersheds. Benzo[a]pyrene exposure can also occur to 16

workers involved in the production of aluminum, coke, graphite, sil icon carbide, and in coal tar 17
distillation. The major sources of non

- occupational exposure are cigarettes and food. 18

Assessments by Other National and International Health Agencies 19

Toxicity information on benzo[a]pyrene has been evaluated by California EPA (CalEPA), the 20

World Health Organization, Health Canada, the International Agency for Research on Cancer, and 21

the European Union. The results of these assessments are presented in Appendix A. It is important 22

to recognize that these assessments may have b een prepared for diff erent purposes and may utilize 23

different methods, and that newer studies may be included in the IRIS assessment. 24Toxicological Review of Benzo[a]pyrene

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

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# PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS 2



## 1. Scope of the IRIS Program 3

Soon after EPA was established in 4 1970, it was at the forefront of developing 5 risk assessment as a science and applying it in 6 decisions to protect human health and the 7 environment. The Clean Air Act, for example, 8 mandates that EPA provide “an ample margin 9 of safety to protect public health”; the Safe 10 Drinking Water Act, that “no adverse effects 11 on the health of persons may reasonably be 12 anticipated to occur, allowing an adequate 13 margin of safety.” Accordingly, EPA uses 14
information on the adverse health effects and 15

on exposure levels below which t hese effects 16 are not anticipated to occur. 17 IRIS assessments critically review the 18 publicly available studies to identify adverse 19
health effects from long

- term exposure to 20 chemicals and to characterize exposure

21 response relationships. An assessment may 22

co ver a single chemical, a group of 23 structurally or toxicologically related 24 chemicals, or a complex mixture. Exceptions 25 are chemicals currently used exclusively as 26 pesticides, ionizing and non

- ionizing 27 radiation, and criteria air pollutants listed 28
under sec tion 108 of the Clean Air Act 29 (carbon monoxide, lead, nitrogen oxides, 30 ozone, particulate matter, and sulfur oxides; 31

EPA ’s Integrated Science Assessments 32 evaluate the effects from these pollutants in 33 ambient air). 34 Periodically , the IRIS Program asks 35
other EPA programs and regions, other 36 federal agencies, state government agencie s, 37 and the general public to nominate chemicals 38
and mixtures for future assessment or 39 reassessment. These agents may be found in 40 air, water, soil, or sediment. Selection is based 41

on program and regional office priorities and 42 on availability of adequate information to 43 evaluate the potential for adverse effects. IRIS 44 may assess other agents as an urgent public 45
health need arises. IRIS also reassesses agents 46 as significant new studies ar e published. 47



## 2. Process for D eveloping and P eer 48



## R eviewing IRIS A ssessments 49

The process for developing IRIS 50 assessments (revised in May 2009) involves 51 critical analysis of the pertinent studies, 52 opportunities for public input, and multiple 53
levels of scientific review. EPA revises draft 54 assessments after each review, and external 55 drafts and comments become part of the 56 public record ( U.S. EPA

, 2009). 57

Step 1. Development of a draft 58 Toxicological Review (usually about 59

11

- 1/2 months duration). The draft 60

assessment c onsiders all pertinent 61 publicly available studies and applies 62 consistent criteria to evaluate the studies, 63 identify health effects, weigh the evidence 64 of causation for each effect, identify 65 mechanistic events and pathways, and 66 derive toxicity values. 67

Step 2. Internal review by scientists in EPA 68 programs and regions (2 months). The 69
draft assessment is revised to address 70
comments from within EPA. 71

Step 3. Interagency science consultation 72
with other federal agencies and the 73
Executive Offices of the President 74 (1

- 1/2 months). The draft assessment is 75 revised to address the interagency 76

comments. The science consultation draft, 77 interagency comments, and EPA’s 78 response to major comments become part 79 of the public record. 80

Step 4. External peer review, after public 81

revie w and comment (3

- 1/2 months). 82
EPA releases the draft assessment for 83Toxicological Review of Benzo[a]pyrene

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

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— DO NOT CITE OR QUOTE public review and comment, followed by 1 external peer review. The peer review 2 meeting is open to the public and includes 3 time for oral public comments. The peer 4
reviewers also receive the w ritten public 5

comments. The peer reviewers assess 6
whether the evidence has been assembled 7 and evaluated according to guidelines and 8 whether the conclusions are justified by 9
the evidence. The peer review draft, peer 10 review report, and written public 11

comment s become part of the public 12
record. 13

Step 5. Revision of draft Toxicological 14 Review and development of draft IRIS 15 Summary (2 months). The draft 16 assessment is revised to reflect the peer 17 review comments, public comments, and 18 newly published studies that are critical 19
to the conclusions of the assessment

. The 20 disposition of peer review comments and 21 public comments becomes part of the 22 public record. 23

Step 6. Final EPA review and interagency 24
science discussion with other federal 25 agencies and the Executive Offices of 26
the President (1

- 1/2 months). The draft 27 assessment and summary are revised to 28 address EPA and interagency comments. 29 The science discussion draft, written 30 interagency comments, and EPA’s 31 response to major comments become part 32 of the public record. 33

Step 7

. Completion and posting (1 month). 34 The Toxicological Review and IRIS 35

S ummary are posted on the IRIS website 36 ( http:// www.epa.gov/ iris/ ). 37

The remainder of this Preamble 38 addresses step 1, the development of a draft 39 Toxicological Review. IRIS assessments 40 follow standard practices of evidence 41 evaluation and peer review, many of which 42 are discussed in EPA guidelines ( U.S. EPA

, 43 1986a, 1986b, 1991, 1996, 1998, 2000a, 44 2005a, 2005b) and other methods ( U.S. EPA

, 45 1994, 2000b, 2002, 2006 a
, 2006b, 2011). 46 Transparent application of scientific 47 judgment is of paramount importance. To 48 provide a harmonized approach across IRIS 49 assessments, this Preamble summarizes 50 concepts from these guidelines and 51 emphasizes principles of general 52

ap plicability. 53



## 3. Identifying and S electing 54



## P ertinent S tudies 55



### 3.1. I dentifying studies 56

Before beginning an assessment, EPA 57 conducts a comprehensive search of the 58 primary scientific literature. The literature 59 search follows standard practices and 60
includes the PubMed and ToxNet databases of 61 the National Library of Medicine and other 62 databases listed in EPA’s HERO system 63 (Health and Environmental Research Online, 64 http:// hero.epa.gov/ ). Each assessment 65 specifies the search strategies, keywords, and 66

cut

- off dates of its literature searches. EPA 67 posts the results of the literature search on 68 the IRIS website and requests information 69 from the public on additional studies and 70 ongoing research. 71

EPA also considers studies received 72 through the IRIS Submission Desk and studies 73 (typically unpublished) submitted to EPA 74
under the Toxic Substances Control Act. 75
Material submitted as Confidential Business 76 Information is considered only if it includes 77 health and safety data that can be publicly 78
release d. If a study that may be critical to the 79
conclusions of the assessment has not been 80

peer

- reviewed, EPA will have it peer

81
reviewed. 82
EPA also examines the toxicokinetics 83 of the agent to identify other chemicals (for 84 example, major metabolites of the agent) to 85 include in the assessment if adequate 86 information is available, in order to more 87 fully explain the toxicity of the agent and to 88 suggest dose metrics for subsequent 89 modeling. 90 In assessments of chemical mixtures, 91 mixture studies are preferred for their ab ility 92 to reflect interactions among components. 93 The literature search seeks, in decreasing 94 order of preference (U.S. EPA, 1986a, 2000a) : 95Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE – Studies of the mixture being assessed. 1

– Studies of a sufficiently similar mixture. 2 In evaluating similarity, the assessm ent 3
considers the alteration of mixtures in 4 the environment through partitioning 5
and transformation. 6

– Studies of individual chemical 7 components of the mixture, if there are 8 not adequate studies of sufficiently 9
similar mixtures. 10



### 3.2. Selecting pertinent epidemiologic 11


### studies 12

Study design is the key consideration 13 for selecting pertinent epidemiologic studies 14
from the results of the literature search. 15

– Cohort studies and case

- control studies 16 provide the strongest epidemiologic 17 evidence, as they collect infor mation 18 about individual exposures and effect

. 19

– Ecologic studies (geographic 20 correlation studies) relate exposures 21
and effects by geographic area. They 22 can provide strong evidence if there are 23 large exposure contrasts between 24 geographic areas, relatively lit tle 25 exposure variation within study areas, 26 and population migration is limited. 27

– Case reports of high or accidental 28 exposure lack definition of the 29 population at risk and the expected 30
number of cases. They can provide 31
information about a rare effect or abou t 32 the relevance of analogous results in 33
animals. 34

The assessment briefly reviews 35 ecologic studies and case reports but reports 36 details only if they suggest effects not 37 identified by other epidemiologic studies. 38



### 3.3. Selecting pertinent experimental 39


### studies 40

Expos ure route is a key design 41 consideration for selecting pertinent 42 experimental studies from the results of the 43
literature search. 44 – Studies of oral, inhalation, or dermal 45 exposure involve passage through an 46 absorption barrier and are considered 47 most pertinent to human 48 environmental exposure. 49

– Injection or implantation studies are 50 often considered less pertinent but may 51 provide valuable toxicokinetic or 52
mechanistic information. They also may 53 be useful for identifying effects in 54 animals if deposition or absorption is 55 problematic (for example, for particles 56 and fibers). 57

Exposure duration is also a key design 58 consideration for selecting pertinent 59 experimental studies. 60

– Studies of effects from chronic exposure 61 are most pertinent to lifetime human 62

exposure. 63

– Studies of e ffects from less

- than

64 chronic exposure are pertinent but less 65 preferred than studies of chronic 66

exposure. 67

Short

- duration studies involving 68 animals or humans may provide toxicokinetic 69
or mechanistic information. Research 70 involving human subjects is consider ed only 71 if conducted according to ethical principles. 72 For developmental toxicity and 73 reproductive toxicity, irreversible effects may 74 result from a brief exposure during a critical 75 period of development. Accordingly, 76 specialized study designs are used for t hese 77 effects ( U.S. EPA

, 1991, 1996, 1998 , 2006b ). 78



## 4. Evaluating the Q uality of 79



## I ndividual S tudies 80



### 4.1. Evaluating the quality of 81 

### epidemiologic studies 82

The assessment evaluates design and 83 methodologic aspects that can increase or 84 decrease the weight given to each 85

e pidemiologic study in the overall evaluation 86 ( U.S. EPA

, 1991, 1994, 1996, 1998, 2005a): 87Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE – Documentation of study design, 1 methods, population characteristics, 2
and results. 3

– Definition and selection of the study 4 group and comparison group. 5

– Ascertainment of expo sure to the 6
chemical or mixture under 7
consideration. 8

– Ascertainment of disease or health 9
effect. 10

– Duration of exposure and follow

- up and 11 adequacy for assessing the occurrence 12
of effects. 13

– Characterization of exposure during the 14 critical period s

. 15

– Sample size and statistical power to 16 detect anticipated effects. 17

– Participation rates and potential for 18
selection bias as a result of the achieved 19 participation rate. 20

– Potential confounding and other 21 sources of bias addressed in the study 22 design or in the analysis of re sults. The 23 basis for consideration of confounding 24 is a reasonable expectation that the 25 confounder is related to both exposure 26
and outcome. 27

For developmental toxicity, 28 reproductive toxicity, neurotoxicity, and 29 cancer there is further guidance on the 30

nuances of evaluating epidemiologic studies 31 of these effects ( U.S. EPA

, 1991, 1996, 1998, 32 2005a). 33



### 4.2. Evaluating the quality of 34 

### experimental studies 35

The assessment evaluates design and 36 methodologic aspects that can increase or 37 decrease the weight given to each 38 experimental study in the overall evaluation 39 ( U.S. EPA

, 1991, 1994, 1996, 1998, 2005a): 40

– Documentation of study design, animals 41 and methods, basic data, and results. 42 – Relevance to humans of the animal 43 model and the experimental methods. 44

– Characterization of t he nature and 45 extent of impurities and contaminants 46
of the administered chemical or 47

mixture. 48

– Characterization of dose and dosing 49 regimen (including age at exposure) 50 and their adequacy to elicit adverse 51
effects. 52

– Numbers of animals and statistical 53 power to d etect dose

- related 54
differences or trends. 55

– Ascertainment of survival, vital signs, 56 disease or effects, and cause of death. 57

– Control of other variables that could 58
influence the occurrence of effects. 59

The assessment uses statistical tests 60
to evaluate whether t he observations may be 61
due to chance. The standard for determining 62 statistical significance of a response is a trend 63 test or comparison of outcomes in the 64 exposed group with concurrent controls. In 65 some situations, examination of historical 66
control data fr om the same laboratory within 67 a few years of the study may improve the 68 analysis. For an uncommon effect that is not 69 statistically significant compared with 70 concurrent controls, historical controls may 71 show that the effect is unlikely to be due to 72
chance. F or a response that appears 73 significant against a concurrent control 74 response that is unusual, historical controls 75 may offer a different interpretation ( U.S. EPA

, 76 2005a). 77 For developmental toxicity, 78 reproductive toxicity, neurotoxicity, and 79
cancer there is further guidance on the 80 nuances of evaluating experimental studies of 81 these effects ( U.S. EPA

, 1991, 1996, 1998, 82 2005a). In multi

- generation studies, agents 83 that produce developmental effects at doses 84
that are not toxic to the maternal animal are 85 of specia l concern. Effects that occur at doses 86 associated with mild maternal toxicity are not 87 assumed to result only from maternal 88 toxicity. Moreover, maternal effects may be 89Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE reversible, while effects on the offspring may 1 be permanent ( U.S. EPA

, 1991, 1998). 2



### 4.3. Repor ting study results 3

The assessment uses evidence tables 4

to summarize details of the design and key 5 results of pertinent studies. There may be 6 separate tables for each site of toxicity or type 7 of study. 8 If a large number of studies observe 9 the same effect, t he assessment considers the 10 study characteristics in this section to identify 11 the strongest studies or types of study. The 12 tables report details from these studies, and 13 the assessment explains the reasons for not 14 reporting details of other studies or group s of 15
studies that do not add new information. 16 Supplemental material provides references to 17 all studies considered, including those not 18
summarized in the tables. 19 The assessment discusses strengths 20 and limitations that affect the interpretation 21 of each study

. If the interpretation of a study 22
in the assessment differs from that of the 23 study authors, the assessment discusses the 24
basis for the difference. 25
As a check on the selection and 26 evaluation of pertinent studies, EPA asks peer 27 reviewers to identify studies that were not 28 adequately considered. 29



## 5. Weighing the O verall E vidence of 30



## E ach E ffect 31



### 5.1. Weighing epidemiologic evidence 32

For each effect, the assessment 33
evaluates the evidence from the 34 epidemiologic studies as a whole to 35 determine the extent to which any observed 36 associations may be causal. Positive, negative, 37 and null results are given weight according to 38 study quality. This evaluation considers 39 aspects of an association that suggest 40 causality, discussed by Hill (1965) and 41 elaborated by Rothman and Greenl and 42 (1998) ( U.S. EPA

, 1994, 2002, 2005a; DHHS

, 43 2004). 44 Strength of association: The finding of a 45 large relative risk with narrow confidence 46 intervals strongly suggests that an 47 association is not due to chance, bias, or 48
other factors. Modest relative risks, 49 however, may reflect a small range of 50 exposures, an agent of low potency, an 51

increase in a n e ffect that is common, 52 exposure misclassification, or other 53
sources of bias. 54

Consistency of association: An inference of 55 causality is strengthened if elevated risks 56 are observed in independent studies of 57 different populations and exposure 58

scenarios. Reproducibility of findings 59 constitutes one of the strongest 60 arguments for causality. Discordant 61
results sometimes reflect differences in 62 study design, exposure
, or confo unding 63
factors. 64

Specificity of association: As originally 65 intended, this refers to one cause 66
associated with one effect

. Current 67 understanding that many agents cause 68 multiple effect s and many effect s have 69 multiple causes make this a less 70 informative aspect of causality, unless the 71 effect is rare or unlikely to have multiple 72

causes. 73

Temporal relationship: A causal 74 interpretation requires that exposure 75 precede development of the e ffect

. 76

Biologic gradient (exposure

- response 77 relationship): Exposure

- response 78 relationships strongly suggest causality. A 79 monotonic increase is not the only 80 pattern consistent with causality. The 81 presence of an exposure

- response 82 gradient also weighs against bias and 83 confounding as the source of an 84

association. 85

Biologic plausibility: An inference of 86 causality is strengthened by data 87 demonstrating plausible biologic 88 mechanisms, if available. 89

Coherence: An inference of causality is 90 strengthened by supportive results from 91Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE animal experiments, toxicokinetic studies, 1
and short

- term tests. C oherence may also 2 be found in other lines of evidence, such 3 as changing disease patterns in the 4 population. 5

“Natural experiments”: A change in 6 exposure that brings about a change in 7 disease frequency provides strong 8 evidence of causality , for example, an 9 intervention to reduce exposure in the 10 workplace or environment that is 11 followed by a reduction of an adverse 12
effect

. 13

Analogy: Information on structural 14 analogues or on chemicals that induce 15 similar mechanistic events can provide 16 insight into causality. 17

Th ese considerations are consistent 18 with guidelines for systemic reviews that 19 evaluate the quality and strength of evidence. 20 Confidence is increased if the magnitude of 21 effect is large, if there is evidence of an 22

exposure

- response relationship, or if an 23

asso ciation was observed and the plausible 24 biases would tend to decrease the reported 25
effect. Confidence is decreased for study 26 limitations, inconsistency of results, 27 indirectness of evidence, imprecision, or 28 reporting bias (Guyatt et al

., 2008a,b). 29
To make cl ear how much the 30 epidemiologic evidence contributes to the 31 overall weight of the evidence, the 32 assessment may choose a descriptor such as 33 sufficient evidence, suggestive evidence, 34 inadequate evidence, or evidence suggestive 35 of no causal relationship to cha racterize the 36 epidemiologic evidence of each effect (DHHS

, 37 2004). 38



### 5.2. Weighing experimental animal 39


### evidence 40

For each effect, the assessment 41
evaluates the evidence from the animal 42 experiments as a whole to determine the 43 extent to which they indicate a potential for 44
effects in humans. Consistent results across 45 various species and strains increase 46
confidence that similar results would occur in 47 humans. S everal concepts discussed by Hill 48 (1965) are pertinent to the weight of 49 experimental results: consistency of 50

resp onse, dose

- response relationships, 51 strength of response, biologic plausibility, and 52 coherence ( U.S. EPA

, 1994, 2002, 2005a). 53 In weighing evidence from multiple 54 experiments, U.S. EPA (2005a) distinguishes 55

Conflicting evidence (that is, mixed positive 56
and n egative results in the same sex and 57 strain using a similar study protocol) 58
from 59

Differing results (that is, positive results and 60 negative results are in different sexes or 61 strains or use different study protocols). 62

Negative or inconclusive results do 63

not invalidate positive results in a different 64 experimental system. EPA regards both as 65
valid observations and looks to 66 methodological differences or, if available, 67 mechanistic information to reconcile differing 68
results. 69
It is well established that there are 70 critical periods for some developmental and 71 reproductive effects. Accordingly, the 72
assessment determines whether critical 73 periods have been adequately investigated 74 ( U.S. EPA

, 1991, 1996, 1998, 2005a, 2005b

, 75
2006b ). Similarly, the assessment determines 76 whether the database is adequate to evaluate 77
other critical sites and effects. 78 In evaluating evidence of genetic 79 toxicity: 80

– Demonstration of gene mutations, 81 chromosome aberrations, or aneuploidy 82 in humans or experimental mammals 83 ( in vivo ) provides the stron gest 84
evidence. 85

– This is followed by positive results in 86 lower organisms or in cultured cells 87 ( in vitro ) or for other genetic events. 88

– Negative results carry less weight, 89 partly because they cannot exclude the 90 possibility of effects in other tissues 91 (IARC 200 6). 92Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE For germ

- cell mutagenicity, EPA has 1 defined categories of evidence, ranging from 2 positive results of human germ

- cell 3 mutagenicity to negative results for all effects 4 of concern (EPA 1986b). 5



### 5.3. Characterizing modes of action 6

For each effect, the assessment 7
discusses the available information on its 8 modes of action and associated key events 9 (key events being empirically observable, 10 necessary precursor steps or biologic 11 markers of such steps; mode of action being a 12 series of key events involving interaction w ith 13 cells, operational and anatomic changes, and 14 resulting in disease). Pertinent information 15 may also come from studies of metabolites or 16 of compounds that are structurally similar or 17 that act through similar mechanisms. The 18 assessment addresses several q uestions 19 about each hypothesized mode of action ( U.S. 20

EPA

, 2005a). Information on mode of action 21 is not required for a conclusion that an effect 22 is causally related to an agent (EPA 2005a). 23

1) Is the hypothesized mode of action 24 sufficiently supported in test animals? 25 Strong support for a key event being 26 necessary to a mode of action can come 27 from experimental challenge to the 28 hypothesized mode of action, in which 29
studies that suppress a key event observe 30

suppress ion of the e ffect

. Support for a 31
mode of action is meaningfully 32 strengthened by consistent results in 33 different experimental models, much 34
more so than by replicate experiments in 35
the same model. The assessment may 36 consider various aspects of causality in 37 addressing this question. 38

2) Is the hypothesized mo de of action 39
relevant to humans? The assessment 40 reviews the key events to identify critical 41
similarities and differences between the 42
test animals and humans. Site 43
concordance is not assumed between 44 animals and humans, though it may hold 45
for certain effects or modes of action. 46 Information suggesting quantitative 47 differences in doses where effects would 48
occur in animals or humans is considered 49

in the dose

- response analys i s but is not 50
used to determine relevance. Similarly, 51 anticipated levels of human exposure are 52
not used to determine relevance. 53

3) Which populations or life

- stages can 54 be particularly susceptible to the 55 hypothesized mode of action? The 56 assessment reviews the key events to 57 identify populations and life

- stages that 58 might be susceptible to their occu rrence. 59 Quantitative differences may result in 60 separate risk estimates for susceptible 61 populations or life

- stages. 62

The assessment discusses the 63 likelihood that an agent operates through 64 multiple modes of action. An uneven level of 65 support for different modes of action can 66 reflect disproportionate resources spent 67 investigating them ( U.S. EPA

, 2005a). It 68 should be noted that in clinical reviews, the 69 credibility of a series of studies is reduced if 70 evidence is limited to studies funded by one 71
interested sec tor (Guyatt et al

., 2008b). 72 For cancer, the assessment evaluates 73 evidence of a mutagenic mode of action to 74 guide extrapolation to lower doses and 75 consideration of susceptible lifestages. Key 76 data include the ability of the agent or a 77
metabolite to react wi th or bind to DNA, 78 positive results in multiple test systems, or 79 similar properties and structure

- activity 80 relationships to mutagenic carcinogens (EPA 81 2005a). 82



### 5.4. Characterizing the overall weight 83


### of the evidence 84

After weighing the epidemiologic and 85 experiment al studies pertinent to each effect, 86 the assessment may select a standard 87 descriptor to characterize the overall weight 88
of the evidence. For example, the following 89 standard descriptors combine epidemiologic, 90 experimental, and mechanistic evidence of 91

carcin ogenicity ( U.S. EPA

, 2005a). 92

Carcinogenic to humans: There is 93 convincing epidemiologic evidence of a 94Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE causal association (that is, there is 1
reasonable confidence that the 2 association cannot be fully explained by 3 chance, bias, or confounding), or there is 4

st rong human evidence of cancer or its 5 precursors, extensive animal evidence, 6 identification of key precursor events in 7 animals, and strong evidence that they 8 are anticipated to occur in humans. 9

Likely to be carcinogenic to humans: The 10
evidence demonstrates a potential hazard 11
to humans but does not meet the criteria 12 for carcinogenic. There may be a plausible 13 association in humans, multiple positive 14 results in animals, or a combination of 15 human, animal, or other experimental 16
evidence

. 17

Suggestive evidence of ca rcinogenic 18 potential: The evidence raise s concern 19
for effects in humans but is not sufficient 20 for a stronger conclusion. This descriptor 21 covers a range of evidence, from a 22 positive result in the only available study 23 to a single positive result in an extens ive 24 database that includes negative results in 25 other species. 26

Inadequate information to assess 27 carcinogenic potential: No other 28 descriptors apply. Conflicting evidence can 29
be classified as inadequate information if 30 all positive results are opposed by 31
negat ive studies of equal quality in the 32
same sex and strain. Differing results

, 33 however, can be classified as suggestive 34
evidence or as likely to be carcinogenic. 35

Not likely to be carcinogenic to humans: 36
There is robust evidence for concluding 37
that there is no basis for concern. There 38 may be no effects in both sexes of at least 39 two appropriate animal species; positive 40 animal results and strong, consistent 41
evidence that each mode of action in 42 animals does not operate in humans; or 43 convincing evidence that effects are not 44 likely by a particular exposure route or 45
below a defined dose. 46 Multiple descriptors may be used if 47 there is evidence that carcinogenic effects 48 differ by dose range or exposure route (EPA 49 2005a). 50 EPA is investigatin g and may on a trial 51 basis propose standard descriptors to 52 characterize the overall weight of the 53
evidence for effects other than cancer. 54



## 6. Selecting S tudies for D erivation of 55



## T oxicity V alues 56

For each effect where there is credible 57
evidence of an association with the agent, the 58 assessment derives toxicity values if there are 59 suitable epidemiologic or experimental data. 60 The decision to derive toxicity values may be 61 linked to the weigh

- of

- evidence descriptor. 62 For example, EPA typically derives toxicity 63
values for agents classified as carcinogenic to 64
humans or as likely to be carcinogenic (U.S. 65 EPA, 2005a). 66

Dose

- response analysis requires 67
different information than is needed to 68 identify the occurrence of effects. Although 69
dose

- response relationships may contrib ute 70 to a qualitative assessment quantitative 71 measures of dose and response are essential 72
to dose

- response analysis. Then, other factors 73 being equal ( U.S. EPA

, 1994, 2005a): 74

– Epidemiologic studies are preferred 75 over animal studies, if quantitative 76

measures o f exposure are available and 77
estimated effects can be attributed to 78 the agent. 79

– Among experimental animal models, 80 those that respond most like humans 81 are preferred, if the comparability of 82 response can be determined. 83

– Studies by a route of human 84

environmenta l exposure are preferred, 85 although a validated toxicokinetic 86 model can also be used to extrapolate 87
across exposure routes. 88

– Studies of longer exposure duration and 89
follow

- up are preferred, to minimize 90 uncertainty about whether effects 91 change with time. 92Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE – Stud ies with multiple exposure levels 1 are preferred for their ability to provide 2 information about the shape of the 3

exposure

- response curve. 4

– S tudies with adequate power to detect 5 effects at lower exposure levels are 6 preferred, to minimize the extent of 7
extrapo lation to levels found in the 8

environment. 9

Studies with non

- monotonic 10

exposure

- response relationships are not 11 necessarily excluded from the analysis. A 12 diminished effect at higher exposure levels 13 may be satisfactorily explained by factors 14 such as competing toxicity, saturation of 15 absorption or metabolism, exposure 16 misclassification, or selection bias. 17 If a large number of studies are 18
suitable for dose

- response analysis, the 19 assessment considers the study 20
characteristics in this section to focus on the 21

most informative data. The assessment 22 explains the reasons for not analyzing other 23 groups of studies. As a check on the selection 24
of studies for dose

- response analysis, EPA 25 asks peer reviewers to identify studies that 26 were not adequately considered. 27



## 7. Deriv ing T oxicity V alues 28



### 7.1. General framework for dose

29 

### response analysis 30

EPA uses a two

- step approach that 31 distinguishes analysis of the observed dose

32 response data from inferences about lower 33 doses ( U.S. EPA

, 2005a). 34 Within the observed range, the 35 preferred approach is to use modeling to 36 incorporate a wide range of data into the 37 analysis. The modeling yields a point of 38 departure (an exposure level near the lower 39 end of the observed range, without significant 40 extrapolation to lower doses) ( S ections 7.2 41
and 7.3). 42 Extrap olation to lower doses 43
considers what is known about the modes of 44 action for each effect ( S ections 7.4 and 7.5). 45 When response estimates at lower doses are 46 not required, a n alternative is to derive 47 reference values , which are calculated by 48 applying factors that account for sources of 49 uncertainty and variability to the point of 50 departure ( S ection 7.6). 51 For a group of agents that induce an 52 effect through a common mode of action, the 53
dose

- response analysis may derive a relative 54 potency factor for each agent. A full dose

55 response analysis is conducted for one well

56
studied index chemical in the group, and then 57 the potencies of other members are 58 expressed in relative terms based on relative 59 toxic effects, relative absorption or metabolic 60 rates, quantitative struct ure

- activity 61 relationships, or receptor binding 62 characteristics (EPA 2000a, 2005a). 63 Increasingly, EPA is basing toxicity 64 values on combined analyses of multiple data 65 sets or multiple responses. EPA also 66 considers multiple dose

- response approaches 67 when they can be supported by robust data. 68



### 7.2. Modeling dose 69

The preferred approach for analysis 70 of dose is toxicokinetic modeling because of 71 its ability to incorporate a wide range of data. 72 The preferred dose metric would refer to the 73 active agent at the site of its b iologic effect or 74 to a close, reliable surrogate measure. The 75 active agent may be the administered 76
chemical or a metabolite. Confidence in the 77 use of a toxicokinetic model depends on the 78 robustness of its validation process and on 79
the results of sensitivit y analyses ( U.S. EPA

, 80 1994, 2005a, 2006 a ). 81 Because toxicokinetic modeling can 82 require many parameters and more data than 83 are typically available, EPA has developed 84 standard approaches that can be applied to 85 typical data sets. These standard approaches 86
also facilitate comparison across exposure 87 patterns and species. 88

– Intermittent study exposures are 89 standardized to a daily average over the 90 duration of exposure. For chronic 91 effects, daily exposures are averaged 92 over the lifespan. Exposures during a 93 critical pe riod, however, are not 94Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE averaged over a longer duration ( U.S. 1

EPA

, 1991, 1996, 1998, 2005a). 2

– Doses are standardized to equivalent 3 human terms to facilitate comparison of 4 results from different species: 5

Oral doses can be scaled 6 allometrically using mg/kg 3/4

- d as the 7 equivalent dose metric across species. 8

A llometric scaling pertains to 9 equivalence across species, not across 10 lifestages, and is not used to scale 11
doses from adult humans or mature 12
animals to infants or children ( U.S. 13

EPA

, 2005a, 2011). 14

Inhalation exposures are scaled using 15 dosimetry models that apply species

16 specific physiologic and anatomic 17
factors and consider whether the 18
effect occurs at the site of first contact 19 or after systemic circulation ( U.S. EPA

, 20 1994). 21

It can be informative to convert d oses 22 to target sites across different exposure 23

routes. If this approach is followed, the 24 assessment describes the underlying data, 25 algorithms, and assumptions ( U.S. EPA

, 26 2005a). 27 In the absence of study

- specific data 28 on, for example, intake rates or body we ight, 29 EPA has developed recommended values for 30
use in dose

- response analysis (EPA 1988). 31



### 7.3. Modeling response in the range of 32


### observation 33

Toxicodynamic (“biologically based”) 34 modeling can incorporate data on biologic 35 processes leading to a n effect

. Such model s 36 require sufficient data to ascertain a mode of 37 action and to quantitatively support model 38 parameters associated with its key events. 39 Because different models may provide 40 equivalent fits to the observed data but 41 diverge substantially at lower doses, criti cal 42 biologic parameters should be measured 43 from laboratory studies, not by model fitting. 44 Confidence in the use of a toxicodynamic 45 model depends on the robustness of its 46 validation process and on the results of 47 sensitivity analyses. Peer review of the 48 scientific basis and performance of a model is 49 essential ( U.S. EPA

, 2005a). 50 Because toxicodynamic modeling can 51 require many parameters and more 52 knowledge and data than are typically 53 available, EPA has developed a standard set 54 of empirical (“curve

- fitting”) models (http:// 55 www.epa.gov/ ncea/ bmds/) that can be 56 applied to typical data sets, including those 57
that are nonlinear. EPA has also developed 58 guidance on modeling dose

- response data, 59 assessing model fit, selecting suitable models, 60 and reporting modeling results ( U.S. EPA

, 61 2000b). Additional judgment or alternative 62 analyses are used when the procedure fails to 63 yield reliable results, for example, if the fit is 64 poor, modeling may be restricted to the lower 65 doses, especially if there is competing toxicity 66

at higher doses ( U.S. EPA

, 2005a). 67 Modeling is used to derive a point of 68 departure (U.S. EPA, 2000b, 2005a)

. (See 69

S ection 7.6 for alternatives if a point of 70 departure cannot be derived by modeling.) 71

– For dichotomous responses, the point 72 of departure is often the 95% lower 73
bound on the dose associated with a 74 10% response, but a lower response 75 that falls within the observed range 76 may be used instead. For example, 77 reproductive or developmental studies 78 often have power to detect a 5% 79 response; epidemiologic studie s, 1% or 80
lower

. 81

– For continuous responses, the point of 82 departure is ideally the dose where the 83
effect becomes biologically significant

. 84 In the absence of such definition, both 85 statistical and biologic factors are 86
considered

. 87



### 7.4. Extrapolating to lower doses 88

The purpose of extrapolating to lower 89 doses is to estimate risks from exposures 90
below the observed data. Low

- dose 91 extrapolation is typically used for known and 92 likely carcinogens. Low

- dose extrapolation 93Toxicological Review of Benzo[a]pyrene

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xxiii DRAFT

— DO NOT CITE OR QUOTE considers what is known about modes of 1 action ( U.S. E PA

, 2005a). 2

1) If a biologically based model has been 3 developed and validated for the agent, 4 extrapolation may use the fitted model 5
below the observed range if significant 6 model uncertainty can be ruled out with 7
reasonable confidence. 8

2) Linear extrapolation is used if the dose

9 response curve is expected to have a 10 linear component below the point of 11 departure. This includes: 12

– Agents or their metabolites that are 13

DNA

- reactive and have direct 14 mutagenic activity. 15

– Agents or their metabolites for which 16 human exposures or body burdens 17 are near doses associated with key 18 events leading to an effect. 19

Linear extrapolation is also used if the 20
evidence is insufficient to establish a 21
mode of action. 22

The result of linear extrapolation is 23 described by an oral slope factor or an 24
inhalation unit risk , which is the slope of 25
the dose

- response curve at lower doses 26 or concentrations, respectively

. 27

3) Nonlinear extrapolation is used if there 28
are sufficient data to ascertain the mode 29
of action and to conclude that it is not 30
linear at lower doses, and the agent does 31 not demonstrate mutagenic or other 32 activity consistent with linearity at lower 33
doses. If nonlinear extrapolation is 34 appropriate but no model is developed, 35
an alternative is to calculate reference 36
values. 37

If linear extrapolation is used, the 38 assessment develops a candidate slope factor 39
or unit risk for each suitable data set. These 40 results are arrayed, using common dose 41 metrics, to show the distribution of relative 42 potency across various effects and 43 experimental systems. The assessm ent then 44 calculates an overall slope factor and an 45 overall unit risk for the agent, considering the 46 various dose

- response analyses, the study 47 preferences discussed in Section 6, and the 48 possibility of basing a more robust result on 49 multiple data sets. 50



### 7.5. Cons idering susceptible 51 

### populations and life

- stages 52

The assessment analyzes the available 53 information on populations and life

- stages 54 that may be particularly susceptible to each 55
effect. If adequate data are available, the 56 assessment derives separate risk estim ates 57 for susceptible populations or life

- stages. A 58 tiered approach is used ( U.S. EPA

, 2005a). 59

1) If an epidemiologic or experimental study 60 reports quantitative results for a 61 susceptible population or life

- stage, these 62 data are analyzed to derive separate risk 63 estimates for susceptible individuals. 64

2) If data on risk

- related parameters allow 65 comparison of the general population and 66 susceptible individuals, these data are 67 used to adjust the general

- population risk 68 estimate for application to susceptible 69
individuals

. 70

3) In the absence of chemical

- specific data, 71 EPA has developed age

- dependent 72 adjustment factors early

- life exposure to 73 suspected carcinogens that have a 74 mutagenic mode of action

. There is 75 evidence of early

- life susceptibility to 76 various carcinogenic agents, but most 77 epidemiologic studies and cancer 78 bioassays do not include early

- life 79

exposure. To address the potential for 80 early

- life susceptibility, EPA recommends 81 (U.S. EPA, 2005b) : 82

– 10

- fold adjustment for exposures 83 before age 2 years. 84

– 3

- fold adjustment for ex posures 85 between ages 2 and 16 years. 86



### 7.6. Reference values and uncertainty 87


### factors 88

An oral reference dose or an inhalation 89 reference concentration is an estimate of an 90 exposure (including in susceptible 91Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE subgroups) that is likely to be without an 1 appreciable ris k of adverse health effects over 2 a lifetime ( U.S. EPA

, 2002). Reference values 3 are typically calculated for effects other than 4 cancer and for suspected carcinogens if a well 5
characterized mode of action indicates that a 6

necessary key event does not occur b elow a 7 specific dose

. Reference values provide no 8
information about risks at higher exposure 9
levels

. 10
The assessment characterizes effects 11
that form the basis for reference values as 12 adverse, considered to be adverse, or a 13 precursor to an adverse effect. Fo r 14 developmental toxicity , reproductive toxicity

, 15 and neurotoxicity there is guidance on 16 adverse effects and their biologic markers 17 ( U.S. EPA

, 1991, 1996, 1998). 18 To account for uncertainty and 19 variability in the derivation of a lifetime 20 human exposure where effects are not 21 anticipated to occur, reference doses and 22 reference concentrations are calculated by 23 applying a series of uncertainty factors to the 24 point of departure

. If a modeled point of 25 departure is not available, a no

- observed

26
adverse

- effect level or a lowest

- observed

27
adverse

- effect level is used instead

. The 28
assessment discusses scientific 29 considerations involving several areas of 30 variability or uncertainty. 31

Human variation. A factor of 10 is applied to 32 account for variation in susceptibility 33

acros s the human population and the 34 possibility that the available data may not 35 be representative of individuals who are 36 most susceptible to the effect. This factor 37 is reduced only if the point of departure is 38 derived specifically for susceptible 39 individuals (n ot for a general population 40 that includes both susceptible and non

41 susceptible individuals) ( U.S. EPA

, 1991, 42 1994, 1996, 1998, 2002). 43

Animal

- to

- human extrapolation. A factor of 44 10 is applied if animal results are used to 45
make inferences about humans. This 46 factor is often regarded as comprising 47 toxicokinetics and toxicodynamics in 48 equal parts. Accordingly, if the point of 49 departure is based on toxicokinetic 50 modeling, dosimetry modeling, or 51 allometric scaling across species, a factor 52
of 10 1/2 (rounded to 3) i s applied to 53 account for the remaining uncertainty 54 involving toxicodynamic differences. An 55
animal

- to

- human factor is not applied if a 56 biologically based model adjusts fully for 57 toxicokinetic and toxicodynamic 58 differences across species ( U.S. EPA

, 59 1991, 199 4, 1996, 1998, 2002 , 2011 ). 60

Adverse

- effect level to no

- observed

61
adverse

- effect level. If a point of 62 departure is based on a lowest

- observed

63
adverse

- effect level, the assessment must 64
infer a dose where such effects are not 65 expected. This can be a matter of great 66 uncertainty, especially if there is no 67
evidence available at lower doses. A 68 factor of 10 is applied to account for the 69 uncertainty in making this inference. A 70 factor other than 10 may be used, 71 depending on the magnitude and nature 72 of the response and the shape of the dose

73 response curve ( U.S. EPA

, 1991, 1994, 74 1996, 1998, 2002). 75

Subchronic

- to

- chronic exposure

. If a point 76 of departure is based on subchronic 77 studies, the assessment considers 78 whether lifetime exposure could have 79
effects at lower levels of exposure

. A 80 factor of 10 is applied to account for the 81 uncertainty in using subchronic studies to 82 make inferences about lifetime exposure. 83 This factor may also be applied for 84 developmental or reproductive effects if 85 exposure covered less than the full cri tical 86 period. A factor other than 10 may be 87 used, depending on the duration of the 88 studies and the nature of the response 89 ( U.S. EPA

, 1994, 1998, 2002). 90

Incomplete database. If an incomplete 91
database raises concern that further 92 studies might identify a more sensitive 93 effect, organ system, or life

- stage, the 94 assessment may apply a database 95 uncertainty factor ( U.S. EPA

, 1991, 1994, 96 1996, 1998, 2002). The size of the factor 97Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE depends on the nature of the database 1 deficiency. For example, EPA typically 2
follows the suggestion that a factor of 10 3 be applied if both a prenatal toxicity study 4
and a two

- generation reproduction study 5 are missing, and a factor of 10 1/2 if either 6 is missing ( U.S. EPA

, 2002). 7

In this way, the assessment derives 8
candidate reference values fo r each suitable 9
data set and effect that is credibly associated 10 with the agent. These results are arrayed, 11 using common dose metrics, to show where 12 effects occur across a range of exposures ( U.S. 13

EPA

, 1994). The assessment then selects an 14
overall reference dose and an overall 15 reference concentration for the agent to 16 represent lifetime human exposure levels 17 where effects are not anticipated to occur. 18 The assessment may also report 19
reference values for each effect. This would 20 facilitate subsequent cumulative risk 21

assessments that consider the combined 22
effect of multiple agents acting at a common 23 site or through common mechanisms ( U.S. 24

EPA

, 2002). 25



### 7.7. Confidence and uncertainty in the 26


### reference values 27

The assessment selects a standard 28 descriptor to characterize the level of 29 confidence in each reference value, based on 30 the likelihood that the value would change 31 with further testing. Confidence in reference 32 values is based on quality of the studies used 33 and completeness of the database, with more 34 weight given to the l atter. The level of 35
confidence is increased for reference values 36 based on human data supported by animal 37 data ( U.S. EPA

, 1994). 38

High confidence: The reference value is not 39 likely to change with further testing, 40 except for mechanistic studies that might 41
af

fect the interpretation of prior test 42
results. 43

Medium confidence: This is a matter of 44 judgment, between high and low 45
confidence. 46 Low confidence: The reference value is 47 especially vulnerable to change with 48 further testing. 49

These criteria are consistent with 50 guidelines for systematic reviews that 51 evaluate the quality of evidence. These also 52
focus on whether further research would be 53 likely to change confidence in the estimate of 54 effect (Guyatt et al

., 2008a). 55 All assessments discuss the significant 56 uncertainties encountered in the analysis. 57 EPA provides guidance on characterization of 58 uncertainty ( U.S. EPA

, 2005a). For example, 59 the discussion distinguishes model 60 uncertainty (lack of knowledge about the 61 most appropriate experimental or analytic 62 model) and parameter uncertainty (lack of 63 knowledge about the parameters of a model)

. 64
Assessments also discuss human variation 65 (interpersonal differences in biologic 66 susceptibility or in exposures that modify the 67 effects of the agent). 68

69

DHHS (2004) The health c onsequences of 70 smoking: a report of the Surgeon 71 General. Dept of Health and Human 72 Services, http:// www.cdc.gov/ 73 tobacco/ data_statistics/ sgr/ 2004/ 74
index.htm

. 75

Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck

76

Ytter Y, Alonso

- Coello P, 77
Sch ü nemann HJ (2008a) GRA DE: an 78 emerging consensus on rating quality 79 of evidence and strength of 80
recommendations. British Medical 81 Journal 336: 924

- 926, http:// 82 www.bmj.com/ content/ 336/ 7650/ 83
924.full

. 84

Guyatt GH, Oxman AD, Kunz R, Vist GE, Falck

85

Ytter Y, Sch ü nemann HJ (2008b) 86

GR ADE: what is “quality of evidence” 87 and why is it important to clinicians? 88 British Medical Journal 336: 995

- 998, 89 http:// www.bmj.com/ content/ 336/ 90 7651/ 995.full

. 91

Hill AB (1965) The environment and disease: 92 association or causation? Proceedings 93Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE of the Royal Society of Medicine 58: 1
295

- 300. 2

IARC (2006) Preamble to the IARC 3 Monographs. 4 http://monographs.iarc.fr/. 5

Rothman KJ, Greenland S (1998) Modern 6 Epidemiology. Philadelphia: 7 Lippincott Williams and Wilkins. 8

U.S. EPA (1986a) Guidelines for the Health 9

Ri sk Assessment of Chemical 10 Mixtures. EPA/630/R

- 98/002. 11

U.S. EPA (1986b) Guidelines for Mutagenicity 12 Risk Assessment. EPA/630/R

13 98/003. 14

U.S. EPA (1988) Recommendations for and 15 Documentation of Biological Values 16
for Use in Risk Assessment. 17 EPA/600/6

- 87/008. 18

U .S. EPA (1991) Guidelines for Developmental 19 Toxicity Risk Assessment. 20 EPA/600/FR

- 91/001. 21

U.S. EPA (1994) Methods for Derivation of 22
Inhalation Reference Concentrations 23 and Application of Inhalation 24 Dosimetry. EPA/600/8

- 90/066F. 25

U.S. EPA (1996) Guidelines fo r Reproductive 26 Toxicity Risk Assessment. 27 EPA/630/R

- 96/009. 28

U.S. EPA (1998) Guidelines for Neurotoxicity 29 Risk Assessment. EPA/630/R

30 95/001F. 31

U.S. EPA (2000a) Supplementary Guidance 32 for Conducting Health Risk 33
Assessment of Chemical Mixtures. 34 EPA/630/R

- 00/002

. 35

U.S. EPA (2000b) Benchmark Dose Technical 36 Guidance Document. EPA/630/R

37 00/001 (external review draft). 38

U.S. EPA (2002) A Review of the Reference 39
Dose and Reference Concentration 40 Processes. EPA/630/P

- 02/002F. 41

U.S. EPA (2005a) Guidelines for Carcinogen 42

Ris k Assessment. EPA/630/P

43 03/001F. 44 U.S. EPA (2005b) Supplemental Guidance for 45 Assessing Susceptibility from Early

46 Life Exposure to Carcinogens. 47 EPA/630/R

- 03/003F. 48

U.S. EPA (2006a) Approaches for the 49 Application of Physiologically Based 50 Pharmacokinetic (PBPK) Models and 51 Supporting Data in Risk Assessment. 52 EPA/600/R

- 05/043F. 53

U.S. EPA (2006b) A Framework for Assessing 54
Health Risks of Environmental 55 Exposures to Children. EPA/600/R

56 05/093F. 57

U.S. EPA (2009) IRIS Process. 58 http://www.epa.gov/iris/process.ht 59

m

. 60

U.S. EPA (2011) Recommended Use of Body 61 Weight 3/4 as the Default Method in 62
Derivation of the Oral Reference Dose. 63 EPA/100/R11/0001. 64

65

May 2012 version 66

67Toxicological Review of Benzo[a]pyrene

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# E XECUTIVE SUMMARY

Occurrence and Health Effects 1

2 Benzo[a]pyrene is a five ring polycyclic aromatic hydrocarbon (PAH). 3 Benzo[a]pyrene (along with other PAHs) is released into the atmosphere as a 4 component of smoke from forest fires, industrial processes, vehicle exhaust, 5 cigarettes, and through the burni ng of fuel (such as wood, coal, and petroleum 6 products). Oral exposure to benzo[a]pyrene can occur by eating foods grown in 7 areas contaminated with benzo[a]pyrene (from the air and soil) or by eating certain 8 food products, such as charred meats, where benz o[a]pyrene is formed during the 9 cooking process. Dermal exposure may occur from contact with soils or materials 10 that contain soot, tar, or crude petroleum products or by using certain 11 pharmaceutical products containing coal tars, such as those used to tre at the skin 12 conditions eczema and psoriasis. The magnitude of human exposure to 13 benzo[a]pyrene and other PAHs depends on factors such as lifestyle (e.g., diet, 14 tobacco smoking), occupation, and living conditions (e.g., urban versus rural setting, 15
domesti c heating, and cooking methods). 16 Animal studies demonstrate that exposure to benzo[a]pyrene may be 17 associated with developmental, reproductive, and immunological effects. In 18 addition, epidemiology studies involving exposure to PAH mixtures have reported 19 associations between adverse birth outcomes (including reduced birth weight, 20 postnatal body weight, and head circumference) and decreased fertility with 21 internal biomarkers of exposure to benzo[a]pyrene ( benzo[a]pyrene diol epoxide

22 DNA adducts). 23
Studies i n multiple animal species demonstrate that benzo[a]pyrene is 24 carcinogenic at multiple tumor sites (alimentary tract, liver, kidney, respiratory 25 tract, pharynx, and skin) by all routes of exposure. In addition, there is strong 26 evidence of carcinogenicity i n occupations involving exposure to PAH mixtures 27 containing benzo[a]pyrene, such as aluminum production, chimney sweeping, coal 28 gasification, coal

- tar distillation, coke production, iron and steel founding, and 29 paving and roofing with coal tar pitch. An i ncreasing number of occupational 30 studies demonstrate a positive exposure

- response relationship with cumulative 31 benzo[a]pyrene exposure and lung cancer. 32

33

Effects Other Than Cancer Following Oral Exposure 34

In animals, oral exposure to benzo[a]pyrene has bee n shown to result in developmental 35

toxicity, reproductive toxicity, and immunotoxicity. Developmental e ffects in rats and mice include 36

altered spatial learning and memory and cardi ovascular effects following ge s tational exposures. 37

