v>EPA
EPA/635/R-17/003Fa
www.epa.gov/iris
Toxicological Review of Benzo[a]pyrene
[CASRN 50-32-8]
January 2017
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of Benzo[a]pyrene
DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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Toxicological Review of Benzo[a]pyrene
CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS x
PREFACE xiv
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xviii
EXECUTIVE SUMMARY xxvi
LITERATURE SEARCH STRATEGY| STUDY SELECTION xxxiii
1. HAZARD IDENTIFICATION 1-1
1.1. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-1
1.1.1. Developmental Toxicity 1-1
1.1.2. Reproductive Toxicity 1-30
1.1.3. Immunotoxicity 1-50
1.1.4. Other Toxicity 1-61
1.1.5. Carcinogenicity 1-71
1.2. SUMMARY AND EVALUATION 1-101
1.2.1. Weight of Evidence for Effects Other than Cancer 1-101
1.2.2. Weight of Evidence for Carcinogenicity 1-104
2. DOSE-RESPONSE ANALYSIS 2-1
2.1.ORAL REFERENCE DOSE FOR EFFECTS OTHERTHAN 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 Values 2-10
2.1.4. Derivation of Organ/System-Specific Reference Doses 2-15
2.1.5. Selection of the Overall Reference Dose 2-16
2.1.6. Confidence Statement 2-17
2.1.7. Previous IRIS Assessment: Reference Dose 2-17
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER 2-17
2.2.1. Identification of Studies and Effects for Dose-Response Analysis 2-18
2.2.2. Methods of Analysis 2-19
2.2.3. Derivation of Candidate Values 2-23
2.2.4. Derivation of Organ/System-Specific Reference Concentrations 2-26
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2.2.5. Selection of the Reference Concentration 2-27
2.2.6. Confidence Statement 2-27
2.2.7. Previous IRIS Assessment: Reference Concentration 2-28
2.2.8. Uncertainties in the Derivation of the RfD and RfC 2-28
2.3. ORAL SLOPE FACTOR FOR CANCER 2-30
2.3.1. Analysis of Carcinogenicity Data 2-30
2.3.2. Dose-Response Analysis—Adjustments and Extrapolation Methods 2-32
2.3.3. Derivation of the Oral Slope Factor 2-34
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 2-37
2.3.5. Previous IRIS Assessment: Oral Slope Factor 2-39
2.4. INHALATION UNIT RISK FOR CANCER 2-40
2.4.1. Analysis of Carcinogenicity Data 2-40
2.4.2. Dose-Response Analysis—Adjustments and Extrapolation Methods 2-41
2.4.3. Inhalation Unit Risk Derivation 2-42
2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk 2-43
2.4.5. Previous IRIS Assessment: Inhalation Unit Risk 2-47
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS (ADAFs) 2-47
REFERENCES R-l
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TABLES
Table ES-1. Organ/system-specific RfDs and overall RfD for benzo[a]pyrene xxviii
Table ES-2. Organ/system-specific RfCs and overall RfCfor benzo[a]pyrene xxix
Table LS-1. Summary of the search strategy employed for benzo[a]pyrene xxxiii
Table 1-1. Evidence pertaining to developmental effects of benzo[a]pyrene in humans 1-4
Table 1-2. Evidence pertaining to developmental effects of benzo[a]pyrene in animals after oral
or inhalation exposure 1-7
Table 1-3. Evidence pertaining to the neurodevelopmental effects of benzo[a]pyrene from PAH
mixtures 1-18
Table 1-4. Evidence pertaining to the neurodevelopmental effects of benzo[a]pyrene in animals 1-20
Table 1-5. Evidence pertaining to the male reproductive toxicity of benzo[a]pyrene in adult
animals after oral or inhalation exposure 1-34
Table 1-6. Evidence pertaining to the female reproductive effects of benzo[a]pyrene in humans 1-42
Table 1-7. Evidence pertaining to the female reproductive effects of benzo[a]pyrene in adult
animals after oral or inhalation exposure 1-43
Table 1-8. Evidence pertaining to immune effects of benzo[a]pyrene in humans 1-55
Table 1-9. Evidence pertaining to the immune effects of benzo[a]pyrene in animals after oral or
inhalation exposure 1-55
Table 1-10. Evidence pertaining to liver, kidney, and cardiovascular effects of benzo[a]pyrene in
animals after oral or inhalation exposure 1-68
Table 1-11. Evidence pertaining to neurotoxicity following repeated oral or inhalation exposure
to benzo[a]pyrene in adult humans and animals 1-69
Table 1-12. Cancer sites for PAH-related agents reviewed by the International Agency for
Research on Cancer (IARC) 1-74
Table 1-13. Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation
to lung cancer risk: Tier 1 studies 1-75
Table 1-14. Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation
to lung cancer risk: Tier 2 studies 1-76
Table 1-15. Summary of epidemiologic studies of benzo[a]pyrene (direct measures) in relation
to bladder cancer risk 1-79
Table 1-16. Tumors observed in chronic oral animal bioassays 1-82
Table 1-17. Tumors observed in chronic inhalation animal bioassays 1-85
Table 1-18. Tumors observed in chronic dermal animal bioassays 1-87
Table 1-19. Experimental support for the postulated key events for mutagenic mode of action 1-95
Table 1-20. Supporting evidence for the carcinogenic to humans cancer descriptor for
benzo[a]pyrene 1-108
Table 2-1. Summary of derivation of PODs 2-8
Table 2-2. Effects and corresponding derivation of candidate values 2-12
Table 2-3. Organ/system-specific RfDs and overall RfD for benzo[a]pyrene 2-15
Table 2-4. Summary of derivation of PODs 2-22
Table 2-5. Effects and corresponding derivation of candidate values 2-25
Table 2-6. Organ/system-specific RfCs and overall RfCfor benzo[a]pyrene 2-26
Table 2-7. Summary of the oral slope factor derivations 2-34
Table 2-8. Summary of uncertainties in the derivation of benzo[a]pyrene oral slope factor 2-38
Table 2-9. Summary of the inhalation unit risk derivation 2-43
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Table 2-10. Summary of uncertainties in the derivation of cancer risk values for benzo[a]pyrene
(inhalation unit risk) 2-46
Table 2-11. Sample application of ADAFs for the estimation of benzo[a]pyrene cancer risk
following lifetime (70-year) oral exposure 2-48
Table 2-12. Sample application of ADAFs for the estimation of benzo[a]pyrene cancer risk
following lifetime (70-year) inhalation exposure 2-49
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FIGURES
Figure LS-1. Study selection strategy xxxv
Figure 1-1. Exposure-response array for developmental effects following oral exposure to
benzo[a]pyrene 1-11
Figure 1-2. Exposure timing in benzo[a]pyrene studies examining nervous system effects 1-12
Figure 1-3. Exposure-response array for neurodevelopmental effects following oral exposure 1-24
Figure 1-4. Exposure-response array for male reproductive effects following oral exposure in
adult animals 1-38
Figure 1-5. Exposure-response array for female reproductive effects following oral exposure in
adult animals 1-46
Figure 1-6. Exposure-response array for immune effects following oral exposure 1-58
Figure 1-7. Proposed metabolic activation pathways and key events in the carcinogenic mode of
action for benzo[a]pyrene 1-89
Figure 2-1. Candidate values with corresponding PODs and composite UFs 2-14
Figure 2-2. Candidate values with corresponding PODs and composite UFs 2-26
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ABBREVIATIONS
AchE acetylcholine esterase FSH
ADAF age-dependent adjustment factor GABA
Ah aryl hydrocarbon GD
AHH aryl hydrocarbon hydroxylase GI
AhR aryl hydrocarbon receptor GJIC
AIC Akaike's Information Criterion
AKR aldo-keto reductase GSH
AMI acute myocardial infarction GST
ANOVA analysis of variance GSTM1
AST aspartate transaminase hCG
ATSDR Agency for Toxic Substances and HEC
Disease Registry HED
BMC benchmark concentration HERO
BMCL benchmark concentration lower
confidence limit HFC
BMD benchmark dose HPLC
BMDL benchmark dose, 95% lower bound
BMDS Benchmark Dose Software hprt
BMR benchmark response
BPDE benzo[a]pyrene-7,8-diol-9,10-epoxide HR
BPdG benzo[a]pyrene-7,8-diol-9,10-epoxide- Hsp90
N2-deoxyguanosine i.p.
BPQ benzo[a]pyrene-7,8-quinone i.v.
BrdU bromodeoxyuridine IARC
BSM benzene-soluble matter
BUN blood urea nitrogen Ig
BW body weight IHD
CA chromosomal aberration IRIS
CAAC Chemical Assessment Advisory LDH
Committee LH
CASRN Chemical Abstracts Service Registry LOAEL
Number MAP
CERCLA Comprehensive Environmental MCL
Response, Compensation, and Liability MCLG
Act MIAME
CHO Chinese hamster ovary
CI confidence interval MLE
CYP cytochrome MMAD
CYP450 cytochrome P450 MN
DAF dosimetric adjustment factor MPPD
dbcAMP dibutyl cyclic adenosine mRNA
monophosphate MS
DMSO dimethyl sulfoxide NCE
DNA deoxyribonucleic acid NCEA
EC European Commission
EH epoxide hydrolase NK
ELISA enzyme-linked immunosorbent assay NMDA
EPA Environmental Protection Agency NOAEL
EROD 7-ethoxyresorufin-0-deethylase NPL
ETS environmental tobacco smoke NQO
Fe203 ferrous oxide NRC
follicle stimulating hormone
gamma-aminobutyric acid
gestational day
gastrointestinal
gap junctional intercellular
communication
reduced glutathione
glutathione-S-transferase
glutathione-S-transferase Ml
human chorionic gonadotropin
human equivalent concentration
human equivalent dose
Health and Environmental Research
Online
high-frequency cell
high-performance liquid
chromatography
hypoxanthine guanine phosphoribosyl
transferase
hazard ratio
heat shock protein 90
intraperitoneal
intravenous
International Agency for Research on
Cancer
immunoglobulin
ischemic heart disease
Integrated Risk Information System
lactate dehydrogenase
luteinizing hormone
lowest-observed-adverse-effect level
mitogen-activated protein
Maximum Contaminant Level
Maximum Contaminant Level Goal
Minimum Information About a
Microarray Experiment
maximum likelihood estimate
mass median aerodynamic diameter
micronucleus
Multi-Path Particle Deposition
messenger ribonucleic acid
mass spectrometry
normochromatic erythrocyte
National Center for Environmental
Assessment
natural killer
N-methyl-D-aspartate
no-observed-adverse-effect level
National Priorities List
NADPH:quinone oxidoreductase
National Research Council
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NTP
National Toxicology Program
SMR
standardized mortality ratio
OECD
Organisation for Economic
SOAR
Systematic Omics Analysis Review
Co-operation and Development
SOD
superoxide dismutase
OR
odds ratio
SRBC
sheep red blood cells
ORD
Office of Research and Development
SSB
single-strand break
PAH
polycyclic aromatic hydrocarbon
TCDD
2,3,7,8-tetrachlorodibenzo-p-dioxin
PBMC
peripheral blood mononuclear cell
TK
thymidine kinase
PBPK
physiologically based pharmacokinetic
ToxR
Toxicological Reliability Assessment
PCA
Principal Components Analysis
TPA
12-0-tetradecanoylphorbol-13-acetate
PCE
polychromatic erythrocyte
TUNEL
terminal deoxynucleotidyl transferase
PCNA
proliferating cell nuclear antigen
dUTP nick end labeling
PND
postnatal day
TWA
time-weighted average
POD
point of departure
UCL
upper confidence limit
PUVA
psoralen plus ultraviolet-A
UDP-UGT
uridine diphosphate-
RBC
red blood cell
glucuronosyltransferase
RDDRer
regional deposited dose ratio for
UDS
unscheduled DNA synthesis
extrarespiratory effects
UF
uncertainty factor
RfC
inhalation reference concentration
UFa
interspecies uncertainty factor
RfD
oral reference dose
UFd
database deficiencies uncertainty factor
RNA
ribonucleic acid
UFh
intraspecies uncertainty factor
ROS
reactive oxygen species
UFl
LOAEL-to-NOAEL uncertainty factor
RR
relative risk
UFs
subchronic-to-chronic uncertainty
SAB
Science Advisory Board
factor
s.c.
subcutaneous
UVA
ultraviolet-A
see
squamous cell carcinoma
UVB
ultraviolet-B
SCE
sister chromatid exchange
WBC
white blood cell
SD
standard deviation
WT
wild type
SE
standard error
WTC
World Trade Center
SEM
standard error of the mean
XPA
xeroderma pigmentosum group A
SHE
Syrian hamster embryo
SIR
standardized incidence ratio
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Assessment Team
Lyle Burgoon, Ph.D. (formerly with EPA)
Christine Cai, MS
Gene Ching-Hung Hsu, Ph.D. (formerly with
EPA)
Glinda Cooper, Ph.D. (formerly with EPA)
John Cowden, Ph.D.
Louis D'Amico, Ph.D.
Jason Fritz, Ph.D.
Martin Gehlhaus, MHS
Catherine Gibbons, Ph.D.
Karen Hogan, MS
Andrew Kraft, Ph.D.
Kathleen Newhouse, MS (Assessment Manager)
Amanda Persad, Ph.D.
Linda Phillips, Ph.D.
Margaret Pratt, Ph.D.
Keith Salazar, Ph.D.
John Schaum, MS (retired)
John Stanek, Ph.D.
Suryanarayana Vulimiri, DVM
U.S. EPA
Office of Research and Development
National Center for Environmental
Assessment
Chris Brinkerhoff, Ph.D.
Emma McConnell, MS
Oak Ridge Institute for Science and
Education Fellow
Scott Glaberman, Ph.D.
American Association for the
Advancement of Science Fellow
Contributors
John Fox, Ph.D.
Paul White, Ph.D.
Maria Spassova, Ph.D.
U.S. EPA
Office of Research and Development
National Center for Environmental
Assessment
Washington, DC
Lynn Flowers, Ph.D.
U.S. EPA
Office of Research and Development
Office of Science Policy
Washington, DC
Production Team
Taukecha Cunningham
Maureen Johnson
Terri Konoza
Vicki Soto
U.S. EPA
Office of Research and Development
National Center for Environmental
Assessment
Washington, DC
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Contractor Support
Heather Carlson-Lynch, S.M.
Peter McClure, Ph.D.
Megan Riccardi, M.S.
Kelly Salinas, Ph.D.
Joe Santodonato, Ph.D.
Julie Melia, Ph.D.
George Holdsworth, Ph.D.
Lutz W. Weber, Ph.D.
Janusz Z. Byczkowski, Ph.D., D.Sc.
SRC, Inc., Syracuse, NY
Oak Ridge Institute for Science and
Education, Oak Ridge, TN
JZB Consulting, Fairborn, OH
Executive Direction
Kenneth Olden, Ph.D., Sc.D., L.H.D.
(Center Director-Retired)
Michael Slimak, Ph.D. (Acting Center Director)
Vincent Cogliano, Ph.D.
(IRIS Program Director)
Gina Perovich, M.S.
(IRIS Program Deputy Directory)
Samantha Jones, Ph.D.
(IRIS Associate Director for Science)
Jamie B. Strong, Ph.D. (Toxic Effects Branch Chief-
now at Office of Water)
Ravi Subramaniam (Acting Toxic Effects Branch
Chief)
U.S. EPA/ORD/NCEA
Washington, DC
Lynn Flowers, Ph.D.
(Senior Scientist)
U.S. EPA/ORD/OSP
Washington, DC
Internal Reviewers
Stephen Nesnow, Ph.D. (retired)
Rita Schoeny, Ph.D. (retired)
Reviewers
U.S. EPA
National Health and Environmental
Effects Research Laboratory
Research Triangle Park, NC
U.S. EPA
Office of Water
Washington, DC
This assessment was provided for review to scientists in EPA's Program and Region Offices.
Comments were submitted by:
Office of Children's Health 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
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This assessment was provided for review to other federal agencies and the Executive Office of the
President (EOP). A summary and EPA's disposition of major comments from the other federal
agencies and EOP is available on the IRIS website. Comments were submitted by:
Agency for Toxic Substances and Disease Registry, Centers for Disease Control and
Prevention, 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 and
Prevention, Department of Health and Human Services
Office of Management and Budget, Executive Office of the President
White House Council on Environmental Quality, Executive Office of the President
This assessment was released for public comment on August 21, 2013 and comments were due on
November 21, 2013. A summary and EPA's disposition of the comments received from the public is
included in Appendix G of the Supplemental Information to the Revised External Review draft of the
Toxicological Review (dated September 2015). Comments were received from the following
entities:
Utility Solid Waste Activities Group
CDM Smith, on behalf of: Pavement Coatings Technology Council
Electric Power Research Institute
Duke Energy
CH2M Hill
Gradient
American Chemistry Council
Arcadis on behalf of: American Coke and Coal Chemicals Institute, American Fuels and
Petrochemical Manufacturers, American Petroleum Institute, Asphalt Institute, Association of
American Railroads, Beazer East, and Pavement Coatings Technology Council
Agnes Francisco, Alexandria, LA
Melanie Nembhard, Baltimore, MD
This assessment was peer reviewed by independent, expert scientists external to EPA convened by
EPA's Science Advisory Board (SAB), the Chemical Assessment Advisory Committee Augmented for
the IRIS Benzo[a]pyrene Assessment. A peer review meeting was held on April 15 to 17, 2015. The
report of the SAB's review of EPA's Draft Toxicological Review of Benzo[a]pyrene, dated April 5,
2016, is available on the IRIS website. A summary and EPA's disposition of the comments received
from the SAB is included in Appendix F.
Dr. Elaine M. Faustman (chair), Professor and Director, Institute for Risk Analysis and Risk
Communication, School of Public Health, University of Washington, Seattle, WA
Dr. Scott Bartell, Associate Professor, Program in Public Health, University of California - Irvine,
Irvine, CA
Dr. Ronald Baynes, Professor, Population Health & Pathobiology, College of Veterinary Medicine,
North Carolina State University, Raleigh, NC
Dr. Annette Bunge, Professor Emeritus, Chemical & Biological Engineering, Colorado School of
Mines, Golden, CO
Dr. Scott Burchiel, Distinguished Professor, Pharmaceutical Sciences, College of Pharmacy,
University of New Mexico, Albuquerque, NM
Dr. Anna Choi, Research Scientist, Environmental Health, Harvard School of Public Health,
Boston, MA
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Dr. John DiGiovanni, Professor and Coulter R. Sublett Chair in Pharmacy, Division of
Pharmacology and Toxicology and Department of Nutritional Sciences, Dell Pediatric
Research Institute, The University of Texas at Austin, Austin, TX
Dr. Joanne English, Senior Toxicologist, Toxicology Services, NSF International, Ann Arbor, MI
Dr. William Michael Foster, Independent Consultant, Durham, NC
Dr. Chris Gennings, Professor, Department of Biostatistics, Icahn School of Medicine at Mount
Sinai, New York, NY
Dr. Helen Goeden, Senior Toxicologist, Minnesota Department of Health, St. Paul, MN
Dr. Sean Hays, President, Summit Toxicology, Allenspark, CO
Dr. John Kissel, Ph.D., Department of Environmental and Occupational Health Sciences, Public
Health, University of Washington, Seattle, WA
Dr. Edward Levin, Professor, Psychiatry, Duke University Medical Center, Durham, NC
Dr. Maureen Lichtveld, Professor and Chair, Global Environmental Health Sciences, School of
Public Health and Tropical Medicine, Tulane University, New Orleans, LA
Dr. Abby A. Li, Senior Managing Scientist, Health Science Practice, Exponent Incorporated, San
Francisco, CA,
Dr. Barry Mclntyre, Senior Toxicologist, Toxicology Branch, National Toxicology Program,
National Institute of Environmental Health Sciences, Research Triangle Park, NC
Dr. Bhagavatula Moorthy, Professor, Pediatrics, Baylor College of Medicine, Houston, TX
Dr. Miriam Poirier, Head, Carcinogen-DNA Interactions Section, National Cancer Institute,
National Institutes of Health, NIH-NCI, Bethesda, MD
Dr. Kenneth M. Portier, Director of Statistics, Department of Statistics and Evaluation, American
Cancer Society, Atlanta, GA
Dr. Kenneth Ramos, Associate Vice-President of Precision Health Sciences and Professor of
Medicine, Arizona Health Sciences Center, University of Arizona, Tucson, AZ
Dr. Stephen M. Roberts, Professor, Center for Environmental and Human Toxicology, University
of Florida, Gainesville, FL
Dr. Richard Schlesinger, Associate Dean, Dyson College of Arts and Sciences, Pace University,
New York, NY
Dr. Leslie T. Stayner, Director, Epidemiology & Biostatistics, Epidemiology & Biostatistics, School
of Public Health, University of Illinois, Chicago, IL
Dr. Alan Stern, Chief, Bureau for Risk Analysis, Division of Science, Research and Environmental
Health, New Jersey Department of Environmental Protection, Trenton, NJ
Dr. Charles Vorhees, Professor, Pediatrics, Division of Neurology, Cincinnati Children's Research
Foundation/University of Cincinnati, Cincinnati, OH
Dr. Christi Walter, Professor and Chair, Cellular & Structural Biology, School of Medicine,
University of Texas Health Science Center at San Antonio, San Antonio, TX
Designated Federal Officer: Dr. Diana Wong
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PREFACE
This Toxicological Review, prepared under the auspices of the U.S. Environmental
Protection Agency's (EPA's) Integrated Risk Information System (IRIS) program, critically reviews
the publicly available studies on benzo[a]pyrene in order to identify potential adverse health effects
and to characterize exposure-response relationships. Benzo[a]pyrene is found in the environment
and in food. Benzo[a]pyrene occurs in conjunction with other structurally related chemical
compounds known as polycyclic aromatic hydrocarbons (PAHs).1 Benzo[a]pyrene is universally
present in these mixtures and is routinely analyzed and detected in environmental media
contaminated with PAH mixtures; thus, it is often used as an indicator chemical to measure
exposure to PAH mixtures (Bostrom etal.. 20021. It also serves as an index chemical for deriving
relative potency factors to estimate the carcinogenicity of other PAH congeners, such as in EPA's
Relative Potency Factor approach for the assessment of the carcinogenicity of PAHs (U.S. EPA.
1993).
Benzo[a]pyrene is listed as a hazardous substance under the Comprehensive Environmental
Response, Compensation, and Liability Act of 1980 (CERCLA), is found at 524 hazardous waste sites
on the National Priorities List (NPL), and is ranked number 8 out of 275 chemicals on the Priority
List of Hazardous Substances for CERCLA (ATSDR. 20111. This ranking is based on a combination
of factors that include the frequency of occurrence at NPL sites, the potential for human exposure,
and the potential health hazard. Benzo[a]pyrene is also listed as a drinking water contaminant
under the Safe Drinking Water Act and a Maximum Contaminant Level Goal (MCLG) and
enforceable Maximum Contaminant Level (MCL) have been established2. It is also one of the
chemicals included in EPA's Persistent Bioaccumulative and Toxic Chemical Program
(https://www.epa.gov/toxics-release-inventory-tri-program/persistent-bioaccumulative-toxic-
pbt-chemicals-covered-tri). In air, benzo[a]pyrene is regulated as a component in a class of
chemicals referred to as Polycyclic Organic Matter, defined as a Hazardous Air Pollutant by the
1990 amendments to the Clean Air Act
This assessment updates the IRIS assessment of benzo[a]pyrene that was developed in
1987. The previous assessment included a cancer descriptor and an oral slope factor. New
information has become available, and this assessment reviews information on all health effects by
all exposure routes. Organ/system-specific reference values are calculated based on developmental
(including developmental neurotoxicity), reproductive, and immune system toxicity data. These
'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.
2MCLG = 0, MCL = 0.0002 mg/L.
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reference values may be useful for cumulative risk assessments that consider the combined effect of
multiple agents acting on the same biological system.
This assessment was conducted in accordance with EPA guidance, which is cited and
summarized in the Preamble to Toxicological Reviews. Appendices for chemical and physical
properties, toxicokinetic information, and summaries of toxicity studies are provided as
Supplemental Information to this assessment.
In April 2011, the National Research Council (NRC) made recommendations for improving
the development of IRIS assessments. In May 2014, the NRC reviewed the IRIS Program again and
found that EPA had made substantial improvements to the IRIS Program in a short amount of time.
As part of this review, the NRC evaluated the August 2013 public comment draft of benzo[a]pyrene
to gauge EPA's progress in implementing the 2011 NRC recommendations. The NRC stated that
"the new document structure, which is reflected in the toxicological review of benzo[a]pyrene,
leads to better organized and streamlined assessments and reduces redundancies" and that "the
draft assessment shows that the IRIS program has taken several additional steps toward addressing
the recommendations in the 2011 NRC formaldehyde report."
This streamlined assessment uses tables, figures, and appendices to increase transparency
and clarity. It has distinct sections for literature search and study selection, hazard identification,
and dose-response assessment. A comprehensive, systematic literature search and screening
approach is documented in a table (databases, keywords) and flow diagram (inclusion and
exclusion of studies). All references were added to the Health and Environmental Research Online
(HERO) database. Studies were evaluated uniformly for aspects of design, conduct, or reporting
that could affect the interpretation of results and contribution to the synthesis of evidence. A
summary of the evaluation is included in the section on methods for identifying and selecting
studies. The evidence is presented in standardized summary and evidence tables and in exposure-
response arrays.
In the hazard identification and dose-response sections, there are subsections for each
target organ or system. The evidence is synthesized for each dataset, integrated for each target
organ/system, and then integrated across different target organs/systems. The IRIS Program used
existing guidelines to systematically approach the integration of human, animal, and mechanistic
evidence. For each health outcome, the IRIS Program evaluated the consistency of a possible
association, the strength of the association, the presence of a dose-response relationship, whether
the exposure preceded the effect, and the biological plausibility of the response and its relevance to
humans. Thus, this assessment provides a streamlined presentation of information, integrated
hazard identification of all toxic effects, and reference values for each target organ or system.
Additionally, this assessment contains an expanded discussion of the rationale for study selection
and evaluation, as well as other key assessment decisions.
The IRIS program released this assessment for peer review in September 2014, prior to the
program implementing systematic review. The approach to implementation of NRC
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recommendations is to use procedures and tools available at the time, without holding assessments
until new methods become available. Accordingly, the IRIS program conducted literature searches
using tools and documentation standards then available. Problem formulation materials and
protocol development began with assessments starting draft development in 2015, after this
assessment was well into peer review. Implementation of systematic review is a process of
continuous improvement subject to periodic review by the Chemical Assessment Advisory
Committee of the EPA's Science Advisory Board (SAB). This assessment represents a step in the
evolution of the IRIS program.
For additional information about this assessment or for general questions regarding IRIS,
please contact EPA's IRIS Hotline at 202-566-1676 (phone), 202-566-1749 (fax), or
hotline.iris@epa.gov.
Chemical Properties
Benzo[a]pyrene is a five-ring PAH. It is a pale yellow crystalline solid with a faint aromatic
odor. It is relatively insoluble in water and has low volatility. Benzo[a]pyrene is released to the air
from both natural and anthropogenic sources and removed from the atmosphere by photochemical
oxidation; reaction with nitrogen oxides, hydroxy and hydroperoxy radicals, ozone, sulfur oxides,
and peroxyacetyl nitrate; and wet and dry deposition to land or water. In air, benzo[a]pyrene is
predominantly adsorbed to particulates, but may also exist as a vapor at high temperatures
fATSDR. 19951.
Uses and Pathways of Exposure
There is no known commercial use for benzo[a]pyrene; it is only produced as a research
chemical. Benzo[a]pyrene is ubiquitous in the environment primarily as a result of incomplete
combustion emissions. It is found in fossil fuels, crude oils, shale oils, and coal tars fHSDB. 20121. It
is released to the environment via both natural sources (such as forest fires) and anthropogenic
sources including stoves/furnaces burning fossil fuels (especially wood and coal), motor vehicle
exhaust, cigarettes, and various industrial combustion processes (ATSDR. 1995). Benzo[a]pyrene is
also found in soot and coal tars. Several studies have reported that urban run-off from asphalt-
paved car parks treated with coats of coal-tar emulsion seal could account for a large proportion of
PAHs in many watersheds fRowe and O'Connor. 2011: Van Metre and Mahler. 2010: Mahler etal..
20051. Benzo[a]pyrene exposure can also occur to workers involved in the production of
aluminum, coke, graphite, and silicon carbide, and in coal tar distillation. The major sources of non-
occupational exposure are tobacco products, inhalation of polluted air, ingestion of contaminated
food and water, and through cooking processes that involve smoke (HSDB. 2012). Dermal exposure
can occur through contact with materials containing soot, tar, or crude petroleum, including
pharmaceutical products containing coal tar, such as coal tar-based shampoos and treatments for
eczema and psoriasis fCal/EPA. 2010: IARC. 20101.
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Benzo[a]pyrene persists for a long period of time in the atmosphere in the particulate phase
and is thus efficiently transported over long distances. It is lipophilic with low water solubility;
therefore, once deposited in water or sediments, it adsorbs strongly to sediments and particulate
matter and degrades slowly over several years fCal/EPA. 2010: GLC. 20071. Because of its presence
in high concentrations in the waters and sediments of the Great Lakes and St. Lawrence river
ecosystem, it is 1 of the 12 level I substances identified and targeted for reduction in the Great
Lakes Region (GLC. 20071.
Most aquatic organisms metabolize benzo[a]pyrene, eliminating it in days, and thus, it is not
expected to bioconcentrate in these organisms; however, several aquatic organisms such as
plankton, oysters, and some fish cannot metabolize benzo[a]pyrene fU.S. EPA. 2010al. Thus, the
data on benzo[a]pyrene bioconcentration in aquatic organisms varies from low to very high fHSDB.
20121. Bio magnification of benzo[a]pyrene in the food chain has not been reported fATSDR. 19951.
Additional information on benzo[a]pyrene exposure and chemical properties can be found in
Appendix A.
Assessments by Other National and International Health Agencies
Toxicity information on benzo[a]pyrene has been evaluated by the World Health
Organization, Health Canada, the International Agency for Research on Cancer, and the European
Union. The results of these assessments are presented in Appendix B. It is important to recognize
that these assessments were prepared at different times, for different purposes, using different
guidelines and methods, and that newer studies have been included in the IRIS assessment.
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The Preamble summarizes the objectives and scope of the IRIS program, general
principles and systematic review procedures used in developing IRIS assessments; and
the overall development process and document structure.
1. Objectives and Scope of the IRIS
Program
Soon after EPA was established in 1970, it
was at the forefront of developing risk
assessment as a science and applying it in
support of actions to protect human health and
the environment. EPA's IRIS program2
contributes to this endeavor by reviewing
epidemiologic and experimental studies of
chemicals in the environment to identify adverse
health effects and characterize exposure-
response relationships. Health agencies
worldwide use IRIS assessments, which are also
a scientific resource for researchers and the
public.
IRIS assessments cover the hazard
identification and dose-response steps of risk
assessment. Exposure assessment and risk
characterization are outside the scope of IRIS
assessments, as are political, economic, and
technical aspects of risk management. An IRIS
assessment may cover one chemical, a group of
structurally or toxicologically related chemicals,
or a chemical mixture. Exceptions outside the
scope of the IRIS program are radionuclides,
chemicals used only as pesticides, and the
"criteria air pollutants" (particulate matter,
ground-level ozone, carbon monoxide, sulfur
oxides, nitrogen oxides, and lead).
Enhancements to the IRIS program are
improving its science, transparency, and
productivity. To improve the science, the IRIS
program is adapting and implementing
principles of systematic review (i.e., using
explicit methods to identify, evaluate, and
synthesize study findings). To increase
transparency, the IRIS program discusses key
science issues with the scientific community and
the public as it begins an assessment External
peer review, independently managed and in
public, improves both science and transparency.
Increased productivity requires that
assessments be concise, focused on EPA's needs,
and completed without undue delay.
IRIS assessments follow EPA guidance3 and
standardized practices of systematic review.
This Preamble summarizes and does not change
IRIS operating procedures or EPA guidance.
Periodically, the IRIS program asks for
nomination of agents for future assessment or
reassessment. Selection depends on EPA's
priorities, relevance to public health, and
availability of pertinent studies. The IRIS
multiyear agenda4 lists upcoming assessments.
The IRIS program may also assess other agents
in anticipation of public health needs.
2. Planning an Assessment: Scoping,
Problem Formulation, and
Protocols
Early attention to planning ensures that IRIS
assessments meet their objectives and properly
frame science issues.
Scoping refers to the first step of planning,
where the IRIS program consults with EPA's
program and regional offices to ascertain their
needs. Scoping specifies the agents an
2IRIS program website: http: IIwww.epa.gov /iris /.
3EPA guidance documents: http://www.epa.gov/iris/basic-information-about-integrated-risk-information-
svstem#guidance/.
4IRIS multiyear agenda: https: //www.epa.gov/iris/iris-agenda.
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assessment will address, routes and durations of
exposure, susceptible populations and lifestages,
and other topics of interest
Problem formulation refers to the science
issues an assessment will address and includes
input from the scientific community and the
public. A preliminary literature survey,
beginning with secondary sources (e.g.,
assessments by national and international health
agencies and comprehensive review articles),
identifies potential health outcomes and science
issues. It also identifies related chemicals (e.g.,
toxicologically active metabolites and
compounds that metabolize to the chemical of
interest).
Each IRIS assessment comprises multiple
systematic reviews for multiple health
outcomes. It also evaluates hypothesized
mechanistic pathways and characterizes
exposure-response relationships. An
assessment may focus on important health
outcomes and analyses rather than expand
beyond what is necessary to meet its objectives.
Protocols refer to the systematic review
procedures planned for use in an assessment
They include strategies for literature searches,
criteria for study inclusion or exclusion,
considerations for evaluating study methods and
quality, and approaches to extracting data.
Protocols may evolve as an assessment
progresses and new agent-specific insights and
issues emerge.
3. Identifying and Selecting Pertinent
Studies
IRIS assessments conduct systematic
literature searches with criteria for inclusion and
exclusion. The objective is to retrieve the
pertinent primary studies (i.e., studies with
original data on health outcomes or their
mechanisms). PECO statements (Populations,
Exposures, Comparisons, Outcomes) govern the
literature searches and screening criteria.
"Populations" and animal species generally have
no restrictions. "Exposures" refers to the agent
and related chemicals identified during scoping
and problem formulation and may consider
route, duration, or timing of exposure.
"Comparisons" means studies that allow
comparison of effects across different levels of
exposure. "Outcomes" may become more specific
(e.g., from "toxicity" to "developmental toxicity"
to "hypospadias") as an assessment progresses.
For studies of absorption, distribution,
metabolism, and elimination, the first objective
is to create an inventory of pertinent studies.
Subsequent sorting and analysis facilitates
characterization and quantification of these
processes.
Studies on mechanistic events can be
numerous and diverse. Here, too, the objective is
to create an inventory of studies for later sorting
to support analyses of related data. The
inventory also facilitates generation and
evaluation of hypothesized mechanistic
pathways.
The IRIS program posts initial protocols for
literature searches on its website and adds
search results to EPA's HERO database.5 Then
the IRIS program takes extra steps to ensure
identification of pertinent studies: by
encouraging the scientific community and the
public to identify additional studies and ongoing
research; by searching for data submitted under
the Toxic Substances Control Act or the Federal
Insecticide, Fungicide, and Rodenticide Act; and
by considering late-breaking studies that would
impact the credibility of the conclusions, even
during the review process.6
4. Evaluating Study Methods and
Quality
IRIS assessments evaluate study methods
and quality, using uniform approaches for each
group of similar studies. The objective is that
subsequent syntheses can weigh study results on
their merits. Key concerns are potential bias
(factors that affect the magnitude or direction of
an effect) and insensitivity (factors that limit the
ability of a study to detect a true effect).
5Health and Environmental Research Online: https: //hero.epa.gov/hero/.
6IRIS "stopping rules": https: //www.epa.gov/sites/production/files/2014-06/documents/
iris stoppingrules.pdf.
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For human and animal studies, the
evaluation of study methods and quality
considers study design, exposure measures,
outcome measures, data analysis, selective
reporting, and study sensitivity. For human
studies, this evaluation also considers selection
of participant and referent groups and potential
confounding. Emphasis is on discerning bias that
could substantively change an effect estimate,
considering also the expected direction of the
bias. Low sensitivity is a bias towards the null.
Study-evaluation considerations are specific
to each study design, health effect, and agent
Subject-matter experts evaluate each group of
studies to identify characteristics that bear on
the informativeness of the results. For
carcinogenicity, neurotoxicity, reproductive
toxicity, and developmental toxicity, there is EPA
guidance for study evaluation (U.S. EPA. 2005a.
1998b. 1996. 1991c). As subject-matter experts
examine a group of studies, additional agent-
specific knowledge or methodologic concerns
may emerge and a second pass become
necessary.
Assessments use evidence tables to
summarize the design and results of pertinent
studies. If tables become too numerous or
unwieldy, they may focus on effects that are
more important or studies that are more
informative.
The IRIS program posts initial protocols for
study evaluation on its website, then considers
public input as it completes this step.
5. Integrating the Evidence of
Causation for Each Health Outcome
Synthesis within lines of evidence. For
each health outcome, IRIS assessments
synthesize the human evidence and the animal
evidence, augmenting each with informative
subsets of mechanistic data. Each synthesis
considers aspects of an association that may
suggest causation: consistency, exposure-
response relationship, strength of association,
temporal relationship, biological plausibility,
coherence, and "natural experiments" in humans
fU.S. EPA. 1994al fU.S. EPA. 2005al.
Each synthesis seeks to reconcile ostensible
inconsistencies between studies, taking into
account differences in study methods and
quality. This leads to a distinction between
conflicting evidence (unexplained positive and
negative results in similarly exposed human
populations or in similar animal models) and
differing results (mixed results attributable to
differences between human populations, animal
models, or exposure conditions) (U.S. EPA.
2005a).
Each synthesis of human evidence explores
alternative explanations (e.g., chance, bias, or
confounding) and determines whether they may
satisfactorily explain the results. Each synthesis
of animal evidence explores the potential for
analogous results in humans. Coherent results
across multiple species increase confidence that
the animal results are relevant to humans.
Mechanistic data are useful to augment the
human or animal evidence with information on
precursor events, to evaluate the human
relevance of animal results, or to identify
susceptible populations and lifestages. An agent
may operate through multiple mechanistic
pathways, even if one hypothesis dominates the
literature (U.S. EPA. 2005a).
Integration across lines of evidence. For
each health outcome, IRIS assessments integrate
the human, animal, and mechanistic evidence to
answer the question: What is the nature of the
association between exposure to the agent and the
health outcome?
For cancer, EPA includes a standardized
hazard descriptor in characterizing the strength
of the evidence of causation. The objective is to
promote clarity and consistency of conclusions
across assessments (U.S. EPA. 2005a).
Carcinogenic to humans: convincing
epidemiologic evidence of a causal
association; or strong human evidence of
cancer or its key precursors, extensive
animal evidence, identification of mode-of-
action and its key precursors in animals, and
strong evidence that they are anticipated in
humans.
Likely to be carcinogenic to humans: evidence
that demonstrates a potential hazard to
humans. Examples include a plausible
association in humans with supporting
experimental evidence, multiple positive
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Toxicological Review of Benzo[a]pyrene
results in animals, a rare animal response, or
a positive study strengthened by other lines
of evidence.
Suggestive evidence of carcinogenic potential:
evidence that raises a concern for humans.
Examples include a positive result in the only
study, or a single positive result in an
extensive database.
Inadequate information to assess carcinogenic
potential: no other descriptors apply.
Examples include little or no pertinent
information, conflicting evidence, or negative
results not sufficiently robust for not likely.
Not likely to be carcinogenic to humans: robust
evidence to conclude that there is no basis
for concern. Examples include no effects in
well-conducted studies in both sexes of
multiple animal species, extensive evidence
showing that effects in animals arise through
modes-of-action that do not operate in
humans, or convincing evidence that effects
are not likely by a particular exposure route
or below a defined dose.
If there is credible evidence of
carcinogenicity, there is an evaluation of
mutagenicity, because this influences the
approach to dose-response assessment and
subsequent application of adjustment factors for
exposures early in life (U.S. EPA. 2005a ), (U.S.
EPA. 2005bl.
6. Selecting Studies for Derivation of
Toxicity Values
The purpose of toxicity values (slope factors,
unit risks, reference doses, reference
concentrations; see section 7) is to estimate
exposure levels likely to be without appreciable
risk of adverse health effects. EPA uses these
values to support its actions to protect human
health.
The health outcomes considered for
derivation of toxicity values may depend on the
hazard descriptors. For example, IRIS
assessments generally derive cancer values for
agents that are carcinogenic or likely to be
carcinogenic, and sometimes for agents with
suggestive evidence (U.S. EPA. 2005a).
Derivation of toxicity values begins with a
new evaluation of studies, as some studies used
qualitatively for hazard identification may not be
useful quantitatively for exposure-response
assessment. Quantitative analyses require
quantitative measures of exposure and response.
An assessment weighs the merits of the human
and animal studies, of various animal models,
and of different routes and durations of exposure
fU.S. EPA. 1994al. Study selection is not
reducible to a formula, and each assessment
explains its approach.
Other biological determinants of study
quality include appropriate measures of
exposure and response, investigation of early
effects that precede overt toxicity, and
appropriate reporting of related effects (e.g.,
combining effects that comprise a syndrome, or
benign and malignant tumors in a specific
tissue).
Statistical determinants of study quality
include multiple levels of exposure (to
characterize the shape of the exposure-response
curve) and adequate exposure range and sample
sizes (to minimize extrapolation and maximize
precision) (U.S. EPA. 2012b).
Studies of low sensitivity may be less useful
if they fail to detect a true effect or yield toxicity
values with wide confidence limits.
7. Deriving Toxicity Values
General approach. EPA guidance describes
a two-step approach to dose-response
assessment: analysis in the range of observation,
then extrapolation to lower levels. Each toxicity
value pertains to a route (e.g., oral, inhalation,
dermal) and duration or timing of exposure (e.g.,
chronic, subchronic, gestational) (U.S. EPA.
20021.
IRIS assessments derive a candidate value
from each suitable data set Consideration of
candidate values yields a toxicity value for each
organ or system. Consideration of the organ/
system-specific values results in the selection of
an overall toxicity value to cover all health
outcomes. The organ/system-specific values are
useful for subsequent cumulative risk
assessments that consider the combined effect of
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multiple agents acting at a common anatomical
site.
Analysis in the range of observation.
Within the observed range, the preferred
approach is modeling to incorporate a wide
range of data. Toxicokinetic modeling has
become increasingly common for its ability to
support target-dose estimation, cross-species
adjustment, or exposure-route conversion. If
data are too limited to support toxicokinetic
modeling, there are standardized approaches to
estimate daily exposures and scale them from
animals to humans (U.S. EPA. 1994a). (U.S. EPA.
2005a"). fU.S. EPA. 2011. 2006).
For human studies, an assessment may
develop exposure-response models that reflect
the structure of the available data fU.S. EPA.
2005a)- For animal studies, EPA has developed a
set of empirical ("curve-fitting") models7 that
can fit typical data sets (U.S. EPA. 2005al. Such
modeling yields a point of departure, defined as a
dose near the lower end of the observed range,
without significant extrapolation to lower levels
(e.g., the estimated dose associated with an extra
risk of 10% for animal data or 1% for human
data, or their 95% lower confidence limits)(U.S.
EPA. 2005al. fU.S. EPA. 2012bl.
When justified by the scope of the
assessment, toxicodynamic ("biologically
based") modeling is possible if data are sufficient
to ascertain the key events of a mode-of-action
and to estimate their parameters. Analysis of
model uncertainty can determine the range of
lower doses where data support further use of
the model flJ.S. EPA. 2005al.
For a group of agents that act at a common
site or through common mechanisms, an
assessment may derive relative potency factors
based on relative toxicity, rates of absorption or
metabolism, quantitative structure-activity
relationships, or receptor-binding
characteristics (U.S. EPA. 2005a).
Extrapolation: slope factors and unit
risks. An oral slope factor or an inhalation unit
risk facilitates subsequent estimation of human
cancer risks. Extrapolation proceeds linearly
(i.e., risk proportional to dose) from the point of
departure to the levels of interest This is
appropriate for agents with direct mutagenic
activity. It is also the default if there is no
established mode-of-action (U.S. EPA. 2005a).
Differences in susceptibility may warrant
derivation of multiple slope factors or unit risks.
For early-life exposure to carcinogens with a
mutagenic mode-of-action, EPA has developed
default age-dependent adjustment factors for
agents without chemical-specific susceptibility
data fU.S. EPA. 2005al. fU.S. EPA. 2005bl.
If data are sufficient to ascertain the mode-
of-action and to conclude that it is not linear at
low levels, extrapolation may use the reference-
value approach (U.S. EPA. 2005a).
Extrapolation: reference values. An oral
reference dose or an inhalation reference
concentration is an estimate of human exposure
(including in susceptible populations) likely to
be without appreciable risk of adverse health
effects over a lifetime (U.S. EPA. 2002).
Reference values generally cover effects other
than cancer. They are also appropriate for
carcinogens with a nonlinear mode-of-action.
Calculation of reference values involves
dividing the point of departure by a set of
uncertainty factors (each typically 1, 3, or 10,
unless there are adequate chemical-specific
data) to account for different sources of
uncertainty and variability fU.S. EPA. 20021. (U.S.
EPA. 20141.
Human variation: An uncertainty factor covers
susceptible populations and lifestages that
may respond at lower levels, unless the data
originate from a susceptible study
population.
Animal-to-human extrapolation: For reference
values based on animal results, an
uncertainty factor reflects cross-species
differences, which may cause humans to
respond at lower levels.
Subchronic-to-chronic exposure: For chronic
reference values based on subchronic
studies, an uncertainty factor reflects the
likelihood that a lower level over a longer
duration may induce a similar response. This
benchmark Dose Software: http: //www.epa.gov/bmds/.
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factor may not be necessary for reference
values of shorter duration.
Adverse-effect level to no-observed-adverse-effect
level: For reference values based on a lowest-
observed-adverse-effect level, an
uncertainty factor reflects a level judged to
have no observable adverse effects.
Database deficiencies: If there is concern that
future studies may identify a more sensitive
effect, target organ, population, or lifestage, a
database uncertainty factor reflects the
nature of the database deficiency.
8. Process for Developing and Peer-
Reviewing IRIS Assessments
The IRIS process (revised in 2009 and
enhanced in 2013) involves extensive public
engagement and multiple levels of scientific
review and comment. IRIS program scientists
consider all comments. Materials released,
comments received from outside EPA, and
disposition of major comments (steps 3, 4, and 6
below) become part of the public record.
Step 1: Draft development. As outlined in
section 2 of this Preamble, IRIS program
scientists specify the scope of an assessment
and formulate science issues for discussion
with the scientific community and the public.
Next, they release initial protocols for the
systematic review procedures planned for
use in the assessment. IRIS program
scientists then develop a first draft, using
structured approaches to identify pertinent
studies, evaluate study methods and quality,
integrate the evidence of causation for each
health outcome, select studies for derivation
of toxicity values, and derive toxicity values,
as outlined in Preamble sections 3-7.
Step 2: Agency review. Health scientists across
EPA review the draft assessment
Step 3: Interagency science consultation.
Other federal agencies and the Executive
Office of the President review the draft
assessment.
Step 4: Public comment, followed by external
peer review. The public reviews the draft
assessment IRIS program scientists release
a revised draft for independent external peer
review. The peer reviewers consider
whether the draft assessment assembled and
evaluated the evidence according to EPA
guidance and whether the evidence justifies
the conclusions.
Step 5: Revise assessment. IRIS program
scientists revise the assessment to address
the comments from the peer review.
Step 6: Final agency review and interagency
science discussion. The IRIS program
discusses the revised assessment with EPA's
program and regional offices and with other
federal agencies and the Executive Office of
the President
Step 7: Post final assessment. The IRIS
program posts the completed assessment
and a summary on its website.
9. General Structure of IRIS
Assessments
Main text. IRIS assessments generally
comprise two major sections: (1) Hazard
Identification and (2) Dose-Response
Assessment. Section 1.1 briefly reviews chemical
properties and toxicokinetics to describe the
disposition of the agent in the body. This section
identifies related chemicals and summarizes
their health outcomes, citing authoritative
reviews. If an assessment covers a chemical
mixture, this section discusses environmental
processes that alter the mixtures humans
encounter and compares them to mixtures
studied experimentally.
Section 1.2 includes a subsection for each
major health outcome. Each subsection
discusses the respective literature searches and
study considerations, as outlined in Preamble
sections 3 and 4, unless covered in the front
matter. Each subsection concludes with evidence
synthesis and integration, as outlined in
Preamble section 5.
Section 1.3 links health hazard information
to dose-response analyses for each health
outcome. One subsection identifies susceptible
populations and lifestages, as observed in human
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or animal studies or inferred from mechanistic
data. These may warrant further analysis to
quantify differences in susceptibility. Another
subsection identifies biological considerations
for selecting health outcomes, studies, or data
sets, as outlined in Preamble section 6.
Section 2 includes a subsection for each
toxicity value. Each subsection discusses study
selection, methods of analysis, and derivation of
a toxicity value, as outlined in Preamble sections
6 and 7.
Front matter. The Executive Summary
provides information historically included in
IRIS summaries on the IRIS program website. Its
structure reflects the needs and expectations of
EPA's program and regional offices.
A section on systematic review methods
summarizes key elements of the protocols,
including methods to identify and evaluate
pertinent studies. The final protocols appear as
an appendix.
The Preface specifies the scope of an
assessment and its relation to prior assessments.
It discusses issues that arose during assessment
development and emerging areas of concern.
This Preamble summarizes general
procedures for assessments begun after the date
below. The Preface identifies assessment-
specific approaches that differ from these
general procedures.
August 2016
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References
U.S. EPA. (1991). Guidelines for developmental toxicity risk assessment (pp. 1-83). (EPA/600/FR-
91/001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
http: / /cfpub.epa.gov/ncea/cfm /recordisplay.cfm?deid=2 3162
U.S. EPA. (1994). Methods for derivation of inhalation reference concentrations and application of
inhalation dosimetry [EPA Report] (pp. 1-409). (EPA/600/8-90/066F). Research Triangle
Park, NC: U.S. Environmental Protection Agency, Office of Research and Development, Office
of Health and Environmental Assessment, Environmental Criteria and Assessment Office.
https://cfpub.epa. gov/ncea/risk/recordisplay.cfm?deid=71993&CFID=51174829&CFTOKE
N=25006317
U.S. EPA. (1996). Guidelines for reproductive toxicity risk assessment (pp. 1-143). (EPA/630/R-
96/009). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
U.S. EPA. (1998). Guidelines for neurotoxicity risk assessment Fed Reg 63: 26926-26954.
U.S. EPA. (2002). A review of the reference dose and reference concentration processes (pp. 1-192).
(EPA/630/P-02/002F). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, http://www.epa.gov/osa/review-reference-dose-and-reference-
concentration-processes
U.S. EPA. (2005a). Guidelines for carcinogen risk assessment [EPA Report] (pp. 1-166).
(EPA/630/P-03/001F). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, http://www2.epa.gov/osa/guidelines-carcinogen-risk-assessment
U.S. EPA. (2005b). Supplemental guidance for assessing susceptibility from early-life exposure to
carcinogens (pp. 1-125). (EPA/630/R-03/003F). Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum.
U.S. EPA. (2006). Approaches for the application of physiologically based pharmacokinetic (PBPK)
models and supporting data in risk assessment (Final Report) [EPA Report] (pp. 1-123).
(EPA/600/R-05/043F). Washington, DC: U.S. Environmental Protection Agency, Office of
Research and Development, National Center for Environmental Assessment.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=l 57668
U.S. EPA. (2011). Recommended use of body weight 3/4 as the default method in derivation of the
oral reference dose (pp. 1-50). (EPA/100/R11/0001). Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum, Office of the Science Advisor.
https://www.epa.gov/risk/recommended-use-body-weight-34-default-method-derivation-
oral-reference-dose
U.S. EPA. (2012). Benchmark dose technical guidance (pp. 1-99). (EPA/100/R-12/001).
Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
U.S. EPA. (2014). Guidance for applying quantitative data to develop data-derived extrapolation
factors for interspecies and intraspecies extrapolation. (EPA/100/R-14/002F). Washington,
DC: Risk Assessment Forum, Office of the Science Advisor.
http://www.epa.gov/raf/DDEF/pdf/ddef-final.pdf
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EXECUTIVE SUMMARY
Summary of Occurrence and Health Effects
Ilenzi)|ii|pyrenc is a live-ring polycyclic aromatic hydrocarbon (I'AII).
ncn/.i)|ii|p\relie (along with other I',Alls) is released into Ihc iilmcisplifro as a
component of smoke I'roni forest lires, induslriiil processes, vehicle exhaust,
cigarettes, and through the burning ol luel (such as wood, coal, and petroleum
products). Oml exposure to henzo|ii|pyrene can occur by eating cerUiin lood
products, such as charred meats, where hen/.o|ii|pyrene is formed during the cooking
process, or by eating loods grown in areas contaminated with hcn/.o|ii|pyrene (from
the air and soil). Dermal exposure may occur from conUict with soils or materials Unit
conUiin soot, tar, or crude petroleum products or by using cerUiin pharmaceutical
products containing con I Uirs, such as those used to treat the skin conditions, eczema
iind psoriiisis. The magnitude of huniiin exposure to henzo|ii|pyrene iind other I'AI Is
depends on factors such iis lifestyle (e.g., diet, tobacco smoking), occupiition, iind
living conditions (e.g., urban versus ruml selling, domestic heiiling, iind cooking
methods).
Animill studies demonstrate lliiil exposure to henzo|ii|pyrcne is iissociiited
with developmental (including developmental neurotoxicity), reproductive, iind
immunologiciil effects. In iiddition, epidemiology studies involving exposure to I'AII
mixtures have reported iissociiilions between interiiiil biomarkers of exposure to
lien/o|ii|pyrene (henzo|ii|pyrene diol epoxide-DNA iidducts) iind adverse birth
outcomes (including reduced birth weight, posliniliil body weight, iind lieiid
circumference), neurobehiivioml effects, nnd decreased fertility.
Studies in multiple iininiiil species (.lemonslmle Unit ben/.o|ii|pyrene is
ciircinogenic ill multiple tumor sites (alimentary Imcl, liver, kidney, respiratory tmcl,
phiirynx, iind skin) by iill routes of exposure. In iiddition. there is strong evidence of
carcinogenicity in occupations involving exposure to I'AII mixtures contiiining
hen/.o|ii|pyrene, such iis iiluminum production, chimney sweeping, coiil giisificiilion,
coiil-Uir distilliition. coke production, iron iind steel rounding, iind pin ing iind roofing
with coiil tiir pilch. An increiising number of occupiilioiiiil studies (.lemonslmle ii
positive exposure-response relationship with en 111 nkili\e ben/.o|ii|pyrene exposure
iind lungciincer.
Effects Other Than Cancer Observed Following Oral Exposure
In animals, oral exposure to benzo[a]pyrene has been shown to result in developmental
toxicity (including developmental neurotoxicity), reproductive toxicity, and immunotoxicity.
Developmental effects in rats and mice include neurobehavioral changes and cardiovascular effects
following gestational exposures. Reproductive and immune effects include decreased sperm
counts, ovary weight, and follicle numbers, and decreased immunoglobulin and B cell numbers and
thymus weight following oral exposures in adult animals. In humans, benzo[a]pyrene exposure
occurs in conjunction with other PAHs and, as such, attributing the observed effects to
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benzo[a]pyrene is complicated. However, some human studies report associations between
particular health endpoints and internal measures of exposure, such as benzo[a]pyrene-
deoxyribonucleic acid (DNA) adducts, or external measures of benzo[a]pyrene exposure. Overall,
the human studies report developmental, neurobehavioral, reproductive, and immune effects that
are generally analogous to those observed in animals, and provide qualitative, supportive evidence
for hazards associated with benzo[a]pyrene exposure.
Oral Reference Dose (RfD) for Effects Other Than Cancer
Organ- or system-specific RfDs were derived for hazards associated with benzo[a]pyrene
exposure where data were amenable (see Table ES-1). These organ- or system-specific reference
values may be useful for subsequent cumulative risk assessments that consider the combined effect
of multiple agents acting at a common site.
Developmental toxicity, represented by neurobehavioral changes persisting into adulthood,
was chosen as the basis for the overall oral RfD as the available data indicate that developmental
neurotoxicity represents the most sensitive hazard of benzo[a]pyrene exposure. The
neurodevelopmental study by Chen etal. ("20121 was used to derive the RfD. Altered responses in
three behavioral tests (i.e., Morris water maze, elevated plus maze, and open field tests) were
selected to represent the critical effect of abnormal behavior, due to the consistency (i.e., each of
these responses were affected in two separate cohorts of rats, including testing as juveniles and as
adults; similar effects in these behavioral tests were observed across studies) and sensitivity of
these responses, and the observed dose-response relationship of effects across dose groups.
Benchmark dose (BMD) modeling for each of the three endpoints resulted in BMDLisd values that
clustered in the range 0.092-0.16 mg/kg-day. The lower end of this range of BMDLs,
0.092 mg/kg-day, was selected to represent the point of departure (POD) from these three
endpoints for RfD derivation.
The overall RfD was calculated by dividing the POD for altered behavior in three tests of
nervous system function by a composite uncertainty factor (UF) of 300 to account for the
extrapolation from animals to humans (10), for interindividual differences in human susceptibility
(10), and for deficiencies in the toxicity database (3).
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Table ES-1. Organ/system-specific RfDs and overall RfD for benzo[a]pyrene
Effect
Basis
RfD
(mg/kg-d)
Confidence
Developmental
Neurobehavioral changes
Gavage neurodevelopmental study in rats (postnatal days [PNDs]
5-11)
Chen et al. (2012)
3 x 10"4
Medium
Reproductive
Decreased ovarian follicles and ovary weight
Gavage subchronic (60 d) reproductive toxicity study in rats
Xu etal. (2010)
4 x 10"4
Medium
Immunological
Decreased thymus weight and serum IgM
Gavage subchronic (35 d) study in rats
De Jong et al. (1999) and Kroese et al. (2001)
2 x 10"3
Low
Overall RfD
Developmental toxicity (including developmental neurotoxicity)
3 x 10"4
Medium
Confidence in the Overall Oral RfD
The overall confidence in the RfD is medium. Confidence in the principal study (Chen etal..
20121 is medium. The design, conduct, and reporting of this neurodevelopmental study was good
and a wide variety of neurotoxicity endpoints were measured across 40 litters of rats. However,
some uncertainty exists regarding the authors' use of dam rotation across litters (an attempt to
reduce potential nurturing bias) and a within-litter dosing design, by potentially introducing
maternal stress or other unanticipated consequences in the pups, and some informative
experimental details were omitted, including the sensitivity of some assays at the indicated
developmental ages and lack of reporting of individual animal- or gender-specific data for all
outcomes. Several subchronic and developmental studies covering a wide variety of endpoints are
also available; however, a multigeneration toxicity study with exposure throughout development
and across generations is not available, and the available neurotoxicity studies did not
comprehensively evaluate all potentially vulnerable lifestages of nervous system development.
Therefore, confidence in the database is medium.
Effects Other Than Cancer Observed Following Inhalation Exposure
In animals, inhalation exposure to benzo[a]pyrene has been shown to result in
developmental and reproductive toxicity. Studies in rats following inhalation exposure show
decreased embryo/fetal survival and nervous system effects in offspring, and decreased testes
weight and sperm counts in adult animals. Overall, the available human PAH mixtures studies
report developmental and reproductive effects that are generally analogous to those observed in
animals, and provide qualitative, supportive evidence for the hazards associated with
benzo[a]pyrene exposure.
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Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
An attempt was made to derive organ- or system-specific RfCs for hazards associated with
benzo[a]pyrene exposure where data were amenable (see Table ES-2). These organ- or system-
specific reference values may be useful for subsequent cumulative risk assessments that consider
the combined effect of multiple agents acting at a common site.
