*>EPA
EPA/690/R-21/008F | September 2021 | FINAL
United States
Environmental Protection
Agency
Provisional Peer-Reviewed Toxicity Values for
Benzo[e]pyrene (BeP)
(CASRN 192-97-2)
PRO1*
SUPERFUND
U.S. EPA Office of Research and Development
Center for Public Health and Environmental Assessment
-------
A United $ta»s
Environmental Protection
%#UI r% Agency
EPA/690/R-21/008F
September 2021
https://www.epa.gov/pprtv
Provisional Peer-Reviewed Toxicity Values for
Benzo[e]pyrene (BeP)
(CASRN 192-97-2)
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Jeffry L. Dean II, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
CONTRIBUTORS
Michelle Angrish, PhD
Center for Public Health and Environmental Assessment, Research Triangle Park, NC
Roman Mezencev, PhD
Center for Public Health and Environmental Assessment, Washington, DC
PRIMARY INTERNAL REVIEWERS
Deborah Segal, MS
Center for Public Health and Environmental Assessment, Washington, DC
Michele M. Taylor, PhD
Center for Public Health and Environmental Assessment, Research Triangle Park, NC
PRIMARY EXTERNAL REVIEWERS
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
PPRTV PROGRAM MANAGEMENT
Teresa L. Shannon
Center for Public Health and Environmental Assessment, Cincinnati, OH
J. Phillip Kaiser, PhD, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
Questions regarding the content of this PPRTV assessment should be directed to the U.S. EPA
Office of Research and Development (ORD) Center for Public Health and Environmental
Assessment (CPHEA) website at https://ecomments.epa.gov/pprtv.
in
Benzo[e]pyrene
-------
TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS v
BACKGROUND 1
QUALITY ASSURANCE 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVs 2
1. INTRODUCTION 3
2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 7
2.1. HUMAN STUDIES 10
2.1.1. Oral Exposures 10
2.1.2. Inhalation Exposures 10
2.2. ANIMAL STUDIES 10
2.2.1. Oral Exposures 10
2.2.2. Inhalation Exposures 10
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 10
2.3.1. Genotoxi city 10
2.3.2. Supporting Human Studies 24
2.3.3. Supporting Animal Studies 25
2.3.4. Metabolism/Toxicokinetic Studies 42
3. DERIVATION OI PROVISIONAL VALUES 44
3.1. DERIVATION OF PROVISIONAL REFERENCE DOSES 44
3.2. DERIVATION OF PROVISIONAL REFERENCE CONCENTRATIONS 44
3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES 44
3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 45
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES 46
APPENDIX A. SCREENING NONCANCER PROVISIONAL VALUES 47
APPENDIX B. BACKGROUND AND METHODOLOGY FOR THE SCREENING
EVALUATION OF POTENTIAL CARCINOGENICITY 76
APPENDIX C. RESULTS OF THE SCREENING EVALUATION OF POTENTIAL
CARCINOGENICITY 85
APPENDIX D. REFERENCES 109
iv
Benzo[e]pyrene
-------
COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
IVF
in vitro fertilization
ACGIH
American Conference of Governmental
LC50
median lethal concentration
Industrial Hygienists
LD50
median lethal dose
AIC
Akaike's information criterion
LOAEL
lowest-observed-adverse-effect level
ALD
approximate lethal dosage
MN
micronuclei
ALT
alanine aminotransferase
MNPCE
micronucleated polychromatic
AR
androgen receptor
erythrocyte
AST
aspartate aminotransferase
MOA
mode of action
atm
atmosphere
MTD
maximum tolerated dose
ATSDR
Agency for Toxic Substances and
NAG
\-acctyl-(}-D-gliicosaiiiiiiidasc
Disease Registry
NCI
National Cancer Institute
BMC
benchmark concentration
NOAEL
no-observed-adverse-effect level
BMCL
benchmark concentration lower
NTP
National Toxicology Program
confidence limit
NZW
New Zealand White (rabbit breed)
BMD
benchmark dose
OCT
ornithine carbamoyl transferase
BMDL
benchmark dose lower confidence limit
ORD
Office of Research and Development
BMDS
Benchmark Dose Software
PBPK
physiologically based pharmacokinetic
BMR
benchmark response
PCNA
proliferating cell nuclear antigen
BUN
blood urea nitrogen
PND
postnatal day
BW
body weight
POD
point of departure
CA
chromosomal aberration
PODadj
duration-adjusted POD
CAS
Chemical Abstracts Service
QSAR
quantitative structure-activity
CASRN
Chemical Abstracts Service registry
relationship
number
RBC
red blood cell
CBI
covalent binding index
RDS
replicative DNA synthesis
CHO
Chinese hamster ovary (cell line cells)
RfC
inhalation reference concentration
CL
confidence limit
RID
oral reference dose
CNS
central nervous system
RGDR
regional gas dose ratio
CPHEA
Center for Public Health and
RNA
ribonucleic acid
Environmental Assessment
SAR
structure-activity relationship
CPN
chronic progressive nephropathy
SCE
sister chromatid exchange
CYP450
cytochrome P450
SD
standard deviation
DAF
dosimetric adjustment factor
SDH
sorbitol dehydrogenase
DEN
diethylnitrosamine
SE
standard error
DMSO
dimethylsulfoxide
SGOT
serum glutamic oxaloacetic
DNA
deoxyribonucleic acid
transaminase, also known as AST
EPA
Environmental Protection Agency
SGPT
serum glutamic pyruvic transaminase,
ER
estrogen receptor
also known as ALT
FDA
Food and Drug Administration
SSD
systemic scleroderma
FEVi
forced expiratory volume of 1 second
TCA
trichloroacetic acid
GD
gestation day
TCE
trichloroethylene
GDH
glutamate dehydrogenase
TWA
time-weighted average
GGT
y-glutamyl transferase
UF
uncertainty factor
GSH
glutathione
UFa
interspecies uncertainty factor
GST
g 1 u ta t h i o nc -.V-1 ra n s fc ra sc
UFC
composite uncertainty factor
Hb/g-A
animal blood-gas partition coefficient
UFd
database uncertainty factor
Hb/g-H
human blood-gas partition coefficient
UFh
intraspecies uncertainty factor
HEC
human equivalent concentration
UFl
LOAEL-to-NOAEL uncertainty factor
HED
human equivalent dose
UFS
subchronic-to-chronic uncertainty factor
i.p.
intraperitoneal
U.S.
United States of America
IRIS
Integrated Risk Information System
WBC
white blood cell
Abbreviations and acronyms not listed on this page are defined upon first use in the
PPRTV document.
v
Benzo[e]pyrene
-------
EPA/690/R-21/008F
PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
BENZO [E] PYRENE (CASRN 192-97-2)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund program. PPRTVs are derived after a review of the relevant
scientific literature using established U.S. Environmental Protection Agency (U.S. EPA)
guidance on human health toxicity value derivations.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
Currently available PPRTV assessments can be accessed on the U.S. EPA's PPRTV
website at https://www.epa.gov/pprtv. PPRTV assessments are eligible to be updated on a 5-year
cycle and revised as appropriate to incorporate new data or methodologies that might impact the
toxicity values or affect the characterization of the chemical's potential for causing adverse
human-health effects. Questions regarding nomination of chemicals for update can be sent to the
appropriate U.S. EPA Superfund and Technology Liaison
(https://www.epa.gov/research/fact-sheets-regional-science).
QUALITY ASSURANCE
This work was conducted under the U.S. EPA Quality Assurance (QA) program to ensure
data are of known and acceptable quality to support their intended use. Surveillance of the work
by the assessment managers and programmatic scientific leads ensured adherence to QA
processes and criteria, as well as quick and effective resolution of any problems. The QA
manager, assessment managers, and programmatic scientific leads have determined under the
QA program that this work meets all U.S. EPA quality requirements. This PPRTV was written
with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP), the QAPP
titled Program Quality Assurance Project Plan (PQAPP) for the Provisional Peer-Reviewed
Toxicity Values (PPRTVs) and Related Assessments/Documents (L-CPAD-0032718-QP), and the
PPRTV development contractor QAPP titled Quality Assurance Project Plan—Preparation of
Provisional Toxicity Value (PTV) Documents (L-CPAD-0031971-QP). As part of the QA
system, a quality product review is done prior to management clearance. A Technical Systems
Audit may be performed at the discretion of the QA staff.
All PPRTV assessments receive internal peer review by at least two CPHEA scientists
and an independent external peer review by at least three scientific experts. The reviews focus on
whether all studies have been correctly selected, interpreted, and adequately described for the
purposes of deriving a provisional reference value. The reviews also cover quantitative and
qualitative aspects of the provisional value development and address whether uncertainties
associated with the assessment have been adequately characterized.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
1
Benzo[e]pyrene
-------
EPA/690/R-21/008F
limitations of the data. All users are advised to review the information provided in this document
to ensure that the PPRTV used is appropriate for the types of exposures and circumstances at the
site in question and the risk management decision that would be supported by the risk
assessment.
Other U.S. EPA programs or external parties who may choose to use PPRTVs are
advised that Superfund resources will not generally be used to respond to challenges, if any, of
PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the content of this PPRTV assessment should be directed to the
U.S. EPA ORD CPHEA website at https://ecomments.epa.gov/pprtv.
2
Benzo[e]pyrene
-------
EPA/690/R-21/008F
1. INTRODUCTION
Benzo[e]pyrene (BeP), CASRN 192-97-2, belongs to the poly cyclic aromatic
hydrocarbon (PAH) class of chemicals. PAHs are typically complex mixtures of several PAH
compounds that occur naturally in fossil fuels, crude oil, and bituminous coal (Nl.M. 2019a).
BeP is also formed as a product of incomplete combustion of organic matter; it has been found in
smoke from tobacco and marijuana cigarettes and in emissions from burning coal, wood, oil, and
garbage. BeP has also been detected in both fresh and used motor oils, gasolines, and smoked
and cooked food (Nl .M. 2019a). BeP is purified for use in research laboratories but has no other
known uses and is not listed on the U.S. EPA's Toxic Substances Control Act (TSCA) public
inventory (U.S. HP A. 2018b). It is registered with Europe's Registration, Evaluation,
Authorisation and Restriction of Chemicals (REACH) program as a member of the PAH group
of chemicals and has a Harmonised Classification Annex VI of Regulation (EC) No. 1272/2008
(CLP Regulation) listing, which requires manufacturers, importers, or downstream users to
classify, label, and package BeP before placing it on the market (HCHA. 2019a. b).
The empirical formula for BeP is C20H12 and its structure is shown in Figure 1. A table of
physicochemical properties for BeP is provided in Table 1. BeP is a crystalline solid with
negligible water solubility and low vapor pressure. A Henry's law constant of
1.07 x 10 6 atm-m3/mol calculated from the extrapolated vapor pressure of 5.7 x 10 9 mm Hg
and measured water solubility of 1.89 x 10 8 mol/L indicates low volatilization of BeP from
moist soil and water. No volatilization from dry soil is expected based on the vapor pressure. In
the atmosphere, BeP will exist primarily in the particulate phase and to a lesser extent in the
vapor phase. The particulate phase BeP in the atmosphere will be removed by wet and dry
deposition. BeP absorbs ultraviolet (UV) wavelengths >290 nm; however, removal by direct
photodegradation is not expected to be an important pathway based on a measured half-life of
21.1 days in the presence of simulated sunlight (NIST. 2019; Katz. 1979). The rate of
photodegradation is expected to increase in the presence of oxidative species, such as oxygen
and ozone. The chemical structure of BeP lacks functional groups susceptible to hydrolysis under
environmental conditions; therefore, hydrolysis is not expected to be an important degradation
route in aqueous environments. BeP is expected to adsorb suspended solids and sediment in
water and will be immobile in soil based on a predicted Koc value of 4.66 x 105 in soil. Sorption
of PAH compounds, such as BeP, by soil organic matter will limit the potential for
biodegradation (WHO. 1998). Limited biodegradation data for BeP indicate slow removal in
water and soil under aerobic and anaerobic conditions, with a reported half-life of 875 days in
petroleum-contaminated sediment and no removal in an anaerobic sludge digestion study [Kirk
and Lesterm (1990) and Callahan et al. (1979) as cited in NLM (2019a)].
3
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 1. Physicochemical Properties of BeP (CASRN 192-97-2)a
Property (unit)
Value
Physical state
Solid
Boiling point (°C)
469 (predicted average)
Melting point (°C)
178
Density (g/cm3)
1.28 (predicted average)
Vapor pressure (mm Hg)
5.7 x 1(T9
pH (unitless)
NA
Acid dissociation constant (pKa) (unitless)
NA
Solubility in water (mol/L)
1.89 x 1(T8
Octanol-water partition constant (log Kow)
6.44
Henry's law constant (atm-m3/mol)
1.07 x 10 6 (predicted average)
Soil adsorption coefficient (Koc) (L/kg)
4.66 x io5 (predicted average)
Atmospheric OH rate constant (cm3/molecule-sec)
3.45 x 10 11 (predictedaverage)
Atmospheric half-life (d)
21.10 (measured in simulated sunlight)b
Relative vapor density (air =1)
NV
Molecular weight (g/mol)
252.316
Flash point (°C)
230 (predicted average)
aData were extracted from the U.S. EPA CompTox Chemicals Dashboard: Benzo[e]pyrene, CASRN 192-97-2;
https://comptox.epa.gOv/dashboard/dsstoxdb/results7searcliFDTXSID3023764#properties: accessed February 8,
2021. Data presented are experimental averages unless otherwise noted.
bKatz (1979).
BeP = benzo[e]pyrene; NA = not applicable; NV = not available.
A summary of available toxicity values for BeP from U.S. EPA and other
agencies/organizations is provided in Table 2.
4
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 2. Summary of Available Toxicity Values for BeP (CASRN 192-97-2)
Source
(parameter)3'b
Value
(applicability)
Notes
Reference0
Noncancer
IRIS
NV
NA
U.S. EPA (2020a)
HEAST
NV
NA
U.S. EPA (2011b)
DWSHA
NV
NA
U.S. EPA (2018a)
ATSDR (MRL)
NV
No MRLs were derived for
BeP
ATSDR (1995)
IPCS
NV
NA
IPCS (2020)
CalEPA
NV
NA
CalEPA (2019)
OSHA
NV
NA
OSHA (2020): OSHA (2020)
NIOSH
NV
NA
NIOSH (2018)
ACGIH
NV
NA
ACGIH (2020)
Cancer
IRIS
NV
NA
U.S. EPA (2020a)
HEAST
NV
NA
U.S. EPA (2011b)
DWSHA
NV
NA
U.S. EPA (2018a)
NTP
NV
NA
NTP (2016)
IARC (WOE)
Group 3: not classifiable
as to its carcinogenicity
to humans
Based on inadequate evidence
in experimental animals for
carcinogenicity
IARC (2010)
CalEPA
NV
NA
CalEPA (2019)
ACGIH
NV
NA
ACGIH (2020)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; CalEPA = California Environmental Protection Agency; DWSHA = Drinking
Water Standards and Health Advisories; HEAST = Health Effects Assessment Summary Tables;
IARC = International Agency for Research on Cancer; IPCS = International Programme on Chemical Safety;
IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety and Health;
NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration.
Parameters: MRL = minimal risk level; WOE = weight of evidence.
°Reference date is the publication date for the database and not the date the source was accessed.
BeP = benzo[e]pyrene; NA = not applicable; NV = not available.
Literature searches were conducted in August 2019 and updated in August 2021 for
studies relevant to the derivation of provisional toxicity values for BeP, CASRN 192-97-2.
Searches were conducted using U.S. EPA's Health and Environmental Research Online (HERO)
database of scientific literature. HERO searches the following databases: PubMed, TOXLINE1
(including TSCATS1), and Web of Science. The following resources were searched outside of
HERO for health-related values: American Conference of Governmental Industrial Hygienists
'Note that this version of TOXLINE is no longer updated
(https://www.nlm.nih.gov/databases/download/toxlinesubset.html'): therefore, it was not included in the literature
search update from August 2021.
5
Benzo[e]pyrene
-------
EPA/690/R-21/008F
(ACGIH), Agency for Toxic Substances and Disease Registry (ATSDR), California
Environmental Protection Agency (CalEPA), Defense Technical Information Center (DTIC),
European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), European
Chemicals Agency (ECHA), U.S. EPA Chemical Data Access Tool (CDAT), U.S. EPA
ChemView, U.S. EPA Integrated Risk Information System (IRIS), U.S. EPA Health Effects
Assessment Summary Tables (HEAST), U.S. EPA Office of Water (OW), International Agency
for Research on Cancer (IARC), U.S. EPA TSCATS2/TSCATS8e, U.S. EPA High Production
Volume (HPV), Chemicals via IPCS INCHEM, Japan Existing Chemical Data Base (JECDB),
Organisation for Economic Cooperation and Development (OECD) Screening Information Data
Sets (SIDS), OECD International Uniform Chemical Information Database (IUCLID), OECD
HPV, National Institute for Occupational Safety and Health (NIOSH), National Toxicology
Program (NTP), Occupational Safety and Health Administration (OSHA), and World Health
Organization (WHO).
6
Benzo[e]pyrene
-------
EPA/690/R-21/008F
2. REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
As summarized in Tables 3A and 3B, no short-term, subchronic, or chronic studies as
well as reproductive and developmental toxicity studies of BeP in humans or animals exposed by
oral or inhalation routes adequate for deriving provisional toxicity values have been identified.
The phrase "statistical significance" and term "significant," used throughout the document,
indicate ap-value of < 0.05 unless otherwise specified.
7
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 3A. Summary of Potentially Relevant Noncancer Data for BeP (CASRN 192-97-2)
Number of Male/Female, Strain, Species, Study
Critical
Reference
Category
Type, Reported Doses, Study Duration
Dosimetry
Effects
NOAEL
LOAEL
(comments)
Notes
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
BeP = benzo[e]pyrene; LOAEL = lowest-observed-adverse-effect level; ND = no data; NOAEL = no-observed-adverse-effect level.
8
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 3B. Summary of Potentially Relevant Cancer Data for BeP (CASRN 192-97-2)
Category
Number of Male/Female, Strain, Species, Study
Type, Reported Doses, Study Duration
Dosimetry
Critical Effects
Reference
(comments)
Notes
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
BeP = benzo[e]pyrene; ND = no data.
9
Benzo[e]pyrene
-------
EPA/690/R-21/008F
2.1. HUMAN STUDIES
2.1.1. Oral Exposures
No studies have been identified.
2.1.2. Inhalation Exposures
No studies have been identified.
2.2. ANIMAL STUDIES
2.2.1. Oral Exposures
No studies have been identified.
2.2.2. Inhalation Exposures
No studies have been identified.
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Available toxicity data for BeP include human occupational studies of exposure to
complex mixtures that are of limited use for hazard identification and do not provide adequate
exposure data for dose-response assessment. Two case-control studies found no associations
between asthma or lung cancer incidence and serum BeP concentrations following BeP
exposure. There is one subchronic study in apolipoprotein E knockout (apoE-KO) mice and two
studies in avian models evaluating atherosclerosis. An acute oral study evaluated biochemical
markers of hepatic damage. There are also dermal and injection studies evaluating
immunotoxicity and carcinogenicity. Other available data include in vitro assays evaluating
immunotoxic potential and retinal cell damage, genotoxicity assays, and toxicokinetic studies.
Because the overall database for BeP was determined to be inadequate for direct derivation of
oral or inhalation reference values, supporting studies described below were not considered for
point of departure (POD) identification.
2.3.1. Genotoxicity
The genotoxicity of BeP has been evaluated in numerous in vitro studies and in a limited
number of in vivo studies. Available studies are summarized below (see Table 4A for more
details). The data indicate that BeP has mutagenic activity following metabolic activation and is
not mutagenic in the absence of activation. In general, BeP did not cause chromosomal damage
in vitro; however, findings from in vivo studies are mixed and suggest that, under certain
conditions, BeP can cause chromosomal damage. BeP (or a metabolite) forms DNA adducts;
however, most available in vitro and in vivo studies do not indicate that BeP alters DNA
damage/ synthesi s/repair.
Mutagenicity
Of the available mutagenicity studies in Salmonella typhimurium, 15/20 reported that
BeP was positive following metabolic activation in one or more strains (see Table 4A). In
general, BeP yielded borderline or relatively low mutation rates compared with other mutagens
(e.g., other PAHs) (Zeiger et at.. 1992; De Flora etal.. 1984; Dunkel et at.. 1984; Haas et at..
1981; Kaden et at.. 1979; Simmon. 1979; Wood et at.. 1979; Andrews et at.. 1978; McCann et
at.. 1975). One experiment that reported negative results for BeP with metabolic activation was
part of a larger study comparing the results of experiments from four separate laboratories using
the same protocol. The other three experiments all found positive results (Dunkel et at.. 1984).
10
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella typhimurium
TA100
0, 1, 2.5, 5, 10, 25,
50, 100, 250, 500,
1,000 ng/plate
NS
+
Plate incorporation method. Criterion for positive
response was dose-response (significant linear
regression). The study authors reported that BeP
was positive with S9 activation. The number of
revertants was increased >twofold at 1,000 |ig with
S9 activation (data not reported for lower doses).
Results without metabolic activation were not
specified.
Andrews et al. (1978)
Mutation
S. typhimurium TA98,
TA100, and TA104
0,0.1,0.5, 1.0,2.5,
5.0, 10 ng/plate
NDr
+
(TA100)
(TA98,
TA104)
Plate incorporation method. Criterion for positive
response was not reported.13 Metabolic activation
was tested at three S9 concentrations (0.08, 0.22,
and 0.95 mg/plate). Based on graphically presented
data, the number of revertants was increased
>twofold at 10 ng/plate at the highest S9
concentration in TA100 only.
Ball et al. (1991)
Mutation
S. typhimurium TA98
0, 25, 50, 100,
200 nmol/plate
NDr
Plate incorporation method. Criterion for positive
response was not reported.13 No evidence of
mutagenicity without metabolic activation. A
positive control was not used (primary purpose of
study was to evaluate influence of PAHs on
mutagenicity of 1-nitropyrene).
Chemg et al. (1996)
11
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
Mutation
S. typhimurium TA97,
TA98, TA100, TA1535,
TA1537, TA1538
Various dilutions
using geometric
ratio of 2, starting
from solubility or
toxicity limit
+
(TA98, TA100)
±
(TA97, TA1537)
(TA1535, TA1538)
Plate incorporation method. Criterion for positive
response was >threefold increase in revertants
(two- to threefold increase considered weak
positive, 1.5- to twofold increase considered
"reproducible," <1.5-fold considered negative).
Based on this criterion, TA98 and TA100 were
positive, TA97 andTA1537 were "reproducible,"
and TA1535 and TA1538 were negative; S9
activation increased mutagenicity. It is not clear
from data presentation if reported fold-changes are
with or without S9 activation. Mutagenetic
potential inTAlOO =1.6 revertants/nmol with S9
activation.
De Flora et al. (1984)
Mutation
S. typhimurium TA98,
TA100, TA1535,
TA1537, TA1538
0,0.3, 1.0,3.3,
10.0, 33.3, 100.0,
333.3 ng/plate
+
(TA98,
TA100,
TA1538)
±
(TA1535)
(TA1537)
Plate test. IRI laboratory. All strains tested with
metabolic activation (using rat [R], mouse [M], and
hamster [H] liver S9; either uninduced [U] or
Arochlor induced [I]) and without activation.
Criterion for positive response was >twofold
increase at two or more doses; >twofold increase in
revertants at only one dose was considered
equivocal.
BeP was positive with metabolic activation in
TA98 (RI), TA100 (RI), and TA1538 (HU) and
equivocal with metabolic activation in TA1535
(MU). No evidence of mutagenicity without
metabolic activation.
Dunkel et al. (1984)
12
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
Mutation
S. typhimurium TA98,
TA100, TA1535,
TA1537, TA1538
0,0.3, 1.0,3.3,
10.0, 33.3, 100.0,
333.3 ng/plate
Plate test. LBI laboratory. All strains tested with
metabolic activation (RU, RI, MU, MI, HU, HI)
and without activation. Criterion for positive
response was >twofold increase at two or more
doses; >twofold increase in revertants at only one
dose was considered equivocal.
No evidence of mutagenicity with or without
metabolic activation. Results for positive controls
(sodium azide, 9-aminoacridine, 2-nitrofluorene,
2-aminoanthracene) were as expected.
Dunkel et al. (1984)
Mutation
S. typhimurium TA98,
TA100, TA1535,
TA1537, TA1538
0,0.3, 1.0,3.3,
10.0, 33.3, 100.0,
333.3 ng/plate
+
(TA98)
±
(TA1537,
TA1538)
(TA100,
TA1535)
Plate test. NYM laboratory. All strains tested with
metabolic activation (RU, RI, MU, MI, HU, HI)
and without activation. Criterion for positive
response was >twofold increase at two or more
doses; >twofold increase in revertants at only one
dose was considered equivocal.