Reproductive and immune effects include decreased sperm counts

, ovarian weight and follicle 38

numbers

, and decreased immunoglobulin and B

- cell numbers and thymus weight following oral 39

exposures in adult animals. In humans, b enzo[a]pyrene exposure occurs in conjunction with other 40Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE PAHs and, as such, attributing the observed effects to benzo[a]pyrene is complicated. However

, 1

human studies report associations between particular health endpoints and internal measures of 2

exposure, such as benzo[a]pyrene

- DNA adducts, or external measures of benzo[a]pyrene exposure. 3

Overall, t he human studies report developmental and reproductive effects that are generally 4

analogous to those observed in animal s, and provide qualitative, supportive evidence for the 5

hazard s associated with benzo[a]pyrene exposure

. 6

Developmental toxicity, represented by neurodevelopmental impairments, was chosen as 7

the basis for the porposed RfD as the available data indicate that neurodevelopmental effects 8

represent a sensitive hazard of benzo[a]pyrene ex posure. The neurodevelopmental study by Chen 9

et al. ( 2012 ) and the observed impaired spatial learning w ere used to derive the RfD. The endpoint 10

of impaired spatial learning, as measured by the increase in latency time to find a hidden platform 11

in the Mor ris water maze, was selected as the critical effect due to the sensitivity of this endpoint 12

and the observed dose

- response relationship of effects across dose groups. Benchmark dose 13

(BMD) modeling was utilized to derive the BMDL 1SD of 0.06 mg/kg

- day that was used as the point of 14

departure (POD) for RfD derivation. 15
The RfD was calculated by dividing the POD by a composite UF of 3 00 to account for the 16
extrapolation from animals to humans ( 10 ), for interindividual differences in human susceptibility 17

(10), an d for deficiencies in the toxicity database (3) as shown in Table ES

- 1

. 18

Table ES

- 1

. Summary of proposed reference dose (RfD) derivation 19

Critical effect Point of departure UF Chronic RfD

Neurodevelopmental impairment

PND 5

- 11 rat study, gavage (Chen et al., 2012) BMDL 1SD : 0.06 mg/kg

- day 3 00 2 x 10 - 4 mg/kg

- day

Confidence in the Chronic Oral RfD 20

The overall confidence in the RfD is medium. Confidence in the principal study (Chen et al., 21

2012) is medium

- to

- high . The design, conduct, and reporting of this neurodevelopmental study 22

was excellent and a wide variety of neurotoxicity endpoints were measured. Several subchronic 23

and developmental studies covering a wide variety of endpoints are available; however, the lack of 24

a multi

- generation toxicity study with exposure throughout development is not available. 25

Therefore, confidence in the database is medium. 26

Effects Other Than Cancer Following Inhalation Exposure 27

In animals, inhalation exposure to benzo[a]pyrene ha s been shown to result in 28

developmental and reproductive toxicity. Studies in rats following inha la tion exposure show 29

decreased fetal survival and brain effects in offspring, and decreased testes weight and sperm 30

counts in adult animals. Overall, the available human PAH mixtures studies report developmental 31Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE and reproductive effects that are generally analogous to those observed in animals, and provide 1

qualitative, supportive evidence for the hazards associated with benzo[a]pyrene exposure

. 2

Development al toxicity, represented by decreased fetal survival, was chosen as the basis for 3

the proposed RfC as the available data indicate that developmental effects represent a sensitive 4

hazard of benzo[a]pyrene exposure. The developmental inhalation study in rat s by Archibong et al. 5

(2002) and the observed decreased fetal survival following exposure to benzo[a]pyrene on GD 11

- 6

20 w ere used to derive the overall RfC

. The LOAEL of 25 μg/m 3 based on decreased fetal survival 7

was selected as the POD. The LOAEL was ad justed to account for the discontinuous daily exposure 8

to derive the POD ADJ and the human equivalent concentration (HEC) was calculated from the POD ADJ 9
by multiplying by the regional deposited dose ratio (RDDR ER ) for extrarespiratory (i.e., systemic) 10

effec ts

, as described in Methods for Derivation of Inhalation Reference Concentrations and 11
Application of Inhalation Dosimetry (U.S. EPA, 1994b). These adjustments resulted in a POD HEC of 12
4.6 μg/m 3 which was used as the POD for RfC derivation. 13

The RfC was calc ulated by dividin g the POD by a composite UF of 3 000 to account for 14

toxicodynamic differences between animals and humans (3), interindividual differences in human 15

susceptibility (10), LOAEL

- to

- NOAEL extrapolation (10), and deficien cies in the toxicity data base 16
(10 ) as shown in Table ES

- 2

. 17

Table ES

- 2

. Summary of proposed reference concentration (RfC) derivation 18

Critical effect Point of departure* UF Chronic RfC

Decreased fetal survival

GD 11

- 20 rat study (Archibong et al., 2002) LOAEL H EC : 4.6 μ g/m 3 (4.6 x 10 - 3 mg/m 3 ) 3 0 00 2 x 10 - 6 m g/m 3

* The POD was adjusted for continuous daily exposure: PODADJ= POD × hours exposed per day/24 hours, and 19

was further adjusted to a human equivalent concentration by adjusting by the regional deposited dose ratio 20

calculated using MPPD software (see Section 2.2.2. and Appendices) 21

Confidence in the Chronic Ora l RfC 22

The overall confidence in the RfC is low

- to

- medium. Confidence in the principal study 23
(Archibong et al., 2002) is medium. The conduct and reporting of this developmental dietary study 24

were adequate; however, a NOAEL was not identified. Confidence in the database is low due to the 25

lack of a multigeneration toxicity study and the lack of information on diverse toxicity endpoints 26

f ollowing subchronic and chronic inhalation exposure. However, confidence in the RfC is bolstered 27
by consistent systemic effects observed by the oral route (including reproductive and 28

developmental effects) and similar effects observed in human populations exposed to PAH 29

mixtures. 30Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Evidence for Human Carcinogenicity 1

Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), benzo[a]pyrene is 2
“carcinogenic to humans” based on strong and consistent evidence in animals and humans. The 3

evidence in cludes an extensive number of studies demonstrating carcinogenicity in multiple animal 4

species exposed via all routes of administration and increased cancer risks, particularly in the lung 5
and skin, in humans exposed to different PAH mixtures containing be nzo[a]pyr ene . Mechanistic 6

studies provide strong supporting evidence that links the metabolism of benzo[a]pyrene to DNA

7
reactive agents with key mutational events in genes that can lead to tumor development. These 8

events include formation of specific DNA adducts and specific mutations in oncogenes and tumor 9

suppressor genes that have been observed in humans exposed to PAH mixtures. This combination 10

of human, animal, and mechanistic evidence provides the basis for characterizing benzo[a]pyrene 11

as “carcino genic to humans.” 12

Quantitative Estimate of Carcinogenic Risk From Oral Exposure 13

Lifetime oral exposure to benzo[a]pyrene has been associated with forestomach, liver, oral 14
cavity, jejunum or duodenum, and auditory canal tumors in male and female Wistar rats

, 15
forestomach tumors in male and female Sprague

- Dawley rats, and forestomach, esophagus, tongue, 16

and larynx tumors in female B6C3F 1 mice (male mice were not tested). Less

- than

- lifetime oral 17

exposure to benzo[a]pyrene has also been associated with forestom ach tumors in more than 18

10 additional bioassays with several strains of mice. The Kroese et al. (2001) and Beland and Culp 19

(1998) studies were selected as the best available studies for dose

- response analysis and 20

extrapolation to lifetime cancer risk foll owing oral exposure to benzo[a]pyrene. These studies 21
included histological examinations for tumors in many different tissues, contained three exposure 22

levels and controls, contained adequate numbers of animals per dose group(~50/sex/group), 23

treated animal s for up to 2 years, and included detailed reporting methods and results (including 24
individual animal data)

. 25

EPA used the multistage

- Weibull model for the derivation of the oral slope factor because it 26

incorporates the time at which death

- with

- tumor occu rred and can account for differences in 27

mortality observed between the exposure group s . Using linear extrapolation from the BMDL 10 , 28
human equivalent oral slope factors were derived for each gender/tumor site combination (slope 29

factor = 0.1/BMDL 10 ) reporte d by Kroese et al. (2001) and Beland and Culp (1998). The oral slope 30

factor of 1 per mg/kg

- day based on the tumor response in the alimentary tract (forestomach, 31
esophagus, tongue, and larynx) of female B6C3F 1 mice (Beland and Culp, 1998) was selected as the 32

factor with the highest value (most sensitive) among a range of slope factors derived

. 33

Quantitative Estimate of Carcinogenic Risk From Inhalation Exposure 34

Inhalation exposure to benzo[a]pyrene has been ass ociated with squamous cell neoplasia in 35

the larynx, pharynx, trachea, esophagus, and forestomach, of male Syrian golden hamsters exposed 36

to benzo[a]pyrene condensed onto NaCl particles (Thyssen et al., 1981). Supportive evidence for 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE the carcinogenicity of inhaled benzo[a]pyrene comes from additional studies with hamsters 1

exposed to benzo[a]pyrene via intratracheal instillation. The Thyssen et al. (1981) bioassay 2

represents the only available data that exhibits a dose

- response relationship for cancer from 3

inhaled benzo[a]pyrene. 4

A time

- to

- tumor dose

- response model was fit to the time

- weighted average exposure 5
concentrations and the individual animal occurrence data for tumors in the larynx, pharynx, 6
trachea, esophagus, and forestomach. The inhalation unit risk of 5 × 10

- 4 per μg/m 3 was calculated 7
by linear extrapolation (slope factor = 0.1/BMCL 10 ) from a BMCL 10 of 0.20 mg/m 3 for the 8

occurrence of upper respiratory and upper digestive tract tumors in male hamsters chronically 9

exposed by inhalation to benzo[a ]pyrene (Thyssen et al., 1981). 10

Quantitative Estimate of Carcinogenic Risk From Dermal Exposure 11

Skin cancer in humans has been documented to result from occupational exposure to 12
complex mixtures of PAHs including benzo[a]pyrene

, such as coal tar, coal tar pitches, unrefined 13

mineral oils, shale oils, and soot. In animal models, numerous dermal bioassays have demonstrated 14

an increased incidence of skin tumors with increasing de rmal exposure of benzo[a]pyrene in all 15
species tested (mice, rabbits, rats, and gu inea pigs), although most benzo[a]pyrene bioassays have 16

been conducted in mice. The analysis in this assessment focuses on chronic carcinogenicity 17
bioassays in several strains of mice demonstrating increasing incidence of benign and malignant 18

skin tumors following repeated dermal exposure to benzo[a]pyrene for the animals’ lifetime. 19

The Poel (1959) and Sivak et al. (1997) studies were selected as the best available studies 20

for dose

- response analysis and extrapolation to lifetime cancer risk following dermal exposure to 21

benzo[a]pyrene. Both studies included at least three exposure levels (including several low doses ), 22
group sizes of 30−50 mice, and reporting of intercurrent mortality. Following the modeling, the 23

BMDL 10 was adjusted for interspecies differences by allometric scaling. The dermal slope factor of 24

0.005 per μg/day was calculated by linear extrapolation (slope factor = 0.1/BMDL 10

- HED ) from the 25

human equivalent POD for the occurrence of skin tumors in male mice chronically exposed 26

dermally to benzo[a]pyrene. As this slope factor has been developed for a local effect , it is not 27

intended to estimate systemic risk of cancer following dermal absorption of benzo[a]pyrene into 28

the systemic circulation. 29

Susceptible Populations and Lifestages 30

Benzo[a]pyrene has been determined to be carcinogenic by a mutagenic mode of action in 31

this assessment . According to the Supplemental Guidance for Assessing Susceptibility from Early Life 32

Exposure to Carcinogens (U.S. EPA, 2005b), individuals exposed during early life to carcinogens with 33

a mutagenic mode of action are assumed to have an increased risk for cancer. The oral slope factor 34

of 1 per mg/kg

- day, inhalation unit risk of 0.005 per μg/day , and dermal slope factor of 0.004 per 35
μg/day for benzo[a]pyrene, calculated from data applicable to adult exposures, do not reflect 36

presumed ear ly life sus ceptibility to this chemical. Alt hough some chemical

- specific data exist for 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE benzo[a]pyrene that demonstrate increased early life susceptibility to cancer, these data were not 1

considered sufficient to develop separate risk estimates for childho od exposure. In the absence of 2

adequate chemical

- specific data to evaluate differences in age

- specific susceptibility, the 3

Supplemental Guidance (U.S. EPA, 2005b) recommends that ADAFs be applied in estimating cancer 4

risk. The ADAFs are 10

- and 3

- fold ad justments that are combined with age specific exposure 5
estimates when estimating cancer risks from early life (<16 years of age) exposures to 6

benzo[a]pyrene. 7
Regarding effects other than cancer, there are epidemiological studies that report 8

associations between developmental effects (decreased postnatal growth, decreased head 9
circumference and neurodevelopmental delays) and internal biomarkers of exposure to 10

benzo[a]pyrene. 11

Studies in animals also indicate alterations in neurological development and he ighte ne d 12
susceptibility to reproductive effects following gestational or early postnatal exposure to 13

benzo[a]pyrene. 14

Key Issues Addressed in Assessment 15

The dermal slope factor was developed based on data in animals, and because there is no 16

established meth odology for extrapolating dermal toxicity from animals to humans. As such, 17

several alternative approaches were evaluated (See Appendix C in Supplemental Information )

. 18
Allometric scaling using body weight to the 3⁄4 power was selected based on known species 19

differences in dermal metabolism and penetration of benzo[a]pyrene. 20Toxicological Review of Benzo[a]pyrene

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# LITERATURE SEARCH STRATEGY | STUDY SELECTION

The literature search strategy used to identify primary, peer

- reviewed literat ure pertaining 1
to benzo[a]pyrene was conducted using the databases and keywords listed in Table LS

- 1. 2

References that were evaluated in other Agency and international health assessments were also 3

examined. A comprehensive literature search was last condu cted in February 2012. 4

Figure LS

- 1 depicts the literature search

, study selection strategy

, and the number of 5
references obtained at each stage of literature screening. Approximately 20,700 references were 6

identified with the initial keyword search. Ba sed on a secondary keyword search followed by a 7
preliminary manual screen of titles or abstracts by a toxicologist, approximately 1,1 90 references 8
were identified that provided information potentially relevant to characterizing the health effects 9

or physic al and chemical properties of benzo[a]pyrene. A more detailed manual review of titles, 10

abstracts, and/or papers was then conducted . Notable exclusions from the Toxicological Review 11
are large numbers of animal in vivo or in vitro studies designed to iden tify potential therapeutic 12

agents that would prevent the carcinogenicity or genotoxicity of benzo[a]pyrene and toxicity 13

studies of benzo[a]pyrene in nonmammalian species (e.g., aquatic species, plants). 14Toxicological Review of Benzo[a]pyrene

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xxxiv DRAFT

— DO NOT CITE OR QUOTE Table LS

- 1 . Details of the literature search strategy employed 1

Database Keywords a

Pubmed Chemical name (CASRN): benzo[a]pyrene (50

- 32

- 8) a

Syn onyms: benzo[d,e,f]chrysene, benzo[def] chrysene, 3,4

- benzopyrene, 1,2

- benzpyrene, 3,4

- bp, benz(a)pyrene, 3,4

- benzpyren, 3,4

- benzpyrene, 4,5

- benzpyrene, 6,7

- benzopyrene, benzopirene, benzo(alpha)pyrene

Initial keyword search Standard toxicology search Toxicity (including duration, effects to children and occupational exposure); development;
reproduction; teratogenicity; exposure routes; p harmacokinetics; toxicokinetics; metabolism; body fluids; endocrinology; carcinogenicity; genotoxicity; antagonists; inhibitors

Secondary keyword search b

Chemical

- specific keywords Cancer; genotoxicity, neurotoxicity, immunotoxicity, reproductive toxicity , developmental toxicity

Additional keywords lung OR skin combined AND tumor, neoplasm, papilloma, OR carcinoma; leukemia; forestomach; tongue; auditory canal; esophagus; larynx; pharynx; fertility; sperm; epididymis; seminiferous;
testosterone; cervical hyperplasia; corpus luteum; estrous; testicular, ovarian OR thymus with atrophy; weight AND testis, ovary, thymus, spleen, pup; spleen AND cells, lymphocytes; immunoglobulin; immunosuppression; motor; functional observational battery; neurobehavioral;

rota rod; nerve conduction; locomotor; neuromuscular; neurodevelopment; cognitive; learning; memory; righting. Toxcenter

Toxline

TSCATS Searched by chemical names (including synonyms) and CASRNs

ChemID

Chemfinder

CCRIS

HSDB

GENETOX

RTECS

a Keywords and synonyms were applied to the Pubmed, Toxcenter, and Toxline databases.

b Secondary keywords were selected from an understanding of the targets of benzo[a]pyrene toxicity gained from review of papers identified in literature searches conduct ed at the start of document development and

relevant review documents.

2Toxicological Review of Benzo[a]pyrene

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Figure LS

- 1 . Study selection strategy. 2Toxicological Review of Benzo[a]pyrene

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xxxvi DRAFT

— DO NOT CITE OR QUOTE Selection of studies for inclusion in the Toxicological Review was based on consideration of 1

the extent to which the study was informative and relevant to the assessment and general study 2
quality considerations. In general, the relevance of health effect studies was evaluated as outlined 3

in the Preamble and EPA guidance ( A Review of the Reference Dose and Refe rence Concentration 4

Processes (U.S. EPA, 2002) and Methods for Derivation of Inhalation Reference Concentrations and 5
Application of Inhaled Dosimetry (U.S. EPA, 1994)). The reasons for excluding epidemiological and 6
animal studies from the approximately 1, 190 references identified by the keyword search are 7
provided in Figure LS

- 1. 8

The available studies examining the health effects of benzo[a]pyrene exposure in humans 9
are discussed and evaluated in the hazard identification sections of the assessment ( Sectio n 1), with 10

specific limitations of individual studies and of the collection of studies noted. The common major 11
limitation of the human epidemiological studies (with respect to identifying potential adverse 12
health outcomes specifically from benzo[a]pyrene ) is that they all involve exposures to complex 13

mixtures containing other PAHs and other compounds. The evaluation of the epidemiological 14

literature focuses on studies in which possible associations between external measures of exposure 15

to benzo[a]pyrene or biomarkers of exposure to benzo[a]pyrene (e.g., benzo[a]pyene

- DNA adducts 16
or urinary biomarkers) and potential adverse health outcomes were evaluated. Pertinent 17
mechanistic studies in humans (e.g., identification of benzo[a]pyene

- DNA adducts and 18

chara cteristics of mutations in human tumors) were also considered in assessing the weight of 19
evidence for the carcinogenicity of benzo[a]pyrene. 20

The health effects literature for benzo[a]pyrene is extensive. All animal studies of 21

benzo[a]pyrene involving r epeated oral, inhalation, or dermal exposure that were considered to be 22

of acceptable quality, whether yielding positive, negative, or null results, were considered in 23
assessing the evidence for health effects associated with chronic exposure to benzo[a]py rene. In 24

addition, animal toxicity studies involving short

- term duration and other routes of exposure were 25

evaluated to inform conclusions about health hazards. 26
The references considered and cited in this document, including bibliographic information 27

a nd abstracts, can be found on the Health and Environmental Research Online (HERO) website 2 28

( Insert link when available ). 29

2 HERO is a database of scientific studies and other references used to develop EPA’s risk assessments aimed at understanding the health and environmental effects of pollutants and chemicals. It is developed and managed in EPA’s Office of Research and Development (ORD) by the Na tional Center for Environmental Assessment (NCEA). The database includes more than 300,000 scientific articles from the peer

- reviewed literature. New studies are added continuously to HERO.Toxicological Review of Benzo[a]pyrene

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# 1. HAZARD IDENTIFICATION

1.1. Synthesis of Evidence 1

NOTE: In the environment, benzo[a]pyrene occurs in conjunction with other 2 structurally related chemical compounds known as polycyclic aromatic 3 hydrocarbons (PAHs). 3 Accordingly, there are no epidemiologic studies designed to 4 solely investigate the effects of benzo[a]pyrene . There are, however, many 5 epidemiologic studies that have investigated the effects of exposure to PAH 6

mixtures. B enzo[a]pyrene is universally present in these mixtures and is often used 7 as an indicator chemical to measure exposure to PAH mixtures. 8



### 1.1.1. Developmental Toxicity 9

Human and animal studies provide evidence for PAH and benzo[a]pyrene

- induced 10

developmental effe cts. Effects on fetal survival, postnatal growth

, and development have been 11

demonstrated in human populations exposed to PAH mixtures during gestation. Animal studi es 12

demonstrate various effects including changes in pup weight, blood pressure, fertility, reproductive 13

organ weight and histology , and neurological function in gestationally or early postnatally treated 14

animals. 15

Altered Birth Outcomes 16

Human and animal stu dies provide evidence that benzo[a]pyrene exposure may lead to 17

altered outcomes reflecting growth and development in utero or early childhood

. Two cohort 18

studies in pregnant women in China and the United States examined cord blood levels of 19

benzo[a]pyrene

- DNA adducts in relation to measures of child growth following exposure to PAH 20

mixtures (Tang et al.

, 2006; Perera et al., 2005a; 2004) ( Table 1

- 1 ). In the Chinese cohort, high 21

benzo[a]pyrene

- adduct levels were associated with reduced weight at 18, 24, and 30 months of age, 22

but not at birth (Tang et al., 2006). In the U.S. cohort, a n independent effect on birth weight was not 23

observed with either benzo[a]pyene

- adducts or environmental tobacco smoke (ETS); however, a 24
doubling of cord blood adducts in combination with ETS exposure in utero was seen, corresponding 25

to an 8% reduction in birth weight (Perera et al., 2005a ; 2004). Environmental tobacco smoke, also 26

called secondhand smoke, is the smoke given off by a burning tobacco product and the smoke 27

exhaled by a smoker that contains over 7

, 000 chemicals including benzo[a]pyrene. No associations 28
were seen with birth length (or height at later ages) in either of these cohort studies. 29

3 PAHs are a large class of chemical compounds formed during the incomplete combustion of organic matter.Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE In animals (Table 1

- 2) , reduced bodyweight in offspring has been noted in some 1
developmental studies. Decreases in body weight (up to 13%) were observed in mice followi ng 2
prenatal gavage exposure (GD 7

- 16), and as time from exposure increased (PND 20 to 42) the dose 3

at which effects were observed decreased (40 to 10 mg/kg

- day, respectively) (MacKenzie and 4
Angevine, 1981). In addition, decreases in body weight (approximately 10 to 15%) were observed 5

in rats on PNDs 36 a nd 71 following gavage exposure at only 2 mg/kg

- day on PNDs 5

- 11 (Chen et 6
al., 2012). At doses up to 1.2 mg/kg

- day and follow

- up to PND 30, two developmental studies in 7
rats did not observe decrements in pup body weight following treatment from GD 14

- 17 (J ules et al., 8

2012; McCallister et al., 2008). Maternal toxicity was not observed in mouse or rat dams exposed to 9
up to 160 mg/kg

- day benzo[a]pyrene (Jules et al., 2012, McCallister et al., 2008; Brown et al., 2007; 10

Kristensen et al., 1995; MacKenzie and A ngevine, 1981). 11
Decreased fetal survival has also been noted in gestationally treated animals at relatively 12
high doses by the oral and inhalation route. An approximate 40% decrease in fetal survival was 13

noted in mouse dams treated by gavage on GD 7

- 16 at d oses of 160 mg/kg

- day, but no decreases 14

were observed at 10 or 40 mg/kg

- day (MacKenzie and Angev ine, 1981). Several lower dose studies 15

of rats treated on GD 14

- 17 with doses of up to 1.2 mg/kg

- day benzo[a]pyrene did not observe any 16

difference in fetal surv ival (Jules et al., 2012, McCallister et al., 2008; Brown et al., 2007). B y the 17

inhalation route, fetal survival was decreased by 19% following exposure to 25 μg/m 3 18

benzo[a]pyrene on GD 11

- 20 (Archibong et al., 2002) and 65% following exposure to 100 μg/m 3 19

benzo[a]pyrene on GD 11

- 21 in (Wormley et al., 2004) F344 rats. Wu et al. (2003) also evaluated 20

fetal survival as part of a study analyzing metabolites of benzo[a]pyrene and activation of the aryl 21

hydrocarbon receptor ( AhR ) and cytochrome P450 (CYP) 1A1. The study authors reported 22

decreased fetal survival at 75 and 100 μg/m 3 , but not at 25 μg/m 3 following exposure to 23

benzo[a]pyrene on GD 11

- 20. This study did not report number of dams or litters and no numerical 24

data were reported. 25

Fertility in Offs

pring 26

Several studies suggest that gestational exposure to maternal tobacco smoke decreases the 27

future fertility of female offspring (Ye et al., 2010; Jensen et al., 1998; Weinberg et al., 1989) (Table 28

1

- 1) . In animal models, marked effects on the develop ment of male and female reproductive organs 29

and the fertility of animals exposed gestationally has been demonstrated (Kristensen et al., 1995; 30
MacKenzie and Angevine 1981) (Table 1

- 2) . In two studies examining reproductive effects in mice, 31

decreased ferti lity and fecundity in F1 animals was observed following exposure to doses ≥ 10 32

mg/kg

- day during gestation (Kristensen et al., 1995; MacKenzie and Angevine, 1981). When F1 33

females were mated with untreated males, a dose

- related decrease in fertility of > 30% was 34

observed, in addition to a 20% decrease in litter size starting at the lowest dose tested (10 mg/kg

35

day). A dose

- related decrease in fertility was also observed in male mice treated gestationally with 36
benzo(a)pyrene. At the lowest dose tested ( 10 mg/kg

- day)

, a 35% decrease in fertility was 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE observed when gestationally exposed animals were mated with untreated females (MacKenzie and 1
Angevine, 1981). Similar effects on fertility were observed in another developmental study in mice 2
(Kristensen et al., 1995). F1 females (bred continuously for 6 months) in this study had 63% fewer 3

litters, and litters were 30% smaller as compared to control animals. The fertility of male offspring 4

was not assessed in this study. 5

Reproductive Effects in Offspring 6

The above mentioned studies also demonstrated dose

- related effects on male and female 7
reproductive organs in animals exposed gestationally to benzo[a]pyrene (Table 1

- 2) . Testicular 8

weight was decreased and atrophic seminiferous tubules and vacuolization w as increased at ≥ 10 9
mg/kg

- day in male mice exposed to benzo[a]pyrene gestationally from GD 7

- 16; severe atrophic 10

seminiferous tubules were observed at 40 mg/kg

- day (MacKenzie and Angevine, 1981). 11
In female mice treated with doses ≥ 10 mg/kg

- day during g estation, ovarian effects were 12
observed including decreases in ovary weight, numbers of follicles, and corpora lutea (Kristensen et 13

al., 1995; MacKenzie and Angevine, 1981). Specifically, ovary weight in F1 offspring was reduced 14
30% following exposure to 10 mg/kg

- day benzo[a]pyrene (Kristensen et al., 1995) while in another 15
gestational study at the same dose level, ovaries were so drastically reduced in size (or absent) that 16
they were not weighed (MacKenzenie and Angevine 1981). Hypoplastic ovaries with f ew or no 17

follicles and corpora lutea (numerical data not reported)

, and ovaries with few or no small, 18

medium, or large follicles and corpora lutea (numerical data not reported) have also been observed 19
in mouse offspring exposed gestationally to benzo[a]py rene (MacKenzenie and Angevine 1981; 20

Kristensen et al., 1995). 21

Cardiovascular Effects in Offspring 22

Increased systolic and diastolic blood pressure was observed in adult animals following 23
gestational treatment with benzo[a]pyrene (Jules et al., 2012) (Table 1

- 2) . Approximate elevations 24

in systolic and diastolic blood pressure of 20

- 30% and 50

- 80% were noted in the 0.6 mg/kg

- day 25

and 1.2 mg/kg

- day dose groups, respectively. Heart rate was decreased at 0.6 mg/kg

- day, but was 26
increased at 1.2 mg/kg

- day. 27Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 1

. Evidence pertaining to the developmental effects of benzo[a]pyrene 1
in humans 2

Reference and Study Design:

Study Type/Period/Study Size/Location/Exposure Estimate Results

Tang et al., 2006

Pregnancy cohort

150 non

- smoking women that d elivered

babies between March 2002

– June 2002

Tongliang, China

Exposure: mean hours per day exposed to

environmenta l tobacco smoke 0.42 (SD 1.19); l ived within 2.5 km of power plant that operated from December 2001

– May

2002 ; B [ a ] P

- DNA adducts from maternal and cord blood samples ; cord blood mean 0.33 (SD 0.14) (m edian 0.36 ) adducts/10 - 8

nucleotides; maternal blood mean 0.29 (SD 0.13) adducts/10 - 8 nucleotides Relation between cord blood B [ a ] P

- DNA ad ducts and log

tran sformed weight and height

Weight Beta (p

value) Length (Height) Beta (p

- value)

Birth

– 0.007

(0.73) – 0.00 1 (0. 89 )

18 months

– 0.048

(0.03) – 0.005 (0.48 )

24 months

– 0.041

(0.027) – 0.0 07 (0. 28 )

30 months

– 0.040

(0.049) – 0.0 06 (0. 44 )

A djusted for environmental tobacco smoke, sex of child, maternal height, maternal weight, cesarean section delivery, maternal head circumference, and gestational age (for measures at birth)

Perera et al. (2005a; 2004)

Pregnancy cohort

214 African

- American and Dominican non

smoking women that d elivered babies

between April 1998

– October 2002 ; approximately 40% with smoker in th e
home

New York, United States

Exposure: B [ a ] P

- DNA adducts in cord blood samples; mean 0.22 (SD 0.14) adducts/10 - 8

nucleotides; median of detectable values 0.36 adducts/10 - 8 nucleotides) Relation between cord blood B [ a ] P

- DNA ad ducts and log

transformed birth weight and length :

Weight Beta (p

value) Length

Beta (p

- value)

Interaction

term – 0.0 88

(0.05) – 0. 014 (0. 39 )

B [ a ] P

- DNA

adducts – 0.012

(0. 49 ) – 0.048 (0. 64 )

E T S in home

– 0.00 3

(0. 90 ) – 0.0 07 (0. 32 )

A djusted for ethnicity, sex of newborns, maternal body
mass index, dietary PAHs, gestational age. ETS =

environmental tobacco smokeToxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 2

. Evidence pertaining to the developmental effects of benzo[a]pyrene 1
in animals 2

Study Design and Reference Results

Birth outcomes
MacKenzie and Angevine, 1981

CD

- 1 mice, 30 or 60 F0 females/

dose 0, 10, 40, or 160 mg/kg

- d by

gavage

GD 7

– 1 6 ↓ Number of F0 females with viable litters : 46/60, 21/30, 44/60, 13/30*

↓ F1 body weight at PND 20
Response relative to control: 0, 4,

- 5*,

- 13*

↓ F1 body weight at PND 42
Response relative to control: 0,

- 6*,

- 6*,

- 10*

Kristensen et al., 1995 NMRI mice, 9 F0 females/dose 0 or 10 mg/kg

- d by gavage

GD 7

– 16 Exposed F0 females showed no gross signs of toxicity and no effects on fertility (data not reported)

Jules et al., 2012
Long

- Evans rats, 6

- 17 F0

f emales/dose

0, 0.15, 0.3, 0.6, or 1.2 mg/kg

- day by gavage

GD 14

- 17 No overt signs of toxicity in dams or offspring, differences in pup body weight, or number of pups per litter

McCallister et al. (2008)
Long Evans Hooded rats, 5

- 6/group

0 or 0.3 mg/kg

- day by gavage

GD 14

- 17 No difference in number of pups/litter

No overt maternal or pup toxicity

No difference in liver:body weight

Increased brain:body weight ratio at PND15 and 30

Brown et al. (2007) Long Evans Hooded rats, 6/group

0, 0.025 or 0.15 mg/kg

- day by

gavage

GD 14

- 1 7 No difference in number of pups/litter or overt maternal or pup toxicity

Chen et al. (2012)
Sprague

- Dawley rats, 20 pups (10 male and 10 female)/group

0, 0.02, 0.2, or 2 mg/kg

- day by

gavage

PND5

- PND11 Statistically significant decrease in pup bodyweight (approximate 10

- 15% decrease) at 2 mg/kg

- day measured on PND36 and 71

No differences among treatment groups in developmental milestones: incisor eruption, eye opening, development of fur, testis decent or vaginal
opening

Fertility in offspring

MacKenzie and Angevine, 1981

CD

- 1 mice, 30 or 60 F0 females/

dose 0, 10, 40, or 160 mg/kg

- d by

gavage

GD 7

- 16 ↓ Number of F1 females with viable litters : 35/35, 23/35*, 0/55*, 0/20*

↓F1 female fertility index (females pregnant/females exposed to males x
100): 100 , 66*, 0*, 0*

↓ F1 male fertility index (females pregnant/females exposed to males x
100): 80, 52*, 5*, 0*Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE ↓F1 litter size (20%) at 10 mg/kg

- day (no litters were produced at high doses)

↓ size or absence of F1 ovaries (weights not collected)

hypoplastic ovaries with few or no follicles and corpora lutea (numerical data not reported)

↓ testicular weight
% change from control: 0,

- 42,

- 82, ND (statistical significance not reported)

↑ atrophic sem iniferous tubules and vacuolization at ≥ 10 mg/kg

- day; severe atrophic seminiferous tubules at 40 mg/kg

- day (numerical data not reported)
Kristensen et al., 1995 NMRI mice, 9 F0 females/dose 0 or 10 mg/kg

- d by gavage

GD 7

– 16 ↓ N umber of F1 litters (63% )

↓F1 litter size (30%)

↓ ovary weight (31%) in F1 females

Few or no small, medium, or large follicles and corpora lutea (numerical data not reported)
Archibong et al., 2002 F344 rats, 10 females/group 0, 25, 75, or 100 μg/m 3 nose

- only

inhalation for 4 hrs/d ay

GD 11

– 20 ↓ Fetal survival ([pups/litter]/[implantation sites/litter] x 100)

% fetal survival 97, 78*, 38*, and 34*%

↓ pup weight 0, 0, 14*, and 16*%

Wormley et al., 2004 F344 rats, 10 females/group 0 or 100 μg/m 3 nose

- only

inhalation for 4 hrs/d ay

GD 11

– 21 ↓ 65% decrease in pups/litter

Reproductive effects in offspring

MacKenzie and Angevine, 1981

CD

- 1 mice, 30 or 60 F0 females/

dose 0, 10, 40, or 160 mg/kg

- d by

gavage

GD 7

- 16 ↓ size or absence of F1 ovaries (weights not collected)

hypoplastic ovaries with few or no follicles and corpora lutea (numerical data not reported)

↓ testicular weight
% change from control: 0,

- 42,

- 82, ND (statistical significance not reported)

↑ atrophic seminiferous tubules and vacuolization at ≥ 10 m g/kg

- day; severe atrophic seminiferous tubules at 40 mg/kg

- day (numerical data not reported)
Kristensen et al., 1995 NMRI mice, 9 F0 females/dose 0 or 10 mg/kg

- d by gavage

GD 7

– 16 ↓ ovary weight (31%) in F1 females

Few or no small, medium, or large follicles and corpora lutea (numerical data not reported)

Cardiac Effects in Offspring

Jules et al., 2012 ↑ systolic blood pressure (measured at PND53)Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Long

- Evans rats, 6

- 17 F0

f emales/dose 0, 0.15, 0.3, 0.6, or 1.2 mg/kg

- day by gavage

GD 14

- 17 ~20%* increase at 0.6 mg/kg

- day ~50% *increase at 1.2 mg/kg

- day (other dose groups not reported)

↑ diastolic blood pressure (measured at PND53) 33%* increase at 0.6 mg/kg

- day 83% *increase at 1.2 mg/kg

- day (other dose groups not reported)

Statistically significant increase in heart rate at 0.6 mg/kg

- day and statistically significant decrease at 1.2 mg/kg

- day

Figure 1

- 1 . Exposure

- response array for developmental effects following oral 1

exposure 2

3

Neurodevelopment Effects 4

There is evidence in humans and animals that benzo[a]pyrene induces developmental 5
neurotoxicity. In addition to the persistent reductions in cognitive ability observed in epidemiology 6Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE studies of prenatal PAH exposure, the two epid emiology studies that examined benzo[a]pyrene

1
specific measures observed effects on neurodevelopment and behavior in young children. Altered 2
learning and memory, motor activity and anxiety, and electrophysiological changes have also been 3

observed in anima ls following oral and inhalation exposure to benzo[a]pyrene. 4
The mammalian brain undergoes a period of rapid brain growth during the last 3 months of 5
pregnancy through the first 2 years of life in humans (Dobbing and Sands, 1973, 1979) and the first 6

1

– 2 we eks of life in the rat and mouse neonate (Chen et al., 2011a). This period is characterized by 7
the maturation of axonal and dendritic outgrowth and the establishment of neuronal connections. 8

Also during this critical period, animals acquire many new motor and sensory abilities (Kolb and 9
Whishaw, 1989). There is a growing literature of animal studies that shows subtle changes in motor 10

and cognitive function following acute or repeated perinatal or lactation exposure to 11

benzo[a]pyrene (Bouayed et al. , 2009; M cCallister et al. , 2008; Wormley et al. , 2004). 12

Cognitive function 13

Head circumference at birth is associated with measures of intelligence in children, even 14
among term infants (Broekman et al., 2009; Gale et al., 2006). The two pregnancy cohort studies 15

th at examined maternal or cord blood levels of benzo[a]pyrene

- DNA adducts in relation to head 16
circumference provide some evidence of an association, most strongly within the context of an 17
interaction with environmental tobacco smoke (Tang et al., 2006; Perer a et al., 2005a; 2004) ( Table 18

1

- 3 ). One of these studies also examined aspects of neurodevelopment in two

- year old children in 19

China (Tang et al. , 2008). In this follow

- up of 2

- year old children in an area surrounding a coal 20
power plant, average scores on the Gesell Development Scale decreased with increased exposure 21

(measured by benzo[a]pyrene

- DNA adducts in cord blood) with similar effects seen in the motor, 22

adaptive, and language domains (approximately 15 point decrement per unit increase in exposure). 23
Animal studies have also provided evidence of altered learning and memory behaviors 24

following lactational or postnatal exposure to benzo[a]pyr ene (Bouayed et al. , 2009b, Chen et al.

, 25

2012) (Table 1

- 4) . In mice, working memory was measured using the Y

- maze spontaneous 26
alternation test (Bouayed et al , 2009b). This test records alternations between arm entries in a Y

27

shaped maze as a measure of m emory, as rodents typically prefer to investigate a new arm of the 28

maze. This test may also be used to measure exploratory behavior in rodents and thus may be 29

reflective of changes in anxiety

- like behavior. A decrease in working memory was evident in mic e, 30
as exhibited by significant increases in spontaneous alternations in the Y

- maze test in mice on PND 31

40 following lactational exposure to 2 mg/kg

- day benzo[a]pyrene (but not 20 mg/kg

- day) from 32

PND 0 to PND 14 (Bouayed et al. , 2009b). The total number of arm entries in the Y

- maze was 33
unaffected by lactational exposure. In rats, spatial learning and memory was measured using the 34

Morris water maze, which measures the ability of a rat to navigate to a target platform using 35

external spatial cues (Chen et al. , 2012). Increased escape latency, decreased time in the target 36

quadrant, and decreased number of platform crossings were observed in PND 39

– PND 40 rats 37
following postnatal exposure to 2 mg/kg

- day benzo[a]pyrene (Chen et al. , 2012). These effects 38Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE were more pronounced in animals tested at PND 74

– PND 75. No difference in swim speed was 1
observed between treatment groups, suggesting the difference observed is not attributable to 2
general motor impairment. 3

Negative geotaxis and surface righting are discrete endpoints routinely used as part of a 4

neurobehavioral test battery to assess acquisition of developmental milestones. In typical 5
protocols, animals are tested on successive days (usually PND 3

- 7 + and PND 6

- 9 + , respectively) and 6

successful acquisition of these phenotypes is indicated when righting occurs within

~ 2 seconds or 7

orienting 180 ° occurs within

~ 60 seconds (although latency for these events is often reported), 8

respectively; in rats, both phenotypes are nearly always established (aka fully matured) before PND 9

10. Chen et al. (2012) perform ed these tests in a slightly atypical manner as quantitative measures 10

of sensorimotor function at PND 12 and beyond, with control animals already to right within 0.8

- 11

1.8 seconds and able to orient 180 ° within 5

- 9 seconds. Although informative in terms of possible 12

developmental delays, the sensitivity of these measures at these later timepoints has not been well 13

established in the literature. Specifically, statistically significant differences observed by Chen et al. 14
(2012) in the surface righting test were on the order of

~ 0.2

- 0.3 seconds and in the negative 15

geotaxis test,

~ 3

- 4 seconds, with no automated recording of latency (such as use of video 16

recordings). Additionally, male and female rats (which often show d ifferences in the maturation of 17

these developmental landmarks) were pooled for these measures. Due to these uncertainties, EPA 18

considered the elevated plus ma ze and Morris water maze tests to be the most informative and 19

appropriate measures of neurobehavi oral function performed by Chen et al. (2012). T he open field 20

tests were considered less informative as male and female data were pooled and the test paradigm 21

could not separate effects on motor activity from anxiety responses. 22

Neuromuscular function, coo rdination, and motor activity 23

Motor behavior, assessed by locomotion, reaching, balance, comprehension, drawing and 24

hand control was one of the specific domains assessed in the Chinese pregnancy cohort evaluated 25
by Tang et al. (2008). In children aged 2 y ears, decreased scores were seen in relation to increasing 26

benzo[a]pyrene

- DNA adducts measured in cord blood, with a Beta per unit increase in adducts of

– 27

16 (p = 0.004), and an approximate two

- fold increased risk of development delay per unit increase 28

in adducts ( Table 1

- 3 ). 29

In laboratory animals (Table 1

- 4) , impaired neuromuscular function and coordination have 30
been consistently observed in mice lactationally exposed to ≥2mg/kg

- day benzo[a]pyrene from 31

PND 0 to PND 14 (Bouayed et al. , 2009b) and in rat p ups postnatally exposed to ≥0.02 mg/kg

- day 32
benzo[a]pyrene from PND5 to PND11 (Chen et al. , 2012). In the righting reflex test, significant 33

increases in righting time were observed in PND 3

– PND 5 mice and in PND 12

– PND 16 rats. In 34

rats, increased righ ting time did not show a monotonic dose response on PND 12 or PND 14. In 35

another test of neuromuscular function and coordination, dose

- dependent increases in latency in 36

the negative geotaxis test were observed in PND 5

– PND 9 mice and in PND 12

- PND 14 rats. The 37

forelimb grip strength test was also evaluated in both mice and rats, but the results differed 38Toxicological Review of Benzo[a]pyrene

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1

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— DO NOT CITE OR QUOTE between the species. In mice, a dose

- dependent increase in duration of forelimb grip was observed 1

on PND 9 and PND 11 during lactational exposure to benzo[a]pyrene, but not on PND 12. The 2
Water Escape Pole Climbing test was also used to evaluate neuromuscular function and 3

coordination in mice (Bouayed et al. , 2009). No effect on climbing time was observed, whereas 4

increased latency in pole grasping and pole escape in PND 20 male pups was observed . Increased 5
locomotor activity on PND 69, measured using the open field test, has been reported in rats 6
postnatally exposed to ≥0.2 mg/kg

- day benzo[a]pyrene on PNDs 5

- 11 (Chen et al. , 2012). This 7

increase i n locomotor activity was observed at doses that did not cause maternal toxicity and at 8

several weeks post

- exposure, suggesting that the developmental neurotoxic effects of 9
benzo[a]pyrene persist through neurodevelopment and may become exacerbated over time . One 10

issue in interpreting these results, however, is that it is difficult to separate an anxiety response 11

from effects solely on motor function with this test. 12

Anxiety 13

Anxiety, attention, and hyperactivity in children ages 6

– 7 years were examined in re lation 14

to benzo[a]pyrene

- DNA adducts measured at birth in a follow

- up of a pregnancy cohort study 15
conducted in New York City (Perera et al., 2012). The associations were stronger using the 16

measures in cord blood compared with maternal samples, with indica tions of a 4

- fold increased 17

risk (p=0.051) of attention problems ( Table 1

- 3 ). Exposure was treated as a dichotomy (i.e., 18

detectable compared with non

- detectable levels) in these analyses. 19

Decreased anxiety was reported in both rat and mouse pups followin g postnatal oral 20
exposure to benzo[a]pyrene (Bouayed et al. , 2009b, Chen et al. , 2012) (Table 1

- 4) . Anxiety

- related 21

behaviors were measured in both species using an elevated plus maze, where an increase in the 22

time spent in the closed arms of the maze is considered evidence of anxious behavior. In mice, 23
significant increases in the entries and time spent in open arms of the maze, as well as significantly 24

decreased entries into closed arms of the maze, were observed on PND 32 following lactational 25

exposure to ≥2 mg/kg

- day benzo[a]pyrene (Bouayed et al. , 2009b). The mice exhibited decreased 26
latency in the elevated plus maze following lactational exposure to 20 mg/kg

- day benzo[a]pyre ne, 27

while there was no exposure

- related effect on the total number of times the mice entered arms of 28

the maze. Decreased anxiety

- like behaviors were also reported in rats following oral 29

benzo[a]pyrene exposure from PND 5 to PND 11, although sex

- specific differences were observed 30

(Chen et al. , 2012). In females, postnatal exposur e to ≥0.2 mg/kg

- day benzo[a]pyrene was 31

assoc iated with a significant increase in the number of open arm entries and significant decreases 32

in the number of closed arm entries on PND 70. Significantly increased time in open arms of the 33

maze was reported in PND 70 female rats following postnatal exposure to ≥0.02 mg/kg

- day. Male 34

rats also showed decreased anxiety

- like behavior on PND 70, although the doses of benzo[a]pyrene 35

were higher than females. A significant increase in the number of open arm entries a nd significant 36

decreases in the number of closed arm entries were observed in male rat pups exposed to 2 mg/kg

37
day, while a significant increase in time in the open arms of the maze was reported for males 38Toxicological Review of Benzo[a]pyrene

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1

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— DO NOT CITE OR QUOTE exposed to ≥0.2 mg/kg

- day. A significant decrease in latency to enter an open arm of the maze was 1
observed in both male and female rat pups exposed to 2 mg/kg

- day benzo[a]pyrene. These data 2

indicate that oral postnatal exposure to benzo[a]pyrene resulted in decreased anxiety

- like behavior 3

in both mice an d rats, with rats more sensitive than mice and female rats more sensitive than male 4

rats. 5

Electrophysi ologi cal changes 6

Electrophysiological effects of gestational exposure to benzo[a]pyrene have been examined 7
in animal studies through implanted electrodes in the rat cortex (Table 1

- 4) . Inhalation exposure to 8

0.1 mg/m 3 benzo[a]pyrene resulted in reduced long

- term potentiation in the dentate gyrus of male 9
rats between PND 60 to PND 70 (Wormley et al. , 2004). Oral exposure to 0.3 mg/kg

- day 10

benzo[a]pyrene res ulted in decreased evoked neuronal activity in male rats following mechanical 11

whisker stimulation between PND 90 to PND 120 (McCallister et al.