Developmental toxicity, represented by decreased embryo/fetal survival, was chosen as the
basis for the proposed inhalation RfC as the available data indicate that developmental effects
represent a sensitive hazard of benzo[a]pyrene exposure. The developmental inhalation study in
rats by Archibong et al. f20021 and the observed decreased embryo/fetal survival (i.e., increased
resorptions) following exposure to benzo[a]pyrene on gestation days (GDs) 11-20 were used to
derive the overall RfC. The lowest-observed-adverse-effect level (LOAEL) of 25 |ig/m3 based on
decreased embryo/fetal survival was selected as the POD. The LOAEL was adjusted to account for
the discontinuous daily exposure to derive the PODadj and the human equivalent concentration
(HEC) was calculated from the PODadj by multiplying by the regional deposited dose ratio (RDDRer)
for extrarespiratory (i.e., systemic) effects, as described in Methods for Derivation of Inhalation
Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA. 1994bl These
adjustments resulted in a PODhec of 4.6 |ig/m3, which was used as the POD for RfC derivation.
The RfC was calculated by dividing the POD by a composite UF of 3,000 to account for
toxicodynamic differences between animals and humans (3), interindividual differences in human
susceptibility (10), LOAEL-to-no-observed-adverse-effectlevel (NOAEL) extrapolation (10), and
deficiencies in the toxicity database (10).
Table ES-2. Organ/system-specific RfCs and overall RfC for benzo[a]pyrene
Effect
Basis
RfC (mg/m3)
Confidence
Developmental
Decreased embryo/fetal survival
Developmental toxicity study in rats (GDs 11-20)
Archibong et al. (2002)
2 x 10"6
Low-medium
Reproductive
Reduced ovulation rate and ovary weight
Premating study in rats (14 d)
Archibong et al. (2012)
3 x 10"6
Low-medium
Overall RfC
Developmental toxicity
2 x 10"6
Low-medium
Confidence in the Overall Inhalation RfC
The overall confidence in the RfC is low-to-medium. Confidence in the principal study
fArchibong et al.. 20021 is medium. The conduct and reporting of this developmental inhalation
study were adequate; however, a NOAEL was not identified. Confidence in the database is low due
to the lack of a multigeneration toxicity study and the lack of information on varied toxicity
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endpoints following subchronic and chronic inhalation exposure. However, confidence in the RfC is
bolstered by consistent systemic effects observed by the oral route (including reproductive and
developmental effects) and similar effects observed in human populations exposed to PAH
mixtures.
Evidence for Human Carcinogenicity
Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005al benzo[a]pyrene is
"carcinogenic to humans" based on strong and consistent evidence in animals and humans. The
evidence includes an extensive number of studies demonstrating carcinogenicity in multiple animal
species exposed via all routes of administration and increased cancer risks, particularly in the lung
and skin, in humans exposed to different PAH mixtures containing benzo[a]pyrene. Mechanistic
studies provide strong supporting evidence that links the metabolism of benzo[a]pyrene to DNA-
reactive agents with key mutational events in genes that can lead to tumor development. These
events include formation of specific DNA adducts and characteristic mutations in oncogenes and
tumor suppressor genes that have been observed in humans exposed to PAH mixtures. This
combination of human, animal, and mechanistic evidence provides the basis for characterizing
benzo[a]pyrene as "carcinogenic to humans."
Quantitative Estimate of Carcinogenic Risk From Oral Exposure
Lifetime oral exposure to benzo[a]pyrene has been associated with forestomach, liver, oral
cavity, jejunum or duodenum, and auditory canal tumors in male and female Wistar rats,
forestomach tumors in male and female Sprague-Dawley rats, and forestomach, esophagus, tongue,
and larynx tumors in female B6C3Fi mice (male mice were not tested). Less-than-lifetime oral
exposure to benzo[a]pyrene has also been associated with forestomach tumors in more than
10 additional bioassays with several strains of mice. The Kroese etal. f20011 and Beland and Culp
(1998) studies were selected as the best available studies for dose-response analysis and
extrapolation to lifetime cancer risk following oral exposure to benzo[a]pyrene. These studies
included histological examinations for tumors in many different tissues, contained three exposure
levels and controls, contained adequate numbers of animals per dose group (~50/sex/group),
treated animals for up to 2 years, and included detailed reporting methods and results (including
individual animal data).
Time-weigh ted average (TWA) daily doses were converted to human equivalent doses
(HEDs) on the basis of (body weight [BW])3/4 scaling (U.S. EPA. 1992). EPA then used the
multistage-Weibull model for the derivation of the oral slope factor. This model was used because
it incorporates the time at which death-with-tumor occurred and can account for differences in
mortality observed between the exposure groups. Using linear extrapolation from the BMDLio,
human equivalent oral slope factors were derived for each gender/tumor site combination (slope
factor = 0.1 /BMDLio) reported by Kroese etal. f20011 and Beland and Culp fl9981. The oral slope
factor of 1 per mg/kg-day based on the tumor response in the alimentary tract (forestomach,
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esophagus, tongue, and larynx) of female B6C3Fi mice fBeland and Culp. 19981 was selected as the
factor with the highest value (most sensitive) among a range of slope factors derived.
Quantitative Estimate of Carcinogenic Risk From Inhalation Exposure
Inhalation exposure to benzo[a]pyrene has been associated with squamous cell neoplasia in
the larynx, pharynx, trachea, nasal cavity, esophagus, and forestomach of male Syrian golden
hamsters exposed for up to 130 weeks to benzo[a]pyrene condensed onto sodium chloride
particles (Thvssen et al.. 1981). Supportive evidence for the carcinogenicity of inhaled
benzo[a]pyrene comes from additional studies with hamsters exposed to benzo[a]pyrene via
intratracheal instillation. The Thvssen et al. (1981) bioassay represents the only study of lifetime
exposure to inhaled benzo[a]pyrene.
A time-to-tumor dose-response model was fit to the TWA continuous exposure
concentrations and the individual animal incidence data for the overall incidence of tumors in the
upper respiratory tract or pharynx. The inhalation unit risk of 6 x 10~4 per ng/m3 was calculated
by linear extrapolation (slope factor = 0.1/BMCLio) from a BMCLio of 0.16 mg/m3 for the
occurrence of upper respiratory and upper digestive tract (forestomach) tumors in male hamsters
chronically exposed by inhalation to benzo[a]pyrene (Thyssen et al.. 1981).
Quantitative Estimate of Carcinogenic Risk From Dermal Exposure
Skin cancer in humans has been documented to result from occupational exposure to
complex mixtures of PAHs including benzo[a]pyrene, such as coal tar, coal tar pitches, unrefined
mineral oils, shale oils, and soot In animal models, numerous dermal bioassays have demonstrated
an increased incidence of skin tumors with increasing dermal exposure of benzo[a]pyrene in all
species tested, although mostbenzo[a]pyrene bioassays have been conducted in mice.
Carcinogenicity studies in animals by the dermal route of exposure are available for
benzo[a]pyrene and are supportive of the overall cancer hazard. A quantitative estimate of skin
cancer risk from dermal exposure is not included in this assessment, as methodology for
interspecies extrapolation of dermal toxicokinetics and carcinogenicity are still under development
Susceptible Populations and Lifestages
Benzo[a]pyrene has been determined to be carcinogenic by a mutagenic mode of action in
this assessment. According to the Supplemental Guidance for Assessing Susceptibility from Early Life
Exposure to Carcinogens fU.S. EPA. 2005bl. individuals exposed during early life to carcinogens with
a mutagenic mode of action are assumed to have an increased risk for cancer. The oral slope factor
of 1 per mg/kg-day and inhalation unit risk of 0.0006 per |ig/m3, calculated from data applicable to
adult exposures, do not reflect presumed early life susceptibility to this chemical. Although some
chemical-specific data exist for benzo[a]pyrene that demonstrate increased early life susceptibility
to cancer, these data were not considered sufficient to develop separate risk estimates for
childhood exposure. In the absence of adequate chemical-specific data to evaluate differences in
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age-specific susceptibility, the Supplemental Guidance (U.S. EPA. 2005b) recommends that age-
dependent adjustment factors (ADAFs) be applied in estimating cancer risk. The ADAFs are 10- and
3-fold adjustments that are combined with age specific exposure estimates when estimating cancer
risks from early life (<16 years of age) exposures to benzo[a]pyrene.
Regarding effects other than cancer, there are epidemiological studies that report
associations between developmental effects (decreased postnatal growth, decreased head
circumference, and neurodevelopmental delays), reproductive effects, and internal biomarkers of
exposure to benzo[a]pyrene. Studies in animals also indicate alterations in neurological
development and heightened susceptibility to reproductive effects following gestational or early
postnatal exposure to benzo[a]pyrene. More preliminary data suggest that effects on
cardiovascular, kidney, pulmonary, and immune system development may result from early life
exposures, although few in vivo developmental studies exist to confirm these findings.
Key Issues Addressed in Assessment
The overall RfD and RfC were developed based on effects observed following exposure to
benzo[a]pyrene during a critical window of development. The derivation of a general population
toxicity value based on exposure during development has implications regarding the evaluation of
populations exposed outside of the developmental period and the averaging of exposure to
durations outside of the critical window of susceptibility. Discussion of these considerations is
provided in Sections 2.1.5 and 2.2.5.
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LITERATURE SEARCH STRATEGY| STUDY SELECTION
The literature search strategy used to identify primary, peer-reviewed literature pertaining
to benzo[a]pyrene was conducted using the databases listed in Table LS-1 (see Appendix C for the
complete list of keywords). References from previous assessments by the U.S. Environmental
Protection Agency (EPA) and other national and international health agencies were also examined
to ensure that critical studies were not missed by the literature search. EPA conducted a
comprehensive, systematic literature search for benzo[a]pyrene through February, 2012. The
literature search results were shared on the EPA Docket fhttps: //www.gpo.gov/fdsys/pkg/FR-
2012-07-16/html/2012-17145.html. and the public was invited to review the literature search
results and submit additional information to EPA (e.g., unpublished studies or other primary
technical sources that are not available through the open literature). The inclusion/exclusion
criteria that were applied to the 2012 and 2014 literature searches conducted prior to external
peer review are presented in Table LS-1.
Following external peer review, the literature search was updated (August 2016).
Consistent with the Integrated Risk Information System (IRIS) Stopping Rules
fhttp://www.epa.gov/sites/production/files/2014-06/documents/iris stoppingrules.pdf). manual
screening of the literature search update focused on identifying new studies that might change a
major conclusion of the assessment Upon review, the potentially pertinent references identified in
the post-peer review literature search did not impact the assessment's conclusions; thus, these
studies were not added to the assessment The references identified with the latest update,
including bibliographic information and abstracts, can be found on the Health and Environmental
Research Online (HERO) website fhttp://hero.epa.gov/benzoapyrenel and are tagged as "August
2016 Update."
Table LS-1. Summary of the search strategy employed for benzo[a]pyrene
Database
Keywords
Pubmed
Toxcenter
Toxline
Chemical name (CASRN): benzo[a]pyrene (50-32-8)
Synonyms: 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
Standard toxicology search keywords
Toxicity (including duration, effects to children and occupational exposure); development;
reproduction; teratogenicity; exposure routes; pharmacokinetics; toxicokinetics; metabolism; body
fluids; endocrinology; carcinogenicity; genotoxicity; antagonists; inhibitors
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Database
Keywords
TSCATS
Searched by CASRNs and chemical names (including synonyms)
ChemID
Chemfinder
CCRIS
HSDB
GENE-TOX
RTECS
aPrimary and secondary keywords used for the Pubmed, Toxcenter, and Toxline databases can be found in the
Supplemental Information.
CASRN = Chemical Abstracts Service Registry Number; CCRIS = Chemical Carcinogenesis Research Information
System; GENE-TOX = Genetic Toxicology Data Bank HSDB = Hazardous Substances Data Bank; RTECS = Registry of
Toxic Effects of Chemical Substances; TSCATS = Toxic Substances Control Act Test Submissions
Figure LS-1 depicts the 2012 literature search, study selection strategy, and number of
references associated with each stage of literature screening. Approximately 20,700 references
were identified with the initial keyword search. Based on a secondary keyword search followed by
a preliminary manual screen of titles or abstracts by a toxicologist, approximately 1,190 references
were identified that provided information potentially relevant to characterizing the health effects
or physical and chemical properties of benzo[a]pyrene. A more detailed manual review of titles,
abstracts, and/or papers was then conducted. Notable exclusions from the Toxicological Review
are large numbers of animal in vivo or in vitro studies designed to identify potential therapeutic
agents that would prevent the carcinogenicity or genotoxicity of benzo[a]pyrene and toxicity
studies of benzo[a]pyrene in nonmammalian species (e.g., aquatic species, plants).
For the updated literature search conducted for the timeframe January 2012 through
August 2014, the search terms included benzo(a)pyrene AND (rat OR mouse OR mice) and results
were screened manually by title, abstract, and/or full text using the exclusion criteria outlined in
Figure LS-1. Relevant studies that could potentially impact the hazard characterization and dose-
response assessment were identified and considered. Several pertinent studies, published since the
last comprehensive literature search (i.e., 2012), were identified and incorporated into the text
where relevant
In addition to the comprehensive literature search, more iterative literature searches were
conducted throughout the draft development process. For example, specialized searches were
conducted during draft development to provide additional context for potential mechanisms of
hazards identified from in vivo subchronic, chronic, or developmental studies. Additional literature
may be sought to fill in data gaps and to help inform a responses to peer review comments. Any
references from the iterative searches were captured and documented in the updated literature
searches of 2014 and 2016.
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Figure LS-1. Study selection strategy.
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Selection of studies for inclusion in the Toxicological Review was based on consideration of
the extent to which the study was informative and relevant to the assessment and general study
quality considerations. In general, the relevance of health effect studies was evaluated as outlined
in the Preamble and EPA guidance (A Review of the Reference Dose and Reference Concentration
Processes fU.S. EPA. 20021 and Methods for Derivation of Inhalation Reference Concentrations and
Application of Inhaled Dosimetry fU.S. EPA. 1994al. The reasons for excluding epidemiological and
animal studies from the references identified by the keyword search are provided in Figure LS-1.
The available studies examining the health effects of benzo[a]pyrene exposure in humans
are discussed and evaluated in the hazard identification sections of the assessment (Section 1), with
specific limitations of individual studies and of the collection of studies noted. The common major
limitation of the human epidemiological studies (with respect to identifying potential adverse
health outcomes specifically from benzo[a]pyrene) is that they all involve exposures to complex
mixtures containing other polycyclic aromatic hydrocarbons (PAHs) and other compounds. The
evaluation of the epidemiological literature focuses on studies in which possible associations
between external measures of exposure to benzo[a]pyrene or biomarkers of exposure to
benzo[a]pyrene (e.g., benzo[a]pyrene-DNA adducts or urinary biomarkers) and potential adverse
health outcomes were evaluated. Pertinent mechanistic studies in humans (e.g., identification of
benzo[a]pyrene-DNA adducts and characteristics of mutations in human tumors) were also
considered in assessing the weight of evidence for the carcinogenicity of benzo[a]pyrene.
The health effects literature for benzo[a]pyrene is extensive. All animal studies of
benzo[a]pyrene involving repeated oral, inhalation, or dermal exposure that were considered to be
of acceptable quality, whether yielding positive, negative, or null results, were considered in
assessing the evidence for health effects associated with chronic exposure to benzo[a]pyrene.
These studies were evaluated for aspects of design, conduct, or reporting that could affect the
interpretation of results and the overall contribution to the synthesis of evidence for determination
of hazard potential using the study quality considerations outlined in the Preamble. Discussion of
study strengths and limitations (that ultimately supported preferences for the studies and data
relied upon) were included in the text where relevant.
Animal toxicity studies involving short-term duration and other routes of exposure were
also evaluated to inform conclusions about health hazards, especially regarding mode of action.
The references considered and cited in this document, including bibliographic information and
abstracts, can be found on the HERO website8 fhttp://hero.epa.gov/benzoapyrene).
8HER0 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 National Center for Environmental
Assessment (NCEA). New studies are added continuously to HERO.
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1. HAZARD IDENTIFICATION
1.1. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM
NOTE: In the environment, benzo[a]pyrene occurs in conjunction with other structurally
related chemical compounds known as polycyclic aromatic hydrocarbons (PAHs).9 Accordingly,
there are few epidemiologic studies designed to solely investigate the effects of benzo[a]pyrene.
There are, however, many epidemiologic studies that have investigated the effects of exposure to
PAH mixtures. Benzo[a]pyrene is universally present in these mixtures and is routinely analyzed
and detected in environmental media contaminated with PAH mixtures; thus, it is often used an
indicator chemical to measure exposure to PAH mixtures (Bostrom etal.. 20021.
1.1.1. Developmental Toxicity
Human and animal studies provide evidence for PAH- and benzo[a]pyrene-induced
developmental effects (including developmental neurotoxicity). Effects on embryo/fetal survival,
postnatal growth, neurobehavioral function, and development have been demonstrated in human
populations exposed to PAH mixtures during gestation. Animal studies demonstrate various effects
including changes in embryo/fetal survival, pup weight, blood pressure, fertility, reproductive
organ weight and histology, and nervous system function in gestationally and/or early postnatally
treated animals.
Studies in humans and animal models indicate thatbenzo[a]pyrene and metabolites are
widely distributed in maternal and fetal tissues, supporting placental transfer (Madhavan and
Naidu. 1995: Withevetal.. 1993: NeubertandTapken. 1988: Shendrikova and Aleksandrov. 19741.
In addition, benzo[a]pyrene can be readily detected in human milk, especially in smokers (Yu etal..
2011: Lapole etal.. 2007: Zanieri etal.. 20071. However, benzo[a]pyrene is also readily metabolized
(albeit to reactive metabolites), widely distributed, and eliminated in mammals (see Section D.l in
the Supplemental Information). Evaluations of benzo[a]pyrene distribution and elimination in
lactating animals do not suggest that neonatal lactational doses to benzo[a]pyrene are magnified
over maternal doses (Lapole etal.. 2007: Lavoie etal.. 1987b: West and Horton. 19761.
Altered Birth Outcomes
Human and animal studies provide evidence that benzo[a]pyrene exposure may lead to
altered outcomes reflecting growth and development in utero or in early childhood. Two cohort
studies in pregnant women in China and the United States examined cord blood levels of
benzo[a]pyrene-7,8-diol-9,10 epoxide (BPDE)-deoxyribonucleic acid (DNA) adducts in relation to
9PAHs are a large class of chemical compounds formed during the incomplete combustion of organic matter.
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measures of child growth following exposure to PAH mixtures (Tang etal.. 2006: Perera etal..
2005b: Perera etal.. 20041 (Table 1-1). In the Chinese cohort, high benzo[a]pyrene-adduct levels
were associated with reduced weight at 18, 24, and 30 months of age, but not at birth fTang etal..
20061. In the U.S. cohort, an independent effect on birth weight was not observed with either
benzo[a]pyrene-adducts or environmental tobacco smoke (ETS) exposure; however, a doubling of
cord blood adducts in combination with ETS exposure in utero was seen, corresponding to an 8%
reduction in birth weight (Perera etal.. 2005b). ETS, also called secondhand smoke, is the smoke
given off by a burning tobacco product and the smoke exhaled by a smoker contains over 7,000
chemicals, including benzo[a]pyrene. No associations were seen with birth length (or height at
later ages) in either of these cohort studies.
Two cohort studies in pregnant women in Spain and Norway evaluated the relationship
between the dietary intake of benzo[a]pyrene estimated by food questionnaire and birth weight
and length of offspring (Duarte-Salles etal.. 2013: Duarte-Salles etal.. 20121 (Table 1-1). In the
Spanish cohort, benzo[a]pyrene intake was associated with decreased birth weight, reduced birth
length, and small for gestational age (SGA) among infants born to women with low, but not high,
vitamin C intake (stratified as greater than or less than the mean vitamin C in the diet: 189.41
mg/day) fDuarte-Salles etal.. 20121. These findings were confirmed by the larger Norwegian
cohort, which demonstrated a relationship between dietary benzo[a]pyrene intake and reduced
birth weight and length in offspring of all women including nonsmokers fDuarte-Salles etal.. 20131.
The magnitude of the association was higher in women consuming less than the recommended
vitamin C intake of 85 mg/day, suggesting that vitamin C may exert a protective influence against
the reduced birth weight associated with benzo[a]pyrene in the diet
A Chinese case-control study indicated that PAH exposure may be associated with increased
risk of fetal death fWu etal.. 20101. A strong association was seen between maternal blood
benzo[a]pyrene-DNA adduct levels and risk of delayed miscarriage (fetal death before 14 weeks of
gestation), with a 4-fold increased risk for levels above compared with below the median.
However, no significant difference in adduct levels was detected between fetal tissue from cases
compared to controls.
Decreased embryo or fetal survival, as evidenced by decreased litter size, has also been
noted in pregnant animals treated following implantation by the oral and inhalation routes. An
approximate 40% decrease in litter size was noted in mouse dams treated by gavage on gestation
days (GDs) 7-16 at doses of 160 mg/kg-day, but no decreases were observed at 10 or 40 mg/kg-
day fMackenzie and Angevine. 19811. Several lower dose studies of rats treated on GDs 14-17 with
doses of up to 1.2 mg/kg-day benzo[a]pyrene did not observe any difference in litter size (Tules et
al.. 2012: McCallister et al.. 2008: Brown etal.. 20071. By the inhalation route, a dose-related
increase in embryo/fetal resorptions was observed, with a 19% increase following the lowest
tested exposure of 25 |ig/m3 on GDs 11-20 in F344 rats, and increasing rates of resorptions seen at
the two higher doses fArchibong et al.. 20021.
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Toxicological Review of Benzo[a]pyrene
In animals (Table 1-2 and Figure 1-1), reduced body weight in offspring has also been noted
in some developmental studies. Decreases in body weight (up to 13%) were observed in mice
following prenatal gavage exposure (GDs 7-16), and as time from exposure increased (postnatal
days [PNDs] 20-42), the dose at which effects were observed decreased (from 40 to 10 mg/kg-day,
respectively) fMackenzie and Angevine. 19811. In addition, decreases in body weight
(approximately 10-20%) were observed in rats on PNDs 36 and 71 following gavage exposure at
only 2 mg/kg-day on PNDs 5-11 (Chen etal.. 2012). and on PND 8 following gavage exposure to
10 or 25 mg/kg-day on PNDs 1-7 (Liang etal.. 2012). At doses up to 1.2 mg/kg-day and follow-up
to PND 30, two developmental studies in rats did not observe decrements in pup body weight
following treatment from GD 14 to 17 flules etal.. 2012: McCallister et al.. 20081. Maternal toxicity
was not observed in mouse or rat dams exposed to up to 160 mg/kg-day benzo[a]pyrene (Tules et
al.. 2012: McCallister et al.. 2008: Brown etal.. 2007: Kristensen etal.. 1995: Mackenzie and
Angevine. 1981).
Fertility in Offspring
Several studies suggest that gestational exposure to maternal tobacco smoke decreases the
future fertility of female offspring (Ye etal.. 2010: Tensen etal.. 1998: Weinberg etal.. 1989)
(Table 1-1). In animal models, marked effects on the development of male and female reproductive
organs and the fertility of animals exposed gestationally has also been demonstrated fKristensen et
al.. 1995: Mackenzie and Angevine. 1981) (Table 1-2 and Figure 1-1). In two studies examining
reproductive effects in mice, decreased fertility and fecundity in F1 animals was observed following
exposure to doses >10 mg/kg-day during gestation (Kristensen etal.. 1995: Mackenzie and
Angevine. 1981). When F1 females were mated with untreated males, a dose-related decrease in
fertility of >30% was observed, in addition to a 20% decrease in litter size starting at the lowest
dose tested of 10 mg/kg-day fMackenzie and Angevine. 19811. A dose-related decrease in fertility
was also observed in male mice treated gestationally with benzo[a]pyrene. At the lowest dose
tested (10 mg/kg-day), a 35% decrease in fertility was observed when gestationally exposed
animals were mated with untreated females (Mackenzie and Angevine. 19811. Similar effects on
fertility were observed in another developmental study in mice fKristensen etal.. 1995). F1
females (bred continuously for 6 months) in this study had 63% fewer litters, and litters were 30%
smaller as compared to control animals. The fertility of male offspring was not assessed in this
study.
Reproductive Organ Effects in Offspring
The above-mentioned studies also demonstrated dose-related effects on male and female
reproductive organs in animals exposed gestationally (or during the early postnatal period) to
benzo[a]pyrene (Table 1-2 and Figure 1-1). Testicular weight was decreased and atrophic
seminiferous tubules and vacuolization were increased at >10 mg/kg-day in male mice exposed to
benzo[a]pyrene gestationally from GD 7 to 16; severe atrophic seminiferous tubules were observed
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Toxicological Review of Benzo[a]pyrene
at 40 mg/kg-day (Mackenzie and Angevine. 19811. Testicular weight on PND 8, serum testosterone
on PNDs 8 and 35, testicular daily sperm production on PND 90, and epididymal sperm count on
PND 90 were also statistically significantly decreased in Sprague-Dawley rats treated on PNDs 1-7
at doses >10 mg/kg-day (Liang etal.. 20121. Increased seminiferous tubule vacuolation occurred in
rats exposed to 25 mg/kg-day on PNDs 1-7 (examined on PNDs 8 and 35) fLiang etal.. 20121.
In female mice treated with doses >10 mg/kg-day during gestation, ovarian effects were
observed including decreases in ovary weight, numbers of follicles, and corpora lutea (Kristensen et
al.. 1995: Mackenzie and Angevine. 19811. Specifically, ovary weight in F1 offspring was reduced
31% following exposure to 10 mg/kg-day benzo[a]pyrene (Kristensen et al.. 19951. while in
another gestational study at the same dose level, ovaries were so drastically reduced in size (or
absent) that they were not weighed (Mackenzie and Angevine. 19811. Hypoplastic ovaries with few
or no follicles and corpora lutea (numerical data not reported), and ovaries with few or no small,
medium, or large follicles and corpora lutea (numerical data not reported) have also been observed
in mouse offspring exposed gestationally to benzo[a]pyrene (Kristensen etal.. 1995: Mackenzie and
Angevine. 19811.
Cardiovascular Effects in Offspring
Increased systolic and diastolic blood pressure was observed in adult animals following
gestational treatment with benzo[a]pyrene flules etal.. 20121 (Table 1-2 and Figure 1-1).
Approximate elevations in systolic and diastolic blood pressure of 20-30 and 50-80% were noted
in the 0.6 and 1.2 mg/kg-day dose groups, respectively. Heart rate was decreased at 0.6 mg/kg-day,
but was increased at 1.2 mg/kg-day.
Immune Effects in Offspring
Several injection studies in laboratory animals suggest that immune effects may occur
following gestational or early postnatal exposure to benzo[a]pyrene. These studies are discussed in
Section 1.1.3.
Table 1-1. Evidence pertaining to developmental effects of benzo[a]pyrene in
humans
Study design and reference
Results
Relation between cord blood benzo[a]pyrene-DNA adducts and log-
transformed weight and height
Weight
Length (height)
Beta (p-value)
Beta (p-value)
Birth
-0.007 (0.73)
-0.001 (0.89)
18 mo
-0.048 (0.03)
-0.005 (0.48)
24 mo
-0.041 (0.027)
-0.007 (0.28)
30 mo
-0.040 (0.049)
-0.006 (0.44)
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Study design and reference
Results
Tang et al. (2006) (Tongliang, China)
Adjusted for ETS, sex of child, maternal height, maternal weight, and
gestational age (for measures at birth)
Birth cohort
150 nonsmoking women who delivered
babies between March 2002 and June 2002
Exposure: Mean hours per day exposed to
ETS 0.42 (SD 1.19); lived within 2.5 km of
power plant that operated from December
2001 to May 2002; benzo[a]pyrene-DNA
adducts from maternal and cord blood
samples; cord blood mean 0.33 (SD 0.14)
(median 0.36) adducts/10"8 nucleotides;
maternal blood mean 0.29 (SD 0.13)
adducts/10"8 nucleotides
Perera et al. (2005b); Perera et al. (2004)
Relation between cord blood benzo[a]pyrene-DNA adducts and log-
transformed weight and length
Weight Length
Beta (p-value) Beta (p-value)
Interaction -0.088 (0.05) -0.014 (0.39)
term
Benzo[a]- -0.020 (0.49) -0.005 (0.64)
pyrene-DNA
adducts
ETS in home -0.003 (0.90) -0.007 (0.32)
Adjusted for ethnicity, sex of newborns, maternal body mass index,
dietary PAHs, and gestational age
(New York, United States)
Birth cohort
265 pregnant African-American and
Dominican nonsmoking women who
delivered babies between April 1998 and
October 2002 (214 and 208 for weight and
length analysis, respectively);
approximately 40% with a smoker in the
home
Exposure: Benzo[a]pyrene-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
Wu et al. (2010) (Tianiin, China)
Benzo[a]pyrene adduct levels (/10s 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 benzo[a]pyrene adducts and miscarriage3
OR (95% CI)
Per unit increase in adducts 1.37 (1.12,1.67)
Dichotomized at median 4.56 (1-46,14.3)
Conditional logistic regression, adjusted for maternal education,
household income, and gestational age; age also considered as
potential confounder
Case control study: 81 cases (96%
participation rate)—fetal death confirmed
by ultrasound before 14 wks of gestation;
81 controls (91% participation rate)—
elective abortions; matched by age,
gestational age, and gravidity; excluded
smokers and occupational PAH exposure
Exposure: Benzo[a]pyrene in aborted tissue
and maternal blood samples (51 cases and
controls; two of four hospitals)
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Study design and reference
Results
Duarte-Salles et al. (2012) (Catalonia, Spain)
Birth cohort
657 pregnant women recruited during the
first trimester between July 2004 and July
2006 (614 and 604 available for weight and
length analysis, respectively); 37% passive
smokers, 16% active smokers
Exposure: Dietary benzo[a]pyrene intake
during first trimester from food
questionnaire (ng/d, mean ± SD) of
0.16 ± 0.04 for all women with less than the
mean vitamin C intake and 0.22 ± 0.06 for
all women with greater than the mean
vitamin C intake.
SGA defined as birth weight below the 10th
percentile of a Spanish reference
population
Relation between dietary benzo[a]pyrene intake during the first
trimester and birth weight and length (by vitamin C intake and
smoking status; Beta coefficients are for a 1-SD increase in dietary
benzo[a]pyrene)
Weight Length
Beta (p-value) Beta (p-value)
-101.63 (0.004)
< Mean vitamin C, all
women
< Mean vitamin C,
nonsmokers
> Mean vitamin C, all
women
> Mean vitamin C,
nonsmokers
-88.26 (0.034)
2.05 (0.945)
-3.71 (0.909)
-0.38 (0.017)
-0.37 (0.048)
0.10 (0.439)
0.04 (0.784)
Adjusted for sex of the child; gestational age; nulliparity; tobacco
smoke exposure during pregnancy; maternal region of origin
(European or non-European); and maternal education level, height,
prepregnancy weight, and energy intake
Relation between SGA births and dietary benzo[a]pyrene intake
during the first trimester (all women, by vitamin C intake,
benzo[a]pyrene tertile 3 compared to tertiles 1 and 2)
OR (95% CI)
< Mean vitamin C 3.51 (1-16,10.59)
> Mean vitamin C 0.81 (0.23,2.75)
Adjusted for sex of the child; gestational age; nulliparity; tobacco
smoke exposure during pregnancy; maternal region of origin
(European or non-European); and maternal education level, height,
prepregnancy weight, and energy intake
Duarte-Salles et al. (2013) (Norway)
Birth cohort
50,651 pregnant women recruited between
1998 and 2008; 92% nonsmokers, 4%
occasional smokers, and 4% daily smokers
Exposure: Dietary benzo[a]pyrene intake
from food questionnaire (ng/d, mean ± SD)
of 0.149 ± 0.048 for all women
Relation between dietary benzo[a]pyrene intake during pregnancy
and birth weight and length (Beta coefficients are for a 1-SD increase
in dietary benzo[a]pyrene)
Weight Length
Beta (p-value) Beta (p-value)
All women
All women, vitamin C
<85 mg/d
All women, vitamin C
>85 mg/d
Nonsmokers
Nonsmokers, vitamin C
<85 mg/d
-10.2 (<0.001)
-17.7 (0.003)
-9.1 (<0.001)
-10.3 (<0.001)
-15.7 (0.016)
-0.05 (<0.001)
Not evaluated
Not evaluated
-0.05 (0.001)
Not evaluated
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Toxicological Review of Benzo[a]pyrene
Study design and reference
Results
Nonsmokers, vitamin C -9.6 (<0.001) Not evaluated
>85 mg/d
Adjusted for sex of the child; gestational age, maternal age, weight
gain, parity, and pre-pregnancy body mass index; smoking during
pregnancy; and plausibility of reported energy intake and vitamin C
intake
CI = confidence interval; OR = odds ratio; SD = standard deviation.
Table 1-2. Evidence pertaining to developmental effects of benzo[a]pyrene in
animals after oral or inhalation exposure
Study design and reference
Results
Birth outcomes and postnatal growt
h
Mackenzie and Angevine (1981)
CD-I mice, 30 or 60 F0 females/
dose
0,10, 40, or 160 mg/kg-d by
gavage
GDs 7-16
(developmental study with
continuous breeding protocol)
F1 pups (4/sex/litter) were
allowed to remain with their
mothers until weaning on PND 20
At 6 wks of age, F1 female mice
(n = 20-55/group) were paired
with an untreated male for a
period of 6 mo
At 7 wks of age, F1 male mice
(n = 20-45/group) were mated
with two untreated females for
5-d periods for 25 d (for a total
exposure of 10 untreated
females/Fl male)
4/ number of F0 females with viable litters: 46/60, 21/30, 44/60, and 13/30*
4/ F1 body weight at PND 20
% change from control: 0, 4, -7*, and -13*
4/ F1 body weight at PND 42
% change from control: 0, -6*, -6*, and -10*
(no difference in pup weight at PND 4)
(Gross abnormalities not observed in F1 or F2 animals)
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Study design and reference
Results
Kristensen et al. (1995)
NMRI mice, 9 FO females/dose
0 or 10 mg/kg-d by gavage
GDs 7-16
(developmental study with
continuous breeding protocol)
At 6 wks of age, one F1 female
from each litter (n = 9) was
continuously bred with an
untreated male for 6 mo
Exposed F0 females showed no gross signs of toxicity and no effects on
fertility (data not reported)
F1 females had statistically significant:
4/ number of F2 offspring
4/ number of pups/litter
4/ number of F2 litters
1" number of days between F2 litters
(Gross abnormalities not observed in F1 or F2 animals; alteration in pup
weight not reported for any generation)
F2 offspring were inspected for
gross deformities at birth; weight
and sex were recorded 2 d after
birth
Jules et al. (2012)
Long-Evans rats, 6-17 FO
females/dose
0, 0.15, 0.3, 0.6, or 1.2 mg/kg-d by
gavage
GDs 14-17
No overt signs of toxicity in dams or offspring, differences in pup body
weight, or number of pups/litter
McCallister et al. (2008)
Long-Evans Hooded rats,
5-6/group
0 or 0.3 mg/kg-d by gavage
GDs 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 PNDs 15 and 30 (data not shown)
Brown et al. (2007)
Long-Evans Hooded rats, 6/group
0, 0.025, or 0.15 mg/kg-d by
gavage
GDs 14-17
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-d by
gavage
PNDs 5-11
Statistically significant decrease in pup body weight (approximate 10-15%
decrease) at 2 mg/kg-d measured on PNDs 36 and 71 (no significant
alteration of pup weight during treatment period)
No differences among treatment groups in developmental milestones:
incisor eruption, eye opening, development of fur, testis decent, or vaginal
opening
Liang et al. (2012)
Sprague-Dawley rats,
5-6 litters/dose (12 pups per
litter)
0, 5,10, or 25 mg/kg-d by gavage
PNDs 1-7
4/ body weight at PND 8
% change from control: 0, -0.6, -16*, and -17*
(no difference at PND 35 and PND 90)
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Toxicological Review of Benzo[a]pyrene
Study design and reference
Results
Archibong et al. (2002)
F344 rats, 10 females/group
0, 25, 75, or 100 ng/m3 nose-only
inhalation for 4 hrs/d
GDs 11-20
4/ embryo/fetal survival ([pups/litter]/[implantation sites/litter] x 100)
% embryo/fetal survival: 97, 78*, 38*, and 34*%
Reproductive effects in offspring
Mackenzie and Angevine (1981)
CD-I mice, 30 or 60 F0 females/
dose
0,10, 40, or 160 mg/kg-d by
gavage
GDs 7-16
(developmental study with
continuous breeding protocol)
At 6 wks of age, F1 female mice
(n = 20-55/group) were paired
with an untreated male for a
period of 6 mo
At 7 wks of age, F1 male mice
(n = 20-45/group) were mated
with two untreated females for
5-d periods for 25 d (for a total
exposure of 10 untreated
females/Fl male)
4/ number of F1 females with viable litters: 35/35, 23/35*, 0/55*, and 0/20*
4/F1 female fertility index (females pregnant/females mated with males x
100): 100, 66*, 0*, and 0*
4/ F1 male fertility index (females pregnant/females mated with males x
100): 80, 52*, 5*, and 0*
4/ F2 litter size from F1 dams (20%) at 10 mg/kg-d (no litters were produced
at high doses)
4/ size or absence of F1 ovaries (weights not collected)
hypoplastic ovaries with few or no follicles and corpora lutea
(numerical data not reported)
4/ testicular weight in F1 offspring
% change from control: 0, -42, -82, and ND (statistical significance
not reported)
1" atrophic seminiferous tubules and vacuolization at >10 mg/kg-d; severe
atrophic seminiferous tubules at 40 mg/kg-d (numerical data not reported)
Kristensen et al. (1995)
NMRI mice, 9 F0 females/dose
0 or 10 mg/kg-d by gavage
GDs 7-16
(developmental study with
continuous breeding protocol)
At 6 wks of age, one F1 female
from each litter (n = 9) was
continuously bred with an
untreated male for 6 mo.
4/ number of F2 litters (-63%)
4/ F2 litter size (-30%)
4/ ovary weight (-31%) in F1 females
Few or no small, medium, or large follicles and corpora lutea
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Toxicological Review of Benzo[a]pyrene
Study design and reference
Results
Liang et al. (2012)
Sprague-Dawley rats,
5-6 litters/dose (12 pups per
litter)
0, 5,10, or 25 mg/kg-d by gavage
PNDs 1-7
4/ testes weight at PNDs 8 and 35
% change from control at PND 8: 0, 4, -14*, and -10*
% change from control at PND 35: 0,1, -6, and -17*
(no difference at PND 90)
4/ serum testosterone at PNDs 8, 35, and 90
% change from control at PND 8: 0, -31, -62*, and -62*
% change from control at PND 35: 0, -38, -42*, and -42*
% change from control at PND 90: 0, -29, -52, and -65*
1" seminiferous tubule vacuolization on PND 8 and PND 35 at 25 mg/kg-d
(numerical data not reported). No difference at PND 90, or at any time point
at 5 mg/kg-d. Report does not clearly indicate whether this was observed at
10 mg/kg-d.
4/ testicular daily sperm production on PND 90
Approximate % change from control (data reported graphically): 0,
-20, -30*, and -30*
4/ epididymal sperm count on PND 90
Approximate % change from control (data reported graphically): 0,
-20, -30*, and-20*
Cardiovascular effects in offspring
Jules et al. (2012)
Long-Evans rats, 6-17 F0
females/dose
0, 0.15, 0.3, 0.6, or 1.2 mg/kg-d by
gavage
GDs 14-17
1" systolic blood pressure (measured at PND 53)
15%* increase at 0.6 mg/kg-d
52%* increase at 1.2 mg/kg-d
(other dose groups not reported)
1" diastolic blood pressure (measured at PND 53)
33%* increase at 0.6 mg/kg-d
83% *increase at 1.2 mg/kg-d
(other dose groups not reported)
Altered heart rate
10%* increase at 0.6 mg/kg-d
8%* decrease at 1.2 mg/kg-d
^Statistically significantly different from the control (p < 0.05).
a% change from control calculated as: (treated value - control value)/control value x 100.
SEM = standard error of the mean
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Toxicological Review of Benzo[a]pyrene
Figure 1-1. Exposure-response array for developmental effects following oral
exposure to benzo[a]pyrene.
Neurodevelopmental Effects
Development of the human (and rodent) nervous system is a prolonged process that begins
around mid-gestation and continues in one form or another into early adulthood. Thus, it is
difficult to pinpoint an exact age at which exposure is no longer interpreted as having the potential
to cause neurodevelopmental effects. The majority of oral and inhalation studies examining the
potential for benzo[a]pyrene to cause nervous system effects assessed children or rodents after
gestational or early neonatal exposure; thus, these data are discussed in the context of
developmental effects. One subchronic intraperitoneal (i.p.) exposure study (Tang etal.. 20111
beginning at weaning but with most of the exposure in adulthood is also discussed in the context of
developmental effects, as pronounced changes in brain development (e.g., myelination and synaptic
refinement/strengthening) occur during adolescence.
Additional data on potential nervous system effects involved benzo[a]pyrene exposure in
sexually mature, adult humans and rodents, or in animals exposed beginning around puberty (i.e.,
approximately 5 weeks of age). Although these latter studies included a brief period of pubertal
exposure that may be interpreted as relevant to neurodevelopment, nearly all of the exposure
occurred after the animals reached sexual maturity. Thus, studies in human adults or in rodents
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Toxicological Review of Benzo[a]pyrene
beginning after 5 weeks of age are discussed in Section 1.1.4 in the context of nervous system
effects interpreted to result from "adult" exposure. A summary of the exposure paradigms used in
the database of studies evaluating the potential nervous system effects of benzo[a]pyrene exposure
is illustrated in Figure 1-2.
Human
a
1 c*
b
1 d-
Rodents
(Oral}
l> « 11
I R
I i* 1
h
j-
C K-
i r ¦
Rodents
(Inhalation)
¦
m
t 5 1
Rodents
(i-P-)
1
° 1
1
1
D* 1
q' 1
r--~\
*
Early
Gestation
GD1-11
Late
Gestation
GD12-21
Early
Neonatal
PND0-11
Late
Neonatal
PND 12-21
Pre-
pubertal
PND 21-34
Pube rtal
5-8 wks
"Adult"
> 8 wks
Conception Birth Weaning
Figure 1-2. Exposure timing in benzo[a]pyrene studies examining nervous
system effects.
The available human and animal studies are presented (Note: exposure durations are not to scale): (a) Perera et al.
(2012a): Tang et al. (2006): Tang et al. (2008): (b) Perera et al. (2004): Perera et al. (2005b): Perera et al. (2012b): (c) Qiu et
al. (2013): (d) Niu et al. (2010): (e) Sheng et al. (2010): (f) McCallister et al. (2008): (g) Bouaved et al. (2009a): (h) Chen et
al. (2012): (i) Chenezhi et al. (2011): (j) Bouaved et al. (2009b): (k) Bouaved et al. (2012): (I) Maciel et al. (2014):
(m) Wormlev et al. (2004): (n) Li et al. (2012): (o) Tang et al. (2011): (p) Xia et al. (2011): (q) Qiu et al. (2011): (r) Grova et
al. (2007): (s) Grova et al. (2008). * = studies discussed separately in Section 1.1.4.
There is evidence in humans and animals that benzo[a]pyrene induces developmental
neurotoxicity. Two epidemiology studies that examined benzo[a]pyrene-specific measures
observed effects on neurodevelopment and behavior in young children. Altered learning and
memory motor activity, anxiety-like behavior, and electrophysiological changes have also been
observed in animals following developmental oral and inhalation exposure to benzo[a]pyrene.
Important developmental processes such as neurogenesis and migration are largely
completed during mid-late gestation in humans and rodents, the disruption of which can have
serious adverse consequences. Next, the mammalian brain undergoes periods of rapid brain
growth, particularly during the last 3 months of pregnancy in humans, which has been compared to
the first 1-2 weeks of life in the rat and mouse neonate (Dobbing and Sands. 1979.19731. This
period is characterized by axonal and dendritic outgrowth and the establishment of mature
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Toxicological Review of Benzo[a]pyrene
neuronal connections. Also during this critical period, animals acquire many new motor and
sensory abilities (Kolb and Whishaw. 19891. Complementing the behavioral effects reported in
observational studies of exposed humans, there is a growing literature of animal studies that shows
changes in motor and cognitive function following acute or repeated perinatal or early neonatal
exposure to benzo[a]pyrene fBouaved etal.. 2009a: McCallister etal.. 2008: Wormlev et al.. 20041.
These effects are described below.
Cognitive function
Head circumference at birth is associated with measures of intelligence in children, even
among term infants (Broekman etal.. 2009: Gale etal.. 20061. The two birth cohort studies that
examined maternal or cord blood levels of benzo[a]pyrene-specific DNA adducts in relation to head
circumference provide some evidence of an association, most strongly within the context of an
interaction with ETS (Tang etal.. 2006: Perera etal.. 2005b: Perera etal.. 20041 (Table 1-3). In
these studies, the internal benzo[a]pyrene-specific dose metrics were in general agreement with
personal air sampling monitors worn by the mothers (Perera et al.. 2012b). The cohort in
Tongliang, China also examined intelligence quotient scores at age 5 years (Perera et al.. 2012a). An
interaction with ETS was seen in this analysis, with larger decrements seen on the full scale and
verbal scales with increased benzo[a]pyrene-DNA adduct levels in the presence of prenatal
exposure to ETS compared to the effects seen in the absence of prenatal exposure.
Animal studies have also provided evidence of altered behaviors in learning and memory
tests following lactational or direct postnatal oral exposure to benzo[a]pyrene (Chen etal.. 2012:
Bouaved etal.. 2009a) (Table 1-4). In mice, spatial working memory was measured using the
Y-maze spontaneous alternation test (Bouaved etal.. 2009a). This test records alternations
between arm entries in a Y-shaped maze as a measure of memory, as rodents typically prefer to
investigate a new arm of the maze. To a lesser extent, this test can also reflect changes in sensory
processing, novelty preference, and anxiety-related responses in rodents. An improvement in
performance was evident in mice, as exhibited by significant increases in spontaneous alternations
in the Y-maze test in mice on PND 40 following lactational exposure to 2 mg/kg-day
benzo[a]pyrene (but not 20 mg/kg-day) from PND 0 to 14 (Bouaved et al.. 2009al. The total
number of arm entries in the Y-maze was unaffected by lactational exposure, suggesting that
changes in motor function were not driving this response. In another test of short-term memory,
wild type (WT) CPRlox/lox transgenic mice (a conditional knock-out mouse model designed to allow
for tissue-specific deletion of CYP450 reductase) exhibited deficiencies in novel object recognition
tests months after in utero oral or inhalation exposure to benzo[a]pyrene during late gestation (Li.
etal.. 2012: Shengetal.. 2010). These findings suggest impairment in short-term memory, although
these tests also reflect locomotor exploratory behavior, response to novelty, and attention.
In rats, spatial learning and memory was investigated using the Morris water maze, which
measures the ability of a rat to navigate to a target platform using external spatial cues (Chen etal..
2012: Tang etal.. 20111. Increased escape latency (time to find the hidden platform), as well as
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decreased time in the target quadrant and decreased number of platform crossings during a probe
trial with the platform removed were observed in PND 39-40 rats following postnatal oral
exposure to 2 mg/kg-day benzo[a]pyrene fChen etal.. 20121. These effects were more pronounced
in animals tested at PNDs 74-75, with effects observable at >0.2 mg/kg-day. No difference in swim
speed was observed during the probe trial tests (swim speed did not appear to be analyzed during
the hidden platform trials) between treatment groups, suggesting that the observed changes are
not attributable to general motor impairment Similar findings in Morris water maze performance
were observed in rats exposed to 2.5 and 6.25 mg/kg (but not at 1 mg/kg) benzo[a]pyrene by daily
i.p. exposure beginning at weaning and continuing for 14 weeks, although swim speed or distance
traveled were not measured fTang etal.. 20111. In both rat studies, these decrements in Morris
water maze performance were not attributable to effects of benzo[a]pyrene exposure on learning
and memory processes alone. Specifically, visual examinations of the improvements in escape
latency (slopes) over the 4-5 learning trial days were not noticeably affected by treatment dose,
suggesting that all groups learned at a similar rate, despite treated animals displaying a baseline
impairment in performance from testing day 1. Typically, these nonspecific differences in latency
on trial day 1 reflect noncognitive influences on performance (Corv-Slechta etal.. 20011. In Chen et
al. f20121. four trials were conducted per day and averaged for each animal at each trial day (data
for these multiple daily trials were not provided). Thus, it is unclear whether the dose-related
increases in escape latency already observable at trial day 1 reflect effects on learning across those
same day trials or other effects that were already present prior to testing (e.g., altered motor
function, anxiety, vision, etc.); however, the study by Tang etal. (20111 also noted a baseline
impairment in water maze performance and this study only tested animals once per day, suggesting
that the decrements present on testing day 1 in Chen etal. (20121 did not reflect impaired learning
during the first trial day.
Performance in the Morris water maze probe trials was also affected by benzo[a]pyrene
exposure fChen etal.. 2011: Tang etal.. 20111: however, as it is not clear that the groups learned to
a comparable extent in hidden platform tests prior to testing memory retention, it is unclear how to
interpret these results.
Overall, the dose-dependent decreases in the performance of the benzo[a]pyrene-exposed
rats in the Morris water maze hidden platform trials were shown to persist for weeks to months
after postnatal exposure in two separate cohorts of male and female rats and, although the
behavioral effect cannot clearly be attributed to learning or memory, these data represent a
persistent and adverse neurobehavioral change.
Neuromuscular function, coordination, and sensorimotor development
Motor behavior and coordination, assessed by locomotion, reaching, balance,
comprehension, drawing, and hand control was one of the specific domains assessed in the Chinese
birth cohort evaluated by Tang etal. (20081. In children aged 2 years, decreased scores were seen
in relation to increasing benzo[a]pyrene- DNA adducts measured in cord blood, with a Beta per unit
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increase in adducts of -16 (p = 0.04), and an approximate 2-fold increased risk of development
delay per unit increase in adducts (Table 1-3).
In laboratory animals (Table 1-4 and Figure 1-3), impaired performance in neuromuscular
and sensorimotor tests have been consistently observed in mice lactationally exposed to
>2 mg/kg-day benzo[a]pyrene from PND 0 to 14 fBouaved et al.. 2009al and in rat pups postnatally
exposed to >0.02 mg/kg-day benzo[a]pyrene from PND 5 to 11 fChen etal.. 20121. In the righting
reflex test, significant increases in righting time were observed in PND 3-5 (but not PND 7-9) mice
and in PND 12-16 (but not PND 18) rats. These decrements did not show a monotonic dose
response. In another test of sensorimotor function and coordination, dose-dependent increases in
latency in the negative geotaxis test were observed in PND 5-9 (but not PND 11) mice and in
PND 12-14 (but not PND 16-18) rats. The forelimb grip strength test of neuromuscular strength
was also evaluated in both mice and rats, but alterations were only observed in mice. In mice, a
dose-dependent increase in duration of forelimb grip was observed on PNDs 9 and 11 during
lactational exposure to benzo[a]pyrene. The Water Escape Pole Climbing test was also used to
evaluate neuromuscular function and coordination in mice (Bouaved et al.. 2009a). No effect on
climbing time was observed, suggesting no change in muscle strength. However, increased latency
in pole grasping and pole escape in PND 20 male pups was observed, highlighting potential
decrements in visuomotor integration and/or coordination, although anxiety or fear-related
responses cannot be ruled out Treatment-dependent increases in pup body weight around the
testing period complicate the interpretation of these results.
Chenetal. (2012) observed statistically significant delays on the order of ~0.2-0.3 seconds
in the surface righting test and ~3-4 seconds in the negative geotaxis test The authors found no
effect of gender; therefore, the data for male and female rats were pooled for these measures.
However, it should be noted that differences in the maturation of these developmental landmarks
following challenge have been shown to exist between males and females. In the surface righting
test, differences between groups were generally <0.5 seconds; as these measures were manually
recorded, it is unclear how reliably these small differences could be detected. An additional
uncertainty in interpreting these results involves the lack of consideration of litter effect (i.e., the
tendency of littermates to respond more like each other than like offspring in other litters), such as
through identifying the litter for each individual animal. Consistent with the results of Bouaved et
al. f2009al in mice, Chen etal. f20121 reported effects in these tests at earlier postnatal ages that
did not persist when tested at later postnatal ages. Negative geotaxis and surface righting are
discrete endpoints routinely used as part of a neurobehavioral test battery to assess acquisition of
behavioral reflexes. Chen etal. (2012) used the surface righting and negative geotaxis tests as
quantitative measures of sensorimotor function at PND 12 and beyond. Typically in these tests,
animals are observed on consecutive days (e.g., PNDs 3-12) and time to acquisition of these
phenotypes is measured. Notably, in rats, the functional phenotypes assessed in these assays are
largely acquired by the postnatal ages tested by Chen etal. f20121. Likely due to the exposure
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window tested by Chen etal. (2012). PNDs 5-11, the authors did not measure performance over
consecutive days earlier during development (e.g., PNDs 3-11) in order to identify baseline
acquisition of these behaviors. Although these tests as conducted by Chen etal. f20121 cannot
discern a developmental delay, the data support a transient impairment of sensorimotor function in
animals that have already developed this reflex (e.g., able to orient 180 degrees and able to right
within 2 seconds). Thus, taken together with the results of Bouavedetal. f2009al. these data
indicate that benzo[a]pyrene may affect sensorimotor function at a particular developmental
lifestage(s).
Anxiety and activity
Anxiety/depression and attention/hyperactivity symptoms in children ages 6-7 years were
examined via questionnaire in relation to prenatal air monitoring of benzo[a]pyrene and other
PAHs, and in relation to benzo[a]pyrene-specific DNA adducts measured at birth in a follow-up of a
birth cohort study conducted in New York City (Perera et al.. 2012b! PAH exposure levels (based
on personal air monitoring, n = 253) and benzo[a]pyrene-specific DNA adducts measured in cord
blood samples (n = 138) were both positively associated with symptoms of anxiety/depression and
attention problems (see Table 1-3). Given the limited sample size, however, the cord blood results
are based on relatively sparse data (<5 in the borderline or clinical range in the low exposure
referent group). Associations with maternal blood adducts were similar to or slightly smaller than
those seen with cord blood adducts. Exposure was treated as a dichotomy (i.e., for adducts,
detectable compared with non-detectable levels) in these analyses.
Decreased anxiety-like behavior was reported in both rats and mice weeks to months
following postnatal oral exposure to benzo[a]pyrene (Chen etal.. 2012: Bouavedetal.. 2009a)
(Table 1-4). A decrease in anxiety, indicative of a change in nervous system function, can impair an
organism's ability to react to a potentially harmful situation. This decreased ability of an organism
to adapt to the environment is considered to be an adverse effect according to EPA's neurotoxicity
guidelines (U.S. EPA. 1998a). Anxiety-like behaviors were measured in both species using an
elevated plus maze, where an increase in the time spent in the closed arms of the maze is
considered evidence of anxious behavior while an increased time spent in the open arms reflects
increased risk taking and/or reduced anxiety. Following lactational exposure to >2 mg/kg-day
benzo[a]pyrene, mice exhibited significant increases in the percent open arm entries and percent
time spent in open arms of the maze, as well as significantly decreased entries into closed arms of
the maze (the latter was observed at 2 mg/kg-day, but not 20 mg/kg-day), on PND 32 fBouaved et
al.. 2009a). To rule out potential differences in total activity or general motivation and exploration,
the authors expressed the open arm data as percentages, and they also demonstrated that there
were no exposure-related effects on the total number of arm entries. The mice also exhibited
decreased latency of the first entry into an open arm following lactational exposure to
20 mg/kg-day benzo[a]pyrene. Similar results were reported for rats, with decreased anxiety-like
behavior following oral benzo[a]pyrene exposure from PND 5 to 11, although sex-specific
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differences were observed (Chen etal.. 20121. In females, postnatal exposure to >0.2 mg/kg-day
benzo[a]pyrene was associated with a significant increase in the number of open arm entries and a
significant decrease in the number of closed arm entries on PND 70. Significantly increased time in
open arms of the maze was reported in PND 70 female rats following postnatal exposure to
>0.02 mg/kg-day. Less sensitive effects (i.e., observable at 2 mg/kg-day) were observed in a second
cohort of exposed rats tested at PND 35. Male rats also showed decreased anxiety-like behavior on
PND 70, although the doses needed to detect these responses were higher than females (i.e.,
increases at >2 mg/kg-day for open arm entries and >0.2 mg/kg-day for time spent in open arms).
A significant decrease in latency to enter an open arm of the maze was observed in both male and
female rat pups exposed to 2 mg/kg-day benzo[a]pyrene. Similar to the observations in mice,
exposure did not appear to have an effect on total activity or general motivation of the rats, as total
arm entries were unchanged by treatment.
Increased spontaneous locomotor activity in the open field on PNDs 34 and 69 has been
reported in two cohorts of rats postnatally exposed to 2 and >0.2 mg/kg-day, respectively (Chen et
al.. 2012). but not in mice exposed lactationally to doses up to 20 mg/kg-day and tested on PND 15
(Bouaved et al.. 2009a). Interestingly, no differences in the open field test were observed in rats
that were postnatally exposed and tested on PNDs 18 and 20, suggesting either that longer latencies
between exposure and testing may be required, or that these developmental effects may only
manifest in more mature rats f Chen etal.. 20121. An apparent increased sensitivity of older animals
was also present in the elevated plus maze and Morris water maze tests performed by Chen et al.
(2012). Both Chen etal. (2012) and Bouaved etal. (2009a) measured total horizontal and vertical
activity, increases in which could indicate increased motor activity or decreased anxiety (less fear
of the open spaces/bright lights). However, the relative contributions of these two behavioral
components to the increased activity observed by Chen et al. f 2 0121 could not be separated, as the
authors did not evaluate activity in central versus peripheral regions of the field (i.e., anxious
rodents will spend less time in the center of the field).
Electrophysiological changes
Electrophysiological effects of gestational exposure to benzo[a]pyrene have been examined
in two studies (by the same research group) through implanted electrodes in the rat cortex and
hippocampus, and using in vitro preparations after in vivo exposure (Li etal.. 2012) (Table 1-4).
Maternal inhalation exposure to 0.1 mg/m3 resulted in reduced long-term potentiation in the
dentate gyrus of male offspring between PND 60 and 70 fWormlev etal.. 20041. and decreased
inward currents in cortical neurons isolated on PND 1 and cultured for 7 days (Li etal.. 2012):
however, significant embryo/fetal resorptions at this exposure level and uncertainties in
extrapolating from the ex vivo preparations complicates the interpretation of these results. Oral
exposure of dams to 0.3 mg/kg-day for 4 days during late gestation resulted in decreased evoked
neuronal activity in male offspring following mechanical whisker stimulation between PND 90 and
120 fMcCallister et al.. 20081. Specifically, the authors noted reduced spike numbers in both short
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and long latency responses following whisker stimulation. These effects were observed several
months post-exposure, suggesting that gestational benzo[a]pyrene exposure may have long-lasting
functional effects on neuronal activity elicited by sensory stimuli.