BeP was positive with metabolic activation in
TA98 (RI) and equivocal with metabolic activation
in TA1537 (MI) and TA1538 (HU). No evidence
of mutagenicity without metabolic activation.
Dunkel et al. (1984)
13
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
Mutation
S. typhimurium TA98,
TA100, TA1535,
TA1537, TA1538
0,0.3, 1.0,3.3,
10.0, 33.3, 100.0,
333.3 ng/plate
+
(TA98,
TA100,
TA1537)
±
(TA1538)
(TA1535)
Plate test. SRI laboratory. All strains tested with
metabolic activation (RU, RI, MU, MI, HU, HI)
and without activation. Criterion for positive
response was >twofold increase at two or more
doses; >twofold increase in revertants at only one
dose was considered equivocal.
BeP was positive with metabolic activation in
TA98 (RI, MU), TA100 (RI) and TA1537 (MI)
and equivocal with metabolic activation in TA1538
(MI). No evidence of mutagenicity without
metabolic activation.
Dunkel et al. (1984)
Mutation
S. typhimurium TA98,
TA100
0.2-2.5 ng/plate
(TA98)
0.2-1.5 ng/plate
(TA100)
NDr
+
(TA100)
(TA98)
Plate incorporation method. Criterion for positive
response was not reported, but the study authors
reported a "significant" mutagenic response in
TA100 with S9 activation. No evidence of
mutagenicity in TA98 with S9 activation.
Haas et al. (1981)
Mutation
S. typhimurium TM677
NS
NDr
+
8-Azaguanine resistance forward mutation assay.
Criterion for positive response was >3 SDs from
the background mean. A positive response was
observed at >90 ^M with S9 activation.
Kaden et al. (1979)
Mutation
S. typhimurium TA98,
TA100
0, 10, 20 ng/plate
NDr
+
(TA100)
(TA98)
Plate incorporation method. Criterion for positive
response was not reported.13 Revertants increased
>twofold inTAlOO with S9 activation. No
evidence of mutagenicity in TA98 with S9
activation.
Lavoie et al. (1979)
Mutation
S. typhimurium TA100
10-333 ng/plate
NDr
Plate incorporation method. Criterion for positive
response was reversion frequency >2 times the
spontaneous rate. BeP was negative with metabolic
activation using liver homogenates from mice
pretreated with corn oil, BeP, or TCDD.
Ma et al. C199D
14
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
Mutation
S. typhimurium TA100
NS
NDr
+
Plate incorporation method. Criterion for positive
response was >0.1 revertants/mol, weak response
was <0.1 but >0.01 revertants/mol, and negative
response was <0.01 revertants/mol. BeP mutation
rate with S9 activation was 0.60 revertants/nmol
(143 revertants/60 ng tested).
McCannetal. (1975)
Mutation
S. typhimurium TA1535,
TA1537, TA1538, TA98,
TA100
0.1-1,000 ng/plate
(TA100)
±
(TA100)
Plate incorporation method. Criterion for positive
response was >twofold increase in revertants. A
twofold increase in revertants was observed in
TA100 with S9 activation. Mutagenic response was
reported as highest in TA100; number of
revertants/plate not reported for other strains.
Salamone et al. (1979)
Mutation
S. typhimurium TA97,
TA98, TA100
0, 5, 10, 50,
250 ng/plate
+
Plate incorporation method. Criterion for positive
response was not reported.13 Revertants increased
>twofold in all strains with S9 activation. No
evidence of mutagenicity without S9 activation.
Sakai et al. (1985)
Mutation
S. typhimurium TA1535,
TA1536, TA1537,
TA1538, TA98, TA100
<50 ng/plate
+
(TA1538,
TA98,
TA100)
(TA1535,
TA1536,
TA1537)
Plate incorporation method. Criterion for positive
result was a reproducible, dose-related increase in
the number of revertants. A positive result was
observed for TA1538, TA98, and TA100 with S9
activation. No evidence of mutagenicity without S9
activation.
Simmon (1979)
Mutation
S. typhimurium TA1535,
TA1536, TA1537,
TA1538
0, 50 ng/plate
NDr
Plate incorporation method. Criterion for positive
response was a significant increase in the number
of revertants. BeP was negative with metabolic
activation using liver homogenates from rats
pretreated with phenobarbital. A positive control
was not used, but other tested PAHs induced
significant increases in the number of revertants.
Teranishi et al. (1975)
15
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
Mutation
S. typhimurium TA98,
TA100
10 nmol/plate
NDr
±
Plate incorporation method. Criterion for positive
response was not reported, but BeP was reported as
weakly mutagenic in TA100 with S9 activation (rat
liver microsomes), although not when incubated
with purified rat hepatic CYP450. BeP was also
weakly mutagenic in TA98 when activated by
purified rat hepatic CYP450, but not S9.
Wood et al. (1979)
Mutation
S. typhimurium TA98
and its NR-deficient
strains TA98NR and
TA98/I,8-DNP6
0, 10 ng/plate
NDr
Plate incorporation assay. Criterion for positive
response was >twofold increase in revertants.
Mutagenic potential tested with and without
irradiation (artificial sunlight or cool-white light).
No evidence of mutagenicity with or without
irradiation. Positive control (BaP) produced
expected increase in revertants without irradiation
(not tested with irradiation).
White et al. (1985a)
Mutation
S. typhimurium TA98,
TA100
0, 3.3, 10, 33, 100,
333, 1,000,
2,000 ng/plate
+
Preincubation assay. Criterion for a positive
response was a reproducible, dose-related response
in replicate trials. BeP was positive inTA98 and
TA100 with rat S9 activation. No evidence of
mutagenicity without metabolic activation or with
hamster S9 activation.
Zeieeretal. (1992)
Mutation
Escherichia coli WP2
uvrA
0,0.3, 1.0,3.3,
10.0, 33.3, 100.0,
333.3 ng/plate
Plate test. E. coli was tested with metabolic
activation (RU, RI, MU, MI, HU, HI) and without
activation at four separate laboratories (IRI, LBI,
NYM, SRI).
All laboratories: No evidence of mutagenicity with
or without metabolic activation.
Dunkel et al. (1984)
DNA repair
E. coli WP2, WP67,
C871
<50 |ig (eight
twofold dilutions)
~
~
Liquid micromethod. The minimal inhibitory
concentration was >50 |ig with or without S9
activation.
De Flora et al. (1984)
16
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
DNA repair
E. coli WP2, WP67,
C871
<50 ng/103 bacteria
±
2-h preincubation assay (treat-and-plate method).
Survival did not differ between repair-deficient and
wild-type strains without metabolic activation.
Findings were equivocal with S9 activation.
De Flora et al. (1984)
DNA damage
E. coli PQ37
0,0.156, 0.625, 2.5,
10.0 ng/assay
±
SOS chromotest. 1.9-fold increase in DNA damage
with S9 activation (>twofold considered positive).
Findings were negative without S9 activation.
Mersch-Sundermann et
al. (1993); Mersch-
Sundermann et al.
(1992)
Genotoxicity studies in mammalian cells—in vitro
Mutation
Human
B-lymphoblastoid cells
(hlAlv2); RPMI1640
medium with 9% horse
serum
0, 10, 100, 1,000,
10,000 ng/mL
+
NDr
Forward mutation assay (thymidine kinase locus).
A twofold increase in mutations was observed at
10,000 ng/mL; this dose was associated with 33%
cytotoxicity. The minimum mutagenic
concentration was calculated to be 8,000 ng/mL
(dose that exceeds the 99% upper confidence limit
of historical negative control). No exogenous
metabolic activation was used, but hi Alv2 cells
constitutively express cytochrome P4501A1.
Durant et al. (1996)
Mutation
Adult rat liver epithelial
(ARL18) cells; culture
media NS
0, 100 (iM
NDr
Hgprt forward mutation assay. No evidence of
mutagenicity without exogenous metabolic
activation (ARL18 cells have endogenous
metabolic capabilities). Cell survival was not
reported.
Ved Brat et al. (1983)
Mutation
Chinese hamster lung
fibroblast (V79 cells);
Eagle's medium and
5-10% FBS
0, 1 ng/mL
NDr
Cell-mediated mutagenesis assay. No evidence of
mutagenicity with metabolic activation (V79 cells
were co-cultured with metabolically competent
rodent cells to produce reactive metabolites during
exposure). Cell survival was not reported.
Huberman (1977)
SCE
Adult rat liver epithelial
(ARL18) cells; culture
media NS
0, 10 (iM
~
NDr
No evidence of SCE induction without exogenous
activation (ARL18 cells have endogenous
metabolic capabilities).
Ved Brat etal. (1983)
17
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
SCE
SHE cells; DMEM with
0, 10, or 40%FBS
0, 0.1 ng/mL
±
NDr
SHE cells were exposed to 10 |ig BeP for 24 hours;
postexposure, the organic phase was extracted to
the culture medium and added to V79 cells (BeP
was not metabolized by SHE cells during organic
phase). A 1.4-fold increase in SCEs was observed
when cells were exposed to organic phase
extraction with 0% serum in the media; SCE
induction was prevented with 10-40% FBS in the
media. The study authors propose the observed
decrease in cellular uptake was due to binding of
BeP to serum proteins.
Coulomb etal. (1981)
SCE
Chinese hamster V79
cells; serum-free M199
medium
0, 1 ng/mL
No induction of SCEs with or without metabolic
activation (V79 cells were cocultured with
metabolically competent rat mammary epithelial
cells to produce reactive metabolites during
exposure).
Mane et al. (1990)
MN
Wistar rat skin
fibroblasts (UGT-normal
or UGT-deficient);
DMEM with 13% FBS
0, 10 nM
(UGT-
deficient)
(UGT-
normal)
No induction of MN in UGT-normal or
UGT-deficient fibroblasts.
Yienneau et al. (1995)
DNA damage
Human skin fibroblasts;
MEM with 20% FBS
0, 13 (iM
-
NDr
No significant induction of DNA breaks.
Milo etal. (1978)
DNA damage
Chinese hamster V79
cells; DMEM with 5%
FBS
0, 10, 20, 50,
100 nM
+
NDr
Comet test (alkaline version). DNA strand breaks
increased >twofold without metabolic activation at
100 nM.
Piatt et al. (2008)
DNA synthesis
Human skin fibroblasts;
MEM with 20% FBS
0, 13 (iM
-
NDr
3HTdR incorporation assay. No significant
inhibition of scheduled DNA synthesis.
Milo etal. (1978)
DNA synthesis
Rat mammary epithelial
cells; serum-free M199
medium
5 iig/mL
~
NDr
3HTdR incorporation assay. No significant
inhibition of scheduled DNA synthesis.
Mane et al. (1990)
18
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
DNA synthesis
F344 rat hepatocytes;
MEM with 25% HPRS
0, 1, 10, 100 (1M
-
NDr
3HTdR incorporation assay. No significant
inhibition of scheduled DNA synthesis.
Novicki et al. (1985)
DNA synthesis
Sprague Dawley rat
hepatocytes; L-15
medium with 10% FBS
0, 1, 100, 1,000 nM
NA
3HTdR incorporation assay. No significant
inhibition of scheduled DNA synthesis.
Zhao and Ramos
(1995)
UDS
Adult rat hepatocytes;
serum-free William's
medium E
<100 nmol/mL
NDr
Hepatocyte primary culture-DNA repair test. No
evidence of increased UDS in the absence of
exogenous metabolic activation (hepatocytes are
metabolically competent).
Probst et al. (1981)
UDS
Adult mouse lung
fibroblasts; DMEM with
10% FBS
0, 1, 10, 100 nM
No evidence of increased UDS with or without S9
activation.
Schmitt et al. (1984)
UDS
SHE cells; serum- and
arginine-free medium
0, 2.5, 5, 10,
20 ng/mL
-
NDr
No evidence of increased UDS.
Casto et al. (1976)
UDS
Syrian hamster tracheal
organ cultures;
serum-free CMRL
medium 1066
0, 0.1, 1.0 ng/mL
NDr
No evidence of increased UDS in the absence of
exogenous metabolic activation (tracheal organ
cultures are metabolically competent).
Schiff et al. (1983)
Cell
transformation
SHE cells; DMEM with
10% FBS
0, 10 ng/mL
±
NDr
BeP treatment produced 0.6% transformation and
was considered "slightly effective." Cloning
efficiency (6.8%) was comparable to control
(7.0%).
DiPaolo et al. (1969)
Genotoxicity studies—in vivo
CA (bone
marrow)
Female Long-Evans rats
exposed to BeP via i.v.
infusion and sacrificed
24 h postinjection
0, 40 mg/kg
+
NA
>Threefold increase in chromosomal breaks at 24 h
postinjection.
Rees et al. (1970)
19
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
CA (bone
marrow)
Male and female Chinese
hamsters were exposed to
BeP via i.p. injection;
hamsters were
administered two
injections, 24 h apart;
hamsters were sacrificed
24 h after 2nd injection
0, 450 mg/kg
NA
No significant induction of CAs.
Roszinskv-Kocher et
al. (1979)
SCE (bone
marrow)
Male and female Chinese
hamsters were exposed to
BeP via i.p. injection;
hamsters were
administered two
injections, 24 h apart;
hamsters were sacrificed
24 h after 2nd injection
0, 450 mg/kg
+
NA
Significant 1.4-fold increase in SCEs per
metaphase.
Roszinskv-Kocher et
al. (1979)
MN (bone
marrow)
Female mice (C57BL/6,
BALB/c, DBA/2, BDF1,
and CDF1 hybrids) were
exposed once to BeP via
i.p. injection and
sacrificed 24, 48, or 72 h
postinjection
0, 100 mg/kg
NA
No significant induction of MN in any mouse
strain at any time point.
Sato etal. (1987)
DNA damage
(liver)
Female Sprague Dawley
rats exposed twice to BeP
via gavage in corn oil;
the 1st dose was
administered 21 h prior
to sacrifice and the 2nd
dose was administered
4 h prior to sacrifice
0, 18 mg/kg
NA
Alkaline elution assay. No significant hepatic DNA
damage.
Kitchin et al. (1993):
Kitchin et al. (1992)
20
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
DNA synthesis
(thymus,
spleen, bone
marrow)
Male Wistar rats exposed
to BeP via i.p. injection
in olive oil and sacrificed
at 24 and 48 h
postinjection. 3HTdR i.m.
injections given 30 min
prior to sacrifice
0.19 |imol/g
+
NA
3HTdR incorporation analysis. DNA synthesis was
significantly decreased by 64.5-67.8% in thymus
and 31.0-50.3% in spleen at 24- and 48-h
postexposure. DNA synthesis was significantly
decreased by 21.2% in bone marrow at 24-h
postexposure only. The study authors note that
decreased 3HTdR incorporation may reflect
cytotoxicity instead of decreased DNA synthesis.
Prodi et al. (1975)
DNA synthesis
(liver)
Male Wistar rats exposed
to BeP via i.p. injection
in olive oil 2 h after a
partial hepatectomy; rats
sacrificed 22 h
postexposure; 3HTdR
i.m. injections given
30 min prior to sacrifice
0.19 |imol/g
+
NA
3HTdR incorporation analysis. DNA synthesis was
significantly decreased by 39.4% in regenerating
liver following BeP exposure. The study authors
note that decreased 3HTdR incorporation may
reflect cytotoxicity instead of decreased DNA
synthesis.
Prodi et al. (1975)
DNA adducts
Female BALB/c mice
exposed to BeP via four
topical applications at 0,
6, 30, and 54 h;
sacrificed 24 h after last
treatment
1.2 |imol
+
NA
32P-postlabeling analysis. Five adducts were
detected in mouse skin at a level of 1 adduct per
>107 normal nucleotides.
Reddv et al. (1984)
21
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4A. Summary of BeP (CASRN 192-97-2) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation"
Results
with
Activation"
Comments
References
Genotoxicity studies in invertebrates-in vivo
DNA repair
Drosophila
melanogaster; Rec~
males and Rec females
were exposed as larvae
and scored as adults
Up to 100 mg/mL
NA
A reduction in Rec larvae would indicate DNA
damage from the test compound. There was no
significant reduction in the Rec :Rec ratio.
Fujikawa etal. (1993)
Genotoxicity studies in subcellular systems
DNA adducts
Human DNA
100 nM
NDr
+
32P-postlabeling assay with nuclease PI treatment
or butanol extraction for enrichment of adducts.
BeP adducts were detected with both procedures,
but -50% more adducts were detected with butanol
extraction.
Sesetback and Vodicka
(1993)
a+ = positive; ± = weakly positive/equivocal; - = negative.
bFor bacterial mutagenicity studies that did not report criteria for positive results, an induction of revertants >twofold was considered positive for the purposes of this
review.
3HTdR = [3H]Thymidine; BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; CA = chromosomal aberration; CMRL = Connaught Medical Research Laboratories Medium
1066; CYP450 = cytochrome P450; DMEM = Dulbecco's modified minimal essential medium; DNA = deoxyribonucleic acid; FBS = fetal bovine serum;
HI = Arochlor-induced hamster liver S9 protein; HPRS = hepatectomized rat serum; HU = uninduced hamster liver S9 protein; i.m. = intramuscular;
i.p. = intraperitoneal; IRI = Inveresk Research International; i.v. = intravenous; LBI = Litton Bionetics, Inc.; MEM = minimal essential medium; MI = Arochlor-induced
mouse liver S9 protein; MN = micronuclei; MU = uninduced mouse liver S9 protein; NA = not applicable; NDr = not determined; NR = nitroreductase; NS = not
specified; NYM = New York Medical College; PAH = polycylie aromatic hydrocarbon; RI = Arochlor-induced rat liver S9 protein; RU = uninduced rat liver S9 protein;
SCE = sister chromatid exchange; SD = standard deviation; SHE = Syrian hamster embryo; SRI = SRI International; TCDD = 2,3,7,8-tetrachlorodibenzo-/?-dioxin;
UDS = unscheduled DNA synthesis; UGT = uridine 5'-diphospho-glucuronosyltransferase.
22
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Of the remaining studies, BeP was not mutagenic when metabolic activation was from liver
homogenates of rats pretreated with phenobarbital (Teranishi et al.. 1975) or corn oil, BeP, or
tetrachlorodibenzo-p-dioxin (TCDD) (Ma et al.. 1991) as alternatives to the standard
Arochlor-induced S9 liver fraction. In the absence of metabolic activation, BeP was consistently
reported as nonmutagenic in S. typhimurium (Chemg et al.. 1996; Zeiger et al.. 1992; Sakai et al..
1985; White et al.. 1985a; Dunkel et al.. 1984; Sal am one et al.. 1979; Simmon. 1979). BeP was
not mutagenic in Escherichia coli with or without metabolic activation (Dunkel et al.. 1984).
Mutagenicity findings in mammalian cells were negative at noncytotoxic concentrations.
In metabolically competent human B-lymphoblastoid cells, forward mutations were induced at
the thymidine kinase (Ik) locus at a BeP concentration associated with >30% cytotoxicity
(10,000 ng/mL); mutations were not observed at noncytotoxic concentrations <1,000 ng/mL
(Durant et al.. 1996). In other assays, BeP did not induce forward mutations in metabolically
competent adult rat liver epithelial (ARL18) cells at the hypoxanthine-guanine
phosphoribosyltransferase (Hgprt) locus (Ved Brat et al .. 1983) or cell-mediated mutagenicity in
Chinese hamster lung fibroblasts (V79 cells) cocultured with metabolically competent rodent
cells (Huberman. 1977).
Clastogenicity
Evidence of clastogenicity from in vitro studies in mammalian cells is predominately
negative. Coulomb et al. (1981) reported a weak induction of sister chromatid exchanges (SCEs)
by BeP in Chinese hamster V79 cells in serum-free culture media, but not in media containing
10-40% fetal bovine serum (FBS). The study authors proposed that the presence or absence of
serum in the media may influence test results, because dose-related decreases in cellular uptake
of BeP by Syrian hamster embryo (SHE) cells were observed with increasing levels of FBS in
culture media (possibly due to BeP binding to serum lipoproteins) (Coulomb et al.. 1981).
However, Mane et al. (1990) reported that SCEs were not induced by BeP in Chinese hamster
V79 cells with or without metabolic activation in serum-free medium. SCEs were also not
induced by BeP in metabolically competent ARL18 cells (culture medium composition not
reported) (Ved Brat et al.. 1983). Micronuclei (MN) were not induced by BeP in normal rat skin
fibroblasts or uridine 5'-diphospho-glucuronosyltransferase (UGT)-deficient rat skin fibroblasts
cultured with 13% FBS (Vienneau et al.. 1995).
Evidence of clastogenicity from in vivo studies in rodents is mixed. BeP induced
chromosomal aberrations (CAs) in rat bone marrow following a single intravenous (i.v.)
injection of 40 mg/kg (Rees et al.. 1970). And in hamsters, BeP induced SCEs (but not CAs) in
bone marrow cells following a single i.p. injection of 450 mg/kg (Roszinskv-Kocher et al..
1979). In mice, BeP did not increase micronucleated erythrocytes in bone marrow following i.p.
injection of 100 mg/kg (Sato et al.. 1987).
DNA Damage and Repair
Findings were generally negative in in vitro assays of DNA damage and repair. In E. coli,
BeP did not induce DNA damage or repair activity without metabolic activation; with activation,
findings were negative or equivocal/borderline (Mersch-Sundermann et al.. 1993; Mersch-
Sundermann et al.. 1992; De Mora et al.. 1984). In mammalian cells, DNA strand breaks were
reported in Chinese hamster V79 cells following exposure to BeP in media containing 5% FBS
(Piatt et al.. 2008); however, DNA strand breaks were not induced by BeP in human skin
fibroblasts in media containing 20% FBS (Milo et al.. 1978). As discussed above, the higher
levels of serum in the human cell assay could have affected test results due to decreased cellular
23
Benzo[e]pyrene
-------
EPA/690/R-21/008F
uptake of BeP with increasing serum levels in culture media (Coulomb et al.. 1981).
Additionally, the presence of serum (a mitogen) may skew the utilization of alternative DNA
damage-and-repair pathways that are only active in actively cycling cell populations. It is unclear
how the use of different molecular DNA damage-and-repair pathways may influence the
dynamics of these repair processes.
In other mammalian cell studies, BeP did not inhibit scheduled DNA synthesis in human
or rat cells with serum present in the culture media or rat cells in the absence of serum (Zhao and
Ramos. 1995; Mane et al.. 1990; Novicki et al.. 1985; Milo et al.. 1978). Similarly, BeP did not
induce unscheduled DNA synthesis in rat or hamster cells in serum-free media or in mouse lung
fibroblasts in the presence of serum (Schmitt et al.. 1984; Schiff et al.. 1983; Probst et al.. 1981;
Casto et al.. 1976).
Findings were also generally negative in in vivo assays of DNA damage and repair.
Drosophila melanogaster did not show increased rates of DNA repair after exposure to BeP
(Fujikawa et al.. 1993). No evidence of hepatic DNA damage was observed in rats following two
gavage doses of 18 mg/kg (Kitchin et al .. 1993; Kitchin et al .. 1992). Another rat study reported
inhibited 3H-thymidine uptake in thymus, spleen, bone marrow, and regenerating liver
24-48 hours after i.p. exposure to BeP, suggesting decreased rates of DNA synthesis (Prodi et
al.. 1975). However, the study authors noted that findings may reflect general cytotoxicity of
BeP, rather than decreased DNA synthesis.
DNA adducts were identified in mouse skin following repeated topical application of BeP
to shaved skin (four exposures over 54 hours) (Reddv et al.. 1984). Isolated human DNA has
also been shown to form BeP DNA adducts in vitro (Segerback and Vodicka. 1993).
Cell Transformation
BeP was a weak inducer of cell transformation in SHE cells in the absence of metabolic
activation in medium containing 10% FBS (PiPaolo et al.. 1969).
2.3.2. Supporting Human Studies
Several occupational studies examined populations with exposures to multiple
compounds in the absence of BeP-specific exposure analyses. These studies are of limited use
for BeP hazard identification and do not provide data for dose-response analysis. Reported
effects include subjective health complaints (skin irritation, headaches, shortness of breath,
nausea, cough, phlegm) and altered markers of immune function in coke oven workers exposed
to various PAHs in coal tar sludge and coal dust (Winker et al.. 1997; NIOSH. 1987. 1986).
Other effects include skin phototoxicity and skin and respiratory tract irritation in petroleum
refining workers exposed to various PAHs in petroleum pitch (Vandervort and Lucas. 1974).
impaired lung function (e.g., increased residual volume, reduced total lung capacity) in rubber
factory workers exposed to suspended particulate matter containing various PAHs (Gupta et al..