, 2008). The authors also noted 12

reduced spike numbers in both the short and long latency responses to whisker st imulation, 13
although no quantitative data were presented by the study authors. These effects were observed 14

several months post

- exposure, suggesting that gestational benzo[a]pyrene exposure has long

15
lasting functional effects on neuronal activity. 16

Table 1

- 3

. Evidence pertaining to the neurodevelopmental effects of 17 benzo[a]pyrene in humans 18

Reference, study design Results
Tang et al. (2008, 2006)

Tongliang, China

Pregnancy cohort

150 non

- smoking women (110 for Developmental Quotient analysis), delivered

March 2002

– June 2002; lived within 2.5 km of power plant that operated from December

2001

– May 2002

Outcomes: head circumference at birth; Gesell Developmental Schedule, administered by physicians at 2 years o f age (4 domains a : motor, adaptive, language, and social); standardized mean score = 100 ± SD 15 (score < 85 = developmental delay)

Exposure: B [ a ] P

- DNA adducts from maternal and cord blood samples; cord blood mean 0.32 (SD 0.14), range 0.125

– 0.812

adduct s/10 - 8 nucleotides Relation between cord blood B [ a ] P

- DNA adducts and log

- transformed

head circumference
Beta (p

- value)

Birth

– 0.011 (0.057)

18 months

– 0.012 (0.085)

24 months

– 0.006 (0.19)

30 months

– 0.005 (0.31)

High versus low, dichotomized at median, adjusted for environmental tobacco smoke, sex of child, maternal height, maternal weight, cesarean section delivery, maternal head circumference, and gestational age (for measures at birth)

Association between B[a]P adducts and development

Beta (95% CI) a OR (95% CI) b

Motor

– 16.0 (

– 31.3,

– 0.72) * 1.91 ( 1.22, 2.97) *

Adaptive

– 15.5 (

– 35.6, 4.61) 1.16 (0.76, 1.76)
Language

– 16.6 (

– 33.7, 0.46) 1.31 (0.84, 2.05)

Social

– 9.29 (

– 25.3, 6.70) 1.52 (0.93, 2.50)

Average

– 14.6 (

– 28.8,

– 0.37) * 1.67 0.93, 3.00)

a Linear regression of change in Developmental Quotient per unit increase
in B[a]P adducts

b Logistic regression of risk of developmental delay ( defined as normalized score < 85) per 1 unit (0.1 adducts/10 - 8 nucleotides) increase in adducts Both analyses adjusted for sex, gestational age, maternal education,
environmental tobacco smoke, and cord lead levels.Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Reference, study design Results
Perera et al. (2012; 2005a; 2004)

United States, New York

Pregnancy cohort

214, non

- smoking women (253 for behavior analysis), African

- American and Dominican, delivered April 1998

– October 2002

Outcomes: head circumference at birth; Child Behavior Checklist (118 items), completed by mothers for children ages 6

– 7 years. Two domains: anxious/depression, attention problems (normalized T

- score ≤ 65 = borderline or clinical syndrome); also used for scales of anxiety problems and attention

de ficit hyperactivity problems based on DSM

classification

Exposure: B [ a ] P

- DNA adducts from maternal and cord blood samples; mean 0.22 (SD 0.14) adducts/10 - 8 nucleotides; median of detectable values 0.36 adducts/10 - 8

nucleotides. Relation between cord blood B [ a ] P

- DNA adducts and log

- transformed

head circumference
Beta (p

- value)

Interaction term

– 0.088 (0.05)

B [ a ] P

- DNA adducts

– 0.012 (0.49)

ETS in home

– 0.003 (0.90)
High versus low, dichotomized at than 0.36 adducts/10 - 8 nucleotides, adjusted for ethnicity, sex of newborns, maternal body mass index, dietary PAHs, gestational age. ETS = environmental tobacco smoke

Logistic regression of risk of borderline or clinical status in relation to
detectable levels of B[a]P adducts

Maternal Cord Blood

Prevalence OR (95% CI) OR (95% CI) Anxious/depressed 6.32 % 1.4 (0.38, 5.4) 2.6 (0.69, 9.4)
Attention problems 6.72% 2.2 (0.74, 6.8) 4.1 (0.99, 16.6) Anxiety (DSM) 9.48% 2.2 (0.79, 6.1) 2.5 (0.84, 7.7)

Attention deficit

– hyperactivity (DSM) 7.91% 1.8 (0.66, 5.1) 2.6 (0.68, 10.3)

Exposure dichotomized as detectable (n=87 maternal, 56 cord blood samples) versus non

- detectable ; adjusted for sex, gestational age, maternal education, maternal IQ, prenatal environmental tobacco smoke, ethnicity, age, heating season, prenatal demoralization, and HOME inventory

Table 1

- 4

. Evidence pertaining to the neuro developmental effects of 1 benzo[a]pyrene in animal s 2

Study Design and Reference Results

Cognitive function

Chen et al. (2012)
Sprague

- Dawley rats, 20 pups (10 male and 10 female)/group 0, 0.02, 0.2, or 2 mg/kg

- day by

gavage

PND5

- PND11 E scape latency in the Morris water maze test : PND39: significant increase in escape latency at 2 mg/kg

- day only PND74: significant increase in escape latency at ≥ 0.2 mg/kg

- day

Similar differences were seen at PND 36

- 38 or PND 71

- 73 time points ; not statistically significantly different from control

T ime spent in the target quadrant in the Morris water maze test : PND40: significant decrease at 2 mg/kg

- day only

PND75: significant decrease at ≥ 0.2 mg/kg

- day

N umber of platform crossings in the Morris water maze test : PND40: significant decrease at 2 mg/kg

- day only PND75: significant decrease at ≥ 0.2 mg/kg

- day (in females) and 2 mg/kg

- day (in males)
Bouayed et al. (2009b) Female Swiss albino mice, 5/group 0, 2, or 20 mg/kg

- day maternal

gavage

PND 0

– 14 (lactational exposure) Significant increase in the percent of spontaneous alternations in the Y

maze alternation test at 2 mg/kg

- day but not at 20 mg/kg

- day

No eff ect on the total number of arm entries in the Y

- maze alternation testToxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Neuromuscular function and coordination
Chen et al. (2012)
Sprague

- Dawley rats, 20 pups (10 male and 10 female)/group 0, 0.02, 0.2, or 2 mg/kg

- day by

gavage

PND5

- PND11 L atency in the surface righting reflex test PND12: significant increase at 0.2 mg/kg

- day only PND14: Significant increase at 0.02 and 2 mg/kg

- day only PND16 Significant difference at 2 mg/kg

- day only PND 18 No significant difference

L atency in the negative geotaxis test PND12: significant increase at all doses PND14: Significant increase at 2 mg/kg

- day only PND16 and 18: No significant difference

No effect on duration of forelimb grip in forelimb grip strength test
Bouayed et al. (2009b)

Female Swiss albino mice, 5/group 0, 2, or 20 mg/kg

- day maternal

gavage

PND 0

– 14 (lactational exposure) Significant increase in righting time in the surface righting reflex test at both doses on PNDs 3 and 5 (but not PNDs 7 and 9)
Significant increase in late ncy in the negative geotaxis time for 20 mg/kg

day dose group at PNDs 5, 7, and 9 (no significant difference at PND 11)
Significant increase in duration of forelimb grip in forelimb grip strength test at both dose groups on PND 9 (statistically significant at PND 11 only at high dose)
Significant increase in pole grasping latency in male pups in the water escape pole climbing test at 20 mg/kg

- day
No effect on climbing time in the water escape pole climbing test
Significant increase in pole escape latency in the water escape pole climbing test in male rats at 20 mg/kg

- day

Anxiety and/or motor activity

Chen et al. (2012)
Sprague

- Dawley rats, 20 pups (10 male and 10 female)/group 0, 0.02, 0.2, or 2 mg/kg

- day by

gavage

PND5

- PND11 Significant increase in the number of entries into open arms in the elevated plus maze at PND 70 at ≥ 0.2 mg/kg

- day (in females) and ≥ 2 mg/kg

- day (in males) (no difference at PND 35)
Significant decrease in the number of entries into closed arms in the elevated plus maze at PND 70 a t ≥ 0.2 mg/kg

- day (in females) and ≥ 2 mg/kg

- day (in males) (no difference at PND 35)
Significant increase in the time spent in open arms in the elevated plus maze at PND35 at ≥ 2 mg/kg

- day in females and at PND70 at doses ≥ 0.02 mg/kg

- day in females and ≥ 0.2 mg/kg

- day in males
Significant decrease in latency time to first entry an open arm in the elevated plus maze on PND 70 at ≥ 0.2 mg/kg

- day (no difference at PND 35)
Significant increase in the number of squares crossed in the open

- field
activity test:

PND34

- significant increase at 2 mg/kg

- day; PND69

- significant increase at ≥ 0.02 mg/kg

- day (no difference at PND18 and 20)
Significant increase in rearing activity in open

- field activity at 0.2 mg/kg

day on PND 69 (no difference at PNDs 18, 20, and 34)
Bouayed et al. (2009b) Female Swiss albino mice, 5/group

0, 2 or 20 mg/kg

- day maternal

gavage

PND 0

– 14 (lactational exposure) Significantly increased time in open arms in the elevated plus maze at ≥ 2 mg/kg

- day
Significantly increased percentage of entries into open arms in the elevated plus maze at ≥ 2 mg/kg

- day
Significantly decreased entries into closed arms in the elevated plus maze at 2 mg/kg

- day, but not at 20 mg/kg

- day
Significantly decreased latency time in the elevated plus maze at 20Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE mg/kg

- day
No effect on the total number of arm entries in the elevated plus maze
No effect on the latency to retract from the edge in the cliff aversion test

Electrophysiological changes

McCallister et al. (2008)
Long Evans Hooded rats, 5

- 6/group

0 or 0.3 mg/kg

- day by gavage

GD14

- 17 Statistically significant decreases in stimulus

- evoked cortical neuronal
activity on PND90

– PND120 Reduction in the number of spikes in both the short and long latency
periods on PND90

– PND120 (no quantitative data presented by authors)

Wormley et al., 2004 F344 rats, 10 females/group 0 or 100 μg/m 3 nose

- only inhalation for 4 hrs/day

GD 11

– 21 Electrophysiological changes in the hippocampus: Consistently lower LTP following g estational exposure (statistical analysis

not reported)
Response relative to control:

- 26%

Figure 1

- 2 . Exposure

- re sponse array for neurodevelopmental effects 1 following oral exposure 2

3Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Summary of Developmental Effects 1

Developmental effects following in utero exposure to benzo[a]pyrene have been reported in 2

human and in animal models. In human populations, decreased head circumference, decreased 3

birth weight, and decreased postnatal weight have been reported (Tang et al ., 2006; Perera et al., 4
2005a, b). Analogous effects in laboratory animals, including decreased pup weight and decreased 5
fetal survival, have been noted following gestational or early postnatal exposure to benzo[a]pyrene 6
by the oral or inhalation route ( Chen et al., 2012; Archibong et al., 2002; MacKenzie and Angevine, 7
1981). Reproductive function is also altered in mice treated gestationally with benzo[a]pyrene 8

(Kristensen et al., 1995; MacKenzie and Angevine, 1981). These effects include impaired 9

repro ductive performance in F1 offspring (male and female) and alterations of the weight and 10

histology of reproductive organs (ovaries and testes). 11

The available human and animal data also support the conclusion that benzo[a]pyrene is 12

developm ental neurotoxi c ant . Human studies of environmental PAH exposure in two cohorts have 13

observed neurotoxic effects, including suggestions of reduced head circumference (Tang et al., 14

2006; Perera et al., 2005a; 2004), i mpaired cognitive ability (Tang et al., 2008, Perera e t al. , 2009), 15
impaired neuromuscular function (Tang et al. , 2008), and increased attention problems following 16
prenatal exposure (Perera et al., 2012). These effects were seen in pregnancy cohort studies in 17

different populations (New York City and China), in studies using specific benzo[a]pyrene measures 18

(i.e., adduct levels measured in cord blood samples) (Tang et al., 2008; 2006; Perera et al., 2012; 19
2005a; 2004). This type of measure covers a relevant time window of exposure with respect to 20

gestational development. The analytical method was similar in the two studies (with a common set 21

of investigators). The coefficient of variation of the exposure measures was relatively small (12%), 22

but a high proportion of samples were below the detection limit and thus these studies were 23
limited in terms of ability to examine a broad range of exposure. The available evidence from mice 24

and rats also demonstrates significant and persistent developmental impairments following 25

exposure to benzo[a]pyrene. Impaired lea rning and memory behaviors and impaired 26
neuromuscular function were consistently observed in multiple neurobehavioral tests in two 27

separ ate species at comparable doses in the absence of maternal or fetal toxicity (Bouayed et al.

, 28

2009b; Chen et al. , 2012). 29

In conclusion, the available human and animal data suggest that developmental toxicity and 30
developmental neurotoxicity is a hazard of benzo[a]pyrene exposure. 31

Susceptible Populations and Lifestages 32

Childhood susceptibility to benzo[a]pyrene toxicity is indicated by epidemiological studies 33
reporting associations between adverse birth outcomes and developmental effects and internal 34

biomarkers of exposure to benzo[a]pyrene, presumably via exposure to complex PAH mixtures 35

(Tang et al., 2008, 2006; Perera et al., 2005a, b). The occurrence of BPDE

- DNA adducts in maternal 36
and umbilical cord blood in conjunction with exposure to ETS was associated with reduced birth 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE weight and head circumference in pregnant women living in the vicinity of fires from the 1

09/11/2 001 disaster site in New York City (Perera et al., 2005a). In other studies, elevated levels of 2

BPDE

- DNA adducts in umbilical cord blood were associated with: (1) reduced birth weights or 3

reduced head circumference in the offspring of 529 Dominican or Af rican

- American nonsmoking 4

women (Perera et al., 2005b); and (2) decreased body weight at 18, 24, and 30 months and deficits 5
in several areas of development as assessed by the Gesell Developmental Schedules at 24 months in 6
the offspring of nonsmoking Chines e women living in the vicinity of a coal

- fired power plant (Tang 7

et al., 2008, 2006). 8

Studies in humans and experimental animals indicate that exposure to PAHs in general, and 9
benzo[a]pyrene in particular, may impact neurological development. Observationa l studies in 10

humans have suggested associations between gestational exposure to PAHs and later measures of 11
neurodevelopment (Perera et al., 2009; Tang et al., 2008). An observational study of a Chinese 12
population living in close proximity to a coal fired power plant found increased levels of 13

benzo[a]pyrene

- DNA adducts in cord blood were associated with decreased developmental 14
quotients in offspring (Tang et al., 2008). 15
Evidence in animals of the effects of benzo[a]pyrene on neurological development inclu des: 16
1) decreased electrophysiological response to electrical stimulation of the dentate gyrus of the 17
hippocampus and increased brain concentrations of benzo[a]pyrene metabolites in offspring of 18

F344 rats exposed by inhalation to benzo[a]pyrene:carbon bla ck aerosols on GD 11

- 21 (Wormley et 19
al., 2004; Wu et al., 2003); 2) decreased evoked response in the field cortex and decreased 20

cerebrocortical levels of mRNA for the NMDA receptor subunit in offspring of Long Evans rats 21

exposed to 300 μg/kg on GD 14

- 17 (M cCallister et al., 2008); and 3) decreased righting reflex and 22

altered disinhibition behavior in offspring of lactating rats exposed to oral doses of 2 or 20 mg/kg

23

day on PND 1

- 14 (Bouayed et al., 2009 b ). 24



### 1.1.2. Reproductive Toxicity 25

Human and animal studies prov ide evidence for benzo[a]pyrene

- induced male and female 26

reproductive toxicity. Effects on sperm quality and male fertility have been demonstrated in human 27

populations highly exposed to PAH mixtures (Soares and Melo, 2008; Hsu et al., 2006). The use of 28

in ternal biomarkers of exposure in humans (e.g. , BPDE

- DNA adducts) support associations between 29

benzo[a]pyrene exposure and these effects. In females, numerous epidemiological studies indicate 30

cigarette smoking reduces fertility; however, few studies have s pecifically examined levels of 31

benzo[a]pyrene exposure and female reproductive outcomes (see Table 1

- 6 ). Animal studies 32
demonstrate decrements in sperm quality, changes in testicular histology, and hormone alterations 33

following benzo[a]pyrene exposure in adult male animals, and decreased fertility and ovotoxic 34

effects in adult females following exposure to benzo[a]pyrene. 35Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Male Reproductive Effects 1

Fertility 2

Effects on male fertility have been demonstrated in populations exposed to mixtures of 3

PAHs. Sperma tozoa from smokers have reduced fertilizing capacity, and embryos display lower 4

implantation rates (Soares and Melo, 2008). Occupational PAH exposure has been associated with 5
higher levels of PAH

- DNA adducts in sperm and male infertility (Gaspari et al., 2003). In addition, 6
men with higher urinary levels of PAH metabolites have been shown to be more likely to be infertile 7

( Xia et al., 2009 ). Studies were not identified which directly examined the reproductive capacity of 8

adult animals following benzo[a] pyrene exposure. However, a dose

- related decrease in fertility 9

was observed in male mice treated in utero with benzo[a]pyrene, as discussed in Section 1.1. 1

. 10

Sperm parameters 11

Effects on semen quality have been demonstrated in populations exposed to mixtures of 12

PAHs including coke oven workers and smokers (Soares and Melo, 2008; Hsu et al., 2006). Coke 13
oven workers had higher frequency of oligospermia (19 vs. 0% in controls) and twice the number 14
of morphologically abnormal sperm (Hsu et al., 2006). El evated levels of BPDE

- DNA adducts have 15

been measured in the sperm of populations exposed to PAHs occupationally (Gaspari et al., 2003) 16

and through cigarette smoke (Phillips, 2002; Zenzes et al., 1999). A higher concentration of BPDE

17

DNA adducts was observ ed in sperm not selected for intrauterine insemination or IVF based on 18

motility and morphology in patients of fertility clinics (Perrin et al., 2011a, b). A n association 19

between benzo[a]pyrene exposure levels and increased sperm DNA fragmentation using th e sperm 20
chromatin structure assay (SCSA) was observed by Rubes et al. (2012) . However, i t is currently 21

unclear whether the SCSA, which measure s sperm fragmentation following denaturation , is 22

predictive of fertility ( Sakkas et al., 2009; American Society o f Reproductive Medicine 2008 ). 23

In several studies in rats and mice, decreased sperm count, motility, and production, and an 24

increase in morphologically abnormal sperm have been observed (Table 1

- 5)

. Alterations in these 25

sperm parameters have been obser ved in different strains of rats and mice and across different 26

study designs and routes of exposure. 27
Decreases in epididymal sperm (25 to 50% compared to controls) counts have been 28

observed in Sprague

- Dawley rats and C57BL6 mice treated with 1

- 5 mg/kg

- d ay benzo[a]pyr ene 29

following oral exposure 42 or 90 days (Chen et al., 2011 a ; Mohamed et al., 2010). Additionally, a 30

15% decrease in epididymal sperm count was observed at a dose 100

- fold lower in Sprague

31

Dawley rats exposed to benzo[a]pyrene for 90 days (Chung et al., 2011). However, confide nce in 32

this study is limited as the authors dosed animals with 0.001, 0.01, and 0.1 mg/kg

- day 33

benzo[a]pyrene

, but only reported on sperm parameters at the mid

- dose and no other available 34
studies demonstrated findings in the range of the mid

- and high

- dose. A short term study in mice 35

and a subchronic inhalation study in rats lend support for the endpoint of decreased sperm count 36

( Arafa et al., 2009; Archibong et al., 2008; Ramesh et al., 2008)

. Significantly decreased sperm 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE count and daily sperm production (~40% decrease from control in each parameter) were observed 1
following 10 days of gavage exposure to 50 mg/kg

- day benzo[a]pyrene in mice (Arafa et al., 2009). 2

In addition, decrements in sperm count were observed fol lowing inhalation exposure to 3

benzo[a]pyrene in rats for 60 days to 75 μg/m 3 (Archibong et al., 2008; Ramesh et al., 2008). 4

B oth oral and inhalatio n exposure to benzo[a]pyrene have been shown to lead to decreased 5
epididymal sperm motility and altered morphology in rodents. Decreased motility of 20 to 30% 6

compared to controls was observed in C57BL6 mi ce (≥ 1mg/kg

- day) and Sprague

- Dawley rats ( at 7
0.01 mg/kg

- day) (Chung et al. 2011; Mo hamed et al., 2010). However, the effective doses spanned 8

two degrees of magnitude and as noted above, confidence in the study observing effects at 0.01 9
mg/kg

- day benzo[a]pyrene (Chung et al., 2011) is limited by poor reporting. A short term oral 10

study in mice also reported a s ignificantly decreased number of motile sperm (~40% decrease) 11
following 10 days of gavage exposure to 50 mg/kg

- day benzo[a]pyrene in rats (Arafa et al., 2009). 12

In addition, decreased sperm motility was observed following inhalatio n exposure to 13

benzo[a]pyrene in rats for 60 days to 75 μg/m 3 (Archibong et al., 2008; Ramesh et al., 2008 ) and for 14
10 days at ≥ 75 μg/m 3 (Inyang et al., 2003). A bnormal sperm morphology was observed in 15

Sprague

- Dawley rats treated with 5 mg/kg

- day benzo[a] pyrene by gavage for 84 days (Chen et al., 16

20 1 1 a ) and in rats exposed to 75 μg/m 3 benzo[a]pyrene by inhalation for 60 days (Archibong et al., 17

2008; Ramesh et al., 2008). 18

Testicular changes 19

Several studies have demonstrated dose

- related effects on male repr oductive organs in 20

adult animals exposed subchronically to benzo[a]pyrene (Table 1

- 5) . Decreases in testicular 21

weight of approximately 35% have been observed in a 10 day gavage study in adult rats exposed to 22

50 mg/kg

- day benzo(a)pyrene (Arafa et al., 2009 ) and following subchronic inhalational exposure 23
of adult F344 rats to 75 μg/m 3 (Archibong et al., 2008; Ramesh et al., 2008). No effects on testes 24

weight were observed in Wistar rats exposed for 35 days to gavage doses up to 36 mg/kg

- day 25

(Kroese et al., 2001) ; F344 rats exposed for 90 days to dietary doses up to 100 mg/kg

- day 26
(Knuckles et al., 2001) ; or Sprague

- Dawley rats exposed for 90 days to gavage doses up to 0.1 27

mg/kg

- day (Chung et al., 2011) . Strain differences may have contributed to the lack of response, 28

however, F344 rats exposed to benzo[a]pyrene via inhalation showed effects on testicular weight 29

(Archibong et al., 2008; Ramesh et al., 2008). In add ition, decreased testicular weight has also been 30
observed in offspring following in utero exposure to benzo[a]pyrene as discussed in Section 1. 1. 1. 31

Histological changes in the testis have often been reported to accompany decreases in 32

testicular weight. A poptosis

, as evident by i ncreases in Terminal deoxynucleotidyl transferase 33
dUTP nick end labeling ( TUNEL ) positive germ cells and increase s in caspase

- 3 staining

, was 34

evident in seminiferous tubules of Sprague

- Dawley rats following 90 days of exposure to ≥0.001 35

and 0.01 mg/kg

- day, respectively, benzo[a]pyrene by gavage (Chung et al., 2011). However, the 36

study authors did not observe testicular atrophy or azospermia in any dose group. S eminiferous 37

tubules were reported to look qualitatively similar betw een controls and animals exposed to 38Toxicological Review of Benzo[a]pyrene

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

1

- 19 DRAFT

— DO NOT CITE OR QUOTE benzo[a]pyrene by inhalation doses of 75 μg/m 3 for 60d ( Archibong et al., 2008; Ramesh et al., 1

2008 ) . However, when histologically examined, statistically significantly reduced tubular lumen 2
size and length were observed in treated animals. Seminiferous tubule diameters also appeared to 3

be reduced in exposed animals, although this difference did not reach statistical significance 4
(Archibong et al., 2008; Ramesh et al., 2008). In addition, histological changes in the seminiferous 5
tubules have also been observed in offspring following in utero exposure to benzo[a]pyrene as 6

discussed in Section 1.1 .1

. 7

Epididymal changes 8

In addition to testicular effects, histological eff ects in the epididymis have been observed 9
following 90

- day gavage exposure to benzo[a]pyrene (Chung et al., 2011) (Table 1

- 5)

. Specifically, 10

s tatistically significant decrease d epididymal tubule diameter (for caput and cauda) was observed 11

at doses ≥ 0.001 mg/kg

- day. At the highest dose tested (0.1 mg/kg

- day) diameters were reduced 12

approximately 25%. No change s in epididymis weight s were observed following an 84 day 13

treatment in Sprague

- Dawley rats of 5 mg/kg

- day benzo[a]pyrene (Chen et al., 2011 a ). 14

Horm one changes 15

Several animal models have reported decreases in testosterone following both oral and 16

inhalation exposure to benzo[a]pyrene (Table 1

- 5) . In male Sprague

- Dawley rats, decreases in 17

testosterone have been observed following 90

- day oral exposures (Chung et al., 2011; Zheng et al., 18

2010). S tatistically significant decreases of 15% in intratesticular testosterone were observed at 5 19

mg/kg

- day in one study (Zheng et al., 2010)

, while a second study in the same strain of rats 20
reported statistically sig nificant decreases of 40% in intratesticular testosterone and 70% in serum 21

testosterone (70%) at 0.1 mg/kg

- day (Chung et al., 2011). S tatistically significant decrease s in 22

intratesticular testosterone (80%) and serum testosterone (60%) were also observed following 23

i nhalation exposure to 75 μg/m 3 benzo[a]pyrene in F344 rats for 60 days (Archibong et al., 2008; 24

Ramesh et al., 2008). Statistically significant increases in serum l uteinizing hormone ( LH ) hav e 25

also been observed in Sprague

- Dawley rats followin g gavage exposure to benzo[a]pyrene at doses 26

of ≥ 0.01 mg/kg

- day and in F344 rats following inhalation exposure to 75 μg/m 3 benzo[a]pyrene 27
for 60 days (Archibong et al., 2008; Ramesh et al., 2008). 28

Table 1

- 5

. Evidence per taining to the male reproductive toxicity of 29 benzo[a]pyrene in animals 30

Study Design and Reference Results

Sperm quality

Mohamed et al., 2010 C57BL/6 mice, 10 males/dose (treated before mating with unexposed females)

0, 1, or 10 mg/kg

- day , daily gavage ↓ epididymal sperm count in F0 mice
Approximate % change from control: 0,

- 50*,

- 70* (numerical data not reported)

↓ epididymal sperm motility in F0 miceToxicological Review of Benzo[a]pyrene

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1

- 20 DRAFT

— DO NOT CITE OR QUOTE Study Design and Reference Results

(F0 males only)
42 days Approximate % change from control: 0,

- 20*,

- 50* (nume rical data not reported)

↓ epididymal sperm count in untreated F1 and F2 generations (numerical data not reported)

No effects were observed in the F3 generation
Chen et al., 2011 a Sprague

- Dawley rats , 10 males/dose

0 or 5 mg/kg

- day by gavage

84d ↓ epididymal sperm count (% change from control)

0,

- 25%*

↑ % abnormal epididymal sperm
5, 8*
Chung et al., 2011
Sprague

- Dawley rats

, 20

- 25 males/dose

0, 0.001, 0.01, 0.1 mg/kg

- day by gavage

90 days ↓ epididymal sperm motility ( % response relative to control;

r eported only for 0.01 mg/kg

- day)

0,

- 30*

N o statistically significant decrease in epididymal sperm count (reported only for 0.01 mg/kg

- day)
Archibong et al., 2008; Ramesh et al.,

2008 F344 rats, 10 males/group

0 or 75 μg/m 3 , 4 hrs/d ay by inhalation 60 days ↓ epididymal sperm motility (% change from control)

0,

- 73*

↓ epididymal sperm count (% change from control)

0,

- 69*

↑ % abnormal epididymal sperm
33, 87*

↓ spermatids/g testis (a pp roximate % change from control; numerical data not reported)

0,

- 45*

Testicular changes (weight, histology)

Mohamed et al., 2010 C57BL/6 mice, 10 males/dose (treated before mating with unexposed females)

0, 1, or 10 mg/kg

- day , daily gavage (F0 males only)
42 days ↓ seminiferous tubules with elongated spermatids (a p proximate % change from control; numerical data not reported) )

0,

- 20*,

- 35*

No statistically significant change in area of seminiferous epithelium

of testis (a p p roximate % change from control; numerical data not reported )

0, 5, 20
Chung et al., 2011
Sprague

- Dawley rats

, 20

- 25 males/dose

0, 0.001, 0.01, 0.1 mg/kg

- day by gavage

90 days ↑ number of apoptotic germ cells per tubule (TUNEL or caspase 3
positive)

No change in testis weight or histology
Chen et al., 2011 a Sprague

- Dawley rats, 10/dose

0 or 5 mg/kg

- day by gavage 84 days ↑ testicular lesions characterized as irregular arrangement of germ
cells and absence of spermatocytes (numerical data not reported)

No change in testis weightToxicological Review of Benzo[a]pyrene

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1

- 21 DRAFT

— DO NOT CITE OR QUOTE Study Design and Reference Results

Archibong et al., 2008; Ramesh et al.,

2008 F344 rats, 10 adult males/group 0 or 75 μg/m 3

, 4 hrs/d ay by inhalation

60 days ↓ decreased testis weight (% change from control)
0, 34*

↓ size of seminiferous tubule lumens and reduced tubular length

No change in % of tubules with elongated spermatids
Kroese et al., 2001 Wistar rats, 10/sex/dose

0, 1.5, 5, 15, or 5 0 mg/kg

- day by gavage 35 days No change in testis weight

Knuckles et al., 2001 F344 rats, 20/sex/dose

0, 5, 50, or 100 mg/kg

- day in diet 90 days No change in testis weight

Epididymal changes (weight, histology)

Chung et al., 2011
Sprague

- Dawley rats

, 20

- 25 males/dose

0, 0.001, 0.01, 0.1 mg/kg

- day by gavage

90 days ↓ diameter of caput epididymal tubule (n=5 ; numerical data not reported)

↓ diameter of cauda epididymal tubule (n=5 ; numerical data not reported)
Chen et al., 2011 a Sprague

- Dawley rats, 10/dose

0 or 5 mg/kg

- day by gavage 84 days No change in epididymis we ight

Hormone changes

Chung et al., 2011
Sprague

- Dawley rats

, 20

- 25 males/dose

0, 0.001, 0.01, 0.1 mg/kg

- day by gavage

90 days ↓ Intratesticular testosterone (a p proximate % change from control; numerical data not reported )

0,

- 12,

- 25,

- 40*

↓ Serum testosterone (a p proximate % change from control ; numerical data not reported )

0, 0,

- 35,

- 70*

↑ serum LH (a pp roximate % change from control; numerical data not reported )
0, 33, 67*, 87*

↓ hCG or dbcAMP

- stimulated testosterone production in Leydig

cells
Zheng et al., 2010
Sprague

- Dawley rats, 8 males/dose

0, 1 or 5 mg/kg

- day by gavage 90 days ↓ Intratesticular testosteron e ( pp roximate % change from control ; numerical data not reported )

0,

- 15,

- 15*

Archibong et al., 2008; Ramesh et al.,

2008 F344 rats, 10 adult males/group 0 or 75 μg/m 3

, 4 hrs/d ay by inhalation

60 days ↓ intratesticular testosterone (a pproximate % change from control ; numerical data not reported )

0,

- 80*

↓ serum testosterone (a ppr oximate % change from control)

0,

- 60*Toxicological Review of Benzo[a]pyrene

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1

- 22 DRAFT

— DO NOT CITE OR QUOTE Study Design and Reference Results

↑ serum LH (a ppr oximate % change from control)
0, 50*

Figure 1

- 3

. Exposure

- response array for male reproductive effects following 1 oral exposure 2

3

Mode of Action Analysis

— Male reproductive effects 4

Data regarding the potential mode of action for male reproductive effects associated with 5
benzo[a]pyrene exposure are limited. Hypothesized modes of action include benzo[a]pyrene 6

mediated DNA damage to mal e germ cells leading to genotoxicity, cytotoxicity, and apoptosis 7Toxicological Review of Benzo[a]pyrene

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1

- 23 DRAFT

— DO NOT CITE OR QUOTE (Chung et al., 2011; Olsen et al., 2010; Perrin et al., 2011 a,b; Revel et al., 2001), compromised 1
function of Leydig and Sertoli cells (Chung et al., 2011; Raychoudhury and Kubinski, 2003), altered 2
androgen hormone regulation (Inyang et al., 2003; Vinggaard et al., 2000), and decreased embryo 3

viability post fertilization associated with sperm DNA damage ( Borini et al., 2006; Seli et al., 2004 ). 4

Female Reproductive Effects 5

Fertility 6

In women
, exposure to cigarette smoke has been shown to affect fertility, including effects 7

related to pregnancy, ovulatory disorders, and spontaneous abortion (as reviewed in Waylen et al., 8

2009; Cooper and Moley, 2008; Soares and Melo, 2008). In addition, severa l studies suggest that in 9
utero exposure to maternal tobacco smoke also decreases the future fertility of female offspring ( Ye 10

et al., 2010; Jensen et al., 1998; Weinberg et al., 1989 ). B enzo[a]pyrene levels in follicular fluid and 11

benzo[a]pyrene

- DNA addu cts in granulosa

- lutein cells and oocytes and in human cervical cells have 12

been associated with smoking status and with amount smoked (Neal et al., 2008; Zenzes et al., 13

1998; Mancini et al., 1999; Melikian et al., 1999; Shamsuddin and Gan , 1988)

. 14

F ew epi demiological studies have e xamin ed the specific i nfluence of components of PAH 15

mixtures on fertility or other reproductive outcomes; EPA identified only two studies with specific 16
data on benzo[a]pyrene (Table 1

- 6). One of these studies addressed the probability of conception 17

among women undergoing in vi tro fertilization ( Neal et al.

, 2008) . Although no association was 18

seen with serum levels of benzo[a]pyrene , f ollicular fluid benzo[a]pyrene levels were significantly 19

higher among the women who did not conceive with women who did get pregnant . The other study 20
examined risk of what was termed a “missed abortion” (i.e., fetal death before 14 weeks gestation), 21

using a case

- control design with controls selected from women undergoing elective abortion (Wu et 22

al., 2010) . B enzo[a]pyrene

- DNA adduct levels were s imilar in the aborted tissue of cases compared 23

with controls , but a strong association was seen between maternal blood benzo[a]pyrene

- DNA 24

adduct levels and risk of missed abortion, with a 4

- fold increased risk for levels above compared 25

with below the media n. 26

Experimental studies in animals also provide evidence that benzo[a]pyrene exposure 27

affects fertility (Table 1

- 7) . Decreased fertility and fecundity (decreased number of F0 females 28

producing viable litters at parturition) was statistically significant ly reduced by about 35% in adult 29

females exposed to 160 mg/kg

- day of benzo[a]pyrene (MacKenzie and Angevine, 1981)

. In another 30
study, F0 females showed no signs of general toxicity or effects on fertility following gavage 31

exposure to 10 mg/kg

- day on GD 7

- 16 (Kristensen et al., 1995). Decrements in fertility were more 32

striking in the offspring from these studies

, as described in Section 1.1. 1

. 33

Ovarian effects 34

Human epidemiological studies which directly relate ovotoxicity and benzo[a]pyrene 35

exposure are n ot available; however, smoking, especially during the time of the peri

- menopausal 36Toxicological Review of Benzo[a]pyrene

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1

- 24 DRAFT

— DO NOT CITE OR QUOTE transition, has been shown to accelerate ovarian senescence (Midgette and Baron, 1990). 1

Benzo[a]pyrene

- induced ovarian toxicity has been demonstrated in animal studies. In adult female 2
rats treated by gavage, statistically significant, dose

- related decreases in ovary weight has been 3
observed in female rats treated for 60 days at doses ≥ 5 mg/kg (2.5 mg/kg

- day adjusted) (Xu et al., 4
2010). At 10 mg/kg in adult rats (5 mg/kg

- d ay adjusted) ovary weight was decreased 15% (Xu et 5
al., 2010). Changes in ovarian weight were not observed in t wo subchronic studies in rats

. 6
Specifically, no changes in ovary weight were seen in Wistar rats exposed for 35 days to gavage 7

doses up to 36 m g/kg

- day (Kroese et al., 2001) or in F344 rats exposed for 90 days to dietary doses 8

up to 100 mg/kg

- day (Knuckles et al., 2001). 9

In adult female rats treated by gavage dose

- related decreases in the number of primordial 10

follicles have been observed in female rats treated for 60 days at doses ≥ 2.5 mg/kg

- day, with a 11
statistically significant decrease of approximately 20% at the high dose (Xu et al., 2010) (Table 1

- 7)

. 12

No notable differences in other follicle populations and corpora lutea were observed. However, in 13

utero studies exposing dams to the same doses produced offspring with few or no follicles or 14
corpora lutea (Kristensen et al., 1995; MacKenzenie and Angevine 1981). Additional support for 15

the alteration of female reproductive endpoints comes from IP experiments in animals and in vitro 16

experiments. Several studies have observed ovarian effects (decreased numbers of ovarian follicles 17
and corpora lutea, absence of folliculogenesis, oocyte degeneration, and decreased fertility) in rats 18

and mice e xposed via intraperitoneal ( i.p. ) injection (Mattison et al., 1980; Swartz and Mattison, 19
1985; Miller et al., 1992; Borman et al., 2000). Further evidence is available from in vitro studies 20

showing i nhibition of antral follicle development and survival, a s well as decreased production of 21

estradiol, was demonstrated in mouse ovarian follicles cultured with benzo[a]pyrene for 13 days 22

(Sadeu and Foster, 2011). Likewise, FSH

- stimulated growth of cultured rat ovarian follicles was 23

inhibited by exposure to benz o[a]pyrene (Neal et al., 2007). 24

Hormone levels 25

Alteration of hormone levels has been observed in female rats following oral or inhalation 26

exposure to benzo[a]pyrene (Table 1

- 7) . Inhalation exposure to benzo(a)pyrene:carbon black 27

particles during gestation resulted in decreases in plasma progesterone, estradiol, and prolactin in 28

pregnant rats (Archibong et al., 2002). In addition, statistically significant, dose

- related decreases 29

in estradiol along with altered estrus cyclicity was observed in female rats treated for 60 days at 30
doses ≥ 2.5 mg/kg

- day by gavage (Xu et al., 2010). Mechanistic experiments have also noted 31

decreased estradiol output in murine ovarian follicles cultured with benzo[a]pyrene in vitro for 13 32

days, but did not find any decrease in pr ogesterone (Sadeu and Foster 2011). 33

Cervical effects 34

One subchronic animal study is available which investigated effects in the cervix following 35

oral exposure to benzo[a]pyrene (Table 1

- 7) . Statistically

- significant dose

- related increases in the 36

incidence of cervical inflammatory cells were observed in mice exposed twice a week for 98 days to 37Toxicological Review of Benzo[a]pyrene

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1

- 25 DRAFT

— DO NOT CITE OR QUOTE benzo[a]pyrene via gavage at doses ≥ 2.5 mg/kg (Gao et al., 2011a, 2010). Cervical effects of 1

increasing severity, including epithelial hyperplasia, atypical hyperpla sia, apoptosis, and necrosis, 2
were observed at higher doses. There are no data on cervical effects in other species or in other 3

mouse strains. Gao et al. (2011) considered the hyperplasia responses to be preneoplastic lesions. 4

Cervical neoplasia was not reported in the available chronic bioassays, but this tissue was not 5
subjected to histopathology examination in either bioassay (Beland and Culp, 1998; Kroese et al., 6
2001). Thus, the relationship of the cervical lesions to potential development of neopl asia is 7

uncertain. 8

Table 1

- 6

. Evidence pertaining to the f emale reproductive effects of 9 benzo[a]pyrene in humans 10

Reference and Study Design: Study Type/Period/Study Size/Location/Exposure

Estimate Results

Probability of Conception

Neal et al., 2008

36 w omen undergoing in vitro fertilization (19 smokers, 7 passive smokers, and 10 non smokers)

Exposure: B[a]P in serum and follicular fluid B[a]P levels (ng/ml) a

Conceived Did not

Conceive (p

- value)

Follicular fluid 0.1 1.7 (< 0.001)

Serum 0.01 0.05 (not reported)

a estimated from Figure 3 of Neal et al., 2008

Fetal Death

Wu et al., 2010

Case control study: 81 cases (96% participation rate)

- fetal death confirmed by ultrasound before 14 weeks gestation; 81 controls (91% participation rate)

- elective abortions); matched by age, gestational age, and gravidity ; excluded smokers and occupational PAH ex posure

Tianjin, China

Exposure: B[a]P in aborted tissue and maternal blood samples (51 cases and controls, 2 of 4 hospitals) B[a]P adduct levels (/10 8 nucleotides), mean (±SD)

Cases Controls (p

- value)

Maternal blood 6.0 (± 4.7) 2.7 (± 2.2) (< 0.001)

Aborted tissue 4.8 (± 6.0) 6.0 (± 7.4) (0.29)
Low correlation between blood and tissue levels (r =

– 0.02 in

cases, r =

– 0.21 in controls)

Association between B[a]P adducts and missed abortion a

OR (95% CI)

Per unit increase in adducts 1.37 (1.12, 1.67)

Dichotomized at median 4.56 (1.46, 14.3)

a Conditional logistic regression, adjusted for maternal education, household income, and gestational age; age also considered as potential confounderToxicological Review of Benzo[a]pyrene

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

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- 26 DRAFT

— DO NOT CITE OR QUOTE Table 1

- 7

. Evidence pertaining to the f emale reproductive effects of 1 benzo[a]pyrene in animal s 2

Study Design and Reference Results

Fertility

MacKenzie and Angevine, 1981

CD

- 1 mice, 30 or 60 F0 females/ dose 0, 10, 40, or 160 mg/kg

- d by gavage

GD 7

– 16 ↓ Number of F0 females with viable litters 46/60, 21/30, 44/60, 13/30*

Kristensen et al., 1995 NMRI mice, 9 females/dose 0 or 10 mg/kg

- d by gavage

GD 7

– 16 No changes in fertility of F0 females

Ovarian effects (weight, histology, follicle numbers)

Xu et al., 2010
Sprague

- Dawley rats, 6 females/

dose

0, 5 or 10 mg/kg by gavage every
other day (2.5 and 5 mg/kg

- day, adjusted)
60 days ↓ Ovary weight (% change from control)
0, 11*, 15*

↓ number of primordial follicles (20%* decrease at high dose)

↑ increased apoptosis of ovarian granulosa c ells (approximate % apoptosis)
2, 24*, 14*
Knuckles et al., 2001 F344 rats, 20/sex/dos e 0, 5, 50, or 100 mg/kg

- d in diet

90 days No changes in ovary weight

Kroese et al., 2001 Wistar rats, 10/sex/dos e 0, 1.5, 5, 15, or 50 mg/kg

- day by gavage 5x/w eek

35 days No changes in ovary weight

Hormone levels

Xu et al., 2010
Sprague

- Dawley rats, 6 females/

dose

0, 5 or 10 mg/kg by gavage every
other day (2.5 and 5 mg/kg

- day, adjusted)
60 days ↓ Serum estradiol ( approximate change from control)

0, 16%, 25%

A ltered estrous cyclicity

Archibong et al., 2002 F344 rats, 10 females/grou p 0, 25, 75, or 100 μg/m 3 by inhalation 4 hrs/d ay

GD 11

– 20 (serum hormones tested
at GD 15 and 17 in 0, 25, and 75 μg/m 3 dose groups) ↓ F0 estradiol, approximately 50% decrease at 75 μg/m 3 at GD 17

↓ F0 prolactin, approximately 70% decrease at 75 μg/m 3 at GD 17 ↑ F0 plasma progesterone approximately 17% decrease at 75 μ g/m 3 at

GD 17

Cervical effectsToxicological Review of Benzo[a]pyrene

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- 27 DRAFT

— DO NOT CITE OR QUOTE Study Design and Reference Results

Gao et al, 2011 ICR mice, 26 females/dose 0, 2.5, 5, or 10 mg/kg by gavage 2 days/w eek 98 days ↑ cervical epithelial hyperplasia: 0/26, 4/26, 6/25*, 7/24*

↑ cervical atypical hyperplasia: 0/26, 0/26, 2/25, 4/24*

↑ inflammatory cells in cervical epithelium: 3/26, 10/26, 12/26*, 18/24*

↑ mortality: 0/26, 0/26, 1/26, 2/26

Figure 1

- 4 . Exposure

- response array for female reproductive effects following 1 oral exposure 2

3

Mode of Action Analysis

— Fem ale reproductive effects 4

Although the mechanisms underlying female reproductive effects following benzo[a]pyrene 5
exposure are not fully established, associations with stimulation of apoptosis, impairment of 6Toxicological Review of Benzo[a]pyrene

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1

- 28 DRAFT

— DO NOT CITE OR QUOTE steroidogenesis, and c ytotoxicity have been made. Ovarian lesions in benzo[a]pyrene

- exposed rats 1

have been associated with increased apoptosis in ovarian granulosa cells and alteration in 2

hormone

- mediated regulation of folliculogenesis (Xu et al., 2010), and results from in vi tro 3

experiments provide support for an association between benzo[a]pyrene exposure and impaired 4
folliculogenesis, steroidogenesis, and oocyte maturation (Sadeu and Foster, 2011; Neal et al., 2007). 5
A growing body of research suggests that benzo(a)pyrene t riggers the induction of apoptosis in 6
oocytes through AhR driven expression of pro

- apoptotic genes including Bax (Kee et al., 2010; Neal 7

et al., 2010; Pru et al., 2009; Matkainen et al., 2002, 2001; Robles et al., 2000). Other proposed 8

mechanisms include the impairment of folliculogenesis from reactive metabolites (Takizawa et al., 9
1984; Mattison and Thorgeirsson, 1979; 1977) or by a decreased sensitivity to FSH

- stimulated 10
follicle growth (Neal et al., 2007). Based on findings that an ERα antagonist count eracted effects of 11
subcutaneously administered benzo[a]pyrene on uterine weight (decreased in neonatal rats and 12
increased in immature rats), interactions with ERα have been proposed, possibly via occupation of 13
ERα binding sites or via AhR

- ER

- crosstalk (Ku mmer et al., 2008; 2007). However, several in vitro 14
studies have demonstrated low affinity binding of benzo[a]pyrene to the estrogen receptor and 15
alteration of estrogen

- dependent gene expression (Liu et al., 2006; Van Lipzig et al., 2005; 16

Vondracek et al ., 2002; Fertuck et al., 2001; Charles et al., 2000); so the role of the ER in 17

benzo[a]pyrene

- induced reproductive toxicity is unclear. 18

Summary of Reproductive Effects 19

Male Reproductive Effects 20

Exposure to benzo[a]pyrene in laboratory animals induces male reproductive effects 21

including decreased levels of testosterone and increased levels of LH, decreased sperm count and 22

motility, histological changes in the testis, and decreased reproductive success. These findings in 23

animals are supported by decrements i n sperm quality and decreased fertility in human 24

populations exposed to PAH mixture s (Soares and Melo, 2008; Hsu et al., 2006). In laboratory 25

animals, male reproductive toxicity has been observed after oral and inhalation exposure to rats or 26

mice. Effect s seen after oral exposure s include impaired fertility, effects on sperm parameters, 27

decreased reproductive organ weight, testicular lesions, and hormone alterations (Chen et al., 28

2011 a ; Chung et al., 2011; Mohamed et al., 2010; Zheng et al., 2010; MacKenz ie and Angevine, 29

1981). In addition to oral exposure, male reproductive effects of benzo[a]pyrene have also been 30
observed following inhalation exposure in rats (Archibong et al., 2008; Ramesh et al., 2008; Inyang 31

et al., 2003). T he male reproductive effe cts associated with benzo[a]pyrene exposure are 32

considered to be biologically plausible and adverse. The evidence for male reproductive toxicity 33

seen across multiple human and animal studies identifies the male reproductive system effects as a 34

potential h azard associated with e xposure to benzo[a]pyrene

. 35Toxicological Review of Benzo[a]pyrene

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1

- 29 DRAFT

— DO NOT CITE OR QUOTE Female Reproductive Effects 1

A large body of mechanistic data, both in vivo and in vitro, suggests that benzo[a]pyene 2

impacts fertility through the disruption of folliculogenensis. This finding is supported, albeit 3

indirectly, by observations of premature ovarian senescence in women exposed to cigarette smoke 4
(Midgette an d Baron, 1990). Evidence for female reproductive toxicity of benzo(a)pyrene comes 5
from studies of human populations exposed to PAH mixtures as well as laboratory animal and in 6

vitro studies. In addition, two human studies observed associations specifical ly between 7
benzo(a)pyrene measures and two fertility

- related endpoints: decreased ability to conceive (Neal 8
et al., 2008, 2007) and increased risk of early fetal death (i.e., before 14 weeks gestation) (Wu et al., 9

2010). Studies in multiple strains of rat s and mice indicate fertility related effects including 10

decreases in ovarian follicle populations and decreased fecundity. Decreased serum estradiol has 11

also been noted in two different strains of rats exposed by oral or inhalation exposure. T he 12

reproduc tive effects associated with benzo(a)pyrene exposure are biologically supported and 13

relevant to humans. In consideration of the evidence from human, animal, and mechanistic studies, 14

female reproductive effects are identified as a potential hazard associat ed with exposure to 15
benzo(a)pyrene. 16

Susceptible Populations and Lifestages 17

Epidemiological studies indicate that exposure to complex mixtures of PAHs, such as 18
through cigarette smoke, is associated with measures of decreased fertility in humans (Neal et a l., 19

2005; El Nemr et al., 1998) and that prenatal exposure to cigarette smoking is associated with 20

reduced fertility of women later in life (Weinberg et al., 1989). A case

- control study in a Chinese 21

population has also indicated that women with elevated l evels of benzo[a]pyrene

- DNA adducts in 22
maternal blood were 4 times more likely to have experienced a missed abortion (Wu et al., 2010). 23

Inhalation exposure of pregnant female rats to benzo[a]pyrene:carbon black aerosols on GD 24

11

- 20 caused decreased fetal survival and number of pups per litter associated with decreased 25
levels of plasma progesterone, estradiol, and prolactin (Archibong et al., 2002). Decreased numbers 26

of live pups were also seen in pregnant mice following i.p. exposure to benzo[a]pyrene (Ma ttison et 27

al., 1980). These results indicate that benzo[a]pyrene exposure can decrease the ability of females 28

to maintain pregnancy. 29
Oral multigenerational studies of benzo[a]pyrene exposure in mice demonstrated effects on 30

fertility and the development of reproductive organs (decreased ovary and testes weight) in both 31

male and female offspring of pregnant mice exposed to 10 to 160 mg/kg

- day on GD 7

- 16 32
(Kristensen et al., 1995; Mackenzie and Angevine, 1981). 33

Reductions in female fertility associated with de creased ovary weight following gestational 34

exposure are supported by observations of: 1) destruction of primordial follicles (Bor man et al., 35

2000; Mattison et a l., 1980) and decreased corpora lutea (Miller et al., 1992; Swartz and Mattison, 36
1985) in adult female mice following i.p. exposure; 2) decreased ovary weight in adult female rats 37Toxicological Review of Benzo[a]pyrene

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1

- 30 DRAFT

— DO NOT CITE OR QUOTE following oral exposure (Xu et al. 2010); 3) stimulation of oocyte apoptosis (Matkainen et al., 2002, 1