Table 1-3. Evidence pertaining to the neurodevelopmental effects of
benzo[a]pyrene from PAH mixtures
Reference and study design
Results
Tang et al. (2008); Tang et al. (2006)
(Tongliang, China)
Birth cohort
150 nonsmoking women, delivered
March 2002-June 2002; lived within
2.5 km of power plant that operated from
December 2001 to May 2002
Outcomes: Head circumference at birth;
Gesell Developmental Schedule,
administered by physicians at 2 yrs of age
(four domains: motor, adaptive, language,
and social); standardized mean
score = 100 ± SD 15 (score
<85 = developmental delay)
Exposure: Mean hrs/d exposed to ETS
0.42 (SD 1.19); lived within 2.5 km of
power plant that operated from
December 2001 to May 2002;
benzo[a]pyrene-DNA adducts from
maternal and cord blood samples; cord
blood mean 0.33 (SD 0.14) (median 0.36)
adducts/10"8 nucleotides; maternal blood
mean 0.29 (SD0.13)
adducts/10"8 nucleotides
Relation between cord blood benzo[a]pyrene-DNA adducts and log-
transformed head circumference
Beta (p-value)
Birth -0.011 (0.057)
18 mo -0.012 (0.085)
24 mo -0.006 (0.19)
30 mo -0.005 (0.31)
High versus low, dichotomized at median, adjusted for ETS, sex of
child, maternal height, maternal weight, Cesarean section delivery,
maternal head circumference, and gestational age (for measures at
birth)
Tang et al. (2008); Tang et al. (2006) (see
above for population and exposure
details)
n = 110 for Developmental Quotient
analysis; no differences between the
110 participants in this analysis and the
nonparticipants with respect to maternal
age, gestational age, birth weight, birth
length, or birth head circumference;
higher maternal education was suggested
(direction of suggestive association not
reported, p = 0.056)
Association between benzo[a]pyrene adducts and development
Beta (95% Cl)a
OR (95% Cl)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)
aLinear regression of change in Developmental Quotient per unit
increase in benzo[a]pyrene adducts
bLogistic regression of risk of developmental delay (defined as
normalized score <85) per 1 unit (0.1 adducts/10"8 nucleotides)
increase in adducts
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Reference and study design
Results
Outcomes: Gesell Developmental
Schedule, administered by physicians at
2 yrs of age (four domains: motor,
adaptive, language, and social);
standardized mean score = 100 ± SD 15
(score <85 = developmental delay)
Both analyses adjusted for sex, gestational age, maternal education,
ETS, and cord lead levels
Perera et al. (2012a); Tang et al. (2008);
ETS measure not correlated with benzo[a]pyrene adduct measures
(i.e., absolute value of Spearman r < 0.10)
Relation between cord blood benzo[a]pyrene-DNA adducts, ETS
exposure, and IQ measures
Beta (95% CI)
Main effect With ETS interaction term
Full scale -2.42 (-7.96,3.13) -10.10 (-18.90,-1.29)
Verbal -1.79 (-7.61,4.03) -10.35 (-19.61,-1.10)
Performance -2.57 (-8.92,3.79) -7.78 (-18.03,2.48)
Beta per 1 unit increase in log-transformed cord adducts, adjusted for
ETS exposure, gestational age, maternal education, cord lead, maternal
age, and gender
Tang et al. (2006) (see above for
population and exposure details)
132 (83%) followed through age 5; 100 of
these had complete data for analysis; no
differences between the 100 participants
in this analysis and the nonparticipants
with respect to adduct levels, ETS
exposure, IQ measures, maternal age,
gestational age, or infant gender; higher
maternal education (60 and 35% with
> high school, respectively, in participants
and nonparticipants, p < 0.05)
Outcomes: Wechsler Preschool and
Primary Intelligence Quotient scale
(Shanghai version)
Perera et al. (2012b); Perera et al.
Relation between cord blood benzo[a]pyrene-DNA adducts, ETS
exposure, and log-transformed head circumference
Beta (p-value)
Interaction term -0.032 (0.01)
Benzo[a]pyrene-DNA adducts -0.007 (0.39)
ETS in home -0.005 (0.43)
High versus low, dichotomized at 0.36 adducts/10-8 nucleotides,
adjusted for ethnicity, sex of newborns, maternal body mass index,
dietary PAHs, and gestational age
(2005b); Perera et al. (2004) (United
States, New York)
Birth cohort
265 pregnant women: African-American
and Dominican nonsmoking women who
delivered babies between April 1998 and
October 2002 (253 and 207 for behavior
and head circumference analysis,
respectively); approximately 40% with a
smoker in the home
Outcomes: Head circumference at birth
Exposure: Benzo[a]pyrene-DNA adducts
from maternal and cord blood samples;
mean 0.22 (SD0.14)
adducts/10"8 nucleotides; median of
detectable values
0.36 adducts/10"8 nucleotides
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Reference and study design
Results
Perera et al. (2012b)
Logistic regression of risk of borderline or clinical status in relation PAH
levels and to detectable levels of benzo[a]pyrene adducts
PAH Cord blood
Prevalence OR (95% CI) OR (95% CI)
Anxious/depressed 6.3% 8.9(1.7,46.5) 2.6(0.69,9.4)
Attention problems 6.7% 3.8(1.1,12.7) 4.1(0.99,16.6)
Anxiety (DSM) 9.5% 4.6 (1.5, 14.3) 2.5 (0.84,7.7)
Attention deficit- 7.9% 2.3 (0.79,6.7) 2.6 (0.68,10.3)
hyperactivity (DSM)
Exposure dichotomized for PAH as above and below median
(2.273 ng/m3) for parent population and for cord blood
benzo[a]pyrene adducts as detectable (n = 56 cord blood samples)
versus non-detectable (n = 92); adjusted for sex, gestational age,
maternal education, maternal IQ, prenatal ETS, ethnicity, age, heating
season, prenatal demoralization, and HOME inventory
n = 215 with outcome data and no missing
covariate data); no differences between
the participants in this analysis and the
nonparticipants with respect to adduct
levels, ETS exposure, maternal age,
gestational age, and socioeconomic
variables; participants more likely to be
female and African-American
Outcomes: Child Behavior Checklist
(118 items), completed by mothers for
children ages 6-7 yrs. Two domains:
anxious/depression, attention problems
(normalized T-score <65 = borderline or
clinical syndrome); also used for scales of
anxiety problems and attention deficit
hyperactivity problems based on DSM
classification
^Statistically significantly different from the control (p < 0.05).
DSM = Diagnostic and Statistical Manual of Mental Disorders; HOME = Home Observation for Measurement of the
Environment; IQ= intelligence quotient.
Table 1-4. Evidence pertaining to the neurodevelopmental effects of
benzo[a]pyrene in animals
Reference and study design
Results3
Cognitive function
Chen et al. (2012)b
Sprague-Dawley rats, 20 pups
(10 male and 10 female)/group
0, 0.02, 0.2, or 2 mg/kg-d by gavage
PNDs 5-11
Hidden Platform test in Morris water maze, escape latency:
Adolescent test period (PNDs 36-39): significant increase at
2 mg/kg-d only
Adult test period (PNDs 71-74): significant increase in at
>0.2 mg/kg-d
Increases in latency were already ~30% greater than controls at
2 mg/kg-d on the first trial day (i.e., on PND 36 or 71)
All experimental groups exhibited similar improvements in escape
latency, as slopes were parallel across the 4 trial days
Probe test in the Morris water maze (trial day 5):
Time spent in the target quadrant:
PND 40: significant decrease at 2 mg/kg-d only
PND 75: significant decrease at >0.2 mg/kg-d
Number of platform crossings:
PND 40: significant decrease at 2 mg/kg-d only
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Reference and study design
Results3
PND 75: significant decrease at >0.2 mg/kg-d (in females) and
2 mg/kg-d (in males)
Bouaved et al. (2009a)
Female Swiss albino mice, 5/group
0, 2, or 20 mg/kg-d maternal
gavage
PNDs 0-14 (lactational exposure)
Significant increase in the percent of spontaneous alternations in the
Y-maze alternation test at 2 mg/kg-d but not at 20 mg/kg-d
No effect on the total number of arm entries in the Y-maze alternation test
Neuromuscular function, coordination, and sensorimotor development
Chen et al. (2012)b
Sprague-Dawley rats, 20 pups
(10 male and 10 female)/group
0, 0.02, 0.2, or 2 mg/kg-d by gavage
PNDs 5-11
Latency in the surface righting reflex test
PND 12: significant increase at 0.2 mg/kg-d only
PND 14: significant increase at 0.02 and 2 mg/kg-d only
PND 16: significant difference at 2 mg/kg-d only
PND 18: no significant difference
Latency in the negative geotaxis test
PND 12: significant increase at all doses
PND 14: significant increase at 2 mg/kg-d only
PNDs 16 and 18: no significant difference
No effect on duration of forelimb grip in forelimb grip strength test
No effect on the latency to retract from the edge in cliff aversion test
Note: Males and females were pooled for all analyses
Bouaved et al. (2009a)
Female Swiss albino mice, 5/group
0, 2, or 20 mg/kg-d by maternal
gavage
PNDs 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 latency in the negative geotaxis time for 20 mg/kg-d
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-d
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-d
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Reference and study design
Results3
Anxiety and/or motor activity
Chen et al. (2012)b
Sprague-Dawley rats, 20 pups
(10 male and 10 female)/group
0, 0.02, 0.2, or 2 mg/kg-d by gavage
PNDs 5-11
Elevated plus maze:
Number of entries into open arms:
PND 35: No difference
PND 70: >26% increase at >0.2 mg/kg-d (females) and 38%
2 mg/kg-d (males)
Significant decrease in the number of entries into closed arms at
PND 70 at >0.2 mg/kg-d (in females) and 2 mg/kg-d (in males) (no
difference at PND 35)
Significant increase in the time spent in open arms at PND 35 at
2 mg/kg-d in females and at PND 70 at doses >0.02 mg/kg-d in
females and >0.2 mg/kg-d in males
Significant decrease in latency time to first enter an open arm on
PND 70 at >0.2 mg/kg-d (no difference at PND 35)
No effect on the total number of arm entries between treatment
groups
(calculated by EPA from graphically reported open and closed arm
entries)
Open field test:
Significant increase in the number of squares: PND 34, significant
increase at 2 mg/kg-d; PND 69, significant increase at
>0.02 mg/kg-d (no difference at PNDs 18 and 20)
Significant increase in rearing activity at 0.2 mg/kg-d on PND 69 (no
difference at PNDs 18, 20, and 34)
Bouaved et al. (2009a)
Female Swiss albino mice, 5/group
0, 2, or 20 mg/kg-d by maternal
gavage
PNDs 0-14 (lactational exposure)
Elevated plus maze:
Significantly increased time in open arms at >2 mg/kg-d
Significantly increased percentage of entries into open arms at
>2 mg/kg-d
Significantly decreased entries into closed at 2 mg/kg-d, but not at
20 mg/kg-d
Significantly decreased latency time to enter an open arm at
20 mg/kg-d
No effect on the total number of arm entries
No significant effect of gender on performance was detected, so
males and females were pooled for analyses
Open field test:
No significant change in activity on PND 15, but data not provided
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Reference and study design
Results3
Electrophysiological changes
McCallister et al. (2008)
Long-Evans Hooded rats, 5-6/group
0 or 0.3 mg/kg-d by gavage
GDs 14-17
Statistically significant decreases in stimulus-evoked cortical neuronal
activity on PNDs 90-120
Reduction in the number of spikes in both the short and long latency
periods on PNDs 90-120 (numerical data not presented)
Wormlev et al. (2004)
F344 rats, 10 females/group
0 or 100 ng/m3 by nose-only
inhalation for 4 hrs/d
GDs11-21
Electrophysiological changes in the hippocampus (PNDs 60-70):
Consistently lower long term potentiation following gestational
exposure (statistical analysis not reported)
% change relative to control: -26%
Note: significant decrease in embryo/fetal survival observed (99% in
controls versus 34% in treated group)
a% change from control calculated as: (treated value - control value)/control value x 100.
Significant denotes statistical significance; instances of statistical significance (p < 0.05) as reported by study
authors.
bAuthors used the Least Significant Difference (LSD) test which can inflate statistical significance. Magnitudes of
effect and overall biological significance were also considerations in developing the weight of evidence across
outcomes and studies.
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Morris
water
maze
Cognitive Function
Neuromuscular Function, Coordination, and Sensorimotor
Development
Anxiety and/or
Motor Activity
Figure 1-3. Exposure-response array for neurodevelopmental effects
following oral exposure.
Mode-of-Action Analysis—Developmental Toxicity and Developmental Neurotoxicity
Data regarding the potential mode of action for the various manifestations of developmental
toxicity associated with benzo[a]pyrene exposure are limited, and the mode of action for
developmental toxicity is not known. General hypothesized modes of action for the various
observed developmental effects include, but are not limited to, genotoxicity and mutagenicity,
altered cell signaling (e.g., through the AhR), cytotoxicity, and oxidative stress.
Benzo[a]pyrene is well established as a mutagen (see Section 1.1.5). It is therefore
plausible that exposure could result in mutations in male and female germ cells, as well as fetal
tissues leading to decreased viability, birth defects, and altered development in offspring. An
increasing body of information suggests that genotoxicity (including fragmentation and strand
breaks) in male germ cells can lead to decreased embryo viability post-fertilization (Borini etal..
2006: Seli etal.. 20041.
It is plausible that developmental effects of benzo[a]pyrene may be mediated by altered cell
signaling through the AhR. Benzo[a]pyrene is a ligand for the AhR, and activation of this receptor
regulates downstream gene expression including the induction of cytochrome (CYP) enzymes
important in the conversion of benzo[a]pyrene into reactive metabolites. Studies in AhR knock-out
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mice indicate that AhR signaling during embryogenesis is essential for normal liver, kidney,
vascular, hematopoietic, and immune development (Schmidt etal.. 1996: Fernandez-Salguero etal..
19951. In experiments in AhR-responsive and less-responsive mice, the mice with the less-
responsive AhR were protected from renal injury as adults following gavage treatment with 0.1 or
0.5 mg/kg-day benzo[a]pyrene from GD 10 to 13. Renal injury was indicated by an increase in
urinary albumin and a decrease in glomerular number fNanez etal.. 20111. Another study at much
higher doses (200 mg/kg-day by i.p. on GD 7,10, or 12) found increased developmental effects in
AhR-responsive C57BL/6 mice as compared to nonresponsive AhR AKR mice (Shum etal.. 19791.
Specifically, resorptions, malformations, and congenital abnormalities as well as decreased fetal
body weight were observed more commonly in AhR-responsive mice. Similar findings were
observed in a developmental toxicity study of two PAHs (3-methylcholanthrene and
7,12-dimethylbenz(a)anthracene) in mice, with increases in stillborns, resorptions, and
malformations in AH-responsive strains, indicating that mechanisms of developmental toxicity may
be related to AhR signaling (Nebertetal.. 19771.
Low birth weight has been associated with prenatal exposure to PAHs in human
populations (Duarte-Salles etal.. 2013: Duarte-Salles etal.. 2012: Pereraetal.. 2005b). Several
epidemiology studies have revealed an inverse association between low birth weight and increased
blood pressure, hypertension, and measures of decreased renal function as adults (Zandi-Neiad et
al.. 20061. It has been hypothesized that this may be attributable to a congenital nephron deficit
associated with intrauterine growth restriction (Nanez etal.. 2011: Zandi-Neiad et al.. 20061.
Oxidative stress, through oxidative DNA and protein damage induced through reactive
metabolites of benzo[a]pyrene, has also been proposed as a contributing mechanism for various
developmental effects (as reviewed by Wells et al.. 19971. Studies in GSH-deficient mice suggest
that oxidative stress may mediate male reproductive effects observed after developmental
exposure (Nakamura etal.. 20121. Some evidence also indicates increased susceptibility to
oxidative lung damage in rats exposed to relatively high i.p. doses of benzo[a]pyrene during
gestation (Thakur etal.. 20141. Several studies investigating mechanisms of developmental
neurotoxicity (Li etal.. 2012: Shengetal.. 20101 also indicate a role for increases in brain oxidative
stress as a contributor to behavioral changes elicited by developmental exposure to benzo[a]pyrene
(discussed further below).
No clear mode(s) of action for the observed neurodevelopmental changes following
benzo[a]pyrene exposure have been demonstrated. General hypothesized mechanisms with
limited support are related to altered central nervous system neurotransmission. These
mechanisms involve altered neurotransmitter gene expression, neurotransmitter levels, and
neurotransmitter receptor signaling in regions associated with spatial learning, anxiety, and
aggression, such as the hippocampus, striatum, amygdala, and hypothalamus (Li etal.. 2012: Oiu et
al.. 2011: Tang etal.. 2011: Xia etal.. 2011: Bouaved etal.. 2009a: Grova etal.. 2008: Brown etal..
2007: Grova etal.. 2007: Stephanou et al.. 19981.
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Mechanistic studies in rodents exposed as adults, which exhibit some of the same
behavioral changes as animals exposed during development (see Section 1.1.4), may also inform
potential mode(s) of action for the observed neurodevelopmental changes. Specifically regarding
potential changes in spatial learning and memory processes, multiple studies in developing (Li et
al.. 2012: Tang etal.. 2011: McCallister et al.. 2008: Wormlev etal.. 20041 and adult (Maciel etal..
2014: Oiu etal.. 2013: Tang etal.. 2011: Grova etal.. 2008: Grova etal.. 20071 rodents suggest that
changes in N-methyl-D-aspartate (NMDA) receptor signaling seen with benzo[a]pyrene exposure
(e.g., changes in expression patterns of NR2A and NR2B subunits) and possibly related effects on
synapse strength and long-term potentiation, may be responsible for behavioral effects in tests of
learning and memory.
Alternatively, or perhaps in concert with changes in NMDA receptor signaling, a series of
experiments by the same lab, using mice exposed to benzo[a]pyrene by inhalation (Li etal.. 20121
or gavage fSheng etal.. 20101 during late gestation (i.e., GDs 14-17), indicate a role for increases in
brain oxidative stress (possibly due to oxidative metabolites of benzo[a]pyrene) as a contributor to
the persistent behavioral changes elicited by developmental exposure. In the WT offspring,
benzo[a]pyrene exposure induced changes in brain markers of glutamate-associated
neurotransmission (i.e., levels of Sp4 transcription factor and the Sp4 target, NR2A in neonates, and
levels of glutamate at PND 100) and oxidative stress (i.e., neonatal F2-isoprostane levels) (Li etal..
20121. which might be related to decrements in short-term memory in the novel object test
observed at either PND 40 fSheng etal.. 20101 or PND 100 fLi etal.. 20121. These changes,
including the decrements in short-term memory, were largely reversed by knocking out brain
NADPH CYP450 reductase fLi etal.. 20121. an enzyme believed to be involved in the oxidative
metabolism of benzo[a]pyrene. Interestingly, the changes induced by benzo[a]pyrene exposure
during late gestation, including increases in oxidative metabolites and associated molecular
markers, were generally highest during the period of active synaptogenesis (e.g., PNDs 3-15).
In relation to potential changes in anxiety-like behaviors (and also relevant to effects on
learning and memory processes), many commonly used anti-anxiety medications work by
increasing brain serotonin levels (e.g., selective serotonin reuptake inhibitors), increasing brain
dopamine levels (e.g., dopamine reuptake inhibitors), or by targeting gamma-aminobutyric acid
(GABA) receptors (e.g., benzodiazepines). Although GABAa receptor messenger ribonucleic acid
(mRNA) in whole-brain homogenates was unchanged following lactational benzo[a]pyrene
exposure, exposure at >2 mg/kg-day from PND 1 to 14 caused dose-dependent decreases in
serotonin receptor (5HTia) expression (Bouaved etal.. 2009a: Stephanou etal.. 19981: however,
with short-term oral exposure in adult mice, 5HTia expression was increased at 2 mg/kg-day
(although it was unchanged at 20 mg/kg-dav: Bouaved etal.. 20121. Additional support for
identifying alterations in monoamine neurotransmitter signaling (serotonin and dopamine
signaling, in particular) as a potential mechanism(s) in the altered anxiety-like behaviors observed
following benzo[a]pyrene exposure is provided in multiple studies of rodents exposed as adults
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(Bouaved etal.. 2012: Oiu etal.. 2011: Xia etal.. 2011: Stephanou etal.. 1998: Tavasekara etal..
19921 and in a single study of blood neurotransmitter levels in occupationally-exposed men (Niuet
al.. 20101. Overall, these data suggest possible effects of benzo[a]pyrene exposure on NMDA
receptor expression and regulation of monoamine neurotransmitters including serotonin and
dopamine, but these findings require additional studies to clarify and extend understanding of
these events.
Summary of Developmental Effects
Developmental effects following in utero exposure to PAH mixtures or benzo[a]pyrene
alone have been reported in humans and in animal models. In human populations, decreased head
circumference, decreased birth weight, and decreased postnatal weight, as well as increased
frequency of miscarriage, have been reported. Analogous effects in laboratory animals, including
decreased pup weight and increased embryo/fetal resorptions, have been noted following
gestational or early postnatal exposure to benzo[a]pyrene by the oral or inhalation route (Chenet
al.. 2012: Archibongetal.. 2002: Mackenzie and Angevine. 19811. Reproductive function is also
altered in mice treated gestationally with benzo[a]pyrene fKristensen etal.. 1995: Mackenzie and
Angevine. 19811. These effects include impaired reproductive performance in F1 offspring (male
and female) and alterations of the weight and histology of reproductive organs (ovaries and testes).
The available human and animal data also support the conclusion thatbenzo[a]pyrene is a
developmental neurotoxicant Human studies of environmental PAH exposure in two cohorts have
observed neurotoxic effects, including suggestions of reduced head circumference fTang etal..
2006: Perera etal.. 2005b: Perera etal.. 20041. impaired cognitive ability fPerera etal.. 2009: Tang
et al.. 20081. impaired neuromuscular function fTangetal.. 20081. and increased attention
problems and anxious/depressed behavior following prenatal exposure (Perera et al.. 2012b).
These effects were seen in birth cohort studies in different populations (New York City and China),
in studies using specific benzo[a]pyrene measures (i.e., adduct levels measured in cord blood
samples) fPerera et al.. 2012b: Tang etal.. 2008: Tang etal.. 2006: Perera etal.. 2005b: Perera etal..
20041.
The available evidence from mice and rats also demonstrates significant and persistent
developmental impairments following exposure to benzo[a]pyrene. The most compelling evidence
derives from postnatal oral exposure studies in rats and mice testing a battery of behavioral tests
and observing consistent deficits in tests of learning, memory, and anxiety-related behaviors,
sensorimotor development, and neuromuscular function, often with effects observed across
multiple parameters tested for each of the individual behavioral tests fChen etal.. 2012: Bouaved et
al.. 2009al. Despite the differences in design of these two studies (e.g., different species; lactational
exposure versus direct gavage, etc.), the results were largely in agreement, with effects observed at
comparable oral doses generally at >0.2 mg/kg-day fChen etal.. 20121 and at >2 mg/kg-day
(Bouaved etal.. 2009a) and in the absence of maternal or neonatal toxicity. In these studies,
behavioral alterations were detectable from early neonatal ages through adulthood, including
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treatment-related changes in weanlings, pre-pubertal juveniles, and sexually mature animals, which
suggests that the neurotoxic effects of developmental benzo[a]pyrene exposure may be
irreversible. The findings of Chen etal. (20121 and Bouavedetal. (2009a) following early postnatal
exposure (i.e., PNDs 5-11 and 0-14, respectively) are supported by a single i.p. exposure study in
weanling rats demonstrating Morris water maze decrements (similar to Chen etal.. 2012: Tang et
al.. 20111. and by a number of more limited studies (e.g., typically single dose) in rats and mice that
observed persistent decrements in short-term memory and electrophysiological changes after oral
or inhalation exposure during late gestation fLi etal.. 2012: Shengetal.. 2010: McCallister etal..
2008: Wormlev et al.. 20041.
While both Chen etal. (20121 and Bouavedetal. (2009a) provide evidence of neurotoxicity
across a variety of traditional behavioral assays, there were notable strengths and weaknesses of
each study. Chen etal. (2012) was a fairly large study, with 40 litters of rats evaluated in multiple
behavioral tests at various ages. The study was designed with the intended goal of reducing bias,
including consideration of litter effects, blinding of manually observed behaviors, and a randomized
order of testing. However, the authors did not report methods for achieving matched pup ages
across the 40 litters and they rotated dams across litters to prevent potential nurturing bias. Dam
rotation is an unproven approach that may have introduced unexpected effects in the pups,
including effects on maternal caretaking (e.g., neglect of high-dose pups) and dosing accuracy (e.g.,
cross-contamination from littermates). Bouaved et al. (2009a) was a smaller and less robust study
that evaluated multiple measures across several ages using five litters of mice per dose group, used
appropriate statistical analyses, and included a detailed evaluation of maternal caretaking
behaviors. However, the study design did not account for potential litter effects (i.e., each litter was
assigned a dose group), treatment-related changes in body weight complicate some interpretations,
and protocols for observer blinding and randomized testing were not reported.
Of the behaviors tested by Chen etal. (2012). the endpoints providing the most convincing
evidence of behavioral toxicity were alterations in open field, Morris water maze, and elevated plus
maze tests, as multiple parameters were affected in each of these tests, the effects were observed
across two cohorts of rats, and altered behavior was demonstrated to persist in juvenile and adult
animals weeks to months after exposure. Bouavedetal. f2009al did not evaluate effects in adult
animals, but of the behaviors tested after exposure, effects on water escape pole climbing in male
weanlings and on elevated plus maze performance in juveniles of both sexes were the most
sensitive treatment-related changes.
In summary, it has been consistently demonstrated that developmental exposure to
benzo[a]pyrene, particularly during late gestation or early postnatal development, causes
persistent neurobehavioral effects that have been observed across two species, multiple strains,
and both sexes of experimental animals, and across several behavioral domains. These data are
supported by observations suggesting developmental neurotoxicity in children exposed to PAH
mixtures, including-altered head circumference and neurobehavioral changes, as well as molecular
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changes in experimental animals which are consistent with altered central nervous system
function. While not every endpoint tested was affected to the same extent, or in the same manner,
across studies and species, all of the identified studies reported at least one nervous system effect
of developmental exposure, demonstrating a high level of consistency within the available database.
In conclusion, although significant exposure gaps remain to be tested (most notably,
benzo[a]pyrene exposures spanning gestation and lactation), EPA identified developmental toxicity
(including developmental neurotoxicity) as a human hazard of benzo[a]pyrene exposure.
Susceptible Populations and Lifestages
Childhood susceptibility to benzo[a]pyrene toxicity is indicated by epidemiological studies
reporting associations between adverse birth outcomes and developmental effects and internal
biomarkers of exposure to benzo[a]pyrene, presumably via exposure to complex PAH mixtures
(Pereraetal.. 2012b: Perera etal.. 2009: Tangetal.. 2008: Tangetal.. 2006: Pereraetal.. 2005b:
Perera etal.. 2005a: Perera etal.. 20041. The occurrence of benzo[a]pyrene-specific DNA adducts in
maternal and umbilical cord blood in conjunction with exposure to ETS was associated with
reduced birth weight and head circumference in offspring of pregnant women living in New York
City (Perera et al.. 2005b). In other studies, elevated levels of BPDE-DNA adducts in umbilical cord
blood were associated with: (1) reduced birth weights or reduced head circumference (Perera et
al.. 2005a: Perera etal.. 2004): and (2) decreased body weight at 18, 24, and 30 months (Tangetal..
2008: Tangetal.. 20061.
Studies in animals exposed during development also support effects on pup growth (Chen
etal., 2012; Liang etal., 2012; Kristensen etal., 1995; Mackenzie and Angevine, 1981),
development of reproductive organs (Kristensen etal., 1995; Mackenzie and Angevine. 1981), and
fertility (Archibongetal., 2002; Kristensen etal., 1995; Mackenzie and Angevine. 1981). Studies
evaluating developmental immunotoxicity following environmentally relevant exposures to
benzo[a]pryene are not available in the database; however, studies utilizing i.p. exposure
paradigms provide a strong indication of potential developmental Immunotoxicity (see
Developmental lmmunotoxicty in Section 1.1.3).
Studies in humans and experimental animals indicate that exposure to PAHs in general, and
benzo[a]pyrene in particular, may impact neurological development. Observational studies in
humans have suggested associations between gestational exposure to PAHs and later measures of
neurodevelopment (Perera etal.. 2009: Tangetal.. 2008). In the Perera etal. (2009) study, the
exposure measures are based on a composite of eight PAHs measured in air. In Tangetal. (2008).
increased levels of benzo[a]pyrene-DNA adducts in cord blood were associated with decreased
developmental quotients in offspring fTang etal.. 20081.
Evidence in animals of the effects of benzo[a]pyrene on neurological development includes:
(1) decrements in reflex-related behaviors associated with neuromuscular coordination and
sensorimotor function (Chen etal.. 2012: Bouaved et al.. 2009a): (2) disrupted learning and/or
short-term memory processes (Chen etal.. 2012: Li etal.. 2012: Shengetal.. 2010: Bouaved etal..
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2009a); and (3) decreased anxiety-related responses (Chen etal.. 2012: Bouaved etal.. 2009a).
Mechanistic studies also support findings of benzo[a]pyrene induced alterations in
electrophysiological response to stimulation of the dentate gyrus of the hippocampus (Wormlev et
al.. 2004: Wu etal.. 2003a) and decreased evoked response in the cortex (McCallister etal.. 20081.
1.1.2. Reproductive Toxicity
Human and animal studies provide evidence for benzo[a]pyrene-induced male and female
reproductive toxicity. Effects on sperm quality and male fertility have been demonstrated in human
populations highly exposed to PAH mixtures fSoares and Melo. 2008: Hsu etal.. 20061. The use of
internal biomarkers of exposure in humans (e.g., BPDE-DNA adducts) supports associations
between benzo[a]pyrene exposure and these effects. In females, numerous epidemiological studies
indicate that cigarette smoking reduces fertility; however, few studies have specifically examined
levels of benzo[a]pyrene exposure and female reproductive outcomes. Animal studies demonstrate
decrements in sperm quality, changes in testicular histology, and hormone alterations following
benzo[a]pyrene exposure in adult male animals, and decreased fertility and ovotoxic effects in adult
females following exposure to benzo[a]pyrene.
Male Reproductive Effects
Fertility
Effects on male fertility have been demonstrated in populations exposed to mixtures of
PAHs. Spermatozoa from smokers have reduced fertilizing capacity, and embryos display lower
implantation rates fSoares and Melo. 20081. Occupational PAH exposure has been associated with
higher levels of PAH-DNA adducts in sperm and male infertility (Gaspari etal.. 20031. In addition,
men with higher urinary levels of PAH metabolites have been shown to be more likely to be infertile
(Xia etal.. 20091. Studies were not identified that directly examined the reproductive capacity of
adult animals following benzo[a]pyrene exposure. However, a dose-related decrease in fertility
was observed in male mice treated in utero with benzo[a]pyrene, as discussed in Section 1.1.1.
Sperm parameters
Effects on semen quality have been demonstrated in populations exposed to mixtures of
PAHs including coke oven workers and smokers (Spares and Melo. 2008: Hsu et al.. 20061. Coke
oven workers had higher frequency of oligospermia (19 versus 0% in controls) and twice the
number of morphologically abnormal sperm (Hsu etal.. 20061. Elevated levels of BPDE-DNA
adducts have been measured in the sperm of populations exposed to PAHs occupationally fGaspari
etal.. 20031 and through cigarette smoke fPhillips. 2002: Zenzes etal.. 19991. A higher
concentration of BPDE-DNA adducts was observed in sperm not selected for intrauterine
insemination or in vitro fertilization based on motility and morphology in patients of fertility clinics
(Perrin etal.. 2011b: Perrin etal.. 2011a). An association between benzo[a]pyrene exposure levels
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and increased sperm DNA fragmentation using the sperm chromatin structure assay was observed
by Rubes etal. f20101. However, it is currently unclear whether the sperm chromatin structure
assay, which measures sperm fragmentation following denaturation, is predictive of fertility
fSakkas and Alvarez. 2010: ASRM. 20081.
In several studies in rats and mice, a decrease in sperm count, motility, and production and
an increase in morphologically abnormal sperm have been reported (Table 1-5 and Figure 1-4).
Alterations in these sperm parameters have been observed in different strains of rats and mice and
across different study designs and routes of exposure.
Decreases in epididymal sperm counts (25-50% compared to controls) have been reported
in Sprague-Dawley rats and C57BL6 mice treated with 1-5 mg/kg-day benzo[a]pyrene by oral
exposure for 42 or 84 days (Chen etal.. 2011: Mohamed etal.. 2010). Another subchronic study
noted a 44% decrease in vas deferens sperm concentration at doses >50 mg/kg-day in Hsd:ICR
(CD1) mice fleng etal.. 20131. Additionally, a 15% decrease in epididymal sperm count was
observed at a much lower dose in Sprague-Dawley rats exposed to benzo[a]pyrene for 90 days
f Chung etal.. 20111. However, confidence in this study is limited because the authors dosed the
animals with 0.001, 0.01, and 0.1 mg/kg-day benzo[a]pyrene, but only reported on sperm
parameters at the mid-dose, and no other available studies demonstrated findings in the range of
the mid- and high-dose. In rats, an oral short-term study and a subchronic inhalation study lend
support for the endpoint of decreased sperm count (Arafa etal.. 2009: Archibongetal.. 2008:
Ramesh etal.. 20081. Significantly decreased sperm count and daily sperm production (20-40%
decrease from control in each parameter) were observed in rats following 10 days of gavage
exposure to 50 mg/kg-day f Arafa etal.. 20091 and following gavage dosing with 10 mg/kg-day on
PNDs 1-7 f Liang etal.. 20121. In addition, a 69% decrease from controls in sperm count was
observed in rats following inhalation exposure to 75 |ig/m:i benzo[a]pyrene for 60 days (Archibong
et al.. 2008: Ramesh et al.. 2008).
Both oral and inhalation exposure of rodents to benzo[a]pyrene have been shown to lead to
decreased epididymal sperm motility and altered morphology. Decreased motility of 20-30%
compared to controls was observed in Hsd:ICR (CD1) mice (>100 mg/kg-day), C57BL6 mice
(>1 mg/kg-day), and Sprague-Dawley rats (0.01 mg/kg-day) following subchronic oral exposure
flengetal.. 2013: Chung etal.. 2011: Mohamed et al.. 20101. The effective doses spanned several
orders of magnitude; however, as noted above, reporting is limited in the study that observed
effects at 0.01 mg/kg-day benzo[a]pyrene (Chung etal.. 2011). A short-term oral study in rats also
reported a significantly decreased number of motile sperm (~40% decrease) following 10 days of
gavage exposure to 50 mg/kg-day benzo[a]pyrene fArafa etal.. 20091. In addition, decreased
sperm motility was observed following inhalation exposure to 75 |ig/m3 benzo[a]pyrene in rats for
60 days f Archibong et al.. 2008: Ramesh etal.. 20081 and to >75 |ig/m:i for 10 days flnvangetal..
2003). Abnormal sperm morphology was observed in Sprague-Dawley rats treated with 5 mg/kg
day benzo[a]pyrene by gavage for 84 days (Chen etal.. 2011). Hsd:ICR (CD1) mice exposed to
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>50 mg/kg-day benzo[a]pyrene by gavage for 60 days fleng etal.. 20131. and F344 rats exposed to
75 |J.g/m3 benzo[a]pyrene by inhalation for 60 days fArchibong et al.. 2008: Ramesh et al.. 20081.
T esticular changes
Several studies have demonstrated dose-related effects on male reproductive organs in
adult animals exposed subchronically to benzo[a]pyrene (Table 1-5 and Figure 1-4). Decreases in
testicular weight of approximately 35% have been observed in a 60-day gavage study in Hsd:ICR
(CD1) mice at 100 mg/kg-day flengetal.. 20131. in a 10-day gavage study in adult Swiss albino rats
at 50 mg/kg-day fArafa etal.. 20091. and following subchronic inhalational exposure of adult F344
rats to 75 |ig/m3 fArchibongetal.. 2008: Ramesh etal.. 20081. No effects on testes weight were
observed in Wistar rats exposed for 35 days to gavage doses up to 50 mg/kg-day (Kroese etal..
20011. F344 rats exposed for 90 days to dietary doses up to 100 mg/kg-day (Knuckles etal.. 20011.
or Sprague-Dawley rats exposed for 90 days to gavage doses up to 0.1 mg/kg-day (Chungetal..
20111. Strain differences may have contributed to differences in response; however, F344 rats
exposed to benzo[a]pyrene via inhalation showed effects on testicular weight fArchibongetal..
2008: Ramesh et al.. 20081. In addition, decreased testicular weight has also been observed in
offspring following in utero and early postnatal exposure to benzo[a]pyrene as discussed in
Section 1.1.1.
Histological changes in the testis have often been reported to accompany decreases in
testicular weight. Apoptosis, as evident by increases in terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) positive germ cells and increases in caspase-3 staining, was evident in
seminiferous tubules of Sprague-Dawley rats following 90 days of exposure to >0.001 and
0.01 mg/kg-day, respectively, benzo[a]pyrene by gavage f Chung etal.. 20111. However, the study
authors did not observe testicular atrophy or azospermia in any dose group. Hsd:ICR (CD1) mice
exposed to 50 or 100 mg/kg-day by gavage for 30 days showed loss of seminiferous tubule integrity
and Sertoli cell fidelity fleng etal.. 20131. Seminiferous tubules were reported to look qualitatively
similar between controls and animals exposed to benzo[a]pyrene by inhalation doses of 75 |ig/m3
for 60 days fArchibongetal.. 2008: Ramesh et al.. 20081. However, when histologically examined,
statistically significantly reduced tubular lumen size and length were observed in treated animals.
Seminiferous tubule diameters also appeared to be reduced in exposed animals, although this
difference did not reach statistical significance fArchibongetal.. 2008: Ramesh et al.. 20081. In
addition, histological changes in the seminiferous tubules have also been observed in offspring
following in utero exposure to benzo[a]pyrene as discussed in Section 1.1.1.
Epididvmal changes
In addition to testicular effects, histological effects in the epididymis have been observed
following 90-day gavage exposure to benzo[a]pyrene fChungetal.. 20111 (Table 1-5 and
Figure 1-4). Specifically, statistically significant decreased epididymal tubule diameter (for caput
and cauda) was observed atdoses >0.001 mg/kg-day. Atthe highestdose tested (0.1 mg/kg-day),
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diameters were reduced approximately 25%. A 60-day gavage study in Hsd:ICR(CDl) mice
observed a 27% decrease in cauda epididymis weight at 100 mg/kg-day fleng etal.. 20131:
however, no change in epididymis weight was observed following an 84-day treatment in Sprague-
Dawley rats of 5 mg/kg-day benzo[a]pyrene (Chen etal.. 20111.
Hormone changes
Several animal models have reported decreases in testosterone following both oral and
inhalation exposure to benzo[a]pyrene (Table 1-5 and Figure 1-4). In male Sprague-Dawley rats,
decreases in testosterone have been observed following 90-day oral exposures f Chung etal.. 2011:
Zheng etal.. 20101. Statistically significant decreases of 15% in intratesticular testosterone were
observed at 5 mg/kg-day in one study (Zheng etal.. 20101. while a second study in the same strain
of rats reported statistically significant decreases of approximately 40% in intratesticular
testosterone and 70% in serum testosterone at 0.1 mg/kg-day (Chung etal.. 20111. In addition,
Sprague-Dawley rats treated with 10 mg/kg-day by gavage on PNDs 1-7 exhibited statistically
significantly decreased serum testosterone (>40%) when examined atPND 8 and PND 35 fLiang et
al.. 20121. Statistically significant decreases in intratesticular testosterone (80%) and serum
testosterone (60%) were also observed following inhalation exposure to 75 |ig/m3 benzo[a]pyrene
in F344 rats for 60 days (Archibong etal.. 2008: Ramesh et al.. 20081. In contrast to these findings,
exposure of Sprague-Dawley rats to 5 mg/kg-day benzo[a]pyrene by gavage produced a transient
increase in serum testosterone at 4 and 8 weeks, which returned to controls levels at 12 weeks of
exposure fChen etal.. 20111. Statistically significant increases in serum luteinizing hormone (LH)
have also been observed in Sprague-Dawley rats following gavage exposure to benzo[a]pyrene at
doses of >0.01 mg/kg-day f Chung etal.. 20111 and in F344 rats following inhalation exposure to
75 |J.g/m3 benzo[a]pyrene for 60 days (Archibong etal.. 2008: Ramesh et al.. 20081.
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Table 1-5. Evidence pertaining to the male reproductive toxicity of
benzo[a]pyrene in adult animals after oral or inhalation exposure
Reference and study design
Results3'15
Sperm quality
Mohamed et al. (2010)
C57BL/6 mice (6 wks old), 10 males/dose
(treated before mating with unexposed
females)
0,1, or 10 mg/kg-d by gavage (F0 males
only)
42 d
Sperm parameters assessed in F0 males
2 wks after cessation of exposure (14 wks
of age); sperm parameters assessed in
untreated Fl, F2, and F3 males at 14 wks
of age
4/ epididymal sperm count in F0 mice
Approximate % change from control (data reported graphically):
0, -50*, and -70*
4/ epididymal sperm motility in F0 mice
Approximate % change from control (data reported graphically):
0, -20*, and -50*
4/ epididymal sperm count in untreated Fl and F2 generations (data
reported graphically)
No effects were observed in the F3 generation
Jeng et al. (2013)
Hsd:ICR (CD1) mice (10 wks old),
8 males/dose
0,1,10, 50,100 mg/kg-d by gavage
30 or 60 d
Spermatozoa were obtained from a
consistent length of vas deferens
4/ Sperm concentration at 30 d (% change from control):
0, -33, -31, -26, -18
4/ Sperm concentration at 60 d (% change from control):
0, -19, 7, -44, -42
4/ Sperm motility at 30 d (% change from control):
0, 1, -16, -39, -16
4/ Sperm motility at 60 d (% change from control):
0, -26, -19, -12, -28*
4/ Sperm vitality at 30 d (% change from control):
0, 9, -15, -33, -11
4/ Sperm vitality at 60 d (% change from control):
0, -3, -2, -4, -6*
'Y % abnormal sperm (abnormal head) at 30 d:
10,11,12,14,12
1" % abnormal sperm (abnormal head) at 60 d:
17, 27, 23, 29*, 34*
Chen et al. (2011)
Sprague-Dawley rats (5-6 wks old),
10 males/dose
0 or 5 mg/kg-d by gavage
28, 56, or 84 d
4/ epididymal sperm count at 84 d (% change from control; no
change at 28 or 56 d)
0 and -29*
1" % abnormal epididymal sperm at 84 d (no change at 28 or 56 d)
5 and 8*
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Toxicological Review of Benzo[a]pyrene
Reference and study design
Results3-15
Chung et al. (2011)
Sprague-Dawley rats (8 wks old),
20-25 males/dose
0, 0.001, 0.01, or 0.1 mg/kg-d by gavage
90 d
4/ epididymal sperm motility (% change relative to control; reported
only for 0.01 mg/kg-d)
0 and -30*
No statistically significant decrease in epididymal sperm count
Ramesh et al. (2008); Archibong et al.
(2008)
F344 rats (12-13 wks old),
10 males/group
0 or 75 ng/m3, 4 hrs/d by inhalation
60 d
(sperm parameters were assessed 72 hrs
after final exposure)
4/ epididymal sperm motility (% change from control)
0 and -73*
4/ epididymal sperm count (% change from control)
0 and -69*
1" % abnormal epididymal sperm
33 and 87*
4/ spermatids/g testis (approximate % change from control;
numerical data not reported)
0 and -45*
Testicular changes (weight, histology)
Mohamed et al. (2010)
C57BL/6 mice (6 wks old), 10 males/dose
(treated before mating with unexposed
females)
0,1, or 10 mg/kg-d by gavage (F0 males
only)
42 d
F0 males sacrificed 2 wks after cessation
of exposure (14 wks of age); unexposed
Fl, F2, and F3 males sacrificed at 14 wks
of age
4/ seminiferous tubules with elongated spermatids in F0 males
(approximate % change from control; numerical data not reported)
0, -20*, and -35*
No statistically significant change in area of seminiferous epithelium
of testis in F0 males (approximate % change from control; numerical
data not reported)
0, 5, and 20
Testicular findings non-significant in Fl and F3 generations, but
significant at the high dose in F2 males.
Jeng et al. (2013)
Hsd:ICR (CD1) mice (10 wks old),
8 males/dose
0,1,10, 50, or 100 mg/kg-d by gavage
30 or 60 d
1" testicular lesions after 30 d of exposure were characterized as
decreased seminiferous tubule integrity and loss of Sertoli cell fidelity
at 50 mg/kg-d; variation in the seminiferous tubule diameter and
reduced organization and integrity, decreased luminal volume of
mature sperm, and uneven Sertoli cell maintenance of the
seminiferous epithelium at 100 mg/kg-d. Histopathology findings
were not reported for the 60 d exposure.
4/ decreased testis weight at 30 d (% change from control):
0, -4, -7,1, -10
4/ decreased testis weight at 60 d (% change from control):
0, 3, -15, -23, -35*
Chung et al. (2011)
Sprague-Dawley rats (8 wks old),
20-25 males/dose
0, 0.001, 0.01, or 0.1 mg/kg-d by gavage
90 d
1" number of apoptotic germ cells per tubule (TUNEL or caspase 3
positive)
No change in testis weight or histology
1-35
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Toxicological Review of Benzo[a]pyrene
Reference and study design
Results3-15
Chen et al. (2011)
Sprague-Dawley rats (5-6 wks old),
10 males/dose
0 or 5 mg/kg-d by gavage
84 d
1" testicular lesions characterized as irregular arrangement of germ
cells and absence of spermatocytes (numerical data not reported)
No change in testis weight
Archibong et al. (2008); Ramesh et al.
(2008)
F344 rats (12-13 wks old),
10 males/group
0 or 75 ng/m3, 4 hrs/d by inhalation
60 d
4/ decreased testis weight (% change from control)
0 and 34*
4/ size of seminiferous tubule lumens and reduced tubular length
No change in % of tubules with elongated spermatids
Animals sacrificed 72 hrs after final
exposure.
Kroese et al. (2001)
Wistar rats (6 wks old), 10/sex/dose
0,1.5, 5,15, or 50 mg/kg-d by gavage,
5 d/wk
35 d
No change in testis weight (data not shown)
Knuckles et al. (2001)
F344 rats (8 wks old), (number of animals
examined unclear)
0, 5, 50, or 100 mg/kg-d in diet
90 d
No change in testis weight (data not shown)
Epididymal changes (weight, histology)
Chung et al. (2011)
Sprague-Dawley rats (8 wks old),
20-25 males/dose
0, 0.001, 0.01, or 0.1 mg/kg-d by gavage
90 d
4/ diameter of caput epididymal tubule (n = 5; numerical data not
reported)
4/ diameter of cauda epididymal tubule (n = 5; numerical data not
reported)
Chen et al. (2011)
Sprague-Dawley rats (5-6 wks old),
10/dose
0 or 5 mg/kg-d by gavage
84 d
No change in epididymis weight
Jeng et al. (2013)
Hsd:ICR (CD1) mice (10 wks old),
8 males/dose
0,1,10, 50,100 mg/kg-d by gavage
30 or 60 d
4/ decreased cauda epididymis weight at 30 d (% change from
control):
0, -16, -26, -26, -20
4/ decreased cauda epididymis weight at 60 d (% change from
control):
0, -13, -13, -17, -27*
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Toxicological Review of Benzo[a]pyrene
Reference and study design
Results3-15
Hormone changes
Chung et al. (2011)
Sprague-Dawley rats (8 wks old),
20-25 males/dose
0, 0.001, 0.01, 0.1 mg/kg-d by gavage
90 d
4/ Intratesticular testosterone (approximate % change from control;
data reported graphically)
0, -12, -25, and -40*
4/ Serum testosterone (approximate % change from control;
numerical data not reported)
0, 0, -35, and -70*
1" serum LH (approximate % change from control; numerical data not
reported)
0, 33, 67*, and 87*
4/ Human chorionic gonadotropin (hCG) or dibutyl cyclic adenosine
monophosphate (dbcAMP)-stimulated testosterone production in
Leydig cells
Chen et al. (2011)
Sprague-Dawley rats (5-6 wks old),
10 males/dose
0 or 5 mg/kg-d by gavage
28, 56, or 84 d
1" serum testosterone at 28 and 56 d only (approximate % change
from control; data reported graphically):
28 d 0,160*
56 d 0,100*
84 d 0, -10
Zheng et al. (2010)
Sprague-Dawley rats (6 wks old),
8 males/dose
0,1, or 5 mg/kg-d by gavage
90 d
4/ Intratesticular testosterone (approximate % change from control;
numerical data not reported)
0, -15, and -15*
Archibong et al. (2008); Ramesh et al.
(2008)
F344 rats (12-13 wks old), 10 adult
males/group
0 or 75 ng/m3, 4 hrs/d by inhalation
60 d
4/ intratesticular testosterone, 72 hrs post exposure (approximate %
change from control; numerical data not reported)
0 and -80*
4/ serum testosterone (approximate % change from control)
0 and -70*
1" serum LH (approximate % change from control)
0 and 50*
^Statistically significantly different from the control (p < 0.05).
a% change from control calculated as: (treated value - control value)/control value x 100.
bEndpoints accessed directly following the exposure period unless otherwise indicated.
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Toxicological Review of Benzo[a]pyrene
¦ LOAEL
ANOAEL
• Doses > LOAEL
o Doses < NOAEL
^ 10
ffl"
¦o
to
JC
I 1
o
Q
0.1
0.01
0.001
A
«H QJ
3 ft
ft
3 ft
3 ft
™ E
(N ^
r T3
-a
«N ^
"D
fN .
. >•
T3
fN .
. >-
T3
fN
. >
T5
u
TO T5
Chen et a
84-day sti
td «
1 7
JI o
U CTl
6 S
% ts
ClO m"
1 7
JZ o
(J CTl
Chung et;
90-day sti
tl
00 sT
g Is
.c O
rsj cti
° S
si- epididymal
si- epididymal
si- epididymal
Irregular
4- TT (serum
t LH
4- TT
sperm count
sperm count
sperm motility
germ cell
and
(intratesticular)
and motility
'fin abnormal
sperm
organization
intratesticular)
Quality
Testicular
Hormone Changes
Effects
td
00 TO*
C T3
't'in abnormal
sperm
00 TO*
C "D
4, epididymal
sperm motility
and vitality
Figure 1-4. Exposure-response array for male reproductive effects following
oral exposure in adult animals.
Mode-of-Action Analysis—Male Reproductive Effects
Exposure to benzo[a]pyrene in laboratory animals induces male reproductive effects
including decreased sperm quality, decreased levels of testosterone, increased levels of LH, and
histological changes in the testis. Hypothesized modes of action of include benzo[a]pyrene-
mediated DNA damage to male germ cells leading to cytotoxicity, apoptosis and decreased embryo
viability post-fertilization, compromised function of Sertoli and Leydig cells, oxidative stress, and
altered regulation of the steroidogenic acute regulatory protein (StAR) promoter.
Decrements in sperm quality has been demonstrated in populations highly exposed to PAH
mixtures (Spares and Melo. 2008: Hsu etal.. 20061 as well as in male animals exposed to
benzo[a]pyrene (see Table 1-5). Genotoxicity in male germ cells has been hypothesized to lead to
cytotoxicity and apoptosis (Chung etal.. 2011: Perrin etal.. 2011b: Perrin etal.. 2011a: Olsenetal..
2010: Revel etal.. 20011 as well as mutations fXu etal.. 20141 and decreased embryo viability post-
fertilization associated with sperm DNA damage fBorini etal.. 2006: Seli etal.. 20041.
A study in tobacco smokers suggests that direct DNA damage from the reactive metabolite
BPDE may decrease sperm motility (Perrin etal.. 201 lal. In this study, motile sperm were
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Toxicological Review of Benzo[a]pyrene
separated from non-motile sperm using a "swim-up" self-migration technique. The investigators
found that the motile sperm selected by this method had significantly fewer BPDE-adducts than
non-selected sperm.
Numerous studies have indicated thatbenzo[a]pyrene reduces testosterone levels in adult
animals following oral or inhalation exposure f Chung etal.. 2011: Zheng etal.. 2010: Archibong et
al.. 2008: Ramesh et al.. 20081. It is plausible that the effects on sperm quality and histological
changes of the reproductive organs are secondary to an insufficiency of testosterone flnvangetal..
20031. One study hypothesized that benzo[a]pyrene perturbs the production of testosterone by
Leydig cells (Chung etal.. 20111. This study found a statistically significant reduction in testicular
testosterone in rats treated with 0.1 mg/kg-day benzo[a]pyrene for 90 days and found that
testosterone production in isolated Leydig cells was also inhibited approximately 50%, even in
cultures stimulated with human chorionic gonadotropin and dibutryl cyclic adenosine
monophosphate.
Leydig cell function is thought to be regulated by testicular macrophages (Hales. 20021.
When testicular macrophages are activated and produce inflammatory mediators, Leydig cell
testosterone production is inhibited (Hales. 20021. Zheng etal. (20101 treated rats with
5 mg/kg-day benzo[a]pyrene for 90 days and reported a statistically significant increase in ED-1
type testicular macrophages and a statistically significant decrease in intratesticular testosterone.
Some studies suggest that male reproductive effects may be secondary to increased
oxidative stress. Arafa etal. (2009) reported that male reproductive effects observed following
benzo[a]pyrene exposure could be ameliorated by antioxidant pre-treatment This study reported
decreased sperm count, motility, and production, in addition to decreased testis weight following a
10-day oral administration in rats of 50 mg/kg-day benzo[a]pyrene. Pretreatmentwith the citris
flavonoid hesperidin protected rats from all of these effects except the decrease in sperm motility.
In addition, studies in GSH-deficient mice suggest that oxidative stress may mediate male
reproductive effects observed after developmental exposure fNakamura etal.. 20121.
Female Reproductive Effects
Fertility
In women, exposure to cigarette smoke has been shown to affect fertility, including effects
related to pregnancy, ovulatory disorders, and spontaneous abortion (as reviewed in Wavlen etal..
2009: Cooper and Molev. 2008: Spares and Melo. 20081. In addition, several studies suggest that in
utero exposure to maternal tobacco smoke also decreases the future fertility of female offspring (Ye
etal.. 2010: Tensen etal.. 1998: Weinberg etal.. 19891. Benzo[a]pyrene levels in follicular fluid and
benzo[a]pyrene-DNA adducts in granulosa-lutein cells and oocytes and in human cervical cells have
been associated with smoking status and with amount smoked (Neal etal.. 2008: Mancini etal..
1999: Melikian etal.. 1999: Zenzes etal.. 1998: Shamsuddin and Gan. 19881.
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Toxicological Review of Benzo[a]pyrene
Few epidemiological studies have examined the specific influence of components of PAH
mixtures on fertility or other reproductive outcomes; EPA identified only two studies with specific
data on benzo[a]pyrene (Table 1-6). One of these studies addressed the probability of conception
among women undergoing in vitro fertilization (Neal etal.. 20081. Follicular fluid benzo[a]pyrene
levels were significantly higher among the women who did not conceive compared with women
who did get pregnant No association was seen between conception and serum levels of
benzo[a]pyrene. The other study examined risk of delayed miscarriage (fetal death before
14 weeks of gestation), using a case-control design with controls selected from women undergoing
elective abortion (Wu etal.. 2010). A strong association was seen between maternal blood
benzo[a]pyrene-DNA adduct levels and risk of miscarriage, with a 4-fold increased risk for levels
above compared with below the median. Benzo[a]pyrene-DNA adduct levels were similar in the
aborted tissue of cases compared with controls.
Experimental studies in mice also provide evidence thatbenzo[a]pyrene exposure affects
fertility (see Table 1-7 and Figure 1-5). Decreased fertility and fecundity (decreased number of F0
females producing viable litters at parturition) was statistically significantly reduced by about 35%
in adult females exposed to 160 mg/kg-day of benzo[a]pyrene (Mackenzie and Angevine. 1981). In
another study, F0 females showed no signs of general toxicity or effects on fertility following gavage
exposure to 10 mg/kg-day on GDs 7-16 (Kristensen et al.. 1995). Decrements in fertility were
more striking in the offspring exposed during development, as described in Section 1.1.1
(Developmental Toxicity), as exposure in utero appears to impair the development of reproductive
organs.
One study suggests that exposure to benzo[a]pyrene prior to mating decreases the
ovulation rate in female rats treated by inhalation to 50, 75, or 100 |a,g/m3 for 4 hours/day for
14 days prior to mating (Archibong etal.. 2012). A dose-related decrease in ovulation rate and a
corresponding decrease in the number of pups born per litter was seen starting at the lowest dose.
At the highest concentration tested, a decrease in litter size was seen, which was only partially
explained by the decreased ovulation rate.
Ovarian effects
Human epidemiological studies that directly relate ovotoxicity and benzo[a]pyrene
exposure are not available; however, smoking, especially during the time of the peri-menopausal
transition, has been shown to accelerate ovarian senescence fMidgette and Baron. 19901.
Benzo[a]pyrene-induced ovarian toxicity has been demonstrated in animal studies. In adult female
rats treated by gavage, statistically significant, dose-related decreases in ovary weight have been
observed in female rats treated for 60 days at doses >5 mg/kg (2.5 mg/kg-day adjusted) fXu etal..
2010). At 10 mg/kg in adult rats (5 mg/kg-day adjusted), ovary weight was decreased 15% (Xu et
al.. 2010). Apparent decreases in ovary weight of approximately 10-13% were also observed in a
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Toxicological Review of Benzo[a]pyrene
14-day inhalation study in F344 rats treated with 50, 75, or 100 |a,g/m3 benzo[a]pyrene (Archibong
etal.. 20121.
Two additional studies in adult rats, not designed to investigate reproductive endpoints, did
not detect changes in ovary weight. Specifically, ovary weight was investigated, but significant
findings were not reported (data not shown) in Wistar rats (n = 10) exposed for 35 days to gavage
doses up to 50 mg/kg-day fKroese etal.. 20011. In a study in F344 rats exposed for 90 days to
dietary doses up to 100 mg/kg-day, ovarian weight was reportedly measured, but was not among
the organs with significant weight changes. However, data for this endpoint, as well as the number
of animals examined, were not reported for this exposure duration (Knuckles etal.. 20011.
As discussed in Section 1.1.1 (Developmental Toxicity), severe reductions in ovarian weight
of offspring gestationally treated with benzo[a]pyrene were reported in mice (Kristensen etal..
1995: Mackenzie and Angevine. 19811. Specifically, ovary weight in F1 offspring was reduced 31%
following exposure to 10 mg/kg-day benzo[a]pyrene fKristensen etal.. 19951. while in another
gestational study at the same dose level, ovaries were so drastically reduced in size (or absent) that
they were not weighed (Mackenzie and Angevine. 19811.
In adult female rats treated by gavage, dose-related decreases in the number of primordial
follicles have been observed in female rats treated for 60 days at doses >2.5 mg/kg-day, with a
statistically significant decrease of approximately 20% at the high dose (Xu etal.. 20101 (T able 1-7
and Figure 1-5). No notable differences in other follicle populations and corpora lutea were
observed. However, in utero studies exposing dams to the same doses produced offspring with few
or no follicles or corpora lutea (Kristensen etal.. 1995: Mackenzie and Angevine. 19811. Additional
support for the alteration of female reproductive endpoints comes from i.p. experiments in animals
and in vitro experiments. Several studies have observed ovarian effects (decreased numbers of
ovarian follicles and corpora lutea, absence of folliculogenesis, oocyte degeneration, and decreased
fertility) in rats and mice exposed via i.p. injection (Borman etal.. 2000: Miller etal.. 1992: Swartz
and Mattison. 1985: Mattison etal.. 19801. Further evidence is available from in vitro studies
showing inhibition of antral follicle development and survival, as well as decreased production of
estradiol, in mouse ovarian follicles cultured with benzo[a]pyrene for 13 days (Sadeu and Foster.
20111. Likewise, follicle stimulating hormone (FSH)-stimulated growth of cultured rat ovarian
follicles was inhibited by exposure to benzo[a]pyrene (Neal etal.. 20071.