1998; Gupta et al.. 1994). and increased markers of oxidative deoxyribonucleic acid (DNA)
damage in roofers exposed to various PAHs in hot asphalt (Serdar et al.. 2016). Another study
reported elevated biomarkers of chromosomal and DNA damage in nonsmoking female city hall
clerks employed and living in the most polluted urban region of Silesia, Poland (high PAH
exposure levels), compared with nonsmoking female controls living in a less polluted urban
region of Bialystok, Poland (low PAH exposure levels) (Motvkiewicz et al.. 1998).
24
Benzo[e]pyrene
-------
EPA/690/R-21/008F
In population-based, case-control studies, no significant differences were observed in
serum BeP levels between control and asthmatic children in Saudi Arabia (Ai-Daghri et al..
2014) or lung tissue BeP levels between patients with tuberculosis (control) and patients with
lung carcinoma in 1961 — 1962 or 1991-1996 in Japan (Tokiwa ct al.. 1998).
2.3.3. Supporting Animal Studies
Acute Toxicity
Kitchin ct al. (1993) and Kitchin ct al. (1992) evaluated hepatic biochemical endpoints in
female rats following acute oral exposure to BeP. The rats were exposed twice to BeP at 0 or
18 mg/kg via gavage in corn oil; the first dose was administered 21 hours prior to sacrifice and
the second one 4 hours prior to sacrifice. All rats survived. No exposure-related changes in
serum alanine aminotransferase (ALT) activity, hepatic ornithine decarboxylase activity, or
hepatic cytochrome P450 (CYP450) content were observed.
Cardiovascular Toxicity
There is a body of evidence describing cardiotoxicity induced by environmentally
relevant PAHs in non-human model systems (Brcttc ct al.. 2017). Specifically, there is concern
that BeP may cause cardiovascular effects because the U.S. EPA (2017c) concluded that
available animal studies for benzo[a]pyrene (BaP), a PAH with similar structure and
physicochemical properties, provided suggestive evidence of cardiovascular toxicity
(i.e., atherosclerosis). Curfs ct al. (2005) conducted a study directly comparing BeP- and
BaP-induced atherosclerotic plaque formation in mice genetically predisposed to develop
plaques. Both compounds showed a similar capability to promote atherosclerosis. Following oral
exposure to BaP or BeP (5 mg/kg-day) via once weekly gavage for 24 weeks, the location and
total number of initial and advanced plaques (combined) per aortic arch were similar in treated
and control mice, but the total area of advanced plaques/arch was significantly (p<0.05)
increased by 1.4- to 1.5-fold in BaP- and BeP-treated mice compared with controls. In the
thoracic aorta, there were no exposure-related changes in number or size of plaques; however,
both BaP- and BeP-treated groups exhibited significantly increased plaque total inflammatory
cells (CD45-positive cells) and plaque T lymphocytes (CD3-positive cells). The only clear
difference between BaP- and BeP-treated mice was significant DNA-adduct formation in the
lung of BaP-treated mice compared with BeP-treated and control mice. This suggests potential
differences in distribution and/or potency in lung tissue; however, the relevance of this data to
human PAH-mediated cardiotoxicity is unclear due to the use of transgenic knockout mice.
Immunostaining for TGFpi in aortic arch plaques—a phenomenon observed in stable
atherosclerotic lesions—was also increased in BaP- and BeP-treated groups compared with
controls (Tom a and McCaffrey. 2012). In contrast, in vitro treatment of the murine
monocyte/macrophage RA W364.7 cell line with BaP or BeP revealed no apparent PAH
treatment-related effect on release of tumor necrosis factor-alpha (TNF-a) or expression of
transforming growth factor beta 1 (TGFpi).
Avian models have also been used to study the promotion of atherosclerosis by PAH
compounds. Penn and Snvder (1988) demonstrated that both BaP (40 mg/kg-day) and BeP
(20 mg/kg-day) promoted atherosclerotic plaque development parameters in cockerels following
weekly intramuscular (i.m.) injection for 16 weeks. A study in pigeons, however, reported an
increase in aortic plaque size and number, for BaP only, following weekly i.m. injections of
100 mg/kg BaP or BeP for 3 or 6 months (Revis ct al.. 1984).
25
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Immunotoxicity
The immunotoxic potential of BeP has been evaluated in numerous in vivo and in vitro
studies (see Table 4B for more details). Most of the in vivo studies reported lack of
immunosuppressive effects of BeP on parameters of humoral or cell-mediated immunity in mice
or rats given BeP via subcutaneous (s.c.) or intraperitoneal (i.p.) injection. Effects measured
included antibody responses to antigens (Munson and White. 1992. 1986; White et at.. 1985b;
White and Hotsappte. 1984; Dean et at.. 1983; Munson and White. 1983); delayed
hypersensitivity (White et at.. 2012; Munson and White. 1992. 1986. 1983); lymphoproliferative
response to mitogens (Munson and White. 1992; Woidani and Alfred. 1984; Dean et at.. 1983;
Munson and White. 1983); macrophage function (Munson and White. 1992. 1983); host
resistance to bacteria, viruses, or tumor cells (Munson et at.. 1985); and cell-mediated
cytotoxicity (Woidani and Alfred. 1984; Woidani et at.. 1984). Exposure to BeP reduced the
T cell-dependent antibody response to sheep red blood cells (sRBCs) in spleen cells cultured
from exposed mice in one study (Btanton et at.. 1986). Each of these studies compared the
response of BeP to the immunosuppressive response of the canonical PAH BaP administered at
the same dose levels (highest dose of 40 mg/kg-day). It is unclear whether BeP would have
elicited an immunosuppressive response if administered at higher doses. Dermal exposure to BeP
was shown to elicit a contact hypersensitivity response in mouse skin (Anderson et at.. 1995).
In vitro studies provided some evidence of BeP immunotoxicity, including inhibition of
mitogenesis in human lymphocytes (Davita et at.. 1996) and decreased B-cell lymphopoiesis in
murine bone marrow (Hardin et at.. 1992). Inconsistent findings, however, were reported for
inhibition of T-cell and B-cell antibody responses in mouse spleen cells (Btanton et at.. 1986;
White and Hotsappte. 1984). and no effect was reported after BeP exposure on the differentiation
of human monocytes into macrophages (van Grevenvnghe et at.. 2003). Mechanistic studies have
investigated the possible involvement of altered protein tyrosine kinase activity, calcium uptake
and/or retention, glutathione (GSH) depletion, or aryl hydrocarbon hydrolase (AHH) activity;
however, no clear relationship between these parameters and BeP immunotoxicity was reported
(Davila et al.. 1999; Romero et at.. 1997; Krieger et at.. 1995; Krieger et at.. 1994; Gurtoo et at..
1979).
26
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4B. Immunotoxicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results
Conclusions
References
In vivo animal studies
Contact
hypersensitivity
C3H/HeN mice (3-5/group, sex not reported);
1,000 |ig BeP was applied to the shaved abdominal
skin (covered by permeable membrane); 5 d later,
200 |ig BeP was applied to the dorsal aspect of the
ear; ear thickness was measured immediately
following challenge and daily thereafter for up to
5 d; negative controls received challenge
application only; AHH activity was measured in
microsomes isolated from abdominal skin of two
mice 24 h following application of 1,000 |ig BeP.
Maximum ear thickness was
increased by 3.2 x 10 2 mm over
negative controls; AHH activity
was 5% higher than controls.
BeP produced a contact
hypersensitivity response in
mouse skin but was a poor
inducer of AHH activity.
Anderson et al. (1995)
T cell-dependent and
independent antibody
responses
Female B6C3F1 mice (number per group not
reported) received daily s.c. injections containing
0 (polyvinylpyrrolidone vehicle) or 40 mg/kg-d for
7 or 14 d; spleen cell responses to sRBCs or LPS
were measured in culture (measured as PFCs).
Response to sRBCs was reduced
by 48% at 7 d and 51% at 14 d;
slight inhibition of antibody
response to LPS (data not
shown).
Exposure to BeP reduced the
T cell-dependent antibody
response to sRBCs in spleen
cells cultured from exposed
mice; inhibition of the
B cell-mediated antibody
response was minimal.
Blantonetal. (1986)
T cell-dependent and
independent antibody
responses;
lymphoproliferative
response to mitogens
Female B6C3F1 mice (6-10/group per assay)
received daily s.c. injections containing 0 (corn oil
vehicle), 5, 20, or 40 mg/kg-d for 14 d; parameters
evaluated included body and organ weights,
histopathology, hematology, antibody PFC
response to sRBCs and LPS administered
intravenously, and lymphoproliferative responses
to mitogens (PHA, LPS).
No effects on body or organ
weights or hematological
parameters; no effects on
antibody PFC responses or
lymphoproliferative responses to
mitogens.
BeP did not affect the
T cell-dependent antibody
response to sRBCs, the B-cell
response to LPS, or the
lymphoproliferative response to
mitogens in mice.
Deanetal. (1983)
27
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4B. Immunotoxicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results
Conclusions
References
Multiple assays of
humoral and
cell-mediated
immunity and
macrophage function
Male and female B6C3F1 mice
(72-140/sex/group) received daily s.c. injections
containing 0 (corn oil vehicle), 5, 20, or
40 mg/kg-d for 14 d; endpoints evaluated included
body and organ weights, hematology, humoral
immunity evaluations (spleen IgM response to
sRBCs, serum immunoglobulins and complement,
and spleen B-cell response to LPS), cell-mediated
immunity evaluations (delayed hypersensitivity
response to KLH and sRBCs, popliteal lymph node
response to sRBCs, spleen T-cell response to
Con A and acute inflammatory response to
carrageenan), and macrophage function (vascular
clearance and uptake of radiolabeled sRBCs).
Absolute and relative liver
weights increased by 19 and 7%,
respectively, in males at
40 mg/kg-d; absolute spleen
weight increased by 26 and 18%
in males at 20 and 40 mg/kg-d,
respectively; absolute spleen
weight decreased by 36% in
females at 40 mg/kg-d (no
changes in relative spleen
weight); and relative lung weight
decreased by 11-12% in males at
all doses (no change in absolute
lung weight). Leukocyte count
decreased by 68% in males at
40 mg/kg-d. No other significant
treatment-related effects were
observed.
BeP did not impair humoral
immunity, cell-mediated
immunity, or macrophage
function in mice.
Munson and White
(1992); Munson and
White (1983);
Munson and White
(1983) (unpublished)
Host resistance assays
Female B6C3F1 mice (8/group) received daily s.c.
injections containing 0 (corn oil vehicle) or
40 mg/kg-d for 14 d; Listeria monocytogenes,
Streptococcus pneumoniae, herpes simplex type 2,
influenza A2, orB16F10 melanoma cells injected
on Day 15 (four concentrations of each pathogen);
host resistance measured as percent mortality or
incidence and multiplicity of tumors.
No change in mortality incidence
in response to L. monocytogenes,
S. pneumoniae, herpes simplex
type 2, or influenza A2; no
change in tumor incidence and
multiplicity following injection
of B16F10 melanoma cells.
BeP did not alter host resistance
to bacteria, viruses, or tumor
cells in mice.
Munson etal. (1985)
(unpublished)
28
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4B. Immunotoxicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results
Conclusions
References
T cell-dependent
antibody response and
delayed
hypersensitivity
response
Male F344 rats (8/group) received daily s.c.
injections containing 0 (corn oil vehicle) or
20 mg/kg-d for 14 d; parameters evaluated
included body and organ weights, hematology,
spleen IgM response to sRBCs, and delayed
hypersensitivity response to KLH.
No change in body weight; 18%
increase in relative liver weight
(no other organ-weight changes);
26% decrease in leukocytes, 67%
decrease in monocytes; no
change in the spleen IgM
response to sRBCs; and no
change in the delayed
hypersensitivity response to KLH
and sRBCs.
BeP did not alter
T cell-dependent antibody
response to sRBCs or the
delayed hypersensitivity
response to KLH or sRBCs.
Munson and White
(1986) (unpublished)
T cell-dependent
antibody response
Female B6C3F1 mice (8/group) received daily s.c.
injections containing 0 (corn oil vehicle), 5, 20, or
40 mg/kg-d for 14 d; spleen IgM response was
evaluated 4 d after injection with sRBCs (PFCs).
No change in the spleen IgM
response to sRBCs.
BeP did not impair humoral
immunity in mice.
White and Holsapple
(1984)
T cell-dependent
antibody response
Female B6C3F1 mice (8/group) received daily s.c.
injections containing 0 (corn oil vehicle) or
40 mg/kg-d (160 |imol/kg-d) for 14 d; spleen IgM
response was evaluated 4 d after injection with
sRBCs (spleen weight, cellularity, and number of
PFCs).
No change in the spleen IgM
response to sRBCs.
BeP did not impair humoral
immunity in mice.
White et al. (1985b)
Candida albicans DTH
test
Female B6C3F1 mice (7-8/group) received daily
s.c. injections containing 0 (corn oil vehicle), 5, 20,
or 40 mg/kg-d for 28 d; the C. albicans DTH test
was performed; C. albicans was injected into the
right flank of mice on Day 21; mice were
challenged with injection of C. albicans antigen
(chitosan) into the right footpad; footpad thickness
was measured pre- and postchallenge.
BeP did not increase footpad
swelling above vehicle controls.
BeP did not impair cell-mediated
immunity in mice.
White et al. (2012)
29
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4B. Immunotoxicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results
Conclusions
References
Multiple assays of
cell-mediated
immunity
C3H, C57BL/6, and DBA mice (6/group; sex not
reported) were given an i.p. injection of PHA; 96 h
later, BeP was administered by i.p. injection at
doses of 0 (corn oil), 2.5, 10, or 50 mg/kg; mice
were sacrificed 24 h later; cell-mediated immunity
parameters included blastogenesis of splenic
lymphocytes, cell-mediated cytotoxicity (percent
51 Cr release from target cells), and percent of
monocyte-macrophages in spleen cell suspensions.
BeP did not induce
lymphoblastogenesis or increase
cell-mediated cytotoxicity or the
percent monocyte-macrophages
in spleen cell suspensions.
BeP did not impair cell-mediated
immunity in mice.
Woidani and Alfred
(1984)
Cytotoxic lymphocyte
response
C3H and C57BL/6 mice (6/group, sex not
reported) were preimmunized with P815 tumor
cells (i.p.) and treated 10 d later with a single i.p.
injection of BeP at doses of 0 (corn oil), 0.5, 5, or
50 mg/kg; after 24 h, splenic lymphocytes and
peritoneal exudate lymphocytes were isolated and
used to measure binding and killing of P815 target
cells.
BeP reduced percent binding and
percent killing by splenic and
peritoneal lymphocytes in both
mouse strains at 5 and 50 mg/kg;
however, effects were small in
magnitude.
BeP produced minimal effects on
cell-mediated immunity in mice.
Woidani et al. (1984)
In vitro studies
T cell-dependent and
polyclonal B-cell
antibody responses
Spleen cells from B6C3F1 mice cultured with and
without sRBCs were exposed to 0
(polyvinylpyrrolidone vehicle), 0.2, 2, 20, or
200 |ig/mL BeP; T cell-dependent antibody
response to sRBCs; polyclonal antibody responses
to LPS and PPD (B cell); measured on Day 5.
Dose-dependent reduction in
response to sRBCs (79 and 100%
inhibition at 20 and 200 ng/mL,
respectively); reduction in
response to LPS (83% decrease)
and PPD (46% decrease) at
20 ng/mL.
BeP inhibited both
T cell-dependent and polyclonal
antibody responses in spleen
cells in vitro.
Blanton et al. (1986)
Lymphocyte
mitogenesis
Human peripheral blood mononuclear cells were
incubated with PHA in the presence of BeP at
concentrations of 0 (DMSO vehicle), 0.1, 1, or
10 |iIVI for 72 h; lymphocyte proliferation
(i.e., mitogenesis) was measured for each donor
(4-8 donors/group).
Lymphocyte mitogenesis was
significantly reduced in 1/4 (94%
of control), 3/8 (94% of control),
and 7/8 (76% control) donors at
0.1, 1, or 10 nM, respectively.
BeP inhibited mitogenesis in
human lymphocytes.
Davila et al. (1996)
30
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4B. Immunotoxicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results
Conclusions
References
PTK activation in
human T cells
Human HPB-ALL T cells (a leukemia cell line)
were exposed to 0 (DMSO vehicle) or 10 |iIVI BeP;
total and Fyn- and Zap-70-specific PTK activity
were measured in cell lysates after 10 min
exposure; total PTK activity was measured in a
time course experiment at 5 min, 30 min, and 18 h.
Total PTK activity was increased
at 18 h only; total and Fyn- and
Zap-70-specific PTK activity
were similar to control after
10 min of exposure.
BeP significantly increased total
PTK activity in human
HPB-ALL T cells after 18 h
exposure; the role of increased
total PTK activity in BeP
immunotoxicity is unclear from
the data presented.
Davilaetal. (1999)
AHH activity in fresh
mitogen activated
human lymphocytes
Lymphocytes from a single human donor were
suspended in a medium containing pokeweed
mitogen and PHA; cells were exposed to 0, 10 .
10 6. or 10 s M BeP for 24 h and AHH activity was
measured immediately thereafter.
10 s M BeP inhibited AHH
activity in human lymphocytes in
vitro (-40% of control, data
shown graphically); no effect
was observed at lower
concentrations (10 or 10 6 M).
The role of AHH inhibition in
BeP immunotoxicity is unclear
from the data presented.
Gurtoo etal. (1979)
B-cell lymphopoiesis
C57BL/6 murine bone marrow cells were cultured
under conditions that favor the growth of pre-B
cells; cultures were treated with 0 (acetone
vehicle), 10~7,10~6, 10~5, or 10~4 M BeP, and pre-B
cells were recovered and counted after 2 and 7 d.
10 4 M BeP decreased the
number of pre-B cells after 7 d
(-65% of control lymphocytes,
data shown graphically); no
effect was observed at lower
concentrations (10 . 10 6. or
10~5 M).
BeP inhibited B-cell
lymphopoiesis.
Hardin etal. (1992)
Calcium elevation in
human T cells
Human HPB-ALL T cells (a leukemia cell line)
were exposed to 0 (DMSO vehicle) or 10 |iIVI BeP
for 3 min or 4 h; calcium mobilization was
determined by a fluo-3 fluorescence flow
cytometry assay.
BeP did not induce calcium
mobilization in human T cells
after 3 min or 4 h.
Calcium elevation was not
identified as a mechanism of
immunotoxicity in human T cells
in this study.
Krieeeret al. (1994)
Calcium uptake and
activity of calcium
ATPases in vesicles
prepared from human
T cells
ATP-dependent 45Ca2+ uptake was measured in
microsomes prepared from human HPB-ALL
T cells after 5 min of incubation with 0 (DMSO
vehicle), 0.1, 1, or 10 ^MBeP; microsomal
Ca2+-ATPase activity was measured 30 min
following incubation with 10 |iIVI BeP.
Microsomal calcium uptake was
inhibited by 19, 25, and 20% of
control at 0.1, 1, or 10 ^M BeP;
no effect was observed on
Ca2+-ATPase activity.
BeP inhibited calcium uptake in
T-cell microsomes; however, the
effect was not treatment related.
No effect was observed on
Ca2+-ATPase activity in human
T-cell microsomes.
Krieeeret al. (1995)
31
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4B. Immunotoxicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results
Conclusions
References
GSH levels in human
peripheral blood
mononuclear cells
(lymphocytes)
Human peripheral blood mononuclear cells
obtained from five donors were incubated with
0 (DMSO), 1, or 10 for 6, 48, or 72 h.
BeP did not significantly affect
GSH in human lymphocytes.
GSH depletion was not identified
as a mechanism of
immunotoxicity.
Romero et al. (1997)
Differentiation of
human monocytes into
macrophages
Monocyte cultures prepared from human
peripheral blood were exposed to 10 |iIVI BeP for
6 d; formation of adherent macrophages and
expression of phenotypic markers (CD71) were
monitored.
BeP did not decrease the number
of adherent cells or increase the
expression of CD71.
BeP did not alter differentiation
of human monocytes into
macrophages.
van Grevenvnghe et
al. (2003)
Antibody production
by spleen cells in vitro
Spleen cell cultures were prepared from untreated
female B6C3F1 mice; cells were incubated with
0 (corn oil vehicle), 0.5, 5, or 50 |ig/culturc BeP
for 4 d; antibody response to sRBCs was measured
as AFCs/106 spleen cells (with or without
metabolic activation with Arochlor-induced S9).
BeP did not alter the antibody
response to sRBCs (with or
without metabolic activation).
No effect on humoral immunity
in vitro.
White and Holsapple
(1984)
AFC = antibody-forming colony(ies); AHH = aryl hydrocarbon hydroxylase; ATP = adenosine triphosphate; BeP = benzo[e]pyrene; Con A = Concanavalin A;
DMSO = dimethylsulfoxide; DTH = delayed-type hypersensitivity; GSH = reduced glutathione; IgM = immunoglobin M; i.p. = intraperitoneal; KLH = keyhole limpet
hemocyanin; LPS = lipopolysaccharide; PFC = plaque-forming cell; PHA = phytohemagglutinin; PPD = purified protein derivative; PTK = protein tyrosine kinase;
s.c. = subcutaneous; sRBC = sheep red blood cell.
32
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Effects on Retinal Cells
Several in vitro studies have been performed in retinal cell preparations to evaluate
potential PAH-mediated mechanisms for increased risk of age-related macular degeneration in
cigarette smokers, because cigarette combustion is a relevant source of exposure to complex
PAH mixtures. BaP, the most well-studied PAH, is demonstrated to be metabolized into its diol
epoxide by bovine retinal pigment epithelial (RPE) cells, resulting in concomitant DNA-adduct
formation and subsequent inhibition of cell growth and replication (Pattern et al.. 2002). Because
the structurally related compound BeP has been characterized at higher concentrations in
cigarette smoke than BaP (Patil et al.. 20091 several studies were conducted to specifically
evaluate the effects of BeP exposure on retinal cell preparations. BeP exposure produced a
concentration-dependent reduction in the viability of human retinal pigment epithelial
(ARPE-19) cells after 24 hours of treatment (percent viability was 96, 59, 36, and 20% at
concentrations of 100, 200, 400, and 1,000 |iM, respectively) (Sharma et al.. 2008). Apoptosis
was induced through the activation of multiple caspase pathways, and inhibition of caspase
activation by preincubation with genistein, resveratrol, and memantine was shown to protect the
viability of the ARPE-19 cells (Mansoor et al.. 2010; Sharma et al.. 2008). These data suggest
that the decreased viability of human retinal pigment epithelial cells exposed to BeP was due to
increased apoptosis mediated through increased caspase signaling.
Pretreatment of ARPE-19 cells with 17P-estradiol was also shown to protect against
cytotoxicity of BeP and to reduce markers of apoptosis after BeP exposure. Estrago-Franco et al.
(2016) reported that reactive oxygen/reactive nitrogen species (ROS/RNS) and inflammatory
cytokines (i.e., IL-6, GM-CSF) were increased in ARPE-19 cells exposed to 200 |iM BeP for
24 hours. BeP treatment for 24 hours also reduced cell viability in human microvascular
endothelial cells (HMVECs) at concentrations >200 |iM and in retinal neurosensory (R28) cells
at concentrations >400 |iM (Patil et al.. 2009). The mechanism of cytotoxicity differed by cell
type and concentration (i.e., no caspase activation in HMVECs; activation of caspases at 100 and
200 |iM only in R28 cells) (Patil et al.. 2009). Given the multiple modes of genotoxic activity
exhibited by PAHs and the complex interplay of DNA damage, metabolic CYP activity, and
apoptotic cell death, it is unclear how the cell-specific activity of these molecular pathways will
influence cell fate determination described across the different model systems.
In an ex vivo study using porcine retinal arterioles, intraluminal administration of
100 |iM BeP for 180 minutes reduced endothelium-dependent, nitric oxide-induced vasodilation
via a mechanism involving superoxide production (Kamiva et al.. 2017).
Cancer
The dermal, lung implantation, and i.p. injection studies that evaluated the carcinogenic
potential of BeP in rodents are summarized in Table 4C.