2001) or by a decreased sensitivity to FSH

- stimulated follicle growth (Neal et al., 2007). 2
Reductions in male fertility associated with decreased testes weight following gestational 3

exposure are supported by observations of: 1) decreased sperm count, altered serum testosterone 4

levels, testicular lesions, and/or increased num bers of apoptotic germ cells in adult rats following 5
repeated oral exposure to benzo[a]pyrene (Chung et al., 2011; Chen et al., 2010; Zheng et al., 2010; 6
Arafa et al., 2009); 2) decreased epididymal sperm counts in adult F0 and F1 generations of male 7

mice following 6 weeks oral exposure of the F0 to benzo[a]pyrene (Mohammed et al., 2010); and 3) 8

decreased testis weight, decreased testicular or plasma testosterone levels, and/or decreased 9
sperm production, motility and density in adult male rats following re peated inhalation exposure to 10

aerosols of benzo[a]pyrene:carbon black (Archibong et al., 2008; Ramesh et al., 2008; Inyang et al., 11

2003). 12



### 1.1.3. Immun otoxicity 13

Human studies evaluating immune effects following exposure to benzo[a]pyrene alone are 14

not available fo r any route of exposure. However, a limited number of occupational human studies, 15
particularly in coke oven workers, show effects on immune parameters associated with exposure to 16

PAH mixtures. These studies are of limited utility because effects associa ted specifically with 17
benzo[a]pyrene cannot be distinguished from other constituents of the PAH mixture. Subchronic 18

and short

- term animal studies have reported immunotoxic effects of benzo[a]pyrene by multiple 19

routes of exposure (Table 1

- 8) . Effects incl ude changes in thymus weight and histology, decreased 20

B cell percentages and other alterations in the spleen, and immune suppression. Data obtained 21

from subchronic oral gavage studies are supported by short term, intraperitoneal (i.p.), 22
intratracheal (i.t .), and subcutaneous (s.c.) studies. Additionally, there is evidence in animals for 23

effects of benzo[a]pyrene on the developing immune system. No studies were located that 24

examined immune system endpoints following inhalation exposure of animals to benzo [a]pyrene. 25

Thymus Effects 26

Decreased thymus weight s ( up to 62% compared to controls) were observed in male and 27

female Wistar rats exposed by gavage to 3

- 90 mg/kg

- day benzo[a]pyrene for 35 or 90 days (Kroese 28
et al., 2001; De Jong et al., 1999). This effect may be due to thymic atrophy. The incidence of slight 29

thymic atrophy was increased in males (6/10) and females (3/10) at a dose of 30 mg/kg

- day in a 30

90

- day study, although there was no evidence of atrophy at any lower dose (Kroese et al., 2001). 31

Addition ally, at the highest dose tested (90 mg/kg

- day) in one of the 35

- day studies, the relative 32
cortex surface area of the thymus and thymic medullar weight were significantly reduced (De Jong 33

et al., 1999). Other histopathological changes in the thymus (increa sed incidence of brown 34

pigmentation of red pulp; hemosiderin) were observed in Wistar rats of both sexes at 50 mg/kg

35

day in a 35

- day study ; however

, this tissue was not examined in intermediate

- dose groups (Kroese 36Toxicological Review of Benzo[a]pyrene

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- 31 DRAFT

— DO NOT CITE OR QUOTE et al., 2001). Consistent with the effect s observed in these studies, decreased thymus weights and 1

reduced thymic cellularity w ere observed in i.p. injection studies that exposed mice to doses 2
ranging from 50 to 150 mg/kg in utero (Holladay and Smith 1995, 1994; Urso and Johnson, 1988). 3

Spleen Ef

fects 4

Reduced splenic cellularity indicated by decreased relative and absolute number of B cells 5

in the spleen (decreased 13

- 41% and 61% compared to controls, respectively) and decreased 6
absolute number of splenic cells (31% decrease at the highest dose) was observed in a subchronic 7

study in male Wistar rats administered 3

- 90 mg/kg

- day benzo[a]pyrene by gavage for 35 days (De 8

Jong et al., 1999). While the effect on the relative number of B cells was dose

- related, the lower 9

doses did not affect the number of B cells or the absolute splenic cell number. The reduced splenic 10

cell counts were attributed by the study authors to the decreased B cells, and suggest a possible 11

selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The spleen effects 12
observed in the single subchronic study are supported by observations of reduced spleen cellularity 13

and decreased spleen weights following i.p. injection or in utero benzo[a]pyrene exposure to doses 14
ranging from 50 to 150 mg/kg (Holladay and Smith , 1995; Urso and Johnson, 1988). 15

In addition to physical effects on the spleen, several studies have demonstrated functional 16
suppression of the spleen following benzo[a]pyrene exposure. Dose

- related decreases in sheep red 17

blood cell (SRBC) specific ser um IgM levels after SRBC challenge were reported in rats (10 or 40 18

mg/kg

- day) and mice (5,20,or 40 mg/kg

- day) following s.c. injection of benzo[a]pyrene for 14 days 19
(Temple et al., 1993). Similarly, reduced spleen cell responses, including decreased numbe rs of 20

plaque forming cells and reduced splenic phagocytosis to SRBC and lipopolysaccharide challenge, 21

were observed in B6C3F 1 mice exposed to doses ≥40 mg/kg

- day benzo[a]pyrene by i.p. or s.c. 22

injection for 4

- 14 days (Lyte and Bick, 1985; Dean et al., 1983 ; Munson and White, 1983) or by 23
intratracheal instillation for 7 days (Schnizlein et al., 1987). 24

Immunoglobulin Alterations 25

Alterations in immunoglobulin levels have been associated with exposure to PAH mixtures 26

in a limited number of human studies. Some occupational studies have reported evidence of 27

immunosuppression following PAH exposure. For example, reductions in serum IgM and/or IgA 28

titers were reported in coke oven workers (Szczeklik et al., 1994; Wu et al., 2003). Conversely, 29

immunostimulation o f immunoglobulin levels has also been observed in humans, specifically 30
elevated IgG (Karakaya et al., 1999) and elevated IgE (Wu et al., 2003) following occupational PAH 31

exposure. 32

Decreases in serum IgM (13 to 33% compared to controls) and IgA levels ( 22

- 61% 33
compared to controls) were observed in male Wistar rats exposed to 3

- 90 mg/kg

- day 34

benzo[a]pyrene by gavage for 35 days (De Jong et al., 1999); however, these reductions were not 35

dose

- dependent. Similarly, reductions in IgA (9

- 38% compared to contro ls) were also observed in 36Toxicological Review of Benzo[a]pyrene

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- 32 DRAFT

— DO NOT CITE OR QUOTE male and female B6C3F 1 mice exposed to doses of 5

- 40 mg/kg benzo[a]pyrene by s.c. injection for 1
14 days (Munson and White, 1983). Reductions in serum IgG levels of 18

- 24%, although not 2
statistically significant, were observed in female B6C3F 1 mice exposed to doses ≥50 mg/kg 3

benzo[a]pyrene by i.p. injection for 14 days (Dean et al., 1983). 4

Immune Suppression and Sensitization 5

Some occupational studies of coke oven emissions have reported evidence of 6
immunosuppression following PAH exposure. Reduced mitogenic responses in T cells (Winkler et 7

al., 1996) and reduced T

- lymphocyte proliferative responses (Karakaya et al., 2004) have been 8

observed following occupational exposure to PAH. Increased levels of apoptosis were observed in 9

th e peripheral blood mononuclear cells (a population of lymphocytes and monocytes) of 10

occupationally exposed coke oven workers; a response that may contribute to immunodeficiency in 11
this population (Zhang et al. 2012). However, a limitation of this study is that it does not attribute 12
the proportion of apoptotic activity to a specific class of cells and does not include assessment of 13

other potential markers of immunotoxicity in peripheral blood. 14

Results of functional immune assays in laboratory animals foll owing short term i.p. and s.c. 15

exposures add to the evidence for benzo[a]pyrene immunotoxicity. Resistance to Streptococcus 16

pneumonia or Herpes simplex type 2 was dose dependently reduced in B6C3F 1 mice following s.c. 17
injection of ≥5mg/kg

- day benzo[a]pyrene for 14 days (Munson et al., 1985). Reduced cell 18

proliferation, IFN

- γ release, and IL

- 4 release were observed in male and female C56BL/6 mice 19
following short

- term exposure to a gavage dose of 13 mg/kg benzo[a]pyrene as measured in a 20

modified local lymph node assay (van den Berg et al., 2005). A statistically significant decrease in 21

NK cell activity was observed in male Wistar rats (Effector:Target cell ratio was 40.9 ± 28.4% that 22

of controls) exposed to 90 mg/kg

- day by gavage for 35 days (De Jong et al., 1999); however, splenic 23
NK cell activity was not affected in B6C3F 1 mice after s.c. injection of 40mg/kg

- day benzo[a]pyrene 24

for 14 days (Munson et al., 1985). The magnitude of the dose and duration of the exposure may 25

account for the discrep ancy between these two studies. Single i.p. injections of 50 mg/kg 26

benzo[a]pyrene decreased pro and/or pre B

- lymphocytes and neutrophils in the bone marrow of 27

C57BL/6J mice without affecting the numbers of immature and mature B

- lymphocytes or GR

- 1+ 28

mye loid cells (Galvan et al., 2006). 29

In contrast to studies that have shown immunosuppression, benzo[a]pyrene may also 30
induce sensitization responses. Epicutaneous application of benzo[a]pyrene (100 μg 31

benzo[a]pyrene to C3H/HeN mice followed by ear challenge with 20 μg benzo[a]pyrene 5 days 32

later) produced a contact hypersensitivity (a significant ear swelling) response (Klemme et al., 33
1987). 34Toxicological Review of Benzo[a]pyrene

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- 33 DRAFT

— DO NOT CITE OR QUOTE Developmental Immunotoxicity 1

As noted above, several i.p. injection studies suggest that cell

- mediated and humoral 2
immunity may be altered by exposure to high doses of benzo[a]pyrene during gestation. 3

Suppression of the mixed lymphocyte response, the graft

- versus

- host response, and suppression of 4

the plaque

- forming cell response to SRBCs was observed in mice exposed i n utero to 150 mg/kg 5
during mid ( GD 11

- 13), late ( GD 16

- 18), or both (GD 11

- 17) stages of gestation; these effects 6
persisted until 18 months of age (Urso and Gengozian, 1984, 1982, 1980). Fetal thymic atrophy, as 7
assessed by reductions in cellularity (74

- 9 5%, compared to controls), was observed in mice 8

exposed to 50

- 150 mg/kg benzo[a]pyrene from GD 13

- 17, when examined on GD18 (Holladay and 9
Smith 1994). Analysis of cell surface markers (e.g., CD4, CD8) from the same study indicate that 10

benzo[a]pyrene may in hibit and/or delay thymocyte maturation, possibly contributing to the 11
observed thymic atrophy (Holladay and Smith 1994). Consistent with these findings, several other 12
studies have noted decreased thymocyte numbers and disrupted T cell maturation after in utero 13

exposure to benzo[a]pyrene (Rodriguez et al., 1999; Holladay and Smith, 1995; Lummus and 14
Henningsen 1995, Urso et al., 1992; Urso and Johnson, 1987). 15
The fetal liver is the primary hematopoietic organ during gestation and a major source of 16

thymocyt e precursors beginning around GD 10 or 11 in mice (Landreth and Dodson, 2005; Penit 17
and Vasseur 1989). Statistically significant reductions in total cellularity in the fetal liver of 54% 18

and 67% were reported in offspring after gavage exposures of 50 or 1 00 mg/kg benzo[a]pyrene to 19

the dams on GD 13

- 17, respectively (Holladay and Smith

, 1994). The decreased fetal liver 20

cellularity was accompanied by decreased expression of terminal deoxynucleotidyl transferase and 21

CD45R cellular markers, which are known to be present in cortical thymocyte progenitors in the 22

fetal liver (Holladay and Smith, 1994; Fine et al., 1990; Silverstone et al., 1976). These data also 23
suggest that benzo[a]pyrene disrupts liver hematopoiesis during gestation and may interfere with 24

prol ymphoid seeding of the thymus, possibly contributing to thymic atrophy and cell

- mediated 25

immunosuppression. Decreased numbers of CD4 + T

- cells have been reported in the spleen of 1

- 26

week

- old mice following in utero benzo[a]pyrene exposure by i.p. injection to the dams, 27

demonstrating the potential for downstream effects on T cell development (Rodriguez et al., 1999). 28

The decreased numbers of CD4 + T

- cells correspond with observations of decreased proliferation in 29

the presence of ConA and a weak response compa red to controls in an allogeneic mixed lymphocyte 30
reaction assay (Urso et al., 2008). 31

Table 1

- 8

. Evidence pertaining to the immune effects of benzo[a]pyrene in 32
animals 33

Study Design and Reference Results

Thym us Effects

Kroese et al., 2001 Wistar rats, 10/sex/dose 0, 3, 10, 30 mg/kg

- d by gavage 5 days/week ↓ thymus weight Females (% change from controls): 0,

- 3,

- 6,

- 28* Males (% change from controls): 0, 0,

- 13,

- 29*Toxicological Review of Benzo[a]pyrene

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- 34 DRAFT

— DO NOT CITE OR QUOTE 90 d ays ↑ slight thymic atrophy
Females (i ncidence): 0/10, 0/10, 0/10, 3/10
Males (i ncidence): 0/10, 2/10, 1/10, 6/10*
De Jong et al., 1999 Wistar rats, 8 males/dose

0, 3, 10, 30, 90 mg/kg

- d by gavage 5 days/week

35 days ↓ thymus weight % change from control: 0,

- 9,

- 15*,

- 25*,

- 62*

Kroese et al., 2001 Wistar rats, 10/sex/dose

0, 1.5, 5 , 15, 50 mg/kg

- d by gavage 5 days/week
35 days ↓ thymus weight Females (% change from controls): 0, +13, +8,

- 3,

- 17* Males (% change from controls): 0,

- 8,

- 11,

- 27*,

- 33*

Spleen Effects

De Jong et al., 1999 Wistar rats, 8 males/dose 0, 3, 10, 30, 90 mg/kg

- d by gavage 5 d ays /w ee k 35 days ↓ in relative number (%) of B cells in spleen
% change from control: 0,

- 8,

- 13*,

- 18*,

- 41*

↓ in total number of B cells in spleen
% change from co ntrol: 0, +13,

- 13,

- 13,

- 61*

Change in total cell number in the spleen % change from control: 0, +20, 0, +7,

- 31*
Immunoglobulin Alterations

De Jong et al., 1999 Wistar rats, 8 males/dose

0, 3, 10, 30, 90 mg/kg

- d by gavage 5 days/week

35 d ays ↓ Serum IgM % change from control: 0,

- 13,

- 14,

- 33*,

- 19

↓ Serum IgA
% change from control: 0,

- 27,

- 22,

- 28,

- 61*

* Statistically significant (p<0.05) as reported by study authors. 1Toxicological Review of Benzo[a]pyrene

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1

- 35 DRAFT

— DO NOT CITE OR QUOTE Figure 1

- 5

. Exposure

- response array for immune effects following oral 1

exposure 2

3

Mode of Action Analysis

— Immune Effects 4

Exposure to benzo[a]pyrene induces immunosuppressive effects such as decreased 5
numbers of B cells in the spleen and decreased thymus weight and cell ularity following oral, i.p., 6

s.c., or i.t. exposure in experimental animals. However, the key events underlying benzo[a]pyrene 7
immunotoxicity have not been identified. 8
Benzo[a]pyrene is a well known ligand for the aryl hydrocarbon receptor (AhR) (Okey et al., 9

1994; Nebert et al., 1993; Postlind et al., 1993). Ligands of the AhR have been shown to have a role 10

in regulating hematopoietic stem cells (HSC) in the bone marrow, a major site of B cell proliferation 11
and antibody production (Esser et al 2009). Benzo[a]pyrene induces greater reductions in bone 12

marrow cultures from Ah

- responsive (C57BL/6) mice compared to Ah

- nonresponsive (DBA/2) 13

mice (Hardin et al. 1992). Furthermore, B cell lymphopoiesis in Ah

- responsive mice was dose 14Toxicological Review of Benzo[a]pyrene

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1

- 36 DRAFT

— DO NOT CITE OR QUOTE dependently suppressed but not in Ah

- nonresponsive mice (Hardin et al., 1992). Addition of the 1

AhR antagonist and cytochrome P450 ( CYP450 ) inhibitor, α

- NF, prevented the benzo[a]pyrene

2
induced inhibition of B cell lymphopoiesis in a concentration

- dependent fashion. Similarly, the 3

CYP1A1 inhibitor, 1

- (1

- propynyl) pyrene, blocked benzo[a]pyrene

- induced B

- cell growth 4
inhibition, but not through the metabolite, BPDE (Allen et al., 2006). Altogether, these data suggest 5
that benzo[a]pyrene may regulate B cell proliferation and antibody production in the bone marrow 6

via the AhR. 7

Summary of Immune Effects 8

Immunotoxic effects of benzo[a]pyrene exposure are based on data from animal studies 9
that vary in route and duration of exposure. There are no human epidemiological studies that 10

provide s pecific support for benzo[a]pyrene immunotoxicity; however, immunosuppression has 11

been observed in studies following occupational exposure to PAH mixtures. However, these 12
findings are limited by co

- exposures to other constituents of PAH mixtures. 13

Each of the immune effects reported in anima l bioassays for benzo[a]pyrene provide 14
equivocal evidence of immunotoxicity; functional assays provide greater support for 15
immunotoxicity than observational findings such as organ weight, hematological, or 16

histopatho logical measures (WHO, 2012). Although the overall database for benzo[a]pyrene 17

immunotoxicity in experimental animals is limited and the key events underlying the mod e of 18

action are not established, t here is evidence of physical alterations to tissues/org ans of the immune 19
system, as well as decreases in immune functi on. Evidence of benzo[a]pyrene

- associated 20

immunotoxicity is supported by consistent thymic effects observed in two oral studies, as well as 21

splenic effects, and varying immunosuppressive respo nses observed in short

- term or in vitro tests. 22

Overall, the weight of evidence in animals indicates that immunotoxicity may be a potential hazard 23

associated with benzo[a]pyrene exposure , but the immune system does not appear to be a sensitive 24

target for b enzo[a]pyrene

- induced toxicity. 25

Susceptible Populations and Lifestages 26

The severity and persistence of immune effects observed during in utero studies suggests 27

that immunotoxicity may be greater during gestation than adulthood (Dietert and Pieperbrink, 28

20 06; Holladay and Smialowicz, 2000). Urso and Gengozian (1982) provide experimental support 29

demonstrating immunosuppression from benzo[a]pyrene exposure during gestation was greater 30

than for mice exposed after birth to a 25

- fold higher dose. There is also substantial literature 31

indicating that disruption of the immune system during certain critical periods of development 32

(e.g., initiation of hematopoiesis; migration of stem cells; expansion of progenitor cells) may have 33
significant and lasting impacts on l ifetime immune function (e.g., Burns

- Naas et al., 2008; Dietert, 34

2008; Landreth et al., 2002; Dietert et al., 2000), as well as more specific studies showing increased 35Toxicological Review of Benzo[a]pyrene

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- 37 DRAFT

— DO NOT CITE OR QUOTE dose sensitivity and disease persistence from developmental versus adult chemical exposu re 1

(reviewed in Luebke et al., 2006). 2
Thymus toxicity is a sensitive and specific effect of benzo[a]pyrene and has been observed 3

in both prenatal and adult exposure studies. The thymus serves as a major site of thymocyte 4

proliferation and selection for ma turation, and impairment can lead to cell

- mediated immune 5
suppression (Kuper, 2002, 1992; De Waal et al., 1997). The thymus is believed to be critical for T 6
lymphocyte production during early life and not in adulthood (Hakim et al., 2005; Schonland et al.

, 7
2003; Petrie et al., 2002; Mackall et al., 1995). Therefore, the decreases in thymus weight observed 8

in studies of adult animals exposed to benzo[a]pyrene suggest that immunosuppression may be a 9
heightened concern for individuals developmentally exposed to benzo[a]pyrene. 10



### 1.1.4. Other Toxicological Effects 11

There is some evidence that benzo[a]pyrene can produce systemic effects in several organ 12

systems including the forestomach, liver, kidney, and cardiovascular system, as well as alter 13

hematological parameters. However, there is less evidence for these effects compared to organ 14

systems described earlier in Section 1.1. 15

Forestomach effects 16

Lesions have been observed in the forestomach following subchronic and chronic oral 17

exposure to benzo[a]pyrene (Table 1

- 9)

. Increases in the incidence of forestomach hyperplasia 18
have been observed in Wistar rats following shorter

- term, subchronic, and chronic gavage exposure 19

(Kroese et al., 200; De Jong et al., 1999) and in B6C3F1 mice following chronic dietary exposure 20

(Belan d and Culp, 1998; Culp et al., 1998). 21

Following chronic gavage exposure, increased incidences of forestomach hyperplasia were 22
observed in male and female rats at 3 and 10 mg/kg

- day ; at the highest dose

, limited hyperplasia 23

was reported (Kroese et al., 20 01). Only the highest level lesion (hyperplasia, papilloma, or 24

carcinoma) observed in each organ was scored, such that hyperplasia observed in the forestomach 25
in which tumors were also observed was not scored. The majority of animals in the high dose 26

gro up exhibited forestomach tumors; therefore, the hyperplasia was not scored and the incidence 27

of forestomach hyperplasia in the study is largely uncharacterized at the highest dose. Shorter term 28

studies (Kroese et al., 2001; De Jong et al. 1999) showed dos e

- related increases in forestomach 29
hyperplasia at doses ≥10 mg/kg

- day in Wistar rats . In addition, f ollowing chronic dietary exposure, 30

a dose

- dependent increase in the incidence of forestomach hyperplasia and hyperkeratosis was 31

observed in female mice at ≥0.7 mg/kg

- day (Beland and Culp, 1998; Culp et al., 1998). Forestomach 32

tumors were also observed at ≥0.7 mg/kg

- day by Beland and Culp (1998) and Culp et al. (1998). 33Toxicological Review of Benzo[a]pyrene

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- 38 DRAFT

— DO NOT CITE OR QUOTE Table 1

- 9

. Evidence pertaining to the f ores tomach effects of benzo[a]pyrene in 1
animal s 2

Study design and reference Results

Kroese et al. (2001) Wistar (Riv:TOX) rats: male and female (52/sex/dose group)

0, 3, 10, or 30 mg/kg

- day by gavage 5 days/week

104 weeks (chronic)

Wistar (Riv:TOX) rats: male and female (10/sex/dose group)

0, 3, 10, or 30 mg/kg

- day by gavage 5 days/week 90 days (subchronic)

Wistar (specific pathogen

- free Riv:TOX) rats (10/sex/dose group)

0, 1.5, 5, 15, or 50 mg/kg bw by gavage

5 days/week

5 weeks (shorter

- term) Chronic:

forestomach hyperplasia

– (basal cell hyperplasia)

Incidences: M: 2/50; 8/52; 8/52; 0/52 F: 1/52; 8/51; 13/51; 2/52

Subchronic: forestomach hyperplasia

– (slight basal cell hyperplasia)

Incidences: M: 2/10; 0/10; 6/10; 7/10 F: 0/10; 2/10; 3/10; 7/10

Short term:

forestomach hyperplasia

– (basal cell hyperplasia)

Incidences: M: 1/10; 1/10; 4/10; 3/10; 7/10 F: 0/10; 1/10; 1/10; 3/10; 7/10*

Beland and Culp (1998); Culp et al. (1998)

B6C3F 1 mice: female (48/dose group)

0 , 5, 25, or 100 ppm in the diet (average daily

doses a : 0, 0.7, 3.3, and 16.5 mg/kg

- day)
2 years forestomach hyperplasia Incidences: 13/48; 23/47; 33/46*; 38/47*

De Jong et al. (1999)

Wistar rats: male (8/ dose group)

0, 3, 10, 30, or 90 mg/kg

- day by gavage 5 days/week

5 weeks forestomach hyperplasia

– (basal cell hyperplasia) statistically significantly increased incidences at 30 and 90 mg/kg

- day were reported, but incidence data were not provided

* indicates statistical significance as identified in study

a Based on the assumption that daily benzo[a]pyrene intake at 5 ppm was one

- fifth of the 25

- ppm intake (about

21 μg/day) and using time

- weighted average (TWA) body weights of 0.032 kg for the control, 5

- and 25

- ppm groups and 0.026 kg for the 100

- ppm groupToxicological Review of Benzo[a]pyrene

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- 39 DRAFT

— DO NOT CITE OR QUOTE Figure 1

- 6 . Exposure

- response array for forestomach hype rplasia following 1 oral exposure 2

3

Mode of Action Analysis

— Forestomach Effects 4

Mechanistic investigations suggest that bioactivation of benzo[a]pyrene leads to reactive 5

intermediat es that can lead to mutagenic events, as well as to cytotoxic and apoptotic events. The 6

available human, animal, and in vitro evidence best supports a mutagenic mode of action as the 7
primary mode by which benzo[a]pyrene induces carcinogenesis. Available data indicate that 8
forestomach hyperplasia may be a histological precursor to neoplasia observed at this site after 9

chronic exposure to benzo[a]pyrene (Kroese et al., 2001; DeJong et al., 1999). Dose

- response data 10

show that forestomach hyperplasia occurs at shorter durations and at lower doses than tumors in 11

rats and mice exposed to benzo[a]pyene for up to 2 years (Kroese et al., 2001; Beland and Culp, 12
1998). Kroese et al. (2001) reported that the forestomach lesions demonstrated a progression over 13

the c ourse of intercurrent sacrifices; the authors described early lesions as focal or confluent basal 14

hyperplasia, followed by more advanced hyperplasia with squamous cell papilloma, and 15
culminating in squamous cell carcinoma. The description of the progressi on of forestomach lesions 16Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE provided by Kroese et al. (2001), coupled with the observation that hyperplasia occurs before 1
tumors and at lower doses than tumors suggests that forestomach hyperplasia induced by 2

benzo[a]pyrene is likely a preneoplastic lesion. 3

Summary of Forestomach Effects 4

A dose

- dependent increased incidence of forestomach hyperplasia was observed in both 5
rats and mice and in multiple studies. Evidence for forestomach hyperplasia after both gavage and 6

dietary administration demonstrates conco rdance of the effect across different modes of 7
administration and illustrates that the hyperplasia is not dependent on bolus dosing or mechanical 8

irritation associated with gavage administration. The increase in forestomach hyperplasia was also 9

consistent across multiple exposure regimens, including chronic, subchronic, sh ort

- term, and acute 10

exposures. 11

Hematological Effects 12

Altered hematological parameters

, including decreases in RBC count, hemoglobin, and 13

hematocrit have been observed in laboratory animal s following benzo[a]pyrene exposure (Table 1

- 14

10) . Statistically significant decreases in RBC count, hemoglobin, and hematocrit were observed in 15

male Wistar rats at doses greater than 10 mg/kg

- day for 35 days (De Jong et al. 1999). A minimal, 16

but statisti cally significant increase in mean cell volume and a decrease in mean cell hemoglobin 17
were observed at the highest dose (90 mg/kg

- day), which may indicate dose

- related toxicity for the 18
RBC’s and/or RBC precursors in the bone marrow (DeJong et al., 1999). Similarly, male and female 19

F344 rats also showed statistically significant decreases in RBC counts and hematocrit level, along 20
with decreased hemoglobin levels in a 90 day dietary study (Knuckles et al. 2001). Statistically 21

significant decreases were obse rved in the two highest doses (50 and 100 mg/kg

- day) in male rats, 22

while a maximum decrease of 12% was only observed at the highest dose in female rats at 23
100mg/kg

- day. Small, but not statistically significant decreases in RBC counts and hemoglobin 24

were o bserved in both a 35

- day and 90

- day study in Wistar rats (Kroese et al. 2001). It should be 25

noted that when observed, the magnitudes of the decreases in RBC, hemoglobin, and hematocrit 26
were generally small; about 18% at 90 mg/kg

- day and <10% at lower dose s (De Jong et al., 1999) 27
and about 10% in F344 rats (Knuckles et al., 2001). A decrease in WBC’s, attributed to reduced 28

numbers of lymphocytes and eosinophils, was also observed at 90 mg/kg

- day following gavage 29

exposure for 35 days (DeJong et al., 1999). The mode of action by which benzo[a]pyrene exposure 30

may lead to altered hematological parameters is undetermined. 31

Liver Effects 32

Liver effects other than cancer associated with benzo[a]pyrene exposure primarily include 33
changes in liver weight and abnormal histopathology (Table 1

- 10) . Increased liver weight was 34

reported in a 90

- day study in both male and female Wistar rats given benzo[a]pyrene by gavage 35

(Kroese et al., 2001). Both females (17% increase) and males (29% increase) demonstrated 36Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE statistically s ignificant increased liver weights at the highest dose tested (30 mg/kg

- day); a 1
statistically significant increase (15%) was also reported in males at 10 mg/kg

- day. Similar to the 2
findings in the 90

- day study by Kroese et al. (2001), increased liver:body weight ratios were 3

observed at the highest dose in a 90

- day dietary study in male F344 rats, although there was no 4
change observed in female liver weights (Knuckles et al. 2001). Increased liver:body weight ratios 5
were also observed in both sexes at high doses (600 and 1000 mg/kg) in an accompanying acute 6
study (Knuckles et al. 2001). A statistically significant increase in liver weight was also observed in 7
male Wistar rats given 90 mg/kg

- day benzo[a]pyrene by gavage for 35 days (De Jong et al., 1999). 8

C onsistent with the findings by De Jong et al. (1999), a statistically significant increased liver weight 9
(about 18%) was also observed in both male and female Wistar rats at the highest dose (50 mg/kg

10

day) given by gavage in a 35

- day study (Kroese et al., 2001). 11

Limited exposure

- related differences in clinical chemistry parameters associated with liver 12
toxicity were observed; no differences in alanine aminotransferase (ALT) or serum aspartate 13

transaminase (AST) levels were observed, and a small dose

- relat ed decrease in γ

- glutamyl 14

transferase (GGT) was observed in males only exposed to benzo[a]pyrene for 90 days (Kroese et al., 15

2001). 16

Treatment

- related lesions in the liver (oval cell hyperplasia) were reported following 17

exposure to 90 mg/kg

- day benzo[a]py rene for 35 days, however incidence data were not reported 18

(De Jong et al., 1999) . Other histopathological changes observed in the liver include an increased 19
incidence of liver clear cell foci of alteration in males and females during a 2

- year carcinogeni city 20

study (Kroese et al., 2001), however organs with tumors were not evaluated. Since many of the 21

animals in the top two doses developed liver tumors, the dose responsiveness of the increased 22

incidence of clear cell foci is unclear. 23

A dose

- dependent i ncrease in liver microsomal EROD activity, indicative of CYP1A1 24
induction, was observed in both sexes at doses ≥1.5 mg/kg

- day (Kroese et al. 2001). However, at 25

the highest dose tested, with the greatest fold induction in EROD activity, there was no eviden ce of 26
associated adverse histopathologic findings. The finding of increased liver weight across multiple 27

studies of varying exposure durations, as well as histopathological changes in the liver provide 28

evidence of the liver as a target of benzo[a]pyrene

- induced toxicity. The mode of action by which 29

benzo[a]pyrene induces these effects is unknown

. 30

Kidney Effects 31

There is minimal evidence of kidney toxicity following exposure to benzo[a]pyrene (Table 32

1

- 10) . A statistically significant decr ease in kidney weight was observed in the highest dose tested 33
(90 mg/kg

- day) in a 35

- day gavage study in male Wistar rats (De Jong et al. 1999). Decreases in 34

kidney weight at 3 and 30 mg/kg

- day were also observed, but these changes were not dose

35

dependent . Additionally, kidney weights were not affected by following exposure to 36
benzo[a]pyrene for 35 days (Kroese et al. 2001). Histopathological analysis of kidney lesions 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE revealed an apparent dose responsive increase in the incidence of abnormal tubular cas ts in the 1

kidney in male F344 rats exposed by diet for 90 days (Knuckles et al 2001). The casts were 2
described as molds of distal nephrons lumen and were considered by the study authors to be 3

indicative of renal dysfunction. However, the statistical sig nificance of the kidne y lesions is 4

unclear. Several gaps and inconsistencies in the reporting make interpretation of the kidney effects 5
difficult, including 1) no reporting of numerical data, 2) no indication of statistical significance in 6

the accompanyin g figure for kidney lesions, 3) discrepancies between the apparent incidences and 7
sample sizes per dose group, and 4) uncertainty in how statistical analysis of histopathological data 8

was applied. As such, the significance of the abnormal tubular casts i s unclear. While there are 9
some findings to suggest that the kidneys may be affected by benzo[a]pyrene exposure, overall 10

there is insufficient data to suggest that the kidneys may be a primary target of benzo[a]pyrene

11

induced toxicity. 12

Cardiovascular Effe cts 13

Atherosclerotic vascular disease and increased risk of cardiovascular mortality has been 14
associated with cigarette smoking (Ramos and Moorthy, 2005; Miller and Ramos, 2001; Thirman et 15
al., 1994) and, to a more limited degree, occupational exposure to P AH mixtures ( Friesen et al., 16

2010, 2009 ; Burstyn et al., 2005; Chau et al., 1993 ). Elevated mortality due to cardiovascular 17

disease was observed in a PAH

- exposed occupational population (coke oven plant workers), but 18

elevated cardiovascular mortality was also observed in the non

- exposed or slightly exposed 19

populations as well (Chau et al., 1993). Elevated risks of ischemic heart disease (IHD) were 20

associated with past cumulative benzo[a]pyrene exposure (with a 5

- year lag), although the trend 21

was not stati stically significant; there was no observed association with more recent 22

benzo[a]pyrene exposure (Friesen et al., 2010). Elevated risk of mortality from IHD was also 23
associated with cumulative benzo[a]pyrene exposure in a cohort of male asphalt workers (al though 24

not statistically significant); the trend in average benzo[a]pyrene exposure and association with 25

IHD was statistically significant, with an approximately 60% increase in risk between the lowest 26
and highest exposure groups (Burstyn et al., 2005). The two studies which associate 27

benzo[a]pyrene exposure with cardiovascular effects (Friesen et al., 2010; Burstyn et al., 2005) rely 28

on statistical models to create exposure groups based on previously gathered benzo[a]pyrene air 29

samples that may or may n ot have included members of the cohort under examination. 30
Additionally, while these studies used benzo[a]pyrene exposure groupings for analysis, they cannot 31

address co

- exposures that may have occurred in the occupational setting (asphalt or aluminum 32

smelt ers) or exposures that o ccurred outside the workplace. 33
Increased systolic and diastolic blood pressure has been observed in the offspring of dams 34

exposed to increasing concentrations of benzo[a]pyrene (Jules et al., 2012) (Table 1

- 10) . At the 35

highest dose tested (1200 μg/kg BW by gavage to the dams), systolic pressures were elevated 36

approximately 50% and diastolic pressures were elevated approximately 80% above controls. 37Toxicological Review of Benzo[a]pyrene

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- 43 DRAFT

— DO NOT CITE OR QUOTE Reduced endothelial integrity and increased smooth muscle cell mass, both relate d to 1

atherosclerosis, have been observed in Sprague

- Dawley rats exposed to 10mg/kg benzo[a]pyrene 2

by i.p. injection (once/week for 8 weeks) (Zhang and Ramos, 1997). The molecular mechanisms 3

underlying PAH

- induced vascular injury, and the development of at herosclerosis are not well 4
established, but current hypotheses include cell proliferative responses to injury of endothelial cells 5
from reactive metabolites (including ROS) and genomic alterations in smooth muscle cells from 6
reactive metabolites leading to transformed vasculature cells and eventual plaque formation 7
(Ramos and Moorthy, 2005). However, while the link between PAHs and atherosclerotic disease 8

have been studied, experiments specifically looking at the relationship between levels of exposure 9

to benzo[a]pyrene (via environmentally relevant routes), and the development of aortic wall 10

lesions related to atherosclerosis have not generally been performed. 11

One exception to this observation comes from a series of experiments on Apolipoprotein E 12

knock

- out (ApoE

- /

- ) mice exposed orally to benzo[a]pyrene (Knaapen et al., 2007; Curfs et al., 13

2005, 2004; Godschalk et al., 2003). ApoE

- /

- mice develop spontaneous atherosclerosis, which is 14
thought to be due to enhanced oxidative stress from the lack o f ApoE (Godschalk et al., 2003). 15

Overall, these studies suggest that benzo[a]pyrene exposure in ApoE

- /

- mice enhances the 16
progression of atherosclerosis through a general local inflammatory process. 17

Neurological Effects 18

Impaired learning and memory, as well as neurochemical alterations, have been observed in 19
humans following occupational exposure to PAH mixtures (Niu et al, 2009). Male coke oven 20

workers were analyzed for alterations in neurobehavioral function using the World Health 21

Organization Neur obehavioral Core Test Battery (WHO

- NCTB), as well as changes in 22

neurotransmitter concentrations in blood. Urinary levels of the PAH metabolite 1

- hydroxypyrene 23

were used as markers of PAH exposure. In the WHO

- NCTB, coke workers had lower scores in the 24

dig it span and forward digit span tests than matched control subjects, suggesting that short

- term 25

memory was impaired. The authors also reported that the digit span and forward digit span scores 26
cores significantly decreased with increasing 1

- hydroxypyrene l evels in urine. PAH exposure also 27

altered the blood levels of several neurotransmitters. As in the functional assays, the authors 28

reported that alterations in neurochemical measures were associated with urinary levels of 1

- 29

hydroxypyrene. 30

Reductions in neuromuscular, autonomic, sensorimotor, and electrophysiological endpoints 31

have been reported in rats and mice following acute or short

- term exposure to benzo[a]pyrene 32

(Saunders et al., 2006, 2002, 2001; Liu et al., 2002; Grova et al., 2007, 2008; Bouayed et al., 2009a). 33
Impaired Morris water maze performance was observed following subchronic oral gavage in adult 34

rats (Chen et al. 2011 b ); however, the study was conducted with one dose group (Table 1

- 10)

. 35

These data suggest that longer durations of benzo[a] pyrene exposure could be neurotoxic; 36
however, only limited data are available to inform the neurotoxic potential of repeated sub

- chronic 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE or chronic exposure to benzo[a]pyrene. The available data from the human and chronic and 1

subchronic animal studies (Kro ese et al., 2001; Beland and Culp 1998) provide limited support for 2

benzo[a]pyrene as a neurotoxicant in adults. 3

Table 1

- 10

. Evidence pertaining to o ther systemic effects of benzo[a]pyrene in 4
animals 5

Study Design and Reference Results

Hematological Parameters

Kroese et al., 2001 Wistar rats, 10/sex/dose

0, 3, 10, 30 mg/kg

- d by gavage 5 days /w eek 90 days RBC count and hemoglobin changes not statistically significant in males or

females at any dose (numerical data not reported)

Knuckles et al., 2001 F344 rats, 20/sex/dose 0, 5, 50, 100 mg/kg

- d by diet

90 d ays ↓ RBC count Females (% change from controls): statistically significant at 100mg/kg

- d (numerical data not reported)

Males (% ch ange from controls): statistically significant at 50 and 100mg/kg

- d (numerical data not reported)

↓ hematocrit Females (% change from controls): statistically significant at 100mg/kg

- d (numerical data not reported)

Males (% change from controls): statistically significant at 50 and 100mg/kg

- d (numerical data not reported)

↓ hemoglobin

Females: statistically significant at 100mg/kg

- d (numerical data not reported)Males: statistically significant at 100mg/kg

- d (numerical data not reported)

De Jong e t al., 1999 Wistar rats, 8 males/dose

0, 3, 10, 30, 90 mg/kg

- d by gavage 5 days/week

35 d ays ↓ RBC count
% change from controls: 0,

- 1,

- 5*,

- 10*,

- 18*

↓ hemoglobin
% change from controls: 0,

- 1,

- 7*,

- 10*,

- 18*

↓ hematocrit
% change from controls: 0, 0,

- 6*,

- 8*,

- 14*

↓ WBC count
% change from controls: 0,

- 8,

- 9,

- 9,

- 43*

↑ mean cell volume
% change from controls: 0, 0,

- 3, 0, +3*

↓ mean corpuscular hemoglobin concentration
% change from controls: 0,

- 1,

- 1,

- 1,

- 3*Toxicological Review of Benzo[a]pyrene

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- 45 DRAFT

— DO NOT CITE OR QUOTE Study Design and Reference Results

Kroese et al., 2001 Wistar rats, 10/sex/dose 0, 1.5, 5, 15, 50 mg/kg

- d by gavage 5 days/week
35 days RBC count: not statistically significant (numerical data not reported)

hemoglobin: not statistically significant (numerical data not reported)

Liver Effects

Kroese et al., 2001 Wistar rats, 10/sex/dose

0, 3, 10, 30 mg/kg

- d by gavage 5 days /w eek 90 days ↑ liver weight Females (% change from controls): 0,

- 2, +4, +17* Males (% change from controls): 0, +7, +15*, +29*

Liver histopathology: no effects reported

Knuckles et al., 2001 F344 rats, 20/sex/dose 0, 5, 50, 100 mg/kg

- d by diet 90 days ↑ liver:body weight ratio

Females: no change (numerical data not reported)

Males (% change from controls): 23% change reported at 100mg/kg

- d (numerical data not reported)
De Jong et al., 1999 Wistar rats, 8 males/dose

0, 3, 10, 30, 90 mg/kg

- d by gavage 5 days/week

35 d ays ↑ liver weight
% change from controls: 0,

- 9, +7, +5, +15*

↑ liver oval cell hyperplasia (numerical data not reported)

Kroese et al., 2001

0, 1.5, 5, 15, 50 mg/kg

- d by gavage 5d/wk for 35d Wistar rats, 10/sex/dose ↑ liver weight Females (% change from controls): 0, +3, +2, +9, +18* Males (% change from controls): 0, +2, +1, +3, +18*

Liver histopathology: no effects reported

Kidney Effects

Knuckles et al., 2001 F344 rats, 20/sex/dose 0, 5, 50, 100 mg/kg

- d by diet 90 days ↑ abnormal tubular casts
Females: not statistically significant (numerical data not reported) Males: apparent dose

- dependent increase (numerical data not reported)

De Jong et al., 1999 Wistar rats, 8 males/dose

0, 3, 10, 30, 90 mg/kg

- d by gavage 5 days/week
35 days ↓ kidney weight
% change from controls: 0,

- 11*,

- 4,

- 10*,

- 18*

Kroese et al., 2001 Wistar rats, 10/sex/dose

0, 1.5, 5, 15, 50 mg/kg

- d by gavage 5 days/week
35 days kidney weight: no change (data not reported)

Neurological EffectsToxicological Review of Benzo[a]pyrene

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- 46 DRAFT

— DO NOT CITE OR QUOTE Study Design and Reference Results
Chen et al. (2011 b )
Sprague

- Dawley rats, male, 32/dose

0 or 2 mg/kg

- day by gavage

90 d ays ↑ time required for treated rats to locate platform in water maze (data reported graphically)

Bouayed et al. (2009a) Male Swiss albino mice, 9/group

0, 0.02, 0.2 mg/kg by gavage

28 d ays Significant decrease in latency to attack and increase in the number of

attacks in the resident

- intruder test at 0.02 mg/kg

- day (but not at high dose)

Significant increase in mount number in the copulatory behavior test at 0.02 mg/kg

- day (but not at high dose)

* Statistically significant. 1



### 1.1.5. Carcinogenicity 2

Evidence in Humans 3

There are many epidemiologic studies involving exposure to PAH mixtures that contain 4
benzo[a]pyrene (e.g.

, studies of coke oven workers, asphalt workers) . T his discussion primarily 5
focuses on epidemiologic studies that included a direct measure of benzo[a]pyrene exposure

. 4 The 6

identified studies were separated into tiers according to extent and quality of the exposure analysis 7
and other study design features: 8

Tier 1: Detailed exposure assessment conducted, large sample size (greater than ~50 9

expos ed cases), and adequate follow

- up period to account for expected latency (e.g., 10

greater than 20 years for lung cancer). 11
Tier 2: More limited exposure assessment, or sample size or follow

- up period did not meet 12

the criteria for Tier 1, or only a single

- esti mate exposure analysis was conducted. 13

For lung cancer, each of the Tier 1 studies observed increasing risks of lung cancer with 14
increasing cumulative exposure to benzo[a]pyrene (measured in μg/m 3

- years), and each of these 15

studies addressed in the analysis the potential for confounding by smoking (Armstrong et al., 2009; 16

Spinelli et al., 2006; Xu et al., 1996) ( Table 1

- 12 ). These three studies represent different 17
geographic locations and two different industries. The pattern of results in the Tier 2 studie s was 18

mixed, as would be expected for studies with less precise exposure assessments or smaller sample 19

sizes: one of the SMR estimates was < 1.0, with the other 8 estimates ranging from 1.2 to 2.9 ( Table 20

1

- 13 ). In considering all of the available studies , particularly those with the strongest methodology, 21

there is considerable support for an association between benzo[a]pyrene exposure and lung cancer, 22

although the relative contribution benzo[a]pyrene and of other PAHs cannot be established. 23

For bladder c ancer, the cohort and nested case

- control studies observed a much smaller 24
number of cases compared with lung cancer; this limits their ability to examine exposure

- response 25

4 These studies were identified from PubMed searches (last search conducted May 15, 2012 using terms benzo(a)pyrene AND (cohort OR case

- control) AND ca ncer) and two recent meta

- analyses (Armstrong et al, 2004; Bosetti et al, 2007).Toxicological Review of Benzo[a]pyrene

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- 47 DRAFT

— DO NOT CITE OR QUOTE relationships. Two cohort studies with detailed exposure data, however, had sample sizes of 48 1

and 78 cases (Burstyn et al., 2009; Gibbs et al. 2007a,b,c) (Tier 1 studies, Table 1

- 14 ). Although 2
cumulative exposure (up to approximately 2 μg/m 3

- years) was not related to increasing risk in the 3

study of asphalt workers by Bursyn et al. (201 0), an exposure

- response was seen with the wider 4
exposure range (i.e., above 100 μg/m 3 ) examined in a study in an aluminum smelter workers by 5
Gibbs et al (2007a,b,c). This difference in response is not surprising, given that the highest 6

exposure group in the asphalt worker study corresponded to the exposures seen in the lowest 7
exposure category in the study of aluminum smelter workers. The 6 studies with more limited 8

exposure information or analyses each included between 2 and 16 bladder cancer cases, wit h 9
relative risk estimates ranging from 0.6 to 2.9. None of these individual effect estimates was 10

statistically significant (Table 1

- 14 ). 11

Two of the identified studies contained information on risk of mortality from melanoma. 12

Neither of these studies observed increased risks of this type of cancer, with an SMR of 0.91 (95% 13

CI 0.26, 2.48) [22 cases) in Spinelli et al., 2006 and 0.58 (95% CI 0.12, 1.7) in Gibbs et a. (2007a) [3 14

cases]. 15

Of additional interest is non

- melanoma skin cancer, particularly wi th respect to dermal 16
exposures. The literature pertaining to this kind of cancer and PAH exposure goes back to the 18 th 17
century work of Sir Percival Pott describing scrotal cancer, a squamous cell skin cancer, in chimney 18

sweeps (Brown and Thornton, 1957)

. One of the identified studies reported an increased risk of 19
mortality from non

- melanoma skin cancer among asphalt workers (roofers), with an SMR of 4.0 20

(95% CI: 1.0, 10.9) among workers with 20 or more years. In addition to this study, 3 studies in 21

Scan dinavian countries examined non

- melanoma skin cancer risk in relation to occupations with 22

likely dermal exposure to creosote (i.e., timber workers, brickmakers, power linesmen) using 23
incidence data from population registries ( Tornqvist et al.

, 1986 ; Karleh agen et al.

, 1992 ; Pukkala et 24

al.

, 1995) . The SIR estimates were 1.5 (95% CI: 0.7, 2.6) based on 5 exposed cases, 2.37 (95% CI: 25

1.08, 4.50) [9 cases], and 4.64 (95% CI: 1.51, 10.8) [5 cases], respectively, in Tornqvist et al. (1986), 26
Karlehagen et al. (1 992), and Pukkala et al. (1995). These studies provide additional support for 27

the association between dermal PAH exposure, including benzo[a]pyrene exposure, and skin 28

cancer. 29

Lung, bladder, and skin cancers are the cancers that have been observed in occu pational 30
studies of PAH mixtures (Benbrahim

- Tallaa et al., 2012; IARC, 2010a,b, Secretan et al., 2009; Baan 31

et al., 2009) (see Table 1

- 11 ). The reproducibility of these three sites in different populations and 32

exposure settings adds plausibility to the hy pothesis that common etiologic factors may be 33
operating. The potential role that benzo[a]pyrene may play as a causal agent is further supported 34

by the observation that these same three sites are also increased in the studies that included a 35

direct measure of benzo[a]pyrene. 36Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 11

. Cancer sites for PAH

- related agents reviewed by IARC 1

PAH

- related mixture or

occupation Sites with sufficient

evidence in humans Sites with limited

evidence in humans Reference

Aluminum production Lung Urinary bladder Baan et al (2009)

Carbon electrode manufacture Lung IARC (2010)
Coal gasification Lung Baan et al (2009)

Coal tar distillation Skin Baan et al (2009)
Coal tar pitch (paving and roofing) Lung Urinary bladder Baan et al (2009)
Coke production Lung Baan et al (2009)

Creosotes Skin IARC (2010)

Diesel exhaust Lung Urinary bladder Benbrahim

- Tallaa et al. (2012)

Indoor emissions from household

combustion of biomass fuel (primarily wood) Lung Secretan et al (2009)

Indoor emissions from household

combustion of coal Lung Secretan et al (2009)

Mineral oils, untreated or mildly

treated Skin Baan et al (2009)

Shale oils Skin Baan et al (2009)
Soot (chimney sweeping) Lung

Skin Urinary bladder Baan et al (2009)

Adapted from IARC, 2010.