Hormone levels
Alterations of estrous cyclicity and hormone levels have been observed in female rats
following oral or inhalation exposure to benzo[a]pyrene (Table 1-7 and Figure 1-5). Inhalation
exposure to benzo[a]pyrene:carbon black particles during gestation resulted in decreases in plasma
progesterone, estradiol, and prolactin in pregnant rats (Archibong etal.. 20021. A 14-day inhalation
study in adult F344 rats noted that estrous cycle length was significantly increased in rats in the
high-dose group (100 |a,g/m3). The investigators measured hormone levels in this dose group
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Toxicological Review of Benzo[a]pyrene
during the four different stages of the estrous cycle and noted decreased estrodial during proestrus,
decreased progesterone during diestrus I, increased FSH during all stages, and decreased LH during
proestrus fArchibong etal.. 20121. Similar alterations have been noted following oral exposures
with statistically significant, dose-related decreases in estradiol and altered estrous cyclicity
observed in female rats treated for 60 days at doses >2.5 mg/kg-day by gavage fXu etal.. 20101.
Mechanistic experiments have also noted decreased estradiol output in murine ovarian follicles
cultured with benzo[a]pyrene in vitro for 13 days, but did not find any decrease in progesterone
(Sadeu and Foster. 20111.
Uterine effects
One subchronic animal study is available that investigated effects in the uterine cervix
following oral exposure to benzo[a]pyrene (Table 1-7 and Figure 1-5). Statistically-significant,
dose-related increases in the incidence of cervical inflammatory cells were observed in mice
exposed twice a week for 98 days to benzo[a]pyrene via gavage at doses >2.5 mg/kg (Gao etal..
20111. Uterine cervical effects of increasing severity, including epithelial hyperplasia, atypical
hyperplasia, apoptosis, and necrosis, were observed at higher doses. This study also observed a
depression of body weight (10,15, and 30%) and elevated mortality in the two higher dose groups
(4 and 8%), suggesting potential treatment-related toxicity. Gao etal. f20111 also evaluated effects
on the uterine cervix in separate groups of mice exposed via i.p. injection, and observed similar
responses in these groups of mice. Gao etal. (20111 considered the hyperplasia responses to be
preneoplastic lesions. Cervical neoplasia was not reported in the available chronic bioassays, but
uterine tissue was not subjected to histopathology examination in either bioassay (Kroese etal..
2001: Beland and Culp. 19981. It is plausible that an increase in uterine inflammation during
pregnancy could impact parturition and pre-term birth (as reviewed by Gomez-Lopez etal.. 20141:
however, the relationship of the observed uterine inflammation and hyperplasia in Gao et al.
f20111 to reproductive function is uncertain.
Table 1-6. Evidence pertaining to the female reproductive effects of
benzo[a]pyrene in humans
Reference and study design
Results
Probability of conception
Neal et al. (2008)
36 women undergoing in vitro fertilization
(19 smokers, 7 passive smokers, and
10 nonsmokers)
Exposure: benzo[a]pyrene in serum and follicular
fluid
Benzo[a]pyrene levels (ng/mL)
Did not
Conceived conceive p-value
Follicular fluid 0.1 1.7 <0.001
Serum 0.01 0.05 Not reported
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Toxicological Review of Benzo[a]pyrene
Fetal death
Wu et al. (2010) (Tianiin, China)
Case control study: 81 cases (96% participation
rate)—fetal death confirmed by ultrasound before
14 wks gestation; 81 controls (91% participation
rate)—elective abortions; matched by age,
gestational age, and gravidity; excluded smokers
and occupational PAH exposure
Exposure: benzo[a]pyrene in aborted tissue and
maternal blood samples (51 cases and controls,
2 of 4 hospitals)
Benzo[a]pyrene adduct levels (/10s 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 benzo[a]pyrene adducts and
miscarriage3
OR 95% CI
Per unit increase in adducts 1.37 1.12,1.67
Dichotomized at median 4.56 1.46, 14.3
Conditional logistic regression, adjusted for maternal
education, household income, and gestational age; age also
considered as potential confounder
Table 1-7. Evidence pertaining to the female reproductive effects of
benzo[a]pyrene in adult animals after oral or inhalation exposure
Reference and study design
Results3
Fertility
Mackenzie and Angevine (1981)
CD-I mice, 30 or 60 F0 females/dose
0,10, 40, or 160 mg/kg-d by gavage
GDs 7-16
4/ number of F0 females with viable litters
46/60, 21/30, 44/60, and 13/30*
Kristensen et al. (1995)
NMRI mice, 9 females/dose
0 or 10 mg/kg-d by gavage
GDs 7-16
No changes in fertility of F0 females
Archibong et al. (2012)
Fisher 344 rats, 20 females/dose
0, 50, 75, or 100 ng
benzo[a]pyrene/m3 nose only
inhalation for 4 hrs/d
14 d prior to mating
(carbon black used as carrier particle;
no carrier particle control used)
4/ number of pups born per litter
15, 13.4, 12.3, 4.3**
4/ embryo/fetal survival (%)
98, 96, 96, and 52**
[% Embryo/fetal survival = number of pups/(number of ovulated eggs
from females mated with vasectomized males) x 100]
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Toxicological Review of Benzo[a]pyrene
Reference and study design
Results3
Ovarian effects (weight, histology, follicle numbers)
Xu etal. (2010)
Sprague-Dawley rats, 6 females/
dose
0, 5, or 10 mg/kg by gavage every
other day (2.5 and 5 mg/kg-d,
adjusted)
60 d
4/ ovary weight (% change from control)
0, -11*, and -15*
4/ number of primordial follicles (% change from control; data presented
graphically)
0, -6, -22
1" increased apoptosis of ovarian granulosa cells (approximate
% apoptosis)
2, 24*, and 14*
Knuckles et al. (2001)
F344 rats, (number of animals
examined not reported)
0, 5, 50, or 100 mg/kg-d in diet
90 d
No changes in ovary weight (data not reported)
Kroese et al. (2001)
Wistar rats, 10/sex/dose
0,1.5, 5,15, or 50 mg/kg-d by gavage
5 d/wk
35 d
No changes in ovary weight (data not reported)
Archibong et al. (2012)
Fisher 344 rats, 20 females/dose
0, 50, 75, or 100 ng
benzo[a]pyrene/m3 nose only
inhalation for 4 hrs/d
14 d
(carbon black used as carrier particle;
no carrier particle control used)
4/ ovary weight (% change from control)
0, -10, -13, and -12
4/ ovulation rate (total number of ovulated eggs recovered from both
oviducts and confirmed by the total number of corpora lutea; (% change
from control)
0, -9, -16, and -46*
Hormone levels
Xu etal. (2010)
Sprague-Dawley rats, 6 females/
dose
0, 5, or 10 mg/kg by gavage every
other day (2.5 and 5 mg/kg-d,
adjusted)
60 d
4/ serum estradiol (approximate % change from control)
0, -16, and -25*
Prolonged estrous cycle
Archibong et al. (2002)
F344 rats, 10 females/group
0, 25, 75, or 100 ng/m3 by inhalation
4 hrs/d
GDs 11-20 (serum hormones tested
at GD 15 and 17 in 0, 25, and
75 |jg/m3dose groups)
4/ F0 estradiol, approximately 50% decrease at 75 ng/m3at GD 17
4/ F0 prolactin, approximately 70% decrease at 75 ng/m3 at GD 17
1" F0 plasma progesterone approximately 17% decrease at 75 ng/m3 at
GD 17
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Toxicological Review of Benzo[a]pyrene
Reference and study design
Results3
Archibong et al. (2012)
Fisher 344 rats, 20 females/dose
0, 50, 75, or 100 ng
benzo[a]pyrene/m3 nose only
inhalation for 4 hrs/d
14 d
(carbon black used as carrier particle;
no carrier particle control used)
Serum hormone concentrations measured in highest dose group and
reported by stage of estrous cycle and compared to control:
4/ serum estradiol in proestrus
4/ serum progesterone in diestrus 1
'Y serum FSH at all stages of estrous cycle
4/ serum LH in proestrus
Prolonged estrous cycle in the high dose group
(results presented graphically)
Uterine effects
Gao et al. (2011)
ICR mice, 26 females/dose
0, 2.5, 5, or 10 mg/kg by gavage
2 d/wk
98 d
'Y cervical epithelial hyperplasia: 0/26, 4/26, 6/25*, and 7/24*
1" cervical atypical hyperplasia: 0/26, 0/26, 2/25, and 4/24*
1" inflammatory cells in cervical epithelium: 3/26,10/26,12/25*, and
18/24*
^Statistically significantly different from the control (p < 0.05).
a% change from control calculated as: (treated value - control value)/control value x 100.
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Toxicological Review of Benzo[a]pyrene
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Toxicological Review of Benzo[a]pyrene
that exposure to female germ cells, could result in cellular damage, reducing oocyte and embryo
viability, and potentially transmit mutations to surviving offspring. In female rats exposed to a
single oral dose (5 mg/kg), benzo[a]pyrene-DNA adducts were noted to be higher in ovarian tissue
than the liver (Ramesh et al). In addition, increased BPDE-DNA adducts were found in oocytes and
DNA strand breaks in cumulous cells in female mice exposed to a single oral dose of 13 mg/kg
benzo[a]pyrene (Einaudi et al.. 20141.
Ovarian lesions in benzo[a]pyrene-exposed rats have been associated with increased
apoptosis in ovarian granulosa cells and alteration in hormone-mediated regulation of
folliculogenesis fXu etal.. 20101. and results from in vitro and i.p. experiments provide support for
an association between benzo[a]pyrene exposure and impaired folliculogenesis, steroidogenesis,
and oocyte maturation (Rummer etal.. 2013: Sadeu and Foster. 2011: Neal etal.. 2007: Mattison.
19801. A growing body of research suggests that benzo[a]pyrene triggers the induction of
apoptosis in oocytes through AhR-driven expression of pro-apoptotic genes, including Bax fSadeu
and Foster. 2013: Kee etal.. 2010: Neal etal.. 2010: Pru etal.. 2009: Matikainen etal.. 2002:
Matikainen etal.. 2001: Robles etal.. 20001. Other proposed mechanisms include the impairment of
folliculogenesis from reactive metabolites fTakizawa etal.. 1984: Mattison and Thorgeirsson. 1979.
19771 or by a decreased sensitivity to FSH-stimulated follicle growth (Neal etal.. 20071. Based on
findings that an ERa antagonist counteracted effects of subcutaneously administered
benzo[a]pyrene on uterine weight (decreased in neonatal rats and increased in immature rats),
interactions with ERa have been proposed, possibly via occupation of ERa binding sites or via AhR-
estrogen receptor-crosstalk fKummer etal.. 2008: Kummer et al.. 20071. However, several in vitro
studies have demonstrated low affinity binding of benzo[a]pyrene to the estrogen receptor and
alteration of estrogen-dependent gene expression fLiu etal.. 2006: van Lipzigetal.. 2005:
Vondracek etal.. 2002: Fertuck etal.. 2001: Charles etal.. 20001. so the role of the estrogen receptor
in benzo[a]pyrene-induced reproductive toxicity is unclear.
Some evidence in rodents suggests that decrements in female fertility from benzo[a]pyrene
exposure may be related to disruption in the estrous cycle and the balance of reproductive
hormones fZhao etal.. 2014: Archibongetal.. 20121. One study in rats noted that inhalation
exposure prior to mating resulted in alterations in reproductive hormones, estrous cycle length,
decreased ovulation rate, and production of smaller litters fArchibong etal.. 20121. Another study
dosed mice following mating and prior to implantation (GDs 1-5) and noted that i.p. doses of
benzo[a]pyrene as low as 0.2 mg/kg increased plasma estrogen and progesterone, altered the
morphology of the endometrium, and decreased the number of implantation sites and the overall
pregnancy rate in mice fZhao etal.. 20141.
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Toxicological Review of Benzo[a]pyrene
Summary of Reproductive Effects
Male reproductive effects
Exposure to benzo[a]pyrene in laboratory animals induces male reproductive effects
including decreased levels of testosterone and increased levels of LH, decreased sperm count and
motility, histological changes in the testis, and decreased reproductive success. These findings in
animals are supported by decrements in sperm quality and decreased fertility in human
populations exposed to PAH mixtures (Spares and Melo. 2008: Hsu etal.. 20061. In laboratory
animals, male reproductive toxicity has been observed after oral and inhalation exposure to rats or
mice. Effects seen after oral exposures include impaired fertility, effects on sperm parameters,
decreased reproductive organ weight, testicular lesions, and hormone alterations (Chen etal.. 2011:
Chung etal.. 2011: Mohamed etal.. 2010: Zheng etal.. 2010: Mackenzie and Angevine. 19811. In
addition to oral exposure, male reproductive effects of benzo[a]pyrene have also been observed
following inhalation exposure in rats (Archibong et al.. 2008: Ramesh et al.. 2008: Invangetal..
20031. The male reproductive effects associated with benzo[a]pyrene exposure are considered to
be biologically plausible and adverse.
In conclusion, EPA identified male reproductive system effects as a human hazard of
benzo[a]pyrene exposure.
Female reproductive effects
A large body of mechanistic data, both in vivo and in vitro, suggests that benzo[a]pyrene
impacts fertility through the disruption of folliculogenensis. This finding is supported, albeit
indirectly, by observations of premature ovarian senescence in women exposed to cigarette smoke
(Midgette and Baron. 19901. Evidence for female reproductive toxicity of benzo[a]pyrene comes
from studies of human populations exposed to PAH mixtures as well as laboratory animal and in
vitro studies. In addition, two human studies observed associations specifically between
benzo[a]pyrene measures and two fertility-related endpoints: decreased ability to conceive (Neal et
al.. 2008: Neal etal.. 20071 and increased risk of early fetal death (i.e., before 14 weeks of gestation)
(Wu etal.. 20101. Studies in multiple strains of rats and mice indicate fertility-related effects
including decreases in ovarian follicle populations and decreased fecundity. Decreased serum
estradiol has also been noted in two different strains of rats exposed by oral or inhalation exposure.
The reproductive effects associated with benzo[a]pyrene exposure are biologically supported and
relevant to humans.
In conclusion, EPA identified female reproductive effects as a human hazard of
benzo[a]pyrene exposure.
Susceptible Populations and Lifestages
Epidemiological studies indicate that exposure to complex mixtures of PAHs, such as
through cigarette smoke, is associated with measures of decreased fertility in humans fNeal etal..
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Toxicological Review of Benzo[a]pyrene
2008: El-Nemr etal.. 19981 and that prenatal exposure to cigarette smoking is associated with
reduced fertility of women later in life fWeinbergetal.. 19891. A case-control study in a Chinese
population has also indicated that women with elevated levels of benzo[a]pyrene-DNA adducts in
maternal blood were 4 times more likely to have experienced a miscarriage fWu et al.. 20101.
Evidence from animal models indicate thatbenzo[a]pyrene exposure can decrease the
ability of females to maintain pregnancy. Inhalation exposure of pregnant female rats to
benzo[a]pyrene:carbon black aerosols on GDs 11-20 caused an increase in embryo/fetal
resorptions, as evidenced by decreased litter size in pregnant animals treated after implantation
fArchibongetal.. 20021. Evidence for increased embryo/fetal resorptions was also demonstrated
by decreased litter size following high-dose oral exposure of pregnant mice on GDs 7-16
(Mackenzie and Angevine. 19811. In addition to effects observed following in utero exposure,
decreased production of offspring has also been observed with benzo[a]pyrene administration
prior to mating fArchibong etal.. 2012: Mattison et al.. 19801. Reduced litter sizes at birth was
observed following a 14-day pre-mating inhalation exposure period, and was associated with
decreases in ovulation rate fArchibongetal.. 20121. A continuous breeding study also noted a
decreased production of offspring in mice following a single i.p. exposure to benzo[a]pyrene
2 weeks prior to mating (Mattison etal.. 19801.
Oral multigenerational studies of benzo[a]pyrene exposure in mice demonstrated effects on
fertility and the development of reproductive organs (decreased ovary and testes weight) in both
male and female offspring of pregnant mice exposed to 10-160 mg/kg-day on GDs 7-16
fKristensen etal.. 1995: Mackenzie and Angevine. 19811. Persistent reductions in sperm
parameters have also been observed in SD rats following early gestational exposure (GDs 1-7) to
5-10 mg/kg-day fLiang etal.. 20121.
Reductions in female fertility associated with decreased ovary weight and follicle number
following gestational exposure (as discussed in Section 1.1.1) are supported by observations of:
(1) destruction of primordial follicles (Borman etal.. 2000: Mattison et al.. 19801 and decreased
corpora lutea fMiller etal.. 1992: Swartz and Mattison. 19851 in adult female mice following i.p.
exposure; (2) decreased ovary weight in adult female rats following oral exposure fXu etal.. 20101:
and (3) stimulation of oocyte apoptosis fMatikainenetal.. 2002: Matikainen et al.. 20011 or by a
decreased sensitivity to FSH-stimulated follicle growth fNeal etal.. 20071.
Reductions in male fertility associated with decreased testes weight following gestational
exposure (as discussed in Section 1.1.1) are supported by observations of: (1) decreased sperm
count, altered serum testosterone levels, testicular lesions, and/or increased numbers of apoptotic
germ cells in adult rats following repeated oral exposure to benzo[a]pyrene f Chung etal.. 2011:
Chen etal.. 2010: Zheng etal.. 2010: Arafa etal.. 20091: (2) decreased epididymal sperm counts in
adult F0 and F1 generations of male mice following 6 weeks of oral exposure of the F0 animals to
benzo[a]pyrene (Mohamed etal.. 20101: and (3) decreased testis weight, decreased testicular or
plasma testosterone levels, and/or decreased sperm production, motility, and density in adult male
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Toxicological Review of Benzo[a]pyrene
rats following repeated inhalation exposure to aerosols of benzo[a]pyrene:carbon black (Archibong
etal.. 2008: Ramesh etal.. 2008: Invangetal.. 20031.
1.1.3. Immunotoxicity
No evidence of an association between benzo[a]pyrene exposure in utero and allergic
sensitization was found in the one available human study of developmental immunotoxicity that
provided data specific to benzo[a]pyrene fledrvchowski etal.. 20111 (Table 1-8). No other human
studies evaluating immune effects following exposure to benzo[a]pyrene alone are available for any
route of exposure. However, a limited number of occupational human studies, particularly in coke
oven workers (Zhang etal.. 2012: Wu etal.. 2003b: Winker etal.. 1997: Szczeklik etal.. 19941. show
effects on immune parameters associated with exposure to PAH mixtures. These studies are of
limited utility because effects associated specifically with benzo[a]pyrene cannot be distinguished
from other constituents of the PAH mixture. Subchronic and short-term animal studies have
reported immunotoxic effects of benzo[a]pyrene by multiple routes of exposure (Table 1-9 and
Figure 1-6). Effects include changes in thymus weight and histology, decreased B cell percentages
and other alterations in the spleen, and immune suppression. Data obtained from subchronic
gavage studies are supported by short-term, i.p., intratracheal, and subcutaneous (s.c.) studies.
Additionally, there is evidence in animals for effects of benzo[a]pyrene on the developing immune
system. No studies were located that examined immune system endpoints following inhalation
exposure of animals to benzo[a]pyrene.
Thym us Effects
Decreased thymus weights (up to 62% compared to controls) were observed in male and
female Wistar rats exposed by gavage to 10-90 mg/kg-day benzo[a]pyrene for 35 or 90 days
fKroese etal.. 2001: De long etal.. 19991. This effect may be due to thymic atrophy. The incidence
of slight thymic atrophy was increased in males (6/10) and females (3/10) at a dose of
30 mg/kg-day in a 90-day study, although there was no evidence of atrophy at any lower dose
(Kroese etal.. 20011. Additionally, at the highest dose tested (90 mg/kg-day) in one of the 3 5-day
studies, the relative cortex surface area of the thymus and thymic medullar weight were
significantly reduced (De long et al.. 19991. Other histopathological changes in the thymus
(increased incidence of brown pigmentation of red pulp; hemosiderin) were observed in Wistar
rats of both sexes at 50 mg/kg-day in a 35-day study; however, this tissue was not examined in
intermediate-dose groups (Kroese etal.. 20011. Consistent with the effects observed in these
studies, decreased thymus weights and reduced thymic cellularity were observed in i.p. injection
studies that exposed mice to doses ranging from 50 to 150 mg/kg in utero (Holladav and Smith.
1995.1994: Urso and Tohnson. 19881.
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Toxicological Review of Benzo[a]pyrene
Spleen Effects
Reduced splenic cellularity, indicated by decreased relative and absolute number of B cells
in the spleen (decreased up to 41 and 61% compared to controls, respectively) and decreased
absolute number of splenic cells (31% decrease at the highest dose), was observed in a subchronic
study in male Wistar rats administered 3-90 mg/kg-day benzo[a]pyrene by gavage for 35 days fDe
Tongetal.. 1999). While the effect on the relative number of B cells was dose-related, the lower
doses did not affect the number of B cells or the absolute splenic cell number. The reduced splenic
cell count at the highest dose was attributed by the study authors to the decreased B cells, and
suggests a possible selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow.
The spleen effects observed in De Tongetal. f!9991 are supported by observations of reduced
spleen cellularity and decreased spleen weights following i.p. injection or in utero benzo[a]pyrene
exposure to doses ranging from 50 to 150 mg/kg fHolladav and Smith. 1995: Urso etal.. 19881.
In addition to physical effects on the spleen, several studies have demonstrated functional
suppression of the spleen following benzo[a]pyrene exposure. Dose-related decreases in sheep red
blood cell (SRBC) specific serum IgM levels after SRBC challenge were reported in rats (10 or
40 mg/kg-day) and mice (5, 20,or 40 mg/kg-day) following s.c. injection of benzo[a]pyrene for
14 days fTemple etal.. 19931. Similarly, reduced spleen cell responses, including decreased
numbers of plaque forming cells and reduced splenic phagocytosis to SRBC and lipopolysaccharide
challenge, were observed in B6C3Fi mice exposed to doses >40 mg/kg-day benzo[a]pyrene by i.p.
or s.c. injection for 4-14 days (Lvte and Bick. 1985: Dean etal.. 1983: Munson and White. 1983) or
by intratracheal instillation for 7 days (Schnizlein etal.. 1987).
Immunoglobulin Alterations
Alterations in immunoglobulin levels have been associated with exposure to PAH mixtures
in a limited number of human studies. Some occupational studies have reported evidence of
immunosuppression following PAH exposure. For example, reductions in serum IgM and/or IgA
titers were reported in coke oven workers (Wu etal.. 2003b: Szczeklik et al.. 1994). Conversely,
immunostimulation of immunoglobulin levels has also been observed in humans, specifically
elevated IgG (Karakava etal.. 1999) and elevated IgE (Wu etal.. 2003b) following occupational PAH
exposure.
Decreases in serum IgM (13-33% compared to controls) and IgA levels (22-61% compared
to controls) were observed in male Wistar rats exposed to 3-90 mg/kg-day benzo[a]pyrene by
gavage for 35 days (De long et al.. 1999): however, these reductions were consistent, but not dose-
dependent Similarly, reductions in IgA (9-38% compared to controls) were also observed in male
and female B6C3Fi mice exposed to doses of 5-40 mg/kg benzo[a]pyrene by s.c. injection for
14 days (Munson and White. 1983). Reductions in serum IgG levels of 18-24%, although not
statistically significant, were observed in female B6C3Fi mice exposed to doses >50 mg/kg
benzo[a]pyrene by i.p. injection for 14 days (Dean etal.. 1983).
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Toxicological Review of Benzo[a]pyrene
Hematological Alterations
Altered hematological parameters, including decreases in red blood cell (RBC) count,
hemoglobin, and hematocrit have been observed in laboratory animals following benzo[a]pyrene
exposure (Table 1-9). Statistically significant decreases in RBC count, hemoglobin, and hematocrit
were observed in male Wistar rats at doses >10 mg/kg-day for 35 days fDe long et al.. 19991. A
minimal, but statistically significant, increase in mean cell volume and a decrease in mean cell
hemoglobin were observed at the highest dose (90 mg/kg-day), which may indicate dose-related
toxicity for the RBCs and/or RBC precursors in the bone marrow (De long et al.. 1999). Similarly,
male and female F344 rats also showed maximal decreases in RBC counts, hematocrit, and
hemoglobin levels between 10 and 12% in a 90-day dietary study fKnuckles etal.. 20011. Findings
were significant for RBC counts and hematocrit in males at >50 mg/kg-day, while decreased RBC
counts and hematocrit in females and hemoglobin levels in both sexes were only significant in the
100 mg/kg-day group (Knuckles etal.. 2001). Small, but not statistically significant, decreases in
RBC counts and hemoglobin were observed in both 35- and 90-day studies in Wistar rats (Kroese et
al.. 2001). It should be noted that when observed, the magnitudes of the decreases in RBCs,
hemoglobin, and hematocrit were generally small, about 18% at 90 mg/kg-day and <10% at lower
doses in Wistar rats fDe long et al.. 19991 and about 10% in F344 rats fKnuckles etal.. 20011. A
decrease in white blood cells (WBCs), attributed to reduced numbers of lymphocytes and
eosinophils, was also observed at 90 mg/kg-day following gavage exposure for 35 days (De Tonget
al.. 19991.
Immune Suppression and Sensitization
Some occupational studies of coke oven emissions have reported evidence of
immunosuppression following PAH exposure. Reduced mitogenic responses in T cells (Winker et
al.. 19971 and reduced T-lymphocyte proliferative responses fKarakava et al.. 20041 have been
observed following occupational exposure to PAH. Increased levels of apoptosis were observed in
the peripheral blood mononuclear cells (a population of lymphocytes and monocytes) of
occupationally exposed coke oven workers, which is a response that may contribute to
immunodeficiency in this population (Zhang etal.. 2012). However, a limitation of this study is that
it does not attribute the proportion of apoptotic activity to a specific class of cells and does not
include assessment of other potential markers of immunotoxicity in peripheral blood.
Results of functional immune assays in laboratory animals following short-term i.p. and s.c.
exposures add to the evidence for benzo[a]pyrene immunotoxicity. Resistance to Streptococcus
pneumonia or Herpes simplex type 2 was dose dependently reduced in B6C3Fi mice following s.c.
injection of >5 mg/kg-day benzo[a]pyrene for 14 days (Munson et al.. 1985). Reduced cell
proliferation, IFN-y release, and IL-4 release were observed in male and female C56BL/6 mice
following short-term exposure to a gavage dose of 13 mg/kg benzo[a]pyrene as measured in a
modified local lymph node assay (van den Berg et al.. 2005). A statistically significant decrease in
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Toxicological Review of Benzo[a]pyrene
natural killer (NK) cell activity was observed in male Wistar rats (Effector:Target cell ratio was
40.9 ± 28.4% that of controls) exposed to 90 mg/kg-day by gavage for 35 days (De Tongetal..
19991: however, splenic NK cell activity was not affected in B6C3Fi mice after s.c. injection of
40 mg/kg-day benzo[a]pyrene for 14 days (Munson etal.. 19851. The magnitude of the dose and
duration of the exposure may account for the discrepancy between these two studies. Single i.p.
injections of 50 mg/kg benzo[a]pyrene decreased pro- and/or pre-B-lymphocytes and neutrophils
in the bone marrow of C57BL/6J mice without affecting the numbers of immature and mature
B-lymphocytes or GR-1+ myeloid cells (Galvan etal.. 20061.
In contrast to studies that have shown immunosuppression, benzo[a]pyrene may also
induce sensitization responses. Epicutaneous abdominal application of 100 [igbenzo[a]pyrene to
C3H/HeN mice, followed by ear challenge with 20 [ig benzo[a]pyrene 5 days later, produced a
contact hypersensitivity (a significant ear swelling) response fKlemme etal.. 19871.
Developmental Immunotoxicity
No evidence of an association between benzo[a]pyrene exposure in utero and allergic
sensitization was found in the one available human study of developmental immunotoxicity that
provided data specific to benzo[a]pyrene fledrvchowski etal.. 20111. This birth cohort study found
no statistically significant difference in maternal cord blood levels of benzo[a]pyrene-DNA adducts
between 5-year-old children with or without dermal atopy to one of four common allergens, and no
statistically significant association when the data were analyzed using a logistic regression model
that adjusted for children's gender, maternal age, maternal education, maternal atopy, and ETS
(ledrvchowski etal.. 20111 (T able 1-8). In a New York City birth cohort study, statistically
significant associations were found between serum levels of cockroach IgE (an indicator of
hypersensitivity) in 5- and 9-year-old children and high urinary levels of metabolites of three PAHs
(naphthalene, phenanthrene, and pyrene), but this study did not examine exposure metrics specific
to benzo[a]pyrene flung etal.. 20151. Another birth cohort study found an association between
increased methylation of promoter regions of the interferon gene (IFNy, an important gene in the
etiology of allergic asthma) in DNA from cord WBCs and personal air measures of maternal
exposure to several carcinogenic PAHs including benzo[a]pyrene, but possible associations with
benzo[a]pyrene exposure measures alone were not evaluated (Tang etal.. 20121.
As noted above, several i.p. injection studies suggest that cell-mediated and humoral
immunity may be altered by exposure to high doses of benzo[a]pyrene during gestation.
Suppression of the mixed lymphocyte response, the graft-versus-host response, and suppression of
the plaque-forming cell response to SRBCs was observed in mice exposed in utero to 150 mg/kg
during mid (GDs 11-13), late (GDs 16-18), or both (GDs 11-17) stages of gestation; these effects
persisted until 18 months of age (Urso and Gengozian. 1984.1982.19801. Fetal thymic atrophy, as
assessed by reductions in cellularity (74-95%, compared to controls), was observed in mice
exposed to 50-150 mg/kg benzo[a]pyrene from GD 13 to 17, when examined on GD 18 (Holladav
and Smith. 19941. Analysis of cell surface markers (e.g., CD4, CD8) from the same study indicate
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Toxicological Review of Benzo[a]pyrene
thatbenzo[a]pyrene may inhibit and/or delay thymocyte maturation, possibly contributing to the
observed thymic atrophy (Holladav and Smith. 19941. Consistent with these findings, several other
studies have noted decreased thymocyte numbers and disrupted T cell maturation after in utero
exposure to benzo[a]pyrene (Rodriguez etal.. 1999: Holladav and Smith. 1995: Lummus and
Henningsen. 1995: Urso etal.. 1992: Urso andTohnson. 19871.
The fetal liver is the primary hematopoietic organ during gestation and a major source of
thymocyte precursors beginning around GD 10 or 11 in mice (Landreth and Dodson. 2005: Penit
andVasseur. 19891. Statistically significant reductions in total cellularity in the fetal liver of 54 and
67% were reported in offspring after i.p. exposures of 50 or 100 mg/kg benzo[a]pyrene,
respectively, to the dams on GDs 13-17 fHolladav and Smith. 19941. The decreased fetal liver
cellularity was accompanied by decreased expression of terminal deoxynucleotidyl transferase and
CD45R cellular markers, which are known to be present in cortical thymocyte progenitors in the
fetal liver (Holladav and Smith. 1994: Fine etal.. 1990: Silverstone etal.. 19761. These data also
suggest that benzo [a]pyrene disrupts liver hematopoiesis during gestation and may interfere with
prolymphoid seeding of the thymus, possibly contributing to thymic atrophy and cell-mediated
immunosuppression. Decreased numbers of CD4+ T-cells have been reported in the spleen of
1-week-old mice following in utero benzo[a]pyrene exposure by i.p. injection to the dams,
demonstrating the potential for downstream effects on T-cell development (Rodriguez etal.. 19991.
The decreased numbers of CD4+ T-cells correspond with observations of decreased proliferation in
the presence of Concanavalin A and a weak response compared to controls in an allogeneic mixed
lymphocyte reaction assay (Urso and Kramer. 20081.
Postnatal exposure to benzo [a]pyrene has also been suggested to cause immune effects.
Dose-dependent decreases in erythrocytes (attributed to reduced bone marrow erythropoiesis), as
well as reduced expression of IL-4 and IFN-y, were observed in the pups of Wistar rats exposed to
0.1-10 mg/kg-day benzo[a]pyrene by s.c. injection for 14 days (Matiasovic etal.. 20081. This
finding suggests thatbenzo[a]pyrene may alter the immune response to infection or vaccination in
developing animals.
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Table 1-8. Evidence pertaining to immune effects of benzo[a]pyrene in
humans
Study design and reference
Results
Jedrvchowski et al. (2011) (Krakow, Poland)
Benzo[a]pyrene adduct levels (/10s nucleotides) in cord blood from
mothers of children with and without dermal atopy at 5 yrs of age
Geometric mean (95% CI)
Atopic (N = 37) 0.23 (0.21-0.24)
Non-atopic (N = 187) 0.21 (0.18-0.25)
Association between benzo[a]pyrene-DNA adducts and atopic status3
RR (95% CI)
Relative risk for atopy 0.12 (0.01-1.85)
aRR calculated from a binary outcome logistic regression model,
adjusted for child's gender, parity, maternal age, maternal education,
maternal atopy, and ETS; positive atopic status defined as positive
skin prick test to at least one tested aeroallergen
Birth cohort (5-yr follow-up)
224 women who delivered full-term babies
between January 2001 and February 2004
(Skin prick testing for four aeroallergens
[Dermatophagiodes pteronyssinus,
Dematophagoides farina, dog hair, cat hair]
in children at 5 yrs of age)
Exposure: Benzo[a]pyrene-DNA adducts in
cord blood samples; geometric mean
0.22 (95% CI 0.21-0.24) adducts/
10"8 nucleotides; DNA adducts with
benzo[a]pyrene tetraols were determined
with an HPLC fluorometric assay
Table 1-9. Evidence pertaining to the immune effects of benzo[a]pyrene in
animals after oral or inhalation exposure
Reference and study design
Results3
Thymus effects
Kroese et al. (2001)
Wistar rats, 10/sex/dose
0, 3,10, or 30 mg/kg-d by gavage 5 d/wk
90 d
•i, thymus weight
Females (% change from control): 0, -3, -6, and
-28*
Males (% change from control): 0, 0, -13, and -29*
1" slight thymic atrophy
Females (incidence): 0/10, 0/10, 0/10, and 3/10
Males (incidence): 0/10, 2/10, 1/10, and 6/10*
De Jong et al. (1999)
Wistar rats, 8 males/dose
0, 3,10, 30, or 90 mg/kg-d by gavage 5 d/wk
35 d
•i, thymus weight
% change from control: 0, -9, -15*, -25*, and -62*
Kroese et al. (2001)
Wistar rats, 10/sex/dose
0,1.5, 5,15, or 50 mg/kg-d by gavage 5 d/wk
35 d
•i, thymus weight
Females (% change from control): 0,13, 8, -3, and
-17*
Males (% change from control): 0, -8, -11, -27*,
and -33*
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Toxicological Review of Benzo[a]pyrene
Reference and study design
Results3
Spleen effects
De Jong et al. (1999)
Wistar rats, 8 males/dose
0, 3,10, 30, or 90 mg/kg-d by gavage 5 d/wk
35 d
4/ relative number (%) of B cells in spleen
% change from control: 0, -8, -13*, -18*, and -41*
4/ total number of B cells in spleen
% change from control: 0,13, -13, -13, and -61*
Change in total cell number in the spleen
% change from control: 0, 20, 0, +7, and -31*
Immunoglobulin alterations
De Jong et al. (1999)
Wistar rats, 8 males/dose
0, 3,10, 30, or 90 mg/kg-d by gavage 5 d/wk
35 d
4/ serum IgM
% change from control: 0, -13, -14, -33*, and -19
4/ serum IgA
% change from control: 0, -27, -22, -28, and -61*
Hematological alterations
Kroese et al. (2001)
Wistar rats, 10/sex/dose
0, 3,10, or 30 mg/kg-d by gavage 5 d/wk
90 d
Wistar rats, 10/sex/dose
0,1.5, 5,15, or 50 mg/kg-d by gavage 5 d/wk for
35 d
RBC count and hemoglobin changes not statistically
significant in males or females at any dose (numerical data
not reported)
RBC count: changes not statistically significant (numerical
data not reported)
Hemoglobin: changes not statistically significant (numerical
data not reported)
Knuckles et al. (2001)
F344 rats, 20/sex/dose
0, 5, 50, or 100 mg/kg-d by diet
90 d
4/ RBC count
Females (% change from control): statistically
significant at 100 mg/kg-d (numerical data not
reported)
Males (% change from control): statistically
significant at 50 and 100 mg/kg-d (numerical data
not reported)
4/ hematocrit
Females (% change from control): statistically
significant at 100 mg/kg-d (numerical data not
reported)
Males (% change from control): statistically
significant at 50 and 100 mg/kg-d (numerical data
not reported)
4/ hemoglobin
Females: statistically significant at 100 mg/kg-d
(numerical data not reported)
Males: statistically significant at 100 mg/kg-d
(numerical data not reported)
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Reference and study design
Results3
De Jong et al. (1999)
Wistar rats, 8 males/dose
0, 3,10, 30, or 90 mg/kg-d by gavage 5 d/wk
35 d
4/ RBC count
% change from control: 0, -1, -5*, -10*, and -18*
4/ hemoglobin
% change from control: 0, -1, -7*, -10*, and -18*
4/ hematocrit
% change from control: 0, 0, -6*, -8*, and -14*
4/ WBC count
% change from control: 0, -8, -9, -9, and -43*
1" mean cell volume
% change from control: 0, 0, -3, 0, and 3*
4/ mean corpuscular hemoglobin concentration
% change from control: 0, -1, -1, -1, and -3*
^Statistically significantly different from the control (p < 0.05).
a% change from control calculated as: (treated value - control value)/control value x 100.
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Toxicological Review of Benzo[a]pyrene
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Figure 1-6. Exposure-response array for immune effects following oral
exposure.
Mode-of-Action Analysis—Immune Effects
Exposure to benzo[a]pyrene induces immunosuppressive effects such as decreased
numbers of B cells in the spleen and decreased thymus weight and cellularity following oral, i.p.,
s.c., or intratracheal exposure in experimental animals. The key events underlying benzo[a]pyrene
immunotoxicity have not been determined definitively but likely involve AhR activation and
metabolism, as well as genotoxicity, mutagenicity, cytotoxicity, and apoptosis.
Benzo[a]pyrene is well established as a genotoxic agent (see Section 1.1.5) and
benzo[a]pyrene-DNA adducts are routinely detected in WBCs of humans occupationally exposed to
PAH mixtures (see Table 1-20). Increased apoptosis has been detected peripheral blood
mononuclear cells of coke oven workers highly exposed to PAH mixtures (Zhang etal.. 2012). It is
therefore likely that exposure can result in cellular damage, cell death, and mutations in immune
cell populations, potentially altering immune function.
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Benzo[a]pyrene is also a well-known ligand for the AhR in animal models and human cell
lines fAllan and Sherr. 2005: Okev etal.. 1994: Nebertetal.. 1993: Postlind etal.. 1993: Freedman et
al.. 19791. Ligands of the AhR have been shown to have a role in regulating hematopoietic stem
cells in the bone marrow, a major site of B cell proliferation and antibody production (Esser. 20091.
Benzo[a]pyrene impaired cell expansion and differentiation in a human hematopoietic progenitor
cell line, however, this toxicity was reversed with an AhR antagonist (van Grevenvnghe etal.. 20061.
Benzo[a]pyrene reduced B cell lymphopoiesis in mice at concentrations as low as lOnM (Hardinet
al.. 19921. Furthermore, Ah-responsive (C57BL/6) mice showed greater dose-dependent
reductions in B cell lymphopoiesis than those observed in Ah-nonresponsive (DBA/2) mice (Hardin
etal.. 19921. Addition of the AhR antagonist and CYP450 inhibitor, a-naphthaflavone, inhibited the
benzo[a]pyrene-induced suppression of B cell lymphopoiesis in a concentration-dependent fashion.
Similarly, the CYP1A1 inhibitor, l-(l-propynyl)pyrene, blocked benzo[a]pyrene-induced B cell
growth inhibition but not growth inhibition caused by the benzo[a]pyrene metabolite, BPDE; these
data suggest thata CYP1A1-dependent metabolite of benzo[a]pyrene is responsible for the B cell
growth suppressive effects observed after benzo[a]pyrene exposure fAllan etal.. 20061.
T cells also appear to be similarly sensitive to benzo[a]pyrene. In vitro assays in human
peripheral blood T cell indicate that low concentrations of benzo[a]pyrene (10-100 nM) suppress T
cell mitogenesis (using phytohemagglutinin or concanavalin A) and that the mechanism involves
AhR and P450 related processes (Davila etal.. 1996: Mudzinski. 19931.
Altogether, these data suggest that benzo[a]pyrene may depress B and T cell proliferation
via the AhR and metabolism of benzo[a]pyrene to reactive metabolites.
Summary of Immune Effects
Evidence for immunotoxic effects of benzo[a]pyrene exposure comes from animal studies
that vary in route and duration of exposure. There are no human epidemiological studies that
provide specific support for benzo[a]pyrene immunotoxicity; however, immunosuppression has
been observed in studies following occupational exposure to PAH mixtures. However, these
findings are limited by co-exposures to other constituents of PAH mixtures.
Effects such as altered thymus weight and histology, spleen effects, and altered
immunoglobulin levels observed by the oral route reported in animal bioassays provide some
evidence of immunotoxicity following benzo[a]pyrene exposure; however, in vivo functional assays
provide stronger support for immunotoxicity (WHO. 20121. The immunological changes observed
in the available subchronic gavage studies are supported by a larger database of in vivo studies of
benzo[a]pyrene (by parenteral exposure) indicating functional immunosuppression such as
decreased proliferative responses to antigens and decreased resistance to pathogens or tumor cells
fKongetal.. 1994: Blanton etal.. 1986: Munsonetal.. 1985: White etal.. 1985: Dean etal.. 1983:
Munson and White. 19831.
Few studies are available to inform the effects of benzo[a]pyrene on the developing immune
system. In particular, there is a lack of studies evaluating functional changes in the immune system
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Toxicological Review of Benzo[a]pyrene
following developmental exposure, especially following oral or inhalation exposures. An additional
data gap includes the lack of studies that treated offspring postnatally, up to approximately PND 45,
when the immune system continues to develop (Burns-Naas etal.. 20081. However, the available
i.p. exposure studies of gestationally and early postnatally treated animals provide a strong
indication of potential developmental immunotoxicity and suggest the need for further study.
Although the key events underlying the mode of action of benzo[a]pyrene immunotoxicity
are not firmly established, there is evidence of physical alterations to tissues/organs of the immune
system, as well as decreases in immune function. Evidence of benzo[a]pyrene-associated
immunotoxicity is supported by consistent thymic effects observed in two oral studies, as well as
splenic effects, and varying immunosuppressive responses observed in short-term or in vitro tests.
EPA concluded there was suggestive evidence that immunotoxicity is a potential human
hazard of benzo[a]pyrene exposure.
Susceptible Populations and Lifestages
The severity and persistence of immune effects observed during in utero studies suggest
that immunotoxicity may be greater during early life than adulthood fDietert and Piepenbrink.
2006: Holladav and Smialowicz. 2000: Urso and Gengozian. 19821. Urso and Gengozian (19821
provide experimental support demonstrating that immunosuppression from benzo[a]pyrene
exposure during gestation was greater than for mice exposed after birth to a 2 5-fold higher dose.
There is also substantial literature indicating that disruption of the immune system during certain
critical periods of development (e.g., initiation of hematopoiesis, migration of stem cells, expansion
of progenitor cells) may have significant and lasting impacts on lifetime immune function (e.g.,
Burns-Naas etal.. 2008: Dietert. 2008: Landreth. 2002: Dietert etal.. 20001. In addition, chemical-
specific studies show increased dose sensitivity and disease persistence from developmental versus
adult chemical exposure (reviewed in Luebke et al.. 20061.
Thymus toxicity is a sensitive and specific effect of benzo[a]pyrene and has been observed
in both prenatal and adult exposure studies. The thymus serves as a major site of thymocyte
proliferation and selection for maturation, and impairment can lead to cell-mediated immune
suppression fKuper etal.. 2002: De Waal etal.. 1997: Kuper etal.. 19921. The thymus is believed to
be critical for T lymphocyte production during early life and not in adulthood f Hakim etal.. 2005:
Schonland etal.. 2003: Petrie. 2002: Mackall etal.. 19951. Therefore, the decreases in thymus
weight observed in studies of adult animals exposed to benzo[a]pyrene suggest that
immunosuppression may be a heightened concern for individuals developmentally exposed to
benzo[a]pyrene.
The available studies evaluating immune effects provide limited comparisons for sensitivity
of immune effects of benzo[a]pyrene across species and sexes. Two subchronic oral studies
evaluated immune endpoints in rats fKroese etal.. 2001: De long etal.. 19991: however, no
subchronic or chronic studies were identified in mice by the oral or inhalation route. One study in
rats evaluated immune endpoints in both males and female rats (Kroese etal.. 20011. In this study,
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similar effects were seen in male and female rats, with some indication of effects in the thymus
occurring in males at a slightly lower dose fKroese etal.. 20011.
In addition, few studies are available that directly compare immunotoxicity of
benzo[a]pyrene across species or strains. A study in female F344 rats and B6C3Fi mice noted a
similar degree of immune suppression in rats and mice using the SRBC functional assay after
14-day i.p. exposure to benzo[a]pyrene (Temple etal.. 19931. Another study evaluated
benzo[a]pyrene in several in vitro assays for immunotoxicity in human and rodent cells. The
endpoints assessed included cytoxicity, cytokine release, myelotoxicity, and mitogen
responsiveness. The authors reported that benzo[a]pyrene immunotoxicity was generally similar
in mice, rats, and human cells with IC50 values in the same range (Carfi' etal.. 20071.
1.1.4. Other Toxicity
There is some evidence thatbenzo[a]pyrene can produce noncancer effects in the liver,
kidney, cardiovascular system, and nervous system in animals exposed as adults (effects on these
organ systems following developmental exposure are discussed as part of Section 1.1.1,
Developmental Toxicity). However, there is less robust and consistent evidence for these effects as
compared to organ systems described earlier in Sections 1.1.1-1.1.3. Therefore, atthis time, no
conclusions are drawn regarding these effects as human hazards of benzo[a]pyrene exposure. A
brief overview of the evidence pertaining to these organ systems, with particular focus on findings
from subchronic or chronic oral and inhalation exposures, is included below.
Liver Effects
Liver effects other than cancer associated with benzo[a]pyrene exposure primarily include
changes in liver weight (Table 1-10). Increased liver weight was reported in a 90-day study in both
male and female Wistar rats givenbenzo[a]pyrene by gavage (Kroese etal.. 20011. Both females
(17% increase) and males (29% increase) demonstrated statistically significant increased liver
weights at the highest dose tested (30 mg/kg-day); a statistically significant increase (15%) was
also reported in males at 10 mg/kg-day. Similar to the findings in the 90-day study by Kroese et al.
f20011. increased liver:body weight ratios were observed at the highest dose in a 90-day dietary
study in male F344 rats, although there was no change observed in female liver weights (Knuckles
etal.. 20011. Increased liver:body weight ratios were also observed in both sexes at high doses
(600 and 1,000 mg/kg) in an accompanying acute study (Knuckles etal.. 20011. A statistically
significant increase in liver weight was also observed in male Wistar rats given 90 mg/kg-day
benzo[a]pyrene by gavage for 35 days fDe long et al.. 19991. Consistent with the findings by De
long etal. f 19991. a statistically significant increased liver weight (about 18%) was also observed in
both male and female Wistar rats at the highest dose (50 mg/kg-day) given by gavage in a 35-day
study fKroese etal.. 20011.
Limited exposure-related differences in clinical chemistry parameters associated with liver
toxicity were observed; no differences in alanine aminotransferase or serum aspartate
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transaminase (AST) levels were observed, and a small dose-related decrease iny-glutamyl
transferase was observed in males only exposed to benzo[a]pyrene for 90 days fKroese etal..
20011.
Treatment-related lesions in the liver (oval cell hyperplasia) were identified as statistically
significantly increased following exposure to 90 mg/kg-day benzo[a]pyrene for 35 days; however,
incidence data were not reported (De long et al.. 1999). A 2-year carcinogenicity study (Kroese et
al.. 20011 observed some histopathological changes in the liver; however, organs with tumors were
not evaluated. Since many of the animals in the highest two doses developed liver tumors, the dose
responsiveness of the histological changes is unclear.
A dose-dependent increase in liver microsomal ethoxyresorufin-o-deethylase (EROD)
activity, indicative of CYP1A1 induction, was observed in both sexes at doses >1.5 mg/kg-day in a
35-day study (Kroese etal.. 2001). However, at the highest dose tested, with the greatest fold
induction in EROD activity, there was no evidence of associated adverse histopathologic findings.
Overall, increased liver weight was reported across a few studies of varying exposure durations
providing some evidence of the liver as a target of benzo[a]pyrene exposure; however, these
changes in liver weight do not appear to be substantially supported by histological findings or other
indicators of hepatoxicity. Therefore, at this time, no conclusion is drawn regarding liver toxicity as
a human hazard of benzo[a]pyrene exposure.
Kidney Effects
There is minimal evidence of kidney toxicity following exposure to benzo[a]pyrene
(Table 1-10). Statistically significant decreases in kidney weight were observed at doses of 3, 30,
and 90 mg/kg-day, but not 10 mg/kg-day, in a 35-day gavage study in male Wistar rats fDe long et
al.. 1999). In a 35-day gavage study with a similar dose range in male and female Wistar rats, no
statistically significant changes in kidney weights were observed at any dose (Kroese etal.. 2001).
Histopathological analysis of kidney lesions revealed an apparent dose-responsive increase in the
incidence of abnormal tubular casts in the kidney in male F344 rats exposed by diet for 90 days
fKnuckles etal.. 20011. The casts were described as molds of distal nephrons lumen and were
considered by the study authors to be indicative of renal dysfunction. However, the statistical
significance of the kidney lesions is unclear. Several gaps and inconsistencies in the reporting make
interpretation of the kidney effects difficult, including: (1) no reporting of numerical data; (2) no
indication of statistical significance in the accompanying figure for kidney lesions; (3) discrepancies
between the apparent incidences and sample sizes per dose group; and (4) uncertainty in how
statistical analysis of histopathological data was applied. As such, the significance of the abnormal
tubular casts is unclear. One study indicated that gestational exposure may result in changes in
kidney histology and function in mice with a highly inducible AhR (C57BL/6), but not in another
strain of mice (D2N) with a less inducible AhR fNanez etal.. 20111. At 52 weeks, offspring of
C57BL/6 dams treated with 0.1 or 0.5 mg/kgbenzo[a]pyrene on GDs 10-13 were found to have a
decreased number of podocytes per glomeruli (at both doses) and an increase in urine albumin in
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the high-dose group. Another study in which adult female rats were treated with 10 mg/kg
benzo[a]pyrene by i.p. exposure once a week for 16 weeks demonstrated changes in clinical
indicators consistent with kidney toxicity including elevations in urinary protein, protein/
creatinine ratios, and microalbumin (Nanez etal.. 20051.
Overall, few studies by environmentally relevant routes of exposure are available to inform
the potential of kidney effects after subchronic or chronic exposure to benzo[a]pyrene; thus, at this
time, no conclusion is drawn regarding kidney toxicity as a human hazard of benzo[a]pyrene
exposure.
Cardiovascular Effects
Several studies of cardiovascular effects in populations highly exposed to benzo[a]pyrene as
a component of a complex PAH mixtures are available; however, it is difficult to attribute effects of
these exposures to any one component of the mixture. Very limited information is available that
evaluates the potential cardiovascular toxicity from subchronic or chronic exposure to
benzo[a]pyrene in animal models. Numerous short-term exposure studies, studies by less
environmentally relevant routes of exposure (e.g., injection, instillation), and in vitro studies were
identified in the literature, and while these studies may be useful in understanding potential
mechanisms of toxicity, the ability of these data to predict chronic health effects is uncertain.
Atherosclerotic vascular disease and increased risk of cardiovascular mortality have been
associated with cigarette smoking fRamos and Moorthv. 2005: Miller and Ramos. 2001: Thirman et
al.. 19941 and, to a more limited degree, occupational exposure to PAH mixtures fFriesen etal..
2010: Friesen etal.. 2009: Burstvn et al.. 2 0 0 5: Chau etal.. 19931. Elevated mortality due to
cardiovascular disease was observed in a PAH-exposed occupational population (coke oven plant
workers), but elevated cardiovascular mortality was also observed in the non-exposed or slightly
exposed populations (Chau etal.. 19931. Elevated risks of ischemic heart disease (IHD) were
associated with past cumulative benzo[a]pyrene exposure among aluminum smelter workers (with
a 5-year lag), although the trend was not statistically significant; there was no observed association
with more recent benzo[a]pyrene exposure fFriesen etal.. 20101. Elevated risk of mortality from
IHD was also associated with cumulative benzo[a]pyrene exposure in a cohort of male asphalt
workers (although not statistically significant); the trend in average benzo[a]pyrene exposure and
association with IHD was statistically significant, with an approximately 60% increase in risk
between the lowest and highest exposure groups (Burstvn et al.. 20051. The two studies that
associate benzo[a]pyrene exposure with cardiovascular effects fFriesen etal.. 2010: Burstvn etal..
20051 relied on statistical models to create exposure groups rather than direct measurement of the
cohort under examination. Additionally, while these studies used benzo[a]pyrene exposure
groupings for analysis, they cannot address co-exposures that may have occurred in the
occupational setting (asphalt or aluminum smelters) or exposures that occurred outside the
workplace.
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Data on cardiovascular effects of chronic or subchronic benzo[a]pyrene exposure in adult
WT laboratory animals exposed by environmentally-relevant exposure routes (oral, inhalation, or
dermal) are limited. In C57BL/6J mice fed benzo[a]pyrene at an approximate dose of
12.5 mg/kg-day for 15 weeks in conjunction with an atherogenic diet (high in fat and cholesterol),
"modest" atherosclerotic lesions (not further quantified or described, but apparently increased
relative to controls) were seen in the aorta (Uno etal.. 2014). In a shorter duration study, rats
treated by gavage once per week for 2 weeks with 0.05 mg/kg benzo[a]pyrene exhibited
significantly higher serum LDL-cholesterol than controls; serum HDL-cholesterol and
phospholipids were not affected fChung and Tung. 20031.
In a developmental study, increased systolic and diastolic blood pressure was observed in
the offspring of dams exposed to increasing concentrations of benzo[a]pyrene (Tules etal.. 2012)
(see Section 1.1.1, Developmental Toxicity and Table 1-1). At the highest dose tested (1.2 mg/kg
body weight by gavage to the dams), systolic pressures were elevated approximately 50% and
diastolic pressures were elevated approximately 80% above controls. An intranasal exposure of
0.01 mg/kg-day benzo[a]pyrene in adult male rats also produced an increase in blood pressure
following a 7-day exposure fGentner and Weber. 20111.
Few in vivo evaluations of the effect of subchronic or chronic benzo[a]pyrene exposure on
the development of atherosclerosis have been conducted in WT animals, by environmentally
relevant routes of exposure. However, several studies by other routes of exposure and in
genetically predisposed animal models (e.g., Apo E-/- mice) are available. ApoE-/- mice develop
spontaneous atherosclerosis, which is thought to be due to enhanced oxidative stress from the lack
of ApoE, and appears to be enhanced by benzo[a]pyrene exposure and greatly impacted by AhR
binding affinity. Oral exposure to benzo[a]pyrene lead to a 2.5-fold increase over controls in
ethenoDNA adducts (stable biomarkers of oxidative stress) in the aortas of ApoE null mice
(Godschalk etal.. 2003). In a study comparing mice of different AhR inducibility, ApoE-/-mice
expressing the high affinity AhR gene (C57BL/6J) had more aortic segments with plaque and more
extensive plaque area compared with mice expressing low affinity AhR (B6.D2N-v4/jrd/J) after
10 weeks of exposure to 10 mg/kg-day benzo[a]pyrene by gavage fKerley-Hamilton etal.. 20121.
Evidence for the important role of oxidative stress in benzo[a]pyrene-induced atherosclerosis
comes from a study fYang et al.. 20121 comparing cardiovascular effects in ApoE knock-out mice
with normal expression of scavengers (superoxide dismutase [SOD] and catalase) with those
overexpressing human copper/zinc SOD or catalase. Overexpression of either scavenger
significantly inhibited benzo[a]pyrene-induced increases in the area and morphology of
atherosclerotic lesions, aortic lipid peroxidation, measures of reactive oxygen species (ROS), and
endothelial expression of adhesion molecules (VCAM-1 and ICAM-1). The available studies suggest
thatbenzo[a]pyrene exposure in ApoE-/- mice enhances the progression of atherosclerosis
through a general local inflammatory process.
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In an 8-week i.p. study, reduced endothelial integrity and increased smooth muscle cell
mass, both related to atherosclerosis, have been observed in Sprague-Dawley rats exposed to
10 mg/kgbenzo[a]pyrene by i.p. injection (once/week) (Zhang and Ramos. 19971.
A large body of mechanistic data exists for various cardiovascular endpoints in vitro and in
mammalian and non-mammalian animal models. Available mechanistic evidence suggests that
benzo[a]pyrene may influence cardiovascular endpoints through several parallel or interrelated
mechanisms related to AhR activation. These candidate mechanisms include stimulation of
oxidative stress/DNA adducts fYang et al.. 2009: Curfs etal.. 2005: Curfs etal.. 2004: Godschalk et
al.. 2003: Izzotti etal.. 19941. induction of pro-inflammatory genes and genes linked to cardiac
hypertrophy (Huang etal.. 2014: Gan etal.. 2012: Ichihara etal.. 2009: Knaapen etal.. 2007: Curfs et
al.. 2005: Curfs etal.. 2004: Das etal.. 19851. alterations in the metabolism of arachidonic acid to
vasoactive eicosanoids (Aboutabl et al.. 2 011: Aboutabl etal.. 2009: Bugiak and Weber. 20091. and
modulation of myocyte or endothelial cell intracellular calcium via interaction with (3-adrenergic
receptors fMavati etal.. 2012a: Mavatietal.. 2012b: Irigarav et al.. 20061.
While some evidence suggests cardiovascular effects in highly PAH exposed populations
and a large body of animal studies suggests potential mechanisms for cardiovascular effects, issues
of co-exposure in human studies, as well as the lack of experimental animal studies examining
cardiovascular endpoints in WT laboratory animals exposed by environmentally relevant routes for
subchronic or chronic durations, make it difficult to characterize the human hazard from exposure
to benzo[a]pyrene. Overall, the short-duration studies and studies by other routes of exposure (e.g.,
i.p. and intratracheal instillation), as well as studies in genetically modified, highly susceptible
animal strains (e.g., APOE-/- mice) provide suggestive evidence of cardiovascular toxicity
associated with benzo[a]pyrene exposure.
Nervous System Effects following Adult Exposure
Note: The evidence for nervous system effects following exposure during development are evaluated
under Section 1.1.1, Developmental Toxicity.
Two studies of nervous system effects in occupational populations highly exposed to
benzo[a]pyrene as a component of a complex PAH mixtures are available fOiu etal.. 2013: Niu etal..
20101: however, it is difficult to attribute effects of these exposures to any one component of the
mixture. No chronic exposure studies in animal models are available that evaluate nervous system
effects following benzo [a]exposure. However, several shorter duration oral studies are available in
rats and mice (see Table 1-11). Supplemental information is provided by several i.p. studies. While
these studies may be somewhat informative for hazard, the ability of these data to predict chronic
health effects by environmentally relevant routes of exposure is uncertain.
Neurobehavioral function and mood state were evaluated in two studies of men
occupationally exposed to PAH mixtures fOiu et al.. 2013: Niu et al.. 20101. Alterations in
neurobehavioral function were evaluated in coke oven workers using the Neurobehavioral Core
Test Battery (self-reported symptoms by questionnaire). Air concentrations of benzo[a]pyrene and
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urinary levels of the PAH metabolite, 1-hydroxypyrene, were used as markers of PAH exposure. In
both studies, high occupational exposure in coke oven workers compared to controls was
associated with decrements in short-term memory and/or attention in digit span tests. In addition,
Oiu etal. (20131 reported an association between PAH exposure, based on comparisons of coke
oven workers versus controls as well as urinary 1-hydroxypyrene measures, and decrements in
tests related to sensorimotor coordination (i.e., reaction time, digit symbol, and pursuit aiming
tests), as well as lower self-reported health ratings on the profile of mood states questionnaire.