33
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4C. Other Route Carcinogenicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results (tumor incidence)
References
Dermal complete
carcinogenicity study
Female ICR/Ha Swiss mice (100 controls,
50 treated); 0 or 15 |ig (0.06 |imol) per
application (acetone vehicle); 3 times/wk for
53 wk
Skin papillomas
0 (imol = 0/100 (0%)
0.06 (imol BeP = 0/50 (0%)
Van Duuren and
Goldschmidt (1976)
Dermal complete
carcinogenicity study
Female Swiss Millerton (n = 20); 0.1% BeP
(acetone vehicle); 3 times/wk for up to
13 mo; no negative control was used; dose
(|imol) of BeP could not be calculated
because application volume was not reported
A total of 2/20 (10%) mice developed skin papillomas and 3/20
(15%) mice developed skin carcinomas over the course of the
study. The first papilloma was observed at 9 mo, when there
were only eight survivors, and the first carcinoma at 13 mo,
when there were only five survivors. While a negative control
was not used, no tumors were induced in 20 mice similarly
exposed to 0.1% fluoranthene (9 survivors at 14 mo). In
contrast, 19/20 mice similarly treated with 0.01% BaP
developed both skin papillomas and carcinomas (first tumors
observed at 4 mo; all mice died by 11 mo). The authors
characterized their results as showing "very weak" carcinogenic
activity for BeP.
Wvnder and Hoffmann
(1959)
Dermal initiation-promotion
study
Female Swiss ICR/HA mice (20/group);
single application of 1,000 |ig BeP (4 |imol):
starting 2 wk later, 25 |ig croton resin,
3 times/wk, up to 64 wk; two control groups
were used (croton resin only) with no BeP
exposure; 25 |ig croton resin, 3 times/wk, for
60 or 66 wk; both control groups are
presented because neither were exposed for
the exact duration as the BeP group
Skin papillomas
0 |imol (60 wk group) = 1/20 (5%)
0 |imol (66 wk group) = 5/20 (25%)
4 |imo 1 BeP (64 wk) = 2/20 (10%)
Skin carcinomas
0 |imol (60 wk group) = 0/20 (0%)
0 |imol (66 wk group) = 1/20 (5%)
4 |imo 1 BeP (64 wk) = 0/20 (0%)
Van Duuren et al. (1968)
34
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4C. Other Route Carcinogenicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results (tumor incidence)
References
Dermal initiation-promotion
study
Female CD-I mice (30/group); single
application of 0 (acetone/DMSO vehicle),
single application of 1, 2.5, or 6 |imol BeP;
2.5 or 6 (imol BeP 4,5-dihydrodiol; 1, 2.5, or
6 |.uno 1 BeP 9,10-dihydrodiol; 6 nmol BeP
H4-9,10-diol; or 2.5 (imol 9,10-H2 BeP;
starting 1 wk later, 16 nmol TPA 2 times/wk
for 35 wk
Skin papillomas
0 (vehicle control) = 7%
1 (imol BeP = 15%
2.5 nmol BeP = 11%
6 nmol BeP = 14%
2.5 nmol BeP 4,5-dihydrodiol = 14%
6 nmol BeP 4,5-dihydrodiol = 12%
1 nmol BeP 9,10-dihydrodiol = 14%
2.5 nmol BeP 9,10-dihydrodiol = 0%
6 nmol BeP 9,10-dihydrodiol = 11%
6 nmol BeP H4-9,10-diol = 18%
2.5 nmol 9,10-H2 BeP = 67%t
Note: Incidence data reported only as percentages. The study
authors indicated that at least 27 mice/group survived to 25 wk.
Actual incidences could not be estimated due to unknown
animal number/group.
Buening et al. (1980)
Dermal initiation-promotion
study
Female CD-I mice (20/group BeP, 30/group
untreated controls); single application of
10 nmol BeP; starting 1 wk later, 5 nmol
(exposed) or 10 nmol (untreated controls)
TPA 2 times/wk for 35 wk
Skin papillomas
0 nmol = 0/30 (0%)
10 nmol BeP = 17/20 (85%)*
Scribner (1973)
35
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4C. Other Route Carcinogenicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results (tumor incidence)
References
Dermal initiation-promotion
study
Female CD-I mice (30/group); single
application of 0 (TPA only), 100, or 252 |ig
BeP (0, 0.4, or 1 nmol): starting 1 wk later,
10 |ig TPA 2 times/wk for 30 and 40 wk
Skin papillomas at 30 wk:
0 nmol = 4/30 (14%)
0.4 nmol BeP = 0/30 (0%)
1 nmol BeP = 6/30 (19%)
Skin carcinomas at 40 wk:
0 nmol = 0/30 (0%)
0.4 nmol ng BeP = 0/30 (0%)
1 nmol ng BeP = 0/30 (0%)
Note: Incidence data reported only as percentages; actual
incidences estimated from number of animals placed on study.
Slaeaetal. (1979)
Dermal initiation-promotion
study
Female SENCAR mice (30/group); single
application of 0 (acetone), 0.4, or 0.8 nmol
BeP; starting 2 wk later, 3.4 nmol TPA
2 times/wk for 20 wk
Skin papillomas
0 nmol = 6/30 (19%)
0.4 nmol BeP = 4/30 (14%)
0.8 nmol BeP = 5/30 (18%)
Note: Incidence data reported only as percentages; actual
incidences estimated from number of animals placed on study.
Sawveretal. (1987)
Dermal initiation-promotion
study
Female SENCAR mice (30/group); single
application of 0 (TPA only) or 2 nmol BeP,
BeP 9,10-dihydrodiol, or BeP
9,10-diol-ll,12-epoxide; starting 1 wk later,
2 |ig TPA 2 times/wk for 15 wk
Skin papillomas
0 ng = 3/30 (10%)
2 nmol BeP = 5/29 (17%)
2 nmol BeP 9,10-dihydrodiol = 9/28 (32%)
2 nmol BeP 9,10-diol-ll,12-epoxide = 2/29 (7%)
Slaeaetal. (1980)
Lung implantation study
Female Osborne-Mendel rats (35/group);
implantation of 0, 200, 1,000, or 5,000 |ig
BeP (0, 0.8, 4, or 20 nmol) in a
beeswax/trioctanoin pellet; follow-up until
moribund or dead
Luns carcinomas and sarcomas
0 nmol = 0/35 (0%)
0.8 nmol BeP = 0/35 (0%)
4 nmol BeP = 1/30 (3%)
20 nmol BeP = 1/35 (3%)
Deutsch-Wenzel et al.
(1983)
36
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4C. Other Route Carcinogenicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results (tumor incidence)
References
i.p. injection study
Experiment 1: Newborn male and female
Swiss-Webster BLU:HA (ICR) mice
(80/group); total dose of 0 or 2.8 |imol BeP,
BeP 4,5-dihydrodiol, or BeP
9,10-dihydrodiol; injections given on
PNDs 1, 8, and 15; mice were sacrificed
during PNWs 62-66
Experiment 2: Newborn male and female
Swiss-Webster BLU:HA (ICR) mice
(120/group); total dose of 0 or 5.6 |imol BeP;
injections given on PNDs 1, 8, and 15; mice
were sacrificed during PNWs 62-66
Experiment 1:
Pulmonary tumors at PNW 62-66 sacrifice
Females:
0 |imo 1 = 12/21 (57%)
2.8 nmol BeP = 12/30 (40%)
2.8 nmol BeP 4,5-dihydrodiol = 11/23 (48%)
2.8 |imol BeP 9,10-dihydrodiol = 9/32 (28%)
Males:
0 |imo1 = 16/38 (42%)
2.8 nmol BeP = 12/30 (41%)
2.8 nmol BeP 4,5-dihydrodiol = 10/18 (56%)
2.8 |imol BeP 9,10-dihydrodiol = 12/28 (43%)
Hepatic tumors at PNW 62-66 sacrifice
Buening et al. (1980)
Females:
0 |imo 1 = 0/21 (0%)
2.8 nmol BeP = 0/30 (0%)
2.8 nmol BeP 4,5-dihydrodiol = 0/23 (0%)
2.8 nmol BeP 9,10-dihydrodiol = 0/32 (0%)
Males:
0 |imo 1 = 4/38 (11%)
2.8 nmol BeP = 6/30 (21%)
2.8 nmol BeP 4,5-dihydrodiol = 3/18 (17%)
2.8 |imol BeP 9,10-dihydrodiol = 17/28 (61%)*
37
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4C. Other Route Carcinogenicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results (tumor incidence)
References
Continued:
Continued:
Continued:
Experiment 2:
Pulmonary tumors at PNW 62-66 sacrifice
Females:
0 |imo1 = 12/30 (40%)
5.6 nmol BeP = 6/23 (26%)
Males:
0 |imo 1 = 9/29 (31%)
5.6 nmol BeP = 12/25 (48%)
Hepatic tumors at PNW 62-66 sacrifice
Females:
0 |imo 1 = 0/30 (0%)
5.6 nmol BeP = 0/23 (0%)
Males:
0 |inio 1 = 0/29 (0%)
5.6 |imol BeP = 3/25 (12%)
Continued:
38
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4C. Other Route Carcinogenicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results (tumor incidence)
References
i.p. injection study
Newborn male and female Swiss-Webster
BLU:HA (ICR) mice (80 or 100/group); total
dose of 0 |imol. 0.07 |imol BeP
H4-9,10-epoxide or 0.7 |imol BeP, BeP
4,5-oxide, 9,10-H2 BeP, BeP diol epoxide
(diastereomer 1 and 2); injections given on
PNDs 1, 8, and 15; mice were sacrificed
during PNWs 39-43
Pulmonary tumors PNW 39-43 sacrifice
Chang etal. (1981)
Females:
0 |imo 1 = 3/24 (13%)
0.07 nmol BeP H4-9,10-epoxide = 2/19 (11%)
0.7 |inio 1 BeP = 5/25 (20%)
0.7 |imol BeP 4,5-oxide = 7/30 (23%)
0.7 nmol 9,10-H2 BeP = 8/32 (25%)
0.7 |imol BeP diol epoxide 1 = 8/31 (26%)
0.7 J.11110I BeP diol epoxide 2 = 5/21 (24%)
Males:
0 1111101 = 8/37 (22%)
0.07 nmol BeP H4-9,10-epoxide = 2/36 (6%)
0.7 |imol BeP = 5/31 (16%)
0.7 |imol BeP 4,5-oxide = 2/37 (5%)
0.7 nmol 9,10-H2 BeP = 14/42 (33%)
0.7 nmol BeP diol epoxide 1 = 10/21 (48%)
0.7 |imol BeP diol epoxide 2 = 7/28 (25%)
Hcoatic tumors PNW 39-43 sacrifice
Females:
0 1111101 = 0/24 (0%)
0.07 nmol BeP H4-9,10-epoxide = 0/19 (0%)
0.7 nmol BeP = 0/25 (0%)
0.7 |imol BeP 4,5-oxide = 0/30 (0%)
0.7 nmol 9,10-H2 BeP = 0/32 (0%)
0.7 |imol BeP diol epoxide 1=0/31 (0%)
0.7 |imol BeP diol epoxide 2 = 0/21 (0%)
39
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 4C. Other Route Carcinogenicity Studies of BeP (CASRN 192-97-2)
Test
Materials and Methods
Results (tumor incidence)
References
Continued:
Continued:
Continued:
Males:
0 |imo 1 = 0/37 (0%)
0.07 nmol BeP H4-9,10-epoxide = 1/36 (3%)
0.7 nmol BeP = 2/31 (6%)
0.7 |inio 1 BeP 4,5-oxide = 0/37 (0%)
0.7 nmol 9,10-H2 BeP = 2/42 (5%)
0.7 J.11110I BeP diol epoxide 1 = 1/21 (5%)
0.7 nmol BeP diol epoxide 2 = 6/28 (21%)*
Continued:
Reported to be "significant" by study authors, although statistics not shown and data reporting inadequate to support independent analysis.
* Statistically significant at p< 0.05 based on two-tailed Fisher's exact probability test conducted for this review.
9,10-H2 BeP = 9,10-dihydrobenzo[e]pyrene; BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; BeP 4,5-dihydrodiol = /ra«s-4,5-dihydroxy-4,5-dihydrobenzo[e]pyrene;
BeP 4,5-oxide = benzo[e]pyrene 4,5-oxide; BeP 9,10-dihydrodiol = trans-1-). 10-dihydro\y-9.10-dihvdrobcnzo|c|pyrcnc:
BeP 9,10-diol-l 1,12-epoxide = trans-9,10-dihydroxy-anti-l 1,12-epoxy-9,10,11,12-tetrahydrobenzo[e]pyrene;
BeP diol epoxide 1 = (±)-9p,10a-dihydroxy-lip,12p-epoxy-9,10,ll,12-tetrahydrobenzo[e]pyrene;
BeP diol epoxide 2 = (±)-9p,10a-dihydroxy-lla,12a-epoxy-9,10,ll,12-tetrahydro-benzo[e]pyrene;
BeP H4-9,10-diol = trans-1-). lO-dilivdroxY-9.10.11.12-tctralivdrobcnzo|c|pvrcnc: BeP H4-9,10-epoxide = 9,10-epoxy-9,10,ll,12-tetrahydrobenzo[e]pyrene;
DMSO = dimethyl sulfoxide; i.p. = intraperitoneal; PND = postnatal day; PNW = postnatal week; TPA = 12-0-tetradecanoylphorbol-13 acetate.
40
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Available skin painting studies do not indicate that BeP is a complete dermal carcinogen.
Skin tumors were not observed (0/50) following dermal application of 0.06 [j,mol BeP in acetone
to mice 3 times/week for 53 weeks (Van Duuren and Goldschmidt 1976). According to the
authors of an earlier study (Wvnder and Hoffmann. 1959). BeP showed "very weak"
carcinogenic activity. In this study, mice treated dermally with an unknown dose of BeP (0.1% in
acetone) 3 times/week for up to 13 months developed low incidences of skin papillomas (2/20)
and skin carcinomas (3/20 mice). The study was inadequate, however, due to small initial group
size (n = 20), high early mortality unrelated to tumor development (only eight survivors at
9 months, when the first tumor was observed), and lack of a negative control group.
The majority of other BeP cancer assays were also negative. BeP was not a skin tumor
initiator at single exposure doses up to 6 |imol with 12-0-tetradecanoylphorbol-13 acetate (TPA)
as a promoter or up to 4 |imol with croton resin as a promotor (Sawyer et at.. 1987; Buening et
at.. 1980; Staga et at.. 1979; Van Duuren et at.. 1968). One study, however, reported significantly
increased skin papillomas in mice given BeP as an initiator at 10 |imol with TPA as a promoter
(17/20) compared with mice given TPA alone (0/30) (Scribner. 1973). Lung implantation of BeP
did not significantly increase the formation of pulmonary tumors in rats (Deutsch-Wenze! et at..
1983). Similarly, i.p. injections of BeP in newborn Swiss Webster mice (on Postnatal Days
[PNDs] 1, 8, and 15) at total doses up to 5.6 |imol did not result in an increase in pulmonary or
hepatic tumors in adulthood (Chang et at.. 1981; Buening et at.. 1980).
Similar studies evaluating carcinogenic potential of BeP metabolites were also generally
negative. In dermal initiation-promotion studies, only 9,10-dihydrobenzo[e]pyrene was positive,
resulting in an increased number of papillomas (67%) compared with vehicle control (7%)
following promotion with TPA when tested at 2.5 |imol (Buening et at.. 1980). The
9,10-dihydrodiol metabolite resulted in a borderline (p = 0.05) increase in the number of
papillomas (9/28) compared with vehicle control (3/30) when tested at 2 |imol using TPA as a
promoter (Staga et at.. 1980). but was negative when similarly tested at doses up to 6 |imol in a
second study (Buening et at.. 1980). Other tested metabolites (n = 3, see Table 4C) were not
initiators at doses up to 6 |imol using TPA as a promoter (Buening et at.. 1980; Staga et at..
1980). In injection studies in newborn mice, exposure to the 9,10-dihydrodiol or a diol epoxide
on PNDs 1, 8, and 15 (total doses of 2.8 or 0.7 |imol, respectively) resulted in a significant
increase in hepatic tumors in adult males, but not hepatic tumors in females or pulmonary tumors
in either sex (Chang et at.. 1981; Buening et at.. 1980). Other tested BeP metabolites (n = 5; see
Table 4C) did not induce pulmonary or hepatic tumors following neonatal i.p. injection (Chang
et at.. 1981; Buening et at.. 1980).
Taken together, these data suggest that BeP has low carcinogenic potential. BeP is not a
complete dermal carcinogen and did not induce tumors in lung implantation or i.p. studies;
however, 1/6 studies reported that BeP was a tumor initiator when administered dermally at high
doses. Evidence regarding the carcinogenic potential of BeP metabolites is limited; 1/5 tested
metabolites (9,10-dihydrobenzo[e]pyrene) was positive for initiation of skin tumors and 2/7
tested metabolites (a dihydrodiol and a diol epoxide) were positive for induction of tumors
following neonatal injection. The relevance of these findings to the oral and inhalation routes of
exposure is unclear but do suggest that metabolic processing is required for BeP-mediated
carcinogenic activity.
41
Benzo[e]pyrene
-------
EPA/690/R-21/008F
2.3.4. Metabolism/Toxicokinetic Studies
No data on absorption or distribution are available for BeP. Because absorption and
distribution of a chemical in the body are determined largely by physical and chemical properties
related to chemical size and general structure (e.g., lipophilicity, vapor pressure, etc.), it is
reasonable to assume that BeP will be absorbed and distributed similarly to other PAHs of
similar size and structure with similar physical and chemical properties (e.g., BaP).
Based on data for BaP and other similar PAHs (U.S. EPA. 2017b). BeP is expected to be
absorbed through inhalation, oral, and dermal routes of exposure, and generally through
diffusion across cell membranes. For inhalation exposure, BeP is expected to adsorb onto
particulates in the air and deposit in the respiratory tract with the associated carrier particles. As
for other PAHs (I ARC. 2010). particle size, region of deposition, and rate of dissolution or
desorption from particles are all expected to affect the rate and extent of BeP absorption from the
respiratory tract. In general, absorption through airways will likely be biphasic, with rapid
absorption occurring through thin epithelia in the alveoli and slower absorption occurring in
thicker regions of the airways (ATSDR. 1995). A significant proportion of inhaled BeP is likely
to be transported to the gut via the mucociliary escalator (IARC. 2010). For oral exposure,
studies with BaP indicate gastrointestinal absorption ranging from 10 to 60% (U.S. EPA. 2017b;
ATSDR. 1995). Oral absorption of PAHs in animals is enhanced by the presence of lipophilic
compounds, such as oils and fats, and by the presence of bile in the gastrointestinal tract
(ATSDR. 1995). Dermal absorption studies with BaP reported rapid and near complete
absorption in rats, mice, monkeys, and guinea pigs using vehicles, such as crude oil or acetone,
that enhanced absorption (U.S. EPA. 2017b). Dermal absorption was decreased with higher
viscosity oil vehicles and in the presence of soils with high organic carbon content (U.S. EPA.
2017b).
Like BaP (U.S. EPA. 2017c; I ARC. 2010; ATSDR. 1995). BeP is expected to show
widespread, systemic tissue distribution, with initial rapid uptake into highly diffused tissues,
such as lung, kidney, liver, and blood, followed by accumulation and retention in fat, with
subsequent slow release. Exposure of pregnant rats to BaP both orally and via inhalation
indicates that limited placental transfer can occur (U.S. EPA. 2017b; ATSDR. 1995).
PAHs, in general, are metabolized in multiple tissues in the body into more soluble
metabolites, including dihydrodiols, phenols, quinones, and epoxides, that form conjugates with
glucuronide, glutathione (GSH), or sulfate (U.S. EPA. 2017b; I ARC. 2010; ATSDR. 1995). The
metabolism of BeP has been investigated in several in vitro studies using hamster or mouse
embryo cells, rat liver homogenates, or purified microsomal fractions (Jacob et at.. 1985; Jacob
et at.. 1983; MacLeod et at.. 1982; Selkirk et at.. 1982; MacLeod et at.. 1979; Selkirk and
MacLeod. 1979; Wood et at.. 1979; Sims. 1970a. b; Duncan et at.. 1969). BeP is oxidized via the
CYP450 system (Jacob et at.. 1985; Jacob et at.. 1983; Wood et at.. 1979). The primary oxidative
metabolites include the k-region 4,5-dihydrodiol and phenolic metabolites, such as
3-hydroxybenzo[e]pyrene (3-OH-BeP) and other phenols with uncertain identities (1-OH, 4-OH,
9-OH, and 10-OH-BeP); BeP quinones have also been identified (Jacob et at.. 1985; Jacob et at..
1983; MacLeod et at.. 1979; Selkirk and MacLeod. 1979; Sims. 1970a. b). 4,5-Dihydrodiol and
the phenols conjugate with uridine diphosphate glucuronic acid to their respective glucuronide
conjugates (MacLeod et at.. 1982; MacLeod et at.. 1979). In contrast to BaP, there is little
evidence of direct bay-region activation of BeP, although the characteristic bay region is present
within BeP's structure. The 9,10-dihydrodiol metabolite was detected in trace amounts with rat
liver microsomes, generally only after pretreatment with potent monooxygenase inducers,
42
Benzo[e]pyrene
-------
EPA/690/R-21/008F
including BaP (Jacob et al.. 1985; Jacob et al.. 1983; Wood et al.. 1979). When the
9,10-dihydrodiol did form, subsequent oxidation was observed to occur at the 4,5-position to
form 4,5,9,10-tetrahydroxy-4,5,9,10-tetrahydrobenzyo[e]pyrene, and not the carcinogenic
7,8-diol-9,10-epoxide (Jacob et al.. 1985; Jacob et al.. 1983). A study with human liver
microsomes reported a higher percentage of the 9,10-dihydrodiol metabolite, approximately
12% of the total metabolites, compared with only 1% using liver microsomes from rats
pretreated with PAHs (Jacob et al.. 1985). Confounding metabolic potential through pretreatment
with PAHs is likely to result in decreased rates of individual PAH metabolite production due to
the likelihood of other available PAHs acting as a molecular sink for CYP activity. Given the
unique and varied metabolic capacity of individual model systems and the discrepancy in
production of mutagenic metabolites across those model systems, the significance and
contribution of bay region-directed metabolism of BeP remains unclear. Because Jacob et al.
(1985) described an increase in production of the proximal carcinogen (9,10-dihydrodiol) in
human microsomes compared with that from rodent microsomes, future efforts should be
directed at quantifying production of the ultimate diol-epoxide carcinogen within human models
systems, as this metabolic step is likely to underlie the moderate carcinogenic potential observed
for BeP.
In both mouse and hamster embryo cells, metabolism of BeP was essentially complete by
48 hours (MacLeod et al.. 1982; Duncan et al.. 1969). In liver microsomes from non-induced
rats, the total metabolic rate of BeP was low (2 nmol/mg microsomal protein); metabolic rates of
BeP increased significantly in liver microsomes when rats were pretreated with CYP450
inducers (Jacob et al.. 1983; Sims. 1970b). Metabolic profiles were similar between rat liver and
lung microsomes, and between hepatic microsomes from both rats and mice (Jacob et al.. 1985).
Metabolism studies with hamster embryo cells indicate the majority (71.7%) of BeP metabolites
distribute to extracellular space, while smaller amounts (1.1 and 0.18%) are retained in the
cytoplasm and nucleus, respectively (MacLeod et al.. 1979).
Based on studies of BaP in multiple species (U.S. EPA. 2017b; I ARC. 2010; AT SDR.
1995). excretion, mainly of conjugated metabolites, occurs primarily via biliary excretion to
feces, and to a lesser extent, by urine. BaP has also been detected in small amounts in milk (U.S.
EPA. 2017b). Excretion half-lives for BaP ranged from 22 to 30 hours in rats following
inhalation and dermal exposures, respectively (U.S. EPA. 2017b; AT SDR. 1995).
43
Benzo[e]pyrene
-------
EPA/690/R-21/008F
3. DERIVATION OF PROVISIONAL VALUES
3.1. DERIVATION OF PROVISIONAL REFERENCE DOSES
No studies have been located regarding toxicity of BeP to humans by oral exposure.
Animal studies of oral exposure to BeP are limited to an acute study with limited hepatic
endpoints and a subchronic cardiovascular study in apoE-KO mice of inadequate design and
scope to support derivation of a subchronic or chronic provisional reference dose (p-RfD). As a
result of the limitations of the available oral toxicity data for BeP, subchronic and chronic
p-RfDs were not derived directly. Instead, screening subchronic and chronic p-RfDs are derived
in Appendix A using an alternative analogue approach. Based on the overall analogue approach
presented in Appendix A, BaP was selected as the most appropriate analogue for BeP for
deriving a screening subchronic and chronic p-RfD (see Table 5).