Table 1

- 12 . Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation to lung cancer risk: Tier 1 studies

Reference, design Results

Armstrong et al., 2009 ( Quebec, Canada)

Cohort, aluminum smelter workers, 7 plants 16,431 (15,703 men; 728 women) Duration: minimum 1 year, began work 1966

– 1990

Follow

- up: through 1999 (mean ~ 30 years) Smoking information collected from medical records Exposure: Job expo sure matrix ~ 5,000 personal B[a]P

measures from the 1970s to 1999

Related references: Lavoué et al., 2007 (exposure data); Gibbs et al., 2007a,b,c; Armstrong et al., 1994 SMR 1.32 (1.22, 1.42) [677 cases] Lung cancer risk by cumulative B[a]P exposure

Median B[a]P μg/m 3

- years n

cases SMR (95% CI) RR (95% CI)

0 35 0.62 (0.44, 0.87) 1.0 (referent)

10 266 1.09 (0.96, 1.23) 1.75 (1.23, 2.48)

30 70 1.88 (1.47, 2.38) 3.02 (2.01, 4.52)

60 53 1.21 (0.91, 1.59) 1.94 (1.27, 2.97)

120 114 1.93 (1.59, 2.32) 3.09 (2.12, 4.51)

240 116 1.79 (1.48, 2.15) 2.86 (1.96, 4.18)

480 23 2.36 (1.49, 3.54) 3.77 (2.23, 6.38)
No evidence of confounding by smoking Additional modeling as continuous variable: RR 1.35 (95% CI 1.22, 1.51) at 100 μg/m 3

- years (0.0035 per μg/m 3

- years increase); other shapes of exposure

- response curve examined.Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Adapted from IARC, 2010.

Table 1

- 12 . Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation to lung cancer risk: Tier 1 studies

Reference, design Results

Spinelli et al., 2006 ( British Columbia, Canada)

Cohort, aluminum smelter workers 6,423 (all men) Duration: minimum ≥ 3 years; began work 1954

– 1997

Follow

- up: through 1999 (14% loss to follow

- up; mean ~ 24years) Smoking information from self

- administered questionnaire Exposure: Job exposure matrix using 1,275 personal B[a]P measures from 1977 to 2000 (69% for compliance monitoring)

Related references: Friesen et al., 2006 (exposure data);
Spinelli et al., 1991 SMR 1.07 (0.89, 1.28) [120 cases] SIR 1.10 (0.93, 1.30) [147 cases]

Lung cancer risk by cumulative B[a]P exposure
B[a]P μg/m 3

- years n

cases RR (95% CI) a

0 to 0.5 25 1.0 (referent)

0.5 to 20 42 1.23 (0.74, 2.03)

20 to 40 23 1.35 (0.76, 2.40)

40 to 80 25 1.36 (0.78, 2.39)

≥ 80 32 1.79 (1.04, 3.01)

a Adjusting for smoking category; trend p < 0.001

Xu et al.,1996 (China)

Nested case

- control in iron

– steel worker cohort 610 incident cases (96% participation); 959 controls (94% participation) (all men) Duration: data not reported Smoking information collected from interviews; next

- of

- kin interviews with 30% of lung cancer cases and 5% of controls Exposure: Job exposure matrix 82,867 historical monitoring
records, 1956 to 1992 (not sure what proportion were B[a]P) Lung cancer risk by cumulative B[a]P exposure
B[a]P (μg/m 3

- years) n

cases RR (95% CI) a

< 0.84 72 1.1 (0.8, 1.7)

0.85 to 1.96 117 1.6 (1.2, 2.3)

1.97 to 3.2 96 1.6 (1.1, 2.3)

≥ 3.2 b 105 1.8 (1.2, 2.5)

a Adjusting for birth year and smoking category; trend p < 0.004. Referent group is “nonexposed” (employed in

administrative or low

- exposure occupations)

b Table IV of Xu et al. (1996) report unclear; could be >= 3.0 for
this category

1
2Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 13 . Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation to lung cancer risk: Tier 2 studies

Reference, design Results

Limited Follow

- up Period (≤ 20 years)

Friesen et al., 2009 (Australia)

Cohort, aluminum smelter workers 4,316 (all men) Duration: minimum 90 days; began work after 1962

Follow

- up: through 2002, mean 16 years (maximum 20) Smoking information from company records if employed before 1995 and study interview s if employed after 1994 Exposure: Job / task exposure matrix using TWA B[a]P
measures (n=655), 1977

– 2004 (79% from 1990

– 2004) RR 1.2 (0.7, 2.3) [19 cases in exposed; 20 in unexposed] Lung cancer risk by cumulative B[a]P exposure
B[a]P μg/m 3

- years n

c ases RR (95% CI) a

0 20 1.0 (referent)

> 0 to 0.41 6 0.7 (0.3, 1.8)

0.41 to 10.9 6 1.4 (0.6, 3.5)

> 10.9 7 1.7 (0.7, 4.2)

a Poisson regression, adjusting for smoking; trend p = 0.22

Proxy Measure

Olsson et al., 2010 (Denmark, Norway, Finland, Israel)

Nested case

- control, asphalt workers 433 lung cancer cases (65% participation); 1,253 controls (58% participation), matched by year of birth, country (all men) Duration: minimum ≥ 2 seasons, median 8 seasons; began

work 1913

– 1999

Follow

- up: 1980 to 2002

– 2005 (varied by country) Smoking information from interviews Exposure: compilation of coal tar exposure measures, production characteristics, and repeat measures in asphalt industry in each country used to develop exposure matri x

Related references: Burstyn et al., 2000; Boffetta et al., 2003a Lung cancer risk by cumulative coal tar exposure a

Coal tar

unit

- years a n

cases RR (95% CI)

0.39

– 4.29 43 1.31 (0.87, 2.0)

4.30

– 9.42 32 0.98 (0.62, 1.6)

9.43

– 16.88 30 0.97 (0.61, 1.6)

16.89

– 196.48 54 1.60 (1.09, 2.4)

a Adjusting for set, age, country, tobacco pack

- years

b trend p = 0.07

Costantino et al., 1995 (United States

– Pennsylvania)

Cohort, coke oven workers 5,321 and 10,497 unexposed controls (non

- oven steel workers; matched by age, race, date of first employment) (all men) Duration: data not reported; worked in 1953

Follow

- up through 1982 (length data not reported) Exposure: average daily exposure coal tar pitch volatiles: 3.15 mg/m 3 top

- side full

- time jobs, 0.88 mg/m 3 side jobs; used to calculate weighted cumulative exposure index

Related reference: Mazumdar et al., 1991 (exposure data) SMR 1.95 (1.59, 2.33) [255 cases]
Lung cancer risk by cumulative exposure
Coal tar pitch volatiles (mg/m 3

- months) n

cases RR (95% CI) a

0 203 1.0 (referent)

1

– 49 34 1.2 (0.85, 1.8)

50

– 199 43 1.6 (1.1, 2.3)

200

– 349 59 2.0 (1.5, 2.8)

350

– 499 39 2.0 (1.6, 3.2)

500

– 649 27 2.7 (2.0, 4.6)

≥ 650 56 3.1 (2.4, 4.6)

a adjusted for age, race, coke plant, period of follow

- up; trend p < 0.001

Limited Exposure Information

Liu et al., 1997 (China)

Cohort, various carbon plants and aluminum smelter workers 6,635 (all men) Duration: minimum 15 years; began work before 1971

Follow

- up: through 1985 (mean ~ 14 years) Smoking information from questionnaire SMR 2.2 (1.1, 2.8) [50 cases]
Lung cancer risk by exposure category

Exposure
Category Mean B[a]P

μg/m 3 n

cases SMR (95% CI) a

None

-- 13 1.49 (0.83, 2.5)

Low

-- 6 1.19 (0.48, 2.5)Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Exposure: Area samples from one carbon plant, 1986

- 1987 Moderate 0.30 5 1.52 (0.55, 3.4)
High 1.19 26 4.30 (2.9, 6.2)

a Calculated by EPA from data in paper

Berger et al., 1992 (Germany)

Cohort, coke oven workers 789 (all men) Duration: minimum 10 years (mean 27 years); began work

1900 to 1989

Follow

- up through 1989 (length data not reported) Smoking information from plant records and interviews Exposure: mean B[a]P: 28 (range 0.9

– 89) μg/m 3

Related reference: Manz et al., 1983 (exposure information) SMR 2.88 (2.28

– 3.59) [78 cases]

Hansen et al., 1991; 1989 (Denmark)

Cohort, asphalt workers 679 workers (applicators) (all men)
Duration: data not reported; employed 1959 to 1980

Follow

- up to 1986 (mean ~ 11 years) Smoking information from 1982 surveys of industry and general population Exposure: asphalt fume condensate, 35 personal samples during flooring: median 19.7 (range 0.5

– 260) mg/m 3 SMR 2.90 (1.88, 4.3) [25 cases] (ages 40 to 89) SMR 2.46 (1.59, 3.6) [25 cases] (with smoking adjustment)

Gustavsson et al., 1990 (Sweden)

Cohort, gas production (coke oven) workers 295 (all men)
Duration: minimum 1 year, median 15 years; employed 1965

to 1972 Follow

- up 1966 to 1986 (mortality); 1966 to 1983 (incidence; mean ~ 15 years) Smoking information from interviews with older workers Exposure: area sampling

- top of ovens. B[a]P

- 1964 mean 4.3 (range 0.007 to 33); 1965 mean 0.52, (0.021 to 1.29) μg/m 3 SMR 0.82 (0.22, 2.1) [4 cases] (referent group = employed men) SIR 1.35 (0.36, 3.5)[4 cases]

Moulin et al., 1989 (France)

Cohort and nested case

- control, two carbon electrode plants 1,302 in Plant A (all men), employed in 1975; follow

- up 1975

– 1985 (incidence); smoking information from plant records

1,115 in Plant B (all men); employed in 1957; follow

- up 1957

– 1984 (mortality) Duration of employment and follow

- up: data not reported Exposure: B[a]P

- 19 area samples and 16 personal samples in Plant A (personal sample mean 2.7; range 0.59

– 6.2 μg/m 3 );

10 area s amples and 7 personal samples in Plant B; personal sample mean 0.17, range 0.02

– 0.57 μg/m 3 Plant A: SMR 0.79 (0.32, 1.6) [7 cases] Plant B: SMR 1.18 (0.63, 2.0) [13 cases]

Internal Comparison (case

- control), ≥ 1 year duration: Plant A: OR 3.42 (0.3 5, 33.7) [7cases, 21 controls] Plant B: OR 0.49 (0.12. 2.0) [13 cases, 33 controls]

Hammond et al., 1976 (United States)

Cohort, asphalt

– roofers 5,939 (all men) Duration: minimum 9 years, began before 1960

Follow

- up: through 1971 Exposure: 52 personal samples (masks with filters) during specific jobs and tasks. Mean B[a]P 16.7 μg per 7

- hour day SMR 1.6 (1.3, 1.9) a [99 cases] (≥ 20 years since joining union)

a confidence intervals calculated by EPA from data in paperToxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 14 . Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation to bladder cancer risk

Reference, design Results

Tier 1 Studies

Burstyn et al. (2010) (Denmark, Norway, Finland, Israel)

Cohort, asphalt workers 7,298 all men) Duration: minimum ≥ 2 seasons, median 8 seasons; began

work 1913

– 1999

Follow

- up: began around 1960, ended around 2000 (years varied by country); median 21 years Smoking information not collected Exposure: compilation of B[a]P mea sures, production characteristics, and repeat measures in asphalt industry in each country used to develop exposure matrix

Related references: Burstyn et al., 2000; Boffetta et al.,

2003a 48 incident bladder cancer cases (39 cases in analyses with 15

year lag) Bladder cancer risk by cumulative B[a]P exposure a

B[a]P μg/m 3

- years a n

cases RR (95% CI)

(no lag) b RR (95% CI)

(15 year lag) c

0 to 0.253 12 1.0 (referent) 1.0 (referent)

0.253 to 0.895 12 0.69 (0.29, 1.6) 1.1 (0.44, 2.9)

0.895 to 1.665 12 1.21 (0.45, 3.3) 1.7 (0.62, 4.5)

≥ 1.665 12 0.84 (0.24, 2.9) 1.1 (0.30, 4.0)

a Adjusting for age, calendar period, total duration of employment, country; b trend p = 0.9; c trend p = 0.63 Stronger pattern seen with average exposure in 15

- year lag (RR

1.5, 2.7, 1.9 in second through fourth quartile; trend p = 0.15)

Gibbs et al. (2007a,b,c) ( Quebec, Canada)

Cohort, aluminum smelter workers, 7 plants 16,431 (15,703 men; 728 women) Duration: minimum 1 year, began work 1966

– 1990

Follow

- up: through 1999 (mean ~ 30 years) Smoking information collected from medical records Exposure: Job exposure matrix using ~ 5,000 personal B[a]P

measures from the 1970s to 1999

Related references: Lavoué et al., 2007 (exposure data);
Armstrong et al., 1994 ; Gibbs et al. 1979; 1985 Hired before 1950: SMR 2.24 (1.77, 2.79) [78 cases] Bladder cancer risk by cumulative B[a]P exposure
B[a]P μg/m 3

- years a n

cases SMR (95% CI) Smoking

- adjusted

RR b

0 3 0.73 (0.15, 2.1) 1.0 (referent)

10 14 0.93 (0.45, 1.4) 1.11

30 3 1.37 (0.28, 4.0) 1.97

60 1 0.35 (0.9, 1.9) 0.49

120 15 4.2 (2.4, 6.9) 8.49

240 30 6.4 (4.3, 9.2)

480 12 23.9 (12.2, 41.7)

a Category midpoint

b Confidence intervals not reported; highest category is ≥ 80 μg/m 3

- years (n observed = 57).
Mortality risk reduced in cohort hired in 1950

– 1959, SMR=1.23. Similar patterns seen in analysis of bladder cancer incidence.

Tier 2 Studies

Friesen et al., 2009 (Australia)

See Table 1

- Y for study details RR 0.6 (0.2, 2.0) [5 cases in exposed; 8 in unexposed] Bladder cancer risk by cumulative B[a]P exposure
B[a]P μg/m 3

- years n

cases RR (95% CI) a

0 8 1.0 (referent)

> 0 to 0.41 1 0.2 (0.03, 1.9)

0.41 to 10.9 2 0.7 (0.2, 3.7)

> 10.9 2 1.2 (0.2, 5.6)

a Poisson regression, adjusting for smoking category; trend p =

0.22

Spinelli et al., 2006 ( British Columbia, Canada)

See Table 1

- Y for study details SMR 1.39 (0.72, 2.43) [12 cases]

Costantino et al., 1995 (United States

– Pennsylvania)

See Table 1

- Y for study details SMR 1.14 (1.59, 2.33) [16 cases]Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Hammond et al., 1976 (United States)

See Table 1

- Y for study details SMR 1.7 (0.94, 2.8) a [13cases] (≥ 20 years since joining union)

a confidence intervals calculated by EPA from data in paper

Moulin et al., 1989 (France)

See Table 1

- Y for study details Plant A: [0 observed cases; expected < 1.0] Plant B: SMR 1.94 (0.40, 5.0) [3 cases]

Gustavsson et al., 1990 (Sweden)

See Table 1

- Y for study details SMR 2.85 (0.30, 10.3) [2 cases] (referent group = employed men)

Evidence in Animals 1

Oral Exposure 2

Evidence of tumorigenicity following oral exposure to benzo[a]pyrene has been 3

demonstrated in rats and mice. Oral exposure to benzo[a]pyrene has resulted in an increased 4

incidence of tumors in the alimentary tract in male and female rats (Kroese et al., 2001 ; Brune et al., 5

1981) and female mice ( Beland and Culp, 1998; Culp et al., 1998), and liver carcinomas in male and 6

female rats

, and kidney adenomas in male rats (Kroese et al., 2001). 7

Forestomach tumors have been observed in several lifetime cancer bioassays in rats and 8

mice following both gavage and dietary exposure to benzo[a]pyrene at doses ranging from 0.016 9
mg/kg

- day in Sprague

- Dawley rats to 3.3 and 10 mg/kg

- day in B6C3F 1 mice and Wistar rat s, 10

respectively (Kroese et al., 2001; Beland and Culp, 1998; Culp et al., 1998; Brune et al., 1981). In 11

addition, multiple less

- than

- lifetime oral exposure cancer bioassays in mice provide supporting 12

evidence that oral exposure to benzo[a]pyrene is associ ated with an increased incidence of 13
forestomach tumors (Weyand et al., 1995; Benjamin et al., 1988; Robinson et al., 1987; El Bayoumy, 14
1985; Triolo et al., 1977; Wattenberg, 1974; Field and Roe, 1970; Roe et al., 1970; Biancifiori et al., 15

1967; Chouroulink ov et al., 1967; Fedorenko and Yansheva, 1967; Neal and Rigdon, 1967; 16
Berenblum and Haran, 1955). 17

Elsewhere in the alimentary tract, dose

- related increases of benign and malignant tumors 18

were observed. In rats, oral cavity tumors were induced in both se xes and adenocarcinomas of the 19

jejunum were induced in males (Kroese et al., 2001). In mice, tumors were induced in the tongue, 20
esophagus, and larynx of females (males were not tested) (Beland and Culp, 1998; Culp et al., 1998). 21

Chronic oral exposure to b enzo[a]pyrene resulted in a dose

- dependent increased incidence 22

of liver carcinomas in both sexes of Wistar rats (Table 1

- 15) , with the first liver tumors detected in 23
week 35 in high

- dose male rats ; liver tumors were described as complex, with a considerabl e 24

proportion (59/150 tumors) metastasizing to the lungs (Kroese et al., 2001). Hepatocellular 25

tumors were not observed in mice ( Beland and Culp

, 1998 ; Culp et al.

, 1998). 26

A statistically significantly increased incidence of kidney tumors (cortical adenoma s) was 27
observed in male Wistar rats following chronic gavage expo sure (Kroese et al., 2001) ( Table 1

- 15 ). 28

The kidney tumors were observed at the mid

- and high

- dose groups. Kidney tumors were not 29

observed at a lower dose in another study (Brune et al., 19 81). 30
Lung tumors were also obs erved following oral exposure in female AJ mice (Weyand et al., 31

1995 ). 32Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 15 . Tumors observed in chronic oral animal bioassays. 1

Study design and reference Results

Kroese et al. (2001) Wistar (Riv:TOX) rats (52/sex/dose group)

0, 3, 10, or 30 mg/kg

- day by gavage

5 days/week

104 weeks forestomach

– Incidences: M: 0/52; 7/52 * ; 18/52*; 17/52* (papilloma) M: 0/52; 1/52; 25/52*; 35/52* (squamous cell carcinoma) F: 1/52; 3/51; 20/51*; 25/52* (papilloma) F: 0/52; 3/51; 10/51*; 25/52* (squamous cell carcinoma)
oral cavity

– Incidences: M: 0/24; 0/24; 2/37; 10/38 (papilloma) M: 1/24; 0/24; 5/37; 11/38 (squamous cell carcinoma) F: 0/19; 0/21; 0/9; 9/31(papilloma) F: 1/19; 0/21; 0/ 9; 9/31 (squamous cell carcinoma)
jejunum

– (adenocarcinomas)

Incidences: M: 0/51; 0/50; 1/51; 8/49 F: 0/50; 0/48; 0/50; 2/51

duodenum

– (adenocarcinomas)

Incidences: M: 0/51; 0/50; 0/51; 1/49 F: 0/49; 0/48; 0/50; 2/51

liver

– (adenomas and carcinomas)

Incidences: M: 0/52; 3/52; 15/52 * ; 4/52 (adenoma) M: 0/52; 1/52; 23/52 * ; 45/52 * (carcinoma) F: 0/52; 2/52; 7/52 * ; 1/52 (adenoma) F: 0/52; 0/52; 32/52 * ; 50/52 * (carcinoma)
kidney

– (cortical adenoma)

Incidences: M: 0/52; 0/52; 7/52; 8/52

F: increase not observed
auditory canal b (Zymbal’s gland)

– (carcinomas)

Incidences: M: 0/1; 0/0; 2/7; 19/33* F: 0/0; 0/1; 0/0; 13/20*
Beland and Culp (1998); Culp et al. (1998)

B6C3F 1 mice: female (48/dose group)

0, 5, 25, or 100 ppm (average daily

doses a : 0, 0.7, 3.3, a nd 16.5 mg/kg

- day)

in the diet
2 years forestomach

– (papillomas and squamous cell carcinomas)

Incidences: 1/48; 3/47; 36/46*; 46/47*
esophagus

– (papillomas and carcinomas) Incidences: 0/48; 0/48; 2/45; 27/46*

tongue

– (papillomas and carcinomas) Incidences: 0/49; 0/48; 2/46; 23/46*
larynx

– (papillomas and carcinomas) Incidences: 0/35; 0/35; 3/34; 5/38Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Study design and reference Results

Brune et al. (1981)
Sprague

- Dawley rats: male and female (32/sex/dose) Gavage: 0, 6, 18, 39 mg/kg

- yr (0, 0.016, 0.049, 0.107 mg/kg

- day)

Diet: 0, 6 , 39 mg/kg

- yr (0, 0.016, 0.107 mg/kg

- day)

treated until moribund or dead forestomach

– (papillomas and carcinomas b )

– gavage
Incidences: 3/64; 12/64*; 26/64*; 14/64*

forestomach

– (papillomas)

– diet

Incidences: 2/64; 1/64; 9/64*
larynx and esophagus

– (papillomas)

– gavage Incidences: 3/64; 1/64; 0/64; 0/64
larynx and esophagus

– (papillomas)

– diet Incidences: 1/64; 2/64; 1/64 * indicates statistical significance as identified in study 1

a Based on the assumption that daily benzo[a]pyrene intake at 5 ppm was one

- fifth of the 25

- ppm intake 2 (about 21 μg/day) and using time

- weighted average (TWA) body weights of 0.032 kg for the control, 5

- and 3

25

- ppm groups and 0.026 kg for the 100

- ppm group 4

b Auditory canal tissue was not histologically examined in the lower dose groups and the controls 5

c Two malignant forestomach tumors were observed (one each in the mid

- and high

- dose groups) 6

Inhalation Exposure 7

Chronic inhalation exposure to benzo[a]pyren e resulted in the development of tumors in 8

the respiratory tract and pharynx in Syrian golden hamsters (Table 1

- 16) . A dose

- dependent 9

increased incidence of tumors in the upper respiratory tract, including the larynx and trachea, were 10

reported by Thyssen et al. (1981) at ≥10 mg/kg

- day. In addition, a decrease in tumor latency was 11

observed in the larynx and trachea, and nasal cavity tumors were observed at the mid

- and high

12

dose but the incidences were not dose

- dependently increased. A dose

- related increa se in tumors in 13

the upper digestive tract (pharynx and esophagus) was also reported. In addition, a single 14

forestomach tumor was observed at both the mid

- and high

- doses, and forestomach tumors were 15
not observed in control animals. The study authors pres umed that the pharyngeal and esophageal 16

tumors were a consequence of mucociliary particle clearance. 17

Under contract to the U.S. EPA, Clement International Corporation (1990) obtained the 18

individual animal data (including individual animal pathology reports , time

- to

- death data, and 19
exposure chamber monitoring data) collected by Thyssen et al. (1981). A re

- analysis of the 20

individual animal pathology reports from the original study supports the dose

- dependent increased 21

incidence of tumors in the larynx and ph arynx (Clement International Corporation, 1990; EPA, 22
1990). The exposure measurements and individual animal data from Thyssen et al. (1981) were 23

used to calculate average continuous lifetime exposures for each individual hamster. Group 24

averages of indivi dual average continuous lifetime exposure concentrations were 0, 0.25, 1.01, and 25

4.29 mg/m 3 for the control through high

- exposure groups
, as described in Clement International 26

Corporation (1990)

. 27Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 16 . T umors observed in chronic inhalation animal bioassays. 1

Study design and reference Results

Thyssen et al. (1981)
Syrian golden hamsters: male (20

– 30 animals/group)
Target exposure concentrations: 0, 2, 10, or 50 mg/m 3

Average exposure concentrations b : 0, 0.25, 1.01, and

4.29 mg/m 3

Inhalation: for 3

– 4.5 hours/5

- 7 days per week until

hamsters died or became moribund larynx

– Incidences: 0/27; 0/27; 8/26; 13/25
tumor latency a : 107and 67.6 weeks

trachea

– Incidences: 0/27; 0/27; 1/26; 3/25
tumor latency: 115 and 63 weeks nasal cavity

– incidences: 0/27; 0/27; 3/26; 1/25
tumor latency: 116 and 79 weeks

Revised tumor incidence data c

larynx

– Incidences: 0/27; 0/27; 11/26; 12/34
pharynx

– Incidences: 0/27; 0/27; 9/26; 18/34
larynx and pharynx (combined) d

– Incidences: 0/27; 0/27; 16/26; 18/34
pharynx

– Incidences: 0/27; 0/27; 6/26; 14/25
esophagus

– (papillomas and squamous cell carcinomas) Incidences: 0/27; 0/27; 0/27; 2/25

Forestomach

- (papillomas and squamous cell carcinomas) Incidences: 0/27; 0/27; 1/26; 1/25

a tumor latency provided for 10 and 50 mg/m3 dose groups 2

b Duration adjusted inhalation concentrations calculated from exposure chamber monitoring data and 3 exposure treatment times obtained by Clement International Corporation (1990). Daily exposure times: 4.5 4 hours/day, 5 days/week on weeks 1

– 12; 3 hours/day, 5 days/week on weeks 13

– 29; 3.7 hours/day, 5

5 days/week on week 30; 3 hours/day, 5 days/week on weeks 31

– 41; and 3 hours/day, 7 days/week for 6 reminder of the experiment. 7

c Revised tumor incidence data based on original study pathology reports obtained by Cle ment International 8 Corporation (1990). 9

d Nasal, forestomach, esophageal, and tracheal tumors occurred in hamsters that also had tumors in the larynx 10 or pharynx, except for two animals in the mid

- dose group that displayed nasal tumors (one malignant and 11

one benign) without displaying tumors in the pharynx or larynx. 12

Dermal Exposure 13

Repeated application of benzo[a]pyene to skin (in the absence of exogenous promoters) has 14
been demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs . These s tudies have 15
been reviewed by multiple national and international health agencies (IARC, 2010, 1983, 1973; 16

WHO, 1998; ATSDR, 1995). Th e analysis in this document focuses on chronic carcinogenicity 17
bioassays in several strains of mice following repeated der mal exposure to benzo[a]pyrene for the 18

animals’ lifetime (Table 1

- 17) . These studies involved 2

- or 3

- times/week exposure protocols, at 19

least two exposure levels plus controls, and histopathological examinations of the skin and other 20

tissues (Sivak et al. , 1997; Grimmer et al., 1984, 1983; Habs et al., 1984, 1980; Schmähl et al., 1977; 21
Schmidt et al., 1973; Roe et al., 1970; Poel, 1960, 1959). 22

Mice have been the most extensively studied species in dermal carcinogenesis studies of 23

benzo[a]pyrene because o f evidence that they may be more sensitive than other animal species; 24

however, comprehensive comparison s of species differences in sensitivity to lifetime dermal 25Toxicological Review of Benzo[a]pyrene

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- 57 DRAFT

— DO NOT CITE OR QUOTE exposure are not available. S ystemic tumors in benzo[a]pyrene

- treated mice were not observed in 1
lifetime dermal bioassays in which macroscopic examination of internal organs was included 2
(Higginbotham et al., 1993; Habs et al., 1980; Schmahl et al., 1977; Schmidt et al., 1973; Roe et al., 3

1970; Poel, 1959). 4

Table 1

- 17 . Tumor observations in dermal animal bioassays. 5

Study design and reference Results

Poel (1959)

C57L mice: male ( 13

– 56/dose)
0, 0.15, 0.38, 0.75, 3.8, 19, 94, 188, 376, or 752 μg

Dermal

- 3 times/week for up to 103 weeks or

until the appearance of a tumor by gross

examination skin tumors

– (gross skin tumors and epidermoid carcinoma)

– dose

- dependent decreased time of tumor appearance

Incidences: Gross skin tumors: 0/33; 5/55; 11/55; 7/56; 41/49; 38/38; 35/35; 12/14; 14/14; 13/13

Ep idermoid carcinoma: 0/33; 0/55; 2/55; 4/56; 32/49; 37/38; 35/35; 10/14; 12/14; 13/13

Poel (1960) SWR, C3HeB, or A/He mice: male (14

- 25/dose)

0, 0.15, 0.38, 0.75, 3.8, 19.0, 94.0, or 470 μg
Dermal

- 3 times/week until mice died or a skin

tumor was observed skin tumors and dose

- dependent decreased time of first

tumor appearance

Incidences: SWR: 0/20; 0/25; 2/22; 15/18; 12/17; 16/16; 16/17; 14/14 C3HeB: 0/17; 0/19; 3/17; 4/17; 11/18;/ 17/17; 18/18; 17/17 A/He mice: 0/17; 0/18; 0/19; 0/17; 0/17; 21/23; 11/ 16; 17/17

Roe et al. (1970) Swiss mice (50/dose)
0, vehicle, 0.1, 0.3, 1, 3, or 9 μg
Dermal

- 3 times/week for up to 93 weeks skin tumors

– malignant skin tumors were observed in 4/41 and 31/40 mice in the two high dose groups, respectively

Incidences: 0/43; 0/47; 1/42; 0/42; 1/43; 8/41; 34/46

Schmidt et al., 1973 NMRI mice: female (100/group) Swiss mice: female (100/group)

0, 0.05, 0.2, 0.8, or 2 μg
Dermal

- 2/week until spontaneous death

occurred or until an advanced carcinoma was

observed skin tumors

– (carcinomas)

Incidences:

NMRI: 2/100 at 2 μg (papillomas); 2/100 at 0.8 μg and 30/100 at 2 μg (carcinomas)

Swiss: 3/80 at 2 μg (papillomas); 5/80 at 0.8 μg and 45/80 at 2 μg (carcinomas)

Schmähl et al., 1977

NMRI mice: female (100/group)

0, 1, 1.7, or 3 μg
Dermal

- 2 times/week skin tumors

– (papillomas and carcinomas)

Incidences: 0/81; 1/77; 0/88; 2/81 (papillomas) 0/81; 10/77; 25/88; 43/81 (carcinomas)

Habs et al., 1980 NMRI mice: female (40/group)

0, 1.7, 2.8, or 4.6 μg

Der mal

- 2 times/week until natural death or gross
observation of infiltrative tumor growth skin tumors and dose

- dependent increase in age

standardized tumor incidence

Incidences: 0/35; 8/34; 24/35; 22/36

Age

- standarized tumor incidence:

0, 24.8, 89.3, 91.7%

Grimmer et al., 1984, 1983 CFLP mice: female (65

– 80/group) 0, 3.9, 7.7, and 15.4 μg (1983 study) 0, 3.4, 6.7, and 13.5 μg (1984 study) skin tumors

– (papillomas and carcinomas)

– with a decrease in tumor latency

I ncidences: 1983: 0/80; 7/65; 5/64; 2/64 (papillomas)Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Study design and reference Results

Dermal

- 2 times/wk for 104 weeks 0/80; 15/65; 34/64; 54/64 (carcinomas) 1984: 0/65; 6/64; 8/65; 4/65 (papillomas) 0/65; 37/64; 45/65; 53/64 (carcinomas)

Habs et al., 1984

NMRI mice: female (20/group)

0, 2, or 4 μg
Dermal

- 2 times/week for life skin tumors

– (papillomas and carcinomas)

– with a decrease

in mean survival time

Incidences: 0/20; 2/20; 0/20 (papillomas) 0/20; 7/20; 17/20 (carcinomas)
Higginbotham et al., 1993

Ah

- receptor

- responsive Swiss mice: femal e (23

- 23/group)
0, 0.25, 1 or 2 μg

Dermal

- 2 times/wk for 40 weeks Skin tumors were not observed

Sivak et al., 1997 C3H/HeJ mice: male (30/group)
0, 0.05, 0.5, or 5 μg

Dermal

- 2 times/wk for 104 weeks skin tumors

– (papillomas and carcinomas)

Incidences: 0/30; 0/30; 5/30 (2 papillomas, 3 carcinomas); 27/30 (1 papilloma, 28 carcinomas)

Mode of action analysis

— Carcinogenicity 1

The carcinogenicity of benzo[a]pyrene, the most studied PAH, is well documented ( IARC, 2

2010; Xu et al., 2009; Jiang et al ., 2007, 2005; Xue and Warshawsky, 2005; Ramesh et al., 2004; 3
Bostrom et al., 2002; Penning et al., 1999; WHO, 1998; Harvey, 1996; ATSDR, 1995; Cavalieri and 4
Rogan, 1995; U.S. EPA, 1991b). The primary mode of action by which benzo[a]pyrene induces 5

carcino genicity is via a mutagenic mode of action. This mode of action is presumed to apply to all 6

tumor types and is relevant for all routes of exposure. The general sequence of key events 7
associated with a mutagenic mode of action for benzo[a]pyrene are: (1) bioactivation of 8

benzo[a]pyrene to DNA

- reactive metabolites via three possible metabolic activation pathways: a 9

diol epoxide pathway, a radical cation pathway, and an o

- quinone and ROS pathway (2) direct DNA 10
damage by reactive metabolites, including the f ormation of DNA adducts and ROS

- mediated 11
damage; (3) formation and fixation of DNA mutations, particularly in tumor suppressor genes or 12

oncogenes associated with tumor initiation; and (4) clonal expansion of mutated cells during the 13

promotion and progressi on phases of cancer development. These events are depicted in Figure 1

- 7

. 14
Also included in the figure are other processes that may contribute to the carcinogenicity of 15
benzo[a]pyrene via the promotion and progression phases of cancer development, includ ing 16

inflammation, cytotoxicity, and sustained regenerative cell proliferation. The available human, 17
animal, and in vitro evidence supports a mutagenic mode of action as the primary mode by which 18

benzo[a]pyrene induces carcinogenesis. 19

20Toxicological Review of Benzo[a]pyrene

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Figure 1

- 7 . Proposed metabolic activation pathways and key events in the 2

carc inogenic mode of action for benzo[a]pyrene 3

Data in Support of the MOA 4

Summary of Metabolic Activation Pathways 5

Diol epoxide pathway 6 Benzo[a]pyrene diol epoxide metabolites, believed to be the most potent DNA

- binding 7
metabolites of benzo[a]pyrene, are forme d through a series of Phase I metabolic reactions (see 8
Appendix B of the Supplemental Information) . The initial metabolism is carried out primarily by 9

the inducible activities of CYP enzymes including CYP1A1, CYP1B1, and CYP1A2. Further 10

metabolism by epo xide hydrolase and the mixed function oxidase system yields (+)

- anti

11
benzo[a]pyrene

- 7,8

- diol

- 9,10

- epoxide ( BPDE), one of the most potent DNA

- binding metabolites of 12

benzo[a]pyrene. Benzo[a]pyrene diol epoxide metabolites interact preferentially with the exocyclic 13

amino groups of deoxyguanine and deoxyadenine (Geacintov et al., 1997; Jerina et al., 1991). 14

Adducts may give rise to mutations unless these adducts are removed by DNA repair processes 15
prior to replication. The stereochemical nature of the diol epoxide metabolite (i.e., anti

- versus syn

16

diol epoxides) affects the number and type of adducts and mutation that occurs (Geacintov et al., 17 benzo[a]

pyrene diol epoxide

radical cation

o

- quinone and

ROS DNA Adducts

DNA Adducts

DNA Adducts

and oxidative base damage Mutation (Transversion)

K

- Ras, H

- Ras and
p53 targets

Mutation (Depurination):

H

- ras target

Mutation (Depurination):
Oxidative damage

and strand scission Proliferation of

initiated cells Neoplasm

Inflammatory

response

Cytotoxicity HO

OH O

+ Exposure Metabolism

O

O Initiation Promotion Progression Key events in the mode of action for benzo[a]pyrene carcinogenicity

Binding to AhR

Upregulation of genes

related to
biotransformation, growth, and

differentiationToxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE 1997). Transversion mutations (e.g., GC → TA or AT → TA) a re the most common type of mutation 1

found in mammalian cell s following diol epoxide exposure (Bostrom et al., 2002). 2

Radical cation pathway 3
Radical cation formation involves a one

- electron oxidation by CYP or peroxidase enzymes 4
(i.e., horseradish peroxidase, prostaglandin H synthetase) that produce electrophilic r adical cation 5
intermediates (Cavalieri and Rogan, 1995, 1992). Radical cations can be further metabolized to 6
phenols and quinones (Cavalieri et al., 1988d, e), or they can form unstable adducts with DNA that 7
ultimately result in depurination (Cavalieri et al., 2005, 1993; Rogan et al., 1993). The predominant 8

depurinating adducts occur at the N

- 3 and N

- 7 positions of adenine and the C

- 8 and N

- 7 positions of 9
guanine (Cavalieri and Rogan, 1995). 10

o

- Quinone/ROS pathway 11
The o

- quinone metabolites of PAHs are for med by enzymatic dehydrogenation of 12

dihydrodiols (Bolton et al., 2000; Penning et al., 1999; Harvey, 1996; ATSDR, 1995) (see Appendix B 13
of the Supplemental Information) . DHH (dihydrodiol dehydrogenase) enzymes are members of the 14

α

- keto reductase gene supe rfamily. o

- Quinone metabolites are potent cytotoxins, are weakly 15
mutagenic, and are capable of producing a broad spectrum of DNA damage. These metabolites can 16

interact directly with DNA as well as result in the production of ROS (i.e., hydroxyl and super oxide 17
radicals) that may produce further cytotoxicity and DNA damage. The o

- quinone/ROS pathway 18

also can produce depurinated DNA adducts from benzo[a]pyrene metabolites (Jiang et al., 2007, 19

2005; McCoull et al., 1999). In this pathway, and in the presenc e of NAD(P) + , AKR oxidizes 20

benzo[a]pyrene

- 7,8

- diol to a ketol, which subsequently forms benzo[a]pyrene

- 7

- 8

- dione. This and 21

other PAH o

- quinones react with DNA to form unstable, depurinating DNA adducts. In the presence 22

of cellular reducing equivalents, o

- quinones can also activate redox cycles, which produce ROS 23
(Penning et al., 1996). DNA damage in in vitro systems following exposure to benzo[a]pyrene

- 7,8

- 24

dione or other o

- quinone PAH derivatives occurs through the AKR pathway and can involve the 25

formati on of stable DNA adducts (Balu et al., 2004), N

- 7 depurinated DNA adducts (McCoull et al., 26

1999), DNA damage from ROS (8

- oxo

- dG) (Park et al., 2006), and strand scission (Flowers et al., 27
1997, 1996). 28

Summary of Genotoxicity and Mutagenicity 29

The ability o f metabolites of benzo[a]pyrene to cause mutations and other forms of DNA 30

damage in both in vivo and in vitro studies is well documented (see genotoxicity tables in Appendix 31

B in Supplemental Information ). With metabolic activation (e.g., the inclusion o f S9), 32

benzo[a]pyrene is consistently mutagenic in the prokaryotic Salmonella/Ames and Escherichia coli 33

assays . In mammalian in vitro studies, benzo[a]pyrene is consistently mutagenic, clastogenic, and 34

induces cell transformation both with and without met abolic activation. Cytogenetic damage in the 35

form of chromosomal aberrations ( CA )

, micronuclei ( MN )

, sister chromatid exchanges ( SCE ) , and 36

aneuploidy are commonplace following benzo[a]pyrene exposure as are DNA adduct formation, 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE single strand breaks ( SSB ) , and induction of DNA repair and unscheduled DNA synthesis. I n vitro 1

mammalian cell assays have been conducted in various test systems, including human cell lines. 2
In the majority of in vivo studies, benzo[a]pyrene has tested positive in multiple species and 3

strains and under various test conditions for cell transformation, CAs, DNA adducts, DNA strand 4
breaks, MN formation, germline mutations, somatic mutations (H

- ras , K

- ras
, p53

, lacZ

, hprt ), and 5

SCEs. H uman studies are available following exposures to P AH mixture s through cigarette smoke 6

or occupational exposure in which benzo[a]pyrene

- specific DNA adducts have been detected, and it 7
has been demonstrated qualitatively that benzo[a]pyrene metabolites damage DNA in exposed 8

humans. 9

Experimental Support for the Hypothesized Mode of Action 10

EPA’s Cancer G uidelines (Section 2.4; 2005) describe a procedure for evaluating mode of 11

action data for cancer. A framework for analysis of mode of action information is provided and 12

followed below. 13

Strength, consistency, and specificity of association 14 Strong evidence links the benzo[a]pyrene diol epoxide metabolic activation pathway with 15
key mutational events in genes that are associated with tumor initiation (mutations in the p53 16

tumor suppressor gene and H

- ras or K

- ras o ncogenes) (Table 1

- 18) . Results in support of a 17
mutagenic MOA via benzo[a]pyrene diol epoxide include observations of frequent G → T 18

transversion mutations in p53 and ras genes in lung tumors of human cancer patients exposed to 19

coal smoke (Keohavong et al., 2003; DeMarini et al., 2001) or tobacco smoke (Pfeifer and Hainaut, 20
2003; Pfeifer et al., 2002; Hainaut and Pfeifer, 2001, Bennett et al., 1999). These results are 21

consistent with evidence that benzo[a]pyrene diol epoxide is reactive with guanine bases i n DNA 22
(Koreeda et al., 1978; Jeffrey et al., 1976) ; that G → T transversions, displaying strand bias, are the 23

predominant type of mutations caused by benzo[a]pyrene in several biological systems (Liu et al., 24

2005; Hainaut and Pfeifer, 2001; Marshall et al., 1984) ; and that sites of DNA adduction at guanine 25

positions in cultured human HeLa or bronchial epithelial cells exposed to benzo[a]pyrene diol 26

epoxide correspond to p53 mutational hotspots observed in human lung cancers (Denissenko et al., 27

1996; Puisieux et al., 1991). In addition, mice exposed to benzo[a]pyrene in the diet (Culp et al., 28

2000) or by i.p. injection (Nesnow et al., 1998a, b, 1996, 1995; Mass et al., 1993) had forestomach 29
or lung tumors, respectively, showing frequent G → T or C transversions in the K

- ras gene. 30

Supporting evidence includes observations that benzo[a]pyrene diol epoxide [specifically (+)

- anti

31

BPDE] is more potent than benzo[a]pyrene itself, benzo[a]pyrene phenols, or benzo[a]pyrene diols 32

in mutagenicity assays in bacterial and i n vitro mammalian systems (Malveille et al., 1977; Newbold 33

and Brookes, 1976) and in producing lung tumors in newborn mice following i .p. administration 34
(Chang et al., 1987; Buening et al., 1978; Kapitulnik et al., 1978). Other supporting evidence 35

include s observations of elevated BPDE

- DNA adduct levels in respiratory tissue of lung cancer 36

patients (Li et al., 2001; Xu et al., 1997) and white blood cells ( WBC ) of groups of coke oven 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE workers and chimney sweeps, occupations with known elevated risks of cance r (Vineis and Perera, 1
2007; Pavanello et al., 1999), and in lung tissue from tobacco smokers with lung cancer (Rojas et al., 2
2004; 1998; Andreassen et al., 2002; Godschalk et al., 2002; Alexandrov et al., 1992). Several 3

epidemiological studies have indica ted that PAH

- exposed individuals who are homozygous for a 4
CYP1A1 polymorphism, which increases the inducibility of this enzyme (thus increasing the 5
capacity to produce benzo[a]pyrene diol epoxide), have increased levels of PAH or BPDE

- DNA 6
adducts (Bartsch et al., 2000; Aklillu et al., 2005; Alexandrov et al., 2002; Perera and Weinstein, 7

2000). 8

Additional supporting evidence of a mutagenic mode of action for benzo[a]pyrene 9
carcinogenicity is the extensive database of in vitro and in vivo studies demonstratin g the 10

genotoxicity and mutagenicity of benzo[a]pyrene following metabolic activation (Table 1

- 18)

. In 11
vitro studies overwhelmingly support the formation of DNA adducts, mutagenesis in bacteria , yeast, 12

and mammalian cells , several measures of cytogenetic d amage (CA , SCE, MN), and DNA damage. In 13

vivo systems in animal models are predominantly positive for somatic mutations following 14

benzo[a]pyrene exposure. 15
Support for the radical cation activation pathway contributing to tumor initiation through 16

mutagenic events includes observations that depurinated DNA adducts (expected products from 17

reactions of benzo[a]pyrene radical cations with DNA) accounted for 74% of identified DNA 18

adducts in mouse skin exposed to benzo[a]pyrene (Rogan et al., 1993) and 9/13 tumors examined 19
from mice exposed to dermal applications of benzo[a]pyrene had H

- ras oncogene mutations 20

attributed to depurinated DNA adducts from benzo[a]pyrene radical cations (Chakravarti et al., 21

1995). 22

Support for the o

- quinone/ROS pathway contributing to tu mor initiation via mutagenic 23
events includes in vitro demonstration that several types of DNA damage can occur from o

24

quinones and ROS (Park et al., 2006a; Balu et al., 2004; McCoull et al., 1999; Flowers et al., 1997, 25

1996). In addition, benzo[a]pyrene

- 7 ,8

- dione can induce mutations in the p53 tumor suppressor 26
gene using an in vitro yeast reporter gene assay (Park et al., 2008; Shen et al., 2006; Yu et al., 2002), 27

and dominant p53 mutations induced by benzo[a]pyrene

- 7,8

- dione in this system corresponded t o 28

p53 mutational hotspots observed in human lung cancer tissue (Park et al., 2008). 29

Dose

- response concordance and temporal relationship 30 In vivo demonstrations showing that benzo[a]pyrene

- induced mutational events in p53 or 31

ras oncogenes precede tumor formation are not available. I n vitro exposure of human p53 knock

- in 32

murine fibroblasts to 1 μM benzo[a]pyrene for 4 to 6 days induced p53 mutations with similar 33

features to those identified in p53 mutations in human lung cancer ; i.e., predominance of G → T 34

transversions with strand bias and mutational hotspots at codo ns 157

- 158 (Liu et al., 2005). 35

Bennett et al. (1999) demonstrated a dose

- response relationship between smoking 36
history/intensity and the types of p53 mutations associat ed with benzo[a]pyrene (G → T 37

transversions) in human lung cancer patients (Table 1

- 18) . In lung tumors of nonsmokers, 10% of 38Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE p53 mutations were G → T transversions, versus 40% in lung tumors from smokers with >60 pack

1

years of exposure. A dose

- response rel a tionship also has been demonstrated between BPDE

- DNA 2
adduct levels in lung tissue and odds ratios for cancer in a case control study of 221 lung cancer 3

cases and 229 healthy controls (Li et al., 2001). 4

In mice, dose

- response and temporal relationships have been described between the 5

formation of BPDE

- DNA adducts and skin and forestomach tumors (Table 1

- 18) . In a study using 6
mice treated dermally with benzo[a]pyrene once or twice per week for up to 15 weeks (10, 25 or 7
50 nmol benzo[a]pyrene per application) , levels of benzo[a]pyrene

- DNA adducts in the skin, lung, 8

and liver increased with increasing time of exposure and increasing dose levels (Talaska et al., 9
1996). Levels at the end of the exposure period were highest in the skin; levels in the lung and liv er 10

at the same time were 10

- and 20

- fold lower, respectively. Levels of benzo[a]pyrene

- DNA adducts 11

in skin and lung increased in an apparent biphasic manner showing a lower linear slope between 12

the two lowest dose levels, compared with the slope from the middle to the highest dose. 13

Another study examined the dose

- response relationship and the time course of 14

benzo[a]pyrene

- induced skin damage (Table 1

- 18) , DNA adduct formation, and tumor formation in 15

female mice. Mice were treated dermally with 0, 16, 32, or 64 μg of benzo[a]pyrene once per week 16
for 29 weeks (Albert et al., 1991). Indices of skin damage and levels of BPDE

- DNA adducts in skin 17

reached plateau levels in exposed groups by 2

– 4 weeks of exposure. With increasing dose level, 18

levels of BPDE

- DNA a dducts (fmol/μg DNA) initially increased in a linear manner and began to 19
plateau at doses ≥32 μg/week. Tumors began appearing after 12

– 14 weeks of exposure for the 20

mid

- and high

- dose groups and at 18 weeks for the low

- dose group. At study termination 21

(35 weeks after start of exposure), the mean number of tumors per mouse was approximately one 22

per mouse in the low

- and mid

- dose groups and eight per mouse in the high

- dose group. The time

23

course data indicate that benzo[a]pyrene

- induced increases in BPDE

- DN A adducts preceded the 24

appearance of skin tumors, consistent with the formation of DNA adducts as a precursor event in 25

benzo[a]pyrene

- induced skin tumors. 26
Culp et al. (1996a) compared dose

- response relationships for BPDE

- DNA adducts and 27

tumors in female B6 C3F 1 mice exposed to benzo[a]pyrene in the diet at 0, 18.5, 90, or 350 μg/day 28

for 28 days (to examine adducts) or 2 years (to examine tumors) (Table 1

- 18) . The benzo[a]pyrene 29

dose

- tumor response data showed a sharp increase in forestomach tumor incidence between the 30
18.5 μg/day group (6% incidence) and the 90 μg/day group (78% incidence). The BPDE

- DNA 31

adduct levels in forestomach showed a relatively linear dose

- response throughout the 32

benzo[a]pyrene dose range tested. The appearance of increased levels o f BPDE

- DNA adducts in the 33
target tissue at 28 days is temporally consistent with the contribution of these adducts to the 34

initiation of forestomach tumors. Furthermore, about 60% of the examined tumors had mutations 35

in the K

- ras oncogene at codons 12 and 13, which were G → T or G → C transversions indicative of 36

BPDE reactions with DNA (Culp et al., 1996a)

. 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Biological plausibility and coherence 1 The evidence for a mutagenic mode of action for benzo[a]pyrene is consistent with the 2

current understanding that mutat ions in p53 and ras oncogenes are associated with increased risk 3

of tumor initiation (Table 1

- 18) . The benzo[a]pyrene database is internally consistent in providing 4

evidence for BPDE

- induced mutations associated with tumor initiation in cancer tissue from 5
humans exposed to complex mixtures containing benzo[a]pyrene (Keohavong et al., 2003; Pfeifer 6

and Hainaut, 2003; Pfeifer et al., 2002; DeMarini et al., 2001; Hainaut and Pfeifer, 2001, Bennett et 7
al., 1999), in animals exposed to benzo[a]pyrene (Culp et a l., 2000; Nesnow et al., 1998a, b, 1996, 8
1995; Mass et al., 1993), and in in vitro systems (Denissenko et al., 1996; Puisieux et al., 1991). 9

Consistent supporting evidence includes: (1) elevated BPDE

- DNA adduct levels in respiratory tissue 10

of lung cancer patients (Li et al., 2001; Xu et al., 1997) or tobacco smokers with lung cancer (Rojas 11

et al., 2004; 1998; Andreassen et al., 2002; Godschalk et al., 2002; Alexandrov et al., 1992); (2) 12

demonstration of dose

- response relationships between G → T transversions in p53 mutations in 13

lung tumors and smoking intensity (Bennett et al.,1999) and between odds ratios for lung cancer 14

and BPDE

- DNA adduct levels in lung tissue (Li et al., 2001); (3) the extensive database of in vitro 15
and in vivo studies demonstrating the g enotoxicity and mutagenicity of benzo[a]pyrene following 16

metabolic activation; and (4) general concordance between temporal and dose

- response 17

relationships for BPDE

- DNA adduct levels and tumor incidence in studies of animals exposed to 18
benzo[a]pyrene (Culp et al., 1996a; Albert et al., 1991). There is also supporting evidence that 19

contributions to tumor initiation through mutagenic events can be made by the radical cation 20

(Chakravarti et al., 1995; Rogan et al., 1993) and o

- quinone/ROS metabolic activation pathways 21

(Park et al., 2008, 2006a; Shen et al., 2006; Balu et al., 2004; Yu et al., 2002; McCoull et al., 1999; 22
Flowers et al., 1997, 1996). 23Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 18 . Experimental support for the postulated key events for mutagenic 1

MOA 2

1. Bioactivation of benzo[a]pyrene to DNA

- reactive metabolites via three possible metabolic activation pathways: a diol epoxide pathway, a radical cation pathway, and an o

- quinone and ROS pathway.