However, Niu etal. f20101 did not detect these associations using the same test battery. When
comparisons were specific to the air benzo[a]pyrene concentrations defined by work location, Oiu
etal. (20131 found an association between increasing benzo[a]pyrene levels and decrements in
reaction time and pursuit aiming, while Niu etal. (20101 observed that increasing levels were
associated with decreased plasma levels of several neurotransmitters involved in mood, learning,
and/or memory (e.g., norepinephrine, dopamine, 5-hydroxytryptamine, aspartic acid,
y-aminobutyric acid). The extent to which plasma concentrations of neurotransmitters predict
those in the central nervous system is uncertain.
Alterations in neuromuscular, autonomic, sensorimotor, and electrophysiological endpoints
have been reported in adult rats and mice following acute or short-term exposure to
benzo[a]pyrene (Bouavedetal.. 2009b: Grovaetal.. 2008: Grovaetal.. 2007: Saunders etal.. 2006:
Liu etal.. 2002: Saunders etal.. 2002: Saunders etal.. 20011. Impaired performance in tests of
learning and memory (i.e., Morris water maze or novel object recognition) was observed following
subchronic gavage exposure to 2 mg/kg-day benzo[a]pyrene in adult rats fMaciel etal.. 2014: Chen
etal.. 2011: Chengzhi etal.. 20111 and following short-term i.p. exposure in adult mice fOiu etal..
2011: Xia etal.. 2011: Grova etal.. 20071. These findings are somewhat limited, as the oral
exposure studies in rats were conducted with only a single dose group. Further, the results of the
novel object recognition test were complicated by a lack of automated recording and blinding, no
evaluation of total activity, and an apparent preference for the novel object when testing long-term
memory 24 hours after training (recognition index was similar to controls) that was not present at
1.5 hours after training fMaciel etal.. 20141.
Tests of anxiety- and activity-related behaviors were difficult to interpret. Decreased
anxiety-like behavior in the elevated plus maze was observed following short-term i.p. exposure to
the high dose of 200 mg/kg-day (a dose at which there was general toxicity), while overall activity
appeared to be increased in hole board testing at >20 mg/kg-day (Grova etal.. 20081. Relatedly,
while spontaneous locomotor activity in a 15-minute trial was increased after oral exposure to
2 mg/kg-day in rats fMaciel etal.. 20141. no changes were observed during a 5-minute activity trial
in mice orally exposed to 0.02-20 mg/kg-day benzo[a]pyrene fBouaved etal.. 20121.
Effects on depressive-like activity were also mixed: animals orally exposed to 0.02 or
0.2 mg/kg-day for 17 days showed decreased immobility time in the tail suspension test, but no
effect was observed in the two higher doses (2 or 20 mg/kg-day) and changes were not observed at
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any dose in the forced swim test (Bouaved etal.. 20121. In addition, a 28-day gavage study in male
mice observed an increase in consummatory sexual behavior in mice treated with 0.02 and
0.2 mg/kg-day, whereas aggressive behavior (as measured by the resident intruder test) was only
increased in the low-dose group (0.02 mg/kg-day) (Bouaved et al.. 2009b). Interpretation of the
resident intruder data is further complicated because the study authors did not clearly define
attack behavior and failed to indicate whether scorers were blinded to the treatment Effects of oral
benzo[a]pyrene exposure on motor- and sensory-related behaviors in adult rats are supported by a
series of gavage studies testing single, high doses (i.e., >12.5 mg/kg) of benzo[a]pyrene (Saunders
etal.. 2006: Saunders etal.. 2002: Saunders etal.. 20011.
Two studies from the same laboratory reported learning and memory deficits in male
Sprague-Dawley rats exposed to daily i.p. doses of 2.5 mg/kg-day for 13 (Xia etal.. 2011) or
14 weeks (Oiu etal.. 2011: Xia etal.. 2011: Grova etal.. 2007) starting when animals were 5 weeks
old. Notably, the exposure windows for these studies overlapped briefly with pubertal brain
development and, although the bulk of exposure occurred during adulthood, it is difficult to discern
whether the observed effects are developmental or adult in origin. Interestingly, results in the
Morris water maze after subchronic oral fChengzhi etal.. 20111 or i.p. fOiu etal.. 2011: Xia etal..
2011) exposure in "adult" rats identified an effect of exposure across all trial days during hidden
platform testing, indicating lack of an effect on learning and supporting an effect on some other,
unknown behavior(s); short-term i.p. exposure in mice only resulted in increased latencies on trial
day 5 alongside unexplained decreases in latency on day 1 f Grova etal.. 20071. Notably, the
observations from adult rat studies are consistent with apparent effects on noncognitive
behavior(s) in the Morris water maze after developmental exposure (see Section 1.1.1).
Overall, few studies are available to inform the neurotoxic potential of oral or inhalation
exposure to benzo[a]pyrene in adults (Table 1-11). Notably, one subchronic, oral exposure study in
rats (Chengzhi etal.. 2011) indicated effects on Morris water maze performance at 2 mg/kg-day
that were similar to results that have been observed after postnatal oral exposure and i.p. exposure
in weanlings or adult rats. Further, this behavioral alteration is supported by behavioral effects at
2 mg/kg-day in a second oral exposure study in rats fMaciel etal.. 20141. However, two oral
exposure studies in adult mice fBouaved etal.. 2012: Bouaved etal.. 2009bl were difficult to
interpret as supportive evidence, as effects of benzo[a]pyrene exposure on behavior were not
observed at the highest doses tested, namely 0.2 mg/kg-day (Bouaved et al.. 2009b) and
2-20 mg/kg-day (Bouaved etal.. 2012). Similarly, evidence consistent with benzo[a]pyrene
exposure-induced behavioral effects in two studies of male coke oven workers is complicated by co-
exposures to other constituents of PAH mixtures, and the relevance of behavioral effects observed
in several studies of adult rodents following i.p. exposure to oral or inhalation exposure paradigms
is difficult to infer. Thus, while suggestive evidence of neurobehavioral effects following adult
exposure exists, due to the limitations of the oral and inhalation database, a conclusion could not be
drawn regarding the potential for nervous system toxicity to be a human hazard of benzo[a]pyrene
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exposure in adults, and additional studies are warranted. Overall, however, a human neurotoxicity
hazard is identified based on exposure to benzo[a]pyrene during development (see Section 1.1.1).
Table 1-10. Evidence pertaining to liver, kidney, and cardiovascular effects of
benzo[a]pyrene in animals after oral or inhalation exposure
Reference and study design
Results3
Liver effects
Kroese et al. (2001)
Wistar rats, 10/sex/dose
0, 3,10, or 30 mg/kg-d by gavage 5 d/wk
90 d
Wistar rats, 10/sex/dose
0,1.5, 5,15, or 50 mg/kg-d by gavage 5 d/wk for
35 d
1" liver weight
Females (% change from control): 0, -2, 4, and 17*
Males (% change from control): 0, 7,15*, and 29*
Liver histopathology: no effects reported
1" liver weight
Females (% change from control): 0, 3, 2, 9, and 18*
Males (% change from control): 0, 2,1, 3, and 18*
Liver histopathology: no effects reported
Knuckles et al. (2001)
F344 rats, 20/sex/dose
0, 5, 50, or 100 mg/kg-d by diet
90 d
1" liver:body weight ratio
Females: no change (numerical data not reported)
Males (% change from control): 23% change
reported at 100 mg/kg-d (numerical data not
reported)
De Jong et al. (1999)
Wistar rats, 8 males/dose
0, 3,10, 30, or 90 mg/kg-d by gavage 5 d/wk
35 d
1" liver weight
% change from control: 0, -9, 7, 5, and 15*
1" liver oval cell hyperplasia (numerical data not reported)
reported as significant at 90 mg/kg-d
Kidney effects
Knuckles et al. (2001)
F344 rats, 6-8/sex/dose
0, 5, 50, or 100 mg/kg-d by diet
90 d
1" 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, or 90 mg/kg-d by gavage 5 d/wk
35 d
4/ kidney weight
% change from control: 0, -11*, -4, -10*, and -18*
Kroese et al. (2001)
Wistar rats, 10/sex/dose
0,1.5, 5,15, or 50 mg/kg-d by gavage 5 d/wk
35 d
Kidney weight: no change (data not reported)
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Reference and study design
Results3
Cardiovascular effects
Unoetal. (2014)
C57BL/6J mice, male (n not reported)
0 or 12.5 mg/kg-d in diet
(all animals fed an atherogenic diet)
15 wks
"Modest" increase in atherosclerotic lesions were seen in
the aorta (numerical data not reported)
^Statistically significantly different from the control (p < 0.05).
a% change from control calculated as: (treated value - control value)/control value x 100.
Table 1-11. Evidence pertaining to neurotoxicity following repeated oral or
inhalation exposure to benzo[a]pyrene in adult humans and animals
Reference and study design
Results
Human studies
Qiu etal. (2013)
n = 100 male coke oven workers from Chongqing,
China employed for >1 yr, mean age 41 yrs;
100 controls from oxygen plant in same company,
mean age 39 yrs, comparable age, education level,
smoking status, alcohol consumption,
socioeconomic status, and general physical
condition as exposed.
Three air samples (one for controls) of at least 4-hr
duration on successive days, analyzed for
benzo[a]pyrene; control levels: 0.003 ng/m3;
exposed workplace means: 0.028, 0.781, or
2.82 ng/m3 for coke oven bottom (n = 17), side
(n = 34), and top (n = 49) sites, respectively. Post-
shift urine samples analyzed for creatinine-corrected
1-hydroxypyrene; control: 1.89 nmol/mol
creatinine; exposed: 3.61 nmol/mol creatinine.
Outcomes: Neurobehavioral core test battery
(Anger, 2003) including profile of mood states
(POMS) questionnaire, simple reaction time, digit
span, pursuit aiming, digit symbol, visual retention,
and Santa Ana dexterity.
Statistically significant changes (p < 0.05), with direction
and magnitude of change, in exposed compared to control:
POMS scores: tension-anxiety (^26%), fatigue-
inertia (^30%)
Simple reaction time: mean reaction time CM2%)
Digit span: n forward span (4/9%); n total span
(4/6%)
Digit symbol: n correct (4/20%)
Pursuit aiming: n correct (4/10%); n total (4/8%)
Measures reported as not statistically significant:
POMS scores: depression-dejection; anger-hostility
(note: p = 0.087 and ^27%); vigor-activity;
confusion-bewilderment
Digit span: n backward span
Santa Ana dexterity test: n preferred or non-
preferred hand
Pursuit aiming: n errors
Note: Analysis of covariance on neurobehavioral data
stratified by exposure level (coke oven bottom, side, and
top) showed significantly lower performance on simple
reaction time, correct pursuit aiming, and error pursuit
aiming tests with higher levels of airborne benzo[a]pyrene.
Adverse changes in POMS (tension-anxiety, fatigue-inertia,
and confusion-bewilderment), simple reaction time, and
digit span, but not in digit symbol or pursuit aiming, were
significantly associated with higher post-shift urinary
1-hydroxypyrene measures in simple linear regression
analyses.
Niu etal. (2010)
Statistically significant changes (p < 0.05), with direction
and magnitude of change, in exposed compared to control:
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Reference and study design
Results
n = 176 male coke oven workers from Taiyuan,
China employed for >1 yr, mean age 38 yrs;
48 controls from warehouse workers in same
company, mean age 40 yrs; matched by
socioeconomic status, age, education, lifestyle
(smoking and drinking), and health (depression,
family history, and medication)
Air samples of 6-hr duration on three successive
days, analyzed for benzo[a]pyrene; control levels:
0.0102 ng/m3; exposed workplace means: 0.0195,
0.1859, or 1.624 ng/m3 for coke oven bottom, side,
and top sites, respectively. Post-shift urine samples
analyzed for creatinine-corrected 1-hydroxypyrene;
control: 2.77 nmol/mol creatinine; exposed: 3.66
Hmol/mol creatinine.
Outcomes: Neurobehavioral core test battery
(Anger. 2003) including profile of mood states
(POMS) questionnaire, simple reaction time, digit
span, pursuit aiming, digit symbol, visual retention,
and number of dots; plasma levels of
neurotransmitters (norepinephrine, dopamine,
5-hydroxytryptamine, 5-hydroxyindoleacetic acid,
homovanillic acid, y-aminobutyric acid, aspartic acid,
glutamic acid, glycine, and acetylcholine) and
erythrocyte acetylcholinesterase activity.
Digit span: forward span (4/13%); total span
(4/11%)
Measures reported as not statistically significant:
POMS questionnaire (anger-hostility, confusion-
bewilderment, depression-dejection, fatigue-inertia,
tension-anxiety, and vigor-activity); simple reaction
time; digit span: backward span; Santa Ana manual
dexterity; digit symbol; Benton visual retention;
dotting
Note: Statistical analysis methods were not reported;
statistically significant changes (p < 0.05). Stratification of
neurobehavioral data by tertile of creatinine-corrected
urinary 1 hydroxypyrene showed significantly lower
performance on total and forward digit span tests (but no
other tests) with higher levels of urinary 1-hydroxypyrene.
Plasma levels of norepinephrine, dopamine, aspartic acid,
and y-aminobutyric acid were significantly lower in exposed
workers versus controls. Plasma acetylcholine was
significantly increased over controls, and erythrocyte
acetylcholinesterase activity was significantly decreased, in
exposed workers. Stratification by tertile of creatinine-
corrected urinary 1-hydroxypyrene showed significant
negative associations between urinary 1-hydroxypyrene
and plasma norepinephrine, plasma aspartic acid, and
erythrocyte acetylcholinesterase, and a significant positive
association with plasma acetylcholine.
Rodent studies
Chengzhi et al. (2011)
Sprague-Dawley rats, male, 32/dose
0 or 2 mg/kg-d by gavage
90 d
1" time required for treated rats to locate platform in
water maze (data reported graphically) on trial d 1-4
No information regarding swim speed or path length
Bouaved et al. (2009b)
Swiss albino mice, male, 9/dose
0, 0.02, or 0.2 mg/kg-d by gavage
28 d
Significant decrease in latency to attack and increase in the
number of attacks in the resident-intruder test at
0.02 mg/kg-d (but not at high dose)
Significant increase in mount number in the copulatory
behavior test at 0.02 and 0.2 mg/kg-d
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Reference and study design
Results
Bouaved et al. (2009b)
Swiss albino mice, male, 10/dose
0, 0.02, 0.2, 2, or 20 mg/kg-d by gavage
17 d
Open field locomotor activity unaffected
Immobility time increased in the tail suspension test
reduced at 0.02 and 0.2 mg/kg-d (but not at 2 or
20 mg/kg-d)
Immobility time in the forced swimming test unaffected
Maciel et al. (2014)
Wistar rats, male, 12/dose
0 or 2 mg/kg-d by gavage
28 d
1" in total distance traveled in the locomotor activity test
(numerical data not reported)
4/in short-term memory (tested 1.5 hrs after training), but
no change in long-term memory (tested 24 hrs after
training) in the novel object recognition test
(numerical data not reported)
1.1.5. Carcinogenicity
Evidence in Humans
Numerous epidemiologic studies indicate an association between PAH-related occupations
and lung, bladder, and skin cancer (Table 1-12). This discussion primarily focuses on epidemiologic
studies that included a direct measure of benzo[a]pyrene exposure. All identified studies have co-
exposures to other PAHs. The identified studies were separated into tiers according to the extent
and quality of the exposure analysis and other study design features:
Tier 1: Detailed exposure assessment conducted (using a measure of benzo(a)pyrene
exposure), large sample size (>~50 exposed cases), and adequate follow-up period to
account for expected latency (e.g., >20 years for lung cancer).
Tier 2: Exposure assessment, sample size, or follow-up period did not meet the criteria for
Tier 1, or only a single-estimate exposure analysis was conducted.
For lung cancer, each of the Tier 1 studies observed increasing risks of lung cancer with
increasing cumulative exposure to benzo[a]pyrene (measured in [ig/m3-years), and each of these
studies addressed in the analysis the potential for confounding by smoking (Armstrong and Gibbs.
2009: Spinelli etal.. 2006: Xu etal.. 19961 (Table 1-13). These three studies represent different
geographic locations and two different industries. The pattern of results in the Tier 2 studies was
mixed, as would be expected for studies with less precise exposure assessments or smaller sample
sizes: one of the standardized mortality ratio (SMR) estimates was <1.0, with the other eight
estimates ranging from 1.2 to 2.9 (Table 1-14). In considering all of the available studies,
particularly those with the strongest methodology, there is considerable support for an association
between benzo[a]pyrene exposure and lung cancer, although the relative contributions of
benzo[a]pyrene and of other PAHs cannot be established.
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For bladder cancer, the cohort and nested case-control studies observed a much smaller
number of cases compared with lung cancer; this limits their ability to examine exposure-response
relationships. Three cohort studies with detailed exposure data, however, identified 48-90 cases
(Burstyn etal.. 2007: Gibbs and Sevignv. 2007a: Gibbs etal.. 2007: Gibbs and Sevignv. 2007b)
fSpinelli etal.. 20061 (Tier 1 studies, Table 1-15). Although cumulative exposure (up to
approximately 2 [ig/m3-years) was not related to increasing risk in the study of asphalt workers by
Burstynetal. (20071. an exposure-response was seen with the wider exposure range (i.e.,
>80 [ig/m3-years) examined in two studies of aluminum smelter workers by Gibbs etal. (20071:
Gibbs and Sevignv (2007a): Gibbs and Sevignv (2007b): and Spinelli etal. (20061. This difference in
response is not surprising, given that the highest exposure group in the asphalt worker studies
corresponded to the exposures seen in the lowest exposure categories in the studies of aluminum
smelter workers. The five studies with more limited exposure information or analyses each
included between 2 and 16 bladder cancer cases, with relative risk (RR) estimates ranging from
0.6 to 2.9. None of these individual effect estimates was statistically significant (Tier 2 studies,
Table 1-15).
Two of the identified occupational studies contained information on risk of mortality from
melanoma. Neither of these studies observed increased risks of this type of cancer, with an SMR of
0.91 (95% confidence interval [CI] 0.26, 2.48) (22 cases) in Spinelli etal. (20061 and 0.58 (95% CI
0.12,1.7) in Gibbs etal. f20071 f3 cases). These studies did not include information on non-
melanoma skin cancers.
Non-melanoma skin cancer, specifically squamous cell carcinoma (SCC), is of particular
interest with respect to dermal PAH exposures. The literature pertaining to this kind of cancer and
PAH exposure goes back to the 18th century work of Sir Percivall Pott describing scrotal cancer, a
squamous cell skin cancer, in English chimney sweeps fBrown and Thornton. 19571. Recent studies
of chimney sweeps in several Nordic countries have not found increases in non-melanoma skin
cancer incidence fHogstedtetal.. 2013: Pukkala etal.. 2009: Evanoff et al.. 19931. likely due to
greatly reduced exposure associated with better occupational hygiene (IARC. 20121. A study
among asphalt workers (roofers) reported an increased risk of mortality from non-melanoma skin
cancer among asphalt workers (roofers), with an SMR of 4.0 (95% CI 1.0,10.9) among workers
employed >20 years (Hammond et al.. 19761. In addition to this study, two studies in Scandinavian
countries examined non-melanoma skin cancer risk in relation to occupations with likely dermal
exposure to creosote (i.e., timber workers and brick makers) using incidence data from population
registries fKarlehagen etal.. 1992: Tornqvistetal.. 19861. The standardized incidence ratio (SIR)
estimates were 1.5 (95% CI 0.7, 2.6) based on five exposed cases and 2.37 (95% CI 1.08, 4.50)
based on nine cases in Tornqvistetal. (19861 and Karlehagen et al. (19921. Because non-melanoma
skin cancers are rarely fatal if caught early, and the preventative excision of precancerous lesions is
common, the available occupational studies and cancer registries likely underestimate the risk of
SCC fCarae etal.. 2013: Voelter-Mahlknechtetal.. 2007: ONS. 2003: Letzel and Drexler. 19981.
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In addition to cohorts of workers occupationally exposed to PAH mixtures, populations
exposed to benzo[a]pyrene through topical coal tar formulations for the treatment of psoriasis,
eczema, and dermatitis have also been studied fRoelofzen etal.. 2010: Mitropoulos and Norman.
2005: Hannuksela-Svahn etal.. 2000: Stern etal.. 1998: Maier etal.. 1996: Temec and 0sterlind.
1994: Stern and Laird. 1994: Bhate etal.. 1993: Lindelof and Sigurgeirsson. 1993: T orinuki and
Tagami. 1988: Tones etal.. 1985: Pittelkow etal.. 1981: Maughan etal.. 1980: Stern etal.. 19801.
Epidemiological studies examining skin cancer risk in relation to various types of topical tar
exposure are summarized in the Supplemental Information, Table D-6. Case reports, reviews, and
studies that did not include a measure of coal tar use are not included. The available studies
examining therapeutic topical coal tar use and risk of skin cancer were limited by low quality
exposure data with high potential of exposure misclassification (e.g., Roelofzen et al.. 2 010:
Mitropoulos and Norman. 2005: Hannuksela-Svahn etal.. 2000: Maier etal.. 1996: Temec and
0sterlind. 1994: Lindelof and Sigurgeirsson. 19931: potential outcome misclassification (e.g.. Temec
and 0sterlind. 19941: small size (e.g., Temec and 0sterlind. 1994: Torinuki andTagami. 19881: short
duration of follow-up (e.g.. Torinuki andTagami. 19881: choice of referent group (i.e., there was no
referent group or the referent group did not consist of psoriasis patients) (e.g., Temec and 0sterlind.
1994: Bhate etal.. 1993: Tones etal.. 1985: Pittelkow etal.. 1981: Maughan etal.. 19801: and/or
differences in disease ascertainment between cases and the reference population (e.g., Pittelkow et
al.. 1981: Maughan et al.. 19801. Although some clinic-based studies appear to indicate increased
risk with coal tar exposure, these studies used a regimen of coal tar in conjunction with
ultraviolet-B (UVB) therapy or among patients also treated with psoralen plus ultraviolet-A (PUVA),
and thus cannot distinguish the effects of coal tar from the effects of UVB or PUVA (e.g., Stern etal..
1998: Maier etal.. 1996: Stern and Laird. 1994: Lindelof and Sigurgeirsson. 1993: Stern etal..
19801.
There is some uncertainty regarding whether the anatomical properties of psoriatic skin
limit the utility of the available epidemiological studies in coal tar-treated patients for predicting
whether benzo[a]pyrene induces skin cancer in the general population. Psoriatic skin is
characterized by hyperkeratosis caused by abnormally rapid cell proliferation and greatly
increased rates of desquamation (shedding of skin cells). Both hyperkeratosis and desquamation
could be protective with respect to skin cancer risk from dermal PAH exposure. Desquamation can
reduce penetration of compounds past the stratum corneum, so lipophilic chemicals such as
benzo[a]pyrene may not reach the metabolically active layers of the skin (Reddv et al.. 20001.
Reduced absorption of PAHs into the dermally active layers of the skin is consistent with the results
of Roelofzen etal. (20121. which found reduced PAH-DNA adducts in biopsied skin of psoriasis
patients as well as reduced 1-hydroxypyrene levels (a PAH metabolite) in urine as compared to
healthy volunteers following exposure to coal tar ointments.
Therefore, because of the anatomical differences between psoriatic skin and normal skin, as
well as limitations of the above studies with respect to study design and analysis, EPA did not
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consider these studies further in the evaluation of the risk of skin cancer from exposure to
benzo[a]pyrene. Although EPA does not consider the available studies sufficient to evaluate the
risk of skin cancer, acute studies of coal tar treated patients provide in vivo evidence of
benzo[a]pyrene-specific genotoxicity (increased BPDE-DNA adducts) in human skin (Godschalk et
al.. 2001: Roias etal.. 2001: Zhang etal.. 19901. an early key event in the carcinogenic mode of
action of benzo[a]pyrene (see Figure 1-7).
Lung, bladder, and skin cancers are the cancers that have been observed in occupational
studies of PAH mixtures (Benbrahim-Tallaa etal.. 2012: Baan etal.. 2009: Secretan et al.. 20091.
The reproducibility of lung, bladder, and skin cancers in different populations and exposure
settings after occupational exposure to PAH mixtures (see Table 1-12) adds plausibility to the
hypothesis that common etiologic factors may be operating. The potential role that benzo[a]pyrene
may play as a causal agent is further supported by the observation that cancers at these same sites
are also increased in the studies that included a direct measure of benzo[a]pyrene.
Table 1-12. Cancer sites for PAH-related agents reviewed by the International
Agency for Research on Cancer (IARC)
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)
Source: Adapted from IARC (2010).
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Table 1-13. Summary of epidemiologic studies of benzo[a]pyrene (direct
measures) in relation to lung cancer risk: Tier 1 studies
Reference and study design
Results
Armstrong and Gibbs (2009) (Quebec, Canada)
Cohort, aluminum smelter workers, seven plants
16,431 (15,703 men; 728 women); duration
minimum 1 yr, began work 1966-1990; follow-up
through 1999 (mean ~30yrs); smoking information
collected from medical records
Exposure: Job exposure matrix ~5,000 personal
benzo[a]pyrene measures from the 1970s to 1999
Related references: Lavoue et al. (2007) (exposure
data); Armstrong et al. (1994): Gibbs et al. (2007):
Gibbs and Sevignv (2007a): Gibbs and Sevignv
(2007b)
Spinelli et al. (2006) (British Columbia, Canada)
Cohort, aluminum smelter workers; 6,423 (all men);
duration minimum >3 yrs; began work 1954-1997;
follow-up through 1999 (14% loss to follow-up;
mean ~24 yrs); smoking information from self-
administered questionnaire
Exposure: Job exposure matrix using 1,275 personal
benzo[a]pyrene measures from 1977 to 2000 (69%
for compliance monitoring)
Related references: Friesen et al. (2006) (exposure
data); Spinelli et al. (1991)
SMR 1.32 (1.22, 1.42) [677 cases]
Lung cancer risk by cumulative benzo[a]pyrene exposure
Median
benzo[a]-
pyrene
n
Hg/m3-yrs
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 ng/m3-yrs (0.0035 per ng/m3-yrs
increase); other shapes of exposure-response curve
examined.
SMR: 1.07 (0.89, 1.28) [120 cases]
SIR: 1.10 (0.93,1.30) [147 cases]
Lung cancer risk by cumulative benzo[a]pyrene exposure
Benzo[a]pyrene
Hg/m3-yrs
n cases
RR (95% Cl)a
0-0.5
25
1.0
(referent)
0.5-20
42
1.23
(0.74, 2.03)
20-40
23
1.35
(0.76, 2.40)
40-80
25
1.36
(0.78, 2.39)
>80
32
1.79
(1.04, 3.01)
aAdjusting for smoking category; trend p < 0.001.
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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, 1956-1992
Lung cancer risk by cumulative benzo[a]pyrene exposure
Benzo[a]-pyrene
(Hg/m3-yrs)
n cases
RR (95% Cl)a
<0.84
72
1.1(0.8, 1.7)
0.85-1.96
117
1.6 (1.2, 2.3)
1.97-3.2
96
1.6 (1.1, 2.3)
>3.2b
105
1.8(1.2,2.5)
aAdjusting for birth year and smoking category; trend
p < 0.004. Referent group is "nonexposed" (employed in
administrative or low-exposure occupations).
bStudy table IV unclear; could be >3.0 for this category.
Table 1-14. Summary of epidemiologic studies of benzo[a]pyrene (direct
measures) in relation to lung cancer risk: Tier 2 studies
Reference and study design
Results
Limited follow-up period (<20 yrs)
Friesen et al. (2009) (Australia)
Cohort, aluminum smelter workers; 4,316 (all men);
duration minimum 90 d; began work after 1962;
follow-up through 2002, mean 16 yrs (maximum
20 yrs); Smoking information from company records
if employed before 1995 and study interviews if
employed after 1994
Exposure: Job/task exposure matrix using TWA
benzo[a]pyrene measures (n = 655), 1977-2004 (79%
from 1990 to 2004)
RR 1.2 (0.7, 2.3) [19 cases in exposed; 20 in unexposed]
Lung cancer risk by cumulative benzo[a]pyrene exposure
Benzo[a]-pyrene
Hg/m3-yrs n cases RR (95% Cl)a
0 20 1.0 (referent)
>0-0.41 6 0.7 (0.3, 1.8)
0.41-10.9 6 1.4 (0.6,3.5)
>10.9 7 1.7 (0.7,4.2)
aPoisson regression, adjusting for smoking; trend p = 0.22.
Proxy measure
Olsson et al. (2010) (Denmark, Norwav, 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: from
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 matrix
Lung cancer risk by cumulative coal tar exposure3
Coal tar n
unit-yrsa 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)
(trend p-value) (0.07)
aAdjusting for set, age, country, tobacco pack-years.
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Reference and study design
Results
Related references: Boffetta et a I. (2003); Burstvn et
al. (2000)
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/m3 top-side full-time jobs,
0.88 mg/m3 side jobs; used to calculate weighted
cumulative exposure index
Related reference: Dong et al. (1988) (exposure data)
SMR 1.95 (1.59, 2.33) [255 cases]
Lung cancer risk by cumulative exposure
Coal tar pitch
volatiles n
(mg/m3-mo)
cases
RR (95% Cl)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)
aAdjusting 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 yrs;
began work before 1971; follow-up: through 1985
(mean ~14 yrs); smoking information from
SMR 2.2 (1.1, 2.8) [50 cases]
Lung cancer risk by exposure category
Exposure
Mean
benzo[a]-
pyrene
questionnaire
category
Hg/m3
n cases
SMR (95% Cl)a
None
_
13
1.49 (0.83, 2.5)
Exposure: Area samples from one carbon plant,
1986-1987
Low
-
6
1.19 (0.48, 2.5)
Moderate
0.30
5
1.52 (0.55, 3.4)
High
1.19
26
4.30 (2.9, 6.2)
Calculated by EPA from data in paper.
Berger and Manz (1992) (Germany)
Cohort, coke oven workers; 789 (all men); duration
minimum 10 yrs (mean 27 yrs); began work
1900-1989; follow-up through 1989 (length data not
reported); smoking information from plant records
and interviews
Exposure: Mean benzo[a]pyrene: 28 ng/m3 (range
0.9-89 ng/m3)
SMR 2.88 (2.28, 3.59) [78 cases]
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Reference and study design
Results
Hansen (1991); (Hansen, 1989) (Denmark)
Cohort, asphalt workers; 679 workers (applicators)
(all men); duration data not reported; employed
1959-1980; follow-up to 1986 (mean ~11 yrs);
smoking information from 1982 surveys of industry
and general population
Exposure: Asphalt fume condensate, 35 personal
samples during flooring: median 19.7 mg/m3 (range
0.5-260 mg/m3)
SMR 2.90 (1.88, 4.3) [25 cases] (ages 40-89 yrs)
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 yr, median 15 yrs;
employed 1965-1972; follow-up: 1966-1986
(mortality); 1966-1983 (incidence; mean ~15 yrs);
smoking information from interviews with older
workers
Exposure: Area sampling, top of ovens;
benzo[a]pyrene, 1,964 mean 4.3 ng/m3 (range
0.007-33 ng/m3); 1,965 mean 0.52 ng/m3
(0.021-1.29 ng/m3)
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: Benzo[a]pyrene, 19 area samples and
16 personal samples in Plant A (personal sample
mean 2.7 ng/m3; range 0.59-6.2 ng/m3); 10 area
samples and 7 personal samples in Plant B; personal
sample mean 0.17 ng/m3, range 0.02-0.57 ng/m3
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 yr duration:
Plant A: OR 3.42 (0.35, 33.7) [7 cases, 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 yrs, began before 1960; follow-up
through 1971
Exposure: 52 personal samples (masks with filters)
during specific jobs and tasks; mean benzo[a]pyrene
16.7 ng per 7-hr d
SMR 1.6 (1.3,1.9) [99 cases] (>20 yrs since joining union)
(CIs calculated by EPA from data in paper)
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Table 1-15. Summary of epidemiologic studies of benzo[a]pyrene (direct
measures) in relation to bladder cancer risk
Reference and study design
Results
Tier 1 studies
Burstvn et al. (2007) (Denmark, Norwav, 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 yrs; smoking information not collected
Exposure: Compilation of benzo[a]pyrene
measures, production characteristics, and repeat
measures in asphalt industry in each country used
to develop exposure matrix
Related references: Boffetta et al. (2003); Burstvn
et al. (2000)
48 incident bladder cancer cases (39 cases in analyses with
15-yr lag)
Bladder cancer risk by cumulative benzo[a]pyrene exposure3
Benzo[a]-
pyrene RR (95% CI) RR (95% CI)
Hg/m3-yrsa n cases (no lag)b (15-yr lag)0
0-0.253 12 1.0 (referent) 1.0 (referent)
0.253-0.895 12 0.69 (0.29, 1.6) 1.1 (0.44, 2.9)
0.895-1.665 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)
aAdjusting for age, calendar period, total duration of
employment, country.
bTrend p = 0.9.
Trend p = 0.63.
Stronger pattern seen with average exposure in 15-yr lag
(RR 1.5, 2.7,1.9 in second through fourth quartile; trend
p = 0.15)
Gibbs et al. (2007); Gibbs and Sevignv (2007a);
Gibbs and Sevignv (2007b) (Quebec, Canada)
Cohort, aluminum smelter workers, seven plants
16,431 (15,703 men; 728 women); duration
minimum lyr, began work 1966-1990; follow-up:
through 1999 (mean ~30yrs); smoking information
collected from medical records
Exposure: Job exposure matrix using
~5,000 personal benzo[a]pyrene measures from the
1970s to 1999
Related references: Lavoue et al. (2007) (exposure
data); Armstrong et al. (1994); Gibbs (1985); Gibbs
and Horowitz (1979)
Hired before 1950: SMR 2.24 (1.77, 2.79) [78 cases]
Bladder cancer risk by cumulative benzo[a]pyrene exposure
Benzo[a]- Smoking-
pyrene adjusted
Hg/m3-yrsa n cases SMR (95% CI) RRb
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)
aCategory midpoint.
bCls not reported; highest category is >80 ng/m3-yrs
(n observed = 57).
Mortality risk reduced in cohort hired in 1950-1959,
SMR = 1.23.
Similar patterns seen in analysis of bladder cancer
incidence.
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Reference and study design
Results
Spinelli et al. (2006) (British Columbia, Canada)
See Table 1-13 for study details; this study is
considered a "Tier 2") study for bladder cancer
because of the smaller number of bladder cancer
cases (n = 12) compared with lung cancer cases
(n = 120)
SMR 1.39 (0.72, 2.43) [12 cases]
SIR 1.80; CI 1.45-2.21 [90 cases, including in situ]
Bladder cancer risk by cumulative benzo[a]pyrene exposure
Benzo[a]-
pyrene
Hg/m3-years n cases RR (95% Cl)a
0-0.5 17 1.0 (referent)
0.5-20 20 0.83 (0.43, 1.59)
20-40 13 1.16 (0.56,2.39)
40-80 18 1.50 (0.77,2.94)
>80 22 1.92 (1.02,3.65)
aAdjusting for smoking category; trend p < 0.001.
Tier 2 studies
Friesen et al. (2009) (Australia)
RR 0.6 (0.2, 2.0) [five cases in exposed; eight in unexposed]
See Table 1-14 for study details
Bladder cancer risk by cumulative benzo[a]pyrene exposure
Benzo[a]-
pyrene
Hg/m3-yrs n cases RR (95% Cl)a
0 8 1.0 (referent)
>0-0.41 1 0.2 (0.03, 1.9)
0.41-10.9 2 0.7 (0.2,3.7)
>10.9 2 1.2 (0.2,5.6)
aPoisson regression, adjusting for smoking category; trend
p = 0.22.
Costantino et al. (1995) (United States,
Pennsylvania)
SMR 1.14 (0.61, 2.12) (16 cases)
See Table 1-14 for study details
Hammond et al. (1976) (United States)
See Table 1-14 for study details
SMR 1.7 (0.94, 2.8) (13 cases) (>20 yrs since joining union)
(CIs calculated by EPA from data in paper)
Moulin et al. (1989) (France)
See Table 1-14 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 1-14 for study details
SMR 2.85 (0.30,10.3) (2 cases) (referent group = employed
men)
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Evidence in Animals
Oral exposure
Evidence of tumorigenicity following oral exposure to benzo[a]pyrene has been
demonstrated in rats and mice. As summarized in Table 1-16, oral exposure to benzo[a]pyrene has
resulted in an increased incidence of tumors in the alimentary tract in male and female rats (Kroese
etal.. 2001: Brune etal.. 19811 and female mice fBeland and Culp. 1998: Culp etal.. 19981. liver
carcinomas in male and female rats, kidney adenomas in male rats (Kroese etal.. 20011. and
auditory canal tumors in both sexes fKroese etal.. 20011.
Forestomach tumors have been observed in several lifetime cancer bioassays in rats and
mice following both gavage and dietary exposure to benzo[a]pyrene at doses ranging from
0.016 mg/kg-day in Sprague-Dawley rats to 3.3 and 10 mg/kg-day in B6C3Fi mice and Wistar rats,
respectively (Kroese etal.. 2001: Beland and Culp. 1998: Culp etal.. 1998: Brune etal.. 19811. In
addition, multiple less-than-lifetime oral exposure cancer bioassays in mice provide supporting
evidence that oral exposure to benzo[a]pyrene is associated with an increased incidence of
forestomach tumors fWevand etal.. 1995: Benjamin etal.. 1988: Robinson etal.. 1987: El-Bayoumv.
1985: Triolo etal.. 1977: Wattenberg. 1974: Roe etal.. 1970: Biancifiori etal.. 1967: Chouroulinkov
etal.. 1967: Fedorenko and Yansheva. 1967: Neal and Rigdon. 1967: Berenblum and Haran. 19551.
Increases in the incidence of forestomach hyperplasia have also been observed in Wistar
rats following shorter-term, subchronic, and chronic gavage exposure (Kroese etal.. 2001: De long
etal.. 19991 and in B6C3Fi mice following chronic dietary exposure fBeland and Culp. 1998: Culp et
al.. 19981. Forestomach hyperplasia occurred at shorter durations and at lower doses than tumors
in rats and mice exposed to benzo[a]pyrene for up to 2 years fKroese etal.. 2001: Beland and Culp.
19981. Kroese etal. f20011 reported that the forestomach lesions demonstrated a progression over
the course of intercurrent sacrifices; the authors described early lesions as focal or confluent basal
hyperplasia, followed by more advanced hyperplasia with squamous cell papilloma, culminating in
SCC, indicating that forestomach hyperplasia may be a histological precursor to neoplasia observed
in the forestomach after chronic exposure to benzo[a]pyrene.
Although humans do not have a forestomach, similar squamous epithelial tissue is present
in the oral cavity flARC. 2003: Wester and Kroes. 19881: therefore, forestomach tumors observed in
rodents following benzo[a]pyrene exposure are considered relevant for the assessment of cancer
hazard in humans fBeland and Culp. 19981. For further discussion, see Sections 1.2 and 2.3.4.
Elsewhere in the alimentary tract, dose-related increases of benign and malignant tumors
were observed. In rats, oral cavity tumors were induced in both sexes and adenocarcinomas of the
jejunum were induced in males (Kroese etal.. 20011. In mice, tumors were induced in the tongue,
esophagus, and larynx of females (males were not tested) fBeland and Culp. 1998: Culp etal.. 19981.
Chronic oral exposure to benzo[a]pyrene resulted in a dose-dependent increased incidence
of liver carcinomas in both sexes of Wistar rats, with the first liver tumors detected in week 35 in
high-dose male rats; liver tumors were described as complex, with a considerable proportion
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Toxicological Review of Benzo[a]pyrene
(59/150 tumors) metastasizing to the lungs (Kroese etal.. 20011. Treatment-related hepatocellular
tumors were not observed in mice fBeland and Culp. 1998: Culp etal.. 19981.
An increased incidence of kidney tumors (cortical adenomas) was observed in male Wistar
rats following chronic gavage exposure (Kroese etal.. 20011 (Table 1-16). The kidney tumors were
observed at the mid- and high-dose groups. Treatment-related kidney tumors were not observed in
two other chronic studies fBeland and Culp. 1998: Brune etal.. 19811.
Lung tumors were also observed following almost nine months of dietary exposure to
approximately 10 mg/kg-day in female AJ mice (Wevand etal.. 19951. Other lifetime exposure
studies did not report treatment-related increases in lung tumors (Kroese etal.. 2001: Beland and
Culp. 1998: Culp etal.. 19981.
Table 1-16. Tumors observed in chronic oral animal bioassays
Study design and reference
Results
Kroese et al. (2001)
Forestomach
Wistar (Riv:TOX) rats (52/sex/dose
incidences:
group)
M: 0/52; 7/52*; 18/52*; and 17/52* (papilloma)
0, 3,10, or 30 mg/kg-d by gavage
M: 0/52; 1/52; 25/52*; and 35/52* (SCC)
5 d/wk
F: 1/52; 3/51; 20/51*; and 25/52* (papilloma)
2 yrs
F: 0/52; 3/51; 10/51*; and 25/52* (SCC)
Oral cavity
incidences:
M: 0/24; 0/24; 2/37; and 10/38* (papilloma)
M: 1/24; 0/24; 5/37; and 11/38* (SCC)
F: 0/19; 0/21; 0/9; and 9/31*(papilloma)
F: 1/19; 0/21; 0/9; and 9/31* (SCC)
Jejunum (adenocarcinomas)
incidences:
M: 0/51; 0/50; 1/51; and 8/49*
F: 0/50; 0/48; 0/50; and 2/51
Duodenum (adenocarcinomas)
incidences:
M: 0/51; 0/50; 0/51; and 1/49
F: 0/49; 0/48; 0/50; and 2/51
Liver (adenomas and carcinomas)
incidences:
M: 0/52; 3/52; 15/52*; and 4/52 (adenoma)
M: 0/52; 1/52; 23/52*; and 45/52* (carcinoma)
F: 0/52; 2/52; 7/52*; and 1/52 (adenoma)
F: 0/52; 0/52; 32/52*; and 50/52* (carcinoma)
Kidney (cortical adenoma)
incidences:
M: 0/52; 0/52; 7/52*; and 8/52*
F: increase not observed
Auditory canalb (Zymbal gland) (carcinomas)
incidences:
M: 0/1; 0/0; 2/7; and 19/33*
F: 0/0; 0/1; 0/0; and 13/20*
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Study design and reference
Results
Beland and Culp (1998): Culp et al.
(1998)
B6C3Fi mice: female (48/dose group)
0, 5, 25, or 100 ppm (average daily
doses3: 0, 0.7, 3.3, and 16.5 mg/kg-d) in
the diet
2 yrs
Forestomach (papillomas and SCCs)
incidences: 1/48; 3/47; 36/46*; and 46/47*
Esophagus (papillomas and carcinomas)
incidences: 0/48; 0/48; 2/45; and 27/46*
Tongue (papillomas and carcinomas)
incidences: 0/49; 0/48; 2/46; and 23/48*
Larynx (papillomas and carcinomas)
incidences: 0/35; 0/35; 3/34; and 5/38
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-d)
Diet: 0, 6, 39 mg/kg-yr (0, 0.016,
0.107 mg/kg-d)
Treated until moribund or dead
2 yrs
Forestomach (papillomas and carcinomas0); gavage
incidences: 3/64; 12/64*; 26/64*; and 14/64*
Forestomach (papillomas); diet
incidences: 2/64; 1/64; and 9/64*
Larynx and esophagus (papillomas); gavage
incidences: 3/64; 1/64; 0/64; and 0/64
Larynx and esophagus (papillomas); diet
incidences: 1/64; 2/64; and 1/64
* Indicates statistical significance as identified in study.
aBased on the assumption that daily benzo[a]pyrene intake at 5 ppm was one-fifth of the 25-ppm intake (about
21 ng/day) and using TWA body weights of 0.032 kg for the control, 5- and 25-ppm groups and 0.026 kg for the
100-ppm group.
incidences are for number of rats with tumors compared with number of tissues examined histologically.
Auditory canal tissue was examined histologically when abnormalities were observed on macroscopic
examination.
cTwo malignant forestomach tumors were observed (one each in the mid- and high-dose groups).
Inhalation exposure
The inhalation database of benzo[a]pyrene carcinogenicity studies consists of one lifetime
inhalation bioassay in male hamsters fThvssen etal.. 19811. Intratracheal instillation studies in
hamsters are also available (FeronandKruvsse. 1978: Ketkar etal.. 1978: Feron etal.. 1973: Henry
etal.. 1973: Saffiottietal.. 19721.
Several long-term intratracheal instillation studies in hamsters evaluated the
carcinogenicity of benzo[a]pyrene (Feron and Kruvsse. 1978: Feron etal.. 1973: Henry etal.. 1973:
Saffiotti et al.. 19721. These studies treated animals with benzo[a]pyrene once a week in a saline
solution (0.5-0.9%) for >8 months and observed animals for 1-2 years following cessation of
exposure. Tumors in the larynx, trachea, bronchi, bronchioles, and alveoli were observed.
Individual studies also reported tumors in the nasal cavity and forestomach. These intratracheal
instillation studies support the carcinogenicity of benzo[a]pyrene in the respiratory tract; however,
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Toxicological Review of Benzo[a]pyrene
conversion of a dose delivered by intratracheal instillation to an inhalation concentration is
problematic due to different patterns of deposition and retention.
Lifetime inhalation exposure to benzo[a]pyrene resulted in the development of tumors in
the respiratory tract and pharynx in Syrian golden hamsters (Thvssen et al.. 19811. The authors
stated that the rates of tumors of other organs generally corresponded to the rates in controls. U.S.
EPA Q9901 obtained individual animal data from the study authors (including individual animal
pathology reports for the respiratory and upper digestive tracts, time-to-death data, and exposure
chamber monitoring data) (Clement Associates. 19901: this information is summarized in
Table 1-17. Concentration-dependent increased incidences of tumors in the upper respiratory
tract, including the larynx and trachea, were seen at exposure concentrations of >9.5 mg/m3. In
addition, a decrease in mean tumor latency was observed in the larynx and trachea. Nasal cavity
tumors were observed at the mid- and high-concentration, but the incidences were not dose-
dependent. A concentration-related increase in tumors in the upper digestive tract (pharynx and
esophagus) was also reported. In addition, a single forestomach tumor was observed in each of the
mid- and high-concentration groups. The study authors suggested that the upper digestive tract
tumors were a consequence of mucociliary particle clearance. All nasal, forestomach, esophageal,
and tracheal tumors occurred in hamsters that also had tumors in the larynx or pharynx, except in
the mid-concentration group, where two animals with nasal tumors had no tumors in the pharynx
or larynx.
A re-analysis of the individual animal pathology reports and the exposure chamber
monitoring data provided by the study authors yielded estimates of average continuous lifetime
exposures for each individual hamster. Group averages of individual average continuous lifetime
exposure concentrations were 0, 0.25,1.01, and 4.29 mg/m3 for the control through high-exposure
groups fU.S. EPA. 19901.
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Table 1-17. Tumors observed in chronic inhalation animal bioassays
Reference and study design
Resultsad
Thyssen et al. (1981)
Syrian golden hamsters: male
(26-34 animals/group placed on study)
0, 2.2, 9.5, or 46.5 mg/m3 on sodium chloride particles
by nose only inhalation for 3-4.5 hrs, 5-7 d/wk
(TWA exposure concentrations'5: 0, 0.25,1.01, and
4.29 mg/m3)
Treated until moribund or dead (up to 130 wks)
MMAD: not reported
Larynx
incidences: 0/26; 0/21; 11/26; and 11/25
mean tumor latency0: 107 and 68 wks
Pharynx
incidences: 0/23; 0/19; 9/22; and 18/23
mean tumor latency: 97 and 68 wks
Trachea
incidences: 0/27; 0/21; 2/26; and 3/25
mean tumor latency: 115 and 63 wks
Nasal cavity
incidences: 0/26; 0/22; 4/26; and 1/34
mean tumor latency: 116 and 79 wks
Esophagus
incidences: 0/27; 0/22; 0/26; and 2/34
mean tumor latency: 71 wks
Forestomach
incidences: 0/27; 0/22; 1/26; and 2/34
mean tumor latency: 119 and 72 wks
aThvssen et al. (1981) reported only the incidences of malignant tumors, confirmed by comparison with the
original study pathology data (Clement Associates, 1990). The incidences summarized here include relevant
benign tumors (papillomas, polyps, and papillary polyps). The malignant tumors were SCCs, with the exception of
one in situ carcinoma of the larynx and one adenocarcinoma of the nasal cavity, both in the 9.5 mg/m3 group.
Denominators reflect the number of animals examined for histopathology for each tissue. See Section D.4.2 and
Table E-30 in the Supplemental Material for study details and a complete listing of individual data, respectively.
bDuration-adjusted inhalation concentrations calculated from exposure chamber monitoring data and exposure
treatment times. Daily exposure times: 4.5 hours/day, 5 days/week on weeks 1-12; 3 hours/day, 5 days/week on
weeks 13-29; 3.7 hours/day, 5 days/week on week 30; 3 hours/day, 5 days/week on weeks 31-41; and
3 hours/day, 7 days/week for reminder of the experiment.
cMean time of observation of tumor, 9.5 and 46.5 mg/m3 concentration groups.
dThvssen et al. (1981) did not report statistical significance testing. See Section D.4.2.
MMAD = mass median aerodynamic diameter
Dermal exposure
Repeated application of benzo[a]pyrene to skin (in the absence of exogenous promoters)
has been demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs. These studies
have been reviewed by multiple national and international health agencies flARC. 2010: IPCS. 1998:
ATSDR. 1995: IARC. 1983.19731. Mice have been the most extensively studied species in dermal
carcinogenesis studies of benzo[a]pyrene because of evidence that they may be more sensitive than
other animal species; however, comprehensive comparisons of species differences in sensitivity to
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lifetime dermal exposure are not available. Systemic tumors in benzo[a]pyrene-treated mice were
not increased compared to controls in lifetime dermal bioassays in which macroscopic examination
of internal organs was included fHigginbotham etal.. 1993: Habs etal.. 1980: Schmahl etal.. 1977:
Schmidtetal.. 1973: Roe etal.. 1970: Poel. 19591.
The analysis in this document focuses on lifetime carcinogenicity bioassays in several
strains of mice following repeated dermal exposure to benzo[a]pyrene (Table 1-18). These studies
involved 2- or 3-times/week exposure protocols, at least two exposure levels plus controls, and
histopathological examinations of the skin and other tissues (Sivak etal.. 1997: Grimmer etal..
1984: Habs etal.. 1984: Grimmer etal.. 1983: Habs etal.. 1980: Schmahl etal.. 1977: Schmidt etal..
1973: Roe etal.. 1970: Poel. 1963. 19591.
Additional studies in mice observed skin tumors following benzo[a]pyrene exposure and
were considered supportive for hazard identification, but were not considered further in this
assessment because of the availability of the lifetime studies identified above. These studies
included several "skin painting" studies in mouse skin that did not report the doses applied (e.g.,
Wvnder and Hoffmann. 1959: Wvnder etal.. 1957): several less-than-lifetime studies (Albert etal..
1991: Nesnow et al.. 1983: Emmettetal.. 1981: Levin etal.. 1977): initiation-promotion studies
utilizing acute dosing of benzo[a]pyrene followed by repeated exposure to a potent tumor
promoter; and studies involving vehicles expected to interact with or enhance benzo[a]pyrene
carcinogenicity fe.g.. Bingham and Falk. 19691.
One study applied benzo[a]pyrene (topically once a week for 6 months) to immuno-
compromised mice with human skin grafts (n = 10) and did not observe tumors, whereas all three
control mice (mice with no skin grafts) developed skin tumors (Urano etal.. 1995). The authors
concluded that this result indicates that human skin is much less susceptible to benzo[a]pyrene
than mouse skin. Although some studies indicate that the skin grafts maintain some metabolic
function (Das etal.. 1986). it is unclear whether the human skin grafts maintain the same viability,
vascularization, and full metabolic capacity as human skin in vivo fKappes etal.. 20041. In addition,
no control was used to account for the trauma of the surgery and potential loss of viability in the
transplanted skin (i.e., mice with grafted mouse skin were not used as a control). Another concern
is the short amount of time allowed for tumor development. All of the mice with human skin grafts
treated with benzo[a]pyrene died within 6 months of the start of treatment (Urano etal.. 1995).
While 6 months is generally sufficient for the development of tumors in mouse skin, it is unclear
that this much smaller fraction of a human lifetime would be sufficient time for the development of
human skin cancer if the human skin grafts retain human properties (e.g., better DNA repair, slower
rate of cell turnover, and generally slower toxicokinetics) as human latency for SCC in PAH-exposed
occupational cohorts is thought to be >20 years (Young etal.. 2012: Voelter-Mahlknechtetal.. 2007:
Everall and Dowd. 1978). Potent mutagenic carcinogens such as 7,12-dimethylbenz[a]anthracene,
methylcholanthrene, and methylnitronitrosoguanidine also fail to produce skin tumors in this
model system fSoballe etal.. 1996: Urano etal.. 1995: Graem. 19861. Therefore, the ability of this
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model system to predict hazard for human skin cancer risk (particularly from metabolically active
carcinogens) is unclear.
Table 1-18. Tumors observed in chronic dermal animal bioassays
Reference and study design
Results3
Poel (1959)
C57L mice: male (13-56/dose)
0, 0.15, 0.38, 0.75, 3.8, 19, 94, 188, 376, or 752 ng
Dermal; 3 times/wk for up to 103 wks 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; and 13/13
Epidermoid carcinoma: 0/33; 0/55; 2/55; 4/56;
32/49; 37/38; 35/35; 10/14; 12/14; and 13/13
Cytotoxicity: information not provided
Poel (1963)
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 ng
Dermal; 3 times/wk 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;
and 14/14
C3HeB: 0/17; 0/19; 3/17; 4/17; 11/18; 17/17; 18/18;
and 17/17
A/He mice: 0/17; 0/18; 0/19; 0/17; 0/17; 21/23;
11/16; and 17/17
Cytotoxicity: information not provided
Roe et al. (1970)
Swiss mice: female (50/dose)
0, vehicle, 0.1, 0.3,1, 3, or 9 ng
Dermal; 3 times/wk for up to 93 wks
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; and 34/46
Cytotoxicity: information not provided
Schmidt et al. (1973)
NMRI mice: female (100/group)
Swiss mice: female (100/group)
0, 0.05, 0.2, 0.8, or 2 ng
Dermal; 2 times/wk until spontaneous death
occurred or until an advanced carcinoma was
observed
Skin tumors (carcinomas)
incidences:
NMRI:
2/100 at 2 ng (papillomas);
2/100 at 0.8 |jg and 30/100 at 2 ng (carcinomas)
Swiss:
3/80 at 2 ng (papillomas);
5/80 at 0.8 ng and 45/80 at 2 ng (carcinomas)
Cytotoxicity: information not provided
Schmahl et al. (1977)
NMRI mice: female (100/group)
0, 1,1.7, or 3 ng
Dermal; 2 times/wk until natural death or until they
developed a carcinoma at the site of application
Skin tumors (papillomas and carcinomas)
incidences:
0/81; 1/77; 0/88; and 2/81 (papillomas)
0/81; 10/77; 25/88; and 43/81 (carcinomas)
Cytotoxicity: information not provided
Habs et al. (1980)
NMRI mice: female (40/group)
0, 1.7, 2.8, or 4.6 ng
Dermal; 2 times/wk 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; and 22/36
age-standardized tumor incidence:
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Reference and study design
Results3
0, 24.8, 89.3, and 91.7%
Cytotoxicity: information not provided
Grimmer et al. (1984): Grimmer et al. (1983)
CFLP mice: female (65-80/group)
0, 3.9, 7.7, or 15.4 ng (1983 study)
0, 3.4, 6.7, or 13.5 ng (1984 study)
Dermal; 2 times/wk for 104 wks
Skin tumors (papillomas and carcinomas) with a decrease in
tumor latency
incidences:
1983: 0/80; 7/65; 5/64; and 2/64 (papillomas)
0/80; 15/65; 34/64; and 54/64 (carcinomas)
1984: 0/65; 6/64; 8/65; and 4/65 (papillomas)
0/65; 37/64; 45/65; and 53/65 (carcinomas)
Cytotoxicity: information not provided
Habs et al. (1984)
NMRI mice: female (20/group)
0, 2, or 4 ng
Dermal; 2 times/wk for life
Skin tumors (papillomas and carcinomas) with a decrease in
mean survival time
incidences:
0/20; 2/20; and 0/20 (papillomas)
0/20; 7/20; and 17/20 (carcinomas)
Cytotoxicity: information not provided
Sivak et al. (1997); NIOSH (1989)
C3H/HeJ mice: male (30/group)
0, 0.05, 0.5, or 5 ng
Dermal; 2 times/wk for up to 104 wks
Skin tumors (papillomas and carcinomas)
incidences:
0/30; 0/30; 5/30 (1 papilloma, 1 keratoacanthoma,
3 carcinomas); and 27/30 (1 papilloma,
28 carcinomas)
Cytotoxicity: 80% incidence of scabs and sores in highest dose
group; no cytotoxicity noted at lower doses
statistical significance not reported by study authors.
Mode-of-Action Analysis—Carcinogenicity
The carcinogenicity of benzo[a]pyrene, the most studied PAH, is well documented in animal
models flARC. 2010: Xu etal.. 2009: Tiang etal.. 2007: Tiang etal.. 2005: Xue and Warshawskv. 2005:
Ramesh etal.. 2004: Bostrom etal.. 2002: Penning etal.. 1999: IPCS. 1998: Harvey. 1996: ATSDR.
1995: Cavalieri and Rogan. 1995: U.S. EPA. 1991b). The primary mode of action by which
benzo[a]pyrene induces carcinogenicity is via a mutagenic mode of action. This mode of action is
presumed to apply to all tumor types and is relevant for all routes of exposure. The general
sequence of key events associated with a mutagenic mode of action for benzo[a]pyrene is:
(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; (2) direct DNA damage by reactive metabolites, including the formation of DNA adducts
and ROS-mediated damage; (3) formation and fixation of DNA mutations, particularly in tumor
suppressor genes or oncogenes associated with tumor initiation; and (4) clonal expansion of
mutated cells during the promotion and progression phases of cancer development These events
are depicted as stages of benzo[a]pyrene-induced carcinogenesis in Figure 1-7.
Benzo[a]pyrene is a complete carcinogen, in that it can act as both an initiator and a
promoter of carcinogenesis. Initiation via direct DNA damage (key event 2) can occur via all three
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metabolites of benzo[a]pyrene. DNA damage that is not adequately repaired leads to mutation (key
event 3), and these mutations can undergo clonal expansion (key event 4) enabled by multiple
mechanisms also induced by benzo[a"|pyrene, including AhR binding leading to an upregulation of
genes related to biotransformation, growth, and differentiation, and regenerative cell proliferation
resulting from cytotoxicity and a sustained inflammatory response. However, there is not sufficient
evidence that these mechanisms, which contribute to the promotion and progression phases of
cancer development, act independently of DNA damage and mutation to produce benzo[a]pyrene-
induced tumors (see Other possible modes of action, below]. The available human, animal, and in
vitro evidence supports a mutagenic mode of action as the primary mode by which benzo[a]pyrene
induces carcinogenesis.
Key events inthe mode of action for benzo[a]pyrene carcinogenicity
Exposure
Metabolism
Initiation
Promotion Progression
DNA adducts
DNAadducts
Mutation
(transversion)
K-ras, H-rcts,
andp53 targets
Mutation
(depurination):
H-rastarget
BindingtoAhR
Upregulation of genes
related to
biotransformation,
growth, and
differentiation
Proliferation
of initiated
cells
Neoplasm
o-quinone
andROS
DNAadducts
| and oxidative j
base damage
Mutation
I (depurination):
oxidative
damage and
strand scission
Inflammatory
response
Cytotoxicity
Figure 1-7. Proposed metabolic activation pathways and key events in the
carcinogenic mode of action for benzo[a]pyrene.