3.2. DERIVATION OF PROVISIONAL REFERENCE CONCENTRATIONS
No adequate studies have been located regarding toxicity of BeP to humans via inhalation
exposure. Identified studies included occupational studies in workers exposed to complex
mixtures that lacked BeP-specific exposure data and case-control studies of asthma and lung
cancer that did not find associations with biomarkers of BeP exposure. No animal studies of
inhalation exposure to BeP were identified. As a result of the limitations of the available
inhalation toxicity data for BeP, subchronic and chronic provisional reference concentrations
(p-RfCs) were not derived directly. Instead, screening subchronic and chronic p-RfCs are derived
in Appendix A using an alternative analogue approach. Based on the overall analogue approach
presented in Appendix A, BaP was selected as the most appropriate analogue for BeP for
deriving a screening subchronic and chronic p-RfC (see Table 5).
3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES
A summary of the noncancer provisional reference values is shown in Table 5.
44
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 5. Summary of Noncancer Reference Values for
BeP (CASRN 192-97-2)
Toxicity Type
(units)
Species/
Sex
Critical Effect
p-Reference
Value
POD
Method
POD
(HED/HEC)
UFc
Principal
Study
Screening
subchronic
p-RfD
(mg/kg-d)
Rat/M, F
Neurodevelopmental
changes following
early postnatal
exposure
9 x 1(T5
BMDLisd
0.092a
(based on
analogue POD)
1,000
Chen et al.
(2012) as
cited in U.S.
EPA (2017c)
Screening
chronic p-RfD
(mg/kg-d)
Rat/M, F
Neurodevelopmental
changes following
early postnatal
exposure
9 x 1(T5
BMDLisd
0.092a
(based on
analogue POD)
1,000
Chen et al.
(2012) as
cited in U.S.
EPA (2017c)
Screening
subchronic
p-RfC (mg/m3)
Rat/F
Decreased
embryo/fetal survival
2 x 1(T6
LOAEL
0.0046
(based on
analogue POD)
3,000
Archibong et
al. (2002) as
cited in U.S.
EPA (2017c)
Screening
chronic p-RfC
(mg/m3)
Rat/F
Decreased
embryo/fetal survival
2 x 1(T6
LOAEL
0.0046
(based on
analogue POD)
3,000
Archibong et
al. (2002) as
cited in U.S.
EPA (2017c)
aThe POD was not converted into an HED using BW3'4 because it is unknown whether allometric scaling is
appropriate for exposure in early postnatal animals (see Appendix A for more details).
BeP = benzo[e]pyrene; BMDL = benchmark dose lower confidence limit; BW = body weight; F = female(s);
HEC = human equivalent concentration; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect
level; M = male(s); POD = point of departure; p-RfC = provisional reference concentration; p-RfD = provisional
reference dose; SD = standard deviation; UFC = composite uncertainty factor.
3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Although the scientific literature provides information on the mutagenicity and
genotoxicity of BeP, no oral or inhalation studies have been conducted to assess its
carcinogenicity. Available dermal, lung implantation, and i.p. carcinogenicity studies in animals
suggest that BeP has some carcinogenic potential, although findings are inconsistent across
studies, and the relevance of these findings to oral or inhalation exposure is unclear. Under the
U.S. EPA Cancer Guidelines (U.S. EPA. 20051 there is "Inadequate Information to Assess
Carcinogenic Potential" of BeP by oral or inhalation exposure (see Table 6). Within the current
U.S. EPA Cancer Guidelines (U.S. EPA. 20051 there is no standard methodology to support the
identification of a weight-of-evidence (WOE) descriptor and derivation of provisional cancer
risk estimates for data-poor chemicals using an analogue approach. In the absence of an
established framework, a screening evaluation of potential carcinogenicity is provided using the
methodology described in Appendix B. This evaluation determined that there was a qualitative
level of concern for potential carcinogenicity for BeP (see Appendix C).
45
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table 6. Cancer WOE Descriptor for BeP (CASRN 192-97-2)
Possible WOE Descriptor
Designation
Route of Entry (oral,
inhalation, or both)
Comments
"Carcinogenic to Humans"
NS
NA
No human data are available.
"Likely to Be Carcinogenic
to Humans "
NS
NA
No adequate chronic-duration animal cancer
bioassays are available.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
No adequate chronic-duration animal cancer
bioassays are available.
"Inadequate Information
to Assess Carcinogenic
Potential"
Selected
Both
No adequate animal cancer bioassays are
available. Dermal, lung implantation, and
i.p. studies in animals provide limited
evidence of carcinogenic potential.
"Not Likely to Be
Carcinogenic to Humans"
NS
NA
No evidence of noncarcinogenicity is
available. No adequate chronic-duration
animal cancer bioassays are available.
BeP = benzo[e]pyrene; i.p. = intraperitoneal; NA = not applicable; NS = not selected; WOE = weight of evidence.
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES
The absence of suitable data precludes development of cancer risk estimates for BeP (see
Table 7).
Table 7. Summary of Cancer Risk Estimates for BeP (CASRN 192-97-2)
Toxicity Type (units)
Species/Sex
Tumor Type
Cancer Risk Estimates
Principal Study
p-OSF (mg/kg-d) 1
NDr
p-IUR (lng/in3) 1
NDr
BeP = benzo[e]pyrene; NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral
slope factor.
46
Benzo[e]pyrene
-------
EPA/690/R-21/008F
APPENDIX A. SCREENING NONCANCER PROVISIONAL VALUES
Due to the lack of evidence described in the main Provisional Peer-Reviewed Toxicity
Value (PPRTV) document, it is inappropriate to derive provisional toxicity values for
benzo[e]pyrene (BeP) because the limited database on the toxicity of BeP is insufficient to
support direct derivation. However, some information is available for this chemical, which
although insufficient to support deriving a provisional toxicity value under current guidelines,
may be of use to risk assessors. In such cases, the Center for Public Health and Environmental
Assessment (CPHEA) summarizes available information in an appendix and develops a
"screening value." Appendices receive the same level of internal and external scientific peer
review as the provisional reference values to ensure their appropriateness within the limitations
detailed in the document. Users of screening toxicity values in an appendix to a PPRTV
assessment should understand that there could be more uncertainty associated with the derivation
of an appendix screening toxicity value than for a value presented in the body of the assessment.
Questions or concerns about the appropriate use of screening values should be directed to the
CPHEA.
APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH
The analogue approach allows for the use of data from related compounds to calculate
screening values when data for the compound of interest are limited or unavailable. Details
regarding searches and methods for analogue analysis are presented in Wang et al. (2012). Three
types of potential analogues (structural, metabolic, and toxicity-like) are identified to facilitate
the final analogue chemical selection. The analogue approach may or may not be route specific
or applicable to multiple routes of exposure. All information was considered together as part of
the final weight-of-evidence (WOE) approach to select the most suitable analogue both
toxicologically and chemically.
Structural Analogues
An initial analogue search focused on the identification of structurally similar chemicals
with toxicity values from the Integrated Risk Information System (IRIS), PPRTV, the Agency
for Toxic Substances and Disease Registry (ATSDR), or the California Environmental Protection
Agency (CalEPA) databases to take advantage of the well-characterized chemical-class
information. Under Wang et al. (2012). structural similarity for analogues is typically evaluated
using U.S. EPA's DSSTox database (DSSTox. 2018) and the National Library of Medicine's
(NLM) ChemlDplus database (ChemlDplus. 2021). However, DSSTox is no longer available to
the public, and there is no date available for the implementation of its replacement dashboard. In
lieu of DSSTox scores, the Organisation for Economic Co-operation and Development (OECD)
Toolbox was used to calculate structural similarity using the Dice method and default fingerprint
settings. Six structural analogues to BeP that have oral noncancer toxicity values were identified
(benzo[a]pyrene [BaP], fluoranthene, pyrene, anthracene, fluorene, and acenaphthene).
Table A-l summarizes the analogues' physicochemical properties and similarity scores. The
analogues are presented in order of decreasing molecular weight.
47
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-l. Physicochemical Properties of BeP (CASRN 192-97-2) and Candidate Structural Analogues3
Property
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Structure
cx8
eft)
CASRN
192-97-2
50-32-8
206-44-0
129-00-0
120-12-7
86-73-7
83-32-9
Molecular weight
252.316
252.316
202.256
202.256
178.234
166.223
154.212
OECD Toolbox similarity score (%)b
100
85
88.9
88.9
58.8
60.6
50
ChemlDplus similarity score (%)°
100
100
54
94
67
<50
<50
Melting point (°C)
178
177
108
150
215
115
93.9
Boiling point (°C)
469
(predicted average)
495
380
399
340
295
279
Vapor pressure (mm Hg)
5.7 x 10-9
5.49 x 10-9
9.22 x 10-6
4.5 x 10-6
6.53 x 10-6
6.0 x 10-4
2.15 x 10-3
Henry's law constant (atm-m3/mole)
1.07 x 10-6
4.57 x 10-7
8.86 x 10-6
1.19 x 105
5.56 x 10-5
9.62 x 10-5
1.84 x 10-4
Water solubility (mol/L)
1.89 x 10-8
8.40 x 10-9
1.24 x 10-6
6.65 x 10-7
3.38 x 10-7
1.15 x 10-5
4.64 x 10 5
Octanol-water partition coefficient (log Kow)
6.44
6.13
5.16
4.88
4.45
4.18
3.92
Acid dissociation constant (pKa)
NA
NA
NA
NA
NA
NA
NA
aData were extracted from the U.S. EPA CompTox Chemicals Dashboard:
benzo[e]pyrene, CASRN 192-97-2; https://comptox.epa.gov/dashboard/dsstoxdb/results?search DTXSID3023764#properties: accessed February 8, 2021;
benzo[a]pyrene, CASRN 50-32-8; https://comptox.epa. gov/dashboard/dsstoxdb/results?search=DTXSID2020139#properties: accessed February 8, 2021;
fl uo rant lie ne. CASRN 206-44-0; https://comptox.epa.gov/dashboard/dsstoxdb/results?search=DTXSID3024104#properties: accessed February 8, 2021;
pyrene, CASRN 129-00-0; https://comptox.epa.gov/dashboard/dsstoxdb/results?search=DTXSID3024289#properties: accessed February 8, 2021;
anthracene, CASRN 120-12-7; https://comptox.epa.gov/dashboard/dsstoxdb/results?search=DTXSID0023878#properties: accessed February 8, 2021;
fluorene, CASRN 86-73-7; https://comptox.epa.gov/dashboard/dsstoxdb/results?search=DTXSID8024105#properties: accessed February 8, 2021;
acenaphthene, CASRN 83-32-9; https://comptox.epa.gov/dashboard/dsstoxdb/results?search=DTXSID3021774#properties: accessed February 8, 2021;
all presented values are experimental averages unless otherwise noted.
bOECD (2019).
°ChemIDplus advanced similarity scores (ChemlDplus. 20211.
BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; NA = not applicable; OECD = Organization for Economic Co-operation and Development.
48
Benzo[e]pyrene
-------
EPA/690/R-21/008F
All of the candidate structural analogues contain two or more benzene rings and range in
molecular weight from 154.21 to 252.32 g/mol. The analogues are solids that have negligible to
low solubility in water. Measured octanol-water partition coefficient (log Kow) values for the
analogues ranged from 3.92 for acenaphthene to 6.13 for BaP. Log Kow values >4 correspond to
hydrophobic chemicals that are not very soluble in water, and compounds with the highest
log Kow values are more likely to partition to fat compartments in the body following absorption.
Similar to the target compound BeP, the four analogues with higher molecular weights have low
vapor pressures (see Table A-l), so they are expected to have low volatility from dry surfaces
and will exist as particulates in the atmosphere. Chemicals with low vapor pressures of
<1 x 10 6 mm Hg, such as BaP, have low potential for inhalation exposure as gases or vapors.
The two analogues with moderate vapor pressures, acenaphthene and fluorene, are expected to
have some volatility from dry surfaces and may exist in the atmosphere as a gas/particulate
mixture. However, all of the analogues have the potential for moderate volatilization from water
to air based on their reported Henry's law constant values. Differences in absorption and
distribution between the potential analogues are not expected to be significant for the four
candidate structural analogues with higher molecular weights and lower vapor pressures;
however, acenaphthene and fluorene are more volatile and more soluble in water than the other
analogues by a minimum of one order of magnitude. BaP is the preferred structural analogue for
BeP due to its physical and chemical properties that closely resemble BeP.
Metabolic Analogues
Table A-2 summarizes the available toxicokinetic data for BeP and the structurally
similar compounds identified as potential analogues. Absorption occurs via all routes, with the
rate and extent of absorption dependent on the exposure medium (i.e., enhanced in the presence
of oils and fats). Oral absorption appears to be more rapid and extensive for analogues with
lower molecular weight (e.g., anthracene). No absorption data were identified for fluorene and
acenaphthene. Polycyclic aromatic hydrocarbon (PAH) analogues are widely distributed in the
body, with preferential accumulation in fat as suggested by the log Kow values (see above). PAH
analogues undergo oxidative metabolism to diols, dihydroxy, and hydroxy metabolites, which
are common across all analogues. The presence of a bay region in both BeP and BaP indicates
the potential for metabolism to dihydrodiols and diol epoxides, which are electrophilic and
covalently bind to proteins and deoxyribonucleic acid (DNA) (U.S. EPA. 2017b. c); see
"Genotoxicity" summary in the "Other Data" section in the main body of the PPRTV document.
However, experimental data indicate that the potential for BeP to generate these reactive
metabolites is much lower than for BaP (see Table A-2 and "Metabolism" discussion in "Other
Data" section for more details). PAH analogues with lower molecular weight appear to be
excreted more rapidly in the urine (e.g., anthracene) while larger molecular weight compounds
(i.e., BaP) are excreted primarily in feces via the biliary system. BaP is the preferred metabolic
analogue for BeP due to the potential—albeit limited for BeP—to form reactive dihydrodiol and
diol epoxide metabolites. Further, the rates of absorption and excretion are expected to be similar
for BeP and BaP because their chemical and physical properties are comparable because of their
similar structure and identical molecular weight.
49
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-2. Comparison of ADME Data for BeP (CASRN 192-97-2) and Candidate Analogues
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
a2?
o8
C83
COO
CASRN 192-97-2
CASRN 50-32-8
CASRN 206-44-0
CASRN 129-00-0
CASRN 120-12-7
CASRN 86-73-7
CASRN 83-32-9
Absorption
ND
Laboratory animals
(oral, inhalation,
dermal):
• Absorbed by oral,
inhalation, and dermal
exposure
• Rate and extent of
absorption is variable,
depending on exposure
medium (e.g., oral and
dermal absorption
enhanced in presence of
oils and fats; dermal
absorption decreased in
presence of soils with
high organic carbon
content)
• Significant mucociliary
clearance of inhaled
particulate to gut
• Absorption from gut
depends on presence of
bile in intestinal lumen
Rats (oral):
• Peak blood level,
higher than that for
pyrene, achieved ~2 h
after oral dosing
• Absorption from GI
tract enhanced by
administration in
lipophilic vehicle
(dietary fat)
Rodents (oral, i.t.,
dermal):
• Peak blood level
achieved ~1 h after
oral dosing
• Extensive oral
absorption (68-92% in
one study)
• Absorbed through
tracheal epithelium
more rapidly than BaP
following i.t. exposure
• Rapid and extensive
dermal absorption in
acetone
(disappearance
half-time of radiolabel
from skin of
0.5-0.8 d; -50% of
applied radiolabel
recovered in urine and
feces within 6 d of
application)
• Dermal absorption of
94% in guinea pigs
Rats (oral, dermal):
• More extensive oral
absorption than BaP
in one study
(53-74% vs.
38-58%)
• Absorption from gut
much less dependent
on the presence of
bile than BaP
• -52% of applied
radiolabel recovered
in urine, feces, and
tissues within 6 d of
skin application
ND
ND
50
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-2. Comparison of ADME Data for BeP (CASRN 192-97-2) and Candidate Analogues
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Distribution
ND
Laboratory animals (all
Rats (oral, i.v.):
Rats (all routes):
Rats (dermal):
Rats (i.p.):
ND
routes):
• Widely distributed
• Widely distributed
• 6 d after application,
• Widely
• Widely distributed
throughout the body
throughout the body
radiolabel was
distributed
throughout the body
• Initial rapid uptake
• Initial rapid uptake
located primarily in
throughout the
• Initial rapid uptake into
into well-perfused
into well-perfused
liver and kidney
body (radiolabel
well-perfused tissues
tissues (e.g., lung,
tissues (e.g., lung,
found in all
(e.g., lung, kidney,
kidney, liver)
kidney, liver)
tissues examined)
liver)
• Levels in fat and testes
• Subsequent
• Highest amounts
• Subsequent
were lower and
accumulation,
of radiolabel
accumulation, retention,
peaked later
retention, and slow
found in gut, gut
and slow release from
• Coadministration with
release from fat
contents, kidney,
fat
saturated fat extended
• High levels in gut
and liver 1-8 d
• High levels in gut (from
duration of observed
(from any route) due
after injection (fat
any route) due to
peaks relative to
to mucociliary
not tested)
mucociliary clearance
administration with no
clearance from
from respiratory tract
fat (controls) or
respiratory tract and
and hepatobiliary
unsaturated fat
hepatobiliary excretion
excretion of metabolites
of metabolites
• Limited placental
• Limited placental
transfer
transfer
51
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-2. Comparison of ADME Data for BeP (CASRN 192-97-2) and Candidate Analogues
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Metabolism
In vitro:
Laboratory animals (all
Rats (oral):
ND
Rats (oral):
Laboratory
Rats, rabbits
• Metabolism is rapid
routes), in vitro:
• Primary reactive
• Urinary metabolites
animals (oral,
(oral):
(complete within
• Metabolism is rapid and
metabolites identified
include
i.p.):
• Urinary
48 h)
occurs in many tissues
were trans-2,3-
trans-1,2-dihydro-
• Primary urinary
metabolites
• Oxidized via
throughout the body
dihydro-hydroxy-
1,2-dihydroxy- and
metabolites were
include cis- and
CYP450 to the
• Oxidized via CYP450;
fluoranthene and
1,2-dihydroxy-
2- and 9-fluorenol
trans-
k-region
primary metabolites are
trans-2,3-
anthracene and
(hydroxyfluorene)
acenaphthene-1,2-
4,5-dihydrodiol,
9,10-, 7,8-, 4,5-, and
dihydroxy-l,10b-
trans-9,10-dihydro-
glucuronide and
diols and
phenols and quinones
2,3-dihydrodiols and
epoxy-l,2,3,10b-
9,10-dihydroxy-
sulfate conjugates
naphthalene-1,8-
• Oxidative
epoxides, as well as
tetrahydro-
anthracene, excreted
dicarboxylic acid,
metabolism can be
various phenols,
fluoranthene; primary
mainly as sulphate
the latter
induced by CYP450
quinones, and
nonreactive
and glucuronide
indicating fission
inducers
derivatives
metabolites identified
conjugates
of the 5-carbon
• Bay-region
• Oxidative metabolism
were 3-hydroxy -
ring
9,10-dihydrodiol is
can be induced by
fluoranthene and
formed only in small
CYP450 inducers
8-hydroxy-
amounts and then
• Oxidative metabolites
fluoranthene
oxidized at the
conjugated with GSH,
• Levels of reactive
4,5-position, rather
glucuronic acid, and
metabolites in plasma
than forming the
sulfate esters
and tissues were
9,10-expoxide in
slightly higher when
rodent model systems
coadministered with
• Oxidative
saturated fat relative to
metabolites
administration with no
conjugated with
fat (controls) or
glucuronic acid
unsaturated fat
52
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-2. Comparison of ADME Data for BeP (CASRN 192-97-2) and Candidate Analogues
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Excretion
ND
Laboratory animals (all
routes):
• Excretion is rapid, with
half-times of 22-30 h
• Primary route is biliary
excretion to feces; urine
is secondary route
• Excreted mainly as
conjugated metabolites
• Small amounts excreted
in milk
Rodents (oral, i.v.):
• Excretion is rapid
• Coadministration with
saturated fat increased
excretion half-time
relative to
administration with
unsaturated fat
Rodents (oral,
dermal):
• Excretion is rapid
• Excreted in urine and
feces in similar
amounts
Rat (dermal):
• Excreted in slightly
higher amounts in
urine than in feces
Laboratory
animals (i.p.):
• Excretion is rapid
• Excreted
primarily in urine,
with lower
amounts in feces
• Excreted mainly
as conjugated
metabolites
Laboratory
animals (all
routes):
• Excretion is rapid,
with half-times of
22-30 h
• Primary route is
biliary excretion
to feces; urine is
secondary route
• Excreted mainly
as conjugated
metabolites
• Small amounts
excreted in milk
NA
U.S. EPA (2017b): U.S.
Walker etal. (2007);
[ARC (2010): ATSDR
I ARC (2010): ATSDR
Grantham (1963);
Chang and Young
EPA (2017c): [ ARC
Lioniak and Brandvs
(1995); Lioniak and
(1995); Sims (1964)
Neish (1948)
(1943)
(2010); ATSDR (1995)
(1993)
Brandvs (1993)
ADME = absorption, distribution, metabolism, and excretion; BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; CYP450 = cytochrome P450; GI = gastrointestinal;
GSH = glutathione; i.p. = intraperitoneal; i.t. = intratracheal; i.v. = intravenous; NA = not applicable; ND = no data.
53
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Toxicity-Like Analogues—Oral
No adequate studies were located regarding toxicity of BeP via oral exposure. Table A-3
summarizes available oral toxicity values for the potential structural analogues of BeP. The
critical effects for potential analogues include neurodevelopmental effects, hematological effects,
and kidney and liver toxicity. BaP is the most well-studied of the analogue compounds, with
dose-response data available for each of the critical effects (see exposure-response arrays in
Figures A-l, A-2, A-3, and A-4). The oral data for the other analogue compounds are limited to
one or two subchronic gavage studies in mice. These studies provide dose-response data for
hematological effects and kidney and liver toxicity only (see Figures A-l, A-2, A-3, and A-4). A
review of the available dose-response data for the candidate analogues shows the lowest effect
levels for BaP for each critical effect examined.
In developing the oral reference dose (RfD) for BaP, the U.S. EPA compared candidate
values for developmental, reproductive, and immunological effects (U.S. EPA. 2017b. c). The
overall RfD, based on neurodevelopmental effects in rats exposed during the early postnatal
period, was supported by numerous human and animal studies and was considered protective of
all types of health effects (U.S. EPA. 2017b. c). The mode of action (MOA) for
neurodevelopmental effects of BaP is not fully understood; however, possible mechanisms may
include covalent protein binding of oxidative metabolites (I ARC. 2010; ATS DR. 2005).
oxidative stress, and the formation of reactive oxygen species, aryl hydrocarbon (Ah)
receptor-mediated effects on cell growth and differentiation, DNA damage of germ cells,
stimulation of apoptosis, altered neurotransmitter levels, and changes in the balance of
reproductive hormones (U.S. EPA. 2017c). In the absence of repeated-exposure oral toxicity data
for BeP, there is no information with which to clearly identify or rule out candidate analogues
based on toxicity comparisons.
Toxicity-Like Analogues—Inhalation
No adequate studies were located regarding toxicity of BeP via inhalation exposure.
Table A-4 summarizes available inhalation toxicity values for the potential structural analogues
of BeP. BaP is the only potential analogue with an inhalation toxicity value. Candidate reference
concentration (RfC) values were derived for both reproductive and developmental effects of BaP
(U.S. EPA. 2017b. c). The overall RfC, based on decreased embryo/fetal survival following
prenatal inhalation exposure, was considered protective of all types of health effects (U.S. EPA.
2017b. c). The MO A for BaP effects on fertility may involve a decrease in prolactin and decidual
luteotropin levels, leading to decreases in plasma progesterone and estradiol-17p levels
[Archibong et al. (2002) as cited in U.S. EPA (2017c)"|. Fetal mortality may result from a
subsequent decrease in the levels of uterine progesterone receptors. In the absence of
repeated-exposure inhalation toxicity data for BeP, there is no information with which to clearly
identify or rule out BaP as an appropriate analogue based on toxicity comparisons.