Evidence that benzo[a]pyrene metabolites induc e key events:
Metabolism of benzo[a]pyrene via all three pathways has been demonstrated in multiple in vitro

and in viv o studies in humans and animals (see Metabolic Activation Pathways section)
Multiple in vivo studies in humans and animals have demonstrated distribution of reactive metabolites to target tissues Human evidence that key events are necessary for carcinogenesis:
Humans with CYP polymorphisms or lacking a functional GSTM1 gene form higher levels of benzo[a]pyrene diol epoxides, leading to increased BPDE

- DNA adduct formation and increased risk

of cancer (Vineis et al., 2007; Pavanello et al., 2005; 2004; Alexandrov et al., 2002; Perera and Weinstein, 2000)

2. Direct DNA damage by the re active metabolites, including the formation of DNA adducts and ROS

mediated damage.

Evidence that benzo[a]pyrene metabolites induce key events:
Reactive benzo[a]pyrene metabolites have demonstrated genotoxicity in most in vivo and in vitro systems in whic h they have been tested, including the bacterial mutation assay, transgenic mouse assay, dominant lethal mutations in mice, BPDE

- DNA adduct detection in humans and animals, and DNA damage, chromosomal aberrations, micronucleus formation, and sister chromat id exchange in animals ( Appendix B in Supplemental Information )
Multiple in vivo benzo[a]pyrene animal exposure studies have demonstrated DNA adduct formation in target tissues that precede tumor formation and increase in frequency with dose (Culp et al., 1996a; Talaska et al., 1996; Albert et al., 1991)
Benzo[a]pyrene diol epoxide metabolites interact preferentially with the exocyclic amino groups of deoxyguanine and deoxyadenine in DNA (Geacintov et al., 1997; Jerina et al., 1991; Koreeda et al.,

1978; Je ffrey et al., 1976)
Benzo[a]pyrene o

- quinone metabolites are capable of activating redox cycles and producing ROS that cause oxidative base damage (Park et al., 2006a; Balu et al., 2004; McCoull et al., 1999; Flowers et al., 1997, 1996)

Human evidence that key events are necessary for carcinogenesis:
Detection of benzo[a]pyrene diol epoxide

- specific DNA adducts is strongly associated with increased cancer risk in humans that are occupationally exposed ( see Evidence in Humans section )
These benzo[a]pyrene di ol epoxides formed BPDE

- DNA adducts preferentially at guanine residues that have been detected in tissues of humans with cancer that were exposed to PAHs (Li et al., 2001; Xu et al., 1997; Vineis and Perera, 2007; Pavanello et al., 1999; Rojas et al., 2004 ; 1998; Andreassen et al., 2002; Godschalk et al., 2002; Alexandrov et al., 1992)

3. Formation and fixation of DNA mutations, particularly in tumor suppressor genes or oncogenes

associated with tumor initiation.

Evidence that benzo[a]pyrene metabolites indu ce key events:
Several in vivo exposure studies have observed benzo[a]pyrene diol epoxide

- specific mutational spectra (e.g., G  T transversion mutations) in K

- ras, H

- ras, and p53 in forestomach or lung tumorsToxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE (Culp et al., 2000; Nesnow et al., 1998a, b, 199 6, 1995; Mass et al., 1993)
Multiple animal exposure studies have identified benzo[a]pyrene

- specific mutations in H

- ras, K

- ras, and p53 in target tissues preceding tumor formation (Liu et al., 2005; Wei et al., 1999; Culp et al., 1996a)

Human evidence that key events are necessary for carcinogenesis:
DNA adducts formed by the benzo[a]pyrene diol epoxide reacting with guanine bases lead predominantly to G  T transversion mutations; these specific mutational spectra have been

identified in PAH

- associated tumors in humans at mutational hotspots, including oncogenes (K

- ras) and tumor suppressor genes (p53) (Denissenko et al., 1996; Puisieux et al., 1991; Liu et al., 2005; Marshall et al., 1984; Koreeda et al., 1978; Jeffrey et al., 1976; Keohavong et al., 20 03; DeMarini et al., 2001; Pfeifer and Hainaut, 2003; Pfeifer et al., 2002; Hainaut and Pfeifer, 2001; Bennett et al., 1999)

4. Clonal expansion of mutated cells during the promotion and progression phases of cancer development.

Evidence that benzo[a]pyrene metabolites induce key events:
Numerous studies in animals have observed carcinogenesis following exposure to benzo[a]pyrene via all exposure routes (see Evidence in Animals section )
Mice exposed dermally to benzo[a]pyrene for 26 weeks were found to have increased frequencies

of H

- ras mutations in exposure

- induced hyperplastic lesions that were further increased in tumors (Wei et al., 1999) Human evidence that key events are necessary for carcinogenesis:
The frequency of G  T transversions in p53 was found to increase with increasing smoking duration and intensity in human lung tumors (Bennett et al., 1999)
Benzo[a]pyrene

- specific DNA adducts have been detected in preneoplastic tissues in humans exposed to PAH mixtures, e.g., in lung tissue of smokers with lung cancer, and in human skin treated with coal tar containing ointment (Godschalk et al., 2002; Bartsch et al., 1999; Alexandrov et al., 1992)
Detection of benzo[a]pyrene

- specific DNA adducts is strongly associated with increased cancer risk

in coke oven workers and chimney sweeps (Pavanello et al., 1999)

Other Possible Modes of Action 1

The carcinogenic process for benzo[a]pyrene is likely to be related to some combination of 2

molecular events resulting from the formation of several reactive metabolites wh ich interact with 3

DNA to form adducts and produce DNA damage resulting in mutations in cancer

- related genes, such 4
as tumor suppressor genes or oncogenes. These events may reflect the initiation potency of 5

benzo[a]pyrene. However, benzo[a]pyrene possesses promotional capabilities that may be related 6

to AhR affinity, cytotoxicity and the formation of reactive oxygen species, as well as the inhibition of 7
gap junctional intercellular communication. 8

The ability of certain PAHs to act as initiators and promoter s may increase their 9

carcinogenic potency (Andrews et al., 1978). The promotional effects of PAHs appear to be related 10

to AhR affinity and the upregulation of genes related to growth and differentiation (Bostrom et al., 11
2002). The genes regulated by this receptor belong to two major functional groups (i.e., induction 12

of metabolism or regulation cell differentiation and proliferation). PAHs bind to the cytosolic AhR 13Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE in complex with heat shock protein 90. The ligand

- bound receptor is then transported to n ucleus in 1

complex with the AhR nuclear translocator protein. The AhR complex interacts with AhR elements 2

of DNA to increase the transcription of proteins associated with induction of metabolism and 3

regulation of cell differentiation and proliferation. Fo llowing benzo[a]pyrene exposure, disparities 4

have been observed in the tumor pattern and toxicity of Ah

- responsive and Ah

- nonresponsive mice, 5

as Ah

- responsive mice were more susceptible to tumorigenicity in target tissues such as liver, lung, 6
and skin (Ma and Lu, 2007; Talaska et al., 2006; Shimizu et al., 2000). 7
Inflammatory responses to cytotoxicity may contribute to the tumor promotion process; for 8

example, benzo[a]pyrene quinones (1,6

- , 3,6

- , and 6,12

- benzo[a]pyrene

- quinone) generated 9

reactive oxygen sp ecies and increased cell proliferation by enhancing the epidermal growth factor 10

receptor pathway in cultured breast epithelial cells (Burdick et al., 2003). In addition, several 11

studies have demonstrated that exposure to benzo[a]pyrene increases the produ ction of 12
inflammatory cytokines which may contribute to cancer progression (N’Diaye et al., 2006; Tamaki 13

et al., 2004; Garçon et al., 2001a, b) 14
Gap junctions are channels between cells that are crucial for differentiation, proliferation, 15

apoptosis, and cel l death and consequently for the two epigenetic steps of tumor formation, 16
promotion, and progression. Inhibition of gap junctional intercellular communication by 17

benzo[a]pyrene has been observed in vitro (Sharovskaya et al., 2006; Blaha et al., 2002). 18

Con clusions About the Hypothesized Mode of Action 19

There is sufficient evidence to conclude that the major mode of action for benzo[a]py rene 20
carcinogenicity involves mutagenic ity mediated by DNA reactive metabolites. The evidence for a 21

mutagenic mode of actio n for benzo[a]pyrene is consistent with the current understanding that 22

mutations in p53 and ras oncogenes are associated with increased risk of tumor initiation. The 23
benzo[a]pyrene database provides strong and consistent evidence for BPDE

- induced mutation s 24

associated with tumor initiation in cancer tissue from humans exposed to complex mixtures 25

containing benzo[a]pyrene, in animals exposed to benzo[a]pyrene, and in in vitro systems. 26
Supporting evidence suggests that contributions to tumor initiation throu gh potential mutagenic 27

events can be made by the radical cation and o

- quinone/ROS metabolic activation pathways. Other 28

processes may contribute to the carcinogenicity of benzo[a]pyrene via the promotion and 29

progression phases of cancer development (e.g., inflammation, cytotoxicity, sustained regenerative 30

cell prolifera tion)

. 31

Support for the hypothesized mode of action in test animals 32 Benzo[a]pyrene induces gene mutations in a variety of in vivo and in vitro systems and 33

produces tumors in all animal species tested and all routes of exposure (s ee Appendix B in 34

Supplemental Information ). Strong, consistent evidence in animal models supports the postulated 35

key events: the metabolism of benzo[a]pyrene to DNA

- reactive intermediates, the formation of 36Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE DNA adducts , and the subsequent occurrence of mutations in oncogenes and tumor suppressor 1

genes. 2

Relevance of the hypothesized mode of action to humans 3 A substantial database indicates that the postulated key events for a mutagenic mode of 4

action all occur in human t issues. Strong evidence is available from studies of humans exposed to 5
PAH mixtures (including coal smoke and tobacco smoke) indicating a contributing role for 6
benzo[a]pyrene diol epoxide in inducing key mutational events in genes that are associated with 7

tumor initiation (mutations in the p53 tumor suppressor gene and H

- ras or K

- ras oncogenes). The 8

evidence includes observations of a spectrum of mutations in ras oncogenes and the p53 gene in 9
lung tumors of human patients exposed to coal smoke or tobacco smoke) that are similar to the 10

spectrum of mutations caused by benzo[a]pyrene diol epoxide in several biological systems, 11
including tumors from mice exposed to benzo[a]pyrene. Additional supporting evidence includes 12

correspondence between hotspots of p53 mutations in human lung cancers and sites of DNA 13

adduction by benzo[a]pyrene diol epoxide in experimental systems, and elevated BPDE

- DNA 14
adduct levels in respiratory tissue of lung cancer patients or tobacco smokers with lung cancer. 15

Populations or lifesta ges particularly susceptible to the hypothesized mode of action 16

A mutagenic mode of action for benzo[a]pyrene

- induced carcinogenicity is considered 17
relevant to all populations and lifestages. The current understanding of biology of cancer indicates 18

that m utagenic chemicals, such as benzo[a]pyrene, are expected to exhibit a greater effect in early 19

life exposure versus later life exposure (U.S. EPA, 2005b; Vesselinovitch et al., 1979). Although the 20
developing fetus and infants may have lower levels of some bioactivating enzymes than adults (e.g., 21

CYP1A1/1B1), infants or children are expected to be more susceptible. Newborn or infant mice 22

developed liver and lung tumors more readily than young adult mice following acute i.p. exposures 23
to benzo[a]pyrene (Vess elinovitch et al., 1975). These results indicate that exposure to 24

benzo[a]pyrene during early life stages presents additional risk for cancer, compared with 25

exposure during adulthood. The EPA’s Supplemental Guidance for Assessing Susceptibility from 26

Early

- Life Exposure to Carcinogens (U.S. EPA, 2005b) recommends the application of age

- dependent 27
adjustment factors (ADAFs) for carcinogens that act through a mutagenic mode of action. Given 28

that a determination benzo[a]pyrene acts through a mutagenic mode of carcinogenic action has 29

been made, A DAFs should be applied along with exposure information to estimate cancer risks for 30

early

- life exposure

. 31

Population variability in metabolism and detoxification of benzo[a]pyrene, in addition to 32

DNA repair capability, may affect cancer risk. Polymorphic variations in the human population in 33

CYP1A1, CYP1B1, and other CYPs have been implicated as determinants of increased individual 34
lung cancer risk in some studies (Aklillu et al., 2005; Alexandrov et al., 2002; Perera a nd Weinstein, 35

2000). Some evidence suggests that humans lacking a functional GSTM1 gene have higher BPDE

36

DNA adduct levels and are thus at greater risk for cancer (Vineis et al., 2007; Pavanello et al., 2005; 37

2004; Alexandrov et al., 2002; Perera and Wein stein, 2000). In addition, acquired deficiencies or 38Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE inherited gene poly morphisms that affect the efficiency or fidelity of DNA repair may also influence 1
individual susceptibility to cancer from environmental mutagens (Matullo et al., 2003; Shen et al., 2

2 003; Cheng et al., 2000; Perera and Weinstein, 2000; Wei et al., 2000; Amos et al., 1999). In 3

general, however, available support for the role of single polymorphisms in significantly 4
modulating human PAH cancer risk from benzo[a]pyrene or other PAHs is r elatively weak or 5
inconsistent. Combinations of metabolic polymorphisms, on the other hand, may be critical 6

determinants of a cumulative DNA

- damaging dose, and thus indicate greater susceptibility to 7
cancer from benzo[a]pyrene exposure (Vineis et al., 200 7). 8



## 1.2. Summary and Evaluation 9



### 1.2.1. Weight of Evidence for Effects Other Than Cancer 10

The weight of the evidence from human and animal studies indicate that the strongest 11
evidence for potential hazard following benzo[a]pyrene exposure are for developmental and 12

reproductive toxicity and Immunotoxicity. In humans, exposure to PAH mixtures has been shown 13

to result in development al and reproductive toxicity and immunotoxicity. Most of the available 14

human data repor t associations between particular health endpoints and concentrations of 15

benzo[a]pyrene

- DNA adducts, with fewer studies correlating health effects with external measures 16

of exposure. The available human studies report effects that are generally analogous to the effects 17
observed in animal toxicological studies, and provide qualitative, supportive evidence for the effect

18

specific hazards identified in Section 1.1.

. 19

I n animals, evidence of developmental and reproductive toxicity and immunotoxicity ha s 20

been ob served across species and dosing regimens (Figure 1

- 8). The available evidence from mice 21

and rats treated by gavage during gestation or in the early postnatal period demonstrate 22

developmental effects including decreased body weight, decreased fetal surviv al, decreased 23

fertility, atrophy of reproductive organs, and altered neurobehavioral outcomes (Chen et al., 2012; 24

Jules et al., 2012; Bouayed et al. , 2009b; Kristensen et al., 1995; MacKenzie and Angevine 1981)

. 25

M ale and female reproductive toxicity , as e videnced by effects on sperm parameters, decreased 26

reproductive organ weights, histological changes, and hormone alterations, have been observed 27

after oral exposure in rats and mice (Chen et al., 2011; Chung et al., 2011; Mohamed et al., 2010; 28
Zheng et al. , 2010; MacKenzie and Angevine, 1981)

. B enzo[a]pyrene exposure has also been shown 29

to lead to altered immune cell populations and histopathological changes in immune system organs 30

(Kroese et al., 2001; De Jong et al., 1999) , as well as thymic and splenic effects following subchronic 31

oral exposure. V arying immunosuppressive responses are also observed in short term oral and 32

injection studies. The weight of the evidence indicate s that developmental toxicity, reproductive 33

toxicity and i mmunotoxicity are haz ards following oral exposure to benzo[a]pyrene. 34

Following inhalation exposure to benzo[a]pyrene in animals, evidence of developmental 35

and reproductive toxicity has been observed. Decreased fetal survival has been observed in rats 36

exposed to benzo[a]pyrene via i nhalation during gestation (Wormley et al., 2004; Archibong et al., 37Toxicological Review of Benzo[a]pyrene

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. Male reproductive toxicity , as evidenced by effects on sperm parameters, decreased testes 1

weight, and hormone alterations, has also been observed in rats following subchronic i nhalation 2

exposure to benzo[a]pyrene (Archibong et al., 2008; Ramesh et al., 2008)

. Female reproductive 3

toxicity, as evidenced by modified hormone levels in dams, has been observed following inhalation 4

exposure to benzo[a]pyrene during gestation (Archib ong et al., 2002) . The weight of the evidence 5

indicate s that developmental toxicity and reproductive toxicity are hazards following inhalation 6

exposure to benzo[a]pyrene. 7

F orestomach hyperplasia was observed following oral and inhalation exposure; however

, 8

this endpoint most likely reflects early events in the neoplastic progression of forestomach tumors 9
following benzo[a]py rene exposure (see Section 1.1.4 ), and was not carried forward as an effect 10

other than cancer for the derivation of reference values. 11



### 1.2.2. Weight of Evidence for Carcinogenicity 12

Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), benzo[a]pyrene 13
is “carcinogenic to humans.” EPA’s Cancer Guidelines (U.S. EPA, 2005a) emphasize the importance 14
of weighing all of the evidence in reaching conclusions about human carcinogenic potential. The 15
descriptor of “carcinogenic to humans” can be used when the following conditions are met: (a) 16

there is strong evidence of an association between human exposure and either cancer or the key 17

pr ecursor events of the agent's mode of action but not enough for a causal association, (b) there is 18

extensive evidence of carcinogenicity in animals, (c) the mode or modes of carcinogenic action and 19

associated key precursor events have been identified in an imals, and (d) there is strong evidence 20

that the key precursor events that precede the cancer response in animals are anticipated to occur 21

in humans and progress to tumors, based on available biological information. The data supporting 22

these four conditio ns for benzo[a]pyrene are presented below and in Table 1

- 19

. 23

a) Strong human evidence of cancer or its precursors 24

There is a large body of evidence for human carcinogenicity for complex PAH mixtures 25
containing benzo[a]pyrene, including soot, coal tars, coa l

- tar pitch, mineral oils, shale oils, and 26

smoke from domestic coal burning (IARC, 2010a,b; Baan et al., 2009). There is also evidence of 27

carcinogenicity, primarily of the lung and skin, in occupations involving exposure to PAH mixtures 28
containing benzo[a ]pyrene, such as chimney sweeping, coal gasification, coal

- tar distillation, coke 29

production, iron and steel founding, aluminum production, and paving and roofing with coal tar 30

pitch (IARC, 2010a; Baan et al., 2009; Straif et al., 2005). Increased cancer risks have been reported 31

among other occupations involving exposure to PAH mixtures such as carbon black and diesel 32
exhaust (Bosetti et al., 2007). Benzo[a]pyrene is also a notable carcinogenic constituent of tobacco 33

smoke (IARC, 2004). 34

Evidence for a c ontributing role of benzo[a]pyrene to human carcinogenic responses to 35

complex PAH mixtures is available. Elevated BPDE

- DNA adducts have been reported in smokers 36Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE compared to nonsmokers, and the increased adduct levels in smokers are typically increased two

1

fold compared with nonsmokers (Philips, 2002). Elevated BPDE

- DNA adduct levels have been 2
observed in WBCs of groups of coke oven workers and chimney sweeps, occupations with known 3

elevated risks of cancer (Rojas et al., 2000, 1998; Bartsch et al., 1999, 1998; Pavanello et al, 1999), 4
and in lung tissue from tobacco smokers with lung cancer (Rojas et al., 2004; 1998; Godschalk et al., 5

2002; Bartsch et al., 1999; Andreassen et al., 1996; Alexandrov et al., 1992). 6
Although it is likely that multiple carcin ogens present in PAH mixtures contribute to the 7
carcinogenic responses, strong evidence is available from several studies of humans exposed to 8

PAH mixtures supporting a contributing role for benzo[a]pyrene diol epoxide in inducing key 9

mutagenic precursor c ancer events in target tissues. Distinctive mutation spectra have been 10

observed in the tumor suppressor gene p53 and the K

- ras oncogene in tumor tissues taken from 11
lung cancer patients that were chronically exposed to two significant sources of PAH mixtur es: coal 12
smoke and tobacco smoke. Hackman et al. (2000) reported an increase of GC  TA transversions 13

and a decrease of GC  AT transitions at the hprt locus in T

- lymphocytes of humans with lung 14

cancer that were smokers compared to nonsmokers. Lung tumors fr om cancer patients exposed to 15
emissions from burning smoky coal showed mutations in p53 and K

- ras that were primarily G → T 16

transversions (76 and 86%, respectively) (DeMarini et al., 2001). Keohavong et al. (2003) 17

investigated the K

- ras mutational spectra f rom nonsmoking women and smoking men chronically 18

exposed to emissions from burning smoky coal, and smoking men who resided in homes using 19
natural gas; among those with K

- ras mutations, 67, 86, and 67%, respectively, were G → T 20

transversions. Lung tumors fr om tobacco smokers showed a higher frequency of p53 mutations 21

that were G → T transversions compared with lung tumors in nonsmokers (Pfeifer and Hainaut, 22

2003; Pfeifer et al., 2002; Hainaut and Pfeifer, 2001), and the frequency of these types of p53 23

mutation s in lung tumors from smokers increased with increasing smoking intensity (Bennett et al., 24

1999). 25

Similarly, investigations of mutagenesis following specific exposures to benzo[a]pyrene (as 26
opposed to PAH mixtures) have consistently observed that the ben zo[a]pyrene diol epoxide is very 27
reactive with guanine bases in DNA, and that G → T transversions are the predominant type of 28

mutations caused by benzo[a]pyrene diol epoxide in several biological test systems (Pfeifer and 29

Hainaut, 2003; Hainaut and Pfeifer, 2001). Following treatment of human HeLa cells with 30
benzo[a]pyrene diol epoxide, Denissenko et al. (1996) reported that the distribution of BPDE

- DNA 31

adducts within p53 corresponded to mutational hotspots observed in p53 in human lung cancers. 32

Benzo[a]pyr ene exposure induced mutations in embryonic fibroblasts from human p53 “knock

- in” 33
mice that were similar to those found in smoking related human cancers, with a predominance of 34

G → T transversions that displayed strand bias and were also located in the same mutational 35

hotspots found in p53 in human lung tumors (Liu et al., 2005). These results, combined with a 36
mechanistic understanding that mutations in p53 (which encodes a key transcription factor in DNA 37

repair and regulation of cell cycle and apoptosis) may be involved in the initiation phase of many 38Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE types of cancer, are consistent with a common mechanism for mutagenesis following exposures to 1

PAH mixtures and provide evidence of a contributing role of benzo[a]pyrene diol epoxide in the 2
carcinogenic response of humans to coal smoke and tobacco smoke. 3

Therefore, while the epidemiological evidence alone does not establish a causal association 4

between human ex posure and cancer, there is strong evidence that the key precursor events of 5
benzo[a]pyrene’s mode of action are likely to be associated with tumor formation in humans. 6

b) Extensive animal evidence 7

In laboratory animals (rats, mice, and hamsters), exposure to benzo[a]pyrene via the oral, 8

inhalation, and dermal routes have been associated with carcinogenic responses both systemically 9

and at the site of administration. Three 2

- year oral bioassays are available that associate lifetime 10

benzo[a]pyrene exposure with carcinogenicity at multiple sites. These bioassays observed 11
forestomach, liver, oral cavity, jejunum, kidney, auditory canal (Zymbal’s gland), and skin or 12
mammary gland tumors in male and female Wistar rats (Kroese et al., 2001); forestomach tumors 13

i n male and female Sprague

- Dawley rats (Brune et al., 1981); and forestomach, esophagus, tongue, 14

and larynx tumors in female B6C3F 1 mice (Beland and Culp, 1998; Culp et al., 1998). Repeated or 15

short

- term oral exposure to benzo[a]pyrene was associated with forestomach tumors in additional 16
bioassays with several strains of mice (Weyand et al., 1995; Benjamin et al., 1988; Robinson et al., 17

1987; El Bayoumy, 1985; Triolo et al., 1977; Wattenberg, 1974; Field and Roe, 1970; Roe et al., 18

1970; Biancifiori et al., 1967; Chouroulinkov et al., 1967; Fedorenko and Yansheva, 1967; Neal and 19
Rigdon, 1967; Berenblum and Haran, 1955). Chronic inhalation exposure to benzo[a]pyrene was 20

associated with tumors in the larynx and pharynx of male Syrian golden hamsters exposed to 21

benzo[a]pyrene:NaCl aerosols (Thyssen et al., 1981). Additionally, less

- than

- lifetime oral exposure 22

cancer bioassays in mice provide supporting evidence that exposure to benzo[a]pyrene is 23
associated with an increased incidence of lung tumors in mice (We yand et al., 1995; Robinson et al., 24

1987; Wattenberg, 1974). Intratracheal instillation of benzo[a]pyrene was associated with 25

respiratory tract tumors in additional studies with hamsters (Feron and Kruysse, 1978; Ketkar et 26

al., 1978; Feron et al., 1973; H enry et al., 1973; Saffiotti et al., 1972). Chronic dermal application of 27

benzo[a]pyrene (2

– 3 times/ week) has been associated with mouse skin tumors in numerous 28

bioassays (Sivak et al., 1997; Habs et al., 1984, 1980; Grimmer et al., 1984, 1983; Schmähl e t al., 29

1977; Schmidt et al., 1973; Roe et al., 1970; Poel, 1960, 1959). Skin tumors in rats, rabbits, and 30
guinea pigs have also been associated with repeated application of benzo[a]pyrene to skin in the 31

absence of exogenous promoters (WHO, 1998; ATSDR, 19 95; IARC, 1983, 1973). When followed by 32

repeated exposure to a potent tumor promoter, acute dermal exposure to benzo[a]pyrene induced 33
skin tumors in numerous studies of mice, indicating that benzo[a]pyrene is a strong tumor

34

initiating agent in the mouse s kin model (Weyand et al., 1992; Cavalieri et al., 1991, 1981; Rice et 35

al., 1985; El

- Bayoumy et al., 1982; LaVoie et al., 1982; Raveh et al., 1982; Slaga et al., 1980, 1978; 36
Wood et al., 1980; Hoffmann et al., 1972). 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Carcinogenic responses in animals expose d to benzo[a]pyrene by other routes of 1
administration include: (1) liver or lung tumors in newborn mice given acute postnatal i.p. 2
injections (LaVoie et al., 1994, 1987; Busby et al., 1989, 1984; Weyand and LaVoie, 1988; Wislocki 3

et al., 1986; Buening et al., 1978; Kapitulnik et al., 1978); (2) increased lung tumor multiplicity in 4
A/J adult mice given single i.p. injections (Mass et al., 1993); (3) injection site tumors in mice 5
following s.c. injection (Nikonova, 1977; Pfeiffer, 1977; Homburger et al., 197 2; Roe and Walters, 6

1967; Grant and Roe, 1963; Steiner, 1955; Rask

- Nielson, 1950; Pfeiffer and Allen, 1948; Bryan and 7
Shimkin, 1943; Barry et al., 1935); (4) injection site sarcomas in mice following intramuscular 8

injection(Sugiyama, 1973); (5) mammary tum ors in rats with intramammilary administration 9
(Cavalieri et al., 1991, 1988a, b, c); (6) cervical tumors in mice with intravaginal application 10

(Naslund et al., 1987); and (7) tracheal tumors in rats with intratracheal implantation (Topping et 11

al., 1981, N ettesheim et al., 1977). 12
Therefore, the animal database provides extensive evidence of carcinogenicity in animals. 13

c) Identification of key precursor events have been identified in animals 14

There is sufficient evidence to conclude that benzo[a]pyrene carcin ogenicity involves a 15
mutagenic mode of action mediated by DNA reactive metabolites. The benzo[a]pyrene database 16
provides strong and consistent evidence for BPDE

- induced mutations associated with tumor 17

initiation in cancer tissue from humans exposed to com plex mixtures containing benzo[a]pyrene, in 18

animals exposed to benzo[a]pyrene, and in in vitro systems. Other processes may contribute to the 19
carcinogenicity of benzo[a]pyrene via the promotion and progression phases of cancer 20

development (e.g., inflammat ion, cytotoxicity, sustained regenerative cell proliferation, anti

21

apoptotic signaling), but the available evidence best supports a mutagenic mode of action as the 22

primary mode by which benzo[a]pyrene acts. 23

d) Strong evidence that the key precursor events are anticipated to occur in humans 24

Mutations in p53 and ras oncogenes have been observed in tumors from mice exposed to 25

benzo[a]pyrene in the diet (Culp et al., 2000) or by intraperitoneal injection (Nesnow et al., 26
1998a,b, 1996, 1995; Mass et al., 1993) . Mutations in these same genes have also been reported in 27
lung tumors of human cancer patients, bearing distinctive mutation spectra (G → T transversions) 28

that correlate with exposures to coal smoke (Keohavong et al., 2003; DeMarini et al., 2001) or 29

tobacc o smoke ( Pfeifer and Hainaut, 2003; Pfeifer et al., 2002; Hainaut and Pfeifer, 2001, Bennett et 30
al., 1999). 31Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 1

- 19 . Supporting evidence for the carcinogenic to humans cancer 1 descriptor for b enzo[a]pyrene 2

Evidence Reference

a) Strong human evidence of cancer or its precursors

Increased cancer risks in humans exposed to complex PAH mixtures containing benzo[a]pyrene IARC, 2010a,b, 2004

Benzo[a]pyrene

- specific biomarkers detected in humans exposed to PAH mixtures

– BPDE

- DNA adducts in WBCs of coke oven workers and chimney sweeps Rojas et al., 2000, 1998; Bartsch et al., 1999, 1998;
Pavanello et al, 1999

– BPDE

- DNA adducts in smokers Philips, 2002

Benzo[a]pyrene

- specific DNA adducts have been detected in preneoplastic target tissues in humans exposed to PAH mixtures

– BPDE

- DNA adducts in non

- tumor lung tissues of cigarette smokers with lung cancer and in skin eczema patients treated with coal tar Rojas et al., 2004; 1998; Godschalk et al., 2002, 1998;
Bartsch et al., 1999; Andreassen et al., 1996; Alexandrov
et al., 1992

– BPDE

- DNA adduct formation in p53 in human cells in vitro corresponds to mutational hotspots at guanine residues in human lung

t umors Denissenko et al., 1996; Puisieux et al., 1991

Benzo[a]pyrene

- specific mutational spectra

identified in PAH

- associated tumors in humans

– GC  TA transversions and GC  AT transitions at hprt locus in T

- lymphocytes of humans with lung cancer Hackman et al., 2000

– G  T transversions at the same mutational hotspot in p53 from smoking

- related lung

tumors in humans Liu et al., 2005; Pfeifer and Hainaut, 2003; Pfeifer et al.,
2002; Hainaut and Pfeifer, 2001

– G  T transversions at the same mutational hotspot in p53 and K

- ras in human lung tumors associated with smoky coal exposures Keohavong et al., 2003; DeMarini et al., 2001

– Increased percentage of G  T transversions in p53 in smokers vs. nonsmokers Bennett et al., 1999

b) Extensive animal evidence

Oral exposures

Forestomach tumors in male and female rats and in female mice following chronic exposure Kroese et al., 2001; Brune et al., 1981; Beland and Culp, 1998; Culp et al., 1998

Forestomach tumors in mice following less

- than

lifetime exposures Weyand et al., 1995; Benjamin et al., 1988; Robinson et al., 1987; El Bayoumy, 1985; Triolo et al., 1977; Wattenberg, 1974; Field and Roe, 1970; Roe et al., 1970;
Biancifiori et al., 1967; Chouroulinkov et al., 1967; Fedorenko and Yansheva, 1967; Neal and Rigdon, 1967;
Berenblum and Haran, 1955Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Evidence Reference

Alimentary tract and Liver tumors in male and female rats following chronic exposure Kroese et al., 2001

Auditory canal tumors in male and female rats following chronic exposure Kroese et al., 2001

Esophageal, tongue, and laryngeal tumors in female mice following chronic exposure Beland and Culp, 1998; Culp et al., 1998

Lung tumors in mice following less

- than

- lifetime

exposure Weyand et al., 1995 ; Robinson et al., 1987; Wattenberg,

1974

Inhalation exposures

Laryngeal and pharyngeal tumors in male hamsters following chronic exposure Thyssen et al., 1981

Dermal exposures

Skin tumors in mice following chronic exposures without a promoter or acute exposures with a

promoter Sivak et al., 1997; Habs et al., 1984, 1980; Grimmer et al., 1984, 1983; Schmähl et al., 1977; Schmidt et al.,
1973; Roe et al., 1970; Poel, 1960, 1959

Skin tumors in rats, rabbits, and guinea pigs following subchronic exposures WHO, 1998; ATSDR, 1995; IARC, 1983, 1973

Other routes of exposure

Respiratory tract tumors in hamsters following

intratracheal instillation Feron and Kruysse, 1978; Ketkar et al., 1978; Feron et al., 1973; Henry et al., 1973; Saffiotti et al., 1972

Liver or lung tumors in newborn mice given acute postnatal i.p. injections LaVoie et al., 1994, 1987; Busby et al., 1989, 1984; Weyand and LaVoie, 1988; Wislocki et al., 1986; Buening et al., 1978; Kapitulnik et al., 1978
Lung tumor multiplicity in A/J adult mice given
single i.p. injections Mass et al., 1993

c) Identification of key precursor events have been identified in animals

Bioactivation of benzo[a]pyrene to DNA

- reactive metabolites has been shown to occur in multiple species and tissues by all routes of exposure See ‘Experimental Support for Hypothesized Mode of

Action’ section

Direct DNA damage by the reactive metabolites, including the formation of DNA adducts and ROS

mediated damage

Formation and fixation of DNA mutations, particularly in tumor suppressor genes or oncogenes associated with tumor initiation

d) Strong evidence that the key precursor events are anticipated to occur in humans

Mutations in p53 or ras oncogenes have been observed in forestomach or lung tumors from mice exposed to benzo[a]pyrene Culp et al., 2000; Nesnow et al., 1998a,b, 1996, 1995;
Mass et al., 1993

– G→T transversions in ras oncogenes or the p53 gene have been observed in lung tumors of human cancer patients exposed to coal smoke Keohavong et al., 2003; DeMarini et al., 2001

– Higher frequency of G→T transversions in lung

tumors from smokers vs. nonsmokers Pfeifer and Hainaut, 2003; Pfeifer et al., 2002; Hainaut
and Pfeifer, 2001; Bennett et al., 1999Toxicological Review of Benzo[a]pyrene

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# 2. DOSE

- RESPONSE ANALYSIS

2.1. Oral Reference Dose for Effects O ther T han Cancer 1

The RfD (expressed in units of mg/kg

- day) is defined as an estimate (with uncertainty 2

spanning perhaps an order of magnitude) of a daily exposure to the human population (including 3

sensitive subgroups) that is likely to be without an appreciable risk of de leterious effects during a 4

lifetime. It can be derived from a no

- observed

- adverse

- effect level (NOAEL), lowest

- observed

5

adverse

- effect level (LOAEL), or the 95 percent lower bound on the benchmark dose (BMDL), with 6

uncertainty factors (UFs) generally appl ied to reflect limitations of the data used. 7



### 2.1.1. Identification of Studies and Effects for Dose

- Response Analysis 8

In Section 1.2 .1

, developmental, reproductive, and immunological toxicities were 9

highlighted as hazards of benzo[a]pyrene exposure by the oral rou te. Studies within each effect 10
category were evaluated using general study quality characteristics (as discussed in Section 6 of the 11

Preamble) to help inform the selection of studies from which to derive toxicity values

. Rationale s 12

for selecting the stud ies and effects to represent each of these hazards are summarized below. 13

Human studies are preferred over animal studies when quantitative measures of exposure 14

are reported and the reported effects are determined to be associated with exposure. For 15

benzo[ a]pyrene, h uman studies of environmental PAH mixtures across multiple cohorts have 16
observed effects following exposure to complex mixtures of PAHs

. The available human studies 17

that utilized benzo[a]pyrene

- DNA adducts as the exposure metric do not provide external exposure 18

levels of benzo[a]pyrene from which to derive a value. Thus, these studies were not considered 19

because of the contribution to the observed hazard of multiple PAHS across multiple routes of 20

exposure. Animal studies were evaluated to dete rmine which provided the most relevant r outes 21

and durations of exposure; multiple exposure levels to provide information about the shape of the 22

dose response curve; and power to detect effects at low exposure levels. 23

Developmental toxicity 24

Numerous animal studies observed endpoints of developmental toxicity following oral 25

exposure during gestational or early post

- natal development (Chen et al. , 2012; Jules et al., 2012; 26

Bouayed, 2009b; Kristensen et al., 1995; MacKenzie and Angevine 1981 ) and were considere d for 27

dose response analysis based on the above criteria

. Kristensen et al. (1995), with only one dose 28

group, was not considered further given its concordance with MacKenzie and Angevine ( 1981 ) 29

which had multiple groups. From the remaining studies demons trating developmental toxicity, the 30

studies conducted by Chen et al. (2012) and Jules et al. (2012) were identified as the most 31Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE informative studies for dose

- response analysis. The neurodevelopmental study by Chen et al. 1

(2012) was a well conducted study that evaluated multiple neurobehavioral endpoints and 2
measures of neurotoxicity in adolescent and adult animals. T he study was designed with sufficient 3

statistical power and randomized pups with a total of 10 males and 10 females per treatment group 4

and n o more than one male and one female from each litter (i.e., pups from 40 litters were used) for 5
behavioral testing. In addition, the pups were cross

- fostered with dams being rotated among litters 6

every 2

- 3 days to distribute any maternal caretaking differ ences randomly across litters and 7

treatment groups. 8

Chen et al. (2012) observed increased latency in negative geotaxis and increased motor 9
activity or anxiety in the open field test at 0.02 mg/kg

- day

, and decreased anxiety in the elevated 10

plus maze test and impaired spatial learning as measured by an increase in latency time to find a 11
hidden platform in the Morris water maze test at 0.2 mg/kg

- day

. Altered behaviors and locomotion 12
in open field tests can be attributed to anxiety responses due to open spac es and bright light, as 13

well as changes to motor system function. Chen et al. (2012) reported increased quadrants crossed, 14

which could indicate either increased motor activity or decreased anxiety (less fear of the open 15
spaces/ bright lights). Similarly, this study reported the number of open arm entries in the elevated 16

plus maze test and not time spent in the closed arms and open arms which would provide a more 17

s ensitive measurement of the effect

. T he number of open arm entries does serve as an indicat or of 18

decreased anxiety and is unlikely to be confounded by changes in motor activity (as total arm 19

entries appears to be unchanged with treatment), but t he magnitude of response in the elevated 20

plus maze test was not as robust as the response observed in the Morris water maze response. 21

Chen et al. (2012) also observed effects in the surface righting test that were on the order of

~ 0.2

- 22

0.3 seconds and in t he negative geotaxis test of approximately 3

- 4 seconds with no automated 23
recording of latency (such as use of video recordings). Additionally, male and female rats (which 24

often show differences in the maturation of these developmental landmarks) were pool ed for these 25

measur es. Due to these uncertainties a ltered behaviors and locomotion in open field tests

, number 26

of open arm entries in the elevated plus maze test, effects in the surface righting test, and effects in 27

the negative geotaxis test were not con sidered. 28

Impaired spatial learning as measured by an increase in latency time to find a hidden 29

platform in the Morris water maze test was selected for dose response analysis . This endpoint is 30

supported by probe trials from the same study in which the plat form was removed and the 31

duration of time spent in the target quadrant and the number of times the animal crosses the 32

location of the previous platform was measured

. Similar impairments were observed in both the 33

probe tests and water maze escape measures in rats treated with benzo[a]pyrene. Th us, th ese tests 34

confirm ed that the animals were using learned spatial cues to identify the location of the platform 35

and not relying on random, non

- spatial strategies such as circular swimming. In addition, there 36

we re no changes in measured swim speed indicating an effect on long term memory. 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Jules et al. (2012) was also identified for dose

- response analysis. This study was of 1

sufficient dur ation, utilized multiple doses , did not observe maternal toxicity, and eva luated 2

multiple cardiovascular endpoints. The study authors reported increase s in both systolic 3

(approximately 20

- 50%) and diastolic (approximately 33

- 83%) pressure and h eart rate in adult 4
rats that were exposed gestationally to benzo[a]pyrene. A limitation of this study is that the 5
authors only reported effects at the two highest doses. However, given the magnitude of the 6

response and the appearance of these effects in adulthood following gestational exposure, these 7

endpoints were selected for dose

- response analysis because of their sensitivity and biological 8

plausibility. 9

Bouayed et al. (2009b) and MacKenzie and Angevine (1981) were not selected for dose 10

response analysis. Boua yed et al. (2009b) used the same tests as Chen et al. (2012), but at higher 11
doses (2 and 20 mg/kg

- day compared to 0.02, 0.2, and 2 mg/kg

- day, respectively ). Because Chen et 12

al. (2012) reported adverse effects at doses lower than Bouayed (200 9 b), the later study was not 13

selected for dose

- response analysis. Similarly, MacKen z ie and Angevine (1981) demonstrated 14

developmental effects in a multi

- dose study with relevant rout e s and durations of exposure ; 15

however, the doses studied (10

- 160 mg/kg

- day) were much h igher than those evaluated in other 16
developmental toxicity studies ( Chen et al., 2012 ; Jules et al., 2012). 17

Reproductive toxicity 18

Male reproductive toxicity was demonstrated in numerous subchronic studies (Chen et al., 19

2011 a ; Chung et al., 2011; Mohamed et al., 2010; Zheng et al., 2010 ). Chu ng et al. (2011) was not 20

included in the dose

- response analysis because of study reporting limitations (i.e.