Data in Support of the Mode of Action
Summary of metabolic activation pathways
Diol epoxide pathway. Benzo[a]pyrene diol epoxide metabolites, believed to be the most
potent DNA-binding metabolites of benzo[a]pyrene, are formed through a series of Phase I
metabolic reactions (see Section D.1.4 of the Supplemental Information for a more detailed review}.
The initial metabolism is carried out primarily by the inducible activities of CYP enzymes including
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CYP1A1, CYP1B1, and CYP1A2, producing four benzo[a]pyrene epoxides. Further metabolism by
epoxide hydrolase and the mixed function oxidase system yields trans-dihydrodiols, one of which,
benzo[a]pyrene-7,8-diol (formed from benzo[a]pyrene-7,8-oxide), is the metabolic precursor to the
potent DNA-binding metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) (Grover. 19861. The
stereochemical nature of the diol epoxide metabolite (i.e., anti- versus syn-diol epoxides) affects the
number and type of adducts and mutation that occurs; the enantiomer (+)-benzo[a]pyrene-7R,8S-
diol-9S,10R-epoxide [(+)-anti-BPDE] is the most potent DNA-binding metabolite of benzo[a]pyrene
(Geacintov etal.. 19971. Benzo[a]pyrene diol epoxide metabolites interact preferentially with the
exocyclic amino groups of deoxyguanine and deoxyadenine (Geacintovetal.. 1997: Terinaetal..
19911. Adducts may give rise to mutations unless these adducts are removed by DNA repair
processes prior to replication. Transversion mutations (e.g., GC^TA or AT^TA) are the most
common type of mutation found in mammalian cells following diol epoxide exposure fBostrom et
al„ 20021.
Radical cation pathway. Radical cation formation involves a one-electron oxidation by CYP
or peroxidase enzymes (i.e., horseradish peroxidase, prostaglandin H synthetase) that produce
electrophilic radical cation intermediates (Cavalieri and Rogan. 1995.19921. Radical cations can be
further metabolized to phenols and quinones fCavalieri etal.. 1988e: Cavalieri et al.. 1988dl. or they
can form unstable adducts with DNA that ultimately result in depurination. The predominant
depurinating adducts occur at the N-3 and N-7 positions of adenine and the C-8 and N-7 positions of
guanine (Cavalieri and Rogan. 19951.
o-Quinone/ROS pathway. The o-quinone metabolites of PAHs are formed by enzymatic
dehydrogenation of dihydrodiols (Bolton etal.. 2000: Penning etal.. 1999: Harvey. 1996: ATSDR.
19951 (see Appendix D of the Supplemental Information). Dihydrodiol dehydrogenase enzymes are
members of the a-keto reductase gene superfamily. o-Quinone metabolites are potent cytotoxins,
are weakly mutagenic, and are capable of producing a broad spectrum of DNA damage. These
metabolites can interact directly with DNA as well as result in the production of ROS (i.e., hydroxyl
and superoxide radicals) that may produce further cytotoxicity and DNA damage. The
o-quinone/ROS pathway also can produce depurinated DNA adducts from benzo[a]pyrene
metabolites. In this pathway, and in the presence of NAD(P)+, aldo-keto reductase oxidizes
benzo[a]pyrene-7,8-diol to a ketol, which subsequently forms benzo[a]pyrene-7,8-dione. This and
other PAH o-quinones react with DNA to form unstable, depurinating DNA adducts. In the presence
of cellular reducing equivalents, o-quinones can also activate redox cycles, which produce ROS
fPenning etal.. 19961. DNA damage in in vitro systems following exposure to benzo[a]pyrene-
7,8-dione or other o-quinone PAH derivatives occurs through the aldo-keto reductase (AKR)
pathway and can involve the formation of stable DNA adducts (Balu etal.. 20041. N-7 depurinated
DNA adducts (Mccoull etal.. 19991. DNA damage from ROS (8-oxo-7,8-dihydro-2'-deoxyguanosine
adducts) fPark etal.. 20061. and strand scission fFlowers etal.. 1997: Flowers etal.. 19961.
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Summary of genotoxicity and mutagenicity
The ability of metabolites of benzo[a]pyrene to cause mutations and other forms of DNA
damage in both in vivo and in vitro studies is well documented (see genotoxicity tables in
Appendix D in Supplemental Information). With metabolic activation (e.g., the inclusion of S9),
benzo[a]pyrene is consistently mutagenic in the prokaryotic Salmonella/Ames and Escherichia coli
assays. In mammalian in vitro studies, benzo[a]pyrene is consistently mutagenic and clastogenic,
and induces cell transformation both with and without metabolic activation. Cytogenetic damage in
the form of chromosomal aberrations (CAs), micronuclei (MN), sister chromatid exchanges (SCEs),
and aneuploidy are commonplace following benzo[a]pyrene exposure as are DNA adduct
formation, single-strand breaks (SSB), and induction of DNA repair and unscheduled DNA synthesis
(UDS). In vitro mammalian cell assays have been conducted in various test systems, including
human cell lines.
In the majority of in vivo studies, benzo[a]pyrene has tested positive in multiple species and
strains and under various test conditions for cell transformation, CAs, DNA adducts, DNA strand
breaks, MN formation, germline mutations, somatic mutations (H-ras, K-ras, p53, lacZ, hprt), and
SCEs. Human studies are available following exposures to PAH mixtures through cigarette smoke
or occupational exposure in which benzo[a]pyrene-specific DNA adducts have been detected, and it
has been demonstrated qualitatively thatbenzo[a]pyrene metabolites damage DNA in exposed
humans.
Experimental support for the hypothesized mode of action
EPA's Guidelines for Carcinogen Risk Assessment [Section 2.4; (U.S. EPA. 2005a)] describe a
procedure for evaluating mode-of-action data for cancer. A framework for analysis of mode of
action information is provided below, providing context for the key events depicted in Figure 1-7.
Strength, consistencyand specificity of association. An extensive database of in vitro and in
vivo studies demonstrating the genotoxicity and mutagenicity of benzo[a]pyrene following
metabolic activation provides supporting evidence of a mutagenic mode of action for
benzo[a]pyrene carcinogenicity (see Table 1-19 and Section D.5.1 of the Supplemental
Information). In vitro studies overwhelmingly support the formation of DNA adducts, mutagenesis
in bacteria, yeast, and mammalian cells, several measures of cytogenetic damage (CA, SCE, MN), and
DNA damage. In vivo systems in animal models are predominantly positive for somatic mutations
following benzo[a]pyrene exposure.
Strong evidence links the benzo[a]pyrene diol epoxide metabolic activation pathway with
key mutational events in genes that are associated with tumor initiation (i.e., mutations in the p53
tumor suppressor gene and H-ras or K-ras oncogenes) (Table 1-19). The mutagenic potency of the
benzo[a]pyrene diol epoxide BPDE (and specifically (+)-anti-BPDE) has been confirmed in
mutagenicity assays in bacterial and in vitro mammalian systems fMalaveille etal.. 1977: Newbold
and Brookes. 1976). The major BPDE-DNA adducts observed in vivo are formed through reaction
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of BPDE with the exocyclic amino groups of deoxyguanosine and deoxyadenosine, with a
preference for forming the stable benzo[a]pyrene-7,8-diol-9,10-epoxide-N2-deoxyguanosine
(BPdG) adducts fXue and Warshawskv. 2005: Teffrev etal.. 1976: Daudel etal.. 19751. BPdG
adducts form in human cells and mouse skin (Grover etal.. 1976: Osborne etal.. 19761. and were
first chemically confirmed in humans in placental tissues fManchester et al.. 19881.
G^T transversions, displaying strand bias, are the predominant type of mutations caused
by benzo[a]pyrene in several biological systems (Liu etal.. 2005: Hainaut andPfeifer. 2001:
Marshall etal.. 19841 and sites of DNA adduction at guanine positions in cultured human HeLa cells
or plasmids containing the human p53 gene exposed to benzo[a]pyrene diol epoxide correspond to
p53 mutational hotspots observed in human lung cancers fDenissenko etal.. 1996: Puisieux etal..
19911. Mutational hotspots have been linked to regions of inefficient nucleotide excision repair of
BPDE-DNA adducts, both in vitro in hprt in human fibroblasts fWei etal.. 19951 and p53 in human
bronchial epithelial BEAS-2B cells, and in vivo in nontumorous lung tissue from smokers with lung
cancer (Hussain etal.. 20011. Results in support of a mutagenic mode of action via benzo[a]pyrene
diol epoxide include observations of frequent G^T transversion mutations in p53 and ras genes in
lung tumors of human cancer patients exposed to coal smoke (Keohavong etal.. 2003: Demarini et
al.. 20011. In addition, mice exposed to benzo[a]pyrene in the diet fCulp etal.. 20001 or by i.p.
injection (Nesnow et al.. 1998a: Nesnowetal.. 1998b: Nesnowetal.. 1996.1995: Mass etal.. 19931
had forestomach or lung tumors, respectively, showing BPdG adduct formation and frequent G^T
or C transversions in the K-ras gene.
An experimental challenge to the hypothesized mode of action further strengthens the
association between the diol epoxide pathway, DNA adduct formation, and tumorigenesis.
Isothiocyanates, reported to inhibit lung tumorigenesis in mice treated with benzo[a]pyrene, were
also observed to significantly reduce conversion of benzo[a]pyrene by CYP450 enzymes to the
benzo[a]pyrene-7,8-diol, and significantly reduce formation of BPDE-DNA adducts in the lung and
liver of female A/J mice exposed via gavage fSticha etal.. 20001. Other supporting evidence
includes observations of elevated BPDE-DNA adduct levels in WBCs of groups of coke oven workers
and chimney sweeps, occupations with known elevated risks of cancer (Vineis etal.. 2007:
Pavanello etal.. 2006: Pavanello etal.. 2005: Pavanello etal.. 2004: Pavanello etal.. 1999: Roias et
al.. 1998: Roias etal.. 19951. and in lung tissue from tobacco smokers with lung cancer (Roias etal..
2004: Godschalketal.. 2002: Roias etal.. 1998: Andreassenetal.. 1996: Alexandrovetal.. 19921.
Several epidemiological studies have indicated that PAH-exposed individuals who are homozygous
for a CYP1A1 polymorphism, which increases the inducibility of this enzyme (thus increasing the
capacity to produce benzo[a]pyrene diol epoxide), have increased levels of PAH or BPDE-DNA
adducts (Aklillu etal.. 2005: Alexandrov et al.. 2 0 0 2: Bartsch etal.. 2000: Perera and Weinstein.
20001.
Support for the o-quinone/ROS pathway contributing to tumor initiation via mutagenic
events includes in vitro demonstration that several types of DNA damage can occur from
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o-quinones and ROS (Park etal.. 2006: Balu etal.. 2004: Mccoull etal.. 1999: Flowers etal.. 1997:
Flowers etal.. 19961. In addition, benzo[a]pyrene-7,8-dione can induce mutations in the p53 tumor
suppressor gene using an in vitro yeast reporter gene assay fPark etal.. 2008: Shen etal.. 2006: Yu
etal.. 20021. and dominant p53 mutations induced by benzo[a]pyrene-7,8-dione in this system
corresponded to p53 mutational hotspots observed in human lung cancer tissue fPark etal.. 20081.
Dose-response concordance and temporal relationship. Studies in humans demonstrating
thatbenzo[a]pyrene-induced mutational events in p53 or ras oncogenes precede tumor formation
are not available, but there is evidence linking benzo[a]pyrene exposure to signature mutational
events in humans. In vitro exposure of human p53 knock-in murine fibroblasts to 1 |iM
benzo[a]pyrene for 4-6 days induced p53 mutations with similar features to those identified in p53
mutations in human lung cancer; i.e., predominance of G^T transversions with strand bias and
mutational hotspots atcodons 157-158 fLiu etal.. 20051. Anti-BPDE exposure in vitro activated
the cloned human c-Ha-ras-1 proto-oncogene and covalently bound to DNA, preferentially forming
adducts at the N2 position of guanine (Marshall etal.. 19841.
Bennett etal. (19991 demonstrated a dose-response relationship between smoking history/
intensity and the types of p53 mutations associated with benzo[a]pyrene (G^T transversions) in
human lung cancer patients (Table 1-19). In lung tumors of nonsmokers, 10% of p53 mutations
were G^T transversions, versus 40% in lung tumors from smokers with >60 pack-years of
exposure.
Skin and forestomach tumors in mice showed a dose-response relationship, including
temporal variation, with levels of BPDE-DNA adducts (Table 1-19). In a study using mice treated
dermally with benzo[a]pyrene once or twice per week for up to 15 weeks (10, 25, or 50 nmol
benzo[a]pyrene per application), levels of benzo[a]pyrene-DNA adducts in the skin, lung, and liver
increased with increasing time of exposure and increasing dose levels fTalaska etal.. 20061. Levels
at the end of the exposure period were highest in the skin; levels in the lung and liver at the same
time were 10- and 20-fold lower, respectively. The dose-response for benzo[a]pyrene-DNA adducts
in skin and lung increased in an apparent biphasic, curvilinear, manner with increasing steepness at
the mid to high exposure concentrations.
Mice exposed dermally to benzo[a]pyrene followed by the promoter 12-O-tetradecanoyl-
phorbol 13-acetate (TPA) for 26 weeks were found to have c-Ha-ras mutations in normal-looking
and hyperplastic lesions as well as in tumors fWei etal.. 19991. These mutations increased in
frequency from normal to hyperplastic to neoplastic skin samples, indicating that activation of the
c-Ha-ras proto-oncogene may precede tumorigenesis. Although the c-Ha-ras mutation did not
appear in all skin tumors tested (21% were negative), the vast majority of these mutations detected
in tumors (74 and 61% for the low- and high-dose groups, respectively) were G^T transversions.
Another study examined the dose-response relationship and the time course of
benzo[a]pyrene-induced skin damage (Table 1-19), DNA adduct formation, and tumor formation in
female mice. Mice were treated dermally with 0,16, 32, or 64 |ig of benzo[a]pyrene once per week
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for 29 weeks (Albert etal.. 19911. Indices of skin damage and levels of BPDE-DNA adducts in skin
reached plateau levels in exposed groups by 2-4 weeks of exposure. With increasing dose level,
levels of BPDE-DNA adducts (fmol/ng DNA) initially increased in a linear manner and began to
plateau at doses >32 ng/week. Tumors began appearing after 12-14 weeks of exposure for the
mid- and high-dose groups and at 18 weeks for the low-dose group. At study termination
(35 weeks after start of exposure), the mean number of tumors per mouse was approximately one
per mouse in the low- and mid-dose groups and eight per mouse in the high-dose group. The time-
course data indicate thatbenzo[a]pyrene-induced increases in BPDE-DNA adducts preceded the
appearance of skin tumors, consistent with the formation of DNA adducts as a precursor event in
benzo[a]pyrene-induced skin tumors. A follow-up to this study by the same authors f Albert etal..
19961 measured DNA adducts, necrosis, and inflammation (marked by an increase in leukocytes) in
the skin of treated mice after 5 weeks of dermal exposure. In the 64 [ig/week dose group,
statistically elevated levels of DNA adducts, inflammation, and necrosis were reported; however, in
the lower dose group (16 (ig/week), DNA adducts were statistically significantly elevated without
increases in inflammation and necrosis.
Culp and Beland (19941 demonstrated a dose-dependent increase in DNA adducts in the
livers, lungs, and forestomachs of male B6C3Fi mice fed coal tar for 28 days or benzo[a]pyrene for
21 days. Culp and Beland (19941 then compared dose-response relationships for BPDE-DNA
adducts and tumors in female B6C3Fi mice exposed to benzo[a]pyrene in the diet at 0,18.5, 90, or
350 ng/day for 28 days (to examine adducts) or 2 years (to examine tumors) (Table 1-19). The
benzo[a]pyrene dose-tumor response data showed a sharp increase in forestomach tumor
incidence between the 18.5 ng/day group (6% incidence) and the 90 ng/day group (78%
incidence). The BPDE-DNA adduct levels in the forestomach showed a relatively linear dose-
response throughout the benzo[a]pyrene dose range tested. The appearance of increased levels of
BPDE-DNA adducts in the target tissue at 28 days is temporally consistent with the contribution of
these adducts to the initiation of forestomach tumors. Furthermore, about 60% of the examined
tumors had mutations in the K-ras oncogene at codons 12 and 13, which were G^T or G^C
transversions indicative of BPDE reactions with DNA (Culp etal.. 19961. In addition to forestomach
tumors, the 2-year oral exposures to benzo[a]pyrene also resulted in an increased incidence of
tumors of the esophagus, tongue, and larynx in female mice (Beland and Culp. 19981.
Biological plausibility and coherence. The evidence for a mutagenic mode of action for
benzo[a]pyrene is consistent with the current understanding that mutations in p53 and ras
oncogenes are associated with increased risk of tumor initiation (Table 1-19). The benzo[a]pyrene
database is internally consistent in providing evidence for the formation of BPDE-DNA adducts and
BPDE-induced mutations associated with tumor initiation in cancer tissue from humans exposed to
complex mixtures containing benzo[a]pyrene (Keohavongetal.. 2003: Pfeifer and Hainaut. 2003:
Pfeifer etal.. 2002: D emarini et al.. 2 0 01: Hainaut and Pfeifer. 2001: Bennett etal.. 19991. in animals
exposed to benzo[a]pyrene fCulp etal.. 2000: Nesnow etal.. 1998a: Nesnow etal.. 1998b: Nesnow
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etal.. 1996.1995: Mass etal.. 1993). and in in vitro systems (Denissenko etal.. 1996: Puisieux etal..
19911. Consistent supporting evidence includes: (1) elevated BPDE-DNA adduct levels in tobacco
smokers with lung cancer fRoias etal.. 2004: Godschalketal.. 2002: Roias etal.. 1998: Andreassen
etal.. 1996: Alexandrov etal.. 19921: (2) demonstration of dose-response relationships between
G^T transversions in p53 mutations in lung tumors and smoking intensity (Bennett etal.. 19991:
(3) the extensive database of in vitro and in vivo studies demonstrating the ge no toxicity and
mutagenicity of benzo[a]pyrene following metabolic activation; and (4) general consistency
between temporal and dose-response relationships for BPDE-DNA adduct levels and tumor
incidence in studies of animals exposed to benzo[a]pyrene (Culp etal.. 1996: Albert etal.. 19911.
There is also supporting evidence that contributions to tumor initiation through mutagenic events
can be made by the radical cation (Chakravarti et al.. 1995: Rogan etal.. 19931 and o-quinone/ROS
metabolic activation pathways fPark etal.. 2008: Park etal.. 2006: Shen etal.. 2006: Balu etal..
2004: Yu etal.. 2002: Mccoull etal.. 1999: Flowers etal.. 1997: Flowers etal.. 19961.
Table 1-19. Experimental support for the postulated key events for mutagenic
mode of action
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 induce key events:
• Metabolism of benzo[a]pyrene via all three pathways has been demonstrated in multiple in vitro
studies, and the diol epoxide and radical cation metabolic activation pathways have been demonstrated
in in vivo studies in humans and animals (see Summary of 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: Pavanello et al.. 2004: Alexandrov et al.. 2002: Perera
and Weinstein, 2000)
2. Direct DNA damage by the reactive 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 which 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, CAs, MN formation, and SCE in animals (Appendix D 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., 1996:
Talaska et al., 1996: Culp and Beland, 1994: Albert et al., 1991)
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• Treatment with isothiocyanates, which inhibit the biotransformation of benzo[a]pyrene to the 7,8-diol
and BPDE-DNA adduct formation, also inhibits lung tumorigenesis in mice exposed to benzo[a]pyrene
(Sticha et al., 2000)
• 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:
Jeffrey 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.. 2006: Balu et al.. 2004: Mccoull et al.. 1999: Flowers et al..
1997: Flowers et al.. 1996)
Human evidence that key events are necessary for carcinogenesis:
• Detection of benzo[a]pyrene diol epoxide-specific DNA adducts is associated with increased cancer risk
in humans that are occupationally exposed (see Evidence in Humans section)
• These benzo[a]pyrene diol epoxides formed BPDE-DNA adducts preferentially at guanine residues that
have been detected in tissues of humans with cancer who were exposed to PAHs (Vineis and Perera,
2007: Roias et al.. 2004: Godschalk et al.. 2002: Li et al.. 2001: Pavanello et al.. 1999: Roias et al.. 1998:
Andreassen et al.. 1996: 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 induce 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 tumors (Culp et al..
2000: Nesnow et al.. 1998a: Nesnow et al.. 1998b: Nesnow et al.. 1996,1995: Mass et al.. 1993)
• Multiple studies in vivo and in vitro 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.. 1996)
(Chakravarti et al.. 1995: Ruggeri et al.. 1993)
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) (Liu et al.. 2005: Keohavong et al.. 2003: Pfeifer and Hainaut, 2003: Pfeifer et al..
2002: Demarini et al.. 2001: Hainaut and Pfeifer. 2001: Bennett et al.. 1999: Denissenko et al.. 1996:
Puisieux et al.. 1991: Marshall et al.. 1984: Koreeda et al.. 1978: Jeffrey et al.. 1976)
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4. Clonal expansion of mutated cells during the promotion and progression phases of cancer development
Evidence that benzo[a]pyrene metabolites induce key events:
• Benzo[a]pyrene has been shown to be a complete carcinogen, in that skin tumors in mice, rats, rabbits,
and guinea pigs have been associated with repeated application of benzo[a]pyrene to skin in the
absence of exogenous promoters (IPCS. 1998: Sivak et al.. 1997: ATSDR. 1995: Grimmer et al.. 1984:
Habs etal.. 1984: Grimmer et al.. 1983: IARC. 1983: Habs etal.. 1980: Schmahl etal.. 1977: IARC. 1973:
Schmidt et al.. 1973: Roe et al.. 1970: Poel. 1963.1959)
• 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)
• AhR activation by PAHs (including benzo[a]pyrene) upregulates genes responsible for tumor promotion
and increases tumor incidence in mice (Ma and Lu. 2007: Talaska et al.. 2006: Shimizu et al.. 2000)
Other possible modes of action
The carcinogenic process for benzo[a]pyrene is likely to be related to some combination of
molecular events resulting from the formation of several reactive metabolites that interact with
DNA to form adducts and produce DNA damage resulting in mutations in cancer-related genes, such
as tumor suppressor genes or oncogenes. These events may reflect the initiation potency of
benzo[a]pyrene. However, benzo[a]pyrene possesses promotional capabilities that may be related
to AhR affinity, immune suppression, cytotoxicity and inflammation (including the formation of
ROS), as well as the inhibition of gap junctional intercellular communication (GJIC).
The ability of certain PAHs to act as initiators and promoters may increase their
carcinogenic potency. The promotional effects of PAHs appear to be related to AhR affinity and the
upregulation of genes related to growth and differentiation (Bostrom etal.. 20021. The genes
regulated by this receptor belong to two major functional groups (i.e., induction of metabolism or
regulation cell differentiation and proliferation). PAHs bind to the cytosolic AhR in complex with
heat shock protein 90. The ligand-bound receptor is then transported to the nucleus in complex
with the AhR nuclear translocator protein. The AhR complex interacts with AhR elements of DNA
to increase the transcription of proteins associated with induction of metabolism and regulation of
cell differentiation and proliferation. Following benzo[a]pyrene exposure, Ah-responsive mice
were more susceptible to tumorigenicity in target tissues such as liver, lung, and skin as compared
to Ah-unresponsive mice (Ma and Lu. 2007: Talaska etal.. 2006: Shimizu etal.. 20001.
Benzo[a]pyrene has both inflammatory and immunosuppressive effects that may function
to promote tumorigenesis. Inflammatory responses to cytotoxicity may contribute to the tumor
promotion process; for example, benzo[a]pyrene quinones (1,6-, 3,6-, and 6,12-benzo[a]pyrene-
quinone) generated ROS and increased cell proliferation by enhancing the epidermal growth factor
receptor pathway in cultured breast epithelial cells (Burdick et al.. 20031. In addition, several
studies have demonstrated that exposure to benzo[a]pyrene increases the production of
inflammatory cytokines, which may contribute to cancer progression (N'Diave etal.. 2006: T amaki
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etal.. 2004: Garcon etal.. 2001b: Garcon et al.. 2001a: Albert etal.. 1996: Albert etal.. 19911. One of
these studies, Albert etal. (19961. measured DNA adducts, necrosis, and inflammation (marked by
an increase in leukocytes) in the skin of benzo[a]pyrene-treated mice after 5 weeks of dermal
exposure. In the highest dose group, statistically elevated levels of DNA adducts, inflammation, and
necrosis were reported; however, in the lower dose group, DNA adducts were statistically
significantly elevated without increases in inflammation and necrosis. It is likely that inflammation
promotes the formation of tumors at high doses of benzo[a]pyrene.
In addition to inflammation, immunosuppressive effects of benzo[a]pyrene have been noted
(as reviewed in. Zaccaria and Mcclure. 20131. Immune effects of benzo[a]pyrene exposure (see
Section 1.1.3) may provide an environment where tumor cells can evade detection by immune
surveillance mechanisms normally responsible for recognizing and eliminating nascent cancer cells
fHanahan and Weinberg. 20111. In addition, the developing fetus may be even more sensitive to
these effects; Urso and Gengozian (19801 found that mice exposed to benzo[a]pyrene in utero not
only had a significantly increased tumor incidence as adults, but also had a persistently suppressed
immune system.
Gap junctions are channels between cells that are crucial for differentiation, proliferation,
apoptosis, and cell death. Interruption of GJIC is associated with a loss of cellular control of growth
and differentiation, and consequently with the two epigenetic steps of tumor formation, promotion
and progression. Thus, the inhibition of gap junctional intercellular communication by
benzo[a]pyrene, observed in vitro (Sharovskava etal.. 2006: Blaha etal.. 20021. provides another
mechanism of tumor promotion.
In summary, there are tumor-promoting effects of PAH exposures that are not mutagenic.
Although these effects are observed following benzo[a]pyrene-specific exposures, the occurrence of
BPDE-DNA adducts and associated mutations that precede both cytotoxicity and tumor formation
and increase with dose provides evidence that mutagenicity is the primary event that initiates
tumorigenesis following benzo[a]pyrene exposures. A biologically plausible mode of action may
involve a combination of effects induced by benzo[a]pyrene, with mutagenicity as the initiating
tumorigenic event. Subsequent AhR activation and cytotoxicity could then lead to increased ROS
formation, regenerative cell proliferation, and inflammatory responses, which, along with evasion
of immune surveillance and GJIC, would provide an environment where the selection for mutated
cells increases the rate of mutation, allowing clonal expansion and progression of these tumor cells
to occur. However, it was determined that, in comparison to the large database on the mutagenicity
of benzo[a]pyrene, there were insufficient data to develop a separate mode of action analysis for
these promotional effects.
Conclusions about the hypothesized mode of action
There is sufficient evidence to conclude that the major mode of action for benzo[a]pyrene
carcinogenicity involves mutagenicity mediated by DNA reactive metabolites. The evidence for a
mutagenic mode of action for benzo[a]pyrene is consistent with the current understanding that
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mutations in p53 and ras oncogenes are associated with increased risk of tumor initiation. The
benzo[a]pyrene database provides strong and consistent evidence for BPDE-induced mutations
associated with tumor initiation in cancer tissue from humans exposed to complex mixtures
containing benzo[a]pyrene, in animals exposed to benzo[a]pyrene, and in in vitro systems.
Supporting evidence suggests that contributions to tumor initiation through potential mutagenic
events can be made by the radical cation and o-quinone/ROS metabolic activation pathways. Other
processes may contribute to the carcinogenicity of benzo[a]pyrene via the promotion and
progression phases of cancer development (e.g., inflammation, cytotoxicity, sustained regenerative
cell proliferation).
Support for the Hypothesized Mode of Action in Test Animals
Benzo[a]pyrene induces gene mutations in a variety of in vivo and in vitro systems and
produces tumors in all animal species tested and by all routes of exposure (see Appendix D in
Supplemental Information). Strong, consistent evidence in animal models supports the postulated
key events: the metabolism of benzo[a]pyrene to DNA-reactive intermediates, the formation of
DNA adducts, the subsequent occurrence of mutations in oncogenes and tumor suppressor genes,
and the clonal expansion of mutated cells.
Relevance of the Hypothesized Mode of Action to Humans
A substantial database indicates that the postulated key events for a mutagenic mode of
action all occur in human tissues. Evidence is available from studies of humans exposed to PAH
mixtures (including coal smoke and tobacco smoke) indicating a contributing role for
benzo[a]pyrene diol epoxide in inducing key mutational events in genes that are associated with
tumor initiation (mutations in the p53 tumor suppressor gene and H-ras or K-ras oncogenes). The
evidence includes observations of a spectrum of mutations in ras oncogenes and the p53 gene in
lung tumors of human patients exposed to coal smoke or tobacco smoke that are similar to the
spectrum of mutations caused by benzo[a]pyrene diol epoxide in several biological systems,
including tumors from mice exposed to benzo[a]pyrene. Additional supporting evidence includes
correspondence between hotspots of p53 mutations in human lung cancers and sites of DNA
adduction by benzo[a]pyrene diol epoxide in experimental systems, and elevated BPDE-DNA
adduct levels in respiratory tissue of lung cancer patients or tobacco smokers with lung cancer.
Populations or Lifestages Particularly Susceptible to the Hypothesized Mode of Action
A mutagenic mode of action for benzo[a]pyrene-induced carcinogenicity is considered
relevant to all populations and lifestages. The current understanding of biology of cancer indicates
that mutagenic chemicals, such as benzo[a]pyrene, are expected to exhibit a greater effect in early
life exposure versus later life exposure (U.S. EPA. 2005b: Vesselinovitch etal.. 1979). The EPA's
Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens fU.S. EPA.
2005b) recommends the application of age-dependent adjustment factors (ADAFs) for carcinogens
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that act through a mutagenic mode of action. Since a determination thatbenzo[a]pyrene acts
through a mutagenic mode of carcinogenic action has been made, ADAFs should be applied along
with exposure information to estimate cancer risks for early-life exposure.
Toxicokinetic information suggest early lifestages may have lower levels of some CYP
enzymes than adults f Ginsberg etal.. 2004: Cresteil. 19981: thus, lower levels of mutagenic
metabolites may be formed in early lifestages. Though expression of bioactivating enzymes is
believed to be lower in the developing fetus and children, metabolism of benzo[a]pyrene still
occurs, as indicated by the detection of benzo[a]pyrene-DNA or protein adducts or urinary
metabolites (Naufal etal.. 2010: Ruchirawatetal.. 2010: Suter etal.. 2010: Mielzvriska etal.. 2006:
Perera etal.. 2005a: Tang etal.. 1999: Whvatt et al.. 19981. While expression of CYP enzymes is
lower in fetuses and infants, the greater liver to body mass ratio and increased blood flow to liver in
fetuses and infants may compensate for the decreased expression of CYP enzymes f Ginsberg etal..
20041. Activity of Phase II detoxifying enzymes in neonates and children is adequate for sulfation
but decreased for glucuronidation and glutathione conjugation (Ginsberg etal.. 20041. The
conjugation of benzo[a]pyrene-4,5-oxide with glutathione was approximately one-third less in
human fetal liver cytosol compared to adult liver cytosol (Pacifici et al.. 19881.
In addition, newborn or infant mice develop liver and lung tumors more readily than young
adult mice following acute i.p. exposures to benzo[a]pyrene (Vesselinovitch etal.. 19751. These
results indicate that exposure to benzo[a]pyrene during early lifestages presents additional risk for
cancer, compared with exposure during adulthood, despite lower metabolic activity in early
lifestages. Population variability in metabolism and detoxification of benzo[a]pyrene, in addition to
DNA repair capability, may affect cancer risk. Polymorphic variations in the human population in
CYP1A1, CYP1B1, and other CYP enzymes have been implicated as determinants of increased
individual cancer risk in some studies flckstadtetal.. 2008: Aklillu etal.. 2005: Alexandrov etal..
2002: Perera and Weinstein. 20001. Some evidence suggests that humans lacking a functional
GSTM1 gene have higher BPDE-DNA adduct levels and are thus at greater risk for cancer (Binkova
etal.. 2007: Vineis etal.. 2007: Pavanello etal.. 2005: Pavanello etal.. 2004: Alexandrov et al.. 2 0 0 2:
Perera and Weinstein. 20001. In addition, acquired deficiencies or inherited gene polymorphisms
that affect the efficiency or fidelity of DNA repair may also influence individual susceptibility to
cancer from environmental mutagens (Wang etal.. 2010: Ickstadtetal.. 2008: Binkova etal.. 2007:
Matullo etal.. 2003: Shen etal.. 2003: Cheng etal.. 2000: Perera and Weinstein. 2000: Wei etal..
2000: Amos etal.. 19991. In general, however, available support for the role of single
polymorphisms in significantly modulating human PAH cancer risk from benzo[a]pyrene or other
PAHs is relatively weak or inconsistent. Combinations of polymorphisms, on the other hand, may
be critical determinants of a cumulative DNA-damaging dose, and thus indicate greater
susceptibility to cancer from benzo[a]pyrene exposure (Vineis etal.. 20071.
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Analysis of Toxicogenomics Data
An analysis of pathway-based transcriptomic data was conducted to help inform the cancer
mode of action for benzo[a]pyrene (see the Supplemental Information for details of this analysis).
These data support a mutagenic and cellular proliferation mode of action that follows three
candidate pathways: aryl hydrocarbon signaling; DNA damage regulation of the G1 /S phase
transition; and/or Nrf2 regulation of oxidative stress. Specifically, the analysis showed that
benzo[a]pyrene may activate the AhR, leading to the formation of oxidative metabolites and
radicals, which may lead to oxidative damage and DNA damage. Subsequently, DNA damage can
occur and activate p53 and p53 target genes, including p21 and MDM2. In addition, the data
indicate that p53 signaling may be decreased under these conditions, as ubiquitin and MDM2 are
both upregulated, and work together to degrade p53. Furthermore, the transcriptional
upregulation of Cyclin D may result in enough Cyclin D protein to overcome the p21 inhibitory
competition for CDK4, allowing for Gl/S phase transition to occur. The data also support the
hypothesis that an upregulation of proliferating cell nuclear antigen (PCNA) in combination with
the upregulation of ubiquitin indicates that cells are moving towards the Gl/S phase transition.
Although the alterations to the Nrf2 pathway suggest cells are preparing for a pro-apoptotic
environment, there is no transcriptional evidence that the apoptotic pathways are being activated.
There are uncertainties associated with the available transcriptomics data. For instance,
the available studies only evaluate gene expression following benzo[a]pyrene exposure and do not
monitor changes in protein or metabolite expression, which would be more indicative of an actual
cellular state change. Further research is required at the molecular level to demonstrate that the
cellular signaling events being inferred from such data are actually operative and result in
phenotypic changes. In addition, this analysis relied upon two short-term studies that evaluated
mRNA expression levels in a single tissue (liver) and species (mouse) and were conducted at
relatively high doses.
1.2. SUMMARY AND EVALUATION
1.2.1. Weight of Evidence for Effects Other than Cancer
The weight of the evidence from human and animal studies indicates that the strongest
evidence for human hazards following benzo[a]pyrene exposure is for developmental toxicity
(including neurodevelopmental toxicity), reproductive toxicity, and, to a lesser extent,
immunotoxicity. Some supporting studies in humans exposed to PAH mixtures are available, which
utilize benzo[a]pyrene air monitoring data or report associations between particular health
endpoints and concentrations of benzo[a]pyrene-DNA adducts in blood or tissue. In general, the
available human studies report effects that are analogous to the effects observed in animal
toxicological studies (especially those regarding developmental and reproductive effects), and
provide qualitative, supplemental evidence for the effect-specific hazards identified in
Sections 1.1.1-1.1.3. The endpoint categories included in Section 1.1.4, Other Toxicities (i.e., liver,
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kidney, cardiovascular, and nervous system toxicity [in adult animals] had less robust evidence of
hazard from the available chronic or subchronic oral and inhalation exposure studies in the
benzo[a]pyrene database.
In animals, evidence of developmental toxicity (including developmental neurotoxicity) has
been observed across species and dosing regimens (noting that the potential for developmental
toxicity following combined gestational and neonatal exposure has not been tested). The available
evidence from mice and rats treated by gavage during gestation or in the early postnatal period
demonstrate developmental effects including decreased body weight, decreased embryo/fetal
survival, decreased fertility, atrophy of reproductive organs, abnormal neurophysiological
responses, and altered neurobehavioral outcomes (Chen etal.. 2012: Tules etal.. 2012: Sheng etal..
2010: Bouavedetal.. 2009a: McCallister etal.. 2008: Kristensenetal.. 1995: Mackenzie and
Angevine. 1981). Evidence of developmental toxicity has also been observed following inhalation
exposure in animals. Decreased embryo/fetal survival has been observed in rats exposed to
benzo[a]pyrene via inhalation during gestation fWormlev etal.. 2004: Archibong et al.. 20021.
Several studies in animals have indicated that oral exposure to benzo[a]pyrene in early life
may result in altered neurobehavioral outcomes and sensorimotor development fChen etal.. 2012:
Bouaved etal.. 2009a). Following inhalation exposure during gestation, testing at adult ages also
suggested possible changes in neurophysiological measures in rats and mice (Li etal.. 2012:
Wormlev etal.. 2004) and behavioral responses in mice (Li etal.. 2012). Long-lasting behavioral
effects may be irreversible and have been consistently observed after exposure to benzo[a]pyrene
across species, and across exposure and testing paradigms. Although a mode of action has not been
defined, these alterations are supported by mechanistic changes in levels of brain monoamine
neurotransmitters and NMDA receptors, as well as increases in oxidative stress. Overall, the oral
and inhalation data support the conclusion that developmental toxicity (including developmental
neurotoxicity) is a human hazard following exposure to benzo[a]pyrene.
When considering the data on potential nervous system effects across lifestages, findings of
behavioral changes were largely coherent across studies of varied design. For example, similar
decrements were observed in Morris water maze tests after oral exposure to neonatal or adult rats
fChen etal.. 2012: Chengzhi etal.. 20111. i.p. exposure to weanling or pubertal rats fOiu etal.. 2011:
Tang etal.. 2011: Xia etal.. 20111. and i.p. exposure to adult mice fGrova etal.. 20071. Likewise,
consistent changes in elevated plus maze performance were observed after oral exposure to
neonatal rats or mice (Chen etal.. 2012: Bouaved et al.. 2009a) and i.p. exposure to adult mice
(Grova etal.. 2007). While the rodent behavioral alterations may relate to effects observed in
humans after prenatal or adult exposure to PAH mixtures, including changes in mood, short-term
memory, and sensorimotor-related responses, it is recognized that animals and humans may not
necessarily experience the same effects or functional changes fFrancis etal.. 19901. Overall, while
the adult neurotoxicity data are somewhat consistent with developmental neurotoxicity endpoints,
and considered suggestive of a hazard, these data were comparably less robust than the data
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supporting developmental neurotoxicity as a hazard, and additional studies are needed to identify
adult neurotoxicity as a human hazard of benzo[a]pyrene exposure (see Section 1.1.4). However, as
stated above, the data support neurotoxicity, based on developmental neurotoxicity, as a human
hazard of benzo[a]pyrene exposure.
In animals, evidence of reproductive toxicity has been observed across species and dosing
regimens. Male and female reproductive toxicity, as evidenced by effects on sperm parameters,
decreased reproductive organ weights, histological changes, and hormone alterations, have been
observed after oral exposure in rats and mice (Chen etal.. 2011: Chung etal.. 2011: Mohamed etal..
2010: Zheng etal.. 2010: Mackenzie and Angevine. 19811. Evidence of reproductive toxicity has
also been observed following inhalation exposure in animals. Male reproductive toxicity, as
evidenced by effects on sperm parameters, decreased testes weight, and hormone alterations, has
also been observed in rats following subchronic inhalation exposure to benzo[a]pyrene (Archibong
etal.. 2008: Ramesh etal.. 20081. Female reproductive toxicity, as evidenced by modified hormone
levels (Archibong etal.. 20021. as well as decreased ovulation and estrous cycle length (Archibong
etal.. 20121. has been observed following inhalation exposure. Overall, the oral and inhalation data
support the conclusion that reproductive toxicity is a human hazard following exposure to
benzo[a]pyrene.
Benzo[a]pyrene exposure has also been shown to lead to altered immune cell populations
and histopathological changes in immune system organs fKroese etal.. 2001: De long etal.. 19991.
as well as thymic and splenic effects following subchronic oral exposure. Varying immuno-
suppressive responses have also been observed in short-term oral and injection studies. Overall,
the available animal data support the conclusion that immunotoxicity is a potential human hazard
ofbenzo[a]pyrene exposure.
Effects in other organ systems were observed following benzo[a]pyrene exposure including
liver, kidney, and cardiovascular effects (see Section 1.1.4), but had less robust evidence of hazard
from the available chronic or subchronic exposure studies in the benzo[a]pyrene database, and are
discussed below.
Short-duration animal studies and studies by other routes of exposure (e.g., i.p. and
intratracheal instillation), as well as studies in genetically modified, highly susceptible animal
strains (e.g., APOE-/- mice) provide suggestive evidence of cardiovascular toxicity associated with
benzo[a]pyrene exposure. However, the interpretation of hazard is complicated by the paucity of
studies examining cardiovascular endpoints in humans and WT laboratory animals exposed by
environmentally relevant routes for subchronic or chronic durations. In addition, interpretation of
evidence of cardiovascular effects from human studies is complicated by issues of co-exposure in
populations highly exposed to benzo[a]pyrene as a component of a complex PAH mixtures. Thus,
considering the lower confidence in this hazard, the cardiovascular endpoints were not considered
further for dose-response.
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Evidence of liver effects following subchronic to chronic exposure to benzo[a]pyrene was
generally limited to increases in liver weight with little evidence for histological findings or other
indicators of hepatoxicity. Therefore, at this time, no conclusion is drawn regarding liver toxicity as
a human hazard of benzo[a]pyrene exposure induced toxicity.
Few studies are available to inform the potential of kidney effects after subchronic or
chronic exposure to benzo[a]pyrene. Confidence in the single subchronic study that observed an
apparent increase in kidney lesions in one sex of rats (Knuckles etal.. 20011 was decreased by
incomplete reporting of study methods and results (see Section 1.1.4). Therefore, at this time, no
conclusion is drawn regarding kidney toxicity as a human hazard of benzo[a]pyrene exposure.
1.2.2. Weight of Evidence for Carcinogenicity
Under EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005bl. benzo[a]pyrene is
"carcinogenic to humans." This guidance emphasizes the importance of weighing all of the evidence
in reaching conclusions about human carcinogenic potential. The descriptor of "carcinogenic to
humans" can be used when the following conditions are met: (a) there is strong evidence of an
association between human exposure and either cancer or the key precursor events of the agent's
mode of action but not enough for a causal association; (b) there is extensive evidence of
carcinogenicity in animals; (c) the mode or modes of carcinogenic action and associated key
precursor events have been identified in animals; and (d) there is strong evidence that the key
precursor events that precede the cancer response in animals are anticipated to occur in humans
and progress to tumors, based on available biological information. The data supporting these four
conditions for benzo[a]pyrene are presented below and in Table 1-20.
a) Strong Human Evidence of Cancer or its Precursors
There is a large body of evidence for human carcinogenicity for complex PAH mixtures
containing benzo[a]pyrene, including soot, coal tars, coal-tar pitch, mineral oils, shale oils, and
smoke from domestic coal burning (IARC. 2010: Baan etal.. 20091. There is also evidence of
carcinogenicity, primarily of the lung and skin, in occupations involving exposure to PAH mixtures
containing benzo[a]pyrene, such as chimney sweeping, coal gasification, coal-tar distillation, coke
production, iron and steel founding, aluminum production, and paving and roofing with coal tar
pitch flARC. 2010: Baan etal.. 2009: Straifetal.. 20051. Increased cancer risks have been reported
among other occupations involving exposure to PAH mixtures such as carbon black and diesel
exhaust (Benbrahim-T allaa et al.. 2 012: Bosetti etal.. 20071. There is extensive evidence of the
carcinogenicity of tobacco smoke, of which benzo[a]pyrene is a notable constituent The
methodologically strongest epidemiology studies (in terms of exposure assessment, sample size,
and follow-up period) provide consistent evidence of a strong association between benzo[a]pyrene
exposure and lung cancer. Three large epidemiology studies in different geographic areas,
representing two different industries, observed increasing risks of lung cancer with increasing
cumulative exposure to benzo[a]pyrene (measured in [ig/m3-years), with approximately a 2-fold
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increased risk at the higher exposures; each of these studies addressed potential confounding by
smoking (Armstrong and Gibbs. 2009: Spinelli etal.. 2006: Xu etal.. 19961 (Table 1-13). Although
the relative contributions of benzo[a]pyrene and of other PAHs cannot be established, the
exposure-response patterns seen with the benzo[a]pyrene measures make it unlikely that these
results represent confounding by other exposures. Similarly, for bladder cancer, two of the three
cohort studies with detailed exposure data observed an increasing risk with exposures
>80 [ig/m3-years (Gibbs and Sevignv. 2007a: Gibbs etal.. 2007: Gibbs and Sevignv. 2007b: Spinelli
etal.. 20061 (Table 1-15). The exposure range was much lower in the third study (Burstyn etal..
2007: Gibbs and Sevignv. 2007a: Gibbs etal.. 2007: Gibbs and Sevignv. 2007b). such that the highest
exposure group only reached the level of exposure seen in the lowest exposure categories in the
other studies. Data pertaining to non-melanoma skin cancer is limited to studies with more indirect
exposure measures, e.g., based on occupations with likely dermal exposure to creosote (i.e., timber
workers, brick makers, and power linesmen); the RR estimates seen in the four available studies
that provide risk estimates for this type of cancer ranged from 1.5 to 4.6, with three of these four
estimates >2.5 and statistically significant (Pukkala. 1995: Karlehagen etal.. 1992: Tornqvistetal..
1986: Hammond etal.. 1976). These four studies provide support for the association between
dermal PAH exposure, including benzo[a]pyrene exposure, and skin cancer. Although it is likely
that multiple carcinogens present in PAH mixtures contribute to the carcinogenic responses, strong
evidence is available from several studies of humans exposed to PAH mixtures supporting a
contributing role for benzo[a]pyrene diol epoxide in inducing key mutagenic precursor cancer
events in target tissues. Elevated BPDE-DNA adducts have been reported in smokers compared to
nonsmokers, and the increased adduct levels in smokers are typically increased 2-fold compared
with nonsmokers (Phillips. 2002). Elevated BPDE-DNA adduct levels have been observed in WBCs
of groups of coke oven workers and chimney sweeps, occupations with known elevated risks of
cancer (Roias etal.. 2000: Bartsch etal.. 1999: Pavanello etal.. 1999: Bartsch etal.. 1998: Roias et
al.. 19981. and in lung tissue from tobacco smokers with lung cancer fRoias etal.. 2004: Godschalk
etal.. 2002: Bartsch etal.. 1999: Roias etal.. 1998: Andreassenetal.. 1996: Alexandrovetal.. 1992).
Mutation spectra distinctive to diol epoxides have been observed in the tumor suppressor
gene p53 and the K-ras oncogene in tumor tissues taken from lung cancer patients who were
chronically exposed to two significant sources of PAH mixtures: coal smoke and tobacco smoke.
Hackmanetal. f20001 reported an increase of GC^TA transversions and a decrease of GC^AT
transitions at the hprt locus in T-lymphocytes of humans with lung cancer who were smokers
compared to nonsmokers. Lung tumors from cancer patients exposed to emissions from burning
smoky coal showed mutations in p53 and K-ras that were primarily G^T transversions (76 and
86%, respectively) (Demarini et al.. 2001). Keohavong et al. (2003) investigated the K-ras
mutational spectra from nonsmoking women and smoking men chronically exposed to emissions
from burning smoky coal, and smoking men who resided in homes using natural gas; among those
with K-ras mutations, 67, 86, and 67%, respectively, were G^T transversions. Lung tumors from
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tobacco smokers showed a higher frequency of p53 mutations that were G^T transversions
compared with lung tumors in nonsmokers (Pfeifer and Hainaut. 2003: Pfeifer etal.. 2002: Hainaut
and Pfeifer. 20011. and the frequency of these types of p53 mutations in lung tumors from smokers
increased with increasing smoking intensity (Bennett etal.. 19991.
Similarly, investigations of mutagenesis following specific exposures to benzo[a]pyrene (as
opposed to PAH mixtures) have consistently observed that the benzo[a]pyrene diol epoxide is very
reactive with guanine bases in DNA, and that G^T transversions are the predominant type of
mutations caused by benzo[a]pyrene diol epoxide in several biological test (Pfeifer and Hainaut.
2003: Hainaut and Pfeifer. 20011. Following treatment of human HeLa cells with benzo[a]pyrene
diol epoxide, Denissenko etal. f 19961 reported that the distribution of BPDE-DNA adducts within
p53 corresponded to mutational hotspots observed in p53 in human lung cancers. Benzo[a]pyrene
exposure induced mutations in embryonic fibroblasts from human p53 "knock-in" mice that were
similar to those found in smoking-related human cancers, with a predominance of G^T
transversions that displayed strand bias and were also located in the same mutational hotspots
found in p53 in human lung tumors (Liu etal.. 20051. These results, combined with a mechanistic
understanding that mutations in p53 (which encodes a key transcription factor in DNA repair and
regulation of cell cycle and apoptosis) may be involved in the initiation phase of many types of
cancer, are consistent with a common mechanism for mutagenesis following exposures to PAH
mixtures and provide evidence of a contributing role of benzo[a]pyrene diol epoxide in the
carcinogenic response of humans to coal smoke and tobacco smoke.
Therefore, while the epidemiological evidence alone does not establish a causal association
between human exposure and cancer, there is strong evidence that the key precursor events of
benzo[a]pyrene's mode of action are likely to be associated with tumor formation in humans.
bj Extensive Animal Evidence
In laboratory animals (rats, mice, and hamsters), exposures to benzo[a]pyrene via the oral,
inhalation, and dermal routes have been associated with carcinogenic responses both systemically
and at the site of administration. Three 2-year oral bioassays are available that associate lifetime
benzo[a]pyrene exposure with carcinogenicity at multiple sites. These bioassays observed
forestomach, liver, oral cavity, jejunum, kidney, auditory canal (Zymbal gland), and skin or
mammary gland tumors in male and female Wistar rats (Kroese etal.. 20011: forestomach tumors
in male and female Sprague-Dawley rats fBrune etal.. 19811: and forestomach, esophagus, tongue,
and larynx tumors in female B6C3Fi mice fBeland and Culp. 1998: Culp etal.. 19981. Repeated or
short-term oral exposure to benzo[a]pyrene was associated with forestomach tumors in additional
bioassays with several strains of mice (Wevand etal.. 1995: Benjamin et al.. 1988: Robinson etal..
1987: El-Bayoumv. 1985: Triolo etal.. 1977: Wattenberg. 1974: Roe etal.. 1970: Biancifiori etal..
1967: Chouroulinkov etal.. 1967: Fedorenko and Yansheva. 1967: Neal and Rigdon. 1967:
Berenblum and Haran. 19551. EPA has considered the uncertainty associated with the relevance of
forestomach tumors for estimating human risk from benzo[a]pyrene exposure. While humans do
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not have a forestomach, squamous epithelial tissue similar to that seen in the rodent forestomach
exists in the oral cavity and upper two-thirds of the esophagus in humans (IARC. 20031. Human
studies specifically associating exposure to benzo[a]pyrene with the alimentary tract tumors are
not currently available. However, benzo[a]pyrene-DNA adducts have been detected in oral and
esophageal tissue obtained from smokers freviewed by Phillips. 20021 and several epidemiological
studies have identified increased exposure to PAHs as an independent risk factor for esophageal
cancer (Abedi-Ardekani etal.. 2010: Szvmariska etal.. 2010: Gustavsson et al.. 1998: Liu etal..
19971. Thus, EPA concluded that forestomach tumors in rodents are relevant for assessing the
carcinogenic risk to humans.
Lifetime inhalation exposure to benzo[a]pyrene was associated primarily with tumors in
the larynx and pharynx of male Syrian golden hamsters exposed to benzo[a]pyrene:sodium
chloride aerosols fThvssen etal.. 19811. Additionally, less-than-lifetime oral exposure cancer
bioassays in mice provide supporting evidence that exposure to benzo[a]pyrene is associated with
an increased incidence of lung tumors in mice (Wevand etal.. 1995: Robinson etal.. 1987:
Wattenberg. 19741. In additional studies with hamsters, intratracheal instillation of
benzo[a]pyrene was associated with upper and lower respiratory tract tumors (Feron and Kruvsse.
1978: Ketkar etal.. 1978: Feron etal.. 1973: Henry etal.. 1973: Saffiotti et al.. 19721. Dermal
application of benzo[a]pyrene (2-3 times/week) has been associated with mouse skin tumors in
numerous lifetime bioassays fSivak etal.. 1997: Grimmer et al.. 1984: Habs etal.. 1984: Grimmer et
al.. 1983: Habs etal.. 1980: Schmahl etal.. 1977: Schmidt etal.. 1973: Roe etal.. 1970: Poel. 1963.
19591. Skin tumors in rats, rabbits, and guinea pigs have also been associated with repeated
application of benzo[a]pyrene to skin in the absence of exogenous promoters (IPCS. 1998: ATSDR.
1995: IARC. 1983.19731. When followed by repeated exposure to a potent tumor promoter, acute
dermal exposure to benzo[a]pyrene induced skin tumors in numerous studies of mice, indicating
that benzo[a]pyrene is a strong tumor-initiating agent in the mouse skin model (Wevand etal..
1992: Cavalieri etal.. 1991: Rice etal.. 1985: El-Bavoumvetal.. 1982: LaVoie etal.. 1982: Raveh et
al.. 1982: Cavalieri etal.. 1981: Slaga etal.. 1980: Wood etal.. 1980: Slaga etal.. 1978: Hoffmann et
al.. 19721.
Carcinogenic responses in animals exposed to benzo[a]pyrene by other routes of
administration include: (1) liver or lung tumors in newborn mice given acute postnatal i.p.
injections fLaVoie etal.. 1994: Busby etal.. 1989: Wevand and Lavoie. 1988: LaVoie etal.. 1987a:
Wislocki et al.. 1986: Busby etal.. 1984: Buening etal.. 1978: Kapitulnik et al.. 19781: (2) increased
lung tumor multiplicity in A/J adult mice given single i.p. injections fMass etal.. 19931: (3) injection
site tumors in mice following s.c. injection (Nikonova. 1977: Pfeiffer. 1977: Homburger etal.. 1972:
Roe and Waters. 1967: Grant and Roe. 1963: Steiner. 1955: Rask-Nielsen. 1950: Pfeiffer and Allen.
1948: Bryan and Shimkin. 1943: Barry etal.. 19351: (4) injection site sarcomas in mice following
intramuscular injection fSugivama. 19731: (5) mammary tumors in rats with intramammilary
administration f Cavalieri etal.. 1991: Cavalieri et al.. 1988c: Cavalieri etal.. 1988b: Cavalieri etal..
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1988a): (6) cervical tumors in mice with intravaginal application (Naslund etal.. 19871: and
(7) tracheal tumors in rats with intratracheal implantation (Topping etal.. 1981: Nettesheim etal..
19771.
Therefore, the animal database provides extensive evidence of carcinogenicity in animals.
c) Key Precursor Events have been Identified in Animals
There is sufficient evidence to conclude that benzo[a]pyrene carcinogenicity involves a
mutagenic mode of action mediated by DNA-reactive metabolites. The benzo[a]pyrene database
provides strong and consistent evidence for BPDE-induced mutations associated with tumor
initiation in cancer tissue from humans exposed to complex mixtures containing benzo[a]pyrene, in
animals exposed to benzo[a]pyrene, and in in vitro systems. Other processes may contribute to the
carcinogenicity of benzo[a]pyrene via the promotion and progression phases of cancer
development (e.g., inflammation, cytotoxicity, sustained regenerative cell proliferation, anti-
apoptotic signaling), but the available evidence best supports a mutagenic mode of action as the
primary mode by which benzo[a]pyrene acts.
dj Strong Evidence that the Key Precursor Events are Anticipated to Occur in Humans
Mutations in p53 and ras oncogenes have been observed in tumors from mice exposed to
benzo[a]pyrene in the diet (Culp etal.. 2000) or by i.p. injection (Nesnow et al.. 1998a: Nesnowet
al.. 1998b: Nesnow etal.. 1996.1995: Mass etal.. 19931. Mutations in these same genes have also
been reported in lung tumors of human cancer patients, bearing distinctive mutation spectra (G^T
transversions) that correlate with exposures to coal smoke (Keohavongetal.. 2003: Demarini etal..
2001) or tobacco smoke (Pfeifer and Hainaut. 2003: Pfeifer etal.. 2002: Hainautand Pfeifer. 2001:
Bennett etal.. 19991.
Table 1-20. Supporting evidence for the carcinogenic to humans cancer
descriptor for benzo[a]pyrene
Evidence
Reference
a) Strong human evidence of cancer or its precursors
• Increased risk of lung, bladder, and skin cancer
in humans exposed to complex PAH mixtures
containing benzo[a]pyrene
IARC (2004): IARC (2010): Secretan et al. (2009);Baan et
al. (2009): Benbrahim-Tallaa et al. (2012)
• Benzo[a]pyrene-specific biomarkers detected in
humans exposed to PAH mixtures associated
with increased risk of cancer
- BPDE-DNA adducts in WBCs of coke oven
workers and chimney sweeps
Bartsch et al. (1998): Bartsch et al. (1999): Pavanello et al.
(1999): Roias et al. (1998): Roias et al. (2000)
- BPDE-DNA adducts in smokers
Phillips (2002)
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Evidence
Reference
• Benzo[a]pyrene-specific DNA adducts have
been detected in 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 of eczema patients
treated with coal tar
Roias et al. (2004); Alexandrov et al. (1992); Bartsch et al.
(1999); Godschalk et al. (2002); Roias et al. (1998);
Andreassen et al. (1996); Godschalk et al. (1998)
- BPDE-DNA adduct formation in p53 in
human cells in vitro corresponds to
mutational hotspots at guanine residues in
human lung tumors
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 in exposed human-p53
knock-in mouse fibroblasts at the same
mutational hotspot in p53 from smoking-
related lung tumors in humans
Liu et al. (2005)
- G->T transversions at the same mutational
hotspot in p53 and K-ras in human lung
tumors associated with smoky coal
exposures
Demarini et al. (2001); Keohavong et al. (2003)
- Increased percentage of G->T
transversions in p53 in smokers versus
nonsmokers
Hainaut and Pfeifer (2001); Pfeifer et al. (2002); Pfeifer
and Hainaut (2003); Bennett et al. (1999)
b) Extensive animal evidence
Oral exposures
• Forestomach tumors in male and female rats
and in female mice following lifetime exposure
Kroese et al. (2001); Brune et al. (1981); Beland and Culp
(1998); Culpetal. (1998)
• Forestomach tumors in mice following less-
than-lifetime exposures
Beniamin et al. (1988); Berenblum and Haran (1955);
Biancifiori et al. (1967); Chouroulinkov et al. (1967); El-
Bavoumv (1985); Fedorenko and Yansheva (1967); Neal
and Rigdon (1967); Robinson et al. (1987); Roe et al.
(1970); Triolo et al. (1977); Wattenberg (1974); Weyand
et al. (1995)
• Alimentary tract and liver tumors in male and
female rats following lifetime exposure
Kroese et al. (2001)
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Evidence
Reference
• Kidney tumors in male rats following lifetime
exposure
Kroese et al. (2001)
• Auditory canal tumors in male and female rats
following lifetime exposure
Kroese et al. (2001)
• Esophageal, tongue, and laryngeal tumors in
female mice following lifetime exposure
Beland and Culp (1998); Culp et al. (1998)
• Lung tumors in mice following less-than-
lifetime exposure
Robinson et al. (1987); Wattenberg (1974); Wevand et al.
(1995)
Inhalation exposures
• Upper respiratory tract tumors in male
hamsters following chronic exposure
Thvssen et al. (1981)
Dermal exposures
• Skin tumors in mice following lifetime
exposures without a promoter
Grimmer et al. (1984); Grimmer et al. (1983); Habs et al.