54
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-3. Comparison of Available Oral Toxicity Data for BeP (CASRN 192-97-2) and Candidate Structural
Analogues
Type of Data
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Structure
(^0
cx8
<&>
cad
CASRN
192-97-2
50-32-8
206-44-0
129-00-0
120-12-7
86-73-7
83-32-9
Subchronic oral toxicity values
POD (mg/kg-d)
ND
ND
Note: The POD for the
chronic RfD is also
applicable to
subchronic exposure
because it is based on
a developmental study
(see further details
below)
124
75
1,000
125
161
POD type
ND
ND
BMDLio
NOAEL
NOAEL
LOAEL
BMDLio
Subchronic UFC
ND
ND
1,000
(UFa, UFd, UFh)
300
(UFa, UFd, UFh)
1,000
(UFa, UFd, UFh)
300
(UFa, UFh, UFl)
1,000
(UFa, UFd, UFh)
Subchronic
p-RfD/MRL
(mg/kg-d)
ND
ND
1 x 10-1
3 x 10-1
1 x 10°
4 x 10-1
2 x 10-1
55
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-3. Comparison of Available Oral Toxicity Data for BeP (CASRN 192-97-2) and Candidate Structural
Analogues
Type of Data
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Critical effects
ND
ND
Nephropathy at
>250 mg/kg-d
Kidney effects
(renal tubular
pathology,
decreased kidney
weights) at
>125 mg/kg-d
No adverse
effects on
mortality,
clinical signs,
body weight,
food
consumption,
ophthalmology,
hematology,
clinical
chemistry, organ
weights, gross or
microscopic
pathology
Increased liver
weight
Increased relative
liver weight in
female mice
Species
ND
ND
Mouse
Mouse
Mouse
Mouse
Mouse
Duration
ND
ND
13 wk
13 wk
13 wk
13 wk
13 wk
Route (method)
ND
ND
Oral (gavage)
Oral (gavage)
Oral (gavage)
Oral (gavage)
Oral (gavage)
Source
NA
NA
U.S. EPA (2012)
(PPRTV)
U.S. EPA (2007)
(PPRTV)
U.S. EPA (2009)
(PPRTV)
ATSDR (1995)
(Intermediate MRL)
U.S. EPA (2011c)
(PPRTV)
Chronic oral toxicity values
POD (mg/kg-d)
ND
0.092
125
75
1,000
125
175
POD type
ND
BMDLisd
NOAEL
NOAEL
NOAEL
NOAEL
NOAEL
Chronic UFC
ND
300 (UFa, UFd, UFh)
3,000
(UFa, UFd, UFh,
UFS)
3,000
(UFa, UFd, UFh,
UFS)
3,000
(UFa, UFd, UFh,
UFS)
3,000
(UFa, UFd, UFh,
UFS)
3,000
(UFa, UFd, UFh)
Chronic RfD/p-RfD
(mg/kg-d)
ND
3 x 10-4
4 x 10-2
3 x 10-2
3 x 10-1
4 x 10-2
6 x 10-2
56
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-3. Comparison of Available Oral Toxicity Data for BeP (CASRN 192-97-2) and Candidate Structural
Analogues
Type of Data
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Critical effects
ND
Neurodevelopmental
effects (increased open
field crossed squares at
PND 69; elevated plus
maze open arm entries
at PND 70; increased
Morris water maze
hidden platform trial
escape latency at
PNDs 71-74
Nephropathy,
increased absolute
and relative liver
weights,
hematological and
clinical chemistry
alterations
(decreased
albumin: globulin
ratio, increased
ALT, decreased
packed cell
volume and
eosinophils) at
>250 mg/kg-d
Kidney effects
(renal tubular
pathology,
decreased kidney
weights) at
>125 mg/kg-d
No adverse
effects on
mortality,
clinical signs,
body weight,
food
consumption,
ophthalmology,
hematology,
clinical
chemistry, organ
weights, gross or
microscopic
pathology at
<1,000 mg/kg-d
Decreased RBC,
packed cell volume,
and hemoglobin
Hepatotoxicity
accompanied by
increased liver
weight (which was
considered adaptive)
Species
ND
Rat
Mouse
Mouse
Mouse
Mouse
Mouse
Duration
ND
PNDs 5-11
13 wk
13 wk
13 wk
13 wk
13 wk
Route (method)
ND
Oral (gavage)
Oral (gavage)
Oral (gavage)
Oral (gavage)
Oral (gavage)
Oral (gavage)
Source
NA
U.S. EPA (2017b):
U.S. EPA (2017c)
U.S. EPA(1990d)
(IRIS)
U.S. EPA(1990f)
(IRIS)
U.S. EPA
(1990b) (IRIS)
U.S. EPA(1990e)
(IRIS)
U.S. EPA (1990a)
(IRIS)
Acute oral lethality data
Oral LD5o (mg/kg)
ND
ND
800 (mouse)
2,700 (rat)
ND
>17,000 (mouse)
2,000 (rat)
ND
Toxicity at rat LD50
ND
ND
Behavioral effects
(excitement,
muscle
contraction or
spasticity); eye
irritation
ND
Fatty
degeneration of
the liver
ND
ND
57
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-3. Comparison of Available Oral Toxicity Data for BeP (CASRN 192-97-2) and Candidate Structural
Analogues
Type of Data
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Source
NLM (2019e)
NLM (2019d)
U.S. EPA(1990d)
U.S. EPAQ990f)
NLM (2019c)
NLM (2019g)
NLM (2019b)
(IRIS); NLM
(IRIS); NLM
(2019f)
(2019h)
ALT = alanine aminotransferase; BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; BMDL = benchmark dose lower confidence limit; IRIS = Integrated Risk Information
System; LD5o = median lethal dose; LOAEL = lowest-observed-adverse-effect level; MRL = minimal risk level; NA = not applicable; ND = no data;
NOAEL = no-observed-adverse-effect level; PND = postnatal day; POD = point of departure; (p-)RfD = (provisional) reference dose; PPRTV = provisional
peer-reviewed toxicity value; RBC = red blood cell; SD = standard deviation; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database
uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
58
Benzo[e]pyrene
-------
EPA 690 R-21 008F
• Doses > LOAEL
¦ LOAEL
ANOAEL
O Doses < NOAEL
0
u
u
_Q
-Q
_Q
r*>
t—1
ro
*—1
0
CC
0
+j
O
+-<
ro
CC
r\J
_k~"
(N
ro
CC
(N
<
<
<
isT
Q_
LU
r*>
Q_
LU
5
m
Q_
LU
m
on
%
on
on
t—1
Z>
Z>
Z)
>;
_c
¦a
1—
u
X
_c
¦a
2
1—
U
X
_c
¦a
CUD
O
O
(D
+-<
'0
¦a
c
ro
ro
E
to
CQ
(J
to
¦a
to
(D
ro
ro
c
ro
ro
cd"
X
*—1
¦t—i
c
CD
O
c
O
0
CD
O
3
O
+J
T—1
"ro
c
3
0
(j
r\j
"ro
0
u
U
(N
ro
0
£
(D
u
CQ
CC
CQ
+j
(D
(D
(D
O
Ctf)
c
to
_aj
-»
(D
to
2
0
(D
0
O
(D
3
i_
Q
c
Benzo[a]pyrene
0
01
3
o
< 1
Q_ >
lu ro
i
on
Z>
C
^ i
~ 8
u —
m ^
ro q_
cn °
oo .E
CD ^
*—I o
< «
S] <-
00
Z>
Ace-
naph-
thene
(D
O
CD
CD
_c
T3
(D
ro c0
. (J
CD x
00
2 u
CD
< cr:
-A
LU
on
Z>
Fluorene
Subchronic
¦a
o
CD
CD
T—I
<
Q_
3
o
¦a ¦=*
(D m
.ti
(j
to lZT
ro -—-
00 ?5
SS ^
CD
Q_
on
Z>
o
(N
<
Q_
on ro
=5 *
1»
qj ro
¦M *—1
'<-> *
o
o
3
(D
I
-»
o
o
(N
3
c
Fluoranthene
o
(D
_Q
° g
O) o
< >
Q- 5
11 I
m
on ^
Z>
_ U>
¦- _2
o
+-»
ro
0 E
¦a
(D
to
ro _c
CD
00
CD
t—I
<
Q_
on
Z>
Anthra-
cene
_Q
O
O
CD
r—\
< ^
Ss 2
on >;
—» Ctf)
—' O
.E o
¦a ro
S E
"o
-------
EPA 690 R-21 008F
• Doses > LOAEL
¦ LOAEL
ANOAEL
O Doses < NOAEL
o
o
u
_q
_q
r*>
t—i
o
O
r\j
r\j
<
<
Q_
LU
+j
Q_
LU
on
ro
CC
on
Z>
_k~"
Z>
_c
_c
¦a
r*>
¦a
(D
.
tn
ro
(D
ro
CD
CD
C
O
CD
O
T—1
r\j
"ro
"ro
+j
+j
(D
(D
Ctf)
(D
c
tn
o
o
rvj *—I
< ^
Q- _C
LU -M
ro
oo Q-
=> 2
.E a.
-o «
QJ c
>
(D
C
¦a
CD
00
CD
t—I
<
Q_
on J
Z>
Pyrene
(D
O
CD aj
CD Cn
< £
Q- ^
ro
.E ^
¦a -g
QJ >
>.
U Oj
uo C
ro "o
SS a!
3 <
< ia
Q- m
<-
Fluorene
T3
O
^ Si
U O
o ^
< 5
o_ m
11 i H
^ I
=> «
C Q.
-a 2
(D _c
.ti Q.
O
Fluor-
anthene
ro
O
CD
CD
o
r\j
<
Q_
on H
= §
.E i_
T3 °
0J
.ti 5
U >~
tn o
Acenaph-
thene
_Q
O
CD
CD
t—I
<
Q_
on
Z>
_c
¦a
(D
3
o
>~
Ctf)
_o
O
>~
(D
C
¦a
c
o
o
.
-------
EPA 690 R-21 008F
• Doses > LOAEL
¦ LOAEL
ANOAEL
o Doses < NOAEL
-Q
t-H
o
LU fC
. oc:
IS) - -
i*
ro
-o ^
-
t-H
T—1
o
o
rvj
rsl
<
<
Cu
LU
¦4->
ro
G_
UJ
CO
cd
CO
Z>
_*T
z>
c
c
"O
¦O
ro
ctT
T—1
cn
"o3
i_
O
0)
0s) >
.-t; >
cn —
oo 2
<71
< ro
£] <-
Fluorene
ro
o
<71
O
(N
<
Q_
5
co ro
¦£ %
CT) CO
00 -Q
CTi ro
f-
co
Z>
Ace-
naph-
thene
"O
o
>
10 —
ro c
cn °
co G
0s* a)
*=
< o>
Q- o
uj z
CO
z>
Anthra-
cene
Subchronic
Figure A-3. Liver Weight Following Oral Exposure to Candidate Analogues of Benzo[e]pyrene (CASRN 192-97-2)
61
Benzo[e]pyrene
-------
EPA 690 R-21 0081''
rr• 10000
TO
~u
M
J*
OA
£_
LOAEL
¦ LOAEL
ANOAEL
o Doses < NOAEL
100
0.1
_Q
O
P-J
£ ™
£] «=
C
— TO
"O '(/>
CU 03
u &¦
V) -
& •c
2 a?
05 TO
a> o
00
c
o
a)
Q
_Q
r-- +-
*H 05
O CC
2 ^
UJ ro
So
C O
¦a 2
c
on
™
§i
rN O
c
Benzo[a]pyrene
"O
o
CTi o
= s
- £
"O ^
±= G£i
s -
03 »_
- 03
00 —
00 2
(J) ¦£
tH o
< 'E
o
CM
<
ro
-o _o
« o
U to
v) x:
ro »_
a?
w «u
¦s o
Fluoranthene
ro
0
a *
1 o
o i*r
3 5
Q_
ro
I I
u £
£ »-
03
^
Anthra-
cene
tH
<
Q-
^ tH
= £
— o
13 °
.a V)
u 2
C/l
ro OJ
oi-
ro 7
CTl £
rH O
? c
Q- Q1
LU ^
00 «
=)¦ $
Fluo-
rene
o
CTl
cn oj
" I
_Q O
o 5
o i ±2
C
o
t3
£
O)
o
z
Pyrene
Subchronic
JD
T—I
0
-------
EPA 690 R-21 008F
Table A-4. Comparison of Available Inhalation Toxicity Data for BeP (CASRN 192-97-2) and Candidate Structural
Analogues
Type of Data
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Structure
o8
CGQ
cm
CASRN
192-97-2
50-32-8
206-44-0
129-00-0
120-12-7
86-73-7
83-32-9
Subchronic inhalation toxicity values
POD (mg/m3)
ND
ND
Note: The POD for the
chronic RfC may also
be used as a subchronic
POD because it is
based on a
developmental study
(see below)
ND
ND
ND
ND
ND
POD type
ND
ND
ND
ND
ND
ND
ND
Subchronic UFC
ND
ND
ND
ND
ND
ND
ND
Subchronic
p-RfC/MRL (mg/m3)
ND
ND
ND
ND
ND
ND
ND
Critical effects
ND
ND
ND
ND
ND
ND
ND
Species
ND
ND
ND
ND
ND
ND
ND
Duration
ND
ND
ND
ND
ND
ND
ND
Route (method)
ND
ND
ND
ND
ND
ND
ND
Source
NA
U.S. EPA (2017c)
(IRIS)
U.S. EPA (2012)
(PPRTV)
U.S. EPA (2007)
(PPRTV)
U.S. EPA (2009)
(PPRTV)
ATSDR (1995)
(intermediate MRL)
U.S. EPA (2011c)
(PPRTV)
63
Benzo[e]pyrene
-------
EPA 690 R-21 008F
Table A-4. Comparison of Available Inhalation Toxicity Data for BeP (CASRN 192-97-2) and Candidate Structural
Analogues
Type of Data
BeP
BaP
Fluoranthene
Pyrene
Anthracene
Fluorene
Acenaphthene
Chronic inhalation toxicity values
POD (mg/m3)
ND
0.0046
ND
ND
ND
ND
ND
POD type
ND
LOAEL
ND
ND
ND
ND
ND
Chronic UFC
ND
3,000
(UFa, UFh, UFl, UFd)
ND
ND
ND
ND
ND
Chronic p-RfC/MRL
(mg/m3)
ND
2 x 10-6
ND
ND
ND
ND
ND
Critical effects
ND
Decreased embryo/fetal
survival
ND
ND
ND
ND
ND
Species
ND
Rat
ND
ND
ND
ND
ND
Duration
ND
GDs11-20
ND
ND
ND
ND
ND
Route (method)
ND
Inhalation (nose only)
ND
ND
ND
ND
ND
Source
NA
U.S. EPA (2017c)
(IRIS)
U.S. EPA (2012)
(PPRTV)
U.S. EPA (2007)
(PPRTV)
U.S. EPA (2009)
(PPRTV)
ATSDR (1995)
(intermediate MRL)
U.S. EPA (2011c)
(PPRTV)
Acute inhalation lethality data
LCso (mg/m3)
ND
ND
170 mg/m3
ND
ND
ND
ND
Toxicity at LCso
ND
ND
Behavioral effects
(excitement,
muscle contraction
or spasticity); eye
irritation
ND
ND
ND
ND
Source
NLM (2019e)
U.S. EPA (2017c):
NLM (2019d)
U.S. EPA(1990d)
(IRIS); NLM
(2019f)
U.S. EPA
(1990f) (IRIS):
NLM (2019h)
U.S. EPA
(1990b) (IRIS):
NLM (2019c)
U.S. EPA(1990e)
(IRIS); NLM
(2019s)
U.S. EPA (2011c)
(PPRTV); NLM
(2019b)
BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; GD = gestation day; IRIS = Integrated Risk Information System; LC50 = median lethal concentration;
LOAEL = lowest-observed-adverse-effect level; MRL = minimal risk level; NA = not applicable; ND = no data; NOAEL = no-observed-adverse-effect level;
POD = point of departure; (p-)RfC = (provisional) reference concentration; PPRTV = provisional peer-reviewed toxicity value; UFA = interspecies uncertainty factor;
UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor.
64
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Weight-of-Evidence Approach
To select the best analogue chemical based on all of the information from the three
analogue types, the following considerations are used in a WOE approach: (1) lines of evidence
from U.S. EPA assessments are preferred; (2) biological and toxicokinetic data are preferred
over the structural similarity scores; (3) lines of evidence that indicate pertinence to humans are
preferred; (4) chronic studies are preferred over subchronic studies when selecting an analogue
for a chronic value; (5) chemicals with more sensitive toxicity values are of potential concern;
and (6) if there are no clear indications as to the best analogue chemical based on the other
considerations, then the candidate analogue with the highest structural similarity scores may be
preferred.
Oral
The WOE approach used to select the analogue compound for BeP oral exposure is based
on structural similarity, comparable physicochemical properties, and similar oxidative
metabolism to reactive intermediates. BaP is the preferred structural analogue for BeP due its
physicochemical properties, which closely resemble BeP. BaP is also the preferred metabolic
analogue for BeP due to the potential for BeP to form reactive dihydrodiol and diol epoxide
metabolites, acknowledging that the generation of these reactive metabolites is estimated to be
much lower for BeP compared to BaP. Although in vivo toxicological similarity cannot be
assessed due to the absence of data for BeP, metabolism data suggest that the candidate analogue
with bioactivity most similar to BeP is BaP. Furthermore, the neurodevelopmental effects of BaP
provide the most sensitive measure of toxicity among the candidate analogue compounds. Based
on available data, BaP was selected as the most appropriate analogue compound for both
subchronic and chronic effects.
Inhalation
As discussed above for oral exposure, BaP is the preferred structural and metabolic
analogue for BeP. Regarding toxicity, BaP was the only candidate analogue compound with
repeated-dose inhalation toxicity data. Therefore, BaP was selected as the analogue compound
for both subchronic and chronic inhalation exposure.
ORAL NONCANCER TOXICITY VALUES
Derivation of a Screening Subchronic Provisional Reference Dose
Based on the overall analogue approach presented in this PPRTV assessment, BaP is
selected as the analogue for BeP for derivation of screening subchronic and chronic p-RfDs. The
study used for the U.S. EPA screening subchronic and chronic p-RfD values for BeP is an early
postnatal gavage study of BaP in rats. The Toxicological Review of Benzo[a]pyrene
(CASRN 50-32-8): Supplemental Information (U.S. EPA. 2017b") provided the following
summary:
Chen et al. (2012) treated male andfemale neonatal Sprague-Dawley rats
(10/sex/group) with BaP (unspecifiedpurity) dissolved in peanut oil by gavage
daily on PNDs 5 11, at doses of 0.02, 0.2, or 2 mg/kg in 3 ml vehicle/kg body
weight, determined individually based upon daily measurements. This time period
was described as representing the brain growth spurt in rodents, analogous to
brain developmental occurring from the third trimester to 2 years of age in
human infants. Breeding was performed by pairs of 9-week-old rats, with delivery
designated as PND 0. litters were culled to eight pups/dam (four males and four
65
Benzo[e]pyrene
-------
EPA/690/R-21/008F
females, when possible) and randomly redistributed at PND 1 among the nursing
dams; dams themselves were rotated every 2-3 days to control for caretaking
differences, and cage-side observations of maternal behavior were made daily.
One male andfemale from each litter were assigned per treatment group, and the
following physical maturation landmarks were assessed daily in all treatment
groups until weaning at PND 21: incisor eruption, eye opening, development of
fur, testis decent, and vaginal opening.
Neonatal sensory and motor developmental tests were administered to
pups during the preweaningperiod at PNDs 12, 14, 16, and 18, and were
behavioral tests administered to rats as adolescents (PNDs 35 and 36) or as
adults (PNDs 70 and 71): each rat was only tested during one developmental
period. All dosing was performed from 1300 to 1600 hours, and behavioral
testing was during the "dark"periodfrom 1900 to 2300 hours, although tests
were performed in a lighted environment. Pups were observed individually and
weighed daily, the order of testing litters was randomized each day, and all
observations were recorded by investigators blinded to group treatment.
Sensory and motor developmental tests, including the surface righting
reflex test, negative geotaxis test, and cliff aversion test, were performed only
once, while the forelimb grip strength test was assessed during three 60-second
trials on PND 12. Rat movements during the open-field test were recorded by
camera, and two blinded investigators scored movement and rearing separately
during a 5-minute evaluation period. Blinded investigators directly observed
video monitoring of rat movements during the elevated plus maze, and after a
5-minute free exploration period, recorded number of entries into the closed and
open arms, time spent in the open arms, and latency to the first arm entry.
Assessment of the Morris water maze was slightly different, in that the rats were
habituated to the testing pool by a 60-second swim without a platform on the day
prior to testing. The rats were then tested during a 60-second swim with a hidden
platform present at a constant position each day for 4 days; on the 5th day, the
rats were evaluated during a 60-second probe swim without a platform. The
number of times each animal crossed the original platform location and the
duration of time spent in the platform quadrant were recorded during this final
evaluation. One pup/sex/litter were assignedfor behavioral testing to each of four
tracks: Track 1, surface righting reflex test, cliff aversion test, and open-field test
(PNDs 12-18); Track 2, negative geotaxis test, forelimb grip strength test, and
open-field test (PNDs 12-20); Track 3, elevated plus maze, Morris water maze,
and open-field test (PNDs 34-36); and Track 4, elevated plus maze, Morris water
maze, and open-field test (PNDs 69-71). All results were presented in graphic
form only.
No significant effects on pup body weight were observed during the 7-day
treatment period (PNDs 5-11). Three-way ANOVA (time x BaP treatment x sex)
indicated that effects of BaP were not sex-dependent throughout the 71-day
experiment, so both sexes were pooled together. From this pooled analysis, pups
in the 2 mg/kg-day treatment group gained significantly less weight at both
66
Benzo[e]pyrene
-------
EPA/690/R-21/008F
PND 36 and 71. There were no differences among treatment groups in incisor
eruption, eye opening, development of fur, testis decent, or vaginal opening.
For all measurements of neonatal sensory and motor development, results
from both sexes were analyzed together since BaP was reported to have no
significant interaction with sex by 3-way ANOVA. No significant differences were
observed in either the cliff aversion or forelimb grip strength tests. In the surface
righting reflex test, latency was increased in the 0.2 mg/kg-day group at PND 12,
in the 0.02 and 2 mg/kg-day groups at PND 14, and in only the high-dose group
at PND 16; latency was not significantly different in any group at PND 18. At
PND 12, there was a dose-related increase in negative geotaxis latency
associated with 0.02, 2, and 2 mg/kg-day BaP, which was also present in the
2 mg/kg-day group at PND 14, but returned to control levels at PND 16 and 18.
In the open field test, there were no significant differences in either locomotion or
rearing activity at PND 18 or 20. At PND 34, the 2 mg/kg-day group exhibited
significantly increased movement, but increases in rearing were not significant.
At PND 69, increased locomotion was observed in both the 0.2 and 2 mg/kg-day
groups, while rearing was significantly increased in only the 2 mg/kg-day
treatment group.
The elevated plus maze performance was only evaluated in adolescent and
adult rats. Unlike the previous tests, 3-way ANOVA revealed a statistically
significant interaction between neonatal BaP treatment and sex, so male and
female performance was analyzed independently. No significant differences in
PND 35 males were observed, and the only significant observation in PND 35
females was increased time spent in the open maze arms by the 2 mg/kg-day
treatment group. Significantly decreased latency time to first open arm entry was
observed in PND 70 males andfemales in both 0.2 and 2 mg/kg-day treatment
groups; these groups also spent significantly more time in open maze arms, along
with the 0.02 mg/kg-day female group. At PND 70, the 2 mg/kg-day males, along
with the 0.2 and 2 mg/kg-day females, entered more frequently into open arms
and less frequently into closed arms than the vehicle controls. In the Morris water
maze, escape latency (time to reach the platform during each of the four testing
days) was consistently increased in the 2 mg/kg-day treatment group of both
sexes, in both adolescent and adult animals. These increases were statistically
significant in both males and females treated with 2 mg/kg-day BaP at both
PNDs 39 and 74, and were also significantly elevated in 0.2 mg/kg-day animals of
both sexes at PND 74. Likewise, performance during the 5th test day, in the
absence of the escape platform, was significantly adversely affected by both
metrics (decreased time spent in the target quadrant and decreased number of
attempts to cross the platform location) in 2 mg/kg-day rats of both sexes at both
PNDs 40 and 75. PND 75 females treated with 0.2 mg/kg-day BaP also showed
significant decreases in both performance metrics, while PND 75 0.2 mg/kg-day
males only demonstrated significant differences in 'time spent in target quadrant. '
Swim speed was also assessed, but there were no differences among any
treatment group at either age evaluated.
67
Benzo[e]pyrene
-------
EPA/690/R-21/008F
The BMDLi sd of 0.092 mg/kg-day was identified as the point of departure (POD) for
BaP based on the induction of neurobehavioral changes during a susceptible life stage (U.S.
EPA. 2017c). This POD is selected from among a suite of available endpoints because it
represents multiple neurobehavioral endpoints from several behavioral tests. Similar effects were
replicated among numerous additional studies. The U.S. EPA (2017c) did not convert the POD
into a human equivalent dose (HED) using BW3'4 because the critical study evaluated
developmental toxicity in early postnatal animals directly exposed to BaP. BW3'4 scaling was
determined to be inappropriate because (1) it is unknown whether allometric scaling derived
from adult animals is appropriate for extrapolating doses in neonates in the absence of
quantitative toxicokinetic and toxicodynamic differences and (2) differences in temporal patterns
of development across species results in complications for interspecies dose extrapolation.