, only reported 21

significant observations at the mid

- dose ). Chen et al. (2011a) is a subchronic study that a pp l ied 22

only a single dose level. B ecause the study corroborated available multi

- dose studies it was not 23

considered for dose

- response analysis. The studies conducted by Mohamed et al. (2010) and Zheng 24

et al (2010) were identified as the most informative male reproductive toxicity studies for dose

25

response analysis. Decreased sperm count observed by Mohammed et al. (2010) and decreased 26

intratesticular testosterone levels observed by Zheng et al. (2010) were selected for dose

- response 27

analysis as bo th represen t sensitive endpoint s of male reproductive toxicity and are indicator s of 28

potentially decreased fertility . These effects are also consistent with human studies in PAH 29

exposed populations as effects on male fertility and semen quality have been demonstrated in 30
epidemiological studies of smokers (revie wed by Soares and Melo, 2008). 31

Female reproductive toxicity was demonstrated in two subchronic studies ( Gao et al., 2011; 32

Xu et al., 2010). Specifically, these studies demonstrat ed altered ovarian weights and follicle 33
numbers and cervical epithelial cell hyperplasia following oral exposure to benzo[a]pyrene. The se 34

studies were identified as the most informative studies for dose

- response analysis. Gao et al. 35

(2011) identified statistically

- signifi cant dose

- related increases in the incidence of cervical 36
inflammatory cells in mice exposed to low doses of benzo[a]pyrene for 98 days (Gao et al., 2011, 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE 2010). Cervical effects of increasing severity (including epithelial hyperplasia, atypical hyperplasi a, 1

apoptosis, and necrosis) were also observed at higher doses (Gao et al., 2011, 2010). There are no 2
data on cervical effects in other species or in other mouse strains. However, Gao et al. (2011) also 3

evaluated cervical effects in separate groups of mi ce exposed via i.p. injection, and observed similar 4
responses in these groups of mice, providing support for the association between effects in this 5
target organ and benzo[a]pyrene exposure. Epidemiological studies have demonstrated an 6

association between cigarette smoking and increased risk of cervical cancer (Pate Capps et al., 7

2009). In addition, be nzo[a]pyrene metabolites and benzo[a]pyrene

- DNA adducts have been 8

detected in human cervical mucus and cervical tissues obtained from smokers (Melikian et a l., 9
1999; Phillips et al., 2002). 10

Xu et al. (2010) identified biologically and statistically significant decreases in ovary weight, 11
estrogen, and primordial follicles, and altered estrus cycling in treated animals. These reductions in 12

female reproductiv e parameters are supported by a large database of animal studies indicating that 13

benzo[a]pyrene is ovotoxic with effects including decreased ovary weight, decreased primordial 14
follicles, and reduced fertility (Mattison et al., 1980; MacKenzie and Angevine 1981; Swartz and 15

Mattison 1985; Miller et al., 1992; Kristensen et al., 1995; Borman et al, 2000). Additionally, 16
epidemiology studies indicate that exposure to complex mixtures of PAHs, such as through cigarette 17

smoke, is associated with measures of decre ased fertility in humans (El Nemr et al., 1998; Neal et 18

al., 2005). Specific associations have also been made between infertility and increased levels of 19
benzo[a]pyrene in follicular fluid in women undergoing in vitro fertilization (Neal et al., 2008). 20

Im munotoxicity 21

As described in Section 1.1.4, the immune system was identified as a target of 22

benzo[a]pyrene

- induced toxicity based on findings of organ weight and immunoglobulin 23
alterations, as well as effects on cellularity and functional changes in the im mune system in animals. 24

The studies conducted by Kroese et al. (2001) and De Jong et al. (1999) are subchronic studies with 25

multiple exposure levels and adequate power to detect effects. In comparing these two studies, the 26
Kroese et al. (2001) study is pre ferred for dose

- response analysis due to its longer duration (90 27

days). 28

Decreased thymus weight, observed in Kroese et al. (2001), decreased IgM and IgA levels, 29

and decreased numbers of B

- cells, observed in De Jong et al. (1999), were selected for dose

30

res ponse analysis. It is recognized that thymus weight changes on their own have been noted to be 31

less reliable indicators of immunotoxicity (Luster et al., 1992). However, there are converging lines 32

of evidence that support the derivation of a candidate Rf D for benzo[a]pyrene immunotoxicity, 33
including: alterations in immunoglobulin levels have been noted in humans after exposure to PAHs, 34

as well as in animal studies after exposure to benzo[a]pyrene; changes in B cell populations in the 35

spleen provide additi onal evidence of immunotoxicity; and functional effects on the immune 36
system, including dose

- related decreases in SRBC

- specific IgM levels and dose

- dependent decreases 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE in resistance to pneumonia or Herpes simplex type 2 following short

- term s.c. injection (Temple et 1
al. 1993; Munson et al. 1985). The observed decreases in thymus weight, IgM and IgA levels, and 2
number of B cells associated with exposure to benzo[a]pyrene were determined to be 3

representative of immunotoxicity following benzo[a]pyrene exposur e and were selected for dose

4

response analysis. 5



### 2.1.2. Methods of A nalysis 6

N o biologically based dose

- response models are available for benzo[a] pyrene. In this 7

situation, EPA evaluate s a range of dose

- response models thought to be consistent with underlying 8
biological processes to determine how to best empirically model the dose

- response relationship in 9

the range of the observed data. Consistent with this approach, a ll models available as part of EPA’s 10

Benchmark Dose Software ( BMDS ) were evaluated

. Consistent with EPA’s d raft Benchmark Dose 11

Technical Guidance Document (U.S. EPA, 2000b), the BMD and the 95% lower confidence limit on 12

the BMD (BMDL) were estimated using a BMR of 1 SD from the control mean for continuous data or 13

a BMR of 10% extra risk for dichotomous data in the absence of information regarding what level of 14
change is considered biologically significant, and also to facilitate a consistent basis of comparison 15

across endpoints , studies, and assessments. T he estimated BMDLs were used as points of 16

departure (PODs). Further details including the modeling output and gra ph ical results for the best 17

fit model for each endpoint can be found Appendix C of the Supplemental Information

. 18
Among the endpoints identified as representative of the hazards of benzo[a]pyrene 19

exposure , the data for Morris water maze escape latency (Chen et al., 2012), decreased ovary 20

weight (Xu et al., 2010), and decreased thymus weigh t (Kroese et al., 2000) were amenable to dose

21

response modeling. For the water maze escape latency data, t he data for male and female rats were 22

combined for dose

- response analysis because of the strong similarity in responses and the lack of 23

information available suggesting there wou ld be sex

- specific differ ences in the results of this test 24

( see Appendix C of the Supplemental Information for details of statistical analyses ). 25

The data for the remaining endpoints identified in Section 2.1 were not modeled

. 26

Specifically, the data for cardiovascular effects observed in Jules et al. (2012) were limited due to 27

the reporting of results at only the two highest dose groups. T he data for epididymal sperm counts 28
presented in the Mohamed et al. (2010) study were reported graphically only and r equests for the 29

raw data were unsuccessful. The observed decrease in IgM and IgA (De Jong et al., 1999) was 30

inconsistent and not amenable to dose

- response modeling. NOAELs or LOAELs were used as the 31

POD for these endpoints

. 32

H uman equivalent doses (i.e. H EDs) for oral exposures were derived from the PODs 33

estimated from the laboratory animal data as described in EPA’s Recommended Use of Body 34

Weight

3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 2011b). In this 35
guidance, EPA ad vocates a hierarchy of approaches for deriving HEDs from data in laboratory 36

animals, with the preferred approach being physiologically

- based toxicokinetic modeling. Other 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE approaches can include using chemical

- specific information in the absence of a compl ete 1

physiologically

- based toxicokinetic model. As discussed in Appendix B of the Supplemental 2

Information , several animal PBPK models for benzo[a]pyrene have been developed and published, 3

but a validated human PBPK model for benzo[a]pyrene for extrapolating doses from animals to 4

humans is not available

. In lieu of either chemical

- specific models or data to inform the derivation 5

of human equivalent oral exposures, a body weight scaling to the 3⁄4 power (i.e., BW 3/4 ) approach is 6

applied to extrapo late toxicologically equivalent doses of orally administered agents from adult 7
laboratory animals to adult humans for the purpose of deriving a n oral RfD. 8

Consistent with EPA guidance (U.S. EPA, 2011b) , the PODs estimated based on effects in 9

adult animal s are converted to HED s employing a standard dosimetric adjustment factor (DAF) 10

derived as follows: 11

12
DAF = (BW

a 1/4 / BW h 1/4 ), 13

Where 14

BW
a = animal body weight 15

BW h = human body weight 16

17

Using a BW
a of 0.25 kg for rats and 0.035 kg for mice and a BW h of 70 kg for humans (U.S. 18

EPA, 1988), the resulting DAFs for rats and mice are 0.24 and 0.15, respectively. Applying this DAF 19
to the POD identified for effects in adult rats or mice yields a POD HED as follows (see Table 2

- 1): 20

21

POD HED = Laboratory an imal dose (mg/kg

- day) x DAF 22

23

Table 2

- 1 summarizes the sequence of calculations leading to the derivation of a human

24

equivalent POD for each data set discussed above. 25Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 2

- 1 . Summary of Derivation of Point s of Departure 1

Endpoint and

Reference Species/

Sex Model a BMR BMD (mg/kg

- day) BMDL

(mg/kg

- day) POD ADJ b

(mg/kg

- day) POD HE D c

(mg/kg

- day)

Developmental

Neuro developmental
impairments
Chen et al., 2012 Male and

Female
Sprague

Dawley Rats Hill a 1SD 0.11 0.06 0.06 0.06

Cardiovascular effects
Jules et al., 2012 Long

- Evans

rats LOAEL (0.6 mg/kg

- day) 0.6 0.15

Reproductive

Decreased ovarian weight
Xu et al., 2010 Female

Sprague

Dawley rats Linear a 1 SD 2.3 1.5 1. 5 0.37

Decreased sperm

count

Mohamed et al., 2010 Male C57BL/6

mice LOAEL (1 mg/kg

- day) 1 0.15

Cervical epithelial hyperplasia Gao et al. (2011) Female ICR

mice Log

logistic a 10% 0.58 0.37 0.37 0.06

Immunological

Decreased thymus weight
Kroese et al., 2001 Female

Wistar rats Linear a 1SD 10.5 7.6 7.6 1.9

Decreased IgM levels De Jong et al., 1999 Male

Wistar rats N OAEL (10 mg/kg

- day) 7.1 1.7

Decreased IgA levels De Jong et al., 1999 Male

Wistar rats NOAEL (30 mg/kg

- day) 21 5.2

Decreased number of

B cells DeJong et al., 1999 Male

Wistar rats NOAEL (30 mg/kg

- day) 21 5.2

a For modeling details, see Appendix B in Supplemental Information 2

b For studies in which animals were not dosed daily, a dministered doses were adjusted to calculate the time

3
weighted average daily dose s prior to BMD modeling. 4

c HED PODs were calculated using BW 3/4 scaling (US EPA

, 2011 b ) for effects from dosing studies in adult animals 5 ( i.e., Mohamed et al., 2010; Xu et al., 2010; Gao et al., 2011; and De Jong et al., 1999) or for developmental effects 6
resulting from in utero exposures. BW 3/4 scaling was not employed for deriving HEDs from studies in which dos es 7
were administered directly to early postnatal animals ( i.e., Chen et al., 2012) because of the absence of 8

informa tion on whether allometric (i.e., body weight) scaling holds when extrapolating doses from neonatal 9
animals to adult humans due to p resumed toxicokinetic and/or toxicodynamic differences between lifestages (U.S. 10
EPA, 2011b; Hattis et al., 2004)

. 11Toxicological Review of Benzo[a]pyrene

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### 2.1.3. Derivation of Candidate Reference D oses 1

Table 2

- 2 is a continuation of Table 2

- 1 and summarizes the application of uncertainty 2

factors to each POD to derive a candidate RfD for each data set. The selection of uncertainty factors 3

was based on EPA’s A R eview of the Reference Dose and Reference Concentration Processes ( U.S. EPA, 4

2002; Section 4.4.5 ) and is described in Section 7.6 of the Preamble. Figure 2

- 1 presents graphically 5

these candidate RfDs , uncertainty factors, and points of departure, with each bar corresponding to 6

one data set descr ibed in Tables 2

- 1 and 2

- 2. 7

Table 2

- 2

. E ffects and corresponding derivation of candidate RfDs 8

Endpoint and Reference POD HED a POD

type UF A UF H UF L UF S UF D Composite

UF Candidate RfD (mg/kg

- d)

DEVELOPMENTAL

Neurodevelop mental
impairments in rats
Chen et al., 2012 0.0 6 BMDL 1SD 10 10 1 1 3 3 00 2 x 10 - 4

Cardiovascular effects in rats
Jules et al., 2012 0.15 LOAEL 3 10 10 1 3 1000 2 x 10 - 4

REPRODUCTIVE

Decreased ovary weight and

ovarian follicles in rats

Xu et al., 2010 0.37 BMDL 1SD 3 10 1 10 3 1000 4 x 10 - 4

Decreased sperm count in mice
Mohamed et al., 2010 0.15 LOAEL 3 10 10 10 3 10000 Not calculated due

to UF > 3000 a

Cervical epithelial hyperplasia

in mice Gao et al. (2011) 0.06 BMDL 10 3 10 1 10 3 1000 6 x 10 - 5

IMMUNOLOGICAL

Decreased thymus weight in

rats Kroese et al., 2001 1.9 BMDL 1SD 3 10 1 10 3 1000 2 x 10 - 3

Decreased serum IgM in rats De Jong et al., 1999 1.7 NOAEL 3 10 1 10 3 1000 2 x 10 - 3

Decreased serum IgA in rats De Jong et al., 1999 5.2 NOAEL 3 10 1 10 3 1000 5 x 10 - 3

Decreased number of B cells in

rats De Jong et al., 1999 5.2 NOAEL 3 10 1 10 3 1000 5 x 10 - 3Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Endpoint and Reference POD HED a POD

type UF A UF H UF L UF S UF D Composite

UF Candidate RfD (mg/kg

- d)

UF A – A value of 3 (10 0.5 = 3.16, rounded to 3) was applied to account for uncertainty in characterizing toxicodynamic differences between rats and humans when an HED was calculated using BW 3/4 scaling as uncertainty in characterizing toxicokinetic differences was accounted for through calculation of an HED using a standard DAF consistent with EPA guidance (U

. S

. EPA 2011 b)

. A value of 10 was applied when BW 3/4 scaling was not employed to account for uncertainty in extrapolating from laboratory animals to humans because of

the absence of information to characterize either the toxicokinetic or toxicodynamic differences betwee n animals and humans following oral exposure to BaP.

UF H – A value of 10 was applied to account for potentially susceptible individuals because adequate information is not available to quantitatively estimate variability in human susceptibility

. In t he case of benzo[a]pyrene, Insufficient information is available to quantitatively estimate variability in human susceptibility

. UF L – A value of 1 was applied when the POD is based on dose

- response modeling or a NOAEL; 10 when the POD

is a LOAEL

. In the case of benzo[a]pyrene, An UF L of 1 was applied for LOAEL

- to

- NOAEL extrapolation because a BMR of a 1 SD change from the control mean in neurodevelopmental impairments was selected under an assumption that it represents a minimal biologically sign ificant response level.

UF S – A value of 1 was applied when dosing occurred during gestation or the early postnatal period that is relevant to developmental effects (U.S. EPA, 1991a); 10 when the POD is based on a subchronic study (studies

in this table

, other than the developmental toxicity studies, were 42

- 90 days in duration) to account for the
possibi lity that longer exposure may induce effects at a lower dose

. UF D – A value of 3 was applied to account for database deficiencies including the lack of a standard multigenerational study or extended 1

- generation study that includes exposure from premating through lactation, considering that benzo[a]pyrene has been shown to affect fertility in adult male and female animals by multiple routes of exposure (s ee Section 1.1.2). Also, the lack of a study examining functional neurological endpoints following a more comprehensive period of developmental exposure (i.e.

, gestation through lactation) is a data gap, considering human and animal evidence indicating a ltered neurological development (see Section 1.1.1).

a As recommended in EPA's A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA, 2002), the derivation of a reference value that involves application of the full 10

- fold uncertainty factor in four or more areas of extrapolation should be avoided.Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Figure 2

- 1

. Candidate RfDs with corresponding POD and composite UF 1

2



### 2.1.4. Derivation of O rgan/ S ystem

- specific R eference D oses 3

Table 2

- 3 distills the candidate re ference doses from Table 2

- 2 in t o a single value for each 4
organ or system. These organ or system

- specific reference values may be useful for subsequent 5
cumulative risk assessments that consider the combined effect of multiple age nts acting at a 6

common site. 7Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE De velopmental Toxicity 1

The candidate RfD based on neurodevelopmental impairment in rats ( Chen et al.

, 2012) was 2
selected as the organ/system

- specific RfD representing developmental toxicity. This candidate RfD 3

was s elected because it is associated with the application of the smaller composite uncertainty 4

factor and because similar effects were replicated across other studies

. 5

Reproductive Toxicity 6

The candidate RfD based on decreased ovary weight and ovarian follicle number s in rats 7

from the Xu et al. (2010 ) study was selected as the organ /system

- specific RfD representing 8

reproductive toxicity

. The ovarian effects are supported by a large database of animal studies and 9

human studies of exposure to benzo[a]pyrene and PAH mix tures

. The data supporting cervical 10

effects associated with oral benzo[a]pyrene exposure are limited to a single study; however the 11
finding is supported by corollary findings after i.p. exposure and by studies in humans

. 12

Immunotoxicity 13

The candidate RfD s based on decreased thymus weight (Kroese et al., 2001) and serum IgM 14

levels in rats (DeJong et al., 1999 ) were selected as the organ /system

- specific RfD representing 15

immunotoxicity. The observed decreases in thymus weight, IgM and IgA levels, and num ber of B 16
cells associated with exposure to benzo[a]pyrene were determined to be representative of 17

immunotoxicity. In combination, these effects provide more ro bust evidence of i mmunotoxicity. 18

The candidate RfDs for decreased thymus weight (Kroese et al. , 2001) and serum IgM levels in rats 19
(DeJong et al., 1999 ) were equal and provided the most senstitive candidate RfDs, thus these 20

candidate RfDs were selected as the organ/system

- specific RfDs representing immunotoxicity

. 21

Table 2

- 3

. Organ/system

- specific RfDs and proposed overall RfD for 22 benzo[a]pyrene 23

E ffect Basis RfD (mg/kg

- d) Confidence

Developmental Neurodevelopmental impairments 2 x 10 - 4 MEDIUM

Reproductive Decreased ovary weight and ovarian follicles 4 x 10 - 4 MEDIUM

Immunological Decreased thymus weight and serum IgM 2 x 10 - 3 LOW

Proposed Overall RfD Developmental toxicity 2 x 10 - 4 MEDIUM



### 2.1.5. Selection of the P roposed Overall R eference D ose 24

To estimate an exposure level below which the effects identified as potential hazards from 25

benzo[a]pyrene exposure are not expected to occur, the lowest organ/system

- specific RfD (2 x 10

- 4 26

mg/kg

- day) is proposed as the overall reference dose for benzo[a]pyrene. This value, based on 27Toxicological Review of Benzo[a]pyrene

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susceptible lifestage is supported by a large number of animal and human studies. 2



### 2.1.6. Confidence Statement 3

A confidence level of high, medium, or low is assigned to the study used to derive the Rf C

, 4

the overall database, and the Rf C itself, as described in Section 4.3.9.2 of EPA’s Methods for 5
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry ( U.S. EPA, 6

1994 ). 7
Confidence in the principal study (Chen et al., 2012) is medium

- to

- high. The study design 8
included randomized experimental testing, blinded observations, culling of pups to account for 9

nutritional availability, treatment

- randomization ; and controls for litter and nursing bias. S ome 10

informative experimental details were

, however , omitted including the sensitivity of some assays at 11
the indicated developmental ages and lack of reporting gender

- specific data for all outcomes. 12

Notably, these study limitations do not apply to the endpoint chosen to d erive the RfD, and the 13

overall methods and reporting are considered sufficient. Confidence in the database is medium, 14
primarily due to the lack of a multigenerational reproductive toxicity study given the sensitivity to 15

benzo[a] pyrene during development. Reflecting medium

- to

- high confidence in the principal study 16

and medium confidence in the database, confidence in the RfD is medium. 17



### 2.1.7. Previous Reference Dose 18

An RfD was not derived in the previous IRIS assessment. 19Toxicological Review of Benzo[a]pyrene

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## 2.2. Inhalation Reference Concentration for Eff ects O ther T han Cancer 1

The RfC (expressed in units of mg/m 3 ) is defined as an estimate (with uncertainty spanning 2
perhaps an order of magnitude) of a continuous inhalation exposure to the human population 3

(including sensitive subgroups) that is likely to b e without an appreciable risk of deleterious effects 4
during a lifetime. It can be derived from a NOAEL, LOAEL, or the 95 percent lower bound on the 5
benchmark concentration (BMCL), with UFs generally applied to reflect limitations of the data used. 6



### 2.2.1. Identif

ication of Studies and Effects for Dose

- Response Analysis 7

In Section 1.2 .1

, developmental and reproductive toxicities were highlighted as hazards of 8

benzo[a]pyrene exposure by the inhalation route. Studies within each effect category were 9

evaluated using general study quality characteristics (as discussed in Section 6 of the Preamble) to 10

help inform the selection of studies from which to derive toxicity values

. Rationale s for selecting 11

the studies and effects to represent each of these hazards are summarized below. 12

Human studies of environmental PAH mixtures across multiple cohorts have observed 13

developmental and reproductive effects following prenatal exposure ; however, these studies are 14

li mited by exposure to complex mixtures of PAHs and within individual studies there may have 15

been more than one rou te of exposure

. In addition, t he available human studies that utilized 16

benzo[a]pyrene

- DNA adducts as the exposure metric do not provide extern al exposure levels of 17

benzo[a]pyrene from which to derive a reference concentration

. Although preferred for derivation 18

of reference values, human studies were not considered because of the contribution to the observed 19

hazard of multiple PAHs across multip le routes of exposure. 20

Animal studies were evaluated to determine which provided the most relevant r outes and 21

durations of exposure; multiple exposure levels to provide information about the shape of the dose 22

response curve; and power to detect effects at low exposure levels

. The only chronic inhalation 23

study available for benzo[a]pyrene , Thyssen et al. (1981), was designed as a cancer bioassay and 24

did not report other effects . However, several reproductive and developmental toxicity studies are 25

availabl e in which effects on fetal survival and the male reproductive system have been observed. 26

Developmental toxicity 27

Developmental toxicity, as represented by decreased fetal survival and developmental 28

neurotoxicity , was obse rved by Archibong et al. (2002), Wu et al. (2003) , and Wormley et al. 29

(2004)

. Wu et al. (2003) was not considered for dose

- response analysis due to lack of study details 30

related to number of dams and litters per group and lack of reporting of numerical data. From the 31

remaining studies demonstrating developmental toxicity, the studies conducted by Archibong et al. 32

(2002) and Wormley et al. (2004) were identified as the most informative studies for dose

33

response analysis. A rchibong et al. (2002) observed biologically significant effects at the lowest 34

dose tested by the inhalation route (i.e., LOAEL of 25 μg/m 3 ) . This study indicates that the 35
developing fetus is a sensitive target following inhalation exposure to benzo[a]pyrene. The 36Toxicological Review of Benzo[a]pyrene

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(e.g., decreased survival of litters in mice following in utero exposure to benzo[a]pyrene on GD 7

- 2

16 ) (MacKenzie and Angevine, 1981). In addition, a single exposure inhalation study by Wormley 3

et al. (2004) demonstrated developmental toxicity, represented by decreased pups/litter and 4
electrophysiological changes in the hippocampus, as a result of gestational exposure. T he single 5

exposure concentration used by Wormley et al. study was the equivalent of the high

- dose exposure 6
concentration applied in the Archibong et al. (2002) and Wu et al. (2003) studies , thus the endpoint 7
of fetal survival observed by Wormley et al. (2004) was not considered for dose

- response analysis

. 8

However, similar to oral studie s of benzo[a]pyrene exposure, Wormley et al. (2004) observed 9
effects indicative of developmental neurotoxicity and therefore these effects were considered 10

further for dose

- response analysis. 11

Reproductive toxicity 12

Reproductive toxicity, as represented b y reductions in sperm quality, both count and 13

motility, and testis weights in adults, was observed by Archibong et al. (2008) and Ramesh et al. 14
(2008) and Archibong et al. (2002)

. Archibong et al. ( 2008 ) and Ramesh et al. ( 2008) reported the 15

results of a single exposure, subchronic inhalation exposure study in male rats

. This subchronic 16
study was of sufficient duration and possessed adequate power to detect effects, but utilized a 17
single exposure concentration which is less informative for dose

- response a nalysis than a design 18

using multiple exposure concentrations

. The endpoints of decreased testes weight and sperm 19
count and motility reported in Archibong et al. (2008) were selected for dose

- response analysis as 20

both represent sensitive endpoints of male reproductive toxicity and are indicator s of potentially 21

decreased fertility . These effects are also consistent with human studies in PAH exposed 22

populations as effects on male fertility and semen quality have been demonstrated in 23
epidemiological studies o f smokers (revie wed by Soares and Melo, 2008). 24



### 2.2.2. Methods of A nalysis 25

Data for decreased fetal survival (Archibong et al., 2002 ) were not amenable to BMD 26

modeling due to the pattern of variability (heterogeneous variances) in the data set ; the response at 27

t he lowest exposure showed the greatest variability . Th erefore, the LOAEL from this stud y was 28

used as the POD for dose

- response analysis

. Wormley et al. (2004) and Archibong et al. (2008)

, 29

using only one exposure level, were judged not to support dose

- res ponse modeling due to the lack 30

of understanding of the underlying dose

- response relationship (i.e., limited database). LOAELs 31

were also used as the PODs for dose

- respopnse analysis. 32
By definition, the RfC is intended to apply to continuous lifetime exposu res for humans (U.S. 33

EPA, 1994 b ). EPA recommends that adjusted continuous exposures be used for inhalation 34

developmental toxicity studies as well as for studies of longer durations (U.S. EPA, 2002). The 35Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE LOAEL s identified from Archibong et al. (2002), Archibong et al. (2008), and Wormley et al. (2004) 1

were adjusted to account for the discontinuous daily exposure as follows: 2

3

POD ADJ = POD × hours exposed per day/24 hours 4

= LOAEL × (duration of exposure /24 hours) 5

= POD ADJ 6

7

Next, t he human equivalent concentration (HEC) was calculated from the POD ADJ by 8

multiplying by a dosimetric adjustment factor (DAF), which, in this case, was the regional deposited 9
dose ratio (RDDR ER ) for extrarespiratory (i.e., systemic) effects as described in Methods for 10

Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 11

1994b). The observed developmental effects are considered systemic in nature (i.e., 12
extrarespiratory) and the normalizing factor for extrarespiratory effects of particles is body weight. 13

T he RDDR ER was calculated as follows: 14

15

where: 16
BW = body weight (kg) 17

V E = ventilation rate (L/minute) 18

F TOT = total fractional deposition 19

20
The total fractional deposition (F TOT ) includes part icle deposition in the nasal

- pharyngeal, 21

tracheobronchial, and pulmonary regions. F TOT for both animals and humans was calculated using 22

the Multi

- Path Particle Dosimetry model, a computational model used for estimating human and rat 23

airway particle deposition and clearance [Multi

- Path Particle Dosimetry (MPPD); Version 2.0 © 24

2006, publicly available through the Hamner Institute]. F TOT was based on the average particle size 25

of 1.7 ± 0.085 (MMAD ± geometric SD) as reported in Wu et al. (2003 ) for the exposure range 25

- 26

100 um 3 . For the model runs, the Yeh

- Schum 5

- lobe model was used for the human and the 27
asymmetric multiple path model was used for the rat (see Appendix C for MPPD model output). 28

Both models were run under nasal breathing scenarios with the inhalability adjustment selected. A 29

geometric SD of 1 was used as the default by the model because the reported geometric SD of 0.085 30

was ≤1.05. 31

The human parameters used in the model for calculating F TOT and in the subsequent 32

calculation of the POD HEC were as follows: human body weight, 70 kg; V E , 13.8 L/minute; breathing 33

frequency, 16 p er minute; tidal volume, 860 mL; functional residual capacity, 3,300 mL; and upper 34
respiratory tract volume, 50 mL. Although the most sensitive population in Archibong et al. (2002) 35

and Wormley et al. (2004) studies is the developing fetus, the adult rat dams were directly exposed. 36

Thus, adult rat parameters were used in the calculation of the HEC. The parameters used for the rat 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE were body weight, 0.25 kg ( a generic weight for male and female rats ) ; V E , 0.18 L/minute; 1
breathing frequency, 102 per minute ; tidal volume, 1.8 mL; functional residual capacity, 4 mL; and 2
upper respiratory tract volume, 4.42 mL. All other parameters were set to default value s (see 3

Appendix C). 4

Under these conditions, the MPPD model calculated F TOT values of 0.621 for the human and 5
0.181 for the rat. Using the above equation, the RDDR ER was calculated to be 1.1. 6

From this, the POD HEC was calculated as follows: 7

POD HEC = POD ADJ × RDDR ER 8

9

Table 2

- 4 summarizes the sequence of calculations leading to the derivation of a human

10

equi valent concentration for each data set discussed above. 11

Table 2

- 4 . Summary of Derivation of Point s of Departure 12

Endpoint and

Reference Study Design Model BMR BMC (μg/m 3 ) BMCL

(μg/m 3 ) POD ADJ a

(μg/m 3 ) POD HEC b

(μg/m 3 )

DEVELOPMENTAL

Decreased fetal survival Archibong et al., 2002 Pregnant rats GD 11

- 20, 4hrs/d 7d/wk LOAEL (25 μg/m 3 ) 4.2 4.6

Decreased long term
potentiation in hippocampus Wormley et al., 2004 Pregnant rats GD 11

- 21, 4 hrs/d, 7d/wk LOAEL (100 μg/m 3 ) 16.7 18.3

REPRODUCTIVE

Decreased testis weight Archibong et al., 2008 Male rats, 60d, 4hrs/d, 7d/wk LOAEL (75 μg/m 3 ) 12.5 13.8

Decreased sperm count and motility Archibong et al., 2008 Male rats, 60d, 4hrs/d, 7d/wk LOAEL (75 μg/m 3 ) 12.5 13.8

a PODs were ad j usted for continuous daily exposure: POD ADJ = POD × hours exposed per day/24 hours 13

b POD HEC calculated by adjusting the POD ADJ by the regional deposited dose ratio calculated using particle size 14 reported in Hood et al. (2000) using MPPD software as detailed in Section 2.2.2. and Appendix C in the 15 Supplemental Information. 16



### 2.2.3. Derivation of Candidate Reference C oncentrations 17

Table 2

- 5 is a continuation of Table 2

- 4 and summarizes the application of uncertainty 18

factors to each POD to derive a candidate reference concentration for each data set. The selection 19

of uncertainty factors was based on EPA’s A Review of the Reference Dose and R eference 20

Concentration Processes ( U.S. EPA, 2002; Section 4.4.5 ) and is described in the Section 7.6 of the 21

Preamble. Figure 2

- 2 presents graphically these candidate reference doses, uncertainty factors, and 22

points of departure, with each bar corresponding to one data set described in Tables 2

- 4 and 2

- 5. 23Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 2

- 5

. E ffects and corresponding derivation of candidate Rf C s 1

Endpoint POD HEC (μg/m 3 ) POD

type UF A UF H UF L UF S UF D Composite

UF Candidate

RfC (m g/m 3 ) a

DEVELOPMENTAL

Decreased fetal survival

in rats Archibong et al., 2002 4.6 LOAEL 3 10 10 1 10 3000 2 x 10 - 6

Decreased long term
potentiation in the hippocampus of rats Wormley et al., 2004 18.3 LOAEL 3 10 10 1 10 3000 6 x 10 - 6

REPRODUCTIVE

Decreased testis weight

in rats Archibong et al., 2008 13.8 LOAEL 3 10 10 10 10 30000 Not calculated

due to UF >

3000 b

Decreased sperm count and motility in rats Archibong et al., 2008 13.8 LOAEL 3 10 10 10 10 30000 Not calculated

due to UF >

3000 b

UF A – 3 to account for residual toxicodynamic uncertainties when an HEC was calculated by inhalation particle dosimetry methods (U.S. EPA, 1994b) , as is the case with benzo[a]pyrene.

UF H – 10 to account for potentially susceptible individuals when adequate information is not available to quantitatively estimate variability in human susceptibility . In the case of benzo[a]pyrene, Insufficient information is available to quantitatively estimate variability in human susceptibility to benzo(a)pyrene.

UF L – 1 wh en the POD is based on BMD modeling or a NOAEL; 10 or 3 when the POD is a LOAEL . In the case of benzo[a]pyrene, UF L of 10 was applied to account for the use of a LOAEL. A NOAEL was not identified for decreased fetal survival observed by Archibong et al ( 2002). At the lowest dose, benzo[a]pyrene treated dams gave birth to 15% fewer pups compared to dams treated with vehicle alone (carbon black particles). Due to the lack of a NOAEL and the inability to model the data set for decreased fetal survival, a UF of 10 was applied to extrapolate to a NOAEL.

UF S – 1 when dosing occurred during gestation or the early postnatal period that is relevant to developmental effects (U.S. EPA, 1991a); 10 when the POD is based on a subchronic study (studies in this tabl e were 60 days in duration) to account for the possibility that longer exposure may induce effects at a lower dose

UF D – 10 to account for database deficiencies including the lack of a standard multigenerational study or extended

1

- generation study that includes exposure from premating through lactation, considering that benzo[a]pyrene has been shown to affect fertility in adu lt male and female animals by multiple routes of exposure (see Section 1.1.2). According to EPA’s A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA, 2002; Section 4.4.5)

, t he UF D is intended to account for the potential for deriving an underprotective Rf D/RfC as a result of an incomplete characterizat ion of the chemical’s toxicity, but also
including a review of existing data that may also suggest that a lower reference value might result if additional

data were available. In the case of benzo[a]pyrene

, oral exposure studies have demonstrated effects at doses lower than those where mortality was observed in the inhalation stud y by Archibong et al. (2002). The lack of a study examining functional neurological endpoints following a more comprehensive perio d of developmental exposure (i.e. gestation through lactation) is also a data gap, considering human and animal evidence indicating altered neurological development (see Section 1.1.1).

a Candidate RfCs were converted from μg/m 3 to mg/m 3

b As recommended in EPA's A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA, 2002), the derivation of a reference value that involves application of the full 10

- fold uncertainty factor in four or more areas of extrapolation shou ld be avoided.Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Figure 2

- 2

. Candidate RfCs with corresponding POD and composite UF 1

2



### 2.2.4. Derivation of Organ/System

- specific Reference Concentrations 3

Table 2

- 6 distills the candidat e reference concentrations from Table 2

- 5 in t o a single value 4
for each organ or system. These organ or system

- specific reference values may be useful for 5

subsequent cumulative risk assessments that consider the combined effect of multiple a gents acting 6

at a common site. The candidate RfCs for reproductive toxicity derived from Archibong et al. 7

(2008) were not selected to represent reproductive toxicity because as recommended in EPA's A 8

Review of the Reference Dose and Reference Concentration Processes (U.S. EPA, 2002), the derivation 9

of a reference value that involves application of the full 10

- fold uncertainty factor in four or more 10

areas of extrapolation should be avoided. 11Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 2

- 6 . Organ/system

- specific RfCs and proposed overall RfC fo r 1 benzo[a]pyrene 2

Effect Basis RfC (mg/m 3 ) Confidence

Developmental Decreased fetal survival 2 x 10 - 6 low

- to

- medium

Reproductive Reductions in sperm parameters Not calculated NA

Proposed Overall RfC Decreased fetal survival 2 x 10 - 6 low

- to

- medium



### 2.2.5. Selection of the Proposed Overall Reference Concentration 3

The study by Archibong et al. (2002) was selected as the study used for the derivation of the 4

proposed overall RfC

, as it observed biologically significant effects at the lowest dose tested by the 5

i nhalation route. This study indicates that the developing fetus is a sensitive target following 6

inhalation exposure to benzo[a]pyrene and t he observed dec reases in number of pups/litter and 7

fetal survival/litter are the most sensitive noncancer effects ob served following inhalation 8

exposure to benzo[a]pyrene. A dditional support for this endpoint is provided by the o ral studies of 9

benzo[a]pyrene. A developmental/reproductive study conducted via the oral route in mice 10

observed decreased survival of litters , decreased pup weight, and decreased reproductive organ 11
weight following in utero exposure to benzo[a]pyrene on GD 7

– 16 (MacKenzie and Angevine, 12

1981). 13



### 2.2.6. Confidence Statement 14

A confidence level of high, medium, or low is assigned to the study used to derive the Rf C

, 15

the overall database, and the Rf C itself, as described in Section 4.3.9.2 of EPA’s Methods for 16
Derivation of Inhalation Reference Concentrations and Application of Inhalation D osimetry ( U.S. EPA, 17

1994 ). 18

The overall confidence in the RfC is low

- to

- medium. Confidence in the principal study 19
(Archibong et al., 2002) is medium. The conduct and reporting of this developmental dietary study 20

were adequate; however, a NOAEL was not ide ntified. Confidence in the database is low due to the 21

lack of a multigeneration toxicity study, the lack of studies on immune endpoints, and the lack of 22
information regarding subchronic and chronic inhalation exposure. However, confidence in the RfC 23

is b olstered by consistent systemic effects observed by the oral route (including reproductive and 24

developmental effects) and similar effects observed in human populations exposed to PAH 25

mixtures. Reflecting medium confidence in the principal study and low con fidence in the database, 26

confidence in the RfC is low

- to

- medium. 27



### 2.2.7. Previous Reference Concentration 28

An RfC was not derived in the previous IRIS assessment. 29Toxicological Review of Benzo[a]pyrene

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### 2.2.8. Uncertainties in the Derivation of the RfD and RfC 1

The following discussion identifies uncertainties a ssociated with the RfD and RfC for 2

benzo[a]pyrene. To derive the RfD, the UF approach (U.S. EPA, 2000, 1994b) was applied to a POD 3

based on neurodevelopmental impairments in rats treated developmentally. To derive the RfC, this 4

same approach was applied to a POD from a developmental study for the effect of decreased fetal 5
survival. UFs were applied to the POD to account for extrapolating from an animal bioassay to 6

human exposure, the likely existence of a diverse population of varying susceptibilities, a nd 7
database deficiencies. These extrapolations are carried out with default approaches given the lack 8

of data to inform individual steps. 9

The database for benzo[a]pyrene contains limited human data. The observation of effects 10

associated with benzo[a]py rene exposure in humans is complicated by several factors including the 11
existence of benzo[a]pyrene in the environment as one component of complex mixtures of PAHs, 12

exposure to benzo[a]pyrene by multiple routes of exposure within individual studies , and th e 13

difficulty in obtaining accurate exposure information. Data on the effects of benzo[a]pyrene alone 14
are derived from a large database of studies in animal models. The database for oral 15
benzo[a]pyrene exposure includes two chronic bioassays in rats and m ice, two developmental 16

studies in mice, and several subchronic studies in rats. 17
Although the database is adequate for RfD derivation, there is uncertainty associated with 18
the database including that the principal study for the RfD exposed animals during a relatively short 19

period of brain development potentially underestimating the magnitude of resulting neurological 20

effects. Also, the database lacks a comprehensive multi

- generation reproductive/developmental 21

toxicity studies and immune system endpoints wer e not evaluated in the available chronic

- duration 22
or developmental studies. Additionally, the only available chronic studies of oral or inhalational 23

exposure to benzo[a]pyrene focused primarily on neoplastic effects leaving non

- neoplastic effects 24

mostly u ncharacterized. 25
The only chronic inhalation study of benzo[a]pyrene was designed as a lifetime 26

carcinogenicity study and did not examine noncancer endpoints (Thyssen et al., 1981). However, 27

subchronic and short

- term inhalation studies are available, whic h examine developmental and 28
reproductive endpoints in rats. Developmental studies by the inhalation route identified 29

biologically significant reductions in the number of pups/litter and percent fetal survival and 30

possible neurodevelopmental effects (e.g., diminished electrophysiological responses to stimuli in 31

the hippocampus) following gestational exposures. Additionally, a 60

- day oral study in male rats 32
reported male reproductive effects (e.g., decreased testes weight and sperm production and 33

motility), but provides limited information to characterize dose

- response relationships with 34

chronic exposure scenarios. One area of uncertainty pertains to the lack of information regarding 35
fertility in animals exposed gestationally to benzo[a]pyrene, especially i n light of developmental 36

studies by the oral route indicating reduced fertility in the F1 generation and decreased 37

reproductive organ weights. The database also lacks a multigenerational reproductive study via the 38Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE inhalation route. Areas of uncertainty i nclude the lack of chronic inhalation studies focusing on 1

noncancer effects, limited data on dose

- response relationships for impaired male or female fertility 2
with gestational exposure or across several generations, and limited data on immune system 3

endpoi nts with chronic exposure to benzo[a]pyrene. 4

The toxicokinetic and toxicodynamic differences for benzo[a]pyrene between the animal 5

species in which the POD was derived and humans are unknown. PBPK models can be useful for 6

the evaluation of interspecies to xicokinetics; however, the benzo[a]pyrene database lacks an 7

adequate model that would inform potential differences. There is some evidence from the oral 8

toxicity data that mice may be more susceptible than rats to some benzo[a]pyrene effects (such as 9

ovot ocity [Borman et al., 2000]), although the underlying mechanistic basis of this apparent 10

difference is not understood. Most importantly, it is unknown which animal species may be more 11

comparable to humans. 12



## 2.3. Oral Slope Factor for Cancer 13



### 2.3.1. Analysis of Carcinogenicity Data 14

The database for benzo[a]pyrene contains numerous cancer bioassays that identify tumors, 15
primarily of the alimentary tract including the forestomach, following oral exposure in rodents. 16

Three 2

- year oral bioassays are available that a ssociate lifetime benzo[a]pyrene exposure with 17
carcinogenicity at multiple sites: forestomach, liver, oral cavity, jejunum, kidney, auditory canal 18
(Zymbal’s gland) tumors, and skin or mammary gland tumors in male and female Wistar rats 19

(Kroese et al., 2001 ); forestomach tumors in male and female Sprague

- Dawley rats (Brune et al., 20

1 981); and forestomach, esophageal , tongue, and larynx tumors in female B6C3F 1 mice (Beland and 21

Culp, 1998; additional results reported by Culp et al., 1998). 22

In addition to the se 2

- year cancer bioassays, there are studies available that provide 23

supporting evidence of carcinogenicity but are less suitable for dose

- response analysis due to one 24

or more limitation in study design : (1) no vehicle control group

, (2) only one benzo[a]pyr ene dose 25

group
, or (3) a one

- time exposure to benzo[a]pyrene (Benjamin et al., 1988; Robinson et al., 1987; 26

El Bayoumy, 1985; Wattenberg, 1974; Field and Roe, 1970; Roe et al., 1970; Biancifiori et al., 1967; 27

Chouroulinkov et al., 1967; Berenblum and Haran , 1955). Of the controlled, multiple dose

- group, 28

repeat

- dosing studies that remain, most treated animals for <1 year, which is less optimal for 29

extrapolating to a lifetime exposure (Weyand et al., 1995; Triolo et al., 1977; Fedorenko and 30

Yansheva, 1967; N eal and Rigdon, 1967). 31
Brune et al. (1981) dosed rats (32/sex/group) with several concentration s of 32

benzo[a]pyrene dissolved in a 1.5% caffeine solution, sometimes as infrequently as once every 33

ninth day, for up to 2 years and observed increased forestom ach tumors. This study was not 34

selected for quantitation due to the nonstandard treatment protocol in comparison to the studies 35
conducted by Kroese et al. (2001) and Beland and Culp (1998). 36Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE The Kroese et al. (2001) and Beland and Culp (1998) studies were selected as the best 1

available studies for dose

- response analysis and extrapolation to lifetime cancer risk following oral 2
exposure to benzo[a]pyrene. The rat bioassay by Kroese et al. (2001) and the mouse bioassay by 3

Beland and Culp (1998) were conducted in accordance with Good Laboratory Practice principles as 4

established by the Organization for Economic Co

- operation and Development (OECD). These 5
studies included histological examinations for tumors in many different tissues, contained three 6

exposure le vels and controls, contained adequate numbers of animals per dose group 7
(~50/sex/group), treated animals for up to 2 years, and included detailed reporting of methods 8

and results (including individual animal data). 9
Details of the rat (Kroese et al., 2001) and female mouse (Beland and Culp, 1998) study 10

de signs are provided in Appendix B of the Supplemental Information . Dose

- related, statistically 11
significant increasing trends in tumors were noted at the following sites: 12

Squamous cell carcinomas (SCCs) or pa pillomas of the forestomach or oral cavity in male 13

and female rats; 14

SCCs or papillomas of the forestomach, tongue, larynx, or esophagus in female mice; 15

Auditory canal carcinomas in male and female rats; 16

Kidney urothelial carcinomas in male rats; 17

Jejunum/du odenum adenocarcinomas in female and male rats; 18

Hepatocellular adenomas or carcinomas in male and female rats; and 19

SCCs or basal cell tumors of the skin or mammary gland in male rats

. 20

These tumors were generally observed earlier during the study with incre asing exposure 21

levels, and showed statistically significantly increasing trends in incidence with increasing 22

exposure level (Cochran

- Armitage trend test, p ≤ 0.001). These data are summarized in Appendix C 23

of the Supplemental Information . As recommended by NTP (McConnell et al., 1986) and as outlined 24

in EPA’s Cancer Guidelines (U.S. EPA, 2005a) , etiologically similar tumor types (i.e., benign and 25

malignant tumors of the same cell type) were combined for these tabulations when it was judged 26

that the benign tumors could progress to the malignant form,. In addition, when one tumor type 27
occurred across several functionally related tissues, as with squamous cell tumors in the tongue, 28

esophagus, larynx and forestomach, or adenocarcinomas of the jejunum or duode num, these 29

incidences were also aggregated as counts of tumor

- bearing animals. 30
In the rat study (Kroese et al., 2001), the oral cavity and auditory canal were examined 31

histologically only if a lesion or tumor was observed grossly at necropsy. Consequently , dose

32

response analysis for these sites was not straightforward. Use of the number of tissues examined 33

histologically as the number at risk would tend to overestimate the incidence, because the 34

unexamined animals were much less likely to have a tumor. O n the other hand, use of all animals in 35

a group as the number at risk would tend to underestimate if any of the unexamined animals had 36

tumors that could only be detected microscopically. The oral cavity squamous cell tumors were 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE combined with those in the forestomach because both are part of the alimentary tract, recognizing 1
that there was some potential for underestimating this cancer risk. 2
The auditory canal tumors from the rat study were not considered for dose

- response 3

analysis , for several reasons. F irst, the control and lower dose groups were not thoroughly 4

examined, similar to the situation described above for oral cavity tumors. Unlike the oral cavity 5

tumors, the auditory canal tumors were not clearly related to any other site or tumor type, as th ey 6

were described as a mixture of squamous and sebaceous cells derived from pilosebaceous units. 7
The tumors were observed mainly in the high dose groups and were highly coincident with the oral 8

cavity and forestomach tumors. Because only one mid

- dose mal e had an auditory canal tumor 9
which did not also have a forestomach or oral cavity squamous cell tumor, and none were observed 10

in low

- dose male or female rats, the data are insufficient to conclude whether the auditory canal 11

tumors occur independently of o ther tumors . The investigators did not suggest that these tumors 12
were metastases from other sites (in which case the auditory canal tumors would be repetitions of 13

other tumors, or statistically dependent). Therefore dose

- response analysis was not pursue d for 14

this site, either separately or in combination with another tumor type. 15



### 2.3.2. Dose Response Analysis

– Adjustments and Extrapolations Methods 16

EPA ’s Cancer Guidelines (U.S. EPA, 2005a) recommend that the method used to characterize 17

and quantify cancer risk from a chemical is determined by what is known about the mode of action 18
of the carcinogen and the shape of the cancer dose

- response curve. The dose response is assumed 19

to be linear in the low

- dose range, when evidence supports a mutagenic mode of action b ecause of 20

DNA reactivity, or if another mode of action that is anticipated to be linear is applicable. In this 21

assessment, EPA concluded that benzo[a]pyrene carcinogenicity involves a mutagenic mode of 22
action ( as discussed in Section 1.1.5. ). Thus, a lin ear approach to low

- dose extrapolation was used. 23

The high

- dose groups of both the rat and mouse studies were dead or moribund by week 79 24

for female mice, week 72 for female rats, and week 76 for male rats. Due to the occurrence of 25

multiple tumor types, ea rlier occurrence with increasing exposure, and early termination of the 26

high

- dose group in each study, methods that can reflect the influence of competing risks and 27

intercurrent mortality on site

- specific tumor incidence rates are preferred. In this case EPA has 28
used the multistage

- Weibull model which incorporates the time at which death

- with

- tumor 29

occurred as well as the dose. 30

Adjustments for approximating human equivalent slope factors applicable for continuous 31

exposure were applied prior to dose

- respo nse modeling. First, continuous daily exposure for the 32
gavage study in rats (Kroese et al., 2001) was estimated by multiplying each administered dose by 33

(5 days)/(7 days) = 0.71, under the assumption of equal cumulative exposure yielding equivalent 34

outcom es. Dosing was continuous in the mouse diet study (Beland and Culp, 1998), so no 35
continuous adjustment was necessary. Next, consistent with the EPA’s Cancer Guidelines (U.S. EPA, 36

2005a), an adjustment for cross

- species scaling was applied to address toxi cological equivalence 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE across species. Following EPA’s cross

- species scaling methodology, the time

- weighted daily 1
average doses were converted to human equivalent doses (HEDs) on the basis of (body weight) 3/4 2
(U.S. EPA, 1992). This was accomplished by mul tiplying administered doses by (animal body 3

weight (kg)/70 kg) 0.25 (U.S. EPA, 1992), where the animal body weights were TWAs from each 4
group, and the U.S. EPA (1988) reference body weight for humans is 70 kg. It was not necessary to 5

adjust the administere d doses for lifetime equivalent exposure prior to modeling for the groups 6
terminated early, because the multistage

- Weibull model characterizes the tumor incidence as a 7

function of time, from which it provides an extrapolation to lifetime exposure. 8

Details of the modeling can be found in Appendix C of the Supplemental Information

. PODs 9
for estimating low

- dose risk were identified at doses at the lower end of the observed data, 10

generally corresponding to 10% extra risk. The lifetime oral cancer slope factor for humans is 11

defined as the slope of the line from the lower 95% bound on the exposure at the POD to the control 12
response (slope factor = 0.1/BMDL 10 ). This slope, a 95% upper confidence limit (UCL) represents a 13

plausible upper bound on the true risk. 14



### 2.3.3. De rivation of the Oral Slope Factor 15

The PODs estimated for each tumor site are summarized in Table 2

- 7 . Details of the model 16

selection process are provided in Appendix C of the Supplemental Information . Using linear 17

extrapolation from the BMDL 10 , human eq uivalent oral slope factors were derived for each 18
gender/tumor site combination and are listed in Table 2

- 7

. 19Toxicological Review of Benzo[a]pyrene

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- 25 DRAFT

— DO NOT CITE OR QUOTE Table 2

- 7 . Summary of the Oral Slope Factor Derivations

Tumor Species/

Sex Selected

Model BMR BMD

(mg/kg

- d) POD=

BMDL (mg/kg

- d) Slope factor a

(mg/kg

- d) - 1

Forestomach, oral cavity: squamous cell tumors
Kroese et al., 2001 Male

Wistar rats Multistage

Weibull 10% 0.453 0.281 0.4

0.5 b Hepatocellular adenomas or

carcinomas
Kroese et al., 2001 Male

Wistar rats Multistage

Weibull 10% 0.651 0.449 0.2

Jejunum/duodenum

adenocarcinomas
Kroese et al., 2001 Male

Wistar rats Multistage

Weibull 10% 3.03 2.38 0.04

Kidney: urothelial carcinomas
Kroese et al., 2001 Male

Wistar rats Multistage

Weibull 10% 4.65 2.50 0.04

Skin, mammary:

Basal cell tumors Squamous cell tumors
Kroese et al., 2001 Male

Wistar rats Multistage

Weibull 10%

2.86

2.64 2.35

1.77 0.04

0.06

Forestomach, oral cavity: squamous cell tumors
Kroese et al., 2001 Female

Wistar rats Multistage

Weibull 10% 0.539 0.328 0.3

0.3 b Hepatocellular adenomas or

carcinomas Kroese et al., 2001 Female

Wistar rats Multistage

Weibull 10% 0.575 0.507 0.2

Jejunum/duodenum

adenocarcinomas
Kroese et al., 2001 Female

Wistar rats Multistage

Weibull 10% 3.43 1.95 0.05

Forestomach, esophagus, tongue, larynx: squamous cell

tumors Beland and Culp 1998 Female

B6C3F1

Mice Multistage

Weibull 10%

0.127 0.071 1 1

a Human equivalent slope factor = 0.1/BMDL10HED; see Appendix C of the Supplemental Information for details of modeling results.

b Estimates of risk of incurring at least one of the tumor types listed.