(1984); Habs etal. (1980); Poel (1959); Poel (1963); Roe
et al. (1970); Schmahl et al. (1977); Schmidt et al. (1973);
Sivak et al. (1997)
• Skin tumors in rats, rabbits, and guinea pigs
following subchronic exposures
IPCS (1998); ATSDR (1995); IARC (1973); IARC (1983)
Other routes of exposure
• Respiratory tract tumors in hamsters following
intratracheal instillation
Feron et al. (1973); Feron and Kruysse (1978); Henry et al.
(1973); Ketkar et al. (1978); Saffiotti et al. (1972)
• Liver or lung tumors in newborn mice given
acute postnatal i.p. injections
Buening et al. (1978); Busby et al. (1984); Busby et al.
(1989); Kapitulnik et al. (1978); LaVoie et al. (1987a);
LaVoie et al. (1994); Wevand and Lavoie (1988); Wislocki
etal. (1986)
• Lung tumor multiplicity in A/J adult mice given
single i.p. injections
Mass etal. (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
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Evidence
Reference
• 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); Mass et al. (1993); Nesnow et al.
(1998a); Nesnow et al. (1998b); Nesnow et al. (1995);
Nesnow et al. (1996)
- G->T transversions in ras oncogenes or the
p53 gene have been observed in lung
tumors of human cancer patients exposed
to coal smoke
Demarini et al. (2001); Keohavong et al. (2003)
- Higher frequency of G->T transversions in
lung tumors from smokers versus
nonsmokers
Bennett et al. (1999); Hainaut and Pfeifer (2001); Pfeifer
et al. (2002); Pfeifer and Hainaut (2003)
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2. DOSE-RESPONSE ANALYSIS
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER
The oral reference dose (RfD) (expressed in units of mg/kg-day) is defined as an estimate
(with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. It can be derived from a no-observed-adverse-effect level
(NOAEL), lowest-observed-adverse-effect level (LOAEL), or the 95% lower bound on the
benchmark dose (BMDL), with uncertainty factors (UFs) generally applied to reflect limitations of
the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis
In Section 1.2.1, developmental, reproductive, and immunological toxicities were
highlighted as human hazards or potential human hazards of benzo[a]pyrene exposure by the oral
route. Studies within each effect category were evaluated using general study quality
characteristics (as discussed in Section 6 of the Preamble) to help inform the selection of studies
from which to derive toxicity values. Rationales for selecting the studies and effects to represent
each of these hazards are summarized below.
Human studies are preferred over animal studies when quantitative measures of exposure
are reported and the reported effects are determined to be associated with exposure. For
benzo[a]pyrene, human studies of environmental polycyclic aromatic hydrocarbon (PAH) mixtures
across multiple cohorts have observed effects following exposure to complex mixtures of PAHs.
The available data suggest that benzo[a]pyrene exposure may pose health hazards other than
cancer including reproductive and developmental effects such as infertility, miscarriage, and
reduced birth weight (Wu etal.. 2010: Neal etal.. 2008: Tangetal.. 2008: Perera etal.. 2005b:
Perera etal.. 2005a). effects on the developing nervous system (Perera etal.. 2012a: Perera etal..
20091. and cardiovascular effects fFriesen etal.. 2010: Burstvn etal.. 20051. However, the available
human studies that utilized benzo[a]pyrene-deoxyribonucleic acid (DNA) adducts as the exposure
metric do not provide external exposure levels of benzo[a]pyrene from which to derive a value, and
exposure is likely to have occurred by multiple routes. In addition, uncertainty exists due to
concurrent exposure to other PAHs and other components of the mixture (such as metals).
Animal studies were evaluated to determine which provided the most relevant routes and
durations of exposure; multiple exposure levels to provide information about the shape of the dose-
response curve; and power to detect effects at low exposure levels fU.S. EPA. 20021. The oral
database for benzo[a]pyrene includes a variety of studies and datasets that are suitable for use in
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deriving reference values. Specifically, chronic effects associated with benzo[a]pyrene exposure in
animals include observations of organ weight and histological changes and hematological
parameters observed in several oral cancer bioassays fKroese etal.. 2001: Beland and Culp. 19981.
Multiple subchronic studies are available that characterize a variety of effects other than cancer. In
addition, several developmental studies are available that help inform hazards of exposure during
sensitive developmental windows.
Developmental Toxicity
Numerous animal studies observed endpoints of developmental toxicity (including
developmental neurotoxicity) following oral exposure during gestational or early postnatal
development fChen etal.. 2012: Tules etal.. 2012: Bouaved etal.. 2009a: Kristensen etal.. 1995:
Mackenzie and Angevine. 19811 and were evaluated for dose-response analysis based on the above
considerations. As summarized in Section 1.1.1, two studies demonstrated decreased fertility
among gestationally exposed females (Kristensen etal.. 1995: Mackenzie and Angevine. 19811.
Kristensen et al. (19951. with only one dose group, was not considered further given its
concordance with Mackenzie and Angevine (19811. which had multiple groups. Mackenzie and
Angevine (19811 demonstrated developmental effects in a multi-dose study with relevant routes
and durations of exposure; however, the doses studied (10-160 mg/kg-day) were much higher
than doses evaluated in the other available developmental toxicity studies fChen etal.. 2012: Tules
etal.. 20121 and this study was therefore not considered further for RfD derivation. Similarly, the
developmental study by Bouaved et al. (2009a) used the same tests as Chen etal. (20121. but the
doses evaluated were higher (2 and 20 mg/kg-day compared to 0.02, 0.2, and 2 mg/kg-day,
respectively). From the available studies demonstrating developmental toxicity, the studies
conducted by Chen etal. (20121 and Tules etal. (20121 were identified as the most informative
studies for dose-response analysis to characterize effects in the low-dose region.
Tules etal. f20121 reported increases in both systolic (approximately 20-50%) and diastolic
(approximately 33-83%) blood pressure in adult rats that were exposed gestationally to
benzo[a]pyrene. Given the magnitude of the response and the appearance of these effects in
adulthood following gestational exposure, these endpoints were selected for dose-response
analysis; however, some uncertainty exists in the absence of other studies evaluating these
outcomes. The neurodevelopmental study by Chen etal. (20121 was a well-designed and well-
conducted study that evaluated multiple developmental endpoints and measures of neurotoxicity in
neonatal, adolescent, and adult rats after early postnatal exposure (see Section 1.1.1 for more
detail). Chen etal. (20121 observed increased locomotion in the open field test, increased latency in
negative geotaxis and surface righting tests, decreased anxiety-like behaviors in the elevated plus
maze test, and impaired performance in the Morris water maze test at various time points following
neonatal benzo[a]pyrene treatment. While an understanding of the underlying biological
perturbation(s) causing altered performance in these tests remains incomplete (see discussion in
Section 1.1.1), all of the results are interpreted to indicate an effect of benzo[a]pyrene treatment
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during development on neurobehavioral function. Since any behavioral effect observed in animals
is assumed to pose a potential hazard to humans, and because behavioral effects seen in animal
studies may not always be the same as those produced in humans (U.S. EPA. 1998a). all of the
observed behavioral changes are interpreted to be relevant for estimating potential neurotoxic
risks in humans.
Of the neurobehavioral endpoints observed in Chenetal. (20121. three behavioral tests—
the open field activity test, the elevated plus maze, and the Morris water maze—represent the most
compelling evidence of benzo[a]pyrene exposure-induced developmental neurotoxicity (see
discussion in Section 1.1.1) and were thus identified as the most informative neurobehavioral
endpoints for dose-response analysis.
Reproductive Toxicity
Male reproductive toxicity was demonstrated in a number of subchronic studies (Tengetal..
2013: Chenetal.. 2011: Chungetal.. 2011: Mohamed etal.. 2010: Zhengetal.. 20101. Chungetal.
f20111 was not included in the dose-response analysis because numerical data were not reported
or were only reported for the mid-dose of three doses. Chen etal. f20111. a subchronic study that
corroborated other available multi-dose studies, is considered supportive, but was not considered
for RfD derivation due to the use of a single dose level and limited reporting of numerical data. Teng
etal. (20131 observed effects (decreased testicular weight, decreases in sperm parameters) similar
to other studies in the database but at much higher doses. The studies conducted by Mohamed et
al. f20101 and Zheng etal. f20101 were identified as the most informative male reproductive
toxicity studies for dose-response analysis. Decreased sperm count and motility observed by
Mohamed etal. f20101 and decreased intratesticular testosterone levels observed by Zheng et al.
(20101 were selected for dose-response analysis as both represent sensitive endpoints of male
reproductive toxicity and are indicators of potentially decreased fertility. These effects are also
consistent with human studies in PAH-exposed populations, as effects on male fertility and semen
quality have been demonstrated in epidemiological studies of smokers freviewed by Spares and
Melo. 20081.
Female reproductive toxicity was demonstrated in two subchronic oral studies fGao etal..
2011: Xu etal.. 20101. Specifically, Xu etal. f20101 demonstrated decreased ovary weight and
follicle number, and Gao etal. (20111 reported an increase in inflammatory cells and hyperplasia in
the cervix following oral exposure to benzo[a]pyrene. These studies were identified as the most
informative studies on female reproductive toxicity for dose-response analysis.
Gao etal. f20111 identified statistically-significant, dose-related increases in the incidence
of cervical inflammatory cells and cervical hyperplasia in mice exposed to low doses of
benzo[a]pyrene for 98 days. Cervical effects of increasing severity (including apoptosis and
necrosis) were also observed at higher doses fGao etal.. 20111. This study also observed a
depression of body weight (10,15, and 30%) and elevated mortality in the two higher dose groups
(4 and 8%), suggesting potential treatment-related toxicity. Effects in the cervix have not been
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Toxicological Review of Benzo[a]pyrene
reported in other noncancer or cancer bioassays in the database; it is unclear whether the observed
cervical inflammation and hyperplasia are linked to impaired reproductive function. Although
there is some uncertainty in this endpoint, the histological lesion of cervical hyperplasia was the
most sensitive endpoint observed and was considered further for dose-response.
Xu etal. (20101 identified biologically and statistically significant decreases in ovary weight
and primordial follicles in treated animals. These reductions in female reproductive parameters
are supported by a large database of animal studies (including mechanistic studies as well as
studies using other routes of exposure or shorter durations), altogether indicating that
benzo[a]pyrene is ovotoxic with effects including decreased ovary weight, decreased primordial
follicles, and reduced fertility (Borman et al.. 2000: Kristensen etal.. 1995: Miller etal.. 1992:
Swartz and Mattison. 1985: Mackenzie and Angevine. 1981: Mattison et al.. 19801. Additionally,
epidemiology studies indicate that exposure to complex mixtures of PAHs, such as through cigarette
smoke, is associated with measures of decreased fertility in humans fNeal etal.. 2008: El-Nemr et
al.. 19981. Specific associations have also been made between infertility and increased levels of
benzo[a]pyrene in follicular fluid in women undergoing in vitro fertilization fNeal etal.. 20081.
Immunotoxicity
As described in Section 1.1.3, the immune system was identified as a potential human
hazard of benzo[a]pyrene exposure based on findings of decreased thymus weight and
immunoglobulin alterations, as well as effects on cellularity and functional changes in the immune
system in animals, and supporting data from mechanistic studies and short-term assays. The only
available oral repeat exposure studies to support development of an immune RfD were conducted
by Kroese etal. f20011 and De long et al. f!9991. These subchronic studies used multiple exposure
levels and typical sample sizes (10 or 8 rats/group, respectively). For the endpoint of decreased
thymus weight (observed in both studies), the Kroese etal. (20011 study is preferred due to its
longer duration (90 days compared to 35 days with De long etal.. 19991.
Functional evaluations of immune system response, thought to be more sensitive than
observational immune endpoints fWHO. 20121. were not evaluated in the oral, repeat exposure
studies available for benzo[a]pyrene. Therefore, the observational immune endpoints of decreased
thymus weight in Kroese etal. f20011 and decreased IgM and IgA levels and decreased relative
numbers of B cells in De long etal. (19991 were selected. It is recognized that thymus weight
changes on their own have been noted to be less reliable indicators of immunotoxicity (Luster etal..
19921. However, there are converging lines of evidence that support the use of this endpoint,
among others, as representative of benzo[a]pyrene immunotoxicity. Alterations in immunoglobulin
levels have been noted in humans after exposure to PAHs, as well as in animal studies after
exposure to benzo[a]pyrene. Changes in B cell populations in the spleen provide additional
evidence of immunotoxicity. Finally, functional effects on the immune system, including dose-
related decreases in sheep red blood cell (SRBC)-specific IgM levels and dose-dependent decreases
in resistance to pneumonia or Herpes simplex type 2 following short-term subcutaneous (s.c.)
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Toxicological Review of Benzo[a]pyrene
injection have been reported (Temple etal.. 1993: Munson et al.. 19851. The observed decreases in
thymus weight, IgM and IgA levels, and number of B cells associated with exposure to
benzo[a]pyrene were concluded to be representative of immunotoxicity following benzo[a]pyrene
exposure and were selected for dose-response analysis.
2.1.2. Methods of Analysis
Among the endpoints representing the hazards of benzo[a]pyrene exposure, the data for
neurobehavioral changes (i.e., changes in open field activity, elevated plus maze activity, and Morris
water maze performance) (Chen etal.. 2012). decreased ovary weight and follicle count (Xu etal..
2010). increased cervical hyperplasia Gao etal. (2011). and decreased thymus weight (Kroese etal..
20011 were judged to support dose-response modeling.
For the neurobehavioral tests f Chen etal.. 20121. identified as the most informative for
dose-response (i.e., open field activity, elevated plus maze, and Morris water maze), multiple
datasets were available (e.g., results for different lifestages, both sexes, and related metrics from the
same assay). Data for both sexes were modeled. In addition, for these tests, effects observed in
juveniles persisted and were more pronounced in adults; thus, the response measures in adulthood
were preferred. For the open field activity test, effects on horizontal locomotion in the open field
were more sensitive than rearing measures; thus, these data were preferred. In the elevated plus
maze, several related measures were reported, including increased number of open arm entries,
increased time spent in the open arms, and decreased closed arm entries; ultimately, the number of
entries into the open arms was used for dose-response analysis (for further discussion, see
Appendix E.l.l). For the Morris water maze, escape latency in the hidden platform trials was the
preferred measure of impaired performance as differences in probe trial performance were not
considered reliable since the treated animals never reached the same baseline level of proficiency
prior to testing.
As no biologically based dose-response models are available for benzo[a]pyrene, EPA
evaluated a range of dose-response models thought to be consistent with underlying biological
processes to determine how best to empirically model each dose-response relationship in the range
of observed data. In general, the models in EPA's Benchmark Dose Software (BMDS: U.S. EPA.
2012c) relevant to each data type (e.g., continuous or dichotomous) were applied. Additional
models were also considered when feasible. For example, the study design for escape latency in the
Morris water maze test Chen et al. f20121 involved repeated evaluations for individual animals
across several testing days (postnatal days [PNDs] 71-74), and could conceivably support repeated
measures models that would address intraindividual variation; however, the necessary individual
animal data were not available. Consequently, continuous models in BMDS were applied to the
reported results from Chen etal. (2012) for each day of testing (PNDs 71-74) and are included in
Table 2-1.
Consistent with EPA's Benchmark Dose Technical Guidance (U.S. EPA. 2012c). the
benchmark dose (BMD) and the 95% lower confidence limit on the BMD (BMDL) were estimated
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Toxicological Review of Benzo[a]pyrene
using a benchmark response (BMR) of 1 standard deviation (SD) from the control mean for
continuous data or a BMR of 10% extra risk for dichotomous data, in the absence of information
regarding what level of change is considered biologically important, and in order to facilitate a
consistent basis of comparison across endpoints, studies, and assessments. For outcomes with
information available regarding what level of change is considered biologically important, these
considerations informed BMR selection, as summarized below.
For reduced ovary weights, the standard deviation in the control group ofXu etal. (2010)
was approximately 10% of the control mean, similar to variability in historical controls for
Sprague-Dawley rats (e.g.. Marty et al.. 20091. Given that a 1 SD difference from the control mean of
normally distributed data is equivalent to a 10% increase in the number of individuals exceeding
the 2nd percentile of the control distribution (U.S. EPA. 2012c). a 1 SD change from control was
judged to represent a minimally important degree of change in ovary weights, and was used as the
BMR for this outcome.
For reduced follicle counts, a BMR of 10% relative deviation from control levels was judged
to represent a minimally important degree of change, by the following reasoning. There is no
consensus in the scientific community regarding what degree of change in follicle number is
biologically significant; in the absence of these data, it has been suggested that a detectable
decrease in follicle number be considered adverse (Heindel. 19981. Power analyses by Heindel
f!9981 provide a basis for inferring a critical level. More specifically, the power analyses focused
on identifying follicle counts reduced by >20%. Therefore, a 10% change in follicle count was
defined as a minimally important degree of change.
For escape latency, a 1 SD BMR based on the observed variability was used, taking into
account the repeated measures experimental design, following an evaluation of the reported
standard errors (SEs) across trial days and dose groups. Although there was a slight tendency of
the reported mean escape latencies in the control and mid-dose groups to decrease over the
4 successive days, the low- and high-dose groups showed no particular pattern (see Table E-l);
thus, all variability estimates were taken to be equally representative. An overall SD of 9 seconds
resulted both from an average of the control group SDs across trial days and from a grand average
of all of the SDs across trial days and dose groups.
Further details, including the modeling output and graphical results for the selected model
for each endpoint, can be found in Appendix E of the Supplemental Information.
For outcomes with sufficient data for modeling and that were successfully modeled, the
BMDLs were used to define points of departure (PODs). For the overall POD for neurobehavioral
effects, the BMDs of several behavioral tests (from the same study) were closely clustered. These
neurobehavioral endpoints represent behavioral changes in the same group of rats, following early
postnatal exposure to benzo[a]pyrene, which persisted into adulthood. Thus, the BMDLs
representing different behavioral manifestations of neurotoxicity were considered together to
define the POD for neurobehavioral changes.
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Other endpoints identified in Section 2.1.1 had insufficient data to support dose-response
modeling, but allowed identification of a NOAEL or LOAEL for use as PODs. Specifically, the data for
epididymal sperm counts in the Mohamedetal. f20101 study were reported only as percentages of
control responses, without actual control values; the variability in blood pressure measurements
(i.e., SE) in Tules etal. f20121 were reported inconsistently; and the observed decreases in IgM and
IgA fDe long et al.. 19991. although consistently depressed with exposure, showed highly
heterogeneous variability. These datasets were not amenable to dose-response modeling; thus, a
NOAEL or LOAEL was used as the POD.
Human equivalent doses (HEDs) for oral exposures were derived from the PODs estimated
from the laboratory animal data as described in EPA's Recommended Use of Body Weight3/4 as the
Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 20111. In this guidance, EPA
advocates a hierarchy of approaches for deriving HEDs from data in laboratory animals, with the
preferred approach being physiologically-based pharmacokinetic (PBPK) modeling. Other
approaches can include using chemical-specific information in the absence of a complete
pharmacokinetic (i.e., toxicokinetic) model. As discussed in Appendix D of the Supplemental
Information, several animal PBPK models for benzo[a]pyrene have been developed and published,
but a validated human PBPK model for benzo[a]pyrene for extrapolating doses from animals to
humans is not available. In lieu of either chemical-specific models or data to inform the derivation
of human equivalent oral exposures, a body weight scaling to the % power (i.e., BW3/4) approach is
applied to extrapolate toxicologically equivalent doses of orally administered agents from adult
laboratory animals to adult humans for the purpose of deriving an oral RfD.
Consistent with EPA guidance (U.S. EPA. 20111. the PODs estimated based on effects in adult
animals are converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived
as follows:
DAF = (BWa1/4 / BWh1/4),
where
BWa = animal body weight; and
BWh = human body weight.
Using BWa of 0.25 kg for rats and 0.035 kg for mice and BWh of 70 kg for humans fU.S. EPA.
19881. the resulting DAFs for rats and mice are 0.24 and 0.15, respectively. Applying this DAF to
the POD identified for effects in adult rats or mice yields a PODhed as follows (see Table 2-1):
PODhed = PODadj x DAF.
BW3/4 scaling was not employed for deriving HEDs from the developmental toxicity study
by Chen etal. f20121 in which doses were administered directly to early postnatal animals because
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Toxicological Review of Benzo[a]pyrene
of several areas of uncertainty. The first issue was whether allometric (i.e., BW3/4) scaling, derived
from data in adult animals, holds when extrapolating doses in neonatal animals. This uncertainty
arises because of the absence of quantitative information to characterize the toxicokinetic and
toxicodynamic differences between animals and humans in early lifestages (U.S. EPA. 20111. In
addition, interspecies extrapolation across early lifestages is also complicated by differences in
temporal patterns of development across species. U.S. EPA f20111 states that when such an
extrapolation is considered, key developmental processes need to be matched in a species-
dependent manner, because the temporal pattern of development differs across species. In the
study at issue, Chen etal. (20121. neurobehavioral changes were observed in adult rats after dosing
on PNDs 5-11. This postnatal period of brain development in rats is believed to be more akin to
human brain development occurring in the third trimester of pregnancy (Dobbingand Sands. 1979.
19731. thus challenging the suitability of extrapolating from rats directly exposed on PNDs 5-11 to
third trimester humans with transplacental exposure.
Table 2-1 summarizes the sequence of calculations leading to the derivation of a human-
equivalent POD for each data set discussed above.
Table 2-1. Summary of derivation of PODs
Endpoint and
reference
Species/
sex
Model3
BMR
BMD
mg/kg-d
BMDL
mg/kg-d
POD
mg/kg-d
PODadj"
mg/kg-d
PODhedc
mg/kg-d
Developmental
Neurobehavioral
changes: Open field
crossed squares at
PND69
Chen et al. (2012)
Male and
Female
Sprague-
Dawley
rats
Exponential 4
1SD
0.23
0.11
Neurobehavioral
changes: Elevated
plus maze open arm
entries at PND 70
Chen et al. (2012)
Female
Sprague-
Dawley
rats
Exponential 4
1SD
0.21
0.092
0.092d
0.092
0.092
Neurobehavioral
changes: Morris
water maze hidden
platform trial
escape latency at
PNDs 71-74
Chen et al. (2012)
Male and
Female
Sprague-
Dawley
rats
Hill CV
Hill CV
Hill CV
Hill NCV
1SD
(9 sec)
PND71: 0.49
PND72: 0.33
PND73: 0.27
PND74: 0.23
0.16
0.16
0.12
0.13
Cardiovascular
effects at PND 53
Jules et al. (2012)
Long-
Evans
rats
LOAEL (0.6 mg/kg-d)
(15% 1" in systolic blood pressure; 33% T* in
diastolic blood pressure)
0.6
0.6
0.15
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Endpoint and
reference
Species/
sex
Model3
BMR
BMD
mg/kg-d
BMDL
mg/kg-d
POD
mg/kg-d
PODadj"
mg/kg-d
PODhedc
mg/kg-d
Reproductive
Decreased ovary
weight
Xu et al. (2010)
Female
Sprague-
Dawley
rats
Linear
1SD
2.3
1.5
1.5
1.5
0.37
Decreased ovarian
follicle count
Xu et al. (2010)
Female
Sprague-
Dawley
rats
Linear
10%
RD
2.3
1.6
1.6
1.6
0.38
Decreased
intratesticular
testosterone
Zheng et al. (2010)
Male
Sprague-
Dawley
rats
NOAEL (1 mg/kg-d)
(15% 4/ in testosterone)
1
1
0.24
Decreased sperm
count and motility
Mohamed et al.
(2010)
Male
C57BL/6
mice
LOAEL (1 mg/kg-d)
(50% 4/ in sperm count; 20% 4^ in sperm
motility)
1
1
0.15
Cervical epithelial
hyperplasia
Gao et al. (2011)
Female
ICR mice
Log-logistic
10% ER
0.58
0.37
0.37
0.37
0.06
Immunological
Decreased thymus
weight
Kroese et al. (2001)
Female
Wistar
rats
Linear
1SD
10.5
7.6
7.6
7.6
1.9
Decreased thymus
weight
Kroese et al. (2001)
Male
Wistar
rats
Linear
1SD
16.4
11.3
11.3
11.3
2.7
Decreased IgM
levels
De Jong et al. (1999)
Male
Wistar
rats
NOAEL (10 mg/kg-d)
(14% 4, in IgM)
10
7.1
1.7
Decreased IgA levels
De Jong et al. (1999)
Male
Wistar
rats
NOAEL (30 mg/kg-d)
(28% 4, in IgA)
30
21
5.2
Decreased number
of B cells
De Jong et al. (1999)
Male
Wistar
rats
NOAEL (30 mg/kg-d)
(7% ^ in B cells at NOAEL; 31% 4, at LOAEL)
30
21
5.2
aFor modeling details, see Appendix E.l in Supplemental Information.
bFor studies in which animals were not dosed daily, PODs were adjusted to calculate the TWA daily doses following
BMD modeling, with the exception of Xu et al. (2010) and Gao et al. (2011), for which the TWA daily doses were
used for BMD modeling.
CHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011) for effects from dosing studies in adult animals
(i.e., Gao et al., 2011; Mohamed et al., 2010; Xu et al., 2010; De Jong et al., 1999) or for developmental effects
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Toxicological Review of Benzo[a]pyrene
resulting from in utero exposures (Jules et al., 2012). BW3/4 scaling was not employed for deriving HEDs from
studies in which doses were administered directly to early postnatal animals (i.e., Chen et al., 2012).
dThe POD for neurobehavioral changes based on the lower end of the BMDLs (i.e., 0.092-0.16 mg/kg-day) for
three sensitive behavioral measures.
RD = relative deviation; TWA = time-weighted average
ER = extra risk
2.1.3. Derivation of Candidate Values
Under EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
2002: Section 4.4.51. also described in the Preamble, five possible areas of uncertainty and
variability were considered. An explanation follows:
A UF for extrapolation from a LOAEL to NOAEL, UFl, of 1 was applied when the POD was
based on a NOAEL (Zheng etal.. 2010: De long etal.. 19991. A value of 1 was applied when a BMR of
a 1 SD (Chen etal.. 2012: Kroese etal.. 20011 or 10% change (Gao etal.. 20111 from the control was
selected under an assumption that it represents a minimal biologically significant response level. A
NOAEL was not determined for the most sensitive effects observed in Tules etal. (20121 and
Mohamed etal. f20101. At the LOAEL, Tules etal. f20121 observed statistically significant increases
in systolic (15%) and diastolic (33%) blood pressure when measured in adulthood following
gestational exposure. Regarding the study by Mohamed etal. (20101. the authors observed a
statistically significant decrease in sperm count (50%) and motility (20%) in treated F0 males at
the lowest dose tested. The data in this study were not reported sufficiently to enable dose-
response modeling, since the authors did not report a measure of the variability (SD or standard
error on the mean [SEM]) for the control group. Therefore, a UF of 10 was applied to approximate a
NOAEL for studies flules etal.. 2012: Mohamed etal.. 20101 that observed a high magnitude of
response at the LOAEL.
A subchronic-to-chronic uncertainty factor, UFs, of 10 was applied when the POD was based
on a subchronic study (the studies in Table 2-2, other than the two developmental toxicity studies,
were 42-90 days in duration) to account for the possibility that longer exposure may induce effects
at a lower dose. A UFs of 1 was applied when dosing occurred during gestation flules etal.. 20121
or the early postnatal period (Chen etal.. 20121. A UFs of 1 was applied for PODs from
developmental studies. The developmental period is recognized as a susceptible lifestage when
exposure during a time window of development is more relevant to the induction of developmental
effects than lifetime exposure (U.S. EPA. 1991c): therefore, an adjustment for duration is not
warranted.
An interspecies uncertainty factor, UFa, of 3 (101/2 = 3.16, rounded to 3) was applied to all
PODs in Table 2-2 except Chen et al. f20121. because BW3/4 scaling is being used to extrapolate oral
doses from laboratory animals to humans. Although BW3/4 scaling addresses some aspects of cross-
species extrapolation of toxicokinetic and toxicodynamic processes, some residual uncertainty
remains. In the absence of chemical-specific data to quantify this uncertainty, EPA's BW3/4 guidance
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Toxicological Review of Benzo[a]pyrene
(U.S. EPA. 20111 recommends use of a UF of 3. BW3/4 scaling was not employed for deriving HEDs
from studies in which doses were administered directly to early postnatal animals (i.e.. Chen etal..
20121: therefore, a value of 10 was applied to account for the absence of quantitative information to
characterize either the toxicokinetic or toxicodynamic differences between animals and humans at
this lifestage.
An intraspecies uncertainty factor, UFh, of 10 was applied to account for variability and
uncertainty in toxicokinetic and toxicodynamic susceptibility within the subgroup of the human
population most sensitive to the health hazards of benzo[a]pyrene (U.S. EPA. 20021. In the case of
benzo[a]pyrene, the PODs were derived from studies in inbred animal strains and are not
considered sufficiently representative of the exposure and dose-response of the susceptible human
subpopulations (in this case, the developing fetus). In certain cases, the toxicokinetic component of
this factor may be replaced when a PBPK model is available that incorporates the best available
information on variability in toxicokinetic disposition in the human population (including sensitive
subgroups). In the case of benzo[a]pyrene, insufficient information is available to quantitatively
estimate variability in human susceptibility; therefore, the full value for the UFh was retained.
A database uncertainty factor, UFd, 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, useful for understanding the potential for
benzo[a]pyrene exposure to cause reproductive and neurodevelopmental effects. Benzo[a]pyrene
has been shown to affect fertility in adult male and female animals by multiple routes of exposure,
and decreased fertility in adult male and female mice is observed both following premating
exposure and following gestational exposure (see Section 1.1.1. and 1.1.2). Therefore, it is plausible
that exposure occurring over a more comprehensive period of development or over multiple
generations could result in a more sensitive POD than the POD selected for developmental
neurotoxicity.
Some additional uncertainties exist in the benzo[a]pyrene database, including the paucity of
sensitive studies evaluating endpoints of immune and cardiovascular toxicity. The lack of
developmental immunotoxicity studies, especially those examining functional endpoints, is an
uncertainty in the benzo[a]pyrene database. Some consideration was given to cardiovascular
effects through the candidate value derived for developmental effects on the cardiovascular system
flules etal.. 20121.
The POD for the overall RfD was based on several sensitive neurobehavioral endpoints
observed following treatment during a sensitive period of brain development and were among the
lowest effect levels observed in the benzo[a]pyrene database, even among other developmental
studies utilizing low doses of benzo[a]pyrene; thus, application of a full UFd of 10 was not judged to
be warranted. However, because studies following a more comprehensive period of developmental
exposure (i.e., early gestation through lactation, if not through adolescence) were not available, a
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Toxicological Review of Benzo[a]pyrene
UFd of 3 was applied to help address residual uncertainty associated with the potential for effects at
lower doses.
Table 2-2 is a continuation of Table 2-1 and summarizes the application of UFs to each POD
to derive a candidate value for each data set. The candidate values presented in the table below are
preliminary to the derivation of the organ/system-specific reference values. These candidate
values are considered individually in the selection of a representative oral reference value for a
specific hazard and subsequent overall RfD for benzo[a]pyrene.
Table 2-2. Effects and corresponding derivation of candidate values
Endpoint and reference
PODhed
(mg/kg-d)
POD
type
UFl
UFs
UFa
UFh
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Developmental
Neurobehavioral changes in rats
Chen et al. (2012)
0.092
BMDLisd
1
1
10
10
3
300
3 x 10"4
Cardiovascular effects in rats
Jules et al. (2012)
0.15
LOAEL
10
1
3
10
3
1,000
2 x 10"4
Reproductive
Decreased ovary weight in rats
Xu et al. (2010)
0.37
BMDLisd
1
10
3
10
3
1,000
4 x 10"4
Decreased ovarian follicles in
rats
Xu et al. (2010)
0.38
BMDLiord
1
10
3
10
3
1,000
4 x 10"4
Decreased intratesticular
testosterone in rats
Zheng et al. (2010)
0.24
NOAEL
1
10
3
10
3
1,000
2 x 10"4
Decreased sperm count and
motility in mice
Mohamed et al. (2010)
0.15
LOAEL
10
10
3
10
3
10,000
Not
calculated
due to UF
>3,000a
Cervical epithelial hyperplasia in
mice
Gao et al. (2011)
0.06
BMDLio
1
10
3
10
3
1,000
6 x 10"5
Immunological
Decreased thymus weight in rats
Kroese et al. (2001)
1.9
BMDLisd
1
10
3
10
3
1,000
2 x 10"3
Decreased serum IgM in rats
De Jong et al. (1999)
1.7
NOAEL
1
10
3
10
3
1,000
2 x 10"3
Decreased serum IgA in rats
De Jong et al. (1999)
5.2
NOAEL
1
10
3
10
3
1,000
5 x 10"3
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Toxicological Review of Benzo[a]pyrene
Endpoint and reference
PODhed
(mg/kg-d)
POD
type
UFl
UFS
UFa
UFh
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Decreased number of B cells in
rats
De Jong et al. (1999)
5.2
NOAEL
1
10
3
10
3
1,000
5 x 10"3
aAs 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 UF in four or more areas of
extrapolation should be avoided.
Figure 2-1 presents graphically the candidate values, UFs, and PODs, with each bar
corresponding to one data setdescribed inTables 2-1 and 2-2.
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Toxicological Review of Benzo[a]pyrene
>
LU
o
o
3
Q
O
CH
<
y
O
O
Neurodevelopmental alterations in rats
(Chen etal., 2012)
Cardiovascular effects in rats
(Jules et al., 2012)
¦J, Ovary weight in rats
(Xu etal., 2010)
•J, Ovarian follicles in rats
(Xu etal., 2010)
4- Intratesticular testosterone in rats
(Zheng et al. 2010)
4 Sperm count in mice
(Mohamed et al., 2010)
Cervical epithelial hyperplasia
(Gao etal., 2011)
Thymus weight in rats
(Kroese et al., 2001)
4 Serum IgM in rats
(De Jong et al., 1999)
4- Serum IgA in rats
(De Jong et al., 1999)
-J/ Number of B cells in rats
(De Jong et al., 1999)
Composite UF
A Candidate value
• POD(HED)
0.00001 0.0001
0.001 0.01 0.1
Doses (mg/kg-d)
10
Figure 2-1. Candidate values with corresponding PODs and composite UFs.
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2.1.4. Derivation of Organ/System-Specific Reference Doses
Table 2-3 distills the candidate values from Table 2-2 into a single value for each organ or
system. These organ- or system-specific reference values may be useful for subsequent cumulative
risk assessments that consider the combined effect of multiple agents acting at a common site.
Table 2-3. Organ/system-specific RfDs and overall RfD for benzo[a]pyrene
Effect
Basis
RfD (mg/kg-d)
Study exposure
description
Confidence
Developmental
Neurobehavioral changes
3 x 10"4
Critical window of
development (postnatal)
Medium
Reproductive
Ovotoxicity (decreased primordial
follicles and ovary weight)
4 x 10"4
Subchronic
Medium
Immunological
Decreased thymus weight and serum
IgM
2 x 10"3
Subchronic
Low
Overall RfD
Developmental toxicity (including
developmental neurotoxicity)
3 x 10"4
Critical window of
development (postnatal)
Medium
Developmental Toxicity
Candidate values to represent developmental toxicity were derived based on
neurobehavioral changes and cardiovascular effects following developmental exposure. While the
candidate value derived for developmental cardiovascular effects is slightly lower than the
candidate value based on developmental neurotoxicity, the support across the database for
developmental cardiovascular effects is considerably smaller, with one in vivo rodent study
evaluating cardiovascular endpoints. Several in vivo studies in mice and rats, by multiple routes of
exposure, support the findings of neurobehavioral changes following developmental exposure to
benzo[a]pyrene. Therefore, the candidate value based on neurobehavioral changes in rats (Chen et
al.. 20121 was selected as the organ/system-specific RfD representing developmental toxicity. This
candidate value was selected because it is associated with the application of the smaller composite
UF, because it represents multiple neurobehavioral endpoints, and because similar effects were
replicated across numerous additional studies (see Section 1.1.1).
Reproductive Toxicity
Among the adverse reproductive effects associated with oral benzo[a]pyrene exposure,
decrements in sperm parameters, decreases in testosterone, and effects in the ovary were
supported by a large body of evidence. The data supporting cervical effects are limited to a single
study, and were therefore given less weight compared to the other reproductive effects. The
derivation of a candidate value based on decreased sperm count and motility fMohamed etal..
20101 involved too much uncertainty (see Table 2-2) and the study used to derive a candidate value
based on decreased testosterone (Zheng etal.. 20101 did not observe a dose-response relationship
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(a 15% decrease in testosterone was seen at the low and high doses). The study by Xu etal. (2010)
observed dose-response relationships for decreased ovary weight and decreases in primordial
follicle counts. The ovarian effects are supported by a large database of animal studies and human
studies of exposure to benzo[a]pyrene and PAH mixtures. Therefore, the candidate value based on
decreased ovotoxicity in rats from the Xu etal. f 20101 study was selected as the organ/system-
specific RfD representing reproductive toxicity. While evidence in the benzo[a]pyrene database
supports male and female reproductive hazards, there is more confidence in the POD from Xu et al.
(2010) as the basis for an organ/system-specific RfD for reproductive effects.
Immunotoxicity
The candidate values based on decreased thymus weight fKroese etal.. 20011 and serum
IgM levels in rats fDe long et al.. 19991 were the same and were selected as the organ/system-
specific RfD representing immunotoxicity. The observed decreases in thymus weight, IgM and IgA
levels, and number of B cells associated with exposure to benzo[a]pyrene were determined to be
representative of immunotoxicity. In combination, these effects provide more robust evidence of
immunotoxicity. The candidate values for decreased thymus weight (Kroese etal.. 2001) and
serum IgM levels in rats (De long et al.. 1999) were comparable and provided the most sensitive
POD; thus, these candidate values were selected as the organ/system-specific RfD representing
immunotoxicity.
2.1.5. Selection of the Overall Reference Dose
Multiple organ/system-specific reference doses were derived for effects identified as
human hazards or potential hazards from benzo[a]pyrene: developmental toxicity (including
developmental neurotoxicity), male and female reproductive toxicity, and immunotoxicity. To
estimate an exposure level below which effects from benzo[a]pyrene exposure are not expected to
occur, the lowest organ/system-specific RfD (3 x 10"4mg/kg-day) with the highest confidence was
selected as the overall RfD for benzo[a]pyrene. This value, based on induction of neurobehavioral
changes in rats exposed to benzo[a]pyrene during a susceptible lifestage, is supported by numerous
animal and human studies across a variety of exposure paradigms (see Section 1.1.1).
The overall RfD is derived to be protective of all types of effects for a given duration of
exposure and is intended to protect the population as a whole including potentially susceptible
subgroups fU.S. EPA. 20021. This value should be applied in general population risk assessments.
However, decisions concerning averaging exposures over time for comparison with the RfD should
consider the types of toxicological effects and specific lifestages of concern. For example,
fluctuations in exposure levels that result in elevated exposures during development could
potentially lead to an appreciable risk, even if average levels over the full exposure duration were
less than or equal to the RfD. For the endpoint of developmental neurotoxicity supporting the
overall RfD, the study treated rats in the early postnatal period from PND 5-11. This postnatal
period of brain development in rats is believed to parallel human brain development occurring in
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the third trimester of pregnancy (Dobbing and Sands. 1979.19731. However, the mode of action for
benzo[a]pyrene-induced developmental neurotoxicity is not fully understood, thus, the exact
window of susceptibility or the duration of exposure necessary to trigger adverse effects in humans
cannot be determined with the currently available data.
2.1.6. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfD,
the overall database, and the RfD itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA.
1994a). Confidence in the principal study (Chen etal.. 20121 is medium. The study design included
40 litters of rats tested in multiple behavioral assays at multiple ages across four dose levels
(including the control group). The study randomly assigned a total of 10 male and 10 female pups
per treatment group per behavioral endpoint, with no more than 1 male and 1 female from each
litter for behavioral testing. Importantly, all tests were conducted by investigators blinded to
treatment, and test order was randomized each day. In addition, the pups were cross-fostered with
dams being rotated among litters every 2-3 days to distribute any maternal caretaking differences
randomly across litters and treatment groups. Some uncertainty exists regarding the potential for
dam rotation across litters and the within-litter dosing design to introduce maternal stress and thus
unanticipated consequences in the pups (see Section 1.1.1), and some informative experimental
details were omitted, such as the sensitivity of some assays at the indicated developmental ages,
gender-specific data for all outcomes and information on procedures for matching pup ages across
the 40 litters examined. However, the overall methods and reporting for the specific behavioral
endpoints supporting the RfD (i.e., open field locomotion, elevated plus maze activity, and Morris
water maze performance) are considered sufficient and well-characterized. Confidence in the
database is medium, primarily due to the lack of a multigenerational reproductive toxicity study
and lack of a developmental neurotoxicity study with exposure that spanned early gestation
through weaning (or longer), given the sensitivity to benzo[a]pyrene during development.
Reflecting medium confidence in the principal study and medium confidence in the database,
confidence in the RfD is medium.
2.1.7. Previous IRIS Assessment: Reference Dose
An RfD was not derived in the previous IRIS assessment.
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER
The inhalation reference concentration (RfC) (expressed in units of mg/m3) is defined as an
estimate (with uncertainty spanning perhaps an order of magnitude) of a continuous inhalation
exposure to the human population (including sensitive subgroups) that is likely to be without an
appreciable risk of deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or
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the 95 percent lower bound on the benchmark concentration (BMCL), with UFs generally applied to
reflect limitations of the data used.
2.2.1. Identification of Studies and Effects for Dose-Response Analysis
In Section 1.2.1, developmental and reproductive toxicities were identified as hazards of
benzo[a]pyrene exposure by the inhalation route. Studies within each effect category were
evaluated using general study quality characteristics (as discussed in Section 6 of the Preamble) to
help inform the selection of studies from which to derive toxicity values. Rationales for selecting
the studies and effects to represent each of these hazards are summarized below.
Human studies of environmental PAH mixtures across multiple cohorts have observed
developmental and reproductive effects following prenatal exposure. However, these studies are
limited by exposure to complex mixtures of PAHs; and, within individual studies, there may have
been more than one route of exposure. In addition, the available human studies that utilized
benzo[a]pyrene-DNA adducts as the exposure metric do not provide external exposure levels of
benzo[a]pyrene from which to derive an RfC. Although preferred for derivation of reference values,
human studies were not considered because of the contribution to the observed hazard of multiple
PAHs across multiple routes of exposure and uncertainty due to concurrent exposure to other PAHs
and other components of the mixtures (such as metals).
Animal studies were evaluated to determine which provided the most relevant routes and
durations of exposure, multiple exposure levels to provide information about the shape of the dose
response curve, and relative ability to detect effects at low exposure levels. The only chronic animal
inhalation study available for benzo[a]pyrene, Thvssen et al. (1981). was designed as a cancer
bioassay and did not report other effects; however, the inhalation database for benzo[a]pyrene
includes several shorter duration studies that are sufficient for use in deriving reference values
fU.S. EPA. 20021. including one subchronic study and several developmental studies that identify
hazards of exposure during sensitive developmental windows. In addition, a 4-week inhalation
study in rats is available that investigated, but did not detect, lung injury (Wolff etal.. 1989). The
inhalation database for benzo[a]pyrene is less extensive than the database of studies by the oral
route; however, the types of noncancer effects observed are consistent between routes and are
supported by studies in human populations (see Sections 1.1.1,1.1.2, and 1.1.3).
Developmental Toxicity
Developmental toxicity, as represented by decreased embryo/fetal survival and limited
evidence of developmental neurotoxicity, was observed in inhalation studies conducted during
gestation in rats (Wormlev et al.. 2004: Wu etal.. 2003a: Archibongetal.. 2002) and mice (Li etal..
2012). The studies in rodents demonstrated that the developing organism is a sensitive target
following inhalation exposure to benzo[a]pyrene, in terms of fetal survival. The study conducted by
Archibongetal. f20021. with decreased embryo/fetal survival (e.g., increased resorptions)
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observed atthe lowest inhalation exposure on gestation days (GDs) 11-20, was selected for dose-
response analysis.
The neurodevelopmental effects observed by Wormlev et al. (20041 (reduced long-term
potentiation) and Li etal. (20121 (deficits in object discrimination) were not considered as
informative as Archibong et al. (20021 for dose-response analysis. Wormlev etal. (20041 employed
only a single, relatively high exposure (at which a 66% reduction in embryo/fetal survival in rats
was observed) and Li etal. f20121 offers supplemental evidence of developmental neurotoxicity,
but employed the same, single exposure level as Wormlev etal. f20041 and tested genetically
manipulated mice developed for use in conditional knock-out studies, which may or may not
respond comparably to wild type (WT) animals.
Reproductive Toxicity
Male reproductive toxicity, as represented by reductions in sperm quality, both count and
motility, and testis weights in adults, was observed by Archibong et al. f20081. Ramesh etal. f20081.
and Archibong et al. f20021. Archibong et al. f20081 and Ramesh etal. f20081 reported the results
of a 60-day inhalation exposure study in male rats. Although of sufficient duration for developing a
reference value, the study utilized a single exposure concentration, which is less informative for
dose-response analysis than a design using multiple exposure concentrations. However, the effects
in this study are consistent with male reproductive effects observed across multiple studies by the
oral route and with human studies in PAH exposed populations, as effects on male fertility and
semen quality have been demonstrated in epidemiological studies of smokers (see Section 1.1.2).
The endpoints of decreased testes weight and sperm count and motility reported in Archibong et al.
f20081 were selected for dose-response analysis as both represent sensitive endpoints of male
reproductive toxicity and are indicators of potentially decreased fertility.
For female reproductive toxicity, ovotoxicity—as represented by reduced ovulation rate
and ovarian weight—and reduced litter sizes at birth were observed by Archibong etal. (20121
following a 14-day pre-mating exposure period, distinct from the developmental period studied by
Archibong etal. f20021 on GDs 11-20. Decreased ovary weights and ovulation rates were selected
for dose-response analysis. Reduced litter sizes were not considered for reference concentration
derivation because the means reflected ovulation rates at the lower two exposures, and would have
yielded very similar PODs.
2.2.2. Methods of Analysis
As there were no biologically based, dose-response models for inhalation exposure to
benzo(a)pyrene available, the general methods for dose-response analysis for reference value
concentration derivation were the same as for the reference dose (see Section 2.1.2).
The data for decreased embryo/fetal survival from Archibong et al. f20021. reported as
litter mean percent survival at birth and associated SE, did not yield adequate fits when modeled as
continuous data. The underlying responses were dichotomous (alive, not alive), and the resulting
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pattern of variability in percentages across exposure groups was incompatible with the normal
distribution assumption imposed by BMDS continuous models. Ideally, individual offspring data
are needed to address the impact of intralitter correlation on effective sample sizes, but attempts to
obtain individual offspring data from the study authors were unsuccessful.
In an attempt to use BMD modeling for these data, the Rao-Scott transformation was
applied (Fox etal.. 2016: Fung etal.. 1998: Rao and Scott. 19921. Briefly, in each exposure group,
the transformation reflects the ratio of two variances—one under the assumption of complete
independence of offspring and the other characterizing the hierarchical relationship of offspring
within litters—and results in a decreased sample size (effective sample size). Dichotomous models
in BMDS were then applied to the transformed embryo/fetal survival data. See Appendix E.1.2 for
details of the methodology and the transformed data, as well as modeling details.
For decreased embryo/fetal survival, BMRs of 1 and 5% extra risk were considered,
recognizing the severity of the outcome. Extrapolation below the lowest administered exposure is
involved in estimating these BMCs for the Archibong et al. f20021 data, given that the difference in
response between the control and lowest exposure was approximately 20%. Taking into account
the extent of model uncertainty, modeling results are presented for comparative purposes (see
Table E-16) and the lowest exposure was considered a LOAEL and used as the POD for decreased
embryo/fetal survival.
For the endpoint of ovulation rate (Archibong etal.. 20121. a BMR of 1% was considered,
due to the severity of the endpoint, which was correlated directly with reduced litter size.
However, due to model and extrapolation uncertainty associated with the small sample sizes (five
dams per dose group), extrapolation to this BMR was not supported. That is, although the fitted
models showed adequate goodness-of-fit to the observed data, the estimated BMCiS varied over a
16-fold range, and the associated BMCLs ranged up to 30-fold lower. Therefore, a BMR of 1% was
not supported and the lowest exposure was considered a LOAEL.
For ovary weight (Archibong et al.. 2 0121. a BMR of 10% relative deviation was selected,
similar to the relative magnitude of the SD for ovary weight in the Xu etal. f 20101 study used for
developing a reproductive RfD (see Section 2.1.2). However, adequate model fits were not
achieved, likely due in part to the low reported variability in ovary weights in this study (with
standard deviations estimated at about 0.007 g, or about 1% of the control mean in F344 rats; see
Table E-2) relative to historical controls (e.g.. Marty et al.. 2009 reported standard deviations 11 or
13% of mean ovary weights for Wistar or Sprague-Dawley rats, respectively). Therefore, the lowest
exposure, showing a 10% response, was considered a NOAEL.
The study by Archibong et al. f20081/Ramesh etal. f20081 was judged not to support dose-
response modeling due to the use of a single exposure level. Consequently, there was insufficient
information to characterize the underlying dose-response relationship. This study observed high
magnitudes of response at the only dose tested, specifically a 34% decrease in testicular weight and
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a 69% decrease in sperm number. Therefore, LOAELs were used as the PODs for dose-response
analysis.
By definition, the RfC is intended to apply to continuous lifetime exposures for humans fU.S.
EPA. 1994a 1. EPA recommends that adjusted continuous exposures be used for developmental
toxicity studies by the inhalation route as well as for inhalation studies of longer durations fU.S.
EPA. 20021. The PODs were adjusted to account for the discontinuous daily exposure as follows:
PODadj = POD x hours exposed per day/24 hours
= LOAEL x (duration of exposure/24 hours)
= PODadj
Next, the human equivalent concentration (HEC) was calculated from the PODadj by
multiplying by a DAF, which, in this case, was the regional deposited dose ratio (RDDRer) for
extrarespiratory (i.e., systemic) effects as described in Methods for Derivation of Inhalation
Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA. 1994a). The observed
developmental effects are considered systemic in nature (i.e., extrarespiratory) and the normalizing
factor for extrarespiratory effects of particles is body weight (i.e., the equivalent dose across species
is mass deposited in the entire respiratory tract per unit body weight). The RDDRer was calculated
as follows:
RDDRer x(Ve)a x(ftot)a
BWa (Ve)h (Ftot)h
where:
BW = body weight (kg);
Ve = ventilation rate (L/minute); and
Ftot = total fractional deposition.
The total fractional deposition includes particle deposition in the nasal-pharyngeal,
tracheobronchial, and pulmonary regions. Ftot for both animals and humans was calculated using
the Multi-Path Particle Dosimetry (MPPD) model, a computational model used for estimating
human and rat airway particle deposition (MPPD; Version 2.0 © 2006, as accessed through the
former Hamner Institute; now publicly available through Applied Research Associates). Ftot was
based on the average particle size of 1.7 ± 0.085 [im (mass median aerodynamic diameter [MMAD]
± geometric SD) as reported in Wu etal. f2003al for the exposure range 25-100 [im3. For the
model runs, the Yeh-Schum 5-lobe model was used for the human and the asymmetric multiple
path model was used for the rat (see Appendix E for MPPD model output). Both models were run
under nasal breathing scenarios after adjusting for inhalability. A geometric SD of 1 was used as the
default by the model because the reported geometric SD of 0.085 was <1.05.
The human parameters used in the model for calculating Ftot and in the subsequent
calculation of the PODhec were as follows: human body weight, 70 kg; Ve, 13.8 L/minute; breathing
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frequency, 16 per minute; tidal volume, 860 mL; functional residual capacity, 3,300 mL; and upper
respiratory tract volume, 50 mL. Although the most sensitive population in Archibong et al. (2002)
is the developing fetus, the adult rat dams were directly exposed. Thus, adult rat parameters were
used in the calculation of the HEC. The parameters used for the rat were body weight, 0.25 kg (a
generic weight for male and female rats); Ve, 0.18 L/minute; breathing frequency, 102 per minute;
tidal volume, 1.8 mL; functional residual capacity, 4 mL; and upper respiratory tract volume,
0.42 mL. All other parameters were set to default values (see Appendix E).
Under these conditions, the MPPD model calculated Ftot values of 0.621 for the human and
0.181 for the rat. Using the above equation, the RDDRer was calculated to be 1.1.
From this, the PODhec was calculated as follows:
PODhec = PODadj x RDDRer
Table 2-4 summarizes the sequence of calculations leading to the derivation of a human-
equivalent POD for each data set discussed above.
Table 2-4. Summary of derivation of PODs
Endpoint and reference
Species/sex
Model
BMR
BMC
(Hg/m3)
BMCL
(Hg/m3)
PODadj3
(Hg/m3)
PODhec15
(Hg/m3)
Developmental
Decreased embryo/fetal survival
Archibong et al. (2002)
Pregnant
F344 rats
LOAEL (25 ng/m3) 19% 4,
4.2
4.6
Reproductive
Decreased ovulation rate
Archibong et al. (2012)
Female F344
rats
LOAEL (50 ng/m3) 9% 4,
8.3
9.1
Decreased ovary weight
Archibong et al. (2012)
Female F344
rats
NOAEL (50 ng/m3) 10% 4,
8.3
9.1
Decreased testis weight
Archibong et al. (2008)
Male F344
rats
LOAEL (75 ng/m3) 34% 4,
12.5
13.8
Decreased sperm count and motility
Archibong et al. (2008)
Male F344
rats
LOAEL (75 ng/m3)
69% 4^sperm count
73% 4/ sperm motility
54% 1" abnormal sperm
12.5
13.8
aPODs were adjusted for continuous daily exposure: PODadj= POD x hours exposed per day/24 hours.
^PODhec calculated by adjusting the PODadj by the RDDR calculated using particle size reported in Hood et al. (2000)
and Wu et al. (2003a) using MPPD software as detailed above and Appendix E in the Supplemental Information.
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2.2.3. Derivation of Candidate Values
Under EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
2002: Section 4.4.51. also described in the Preamble, five areas of uncertainty and variability were
considered, as follows:
A UFl of 10 was applied when a LOAEL was used as the POD fArchibongetal.. 2012:
Archibong et al.. 20021. because there was a high magnitude of response at these LOAELs (see Table
2-4). Specifically, the LOAEL for embryo/fetal survival observed in Archibong et al. f20021 was
based on a 19% decrease, while Archibong etal. f20081 observed a 34% decrease in testis weight
and a 69% decrease in sperm count Therefore, a full UF of 10 was applied to approximate a NOAEL
for these PODs. For decreased ovary weight (Archibong etal.. 20121. a decrease of 10% was
observed at the lowest dose tested, similar to the ovary weight decrease considered a minimally
important degree of change in the Xu etal. (20101 study in the oral database (see Section 2.1.2).
Therefore, this dose was considered a NOAEL and a 1-fold UF was applied.
A UFs of 1 was applied when dosing occurred during gestation f Archibong etal.. 20021 or
the early postnatal period that is relevant to developmental effects fU.S. EPA. 1991al. The
developmental period is recognized as a susceptible lifestage when exposure during a time window
of development is more relevant to the induction of developmental effects than lifetime exposure
(U.S. EPA. 1991c): therefore, an adjustment for duration is not warranted. A UF of 1 was applied to
effects on ovulation following pre-mating exposure during a sensitive reproductive window
fArchibongetal.. 20121. A value of 10 is applied when the POD is based on a subchronic study to
account for the possibility that longer exposure may induce more severe effects or effects at a lower
dose.
A dosimetric adjustment (RDDR) is applied to adjust for differences in particle deposition
across species. Accordingly, a reduced UFa of 3 (10^2= 3.16, rounded to 3) is applied to account for
uncertainties in characterizing toxicodynamic as well as residual toxicokinetic differences in the
extrapolation from laboratory animals to humans after inhalation exposure to benzo[a]pyrene as
described in EPA's Methods for Derivation of Inhalation Reference Concentrations and Application of
Inhalation Dosimetry fU.S. EPA. 1994al. Residual toxicokinetic uncertainties include the
considerations that the methodology adjusts for interspecies differences in the deposition of the
inhaled dose in the entire respiratory tract ignoring regional differences (e.g., a greater percentage
would be expected to reach the lung in humans as compared to rats) as well as interspecies
differences in clearance. It is reasonable to consider the dose to the entire respiratory tract in
either species since the mode of action for the endpointused as the basis of the RfC (decreased
embryo/fetal survival) is not known. Furthermore, data for modeling species differences in
clearance and metabolism of the deposited particles are not available; therefore, given these
uncertainties, the relevant dose metric is considered to be the mass of benzo[a]pyrene deposited
per day in the entire respiratory tract This metric is thought to be more accurate than using
exposure concentration as the default.
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A UFh of 10 was applied to account for variability and uncertainty in toxicokinetic and
toxicodynamic susceptibility within the subgroup of the human population most sensitive to the
health hazards of benzo[a]pyrene (U.S. EPA. 20021. In the case of benzo[a]pyrene, the PODs were
derived from studies in inbred animal strains and are not considered sufficiently representative of
the exposure and dose-response of the most susceptible human subpopulations (in this case, the
developing fetus). In certain cases, the toxicokinetic component of this factor may be replaced
when a PBPK model is available that incorporates the best available information on variability in
toxicokinetic disposition in the human population (including sensitive subgroups). In the case of
benzo[a]pyrene, insufficient information is available to quantitatively estimate variability in human
susceptibility; therefore, the full value for the UFh was applied.
Although hazards are similar across oral and inhalation databases, fewer studies exist by
the inhalation route to characterize the dose response of inhaled benzo[a]pyrene. A full UFd of 10
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 and that decrements in fertility are greater following developmental
exposure (see Section 1.1.2).
In addition, the general lack of studies examining functional neurological endpoints
following inhalation exposure during development is a significant data gap, considering human and
animal evidence indicating altered neurological development following exposure to benzo[a]pyrene
alone or through PAH mixtures (see Section 1.1.1). An additional uncertainty in the inhalation
database for benzo[a]pyrene includes the lack of studies characterizing immune system toxicity,
especially following developmental exposure.
The most sensitive POD for the inhalation candidate values in Table 2-5 is based on the
endpoint of decreased embryo/fetal survival observed in Archibong et al. (2002). Decreased fetal
survival was also observed in oral exposure studies; however, it was seen at much higher doses
than developmental neurotoxicity. A16% decrease in F1 fetal survival was observed following
treatment with 160 mg/kg-day benzo[a]pyrene, but not at lower doses fMackenzie and An ge vine.
19811: however, other oral studies observed significant neurobehavioral effects at doses of
benzo[a]pyrene around 0.2-2 mg/kg-day fChen etal.. 2012: Bouaved etal.. 2009al. Considering
the relative sensitivity of the systemic health effects observed in the oral database, it is possible that
developmental neurotoxicity would occur at exposure concentrations below the POD for the RfC
based on decreased embryo/fetal survival. According to EPA's A Review of the Reference Dose and
Reference Concentration Processes fU.S. EPA. 2002: Section 4.4.51. the UFd is intended to account for
the potential for deriving an under-protective RfD/RfC as a result of an incomplete characterization
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. Therefore, a UFd of 10 for the
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benzo[a]pyrene inhalation database was applied to account for the lack of a multigenerational
study and the lack of a developmental neurotoxicity study.
Table 2-5 is a continuation of Table 2-4 and summarizes the application of UFs to each POD
to derive a candidate values for each data set. The candidate values presented in the table below
are preliminary to the derivation of the organ/system-specific reference values. These candidate
values are considered individually in the selection of an RfC for a specific hazard and subsequent
overall RfC for benzo[a]pyrene.