The RfD for BaP is derived using a composite uncertainty factor (UFc) of 300, reflecting
10-fold uncertainty factors for interspecies extrapolation and intraspecies variability and a 3-fold
uncertainty factor for database uncertainties (UF UFh, and UFd, respectively) (U.S. EPA.
2017c). Wang et al. (2012) indicated that the uncertainty factors typically applied in deriving a
toxicity value for the chemical of concern are the same as those applied to the analogue unless
additional information is available. To derive the screening subchronic p-RfD for BeP from the
BaP data, the UFd of 3 is increased to 10 to account for the absence of adequate repeated-dose
oral toxicity data for BeP.
Screening Subchronic p-RfD = Analogue POD ^ UFc
= 0.092 mg/kg-day ^ 1,000
= 9 x 10"5 mg/kg-day
Table A-5 summarizes the uncertainty factors for the screening subchronic p-RfD for
BeP.
68
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-5. Uncertainty Factors for the Screening Subchronic p-RfD for
BeP (CASRN 192-97-2)
UF
Value
Justification
UFa
10
A UFa of 10 is applied to account for uncertainty associated with extrapolating from animals to
humans when no cross-species dosimetric adjustment (HED calculation) is performed.
UFd
10
A UFd of 10 is applied to reflect database limitations for the BaP analogue and the absence of toxicity
data for the target chemical (BeP).
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of BeP in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because a developmental study was selected as the principal study. The
developmental period is recognized as a susceptible life stage when exposure during a time window of
development is more relevant to the induction of developmental effects than lifetime exposure (U.S.
EPA, 1991).
UFC
1,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; BMDL = benchmark dose lower confidence limit; HED = human
equivalent dose; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level;
POD = point of departure; p-RfD = provisional reference dose; UF = uncertainty factor; UFA = interspecies
uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies
uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
Derivation of a Screening Chronic Provisional Reference Dose
BaP is also selected as the analogue for BeP for deriving a screening chronic p-RfD. The
key study and calculation of the POD are described above for the subchronic p-RfD. In deriving
the screening chronic p-RfD for BeP, the same uncertainty factors used for the screening
subchronic p-RfD (UFa of 10, UFh of 10, and UFd of 10) are applied. An additional uncertainty
factor for study duration is not applied because a developmental study is used as the principal
study.
Screening Chronic p-RfD = Analogue POD UFc
= 0.092 mg/kg-day ^ 1,000
= 9 x 10"5 mg/kg-day
Table A-6 summarizes the uncertainty factors for the screening chronic p-RfD for BeP.
69
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-6. Uncertainty Factors for the Screening Chronic p-RfD for
BeP (CASRN 192-97-2)
UF
Value
Justification
UFa
10
A UFa of 10 is applied to account for uncertainty associated with extrapolating from animals to
humans when no cross-species dosimetric adjustment (HED calculation) is performed.
UFd
10
A UFd of 10 is applied to reflect database limitations for the BaP analogue and the absence of toxicity
data for BeP.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of BeP in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because a developmental study is selected as the principal study. The
developmental period is recognized as a susceptible life stage when exposure during a time window
of development is more relevant to the induction of developmental effects than lifetime exposure
(U.S. EPA, 1991). The database also contains chronic-duration exposure information, which did not
characterize a more sensitive endpoint.
UFC
1,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BeP = benzo[e]pyrene; BMDL = benchmark dose lower confidence limit; HED = human equivalent dose;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfD = provisional reference dose; UF = uncertainty factor; UFA = interspecies uncertainty factor;
UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor;
UFl = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
INHALATION NONCANCER TOXICITY VALUES
Derivation of a Screening Subchronic Provisional Reference Concentration
Based on the overall analogue approach presented in this PPRTV assessment, BaP is
selected as the analogue for BeP for deriving the screening subchronic and chronic p-RfCs. The
study used for the U.S. EPA screening subchronic and chronic p-RfC values for BeP is a prenatal
inhalation study of BaP in rats [Archibong et al. (2002) as cited in U.S. EPA (2017b. 2017c)].
The supplemental information for the toxicological review of BaP (U.S. EPA. 2017b) provided
the following summary:
Archibong et al. (2002) evaluated the effect of exposure to inhaled
benzo[a]pyrene on fetal survival and luteal maintenance in timed-pregnant F344
rats. Prior to exposure on GD 8, laparotomy was performed to determine the
number of implantation sites, and confirmed pregnant rats were divided into three
groups, consisting of rats that hadfour to six, seven to nine, or more than nine
conceptuses in utero. Rats in these groups were then assigned randomly to the
treatment groups or control groups to ensure a similar distribution of litter sizes.
Animals (10/group) were exposed to benzo[a]pyrene:carbon black aerosols at
concentrations of25, 75, or 100 ng/m3 via nose-only inhalation, 4 hours/day on
GDs 11-20. Control animals were either sham-exposed to carbon black or
remained entirely unexposed. Results ofparticle size analysis of generated
aerosols were reported by several other reports from this laboratory (Inyang et
al., 2003; Ramesh et al., 2001a; Hood et al., 2000). Aerosols showed a trimodal
distribution (average of cumulative mass, diameter) <95%, 15.85 urn; 89%,
70
Benzo[e]pyrene
-------
EPA/690/R-21/008F
<10 /urn; 55%, <2.5 /urn; and 38%, <1 /urn (Inyanget al., 2003). Ramesh et al.
(2001a) reported that theMMAD (± geometric SD) for the 55% mass fraction
with diameters <2.5 fim was 1.7 ± 0.085. Progesterone, estradiol-17fi, and
prolactin concentrations were determined in plasma collected on GDs 15 and 17.
Fetal survival was calculated as the total number of pups divided by the number
of all implantation sites determined on GD 8. Individual pup weights and
crown-rump length per litter per treatment were determined on PND 4
(PND 0 = day of parturition).
Archibong et al. (2002) reported that exposure of rats to benzo[a]pyrene
caused biologically and statistically significant (p <0.05) reductions in fetal
survival compared with the two control groups; fetal survival rates were 78.3,
38.0, and 33.8% per litter at 25, 75, and 100 /Jg/m3, respectively, and 96.7% with
carbon black or 98.8%per litter in untreated controls (see Table D-30).
Consequently, the number of pups per litter was also decreased in a
concentration-dependent manner. The decrease was -50% at 75 ng/m3 and -65%
at 100 /Jg/m3, compared with sham-exposed and unexposed control groups. No
effects on hormone levels were observed on GDs 15 or 17 at the low dose.
Biologically significant decreases in mean pup weights (expressed as gper litter)
of >5% relative to the untreated control group were observed at doses >75 ng/m3
(14 and 16% decreases at 75 and 100 ng/m3, respectively, p < 0.05). There were
no statistically significant differences from the control groups in crown-rump
length (see Table D-30).
Benzo[a]pyrene exposure at 75 ng/m3 caused a statistically significant
decrease in plasma progesterone, estradiol, and prolactin on GD 17; these levels
were not determined in the rats exposed to 100 ng/m3 (Archibong et al., 2002).
Plasma prolactin is an indirect measure of the activity of decidual luteotropin, a
prolactin-like hormone whose activity is necessary for luteal maintenance during
pregnancy in rats. Control levels of prolactin increasedfrom GD 15 to 17, but
this increase did not occur in the rats exposed to 75 ng/m3. Although the
progesterone concentration at 75 ng/m3 was significantly lower than in controls
on GD 17, the authors thought that the circulating levels should have been
sufficient to maintain pregnancy; thus, the increased loss of fetuses was thought
to be caused by the lower prolactin levels rather than progesterone deficiency.
The reduced circulating levels of progesterone and estradiol-17fi among
benzo[ajpyrene-treated rats were thought to be a result of limited decidual
luteotropic support for the corpora lutea. The authors proposed the following
mechanism for the effects of benzo [a]pyrene on fertility: benzo[a]pyrene or its
metabolites decreased prolactin and decidual luteotropin levels, compromising
the luteotropic support for the corpora lutea and thereby decreasing the plasma
levels of progesterone and estradiol-17p. The low estradiol-17fi may decrease
uterine levels ofprogesterone receptors, thereby resulting in fetal mortality.
Based on biologically and statistically significant decreases in pups/litter and
percent fetal survival/per litter, the LOAEL was 25 ng/m3; no NOAEL was
identified.
71
Benzo[e]pyrene
-------
EPA/690/R-21/008F
The lowest-observed-adverse-effect level (LOAEL) of 25 |ig/m3 for decreased
embryo/fetal survival was selected as the POD for BaP (U.S. EPA. 2017c). The POD was
converted into a LOAEL (HEC) of 4.6 |ig/m3 (0.0046 mg/m3) by the U.S. EPA (2017c). The
quotation from U.S. EPA (2017c) continues:
By definition, the RfC is intended to apply to continuous lifetime exposures
for humans (U.S. EPA, 1994a). EPA recommends that adjusted continuous
exposures be usedfor developmental toxicity studies by the inhalation route as
well as for inhalation studies of longer durations (U.S. EPA, 2002). 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 calculatedfrom 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 ofparticles 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 = (BWh/BWa) * ([Ve]a/[Ve]h) * ([Ftot]a/[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).
Frorwas based on the average particle size of 1.7 ± 0.085 urn (mass median
aerodynamic diameter [MMAD] ± geometric SD) as reported in Wu et al.
(2003a) for the exposure range 25-100 um3. For the model runs, the Yeh-Schum
5-lobe model was usedfor the human and the asymmetric multiple path model
was usedfor 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.
72
Benzo[e]pyrene
-------
EPA/690/R-21/008F
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 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 andfemale 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
The RfC for BaP is derived from the LOAELhec of 0.0046 mg/m3 using a UFc of 3,000,
reflecting a 10-fold UFi, UFh, and UFd and a 3-fold UF\ (U.S. EPA. 2017c). Wang et al. (2012)
indicated that the uncertainty factors typically applied in deriving a toxicity value for the
chemical of concern are the same as those applied to the analogue unless additional information
is available. Given the limitations of the current database, the uncertainty factors for BaP were
adopted for BeP unless otherwise noted below in Tables A-7 and A-8.
Screening Subchronic p-RfC = Analogue POD ^ UFc
= 0.0046 mg/m3 3,000
= 2 x 10"6 mg/m3
Table A-7 summarizes the uncertainty factors for the screening subchronic p-RfC
for BeP.
73
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-7. Uncertainty Factors for the Screening Subchronic p-RfC for
BeP (CASRN 192-97-2)
UF
Value
Justification
UFa
3
A UFa of 3 is applied to account for uncertainty associated with extrapolating from animals to
humans when a cross-species dosimetric adjustment (HEC calculation) is performed.
UFd
10
A UFd of 10 is applied to reflect the database limitations for the BaP analogue and absence of toxicity
data for BeP.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of BeP in humans.
UFl
10
A UFl of 10 is applied for LOAEL-to-NOAEL extrapolation because the POD is a LOAEL.
UFS
1
A UFS of 1 is applied because a developmental study is selected as the principal study. The
developmental period is recognized as a susceptible life stage when exposure during a time window
of development is more relevant to the induction of developmental effects than lifetime exposure
(U.S. EPA. 1991).
UFC
3,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; HEC = human equivalent concentration;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfC = provisional reference concentration; UF = uncertainty factor; UFA = interspecies uncertainty
factor; UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty
factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
Derivation of a Screening Chronic Provisional Reference Concentration
BaP is also selected as the analogue for BeP for deriving a screening chronic p-RfC. The
key study and calculation of the POD are described above for the subchronic p-RfC. In deriving
the screening chronic p-RfC for BeP, the same uncertainty factors used for the screening
subchronic p-RfC (UFa of 3, UFh of 10, UFd of 10, UFl of 10) are applied. An additional
uncertainty factor for study duration is not applied because a developmental study is used as the
principal study.
Screening Chronic p-RfC = Analogue POD ^ UFc
= 0.0046 mg/m3 3,000
= 2 x 10"6 mg/m3
Table A-8 summarizes the uncertainty factors for the screening chronic p-RfC for BeP.
74
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table A-8. Uncertainty Factors for the Screening Chronic p-RfC for
BeP (CASRN 192-97-2)
UF
Value
Justification
UFa
3
A UFa of 3 is applied to account for uncertainty associated with extrapolating from animals to
humans when a cross-species dosimetric adjustment (HEC calculation) is performed.
UFd
10
A UFd of 10 is applied to reflect the database limitations for the BaP analogue and absence of toxicity
data for BeP.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of BeP in humans.
UFl
10
A UFl of 10 is applied for LOAEL-to-NOAEL extrapolation because the POD is a LOAEL.
UFS
1
A UFS of 1 is applied because a developmental study is selected as the principal study. The
developmental period is recognized as a susceptible life stage when exposure during a time window
of development is more relevant to the induction of developmental effects than lifetime exposure
(U.S. EPA, 1991). The database also contains chronic-duration exposure information, which did not
characterize a more sensitive endpoint.
UFC
3,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; HEC = human equivalent concentration;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfC = provisional reference concentration; UF = uncertainty factor; UFA = interspecies uncertainty
factor; UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty
factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
75
Benzo[e]pyrene
-------
EPA 690 R-21 008F
APPENDIX B. BACKGROUND AND METHODOLOGY FOR THE SCREENING
EVALUATION OF POTENTIAL CARCINOGENICITY
Due to the lack of evidence described in the main Provisional Peer-Reviewed Toxicity
Value (PPRTV) document, there is inadequate information to assess the carcinogenic potential of
benzo[e]pyrene (BeP). However, some information is available for this chemical which, although
insufficient to support a weight-of-evidence (WOE) descriptor and derivation of provisional
cancer risk estimates under current guidelines, may be of use to risk assessors. In such cases, the
Center for Public Health and Environmental Assessment (CPHEA) summarizes available
information in an appendix and develops a "screening evaluation of potential carcinogenicity."
Appendices receive the same level of internal and external scientific peer review as the
provisional cancer assessments in PPRTVs to ensure their appropriateness within the limitations
detailed in the document. Users of the information regarding potential carcinogenicity in this
appendix should understand that there could be more uncertainty associated with this evaluation
than for the cancer WOE descriptors presented in the body of the assessment. Questions or
concerns about the appropriate use of the screening evaluation of potential carcinogenicity
should be directed to the CPHEA.
The screening evaluation of potential carcinogenicity includes the general steps shown in
Figure B-l. The methods for Steps 1 through 8 apply to any target chemical and are described in
this appendix. Chemical-specific data for all steps in this process are summarized in Appendix C.
STEP 1
Use automated tools
to identify an initial
list of structural
analogues with
genotoxicity and/or
carcinogenicity data
STEP 4
Summarize ADME
data from targeted
literature searches.
Identify metabolites
likely related to
genotoxic and/or
carcinogenic alerts
Apply expert
judgment to refine
the list of analogues
(based on
physicochemical
properties, ADME,
and mechanisms of
toxicity)
Compare
experimental
genotoxicity data (if
any) for the target
and analogue
compounds
STEP 5
Summarizecancer
data and MOA
information for
analogues.
STEP 6
Use computational
tools to identify
common structural
alerts and SAR
predictions for
genotoxicity
and/or
carcinogenicity
STEP 7
Integrate evidence
streams
STEP 8
Assign qualitative
level of concern for
ca rcinogenicity
based on evidence
integration
(potential concern
or inadequate
information)
Figure B-l. Steps Used in the Screening Evaluation of Potential Carcinogenicity
76
Benzo[e]pyrene
-------
EPA 690 R-21 008F
STEP 1. USE OF AUTOMATED TOOLS TO IDENTIFY STRUCTURAL ANALOGUES
WITH CARCINOGENICITY AND/OR GENOTOXICITY DATA
ChemACE Clustering
The U.S. EPA's Chemical Assessment Clustering Engine [ChemACE; U.S. EPA
(2011a)1 is an automated tool that groups (or clusters) a user-defined list of chemicals based on
chemical structure fragments. The methodology used to develop ChemACE was derived from
U.S. EPA's Analog Identification Methodology (AIM) tool, which identifies structural analogues
for a chemical based on common structural fragments. ChemACE uses the AIM structural
fragment recognition approach for analogue identification and applies advanced queries and
user-defined rules to create the chemical clusters. The ChemACE outputs are available in several
formats and layouts (i.e., Microsoft Excel, Adobe PDF) to allow rapid evaluation of structures,
properties, mechanisms, and other parameters, which are customizable based on an individual
user's needs. ChemACE grouping has been successfully used with chemical inventories for
identifying trends within a series of structurally similar chemicals, demonstrating structural
diversity in a chemical inventory, and detecting structural analogues to fill data gaps and/or
perform read-across.
For this project, ChemACE is used to identify potential structural analogues of the target
compound that have available carcinogenicity assessments and/or carcinogenicity data. An
overview of the ChemACE process is shown in Figure B-2.
Create and curate an
inventory of
chemicals with
carcinogenicity
assessments and/or
cancer data
Cluster the target
compound with the
chemical inventory
using ChemACE
Identify structural
analogues for the
target compound
from specific
ChemACE clusters
lists:
Figure B-2. Overview of ChemACE Clustering Process
The chemical inventory was populated with chemicals from the following databases and
Carcinogenic Potency Database [CPDB; CPDB (2011)]
Agents classified by the International Agency for Research on Cancer (IARC)
monographs (IARC. 2018)
National Toxicology Program (NIP) Report on Carcinogens [ROC; NTP (2016)]
NTP technical reports (NTP. 2017)
Integrated Risk Information System (IRIS) carcinogens (U.S. EPA. 2017a)
California prop 65 list (CalEPA. 2017)
European Chemicals Agency (ECHA) carcinogenicity data available in the Organisation
for Economic Co-operation and Development (OECD) Quantitative Structure-Activity
Relationship (QSAR) Toolbox (OECD. 2019)
• PPRTVs for Superfund (U.S. EPA. 2020b)
In total, 2,189 distinct substances were identified from the sources above. For the purpose
of ChemACE clustering, each individual substance needed to meet the following criteria:
77 Benzo[e]pyrene
-------
EPA/690/R-21/008F
1) Substance is not a polymer, metal, inorganic, or complex salt because ChemACE is not
designed to accommodate these substances;
2) Substance has a CASRN or unambiguous chemical identification; and
3) A unique Simplified Molecular Input Line Entry System (SMILES) notation (encoded
molecular structure format used in ChemACE) for the substance can be identified from
one of these sources:
a. Syracuse Research Corporation (SRC) and Distributed Structure-Searchable
Toxicity (DSSTox) lists of known SMILES associated with unique CASRNs (the
combined lists contained >200,000 SMILES) or
b. ChemlDplus, U.S. EPA Chemicals Dashboard, or internet searches.
Of the initial list of 2,189 substances, 254 were removed because they did not meet at
least one of the three criteria. The final inventory of substances contained 1,935 unique
compounds.
Two separate ChemACE approaches were compared for clustering of the chemical
inventory. The restrictive clustering approach, where all compounds in a cluster contain all of the
same fragments and no different fragments, resulted in 235 clusters. The less restrictive approach
included the following rules for remapping the chemical inventory:
• treat adjacent halogens as equivalent, allowing fluorine (F) to be substituted for chlorine
(CI), CI for bromine (Br), Br for iodine (I);
• allow methyl, methylene, and methane to be equivalent;
• allow primary, secondary, and tertiary amines to be equivalent; and
• exclude aromatic thiols (removes thiols from consideration).
Clustering using the less restrictive approach (Pass 2) resulted in 253 clusters. ChemACE
results for clustering of the target chemical within the clusters of the chemical inventory are
described in Appendix C.
Analogue Searches in the OECD QSAR Toolbox (Dice Method)
The OECD QSAR Toolbox (Version 4.1) is used to search for additional structural
analogues of the target compound. Several structural similarity score equations are available in
the toolbox (Dice, Tanimoto, Kulczynski-2, Ochiai/Cosine, and Yule). Dice is considered the
default equation. The specific options that are selected for the performance of this search include
a comparison of molecular features (atom-centered fragments) and atom characteristics (atom
type, count hydrogens attached, and hybridization). Chemicals identified in these similarity
searches are selected if their similarity scores exceeded 50%.
The OECD QSAR Toolbox Profiler is used to identify those structural analogues from
the Dice search that have carcinogenicity and/or genotoxicity data. Nine databases in the OECD
QSAR Toolbox (Version 4.1) provide data for carcinogenicity or genotoxicity (see Table B-l).
Analogue search results for the target chemical are described in Appendix C.
78
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table B-l. Databases Providing Carcinogenicity and Genotoxicity Data in
the OECD QSAR Toolbox (Version 4.1)
Database Name
Toolbox Database Description"
CPDB
The CPDB provides access to bioassay literature with qualitative and quantitative
analysis of published experiments from the general literature (through 2001) and from
the NCI/NTP (through 2004). Reported results include bioassays in rats, mice,
hamsters, dogs, and nonhuman primates. A calculated carcinogenic potency (TD5o) is
provided to standardize quantitative measures for comparison across chemicals. The
CPDB contains 1,531 chemicals and 3,501 data points.
ISSCAN
The ISSCAN database provides information on carcinogenicity bioassays in rats and
mice reported in sources including NTP, CPDB, CCRIS, and IARC. This database
reports a carcinogenicity TD5o. There are 1,149 chemicals and 4,518 data points
included in the ISSCAN database.
ECHA CHEM
The ECHA CHEM database provides information on chemicals manufactured or
imported in Europe from registration dossiers submitted by companies to ECHA to
comply with the REACH Regulation framework. The ECHA database includes
9,229 chemicals with almost 430,000 data points for a variety of endpoints including
carcinogenicity and genotoxicity. ECHA does not verily the information provided by
the submitters.
ECVAM Genotoxicity and
Carcinogenicity
The ECVAM Genotoxicity and Carcinogenicity database provides genotoxicity and
carcinogenicity data for Ames-positive chemicals in a harmonized format. ECVAM
contains in vitro and in vivo bacteria mutagenicity, carcinogenicity, CA,
CA/aneuploidy, DNA damage, DNA damage and repair, mammalian culture cell
mutagenicity, and rodent gene mutation data for 744 chemicals and 9,186 data points.
ISSCTA
ISSCTA provides results of four types of in vitro cell transformation assays including
SHE cells, mouse BALB/c 3T3, mouse C3H/10T1/2, and mouse Bhas 42 assays that
inform nongenotoxic carcinogenicity. ISSCTA consists of 352 chemicals and 760 data
points.
Bacterial mutagenicity
ISSSTY
The ISSSTY database provides data on in vitro Salmonella typhimurium Ames test
mutagenicity (positive and negative) taken from the CCRIS database in TOXNET. The
ISSSTY database provides data for 7,367 chemicals and 41,634 data points.
Genotoxicity OASIS
The Genotoxicity OASIS database provides experimental results for mutagenicity
results from "Ames tests (with and without metabolic activation), in vitro chromosomal
aberrations and MN and ML A evaluated in vivo and in vitro, respectively." The
Genotoxicity OASIS database consists of 7,920 chemicals with 29,940 data points
from 7 sources.
Micronucleus OASIS
The Micronucleus OASIS database provides experimental results for in vivo bone
marrow and peripheral blood MNT CA studies in blood erythrocytes, bone marrow
cells, and polychromatic erythrocytes of humans, mice, rabbits, and rats for
557 chemicals.
79
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table B-l. Databases Providing Carcinogenicity and Genotoxicity Data in
the OECD QSAR Toolbox (Version 4.1)
Database Name
Toolbox Database Description"
ISSMIC
The ISSMIC database provides data on the results of in vivo MN mutagenicity assay to
detect CAs in bone marrow cells, peripheral blood cells, and splenocytes in mice and
rats. Sources include TOXNET, NTP, and the Leadscope FDA CRADA toxicity
database. The ISSMIC database includes data for 563 chemicals and 1,022 data points.
descriptions were obtained from the OECD QSAR Toolbox documentation (Version 4.1).
CA = chromosomal aberration; CCRIS = Chemical Carcinogenesis Research Information System;
CPDB = Carcinogenic Potency Database; CRADA = cooperative research and development agreement;
DNA = deoxyribonucleic acid; ECHA = European Chemicals Agency; ECVAM = European Centre for the
Validation of Alternative Methods; FDA = Food and Drug Administration; IARC = International Agency for
Research on Cancer; ISSCAN = Istituto Superiore di Sanita Chemical Carcinogen; ISSCTA = Istituto Superiore di
Sanita Cell Transformation Assay; ISSMIC = Istituto Superiore di Sanita Micronucleus; ISSSTY = Istituto
Superiore di Sanita Salmonella typhimurium; ML A = mouse lymphoma gene mutation assay; MN = micro nuclei;
MNT = micronucleus test; NCI = National Cancer Institute; NTP = National Toxicology Program;
OECD = Organization for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship; REACH = Registration, Evaluation, Authorization and Restriction of Chemicals; SHE = Syrian
hamster embryo; TD5o = median toxic dose.