1

Oral slope factors derived from rat bioassay data varied by gender and tumor site ( Table 2

- 2

7 ). Values ranged from 0.04 per mg/kg

- day, based on kidney tumors in males, to 0.4 per mg/kg

3
day, based on alimentary tract tumors in males. Slope factors based on liver tumors in male and 4
female rats (0.2 per mg/kg

- day) were only slightly lower than slope factors based on alimentary 5

tract tumors (0.2

- 0.3 per mg/kg

- day) . The oral slope factor for alimentary tract tumors in female 6
mice was highest at 1 per mg/kg

- day ( Table 2

- 7 ), which was approximately fourfold higher than 7
the oral slope factor derived from the alimentary tract tumors in male rats. 8Toxicological Review of Benzo[a]pyrene

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- 26 DRAFT

— DO NOT CITE OR QUOTE Although the time

- to

- tumor modeling helps to account for competing risks associated with 1

decreased survival times and other causes of death including other tumors, considering the tumor 2
sites individually still does not convey the total amount of risk pot entially arising from the 3

sensitivity of multiple sites

— that is, the risk of developing any combination of the increased tumor 4
types, not just the risk of developing all simultaneously. A method involving the assumption that 5
the variability in the slope f actors could be characterized by a normal distribution is detailed in 6

Appendix C of the Supplemental Information

. The resulting composite slope factor for all tumor 7
types for male rats was 0.5 per mg/kg

- day, about 25 % higher than the slope factor based o n the 8

most sensitive tumor site, oral cavity and forestomach, while for female rats, the composite slope 9
factor was equivalent to that for the most sensitive site ( Table 2

- 7 ; see Appendix C of Supplemental 10

Information for composite slope factor estimates). 11

The overall risk estimates from rats and mice spanned about a three fold range. As there are 12
no data to support any one result as most relevant for extrapolating to humans, the most sensitive 13

result was used to derive the oral slope factor. The recommend ed slope factor for assessing human 14

cancer risk associated with chronic oral exposure to benzo[a]pyrene is 1 per mg/kg

- day , based on 15

the alimentary tract tumor response in female B6C3F 1 mice. 16



### 2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 17

The ora l slope factor for benzo[a]pyrene was based on the increased incidence of 18
alimentary tract tumors, including forestomach tumors, observed in a lifetime dietary study in mice 19

(Beland and Culp 1998). EPA has considered the uncertainty associated with the re levance of 20

forestomach tumors for projecting human risk from benzo[a ]pyrene exposure. The rodent 21

forestomach serves to store foods and liquids for several hours before contents continue to the 22
stomach for further digestion (Clayson et al., 1990; Grice et al., 1986). While humans do not have a 23

forestomach, squamous epithelial tissue similar to that seen in the rodent forestomach exists in the 24

oral cavity and upper two

- thirds of the esophagus in humans (IARC, 1999). However, due to the 25
storage function of the forestomach, tissue of the forest o mach may be exposed to benzo[a]pyrene 26

for longer durations than analogous human tissues in the oral cavity and esophagus. This suggests 27

that the rodent forestomach may be quantitatively more sensitive to development o f squamous 28
epithelial tumors in the forestomach compared to oral or esophageal tumors in humans. 29

Human studies, specifically associating exposure to benzo[a]pyrene with the alimentary 30

tract tumors are not currently available. However, benzo[a]pyrene

- DNA adducts have been 31

detected in oral and esophageal tissue obtained from smokers (reviewed by Phillips, 2002) and 32
several epidemiological studies have identified increased exposure to PAHs as an independent risk 33

factor for esophageal cancer ( Abedi

- Ardekani e t al., 2010; Szymanska et al., 2010; Wang et al., 2002; 34

Gustavsson et al., 1998a; Liu et al., 1997 ). 35

Uncertainty in the magnitude of the recommended oral slope factor is reflected to some 36

extent in the range of slope factors among tumors sites and specie s; the lowest and highest slope 37Toxicological Review of Benzo[a]pyrene

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- 27 DRAFT

— DO NOT CITE OR QUOTE factors by individual sites, as listed in Table 2

- 8 , show about a 35

- fold difference. However, 1

consideration of individual sites when multiple sites are affected perhaps overemphasizes an 2

expectation of site concordance. W hile the highest risk estimates were derived from the incidence 3

data for forestomach tumors in both rats and mice, the oral slope factor based on the mouse 4

alimentary tract data was about threefold higher than the overall oral slope factor based on male 5

ra t data ( Table 2

- 8 ). These comparisons show that the selection of target organ, animal species, 6

and interspecies extrapolation can impact the oral cancer risk estimate. However, all of the 7
activation pathways implicated in benzo[a]pyrene carcinogenicity h ave been observed in human 8

tissues, and associations have been made between the spectra of mutations in tumor tissues from 9

benzo[a]pyrene

- exposed animals and humans exposed to complex PAH mixtures containing 10

benzo[a]pyrene (see Section 1.1.5.)

. 11

Table 2

- 8

. Summary of uncertainties in the benzo[a]pyrene oral cancer slope 12
factor (OSF) 13

Consideration Impact on unit

risk Decision Justification

Target organ ↓ OSF, up to

fivefold, if alimentary tract

tumors not selected Alimentary tract tumors

(forestomach, esophagus, tongue, larynx) Tumor site is concordant across rats and mice

, increasing support for its relevance to humans

. As there are no data to support any one result as most relevant for extrapolating to h umans, the

most sensitive result for alimentary tract tumors was used to derive the oral slope factor.

Data set ↓ OSF ~threefold if

rat bioassay were

selected for OSF

derivation Beland and Culp (1998) Beland and Culp (1998) was a well conducted
study and used the lowest HEDs of the available cancer bioassays, reducing low

- dose extrapolation uncertainty

. D ose metric Alternatives could

↓ or ↑ slope factor Administered dose Experimental evidence supports a role for metabolism in toxicity, but actual responsible

metabolites have not been identified.

Cross

- species scaling Alternatives could

↓ or ↑ slope factor
(e.g., 3.5

- fold ↓
[scaling by body weight] or ↑ 2

- fold [scaling by BW 2/3 ]) BW 3/4 scaling (default

approach) There are no data to support alternatives.

Because the dose metric was not an area under
the curve, BW 3/4 scaling was used to calculate equivalent cumulative exposures for estimating equivalent human risks. While the true human correspondence is unknown, this overall approach is expected neither to over

- or underestimate human equivalent risks.

Dose

- response modeling Alternatives could

↓ or ↑ slope factor Multistage

- Weibull

model No biologically based models for benzo[a]pyrene were available. Because the multistage

- Weibull

model could address additional available data (time of death with tumo r, and whether a tumor caused the death of the animal), this model was superior to other available models.

Low

- dose extrapolation ↓ cancer risk

estimate would be expected with the application of Linear extrapolation

from POD (based on
mutagenic mode of

action ) Available mode of action data support linearity (mutagenicity is a primary mode of action of benzo[a]pyrene );Toxicological Review of Benzo[a]pyrene

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- 28 DRAFT

— DO NOT CITE OR QUOTE Consideration Impact on unit

risk Decision Justification

nonlinear low

- dose extrapolation

Statistical
uncertainty at

POD ↓ OSF 1.8

- fold if

BMD used as the

POD rather than

BMDL BMDL (preferred

approach for calculating plausible upper bound slope factor) Limited size of bioassay results in sampling variability; lower bound is 95% confidence interval on administered exposure at 10% extra risk of alimentary tract tumors.

Sensitiv e subpopulations ↑ OSF to unknown

extent ADAFs are

recommended for early life exposures No chemical

- specific data are available to determine the range of human toxicodynamic variability or sensitivity.



### 2.3.5. Previous Oral Slope Factor 1

The previous cancer assessment for benzo[a]pyrene was posted on the IRIS database in 2

1987. At that time, benzo[a]pyrene was classified as a probable human carcinogen (Group B2) 3

based on inadequate data in humans and sufficient data in animals via several routes of exposure. 4
An oral slope factor was derived from the geometric mean of four slope factor estimates based on 5
studies in Sprague

- Dawley rats (Brune et al., 1981) and CFW

- Swiss mice (Neal and Rigdon, 1967). 6
A single slope factor estimate of 11.7 per mg/kg

- day, using a linearized multistage procedure 7
applied to the combined incidence of forestomach, esophageal, and laryngeal tumors, was derived 8

from the Brune et al. (1981) study (see Section 1.1.5.2 for study details). Three modeling 9

procedures were used to derive risk estimates from the Neal and Rigdon (1967) bioassay (see 10
Section 1.1.5.2). In a report commissioned by EPA, Clement International Corporation (1990) fit a 11

two

- stage response model, based on exposure

- dependent changes in both transition rates and 12

growth ra tes of preneoplastic cells, to derive a value of 5.9 per mg/kg

- day. EPA (1991b) derived a 13
value of 9.0 per mg/kg

- day by linear extrapolation from the 10% response point to the background 14

response in a re

- analysis of the Clement model. Finally, using a We ibull

- type model to reflect less

15

than

- lifetime exposure to benzo[a]pyrene, the same assessment (U.S. EPA, 1991b) derived an 16

upper

- bound slope factor estimate of 4.5 per mg/kg

- day. The four slope factor estimates were 17

within threefold of each other and wer e judged to be of equal merit. Consequently, the geometric 18

mean of these four estimates, 7.3 per mg/kg

- day, was recommended as the oral slope factor. 19



## 2.4. Inhalation Unit Risk for Cancer 20



### 2.4.1. Analysis of Carcinogenicity Data 21

The inhalation database demonstrating ca rcinogenicity of benzo[a]pyrene consists of a 22

lifetime inhalation bioassay (Thyssen et al. 1981 ) and intratracheal instillation studies in hamsters 23

( Feron and Kruysse, 1978; Ketkar et al., 1978; Feron et al., 1973; Henry et al., 1973; Saffiotti et al., 24

197 2)

. 25
The bioassay by Thyssen et al. (1981) represents the only lifetime inhalation cancer 26

bioassay available for describing dose

- response relationships for cancer from inhaled 27Toxicological Review of Benzo[a]pyrene

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- 29 DRAFT

— DO NOT CITE OR QUOTE benzo[a]pyrene. Strengths of the study include the following: 1) exposure s was conductd for 2 1

years; 2) histological examination of organs was extensive and adequate; 3) multiple exposure 2

groups were used; and 4) individual animal pathology reports with time of death and tumor 3

detection data were available

. Increased incidences of benign and malignant tumors of the larynx 4

and pharynx were seen with increasing exposure concentration. Survival was decreased relative to 5
control only in the high

- dose exposure group; mean survival times in the 0, 2, and 10 mg/m 3 6

concentration groups wer e 96.4, 95.2, and 96.4 weeks, respectively, and 59.5 weeks in the 50 7
mg/m 3 group animals. Overall, tumors occurred earlier in the highest benzo[a]pyrene exposure 8

group than in the mid

- exposure group. 9
Limitations of the study include the following: (1) only male animals were test ed; (2) 10

particle analysis of aerosols was not reported (i.e., mass median aerodynamic diameter and 11
geometric SD were not reported) ; and (3) benzo[a]pyrene exposure occurred through the 12
inhalation of hygroscopic particles (benzo[a ]pyrene was adsorbed onto sodium chloride aerosols), 13

which may have a different deposition than benzo[a]pyrene adsorbed onto carbonaceous non

14

hygroscopic particles as is more typical in the environment . In addition, reported information 15

regarding the actu al exposure levels achieved during the study was incomplete. Analytic 16
measurements varied more than 20% from the average achieved for each dose group during the 17
first 79 weeks of the study, but were not available for the remaining 52 weeks that it took to 18

complete the experiment

. Despite the limitations, t he strengths of the study were judged to support 19
use of the data to derive an inhalation unit risk for benzo[a]pyrene. 20

The intratracheal instillation studies provide supporting evidence of carcinogenicit y of 21

inhaled benzo[a]pyrene; however, the use of intratracheal dosing alters the deposition, clearance, 22

and retention of substances

, and therefore studies utilizing this exposure technique are not as 23
useful for the quantitative extrapolation of cancer risk from the inhalation of benzo[a]pyrene in the 24

environment (Driscoll et al., 2000). 25



### 2.4.2. Dose Response Analysis

— Adjustments and Extrapolat ions Methods 26

B iologically based dose

- response models for benzo[a]pyene are not available. A simplified 27

version of the two

- stage carcinogenesis model proposed by Moolgavkar and Venzon (1979) and 28

M oolgavkar and Knudson (1981) has been applied to the Thyssen et al. (1981) data (U.S. EPA, 29

1990). However, the simplifications necessary to fit th e tumor incidence data reduced that model 30

to an empirical model , i.e., there were no biological data to inform estimates of cell proliferation 31

rates for background or in itiated cells . There were sufficient data to apply a time

- to

- tumor dose

32

response model, described in detail in Appendix C of the Supplemental Information

. 33

The tumor incidence data used for dose

- response modeling comprised the benign and 34

malignant tumors i n the pharynx and larynx. The pharynx and larynx are associated with the upper 35
digestive tract and the upper respiratory tract, respectively. However, these sites are close 36

anatomically and in some cases where both tissues were affected, the site of orig in could not be 37Toxicological Review of Benzo[a]pyrene

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- 30 DRAFT

— DO NOT CITE OR QUOTE distinguished (U.S. EPA, 1990). In addition, the b enign tumors (e.g., papillom as, polyps and 1

papillary polyps) were considered early stages of the squamous cell carcinoma s in these tissues 2
(U.S. EPA, 1990)

. Following EPA ’s Cancer G uideli nes (Section 2.2.2.1.2 ; U.S. EPA, 2005 a )

, incidence 3

data for animals with malignant or benign tumors originating from the same cell type were selected 4

for dose

- response modeling based on the assumption that the benign tumors could develop into 5
malignan cies

. 6
The availability of the raw chamber air monitoring data and individual times on study 7

allowed the calculation of TWA continuous exposures for each hamster (U.S. EPA, 1990) . Group 8

averages of these concentrations were 0, 0.25, 1.01, and 4.29 mg/m 3

, respectively, for the 0, 2, 10, 9
and 50 mg/m 3 study concentrations. 10

A toxicokinetic model to assist in cross

- species scaling of benzo[a]pyrene inhalation 11

exposure was not available. In addition, default dosimetry adjustments utilized in the 12

benzo[a]pyrene RfC calculation could not be applied because aerosol particle distribution data 13

were not available for the hamster inhalation bioassay by Thyssen et al. (1981). The carrier 14
particle used in Thyssen et al. (1981) was sodium chloride, a soluble hygroscopic particle, and the 15
approaches presented in the RfC methodology guidelines (U.S. EPA 1994b) were developed for 16
insoluble and nonhygroscopic particles. Consequently, without data to inform a basis for 17

extrapolation to humans, it was assumed that equal risk f or all species would be associated with 18

equal concentrations. This is equivalent to assuming that any metabolism of benzo[a]pyene is 19
directly proportional to breathing rate and that the deposition rate is equal between species. 20

A time

- to

- tumor dose

- res ponse model was fit to the TWA exposure concentrations and the 21

individual animal tumor and survival data for tumors in the larynx, pharynx, trachea, esophagus, 22

and forestomach

, using the computer software program MSW (U.S. EPA, 2010) as described in 23

Append ix C of the Supplemental Information

. Unlike in the available oral bioassays, Thyssen et al. 24

did not determine cause of death for any of the animals. Since the investigators for the oral 25

bioassays considered the same tumors to be fatal at least some of t he time, bounding estimates for 26
the Thyssen et al. (1981) data were developed by treating the tumors alternately as either all 27

incidental or all fatal. Model ing results are provided in Appendix C of the Supplemental 28

Information

. 29



### 2.4.3. Derivation of the Inhalation Unit Risk 30

Because benzo[a]pyrene carcinogenicity involves a mutagenic mode of action, linear low 31

dose extrapolation from the BMCL 10 was used (U.S. EPA, 2005a) to derive the inhalation unit risk

. 32

BMC s and BMCL s associated with an extra risk of 10% calculated using the multistage

- Weibull 33

model

, based on the occurrence of upper respiratory and upper digestive tract tumors in male 34

hamsters exposed to aerosols of benzo[a]pyrene for up to 132 weeks , were estimated under two 35
bounding assumptions. Th e results are summarized in Table 2

- 9

. At one extreme, taking the 36

tumors to have been the cause of death of the experimental animals, the BMC 10 and BMCL 10 were 37Toxicological Review of Benzo[a]pyrene

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- 31 DRAFT

— DO NOT CITE OR QUOTE 0.648 and 0.461 mg/m 3 , respectively. Then taking all of the tumors to have been incidental to t he 1

cause of death for each animal, the BMC 10 and BMCL 10 were 0.285 and 0.198 mg/m 3 , respectively, 2
about twofold lower than the first case. Because the tumors were unlikely to have all been fatal, the 3

lower BMDL 10 was selected for estimating the inhalation unit risk. Using linear extrapolation from 4

the BMCL 10 of 0. 198 mg/m 3 , an inhalation unit risk of 0.5 per mg/m 3

, or 5 × 10

- 4 per μg/m 3 5
(rounding to one significant digit)

, was calculated. 6

Table 2

- 9 . Summary of the Inhalation Unit Risk Derivation

Tumor Site and Context Species/

Sex Selected

Model BMR BMC

( mg/m 3 ) POD=

BMC L ( mg/m 3 ) Unit Risk a

( mg/m 3 ) - 1

Upper respiratory and digestive
tracts; all treated as cause of death Thyssen et al., 1981 Male

Hamsters Multistage

Weibull 10% 0.648 0.461 0.22

Upper respiratory and digestive
tracts; all treated as incidental to

death Thyssen et al., 1981 Male

Hamsters Multistage

Weibull 10% 0.285 0.198 0.51

a Human equivalent unit risk = 0.10/BMCL10; see Appendix C for details of modeling results.



### 2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk 7

Only one animal cancer bioassay by the inhalation route is available that describes the dose

8

response r elationship for respiratory tract tumors with chronic inhalation exposure to 9
benzo[a]pyrene (Thyssen et al., 1981). Although corroborative information on dose

- response 10

relationships in other animal species is lacking, the findings for upper respiratory tr act tumors are 11
consistent with findings in other hamster studies with intratracheal administration of 12

benzo[a]pyrene , and with some of the portal of entry effects in oral exposure studies . This study is 13

adequate for dose

- response analysis and derivation o f an inhalation unit risk estimate, but some 14

associated uncertainty includes the inability to apply U.S. EPA (1994b) dosimetry approaches to 15
extrapolate inhaled doses from animals to humans, due to the use of a soluble hygroscopic carrier 16
particle ( sodium chloride ) for the delivery of benzo[a]pyrene. One likely consequence of the use of 17

hygroscopic carrier particles would be t he growth of benzo[a]pyrene

- sodium chloride particles in 18

the humid environment of the respiratory tract resulting in increased parti cle diameter and 19

resulting changes in particle deposition, specifically, increased impaction in the upper respiratory 20
tract (Varghese and Gangamma, 2009; Asgharian, 2004; Ferron, 1994; Xu and Yu, 1985). Exposure 21

to benzo[a]pyrene in the environment predom inantly occurs via non

- soluble , non

- hygroscopic

, 22

carbonaceous particles (such as soot and diesel exhaust particles) . The potential impact of 23
differences in carrier particle on the magnitude of the inhalation unit risk is unknown. 24

The exposure measures ent ailed some uncertainty, in that the exposure concentrations for 25

about half of the study period were documented to vary above and below the average achieved by 26Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE about twofold in all exposed groups. One possibility that cannot be ruled out, due to lack of da ta, is 1

that peak exposure above some concentration may be associated with the observed effects. Use of 2
Haber’s Law (equal cumulative exposures being expected to lead to similar outcomes) to estimate 3

continuous equivalent exposures may be not justified. T here is not enough information available to 4

estimate an impact on the estimated unit risk due to this uncertainty. 5

Table 2

- 10 . Summary of uncertainties in the benzo[a]pyrene cancer inhalation 6
unit risk (IUR) 7

Consideration Impact on unit

risk Decision Justification

Data set and

target organ No IUR if Thyssen

et al. (1981) not

used Respiratory tract

tumors from Thyssen et al. (1981) The Thyssen et al. (1981) bioassay is the only lifetime inhalation cancer bioassay available for describing dose

- response relationships for cancer from inhaled benzo[a]pyrene. Intratracheal implantation study supports the

association of benzo[a]pyrene exp osure with respiratory tract tumors. Oral exposure

resulted in similar tumors.

Dose metric Alternatives could

↓ or ↑ unit risk Administered dose as

time

- weighted average Experimental evidence supports a role for metabolism in toxicity, but actual responsible

metabolites are not identified.

Cross

- species scaling Alternatives could

↓ or ↑ slope

factor The carcinogenicity

observed was portal

- of

entry, but data to implement RfC methodology (EPA, 1994) were lacking.

Cross

- species scaling was not applied. There are no data to support alternatives. Equal risk per μg/m3 is assumed.

Dose

- response modeling Alternatives could

↓ or ↑ slope

factor Multistage

- Weibull

model No biologically based models for benzo[a]pyrene were available. Because the multistage

- Weibull model could address additional available data (time of death with tumor), this model was superior to other
available empirical models

Low

- dose extrapolation ↓ cancer risk

estimate would be expected with the application of

nonlinear low

dose extrapolation Linear extrapolation

from the point of
departure (based on
mutagenic mode of

action ) Available mode of action data support linearity (mutagenicity is a primary mode of action of benzo[a]pyrene);

Statistical
uncertainty at

POD ↓ IUR 1.4

- fold if

BMC used as the

POD rather than

BMCL BMCL (preferred

approach for calculating plausible upper bound unit risk) Limited size of bioassay results in sampling variability; lower bound is 95% confidence interval on administered exposure at 10% extra risk of respiratory tract tumors.Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Sensitive subpopulations ↑ IUR to

unknown extent ADAFs are

recommended for early life exposu res No chemical

- specific data are available to determine the range of human toxicodynamic variability or sensitivity.



### 2.4.5. Previous Inhalation Unit Risk 1

An inhalation unit risk for benzo[a]pyrene was not previously available on IRIS. 2



## 2.5. Dermal Slope Factor for Cancer 3

E viden ce in humans and animal studies demonstrates an increased incidence of skin tumors 4
with increasing dermal exposure to polycyclic aromatic hydrocarbons (PAHs) mixtures or to 5

benzo[a]pyrene alone. Thus

, this assessment for benzo[a]pyren e d erives a dermal slope factor, a 6
quantitative risk estimate that is a plausible upper bound on the estimate of risk per μg/day of 7

dermal exposure. This is the first derivation of a dermal slope factor for the IRIS database. 8



### 2.5.1. Analysis of Carcinogenicity Data 9

Skin cancer in humans has been documented to result from occupational exposure to 10

complex mixtures of PAHs including benzo[a]pyrene, such as coal tar pitches, non

- refined mineral 11
oils, shale oils, and soot (IARC, 2010; Baan et al., 2009; WHO, 1998; Boffet ta et al., 1997; ATSDR, 12

1995); however, studies of human exposure to benzo[a]pyrene alone are not available. Repeated 13

application of benzo[a]pyrene to skin (in the absence of exogenous promoters) has been 14

demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs. However , no lifetime 15
chronic bioassays of dermal benzo[a]pyrene exposure were located in these species

. T herefore, 16

t his analysis focuses on chronic carcinogenicity bioassays in several strains of mice demonstrating 17

increasing in cidence of benign and malignant skin tumors, as well as earlier occurrence of tumors 18
with increasing exposure, following repeated dermal exposure to benzo[a]pyrene for the animals’ 19

lifetime. These studies involved 2

- or 3

- times/week exposure protocols, at least two exposure 20

levels plus controls, and histopathological examinations of the skin and other tissues (Sivak et al., 21

1997; Grimmer et al., 1984, 1983; Habs et al., 1984, 1980; Schmähl et al., 1977; Schmidt et al., 1973; 22

Roe et al., 1970; Poel, 1960, 1 959) (see Tables B

- 15 to B

- 23 in the Supplemental Information )

. 23

Additional carcinogenicity studies in mice were considered, but not used, in the dose

24

response analysis. These studies included: (1) early “skin painting” studies of benzo[a]pyrene 25

carcinoge nicity in mouse skin that did not report sufficient information to estimate the doses 26

applied (e.g., Wynder and Hoffman, 1959; Wynder et al., 1957); (2) initiation

- promotion studies 27

utilizing acute dosing of benzo[a]pyrene followed by repeated exposure to a potent tumor 28
promoter (sometimes benzo[a]pyrene at a lower dose than the initiation step), because they are 29

not as relevant for calculating risks from constant benzo[a]pyrene exposure alone; (3) bioassays 30

with one benzo[a]pyrene dose level or with only d ose levels inducing 90

– 100% incidence of mice 31

with tumors, because they provide relatively little information about the shape of the dose

32
response relationship (e.g., Wilson and Holland, 1988); (4) studies with shorter exposure and 33Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE observation periods (i.e ., <1 year) (Higginbotham et al., 1993; Albert et al., 1991; Nesnow et al., 1
1983; Emmett et al., 1981; Levin et al., 1977) which are less relevant for characterizing lifetime 2
risk; and (5) studies involving vehicles expected to interact with or enhance ben zo[a]pyrene 3

carcinogenicity (e.g., Bingham and Falk, 1969), which precludes assessment of carcinogenic risks of 4

benzo[a]pyrene alone. 5
Study designs and the extent of data reported varied across the studies identified above. 6

Because no particular studies s tood out as superior for developing a dermal slope factor, these data 7
sets were considered as a group in order to assess the overall evidence. Each data set was examined 8

for limitations that would not support dose

- response evaluation. O nly the data from P oel (1960) 9
were not considered further for modeling. The three data sets reported by Poel (1960) 10

demonstrated high mortality and 100% tumor incidence at doses higher than those in any of the 11

other studies under consideration, used the smallest group sizes of the available studies, and did 12
not provide sufficient information to estimate the duration of exposure for the dose groups with 13

less than 100% tumor incidence (see Table B

- 16 in the Supplemental Information ). 14

The remaining studies varied in complete re porting of: incidence of benign and malignant 15
tumors or only malignancies; times of first tumor occurrence, which informs whether earlier 16
mortality impacts the number at risk of tumor development; and duration of exposure by dose 17

group. With the exceptio n of Poel (1959), summarized next, the rest of the studies considered for 18

dose

- response modeling contained groups which survived the majority of a two

- year exposure 19

period. The se studies support dose

- response modeling, with some limitations that indicate the 20

slope factors could be underestimates (e.g., possible over

- estimation of animalas at risk or exposure 21

associated with tumor response) . The study by Poel (1959) included nine dose groups and also 22

demonstrated high mortality and tumor inc idence at higher exposure levels. All mice in dose 23
groups with >3.8 μg/application died by week 44 of the study. Therefore, these five dose groups 24

were omitted prior to dose

- response modeling because of the relatively large uncertainty 25

associated with ch aracterizing lifetime cancer risk from short exposures (< 1 year) . Four dose 26
groups in addition to control remained, all with exposures lasting at least 83 weeks. 27



### 2.5.2. Dose Response Analysis

– Adjustments and Extrapolations Methods 28

As with the oral and inhala tion benzo[a]pyrene carcinogenicity data, benzo[a]pyrene’s 29

dermal exposure carcinogenicity data were generally characterized by earlier occurrence of tumors 30

and increased mortality with increasing exposure level. However, individual animal data were not 31

a vailable for any of the identified studies. Therefore, time

- to

- tumor modeling was not possible. 32
Each of the dermal data sets was modeled using the multis tage model

. 33

F or all studies, administered doses were converted to average daily doses using the 34

equat ion: 35

36

Average dose/day = ( μ g/application) × (number of exposures/week ÷ 7 days/week) 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Next, lifetime equivalent doses were estimated for study groups that were reported to end 1
before 104 weeks by multiplying the relevant average daily doses by (L

e /104) 3

, where L

e is the 2
length of exposure, based on observations that tumor incidence tends to increase with age (Doll, 3

1971). Note that exposure periods <52 weeks would lead to a relatively large adjustment [i.e., 4

(52/104) 3 = 0.125, or an eightfold lower dose t han administered], reflecting considerable 5
uncertainty in lifetime equivalent dose estimates generated from relatively short studies. This 6
adjustment was relevant for Poel (1959), Roe et al. (1970), Habs et al. (1980), and Sivak et al. 7

(1997). 8

Concernin g the incidence data, s ome of these studies reported incidences of skin tumor

9
bearing animals for tumors thought to be malignant (Roe et al., 1970; Poel, 1959) or without clear 10

designation of the relative percentages of animals with carcinomas and papillom as (Habs et al., 11

1980). In the other studies, incidences of animals with skin papillomas and skin carcinomas were 12
clearly reported, showing that skin tumors from life

- time exposure to benzo [a]pyrene were 13

predominately malignant (Sivak et al., 1997; Habs e t al.,1984; Grimmer et al., 1984, 1983; Schmähl 14
et al., 1977; Schmidt et al., 1973). Following EPA ’s Cancer G uidelines (Section 2.2.2.1.2 ; U.S. EPA, 15

2005a ), incidence data for animals with malignant or benign skin tumors were selected for dose

16

response mo deling based on the evidence that skin papillomas can develop into malignant skin 17

tumors. The data sets as modeled are presented in Tables C

- 24 through C

- 27 in the Supplemental 18

Information. 19
The multistage

- cancer model was then fit to each data set. If there was no adequate fit using 20

the multistage

- cancer model, then other dichotomous models were considered. Because 21

benzo[a]pyrene is expected to cause cancer via a mutagenic mode of action, a linear approach to 22

low dose extrapolation from the PODs (i.e., BMDL 10 ) was used (U.S. EPA, 2005a) for candidate 23

dermal slope factors. 24



### 2.5.3. Derivation of the Dermal Slope Factor 25

Adequate model fits were found using the multistage model for all but one of the mouse 26

skin tumor incidence data sets (see Appendix C of the Supp lemental Information )

. T he data from 27

Grimmer et al. (1984) could not be adequately fit by the multistage model initially, and the other 28

dichotomous models available in BMDS were considered. Due to the supralinear shape of the dose

29

response data, only the log

- logistic and dichotomous Hill models provided adequate fits. Also due 30

to the supralinear dose

- response shape, the POD for slope factor derivation was identified n ear the 31

lowest response of ~70%, because of the lack of data to inform the dose

- response relationship at 32

lower doses

. 33

Dermal slope factors, calculated in units of risk per (μg/day) using linear extrapolation from 34

the BMDL values, ranged from 0.25 to 1.8 per μg/day, a roughly sevenfold range ( see Table 2

- 11 ). A 35
number of differences among stu dies may contribute to this range, including solvent choice, sex and 36

strain of mice studied, dose ranges, varying group sizes, and level of detail reported. Mouse strains 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE were not repeated across sexes among these studies, so it cannot be established whet her Swiss or 1

NRMI mice are more or less sensitive than other strains. In addition, different solvents were used 2
in the various studies with varying strain and sex combinations tested. For example, toluene was 3

used in one male study only, and all of the f emale studies used acetone. Thus, any possible impact 4

of the solvents used is not clear. As noted earlier, incomplete mortality information in several of 5

the female mouse studies (Grimmer et al., 1984, 1983; Habs et al., 1984, 1980; Schmähl et al., 1977 ; 6
Schmidt et al., 1973) suggests that the dermal slope factors derived from those studies may 7

underestimate cancer risk. 8

The estimates derived from the two studies in male mice (Poel, 1959; Sivak et al. 1997) 9
were at the higher end of the range of slope f actors derived. The available information is too 10

limited to conclude that males are more sensitive than females, in view of the similar result from 11
the Habs et al. (1984) data and the limitations of the remaining studies that suggested risk might be 12

under estimated. Nevertheless, the studies in male mice were among the stro nger studies. They 13

included at least three exposure levels, the lowest doses tested

, and better reporting of intercurrent 14

mortality. Both studies yielded very similar BMDs, with Poel ( 1959) providing a higher BMDL 10 , 15
associated with its larger group sizes (~50/dose group). Thus these two results were combined by 16
estimating the midpoint between the BMDL 10 s, 0.068 μg/day. 17

Table 2

- 11

. Summar y of Dermal Slope Factor Derivations

Reference Sex/ strain of

mouse S elected

Model a BMR BMD

(μg/d) BMDL

(μg/d) Dermal

slope

factor b
(μg/d) - 1

Sivak et al., 1997 Male C3H/HeJ Multistage 2° 10% 0.11 0.058 1.7

Poel, 1959 Male C57L Multistage 3° 10% 0.12 0.078 1.3

Habs et al., 1984 Female NMRI Multistage 3° 10% 0.078 0.056 1.8

Grimmer et al., 1984 Female CFLP Log

- logistic 70% 1.07 0.48 1.5

Schmahl et al., 1977 Female NMRI Multistage 2° 10% 0.23 0.15 0.67

Schmidt et al., 1973 Female Swiss Multistage 3° 10% 0.28 0.22 0.45

Grimmer et al., 1983 Female CFLP Multistage 1° 10% 0.24 0.21 0.48

Habs et al., 1980 Female NMRI Multistage 3° 10% 0.29 0.22 0.45

Schmidt et al., 1973 Female NMRI Multistage 2° 10% 0.33 0.29 0.34

Roe et al., 1970 Female Swiss Multistage 2° 10% 0.69 0.39 0.25

a See Appendix C for details of modeling results.

b Unadjusted for interspecies differences

18

The BMDL 10 of 0.068 μg/ day, based on the tumor responses in C57L male mice (Poel, 1959) 19

and in C3H/HeJ mal e mice (Sivak et al., 1997), was chosen for developing a human dermal slope 20

factor. 21Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE 

### 2.5.4. Dermal Slope Factor Cross Species Scaling 1

Different methodologies have been established for interspecies scaling of PODs used to 2

derive oral slope factors and inhalation un it risks. Cross

- species adjustment of oral doses is based 3

on allometric scaling using the 3⁄4 power of body weight. This adjustment accounts for more rapid 4

distribution, metabolism, and clearance in small animals (U.S. EPA, 2005 a ). Cross

- species 5
extrapola tion of inhalation exposures is based on standard dosimetry models that consider factors 6

such as solubility, reactivity, and persistence (U.S. EPA, 1994b). No established methodology exists 7
to adjust for interspecies differences in dermal toxicity at the point of contact; however, allometric 8
scaling using body weight to the 3⁄4 power was selected based on known species differences in 9

dermal metabolism and penetration of benzo[a]pyrene . In vitro skin permeation was highest in the 10

mouse, compared to rat, rabb it and human, and was enhanced by induction of CYP enzymes (Kao et 11
al., 1985). Using this approach, rodents and humans exposed to the same daily dose of a 12

carcinogen, adjusted for BW 3/4 , would be expected to have equal lifetime risks of cancer. 13

Alternati ve approaches were also evaluated. A comparison of these alternatives is provided 14

in Appendix D of the Supplemental Information

. 15

The POD M derived from the mouse studies of Poel (1959) and Sivak et al. (1997) is adjusted 16

to a HED as follows: 17

18

POD HED (μg /day) = POD M (μg/day) × (BW H / BW M ) 3/4 19

= 0.068 μg/day × (70 kg / 0.035 kg) 3/4 20

= 20.3 μg/day 21

22
The resulting POD HED is used to calculate the dermal slope factor for benzo[a]pyrene: 23

24

Dermal slope factor = 0.1/POD HED = 0.1/(20.3 μg/day) = 0.005 (μg/day)

- 1 25

26

Note that the dermal slope factor should only be used with lifetime human exposures 27

<20 μg/day, the human equivalent of the POD M , because above this level, the dose

- response 28

relationship may not be proportional to the mass of benzo[a]pyrene applied. 29

Several assumptions are made in the use of this scaling method. First, it is assumed that the 30

toxicokinetic processes in the skin will scale similarly to interspecies differences in whole

- body 31

toxicokinetics. Secondly, it is assumed that the risk at low do ses of benzo[a]pyrene is linear. 32
Although one study indicates that at high doses of benzo[a]pyrene carcinogenic potency is related 33

to mass applied per unit skin and n ot to total mass (Davies, 1969), this may be due to promotional 34

effects, such as inflammation, that are observed at high doses of benzo[a]pyrene. 35

The dermal slope factor has been developed for a local effect and it is not intended to 36

estimate systemic risk of cancer following dermal abso rption of benzo[a]pyrene into the systemic 37

circulation. Although some information suggests that benzo[a]pyrene metabolites can enter 38Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE systemic circulation following dermal exposure in humans (Godschalk et al., 1998), lifetime skin 1

cancer bioassays that hav e included pathological examination of other organs have not found 2
elevated incidences of tumors at distal sites (Higginbotham et al., 1993; Habs et al., 1980; Schmahl 3

et al., 1977; Schmidt et al., 1973; Roe et al., 1970; Poel, 1959). In addition, benzo[a ]pyrene tends to 4
bind to targets within the skin rather than enter the plasma receptor fluid (a surrogate measure of 5
systemic absorption) in in vitro human skin experiments. These data are consistent with 6
benzo[a]pyrene’s metabolism to reactive metabolite s within the viable layers of the skin (Wester et 7
al., 1990). Some studies indicate that the fraction of benzo[a]pyrene left within the viable layers of 8

the skin is a large portion of the applied dose (Moody et al., 2007, 1995). Taken together, these dat a 9
support the conclusion that the risk of skin cancer following dermal exposure likely outweighs 10

cancer risks at distal organs. 11



### 2.5.5. Uncertainties in the Derivation of the Dermal Slope Factor 12

Uncertainty in the recommended dermal slope factor is partly reflecte d in the range of POD 13

values derived from the modeled mouse skin tumor data sets: the lowest and highest BMDL 10 14

values listed in Table 2

- 11 show a seven

- fold d ifference (0.056

– 0.39 μg/day) in magnitude. There 15

is some indication that the recommended derma l slope factor may underestimate cancer risk, due 16

to inadequate data to take the observed decreasing tumor latency with increasing exposure level 17

into account. Reliance on studies with the lowest exposure levels where early mortality due to 18

benzo[a]pyrene exposure was low and exposures continued for approximately 104 weeks tends to 19

minimize this source of uncertainty. 20

Human dermal exposure to benzo[a]pyrene in the environment likely occurs predominantly 21

through soil contact. The available mouse dermal bio assays of benzo[a]pyrene relied on delivery of 22
benzo[a]pyrene to the skin in a solvent solution (typically acetone or toluene). The use of volatile 23

solvent likely results in a larger dose of benzo[a]pyrene available for uptake into the skin 24

(compared to s oil). Consequently, r eliance on these studies may overestimate the risk of skin 25
tumors from benzo[a]pyrene contact through soil; however, cancer bioassays delivering 26

benzo[a]pyrene through a soil matrix are not available. 27

There is uncertainty in extrapola ting from the intermittent exposures in the mouse assays 28

to daily exposure scenarios. All of the dermal bioassays considered treated animals 2

- 3 times a 29

week. This assessment makes the assumption that risk is proportional to total cumulative 30

exposure. H owever, this may overestimate risk if duration adjusted doses are below doses which 31

saturate or slow detoxifying metabolic steps. 32
The available data were not useful to determine which animal species may be the best 33

surrogate for human dermal response to b enzo[a]pyrene. In extrapolation of the animal dermal 34

information to humans, the assumption is made that equal lifetime risks of cancer would follow 35

from exposure to the same dail y dose adjusted for BW 3/4 . Qualitatively, the toxicokinetics and 36

toxicodynam ics in mouse and human skin appear to be similar (Knafla et al., 2011; Bickers et al., 37Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE 1984). Specifically, all of the activation pathways implicated in benzo[a]pyrene carcinogenicity 1

have been observed in mouse and human skin, and associations have been made between the 2
spectra of mutations in tumor tissues from benzo[a]pyrene

- exposed animals and humans exposed 3

to complex PAH mixtures containing benzo[a]pyrene (see Section 1.1.5). 4

The dermal slope factor for benzo[a]pyrene is based on skin cancer and does not represent 5
systemic cancer risk from dermal exposure. It is unclear whether dermal exposure to 6
benzo[a]pyrene would result in elevated risk of systemic tumors. Some studies in humans suggest 7
that although the skin may be responsible for a “first pass ” metabolic effect, benzo[a]pyrene

8

specific adduct levels have been detected in WBCs following dermal exposure to benzo[a]pyrene, 9
indicating that dermally applied benzo[a]pyrene enters systemic circulation (Godschalk et al., 10

1998). Although none of the li fetime dermal bioassays in mice, which included macroscopic 11
examination of internal organs, reported an elevation of systemic tumors in benzo[a]pyrene

12
treated mice compared to controls (Higginbotham et al., 1993; Habs et al., 1980; Schmahl et al., 13

1977; Sc hmidt et al., 1973; Roe et al., 1970; Poel, 1959), most of these studies attempted to remove 14

animals with grossly observed skin tumors from the study before the death of the animal, possibly 15
minimizing the development of more distant tumors with longer lat ency. The risk of 16

benzo[a]pyrene

- induced point

- of

- contact tumors in the skin possibly competes with systemic risk 17

of tumors. Currently, the potential contribution of dermally absorbed benzo[a]pyrene to systemic 18

cancer risk is unclear. 19

Table 2

- 12 . Summary of uncertainties in the benzo[a]pyrene cancer dermal 20 slope factor (DSF) 21

Consideration Impact on unit

risk Decision Justification

Data set ↓ DSF if

alternative data

set were selected Poel (1959) ; Sivak et al.

(1997) Poel (1959) included lowest doses among available studies (where intercurrent mortality was less likely to impact the number at risk) and
used adequate group sizes.

Target organ No DSF if skin

tumor studies not

used Selection of skin

tumors Skin tumors were replicated in numerous

studies of male or female mice. No studies were available indicating that other tumors occur following dermal exposure;

Dose metric Alternatives could

↓ or ↑ slope

factor Administered dose, as

time

- weighted average in μg/day

. Experimental evidence supports a role for metabolism in toxicity, but actual responsible

metabolites are not identified.

Cross

- species scaling Alternatives could

↓ or ↑ slope

factor Total daily dose scaled

by BW3/4 Alternatives discussed in Appendix C . An established methodology does not exist to adjust for interspecies differences in dermal toxicity at the point of contact. Benzo[a]pyrene

metabolism is known to occur in the dermal layer. Viewing the skin as an org an, metabolic processes were assumed to scale allometrically without evidence to the contrary.Toxicological Review of Benzo[a]pyrene

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- 40 DRAFT

— DO NOT CITE OR QUOTE Consideration Impact on unit

risk Decision Justification

Dose

- response modeling Alternatives could

↓ or ↑ slope

factor Multistage model No biologically based models for benzo[a]pyrene were available. The multistage

model is consistent with biological processes and is the preferred model for IRIS cancer

assessments (Gehlhaus et al., 2011)

. Low

- dose extrapolation ↓ cancer risk

estimate would be expected with the application of

nonlin ear low

dose extrapolation Linear extrapolation

from POD (based on mutagenic MOA) Available mode of action data support linearity (mutagenicity is a primary mode of action of benzo[a]pyrene);

Sensitive subpopulations ↑ DSF to

unknown extent ADAFs are

recommended for early life exposures No chemical

- specific data are available to determine the range of human toxicodynamic variability or sensitivity.



### 2.5.6. Previous Dermal Slope Factor 1

A dermal slope factor for benzo[a]pyrene was not previously available on IRIS. 2



## 2.6. Application of Age

- dependent Adjustment Factors (ADAFs) 3

Based on sufficient support in laboratory animals and relevance to humans, benzo[a]pyrene 4
is determined to be carcinogenic by a mutagenic mode of action. According to the Supplemental 5

Guidance for Assessing Susceptibility from Early Life Exposure to Carcinogens (“ Supplemental 6

Guidance ”) (U.S. EPA, 2005b), individuals exposed during early life to carcinogens with a mutagenic 7

mode of action are assumed to have increased risk for cancer. The oral slope factor of 1 per mg/kg

8

day, inhalation unit risk of 0.5 per mg/m 3 , and dermal slope factor of 0.005 per μg/day for 9
benzo[a]pyrene, calculated from data applicable to adult exposures, do not reflect presumed early 10

life susceptibility to this chemi cal. Alt hough chemical

- specific data exist for benzo[a]pyrene that 11

quantitatively demonstrate increased early life susceptibility to can cer (Vesselinovitch et al., 1975 ), 12

these data were not considered sufficient to develop separate risk estimates for childhoo d 13

exposure, as they used acute i.p. exposures (U.S. EPA, 2005b). In the absence of adequate chemical

14
specific data to evaluate differences in age

- specific susceptibility, the Supplemental Guidance (U.S. 15

EPA, 2005b) recommends that ADAFs be applied in esti mating cancer risk. 16

The Supplemental Guidance (U.S. EPA, 2005b) establishes ADAFs for three specific age 17
groups. These ADAFs and their corresponding age groupings are: 10 for individuals exposed at <2 18

years of age, 3 for exposed individuals at 2

– <16 year s of age, and 1 for exposed individuals ≥16 19
years of age. The 10

- and 3

- fold adjustments are combined with age specific exposure estimates 20

when estimating cancer risks from early life (<16 years of age) exposures to benzo[a]pyrene. To 21

illustrate the use of the ADAFs established in the Supplemental Guidance (U.S. EPA, 2005b), sample 22

calculations are presented for three exposure duration scenarios, including full lifetime, assuming a 23
constant benzo[a]pyrene exposure of 0.001 mg/kg

- day (Table 2

- 1 3 ). 24Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 2

- 13 . Application of ADAFs for the estimation of benzo[a]pyrene cancer risk following lifetime (70

- year) oral exposure

Age group ADAF Unit risk Exposure concentration

Duration adjustment Cancer risk for

specific exposure

duration

scenarios (per mg/kg

- d) (mg/kg

- d)

0

– <2 yrs 10 1 0.001 2 yrs/70 yrs 0.0003

2

– <16 yrs 3 1 0.001 14 yrs/70 yrs 0.0006

≥16 yrs 1 1 0.001 54 yrs/70 yrs 0.0007

Total risk 0.002

1

The example exposure duration scenarios include full lifetime exposure (assuming a 70

- 2

year lifespan). Table 2

- 13 lists the four factors (ADAFs, cancer risk estimate, assumed exposure, 3
and duration adjustment) that are needed to calculate the partial cance r risk based on the early 4

age

- specific group. The cancer risk for each age group is the product of the four factors in columns 5

2

– 5. Therefore, the cancer risk following daily benzo[a]pyrene oral exposure in the age group 0

– <2 6
years is the product of the values in columns 2

– 5 or 10 × 1 × 0.001 × 2/70 = 3 × 10

- 4 . The cancer 7
risk for specific exposure duration scenarios that are listed in the last column are added together to 8

get the total risk. Thus, a 70

- year (lifetime) risk estimate for continuous expos ure to 0.001 mg/kg

9

day benzo[a]pyrene is 2 × 10

- 3 , which is adjusted for early

- life susceptibility and assumes a 70

- year 10
lifetime and constant exposure across age groups. 11
In calculating the cancer risk for a 30

- year constant exposure to benzo[a]pyrene at a n 12

exposure level of 0.001 mg/kg

- day for ages 0

– 30 years, the duration adjustments would be 2/70, 13
14/70, and 14/70, and the partial risks for the three age groups would be 3 × 10

- 4 , 6 × 10

- 4 , and 2 × 14

10

- 4 , which would result in a total risk estimate of 1 × 10

- 3

. 15
In calculating the cancer risk for a 30

- year constant exposure to benzo[a]pyrene at an 16

exposure level of 0.001 mg/kg

- day for ages 20

– 50 years, the duration adjustments would be 0/70, 17
0/70, and 30/70. The partial risks for the three groups are 0, 0, and 4 × 10

- 4 , which would result in 18

a total risk estimate of 4 × 10

- 4

. 19

Consistent with the approaches for the oral route of exposure ( Table 2

- 13 ), the ADAFs 20
should also be applied when assessing cancer risks for subpopulations with early life exposures to 21

benzo[a]pyrene via the inhalation and dermal routes (presented in Tables 2

- 14 and 2

- 15 ). 22Toxicological Review of Benzo[a]pyrene

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— DO NOT CITE OR QUOTE Table 2

- 14 . Application of ADAFs for the estimation of benzo[ a]pyrene cancer risk following lifetime (70

- year) inhalat ion exposure

Age group ADAF Unit risk Exposure concentration

Duration adjustment Cancer risk for

specific exposure

duration

scenarios (per μg/m 3 ) (μg/m 3 )

0

– <2 yrs 10 5 × 10 - 4 1 2 yrs/70 yrs 0.0001

2

– <16 yrs 3 5 × 10 - 4 1 14 yrs/70 yrs 0.0003

≥16 yrs 1 5 × 10 - 4 1 54 yrs/70 yrs 0.0004

Total risk 0.0008

Table 2

- 15 . Application of ADAFs for the estimation of benzo[ a]pyrene cancer risk following lifetime (70

- year) dermal exposure

Age

group ADAF Unit risk Exposure

concentration

Duration

adjustment Cancer risk for

specific exposure

duration

scenarios (per μg/d) (μg/d)

0

– <2 yrs 10 0.00 5 0.001 2 yrs/70 yrs 1 × 10 - 6

2

– <16 yrs 3 0.00 5 0.001 14 yrs/70 yrs 3 × 10 - 6

≥16 yrs 1 0.00 5 0.001 54 yrs/70 yrs 4 × 10 - 6

Total risk 8 × 10 - 6Toxicological Review of Benzo[a]pyrene

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- 1 DRAFT

— DO NOT CITE OR QUOTE 

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31 U.S. EPA. (1992) Draft report: a cross

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34 U.S. EPA. (1994a) Interim policy for particle size and limit concentration issues in inhalation toxicity studies. 35 Federal Register 59(206):53799. Available online at http://www.epa.gov/iris/backgrd.html (a ccessed April 36 20, 2010). 37

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12 U.S. EPA. (2002) A review of the reference dose and reference concentration processes. Risk Assessment 13 Forum, Washington, DC; EPA/630/P

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23 U.S. EPA. (2005b) Supplemental guidance for assessing susceptibility from early

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27 U.S. EPA. (2006a) Science policy council handbook: peer review. Third edit ion. Office of Science Policy, Office 28 of Research and Development, Washington, DC; EPA/100/B

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31 U.S. EPA. (2006b) A framework for assessing health risk of environment al exposures to children. National 32 Center for Environmental Assessment, Washington, DC, EPA/600/R

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35 U.S. EPA. (2007) Framework for determinin g a mutagenic mode of action for carcinogenicity: Using EPA's 36 2005 cancer guidelines and supplemental guidance for assessing susceptibility from early

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40 U.S. EPA. (2010) Revised Draft: Multistage Weibull time

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