Table 2-5. Effects and corresponding derivation of candidate values
Endpoint
PODhec
pg/m3
POD
type
UFl
UFs
UFa
UFh
UFd
Composite
UFb
Candidate
value3
mg/m3
Developmental
Decreased embryo/fetal survival in rats
Archibong et al. (2002)
4.6
LOAEL
10
1
3
10
10
3,000
2 x 10"6
Reproductive
Decreased ovulation rate
Archibong et al. (2012)
9.1
LOAEL
10
1
3
10
10
3,000
3 x 10"6
Decreased ovary weight
Archibong et al. (2012)
9.1
NOAEL
1
10
3
10
10
3,000
3 x 10"6
Decreased testis weight in rats
Archibong et al. (2008)
13.8
LOAEL
10
10
3
10
10
30,000
Not
calculated
due to UF
>3,000
Decreased sperm count and motility in
rats
Archibong et al. (2008)
13.8
LOAEL
10
10
3
10
10
30,000
Not
calculated
due to UF
>3,000
Candidate values were converted from ng/m3 to mg/m3.
bAs 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 UF in four or more areas of
extrapolation should be avoided.
Figure 2-2 presents graphically these candidate values UFs and PODs, with each bar
corresponding to one data set described in Tables 2-4 and 2-5.
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Toxicological Review of Benzo[a]pyrene
4, Embryo/fetal survival in rats
(Archibong et al., 2002)
4- Ovulation rate
(Archibong et al,, 2012)
Ovary weight
(Archibong et al,, 2012)
si- Testis weight in rats
(Archibong et al,, 2008)
4, Sperm count and motility
in rats (Archibong et al,, 2008)
0.000 1 0.001 0.01 0.1 1 10 100
Exposure concentration (ng/m3)
Figure 2-2. Candidate values with corresponding PODs and composite UFs.
2.2.4. Derivation of Organ/System-Specific Reference Concentrations
Table 2-6 distills the candidate values from Table 2-5 into a single value for each organ or
system. These organ- or system-specific reference values may be useful for subsequent cumulative
risk assessments that consider the combined effect of multiple agents acting at a common site.
Candidate values for reproductive toxicity (decreased testis weight and decreased sperm count and
motility) from Archibong etal. f20081 were not derived, because as recommended in EPA's A
Review of the Reference Dose and Reference Concentration Processes fU.S. EPA. 20021. the derivation
of a reference value that involves application of the full 10-fold UF in four or more areas of
extrapolation should be avoided.
Table 2-6. Organ/system-specific RfCs and overall RfC for benzo[a]pyrene
Effect
Basis
RfC (mg/m3)
Study exposure
description
Confidence
Developmental
Decreased embryo/fetal
survival
2 x 10"6
Critical window of
development (prenatal)
Low-medium
Reproductive
Decreased ovary weight and
ovulation rate
3 x 10"6
Short-term pre-mating
exposure (14 d)
Low-medium
Overall RfC
Decreased embryo/fetal
survival
2 x 10"6
Critical window of
development (prenatal)
Low-medium
Composite UF
~ Candidate value
• POD(HEC)
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2.2.5. Selection of the Reference Concentration
The derivation of multiple organ/system-specific reference concentrations was considered
for effects identified as human hazards of benzo[a]pyrene inhalation exposure, i.e., developmental
and reproductive toxicity. Organ/system-specific RfCs to represent reproductive toxicity were
calculated based on female reproductive toxicity, specifically decreased ovulation rate. An RfC
based on male reproductive toxicity could not be derived due to high uncertainty (i.e., a composite
UF of >3,000).
An overall RfC of 2 x 10~6 mg/m3 was selected based on the hazard of developmental
toxicity. The study by Archibong etal. ("2002) was selected for the derivation of the overall RfC, as it
observed biologically significant effects at the lowest dose tested by the inhalation route. This
study indicates that the developing organism is a sensitive target following inhalation exposure to
benzo[a]pyrene and the observed decreased embryo/fetal survival (increased resorptions) is the
most sensitive noncancer effect observed following inhalation exposure to benzo[a]pyrene.
Additional support for this endpoint of decreased embryo/fetal survival is provided by another
developmental/reproductive study conducted via the oral route ("Mackenzie and Angevine. 19811.
This overall RfC is derived to be protective of all effects for a given duration of exposure and
is intended to protect the population as a whole, including potentially susceptible subgroups (U.S.
EPA. 2002). This value should be applied in general population risk assessments. However,
decisions concerning averaging exposures over time for comparison with the RfC should consider
the types of toxicological effects and specific lifestages of concern. For example, fluctuations in
exposure levels that result in elevated exposures during these lifestages could potentially lead to an
appreciable risk, even if average levels over the full exposure duration were less than or equal to
the RfC. For the endpoint of decreased embryo/fetal survival (increased resorptions) supporting
the overall RfC, exposure during the first (and potentially second) trimester of pregnancy would be
the likely window of susceptibility. However, the mode of action for benzo[a]pyrene-induced
resorptions is not fully understood, thus, the exact window of susceptibility and the durations of
exposure necessary to trigger this effect in humans cannot be determined with the currently
available data.
2.2.6. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA.
1994a).
The overall confidence in the RfC is low-to-medium. Confidence in the principal study
(Archibong etal.. 2002) is medium. The conduct and reporting of this developmental study were
adequate; however, a NOAEL was not identified. Confidence in the database is low due to the lack
of a multigeneration toxicity study, lack of studies on developmental neurotoxicity and immune
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endpoints, and lack of information regarding subchronic and chronic inhalation exposure.
However, confidence in the RfC is bolstered by consistent systemic effects observed by the oral
route (including reproductive and developmental effects) and similar effects observed in human
populations exposed to PAH mixtures. Reflecting medium confidence in the principal study and low
confidence in the database, confidence in the RfC is low-to-medium.
2.2.7. Previous IRIS Assessment: Reference Concentration
An RfC was not derived in the previous IRIS assessment
2.2.8. Uncertainties in the Derivation of the RfD and RfC
The following discussion identifies uncertainties associated with the RfD and RfC for
benzo[a]pyrene. To derive the RfD, the UF approach fU.S. EPA. 2000.1994a) was applied to a POD
based on neurobehavioral changes in rats treated developmentally. To derive the RfC, this same
approach was applied to a POD from a developmental study for the effect of decreased embryo/
fetal survival. UFs were applied to the POD to account for extrapolating from an animal bioassay to
human exposure, the likely existence of a diverse population of varying susceptibilities, and
database deficiencies. These extrapolations are carried out with default approaches given the lack
of data to inform individual steps.
The database for benzo[a]pyrene contains limited human data. The observation of effects
associated with benzo[a]pyrene exposure in humans is complicated by several factors including the
existence of benzo[a]pyrene in the environment as one component of complex mixtures ofPAHs,
exposure to benzo[a]pyrene by multiple routes of exposure within individual studies, and the
difficulty in obtaining accurate exposure information. Data on the effects of benzo[a]pyrene alone
are derived from a large database of studies in animal models. The database for oral
benzo[a]pyrene exposure includes two lifetime bioassays in rats and mice, two developmental
studies in mice, and several subchronic studies in rats.
Although the database is adequate for RfD derivation, there is uncertainty associated with
the database including that the principal study for the RfD exposed animals during a relatively short
period of brain development, potentially underestimating the magnitude of resulting neurological
effects. Also, the database lacks comprehensive multigeneration reproductive/developmental
toxicity studies, and immune system endpoints were not evaluated in the available chronic-
duration or developmental studies. Additionally, the only available chronic studies of oral or
inhalational exposure to benzo[a]pyrene focused primarily on neoplastic effects, leaving non-
neoplastic effects mostly uncharacterized.
Additional uncertainty remains that the POD for the overall impact of neurodevelopmental
effects might be lower than the selected POD. Specifically, if individual animal data for the Track 4
rats were available, consideration of the changes in any of the three behavioral tests as indicating
an abnormal response for each rat would better represent the total behavioral effect, and could
result in a lower POD. In addition, as altered performance in these three behavioral tests was more
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severe when tested in adult, as compared to juvenile, animals, it is possible that testing animals at
even older ages (i.e., after PND 75) would reveal even more sensitive effects of exposure. However,
experiments addressing these possibilities were not available and these remain unaddressed
uncertainties. Overall, this POD is the best supported value that can be derived using the currently
available information, recognizing the multiple effects observed in the same study.
The only chronic inhalation study of benzo[a]pyrene was designed as a lifetime
carcinogenicity study and did not examine noncancer endpoints (Thvssen etal.. 19811. In addition,
subchronic and short-term inhalation studies are available, which examine developmental and
reproductive endpoints in rats. Developmental studies by the inhalation route identified
biologically significant reductions in the number of pups/litter and percent embryo/fetal survival
and possible neurodevelopmental effects following gestational exposures. A 14-day premating
reproductive study in female rats observed decreased ovulation rate and ovary weight in treated
animals. Additionally, a 60-day oral study in male rats reported male reproductive effects (e.g.,
decreased testes weight and sperm production and motility), but provides limited information to
characterize dose-response relationships with chronic exposure scenarios. The study selected as
the basis of the RfC provided limited information regarding the inhalation exposures of the animals.
Specifically, it is not clear whether the reported concentrations were target values or analytical
concentrations and the method used to quantify benzo[a]pyrene in the generated aerosols was not
provided. Requests to obtain additional study details from the authors were unsuccessful;
therefore, the assumption was made that the reported concentrations were analytical
concentrations.
Results from several different studies indicate that the endpoint of decreased number of
pups per litter may be impacted during different sensitive windows of exposure, likely by different
modes of action. The critical study used for the derivation of the RfC treated dams following
implantation and quantification of conceptuses, from GD 11 to 20 and observed a decrease in
embryos/fetuses per litter fArchibongetal.. 20021. Another study treated female rats for 2 weeks
immediately prior to mating and observed a decrease in ovulation rate (e.g., number of oocytes
released) and a decreased number of pups born per litter (Archibongetal.. 20121. Yet another
study treated animals by intraperitoneal (i.p.) injection with benzo[a]pyrene by on GDs 1-5,
observed a decrease in the number of implantation sites, and hypothesized thatbenzo[a]pyrene
exposure may effect endometrial receptivity fZhao etal.. 20141. These three studies observed
similar effects during different exposure windows, indicating that an exposure that included
treatment prior to mating and through gestation would likely result in an even greater reduction in
the number of pups produced per litter. Therefore, it is possible that the critical study used for the
RfC may have observed a greater reduction of pups born per litter if exposure covered a more
comprehensive duration.
Another area of uncertainty in the database pertains to the lack of information regarding
fertility in animals exposed gestationally to benzo[a]pyrene, especially in light of developmental
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studies by the oral route indicating reduced fertility in the F1 generation and decreased
reproductive organ weights. The database also lacks a multigenerational reproductive study via the
inhalation route. Areas of uncertainty include the lack of chronic inhalation studies focusing on
noncancer effects, limited data on dose-response relationships for impaired male or female fertility
with gestational exposure or across several generations, and limited data on immune system
endpoints with chronic exposure or developmental exposure to benzo[a]pyrene.
The toxicokinetic and toxicodynamic differences for benzo[a]pyrene between the animal
species in which the POD was derived and humans are unknown. PBPK models can be useful for
the evaluation of interspecies toxicokinetics; however, the benzo[a]pyrene database lacks an
adequate model that would inform potential differences. There is some evidence from the oral
toxicity data that mice may be more susceptible than rats to some benzo[a]pyrene effects (such as
ovotoxicity) fBorman etal.. 20001. although the underlying mechanistic basis of this apparent
difference is not understood. Most importantly, it is unknown which animal species may be more
comparable to humans.
2.3. ORAL SLOPE FACTOR FOR CANCER
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question and quantitative estimates of risk from oral and inhalation exposure
may be derived. Quantitative risk estimates may be derived from the application of a low-dose
extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on the estimate
of risk per mg/kg-day of lifetime oral exposure.
2.3.1. Analysis of Carcinogenicity Data
The database for benzo[a]pyrene contains numerous cancer bioassays that identify tumors,
primarily of the alimentary tract including the forestomach, following oral exposure in rodents.
Three 2-year oral bioassays are available that associate lifetime benzo[a]pyrene exposure with
carcinogenicity at multiple sites: forestomach, liver, oral cavity, jejunum, kidney, auditory canal
(Zymbal gland) tumors, and skin or mammary gland tumors in male and female Wistar rats (Kroese
etal.. 20011: forestomach tumors in male and female Sprague-Dawley rats (Brune etal.. 19811: and
forestomach, esophageal, tongue, and larynx tumors in female B6C3Fi mice (Beland and Culp. 1998:
Culp etal.. 19981.
In addition to these 2-year cancer bioassays, there are studies available that provide
supporting evidence of carcinogenicity but are less suitable for slope factor derivation due to one or
more limitations in study design: (1) no vehicle control group; (2) only one benzo[a]pyrene dose
group; or (3) a one-time exposure to benzo[a]pyrene (Benjamin et al.. 1988: Robinson etal.. 1987:
El-Bayoumv. 1985: Wattenberg. 1974: Roe etal.. 1970: Biancifiori etal.. 1967: Chouroulinkov et al..
1967: Berenblum and Haran. 19551. Of the controlled, multiple dose-group, repeat-dosing studies
that remain, most treated animals for <1 year fWevand etal.. 1995: Triolo etal.. 1977: Fedorenko
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and Yansheva. 1967: Neal and Rigdon. 19671. When lifetime studies are not available, shorter
studies such as these can support extrapolation to lifetime exposure, but are less optimal for slope
factor derivation given the availability of chronic studies.
In another lifetime study, Brune etal. (19811 dosed rats (32/sex/group) with
benzo[a]pyrene in the diet or by gavage in a 1.5% caffeine solution, sometimes as infrequently as
once every 9th day, for approximately 2 years and observed increased forestomach tumors. This
study was not selected for quantitation due to the nonstandard treatment protocol in comparison
to the studies conducted by Kroese etal. (20011 and Beland and Culp (19981 and the limited
reporting of study methods.
The Kroese etal. f20011 and Beland and Culp T19981 studies were selected as the best
available studies for dose-response analysis and extrapolation to lifetime cancer risk following oral
exposure to benzo[a]pyrene. The ratbioassav by Kroese etal. f20011 and the mouse bioassay by
Beland and Culp (19981 were conducted in accordance with Good Laboratory Practice (GLP) as
established by the Organisation for Economic Co-operation and Development (OECD). These
studies included histological examinations for tumors in many different tissues, contained three
exposure levels and controls, contained adequate numbers of animals per dose group
(~50/sex/group), treated animals for up to 2 years, and included detailed reporting of methods
and results (including individual animal data).
Details of the rat fKroese etal.. 20011 and female mouse fBeland and Culp. 19981 study
designs are provided in Appendix D of the Supplemental Information. Dose-related increasing
trends in tumors were noted at the following sites:
• Squamous cell carcinomas (SCCs) or papillomas of the forestomach or oral cavity in male
and female rats;
• SCCs or papillomas of the forestomach, tongue, larynx, or esophagus in female mice;
• Auditory canal carcinomas in male and female rats;
• Kidney urothelial carcinomas in male rats;
• Jejunum/duodenum adenocarcinomas in female and male rats;
• Hepatocellular adenomas or carcinomas in male and female rats; and
• SCCs or basal cell tumors of the skin or mammary gland in male rats.
These tumors were generally observed earlier during the study with increasing exposure
levels, and showed statistically significantly increasing trends in incidence with increasing
exposure level (Cochran-Armitage trend test, p < 0.001). These data are summarized in Appendix D
of the Supplemental Information. As recommended by the National Toxicology Program (NTP)
(McConnell et al.. 19861 and as outlined in EPA's Guidelines for Carcinogen Risk Assessment (U.S.
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EPA. 2005a). etiologically similar tumor types (i.e., benign and malignant tumors of the same cell
type) were combined for these tabulations when it was judged that the benign tumors could
progress to the malignant form. In addition, when one tumor type occurred across several
functionally related tissues, as with squamous cell tumors in the tongue, esophagus, larynx, and
forestomach, or adenocarcinomas of the jejunum or duodenum, these incidences were also
aggregated as counts of tumor-bearing animals.
In the rat study (Kroese et al.. 20011. the oral cavity and auditory canal were examined
histologically only if a lesion or tumor was observed grossly at necropsy. Consequently, dose-
response analysis for these sites was not straightforward. Use of the number of tissues examined
histologically as the number at risk would tend to overestimate the proportion with tumors,
because the unexamined animals would have been less likely to have tumors if none were
observable grossly. On the other hand, use of all animals on study in a group as the number at risk
would tend to underestimate if any of the unexamined animals had tumors that could only be
detected microscopically. The oral cavity squamous cell tumors were combined with those in the
forestomach because both are part of the alimentary tract, recognizing that there was some
potential for underestimating this cancer risk.
The auditory canal tumors from the rat study were not considered for dose-response
analysis, for several reasons. Unlike the oral cavity tumors, the auditory canal tumors appeared to
be independent of the alimentary system tumors, as they were described as a mixture of squamous
and sebaceous cells derived from pilosebaceous units, and there was no indication that these
tumors were metastases from other sites (in which case, the auditory canal tumors would be
repetitions of other tumors, or statistically dependent). As with the oral cavity tumors, the
incomplete histological evaluation in the control and lower dose groups did not support dose-
response analysis. Alternatively, even if these tumors were similar enough to be combined with the
oral cavity and forestomach tumors, with only one exception (one female rat in the high dose
group), they were coincident with alimentary tumors, and the joint incidence would be very similar
to that of alimentary system tumors. Therefore dose-response analysis was not pursued for this
site, either separately or in combination with another tumor type.
The incidence data that were modeled are provided in Appendix E (Kroese etal.. 2001:
Beland and Culp. 1998).
2.3.2. Dose-Response Analysis—Adjustments and Extrapolation Methods
EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al recommend that the
method used to characterize and quantify cancer risk from a chemical is determined by what is
known about the mode of action of the carcinogen and the shape of the cancer dose-response curve.
The dose response is assumed to be linear in the low-dose range when the agent is DNA-reactive
and has direct mutagenic activity, or if another mode of action that is anticipated to be linear is
applicable. EPA concluded that benzo[a]pyrene carcinogenicity involves a mutagenic mode of
action (as discussed in Section 1.1.5). Thus, a linear approach to low-dose extrapolation was used.
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The high-dose groups of both the rat and mouse studies were dead or moribund by week 76
for male rats, week 72 for female rats, and week 79 for female mice. Due to the occurrence of
multiple tumor types, earlier occurrence with increasing exposure and early termination of the
high-dose group in each study, methods that can reflect the influence of competing risks and
intercurrent mortality on site-specific tumor incidence rates are preferred. In this case, EPA used
the multistage-Weibull model, which incorporates the time at which death-with-tumor occurred as
well as the dose.
Adjustments for approximating human equivalent slope factors applicable for continuous
exposure were applied prior to dose-response modeling. First, continuous daily exposure for the
gavage study in rats fKroese etal.. 20011 was estimated by multiplying each administered dose by
(5 days)/(7 days) = 0.71, under the assumption of equal cumulative exposure yielding equivalent
outcomes. Dosing was continuous in the mouse diet study fBeland and Culp. 19981. so no
continuous adjustment was necessary. It was not necessary to adjust the administered doses for
lifetime equivalent exposure prior to modeling for the groups terminated early, because the
multistage-Weibull model characterizes the tumor incidence as a function of time, from which it
provides an extrapolation to lifetime exposure.
Next, consistent with the EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al.
adjustment for cross-species scaling was considered to address toxicological equivalence across
species. Despite extensive research into benzo[a]pyrene toxicokinetics (see Section 1.1.6 and
Appendix D Sections D.l and D.2), very little information directly informs estimates of human-
equivalent benzo[a]pyrene doses. Itis understood thatbenzo[a]pyrene carcinogenicity involves a
mutagenic mode of action mediated by DNA-reactive metabolites in the tissues where tumors
appear. While the metabolites are highly reactive, distribution of benzo[a]pyrene to these tissues
may be limited by processes consistent with BW3/4 proportionality.
EPA guidance for oral exposures fU.S. EPA. 19921 asserts that, for a portal-of-entry scenario,
"the most appropriate dose metric would likely be mass of agent per surface area, e.g., mg/cm2," but
that necessary considerations for implementing this approach have yet to be developed (e.g.,
surface areas of the gastrointestinal (GI) tract in rodents and humans, including for an as yet
unidentified human anatomical equivalent to the rodent forestomach; rates and scenarios of
ingestion, including proximal to distal penetration down the GI tract; and diffusion rates). In the
absence of this information, the guidance makes a general recommendation to use the BW3/4
approach for oral portal-of-entry effects. For the Beland and Culp (19981 study, time-weighted
daily average doses were converted to HEDs on the basis of BW3/4.
Kroese etal. (20011 administered benzo[a]pyrene via gavage and observed tumors both in
the alimentary system and systemically (in the kidney, liver, skin and mammary gland). It is not
clear what impact gavage administration has on estimating HEDs of benzo[a]pyrene for the
alimentary system, but the non-alimentary system tumors would be expected to reflect BW3/4
proportionality to exposure. In the absence of information to characterize portal-of-entry
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dosimetry and given the systemic component of the tumor profile, the time-weighted daily average
doses were converted to HEDs on the basis of BW3/4. This was accomplished by multiplying
administered doses by (animal body weight [kg])/70 kg)1/4, where the animal body weights were
TWAs from each group, and the U.S. EPA (19881 reference body weight for humans is 70 kg.
PODs for estimating low-dose risk were identified at doses at the lower end of the observed
data, corresponding to 10% extra risk. Details of the modeling and the model selection process can
be found in Appendix E.2 of the Supplemental Information.
2.3.3. Derivation of the Oral Slope Factor
The PODs estimated for each tumor site are summarized in Table 2-7. The lifetime oral
cancer slope factor for humans is defined as the slope of the line from the lower 95% bound on the
exposure at the BMD to the control response (slope factor = 0.1/BMDLio). This slope represents a
plausible upper bound on the true risk. Using linear extrapolation from the BMDLio, human
equivalent oral slope factors were derived for each gender/tumor site combination and are listed in
Table 2-7.
Oral slope factors derived from rat bioassay data varied by gender and tumor site
(Table 2-7). Values ranged from 0.04 per mg/kg-day, based on kidney tumors in males, to 0.4 per
mg/kg-day, based on alimentary tract tumors in males. Slope factors based on liver tumors in male
and female rats (0.2 per mg/kg-day) were only slightly lower than slope factors based on
alimentary tract tumors (0.2-0.3 per mg/kg-day). The oral slope factor for alimentary tract tumors
in female mice was highest at 1 per mg/kg-day (Table 2-7), which was approximately 2-fold higher
than the oral slope factor derived from the alimentary tract tumors in male rats.
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 factor3
(mg/kg-d)"1
Forestomach, oral cavity: squamous
cell tumors
Kroese et al. (2001)
Male
Wistar rats
Multistage
Weibull
10%
0.453
0.281
0.36
Hepatocellular adenomas or
carcinomas
Kroese et al. (2001)
Male
Wistar rats
Multistage
Weibull
10%
0.651
0.449
0.22
Jejunum/duodenum
adenocarcinomas
Kroese et al. (2001)
Male
Wistar rats
Multistage
Weibull
10%
3.03
2.38
0.042
Kidney: urothelial carcinomas
Kroese et al. (2001)
Male
Wistar rats
Multistage
Weibull
10%
4.65
2.50
0.040
_Q
LO
o
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Tumor
Species/
sex
Selected
model
BMR
BMD
(mg/kg-d)
POD =
BMDL
(mg/kg-d)
Slope factor3
(mg/kg-d)"1
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
0.043
0.056
Forestomach, oral cavity: squamous
cell tumors
Kroese et al. (2001)
Female
Wistar rats
Multistage
Weibull
10%
0.539
0.328
0.3
Hepatocellular adenomas or
carcinomas
Kroese et al. (2001)
Female
Wistar rats
Multistage
Weibull
10%
0.575
0.507
0.2
0.31b
Jejunum/duodenum
adenocarcinomas
Kroese et al. (2001)
Female
Wistar rats
Multistage
Weibull
10%
3.43
1.95
0.05
Forestomach, esophagus, tongue,
larynx (alimentary tract): squamous
cell tumors
Beland and Culp (1998)
Female
B6C3Fi
mice
Multistage
Weibull
10%
0.127
0.071
1.4
1.4
aHuman equivalent slope factor = 0.1/BMDLiohed; see Appendix E of the Supplemental Information for details of
modeling results.
bSlope factor characterizing the risk of incurring at least one of the tumor types listed.
Although the time-to-tumor modeling helps to account for competing risks associated with
decreased survival times and other causes of death including other tumors, considering the tumor
sites individually does not convey the total amount of risk potentially arising from the sensitivity of
multiple sites—that is, the risk of developing any combination of the increased tumor types. A
method for estimating overall risk, involving the assumption that the variability in the slope factors
could be characterized by a normal distribution, is detailed in Appendix E.2.1 of the Supplemental
Information. The resulting composite slope factor for all tumor types for male rats was 0.5 per
mg/kg-day, about 25% higher than the slope factor based on the most sensitive tumor site, oral
cavity and forestomach, while for female rats, the composite slope factor did not increase from that
for the most sensitive site (Tables 2-, E-27).
The overall risk estimates from male and female rats and female mice spanned about a
5-fold range. While EPA's cancer guidelines (U.S. EPA. 2005al suggest "choosing a single datset if it
can be justified as most representative of the overall response in humans," there are no data to
support any one result as most relevant for extrapolating to humans. Under the assumption that
the three data sets are equally relevant for extrapolating to humans, geometric and harmonic mean
of the three slope factors derived here round to 0.60 and 0.50 per mg/kg-day, respectively, about
40% of the highest slope factor. A geometric mean that gives equal weight to rats and mice is
0.74 per mg/kg-day, about 50% of the highest slope factor.
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Another consideration in developing a human-equivalent slope factor is that slope factors
are intended to provide an upper bound on the cancer risk of a randomly selected individual (U.S.
EPA. 2005al. yet EPA's approach to quantifying low-dose cancer risk relies on a 95% upper bound
on the cancer risk that typically only addresses experimental variability in homogeneous laboratory
animals. The NRC f20091 observed that when cancer risk is expected to be linear at low exposures,
as with benzo[a]pyrene, EPA's cancer risk values tend not to address human variability and
susceptibility adequately. Concern for sensitive populations (separate from the consideration of
increased sensitivity at early lifestages; see Section 2.5, Application of Age-Dependent Adjustment
Factors [ADAFs]) suggests interpreting the near-continuous range of risk-estimate confidence
intervals (CIs) from the three data sets (see CIs in Tables E-27 and E-28), of 0-1.4 per mg/kg-day,
to represent a more heterogeneous population and supports use of the high value as a plausible
upper bound.
Potential for model uncertainty in the slope factor estimate is also a relevant consideration
at this stage. Although EPA's practice has been to rely on multistage models (including the
multistage-Weibull model) for carcinogens with a mutagenic mode of action and expected low-dose
linear behavior, some model uncertainty was evaluated by applying the range of dichotomous
models in BMDS to the B6C3Fi mice data fBeland and Culp. 19981. after adjustment for intercurrent
mortality using the poly-3 approach (Bailer and Portier. 19881. Even including less plausible
models that impose nonlinear, low-dose behavior that is inconsistent with a mutagenic mode of
action—i.e., models fit with a slope of 0 risk/dose unit as doses decrease to 0—for each data set, the
resulting BMDio and BMDLio values were found to encompass the corresponding multistage-
Weibull estimate, and to vary overall <2-fold, and less than a factor of 1.5 from the multistage-
Weibull estimate (see Appendix E.2.1). Model uncertainty is minimized through the POD being near
the lowest exposure in each of the data sets.
Given these considerations, EPA selected the most sensitive result to derive the oral slope
factor. The slope factor for assessing human cancer risk associated with lifetime oral exposure to
benzo[a]pyrene is 1 per mg/kg-day, based on the alimentary tract tumor response in female
B6C3Fi mice. Note that the oral slope factor should only be used with lifetime human exposures
<0.1 mg/kg-day, because above this level, the dose-response relationship is nonlinear and plateaus
at 100% at higher exposures. If risk estimates for exposure above 0.1 mg/kg-day would be
needed—that is, corresponding to expected overall cancer risks greater than 10%—the full dose-
response model as provided in Appendix E.2.1 should be consulted.
The oral slope factor for benzo[a]pyrene is derived with the intention that it will be paired
with EPA's relative potency factors for the assessment of the carcinogenicity of PAH mixtures. In
addition, regarding the assessment of early life exposures, because cancer risk values calculated for
benzo[a]pyrene were derived from adult animal exposures, and because benzo[a]pyrene
carcinogenicity occurs via a mutagenic mode of action, exposures that occur during development
should include the application of ADAFs (see Section 2.5).
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EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005al recommend
characterizing expected or central estimates of risk, where practicable, and confidence limits on the
POD. For the available data, note that a central estimate of risk below the POD is not practicable
because the POD determines the low end of the range for which statistical predictions can be
supported. The slope of the linear extrapolation from the BMDio, the central tendency estimate at
the POD, can be calculated [0.1/(0.127 mg/kg-day) = 0.78 per mg/kg-day], but cannot be
considered a central tendency estimate throughout the low dose range (below the POD). For the
recommended slope factor, the 95% confidence limits based on the POD are
0.071-0.179 mg/kg-day.
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor
The oral slope factor for benzo[a]pyrene was based on the increased incidence of
alimentary tract tumors, including forestomach tumors, observed in a lifetime dietary study in mice
(Beland and Culp. 19981. Although humans do not have a forestomach, forestomach effects
observed in rodents are believed to be qualitatively supportive of a human hazard, as humans have
similar squamous epithelial tissue in their oral cavity (IARC. 2003: Wester and Kroes. 19881. EPA
has considered the uncertainty associated with the relevance of forestomach tumors for
quantitatively estimating human risk from benzo[a]pyrene exposure. The rodent forestomach
serves to store foods and liquids for several hours before contents continue to the stomach for
further digestion (Clavson etal.. 1990: Grice etal.. 19861. Thus, tissue of the forestomach in rodents
may be exposed to benzo[a]pyrene for longer durations than analogous human tissues in the oral
cavity and esophagus. This suggests that the rodent forestomach may be quantitatively more
sensitive to the development of squamous epithelial tumors in the forestomach compared to oral or
esophageal tumors in humans.
There appears to be no biological basis for concluding that the mouse study is more
representative of human response than the rat study. However, there is likely greater uncertainty
in the rat results, owing to administration of benzo[a]pyrene by gavage in soy bean oil as compared
with dietary exposure for the mice. Since benzo[a]pyrene is a lipophilic compound, it will partition
differently between the oil and stomach in this case than if it were administered through water.
From studies with other lipophilic compounds administered via corn oil gavage in rats, it is known
that this increases lymphatic uptake, and consequently, delays systemic delivery of the compound
fReddv etal.. 20051. Furthermore, a bolus gavage dose leads to higher peak concentration
compared to ingestion through food or water over the course of a day, which potentially results in
nonlinearities due to metabolic saturation. Corn oil gavage is also thought to result in increased
tumor-promoting ability based on a study in B6C3Fi mice fKlaunig etal.. 19901. Because these
effects are related to the lipophilicity of the compound, similar results may be expected of soy oil
gavage.
Uncertainty in the magnitude of the recommended oral slope factor is reflected to some
extent in the range of slope factors among tumors sites and species; the oral slope factor based on
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the mouse alimentary tract data was about 3-fold higher than the overall oral slope factor based on
male rat data (Table 2-8). These comparisons show that the selection of target organ, animal
species, and interspecies extrapolation can impact the oral cancer risk estimate. However, all of the
activation pathways implicated in benzo[a]pyrene carcinogenicity have been observed in human
tissues, and associations have been made between the spectra of mutations in tumor tissues from
benzo[a]pyrene-exposed animals and humans exposed to complex PAH mixtures containing
benzo[a]pyrene (see Section 1.1.5).
Table 2-8. Summary of uncertainties in the derivation of benzo[a]pyrene oral
slope factor
Consideration and
impact on cancer risk value
Decision
Justification and discussion
Selection of target organ
4/ oral slope factor, up to 5-fold, 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 humans, the
most sensitive result for alimentary tract tumors
was used to derive the oral slope factor.
Selection of data set
4/ oral slope factor ~3-fold if rat
bioassay were selected for oral slope
factor derivation
Beland and Culp (1998)
Beland and Culp (1998) was a well-conducted
study and had the lowest HEDs of the available
cancer bioassays, reducing low-dose
extrapolation uncertainty.
Selection of dose metric
Alternatives could 4^ or T* slope
factor
Administered dose
Experimental evidence supports a role for
metabolism in toxicity, but actual responsible
metabolites have not been identified.
Interspecies extrapolation
Alternatives could 4/ or T* slope
factor (e.g., 3.5-fold 4^ [scaling by
body weight] or T* 2-fold [scaling by
BW2/3])
BW3/4 scaling (default
approach)
There are no data to support alternatives.
Because the dose metric was not an area under
the curve, BW3/4 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 to neither over- nor
underestimate human equivalent risks.
Dose-response modeling
Alternative models considered,
including nonlinear, low-dose models
incompatible with mutagenic mode
of action, yielded slope factors, which
ranged up to 1.5-fold higher than the
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, and whether a tumor
caused the death of the animal), this model was
superior to other available models.
Low-dose extrapolation
4/ cancer risk estimate would be
expected with the application of
nonlinear low-dose extrapolation
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).
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Consideration and
impact on cancer risk value
Decision
Justification and discussion
Statistical uncertainty at POD
4/ oral slope factor 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% CI on administered
exposure at 10% extra risk of alimentary tract
tumors.
Sensitive subpopulations
1" oral slope factor 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 IRIS Assessment: Oral Slope Factor
The previous cancer assessment for benzo[a]pyrene was posted on the IRIS database in
1992. At that time, benzo[a]pyrene was classified as a probable human carcinogen (Group B2)
based on inadequate data in humans and sufficient data in animals via several routes of exposure.
Four slope factors were estimated based on studies of dietary benzo[a]pyrene administered
either for approximately 2 years in 10-week-old Sprague-Dawley rats (Brune etal.. 19811 or for up
7 months in 2-week-old to 5-month-old CFW-Swiss mice, sex unknown (Neal and Rigdon. 19671.
Each slope factor reflected extrapolation to humans assuming surface area equivalence (BW2/3
scaling), for a 2-fold increase in estimated risk relative to EPA's subsequent update to BW3/4 scaling
fU.S. EPA. 19921. A slope factor estimate of 11.7 per mg/kg-day, using a linearized multistage
procedure applied to the combined incidence of forestomach, esophageal, and laryngeal tumors,
was derived from the Brune etal. (19811 study (see Section 1.1.5 for study details).
Three modeling procedures were used to derive risk estimates from the Neal and Rigdon
f!9671 bioassay (see Section 1.1.5), resulting in a 2-fold range of risk estimates from the same data
set:
1) U.S. EPA f!991al fit a two-stage response model, based on exposure-dependent changes in
both transition rates and growth rates of preneoplastic cells, to derive a value of 5.9 per
mg/kg-day.
2) U.S. EPA (1991b) derived a value of 9.0 per mg/kg-day by linear extrapolation from the
10% response point to the background response in a re-analysis of the 1990 model.
3) Using a Weibull-type model to reflect less-than-lifetime exposure to benzo[a]pyrene, the
U.S. EPA (1991b) assessment derived an upper-bound slope factor estimate of 4.5 per
mg/kg-day.
The four slope factor estimates, within 3-fold of each other, were judged in 1992 to be of
equal merit, although based on less-than-optimal datasets. The geometric mean of these four
estimates, 7.3 per mg/kg-day, was previously recommended as the oral slope factor.
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2.4. INHALATION UNIT RISK FOR CANCER
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question and quantitative estimates of risk from oral and inhalation exposure
may be derived. Quantitative risk estimates may be derived from the application of a low-dose
extrapolation procedure. If derived, the inhalation unit risk is a plausible upper bound on the
estimate of risk per |J.g/m3 air breathed for a lifetime.
2.4.1. Analysis of Carcinogenicity Data
The inhalation database demonstrating carcinogenicity of benzo[a]pyrene consists of a
lifetime inhalation bioassay in male hamsters fThvssen etal.. 19811 and intratracheal instillation
studies, also in hamsters fFeron and Kruvsse. 1978: Ketkar etal.. 1978: Feronetal.. 1973: Henry et
al.. 1973: Saffiotti etal.. 19721. The intratracheal instillation studies provide supporting evidence of
carcinogenicity of inhaled benzo[a]pyrene; however, the use of this exposure method alters the
deposition, clearance, and retention of substances, and therefore, studies utilizing this exposure
technique are not as useful for the quantitative extrapolation of cancer risk from the inhalation of
benzo[a]pyrene in the environment fDriscoll etal.. 20001.
The bioassay by Thvssenetal. (19811 represents the only lifetime inhalation cancer
bioassay available for describing exposure-response relationships for cancer from inhaled
benzo[a]pyrene. As summarized in Section 1.1.5, increased incidences of benign and malignant
tumors of the pharynx, larynx, trachea, esophagus, nasal cavity, or forestomach were seen with
increasing exposure concentration. In addition, survival was decreased relative to control in the
high-exposure group; mean survival times in the control, low-, and mid-concentration groups were
96.4, 95.2, and 96.4 weeks, respectively, compared to 59.5 weeks in the high-exposure group
animals fThvssen etal.. 19811. Overall, tumors occurred earlier in the highestbenzo[a]pyrene
exposure group than in the mid-exposure group.
Strengths of the study included exposures until natural death, up to 2.5 years; multiple
exposure groups; histological examination of multiple organ systems; and availability of individual
animal pathology reports with time of death and tumor incidence data by site in the upper
respiratory and digestive tracts. In addition, the availability of weekly chamber air monitoring data
and individual times on study allowed the calculation of TWA lifetime continuous exposures for
each hamster. Group averages of these TWA concentrations were 0, 0.25,1.01, and 4.29 mg/m3
fU.S. EPA. 19901.
Several limitations concerning exposure conditions in the Thvssen et al. (19811 study were
evaluated for their impact on the derivation of an inhalation unit risk for benzo[a]pyrene. These
issues include minimal detail about the particle size distribution of the administered aerosols,
variability of chamber concentrations, and use of a sodium chloride aerosol as a carrier.
First, particle distribution analysis of aerosols, in particular the MMAD and geometric SD,
was not reported, although the investigators did report that particles were within the respirable
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range for hamsters, with >99% of the particles having diameters 0.2-0.5 |im and >80% having
diameters 0.2-0.3 |im.
Second, weekly averages of chamber concentration measurements varied 2- to 5-fold from
the overall average for each group, which exceeds the limit for exposure variability of <20% for
aerosols recommended by OECD f20091. For risk assessment purposes, EPA generally assumes
that cancer risk is proportional to cumulative exposure, and therefore to lifetime average exposure
as estimated here, when there is no information to the contrary. Under this assumption, the
variability of the chamber concentrations has little impact on the estimated exposure-response
relationship. The impacts of alternative assumptions are considered in Section 2.4.4.
Lastly, exposure occurred through the inhalation of benzo[a]pyrene adsorbed onto sodium
chloride aerosols, which might have irritant carrier effects, and will have a different deposition than
benzo[a]pyrene adsorbed onto carbonaceous particles (as is more typical in the environment). The
above study design and reporting issues concerning the particle size composition, exposure
variability, and deposition do not negate the robust tumor response following benzo[a]pyrene
inhalation exposure. Consequently, EPA concluded that the strengths of the study supported the
use of the data to derive an inhalation unit risk for benzo[a]pyrene. See Section 2.4.4 for a
discussion of uncertainties in the unit risk.
2.4.2. Dose-Response Analysis—Adjustments and Extrapolation Methods
Biologically based dose-response models for benzo[a]pyrene are not available. A simplified
version of the two-stage carcinogenesis model proposed by Moolgavkar and Venzon (1979) and
Moolgavkar and Knudson (1981) has been applied to the Thvssen et al. (1981) individual animal
data (U.S. EPA. 1990). However, the simplifications necessary to fit the tumor incidence data
reduced that model to an empirical model (i.e., there were no biological data to inform estimates of
cell proliferation rates for background or initiated cells, which are generally very sensitive
parameters in these cancer models). Sufficient data were available to apply the multistage-Weibull
model, as used for the oral slope factor (described in detail in Appendix E of the Supplemental
Information), specifically the individual times of death for each animal. Unlike in the oral bioassays,
Thvssen etal. (1981) did not determine cause of death for any of the animals. Since the
investigators for the oral bioassays considered some of the same tumor types to be fatal at least
some of the time, bounding estimates of the POD for these Thvssen etal. (1981) data were
developed by treating the tumors alternately as either all incidental to the death of an affected
animal or as causing the death of an affected animal.
The tumor incidence data used for dose-response modeling comprised the benign and
malignant tumors in the pharynx and respiratory tract (see Table E-27). The tumors in these sites
were judged to be sufficiently similar to combine in overall incidences, based on the assumption
that the benign tumors could develop into malignancies, as outlined in EPA's Guidelines for
Carcinogen Risk Assessment (Section 2.2.2.1.2: U.S. EPA. 2005a). Specifically, while the pharynx and
larynx are associated with the upper digestive tract and the upper respiratory tract, respectively,
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these sites are close anatomically and in some cases where both tissues were affected, the site of
origin could not be distinguished (U.S. EPA. 19901. In addition, the benign tumors (e.g., papillomas,
polyps, and papillary polyps) were considered early stages of the SCCs in these tissues fU.S. EPA.
19901. Consequently, the overall incidence of SCCs or benign tumors judged to originate from the
same cell type (papillomas, polyps, or papillary polyps) were selected for dose-response modeling.
A toxicokinetic model to assist in cross-species scaling of benzo[a]pyrene inhalation
exposure was not available. EPA's RfC default dosimetry adjustments (U.S. EPA. 1994a) were
utilized in the benzo[a]pyrene RfC calculation (see Section 2.2.2) but could not be applied to the
aerosols generated for the inhalation bioassay by Thvssenetal. (1981) as the approaches
presented in the RfC methodology guidelines fU.S. EPA. 1994al were developed for insoluble and
nonhygroscopic particles, not the sodium chloride particle used in Thvssenetal. (1981). Further,
relative surface areas of the upper respiratory tract in hamsters and humans were not considered
relevant because hamsters and humans are not necessarily expected to respond in the same areas
of the respiratory tract. Consequently, without data to inform a basis for extrapolation to humans,
it was assumed that equal risk for all species would be associated with equal concentrations in air,
at least at anticipated environmental concentrations, as would be the case for a soluble gas. This is
equivalentto assuming that breathing rate and metabolism of benzo[a]pyrene to DNA-reactive
metabolites both scale across species in proportion to BW3/4.
The multistage-Weibull model was fit to the TWA exposure concentrations and the
individual animal tumor and survival data for tumors in the larynx, pharynx, trachea, or nasal cavity
(tumors of the pharynx and upper respiratory tract), using the software program, MultiStage-
Weibull (U.S. EPA. 2010b). Modeling results are provided in Appendix E.2 of the Supplemental
Information. Because benzo[a]pyrene carcinogenicity involves a mutagenic mode of action, linear
low-exposure extrapolation from the BMCLio was used to derive the inhalation unit risk fU.S. EPA.
2005a).
2.4.3. Inhalation Unit Risk Derivation
The results from modeling the inhalation carcinogenicity data from Thvssenetal. (1981)
are summarized in Table 2-9. Taking the tumors to have been the cause of death for the
experimental animals with tumors, the BMCio and BMCLio values were 0.468 and 0.256 mg/m3,
respectively. Then, taking all of the tumors to have been incidental to the cause of death for each
animal with a tumor, the BMCio and BMCLio values were 0.254 and 0.163 mg/m3, respectively,
about 2-fold lower than the first case. Because the tumors were unlikely to have all been fatal, and
cancer risk estimation focuses on cancer incidence rather than death from cancer, the lower BMCLio
from the incidental deaths analysis, 0.163 mg/m3, is recommended for the calculation of the
inhalation unit risk.
Using linear extrapolation from the BMCLio (0.163 mg/m3), an inhalation unit risk of
0.6 per mg/m3, or 6 x 10"4 per ng/m3 (rounding to one significant digit), was calculated. Note
that the inhalation unit risk should only be used with lifetime human exposures <0.3 mg/m3, the
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human equivalent POD, because above this level, the dose-response relationship is nonlinear and
plateaus at 100% at higher exposures. If risk estimates are needed for exposure above
0.3 mg/m3—that is, corresponding to overall cancer risks >10%—the full dose-response model as
provided in Appendix E.2.2 should be consulted.
The inhalation unit risk for benzo[a]pyrene is derived with the intention that it will be
paired with EPA's relative potency factors for the assessment of the carcinogenicity of PAH
mixtures. In addition, regarding the assessment of early life exposures, because cancer risk values
calculated for benzo[a]pyrene were derived from adult animal exposures, and because
benzo[a]pyrene carcinogenicity occurs via a mutagenic mode of action, exposures that occur during
development should include the application of ADAFs (see Section 2.5).
EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) recommend
characterizing expected or central estimates of risk where practicable, and confidence limits on the
POD. For the available data, note that a central estimate of risk below the POD is not practicable
because the POD determines the low end of the range for which statistical predictions can be
supported. The slope of the linear extrapolation from the BMDio, the central tendency estimate at
the POD, can be calculated [0.1/(0. 254 mg/m3) = 0.39 per mg/m3], but cannot be considered a
central tendency estimate throughout the low dose range (below the POD). For the recommended
slope factor, the 95% confidence limits based on the POD are 0.163-0.324 mg/m3.
Table 2-9. Summary of the inhalation unit risk derivation
Tumor site and context
Species/
sex
Selected
model
BMR
BMC
(mg/m3)
POD =
BMCL
(mg/m3)
Unit risk3
(mg/m3)"1
Upper respiratory tract and pharynx;
all treated as cause of death
Thvssen et al. (1981)
Male
hamsters
Multistage
Weibull, 2°
10%
0.468
0.256
0.4
Upper respiratory tract and pharynx;
all treated as incidental to death
Thvssen et al. (1981)
Male
hamsters
Multistage
Weibull, 2°
10%
0.254
0.163
0.6
aHuman equivalent unit risk = 0.10/BMCLio; see Appendix E for details of modeling results.
2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk
Table 2-10 summarizes uncertainties in the derivation of the inhalation unit risk for
benzo[a]pyrene; further detail is provided in the following discussion. Only one animal cancer
bioassay, in one sex, by the inhalation route is available that describes the exposure-response
relationship for respiratory tract tumors with lifetime inhalation exposure to benzo[a]pyrene
(Thvssen et al.. 1981). Although corroborative information on exposure-response relationships in
other animal species is lacking, the findings for upper respiratory tract tumors are consistent with
findings in other hamster studies with intratracheal administration of benzo[a]pyrene (upper and
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lower respiratory tract tumors), and with some of the portal-of-entry effects in oral exposure
studies.
The hamster inhalation bioassay by Thvssenetal. T19811 observed upper respiratory tract
tumors, but not lung tumors. The lack of a lung tumor response in hamsters, given the strong
association of inhaled PAH mixtures with lung cancer in humans across many studies (see
Section 1.1.5) suggests that this study may not be ideal for extrapolating to humans. Hamsters have
an apparent lower sensitivity to lung carcinogenesis than rats and mice and a tendency to give false
negatives for particles classified as carcinogenic to humans by the International Agency for
Research on Cancer (IARC) (Mauderlv. 1997). However, hamster laryngeal tumors have been used
as an indication of the carcinogenic hazard of cigarette smoke for more than 50 years flARC. 20021.
For example, a large study investigating the inhalation of cigarette smoke in hamsters (n = 4,400)
indicated that the larynx was the most responsive tumor site, which the authors indicated was due
to a large difference in particle deposition between the larynx and the lung (Dontenwill etal.. 1973).
EPA's Guidelines for Carcinogen Assessment (U.S. EPA. 2005a) stress that site concordance between
animals and humans need not always be assumed. Therefore, the robust tumor response in the
upper respiratory tract of Syrian golden hamsters was considered to be supportive of the use of the
Thvssenetal. f 19811 study for the derivation of an inhalation unit risk.
Data from the Thvssenetal. f!9811 study were incomplete; histopathology reports were
completely missing for 4 hamsters in the mid-dose group and for single tissues in 21 other
hamsters over all four groups. The recommended unit risk (0.6 per mg/m3) omitted these animals
altogether, as if they had never been on study. A reanalysis including the animals with partial
histopathology, and assuming no tumors among them, yielded a unit risk of 0.5 per mg/m3, about
20% lower (see Table E-32).
Additional sensitivity analyses included using other dose-response models and different
latency assumptions in the multistage-Weibull model. BMDS dichotomous dose-response models
were applied to poly-3 adjusted incidence data to address intercurrent mortality (see Table E-32).
These adjusted estimates also considered the length of time on study for the animals with
incomplete histopathology. None of these models provided adequate fits. Dropping the high dose
led to a better fit to the low exposure region and an adequate overall fit, with BMDio and BMDLio
values about 20% higher than for the recommended unit risk.
Alternative assumptions for latency in the time-to-tumor model were more ad hoc, due to
lack of information in the scientific literature for respiratory tumors and lack of cause-of-death
information in the Thvssen et al. (1981) data set One approach involved judging which tumors
were or were not the cause of death. For the purpose of this analysis, it was assumed that benign
tumors did not cause death and that all malignant tumors were the cause of death; this approach
yielded a latency estimate of 14.5 weeks (see Table E-32). However, there were only five benign
tumors, all in the mid-exposure group; even if this is an accurate accounting of the tumors'
involvement in cause of death, it is not a strong basis for estimating this parameter for the 25 cases
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observed in the study. Another approach involved fixing latency at a range of values, 2-90 weeks;
these model versions tended not to fit the data as well as the recommended fit to the incidental
tumor data, as shown by larger Akaike's Information Criterion (AIC) values (see Table E-32).
BMD 10 and BMDLio values were higher for the lower four latency values assumed, but were very
similar to the recommended POD when latency was set at 90 weeks. These results suggest some
insensitivity to latency, or possibly that a constant value for latency across exposure levels is not
supported.
An additional uncertainty includes the inability to apply U.S. EPA (1994a) dosimetry
approaches to extrapolate inhaled concentrations from hamsters to humans, due to the use of a
soluble hygroscopic carrier particle (sodium chloride) for the delivery of benzo[a]pyrene. One
likely consequence of the use of hygroscopic carrier particles would be the growth of
benzo[a]pyrene-sodium chloride particles in the humid environment of the respiratory tract
resulting in increased particle diameter and resulting changes in particle deposition, specifically,
increased impaction in the upper respiratory tract and less deposition in the lung (Varghese and
Gangamma. 2009: Asgharian. 2004: Ferron. 1994: Xu and Yu. 1985). In addition, sodium chloride
can be irritating to the respiratory tract, depending on concentration. The Thvssen etal. (1981)
study reported that vehicle controls were exposed to 240 |ig/m3, and it is unclear whether exposure
to this concentration of sodium chloride could have potentiated the tumor response seen in the
mid- and high-concentration benzo[a]pyrene groups. Exposure to benzo[a]pyrene in the
environment predominantly occurs via non-soluble, non-hygroscopic, carbonaceous particles (such
as soot and diesel exhaust particles). The potential impact of differences in carrier particle on the
magnitude of the inhalation unit risk is unknown.
Extrapolation of risk from hamsters to humans may be informed by considering the
equivalent inhaled dose in each species. Using default breathing rates for hamsters and humans of
0.063 and 20 m3/day (U.S. EPA (1994a). and body weights of 0.1 kg (Thvssen etal.. 1981) and
70 kg, the equivalent daily inhaled doses, normalized by body weight, corresponding to the POD of
0.61 mg/m3 are 0.10 and 0.046 mg/kg-day, respectively. This level of agreement is roughly
supports assuming equal risk at equal concentrations in air. Alternatives comprised consideration
of scaling inhaled doses, in mg/kg-day units, by BW3/4 (highlighting allometric differences in
metabolism and clearance rates over their lifetimes) and by BW2/3 (highlighting species differences
proportional to relative surface areas). Both considerations suggest higher risks to humans than to
hamsters at the same exposure level, by about 5- and 8-fold, respectively.
Regarding uncertainty associated with exposure characterization, the individual exposure
chamber measurements varied from about an order of magnitude less than the target concentration
to about 2-fold higher than the target concentration. Weekly average analytical concentrations
were documented to vary by 2-5-fold in all exposed groups, with no particular trends over time.
Continuous time-weighted group average concentrations were used for dose-response modeling
under the assumption that equal cumulative exposures are expected to lead to similar outcomes.
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This assumption is generally expected to lead to an unbiased estimate of risk when there is
incomplete information. However, it is possible that peak exposure above some concentration may
be more associated with the observed effects, or that deposition of particles may have reached a
maximum level or plateau, such as in the high-exposure group. Regarding the role of peak
exposures, the higher exposures for each group were distributed evenly throughout the study for
the most part, suggesting that any association of risk with peak exposures would also be
proportional to cumulative exposure. If particle deposition reached a plateau with the high-
exposure group, there is relatively less impact on the unit risk because the derivation relies on the
dose-response at lower exposure. But the actual dynamics of particle deposition at these or other
exposure levels are not well understood. There is not enough information available to estimate a
more quantitative impact on the estimated unit risk due to these uncertainties.
Table 2-10. Summary of uncertainties in the derivation of cancer risk values
for benzo[a]pyrene (inhalation unit risk)
Consideration and
impact on cancer risk value
Decision
Justification and discussion
Selection of data set and target organ
No inhalation unit risk if Thvssen et al.
(1981) not used
Respiratory tract tumors
from Thvssen et al.
(1981)
The Thvssen et al. (1981) bioassav is the onlv
lifetime inhalation cancer bioassay available for
describing exposure-response relationships for
cancer from inhaled benzo[a]pyrene.
Intratracheal instillation studies support the
association of benzo[a]pyrene exposure with
respiratory tract tumors.
Selection of dose metric
Alternatives could 4, or T* unit risk
Administered exposure
as TWA
Experimental evidence supports a role for
metabolism in toxicity, but actual responsible
metabolites are not identified. The
recommended unit risk is a reasonable estimate
if the proportion of the carcinogenic moiety
remains the same at lower exposures.
Interspecies extrapolation
Alternatives would T* (mg/kg2/3,
mg/kg3/4) unit risk 5-8-fold
Equal risk per ng/m3
(ppm-equivalence) is
assumed. The carrier
particle used was soluble
and hygroscopic;
therefore, the RfC
methodology (U.S. EPA,
1994a) dosimetric
adjustments could not
be applied.
There are no data to support alternatives. Equal
risk per ng/m3 is equivalent to assuming that
intake scales with BW3/4. It does not account for
the rate of production of DNA-reactive
metabolites in the affected tissues, which are
likely to be proportional to BW3/4
Dose-response modeling
Alternatives could 4' or T* unit risk
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.
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Toxicological Review of Benzo[a]pyrene
Consideration and
impact on cancer risk value
Decision
Justification and discussion
Low-dose extrapolation
4/ cancer risk estimate would be
expected with the application of
nonlinear low-dose extrapolation
Linear extrapolation
from the 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).
Statistical uncertainty at POD
4/ inhalation unit risk 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% CI on
administered exposure at 10% extra risk of
respiratory tract tumors.
Sensitive subpopulations
1" inhalation unit risk 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.
2.4.5. Previous IRIS Assessment: Inhalation Unit Risk
An inhalation unit risk for benzo[a]pyrene was not previously available on IRIS.
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS (ADAFs)
Based on sufficient support in laboratory animals and relevance to humans, benzo[a]pyrene
is determined to be carcinogenic by a mutagenic mode of action. According to the Supplemental
Guidance for Assessing Susceptibility from Early Life Exposure to Carcinogens ("Supplemental
Guidance") fU.S. EPA. 2005bl individuals exposed during early life to carcinogens with a mutagenic
mode of action are assumed to have increased risk for cancer. The oral slope factor of 1 per
mg/kg-day, and inhalation unit risk of 0.6 per mg/m3 for benzo[a]pyrene, calculated from data
applicable to adult exposures, do not reflect presumed early life susceptibility to this chemical.
Although chemical-specific data exist for benzo[a]pyrene that quantitatively demonstrate increased
early life susceptibility to cancer (Vesselinovitch etal.. 19751. these data were not considered
sufficient to develop separate risk estimates for childhood exposure, as they used acute i.p.
exposures (U.S. EPA. 2005b). In the absence of adequate chemical-specific data to evaluate
differences in age-specific susceptibility, the Supplemental Guidance (U.S. EPA. 2005b) recommends
that ADAFs be applied in estimating cancer risk.
The Supplemental Guidance (U.S. EPA. 2005b) establishes ADAFs for three specific age
groups. These ADAFs and their corresponding age groupings are: 10 for individuals exposed at
<2 years of age, 3 for exposed individuals at 2-<16 years of age, and 1 for exposed individuals
>16 years of age. The 10- and 3-fold adjustments are combined with age-specific exposure
estimates when estimating cancer risks from early life (<16 years of age) exposures to
benzo[a]pyrene. To illustrate the use of the ADAFs established in the Supplemental Guidance (U.S.
EPA. 2005b). sample calculations are presented for three exposure duration scenarios, including
full lifetime, assuming a constant benzo[a]pyrene exposure of 0.001 mg/kg-day (Table 2-11).
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Toxicological Review of Benzo[a]pyrene
Table 2-11. Sample application of ADAFs for the estimation of benzo[a]pyrene
cancer risk following lifetime (70-year) oral exposure
Age group
ADAF
Unit risk (per
mg/kg-d)
Sample exposure
concentration (mg/kg-d)
Duration
adjustment
Cancer risk for age-
specific exposure
period
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
0.0008
Total risk
0.002
The example exposure duration scenarios include full lifetime exposure (assuming a
70-year lifespan). Table 2-11 lists the four factors (ADAFs, cancer risk estimate, assumed exposure,
and duration adjustment) that are needed to calculate the age-specific cancer risk based on the
early age-specific group. The cancer risk for each age group is the product of the four factors in
columns 2-5. Therefore, the cancer risk following daily benzo[a]pyrene oral exposure in the age
group 0-<2 years is the product of the values in columns 2-5orl0xlx 0.001x2/70 = 3x 10"4.
The cancer risk for specific exposure duration scenarios that are listed in the last column are added
together to get the total risk. Thus, a 70-year (lifetime) risk estimate for continuous exposure to
0.001 mg/kg-day benzo[a]pyrene is 2 x 10~3, which is adjusted for early-life susceptibility and
assumes a 70-year lifetime and constant exposure across age groups.
In calculating the cancer risk for a 30-year constant exposure to benzo[a]pyrene at an
exposure level of 0.001 mg/kg-day for ages 0-30 years, the duration adjustments would be 2/70,
14/70, and 54/70, and the age-specific risks for the three age groups would be 3 x 10~4, 6 x 10"4,
and 2 x 10"4, which would result in a total risk estimate of 1 x 10"3.
In calculating the cancer risk for a 30-year constant exposure to benzo[a]pyrene at an
exposure level of 0.001 mg/kg-day for ages 20-50 years, the duration adjustments would be 0/70,
0/70, and 30/70. The age-specific risks for the three groups are 0, 0, and 4 x 10"4, which would
result in a total risk estimate of 4 x 10"4.
Consistent with the approaches for the oral route of exposure (Table 2-11), the ADAFs
should also be applied when assessing cancer risks for subpopulations with early life exposures to
benzo[a]pyrene via the inhalation route (presented in Table 2-12).
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Toxicological Review of Benzo[a]pyrene
Table 2-12. Sample application of ADAFs for the estimation of benzo[a]pyrene
cancer risk following lifetime (70-year) inhalation exposure
Age group
ADAF
Unit risk
(per pg/m3)
Sample exposure
concentration (pg/m3)
Duration
adjustment
Cancer risk for
age-specific
exposure period
0-<2 yrs
10
6 x 10"4
0.1
2 yrs/70 yrs
0.00002
2-<16 yrs
3
6 x 10"4
0.1
14 yrs/70 yrs
0.00004
>16 yrs
1
6 x 10"4
0.1
54 yrs/70 yrs
0.00005
Total risk
0.00010
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Toxicological Review of Benzo[a]pyrene
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