STEPS 2-5. ANALOGUE REFINEMENT AND SUMMARY OF EXPERIMENTAL
DATA FOR GENOTOXICITY, TOXICOKINETICS, CARCINOGENICITY, AND
MODE OF ACTION
The outcome of the Step 1 analogue identification process using ChemACE and the
OECD QSAR Toolbox is an initial list of structural analogues with genotoxicity and/or
carcinogenicity data. Expert judgment is applied in Step 2 to refine the list of analogues based on
physicochemical properties; absorption, distribution, metabolism, and excretion (ADME); and
mechanisms of toxicity. The analogue refinement process is chemical specific and is described in
Appendix C. Steps 3, 4, and 5 (summary of experimental data for genotoxicity, toxicokinetics,
carcinogenicity, and mode of action [MOA]) are also chemical specific (see Appendix C for
further details).
STEP 6. STRUCTURAL ALERTS AND STRUCTURE-ACTIVITY RELATIONSHIP
PREDICTIONS FOR BEP AND ANALOGUES
Structural alerts and predictions for genotoxicity and carcinogenicity are identified using
six freely available structure-based tools (described in Table B-2).
80
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table B-2. Tools Used to Identify SAs and Predict Carcinogenicity and
Genotoxicity
Name
Description"
OECD QSAR
Toolbox
(Version 4.1)
Seven OECD QSAR Toolbox profiling methods were used, including:
• Carcinogenicity (genotox and nongenotox) alerts by ISS (Version 2.3); updated version of the
module originally implemented in Toxtree. Toxtree is a decision tree for estimating
carcinogenicity based on 55 SAs (35 from the Toxtree module and 20 newly derived).
• DNA alerts for Ames by OASIS (Version 1.4); based on the Ames mutagenicity TIMES
model; uses 85 SAs responsible for interaction of chemicals with DNA.
• DNA alerts for CA and MNT by OASIS (Version 1.1); based on the DNA reactivity of the CA
TIMES model; uses 85 SAs for interaction of chemicals with DNA.
• In vitro mutagenicity (Ames test) alerts by ISS (Version 2.3); based on the Mutagenicity
module in Toxtree. ISS is a decision tree for estimating in vitro (Ames test) mutagenicity,
based on a list of 43 SAs relevant for the investigation of chemical genotoxicity via DNA
adduct formation.
• In vivo mutagenicity (MN) alerts by ISS (Version 2.3); based on the ToxMic rulebase in
Toxtree. The rulebase has 35 SAs for in vivo MN assays in rodents.
• OncoLogic Primary Classification (Version 4.0); "developed by LMC and OECD to mimic
the structural criteria of chemical classes of potential carcinogens covered by the U.S. EPA's
OncoLogic Cancer Expert System for Predicting the Carcinogenicity Potential" for
categorization purposes only, not for predicting carcinogenicity. This tool is applicable to
organic chemicals with at least one of the 48 alerts specified.
• Protein-binding alerts for CAs by OASIS (Version 1.3); based on 33 SAs for interactions with
specific proteins including topoisomerases, cellular protein adducts, etc.
OncoLogic
(Version 7)
OncoLogic is a tool for predicting the potential carcinogenicity of chemicals based on the
application of rules for SAR analysis, developed by experts. Results may range from "low" to
"high" concern level.
ToxAlerts
ToxAlerts is a platform for screening chemical compounds against SAs, developed as an
extension to the OCHEM svstem (httos://ochem.eu). Onlv "approved alerts" were selected, which
means a moderator approved the submitted data. A list of the ToxAlerts found for the chemicals
screened in the preliminary batch is below:
• Genotoxic carcinogenicity, mutagenicity:
o Aliphatic halide (general)
o Aliphatic halide (specific)
o Aliphatic halogens
o Aromatic amine (general)
o Aromatic amine (specific)
o Aromatic amines
o Aromatic and aliphatic substituted primary alkyl halides
o Aromatic nitro (general)
o Aromatic nitro (specific)
o Aromatic nitro groups
o Nitroarenes
o Nitro-aromatic
o Primary and secondary aromatic amines
o Primary aromatic amine, hydroxyl amine, and its derived esters or amine generating
group
• Nongenotoxic carcinogenicity
o Aliphatic halogens
81
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table B-2. Tools Used to Identify SAs and Predict Carcinogenicity and
Genotoxicity
Name
Description"
ToxRead
(Version 0.9)
ToxRead is a tool designed to assist in making read-across evaluations reproducible. SAs for
mutagenicity are extracted from similar molecules with available experimental data in its
database. Five similar compounds were selected for this project. The rule sets included:
• Benigni/Bossa as available in Toxtree (Version 1)
• SARpy rules extracted by Politecnico di Milano, with the automatic tool SARpy
• IRFMN rules extracted by human experts
• CRS4 rules extracted by CRS4 Institute with automatic tools
Toxtree
(Version 2.6.13)
Toxtree estimates toxic hazard by applying a decision tree approach. Chemicals were queried in
Toxtree using the Benigni/Bossa rulebase for mutagenicity and carcinogenicity. If a potential
carcinogenic alert based on any QSAR model or if any SA for genotoxic and nongenotoxic
carcinogenicity was reported, then the prediction was recorded as a positive carcinogenicity
prediction for the test chemical. The output definitions from the tool manual are listed below:
• SA for genotoxic carcinogenicity (recognizes the presence of one or more SAs and specifies a
genotoxic mechanism)
• SA for nongenotoxic carcinogenicity (recognizes the presence of one or more SAs and
specifies a nongenotoxic mechanism)
• Potential Salmonella typhimurium TA100 mutagen based on QSAR
• Unlikely to be a S. typhimurium TA100 mutagen based on QSAR
• Potential carcinogen based on QSAR (assigned according to the output of QSAR8 aromatic
amines)
• Unlikely to be a carcinogen based on QSAR (assigned according to the output of QSAR8
aromatic amines)
• Negative for genotoxic carcinogenicity (no alert for genotoxic carcinogenicity)
• Negative for nongenotoxic carcinogenicity (no alert for nongenotoxic carcinogenicity)
82
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table B-2. Tools Used to Identify SAs and Predict Carcinogenicity and
Genotoxicity
Name
Description"
VEGA
VEGA applies several QSARs to a given chemical, as described below:
• Mutagenicity (Ames test) CONSENSUS model: a consensus assessment is performed based
on predictions of the VEGA mutagenicity models (CAESAR, SARpy, ISS, and &-NN)
• Mutagenicity (Ames test) model (CAESAR): integrates two models, one is attained SVM
classifier, and the other is for FN removal based on SAs matching
• Mutagenicity (Ames test) model (SARpy/IRFMN): rule-based approach with 112 rules for
mutagenicity and 93 for nonmutagenicity, extracted with SARpy software from the original
training set from the CAESAR model; includes rules for both mutagenicity and
nonmutagenicity
• Mutagenicity (Ames test) model (ISS): rule-based approach based on the work of Benigni and
Bossa (ISS) as implemented in the software Toxtree (Version 2.6)
• Mutagenicity (Ames test) model (&-NN/read-across): performs a read-across and provides a
qualitative prediction of mutagenicity on S. typhimurium (Ames test)
• Carcinogenicity model (CAESAR): counter-propagation artificial neural network developed
using data for carcinogenicity in rats extracted from the CPDB
• Carcinogenicity model (ISS): built implementing the same alerts Benigni and Bossa (ISS)
implemented in the software Toxtree (Version 2.6)
• Carcinogenicity model (IRFMN/ANT ARES): a set of rules (127 SAs), extracted with the
SARpy software from a data set of 1,543 chemicals obtained from the carcinogenicity
database of EU-funded project ANT ARES
• Carcinogenicity model (IRFMN/ISSCAN-CGX): based on a set of rules (43 SAs) extracted
with the SARpy software from a data set of 986 compounds; the data set of carcinogenicity of
different species was provided bv Kirkland et al. (2005)
aThere is some overlap between the tools. For example, OncoLogic classification is provided by the QSAR
Toolbox but the prediction is available only through OncoLogic, and alerts or decision trees were used or adapted
in several models (e.g., Benigni and Bossa alerts and Toxtree decision tree).
ANT ARES = Alternative Non-Testing Methods Assessed for REACH Substances; CA = chromosomal aberration;
CAESAR = Computer-Assisted Evaluation of industrial chemical Substances According to Regulations;
CONSENSUS = consensus assessment based on multiple models (CAESAR, SARpy, ISS, and &-NN);
CPDB = Carcinogenic Potency Database; CRS4 = Center for Advanced Studies, Research and Development in
Sardinia; DNA = deoxyribonucleic acid; EU = European Union; FN = false negative; IRFMN = Istituto di Ricerche
Farmacologiche Mario Negri; ISS = Istituto Superiore di Sanita; ISSCAN-CGX = Istituto Superiore di Sanita
Chemical Carcinogen; &-NN = ^-nearest neighbor; LMC = Laboratory for Mathematical Chemistry;
MN = micronucleus; MNT = micronucleus test; OCHEM = Online Chemical Monitoring Environment;
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship; REACH = Registration, Evaluation, Authorisation and Restriction of Chemicals; SA = structural alert;
SAR = structure-activity relationship; SVM = support vector machine; TIMES = The Integrated MARKEL-EFOM
System; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture.
The tool results for the target and analogue compounds are provided in Appendix C.
83
Benzo[e]pyrene
-------
EPA/690/R-21/008F
STEP 7. EVIDENCE INTEGRATION FOR SCREENING EVALUATION OF
CARCINOGENICITY
Available data across multiple lines of evidence from Steps 1-6 (outlined above) are
integrated to determine the qualitative level of concern for the potential carcinogenicity of the
target compound (Step 8). In the absence of information supporting carcinogenic portal-of-entry
effects, the qualitative level of concern for the target chemical should be considered applicable to
all routes of exposure.
Evidence integration for the target compound is provided in Appendix C.
84
Benzo[e]pyrene
-------
EPA/690/R-21/008F
APPENDIX C. RESULTS OF THE SCREENING EVALUATION OF POTENTIAL
CARCINOGENICITY
STEP 1. USE OF AUTOMATED TOOLS TO IDENTIFY STRUCTURAL ANALOGUES
WITH CARCINOGENICITY AND/OR GENOTOXICITY DATA
U.S. EPA's Chemical Assessment Clustering Engine (ChemACE) clustering was
performed as described in Appendix B. Using the most restrictive clustering rules (where
ChemACE assigns a unique definition for each fragment, ensuring that each chemical submitted
for clustering appears in only one ChemACE cluster), benzo[e]pyrene (BeP) appeared in
Cluster 51, which contained seven additional compounds (benzo[g]chrysene, perylene, picene,
benzo[b]chrysene, dibenz[a,c]anthracene, dibenzo[a,j]anthracene, and benzo[a]pyrene [BaP]).
Using the less restrictive clustering option did not change the result (BeP found in a single
cluster with seven other compounds).
The Organisation for Economic Co-operation and Development (OECD) Quantitative
Structure-Activity Relationship (QSAR) Toolbox Profiler was used to identify structural
analogues from the Dice analogue search with carcinogenicity and/or genotoxicity data (see
Step 1 methods in Appendix B). The Dice analogue search identified >1,500 chemicals with
>50% similarity to BeP; therefore, the similarity threshold was raised to 80%. The Dice analogue
search identified 107 compounds with carcinogenicity and/or genotoxicity data and >80%
similarity to BeP, including five of the seven compounds identified by ChemACE. Refinement
of the selection of final analogues is described below.
STEP 2. ANALOGUE REFINEMENT USING EXPERT JUDGMENT
Expert judgment was applied to refine the initial list of 109 potential analogues based on
physicochemical properties; absorption, distribution, metabolism, and excretion (ADME); and
mechanisms of toxicity.
BeP contains five unsubstituted benzene rings. Therefore, compounds were only
considered as potential analogues if they were unsubstituted and contained five benzene rings. Of
the 109 chemicals identified as potential analogues by ChemACE clustering and the OECD
QSAR Toolbox analogue selection tool (Dice), 101 were not selected for further review,
including:
• Polycyclic aromatic hydrocarbon (PAH) compounds with benzene ring number ^ 5
• Compounds containing nonaromatic ring structures (e.g., benzo[b]fluoranthene)
• Substituted PAHs (e.g., alkyl, amino/nitro/nitroso or halogenated substituents)
• Hydroxyl PAH metabolites
Each of these attributes introduce significant differences in bioavailability, reactivity, and
applicable metabolic pathways relative to BeP.
The remaining eight possible analogues for BeP are listed in Table C-l. The existence of
a cancer toxicity value and/or a weight-of-evidence (WOE) determination for cancer is indicated
for each analogue.
85
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-l. Summary of Cancer Assessment Information for Analogues of
BeP (CASRN 192-97-2)
Analogue Name
(CASRN)3
Cancer Risk Estimates
(if available)
WOE Determinations
BaPab (50-32-8)
U.S. EPA (2017c)—OSF. IUR
CalEPA (2011)—OSF, IUR
U.S. EPA (2017c)—"Carcinogenic to Humans"
I ARC (2010)—carcinogenic to humans (Group 1)
BbCab (214-17-5)
None
I ARC (2010)—not classifiable as to their
carcinogenicity to humans (Group 3)
BgCa (196-78-1)
None
I ARC (2010)—not classifiable as to their
carcinogenicity to humans (Group 3)
DBacA3 (215-58-7)
None
I ARC (2010)—not classifiable as to their
carcinogenicity to humans (Group 3)
DBahAb (53-70-3)
CalEPA (1992). CalEPA
(2019)—OSF, IUR
U.S. EPA (1990c)—probable human carcinogen
(Group B2)
I ARC (2010)—probably carcinogenic to humans
(Group 2A)
DBajAa b (224-41-9)
None
I ARC (2010)—not classifiable as to their
carcinogenicity to humans (Group 3)
Peryleneab (198-55-0)
None
U.S. EPA (2007)—"Inadequate Information to Assess
Carcinogenic Potential"
I ARC (2010)—not classifiable as to their
carcinogenicity to humans (Group 3)
Piceneab (213-46-7)
None
I ARC (2010)—not classifiable as to their
carcinogenicity to humans (Group 3)
aFound by ChemACE.
bFound by Dice.
BaP = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene;
DBacA = dibenz[a,c]anthracene; DBahA = dibenz[a,h]anthracene; DBajA = dibenzo[a,j]anthracene;
IUR = inhalation unit risk; OSF = oral slope factor; WOE = weight of evidence.
STEP 3. COMPARISON OF I II I EXPERIMENTAL GENOTOXICITY DATA FOR
BEP AND ANALOGUES
The genotoxicity data available for BeP are described in the "Other Data" section in the
main body of this report. Available data indicate that BeP is weakly mutagenic following
metabolic activation but not mutagenic without activation. Most studies indicate that BeP is not
clastogenic and does not cause deoxyribonucleic acid (DNA) damage; however, BeP (or a
metabolite) is capable of forming DNA adducts, and there is inconsistent evidence from in vivo
studies indicating that BeP is capable of causing chromosomal damage under certain conditions.
Genotoxicity data for the analogue compounds have been extensively reviewed. A summary of
the genotoxicity data for analogue PAHs is provided in Table C-2. Overall data indicate that
these analogues are mutagenic and clastogenic, and capable of binding DNA and causing DNA
damage. Numerous studies have reported a weak genotoxic potential of BeP in vitro compared
with other PAHs, including analogues BaP, dibenz[a,c]anthracene (DBacA),
dibenz[a,h]anthracene (DBahA), dibenz[a,j]anthracene (DBajA), and, in some assays, perylene
(Durant et at.. 1996; Mersch-Sundermann et at.. 1993; Mersch-Sundermann et at.. 1992; De
86 Benzo[e]pyrene
-------
EPA/690/R-21/008F
Flora et al.. 1984; Haas et al.. 1981; Kaden et al.. 1979; Simmon. 1979; Andrews et al.. 1978;
McCann et al.. 1975).
87
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-2. Comparison of Available Genotoxicity Data for BeP (CASRN 192-97-2) and Analogues
Chemical
(CASRN)
Mutagenicity
Clastogenicity
DNA Damage and Adducts
Cell Transformation
Reference
BeP
• Weakly mutagenic in
Salmonella typhimurium with metabolic
activation; not mutagenic without
activation
• Not mutagenic in Escherichia coli
• Mutagenic in mammalian cells with
metabolic activation only at cytotoxic
concentrations
• Predominantly
negative in
mammalian cells in
vitro
• Mixed results in vivo
(induced CAs in rats
but not hamsters,
induced SCEs in
hamsters, negative for
MN in mice)
• Predominantly negative for DNA
damage/repair assays in vitro and
in vivo
• DNA adducts measured in
mammalian cells and isolated
human DNA
• Weak induction of
cell transformation in
mammalian cells in
vitro
See Table 4A
BaP
(50-32-8)
• Mutagenic in S. typhimurium and E. coli
• Mutagenic in mammalian cells in vitro
• Induced somatic and sex-linked
recessive mutations in Drosophila
melanogaster
• Mutagenic in dominant lethal and
transgenic mouse studies
• K-ras and p53 mutations in human
tumor tissues
• Induced SCEs, CAs,
and MNs in
mammalian cells in
vitro and in vivo
• DNA damage/repair in E. coli
• Induced DNA damage/repair in
mammalian cells in vitro and in
vivo
• DNA adducts measured in
mammalian cells
• Induced neoplastic
transformation in
mammalian cells
De Flora et al.
(1984); U.S.
EPA (2017c);
U.S. EPA
(2017b)
BbC
(214-17-5)
ND
ND
ND
ND
NA
BgC
(196-78-1)
• BgC ll,12-diol-13,14-oxides mutagenic
in S. typhimurium and E. coli
• BgC ll,12-diol-13,14-oxides mutagenic
in mammalian cells in vitro
ND
• DNA adducts of BgC 11,12-diols
and BgC ll,12-diol-13,14-oxides
in mouse skin
• CYP-activated BgC 11,12-diols
cause DNA damage in
cDNA-based recombinant (coli
or Trichoplusia ni) systems
ND
I ARC (2010)
88
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-2. Comparison of Available Genotoxicity Data for BeP (CASRN 192-97-2) and Analogues
Chemical
(CASRN)
Mutagenicity
Clastogenicity
DNA Damage and Adducts
Cell Transformation
Reference
DBacA
(215-58-7)
• Mutagenic in S. typhimurium
• Mutagenic in mammalian cells in vitro
• DBacA 10,11-dihydrodiol mutagenic in
bacteria
• Negative for SCE in
mammalian cells in
vitro (single study)
• DNA damage in Bacillus subtilis
• DNA damage in mammalian cells
in vitro
• DNA adducts in mammalian cells
in vitro
• Cell transformation
in mammalian cells
in vitro
I ARC (1983)
DBahA
(53-70-3)
• DBahA, DBahA, 3,4-diol, and DBahA
3,4:10,11-bisdiol mutagenic in
S. typhimurium
• DBahA mutagenic in mammalian cells
in vitro
• Induced MN in
mammalian cells in
vivo and SCEs and
MN in mammalian
cells in vitro
• DNA adducts of DBahA
3,4-diol-l,2-oxide and DBahA
3,4:10,11-bisdiol in vitro and/or in
vivo
• DBahA 3,4-diol and
DBahA
3,4-diol-l,2-oxide
induced
morphological cell
transformation in
mammalian cells
I ARC (2010)
DBajA
(224-41-9)
• Mutagenic in S. typhimurium
• Mutagenic in human lymphoblastoid
cells
• DBajA 3,4-diol-l,2-oxide mutagenic in
E. coli
ND
• DNA adducts of DBajA diols,
bisdiols, and oxides in vivo
ND
I ARC (2010)
Perylene
(198-55-0)
• Mutagenic in S. typhimurium
• Not mutagenic in human
lymphoblastoid cells
• CAs in mammalian
cells in vitro
• Negative for DNA damage/repair
in E. coli
ND
De Flora et al.
(1984): I ARC
(1983)
Picene
(213-46-7)
• Not mutagenic in S. typhimurium
• Not mutagenic in human
lymphoblastoid cells
ND
ND
ND
Durant et al.
(1996): Kaden
et al. (1979)
BaP = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene; CA = chromosomal aberration; DBacA = dibenz[a,c]anthracene;
DBahA = dibenz[a,h]anthracene; DBaj A = dibenz[a,j]anthracene; DNA = deoxyribonucleic acid; MN = micronuclei; ND = no data; SCE = sister chromatid exchange.
89
Benzo[e]pyrene
-------
EPA/690/R-21/008F
STEP 4. TOXICOKINETICS OF BEP AND ANALOGUES
The toxicokinetics of BeP and potential analogues are briefly described in Table C-3.
No data on absorption or distribution are available for BeP or any analogue with the
exception of BaP. BaP absorption and distribution are extensively reviewed by U.S. EPA
(2017b). IARC (2010). and ATSDR (1995). BaP is absorbed via inhalation, oral, and dermal
routes. The rate of inhalation absorption is dependent on numerous factors, including particle
size, region of deposition, and rate of dissolution or desorption from particles, with rapid
absorption occurring through thin alveolar epithelium and slower absorption in thicker regions of
the airways. Inhaled BaP is also transported to the gut via the mucociliary escalator.
Gastrointestinal absorption of BaP ranges from 10 to 60%, with enhanced absorption in the
presence of lipophilic compounds. Dermal absorption is rapid and near complete when
administered in crude oil or acetone, with decreased absorption if administered in high-viscosity
oil vehicles or in soils with high organic carbon content. Following absorption, BaP shows rapid,
widespread systemic distribution followed by accumulation and retention in fat. Because
absorption and distribution of a chemical in the body are determined largely by physical and
chemical properties related to its molecular size and general structure (e.g., lipophilicity, vapor
pressure, etc.), it is reasonable to assume that BeP and other PAH analogues will be absorbed
and distributed similarly to BaP based on similar size, structure, and physicochemical properties.
In general, PAHs (including BeP and analogues) are oxidized by cytochrome P450
(CYP450) in multiple tissues in the body to more soluble metabolites, including dihydrodiols,
phenols, quinones, and epoxides, which then form conjugates with glucuronide, glutathione
(GSH), or sulfate (U.S. EPA. 2017b; I ARC. 2010; ATSDR, 1995). While PAHs share similar
metabolic pathways, there are differences in the primary metabolic products that can affect
carcinogenic potential (see "Metabolism/Toxicokinetic Studies" section and Table C-3 for more
details). For example, in contrast to BaP (the most well-studied carcinogenic PAH), there is
minimal evidence for bay-region activation of BeP in rodent model systems and generation of
the 9,10-dihydrodiol metabolite, and subsequent formation of the DNA reactive epoxide is
described as limited within this model system (Jacob et at.. 1985; Jacob et at.. 1983; Wood et at..
1979). Data support the notion that production of the 9,10-dihydrodiol metabolite is increased in
human model systems, but the extent and consequence of production of the DNA reactive
epoxide metabolite in humans remains uncertain.
No excretion data are available for BeP or any analogue, with the exception of BaP (see
Table C-3). BaP excretion has been extensively reviewed by U.S. EPA (2017b). IARC (2010).
and ATSDR (1995). Excretion of BaP, primarily as conjugated metabolites, is rapid and occurs
primarily via biliary excretion to feces, with small amounts excreted in urine. Because PAHs
share common metabolic profiles, it is reasonable to assume that conjugated metabolites of BeP
and other PAH analogues will be excreted similarly to BaP.
90
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-3. Summary of Toxicokinetic Data for BeP (CASRN 192-97-2) and Analogues
Compound
(CASRN)
Absorption, Distribution, Excretion
Metabolism
References
BeP
(192-97-2)
ND
• Metabolism is rapid (complete within 48 h)
• Oxidized via CYP450 to the k-region
4,5-dihydrodiol, phenols and quinones
• Oxidative metabolism can be induced by CYP450
inducers
• Bay-region 9,10-dihydrodiol is formed in small
amounts and then preferentially oxidized at the
4,5-position, rather than forming the 9,10-expoxide
• Oxidative metabolites conjugated with glucuronic
acid
See "Other Data"
section of this
document.
BaP
(50-32-8)
Absorption:
• Absorbed by oral, inhalation, and dermal exposure
• Rate and extent of absorption is variable, depending on exposure
medium (e.g., oral and dermal absorption enhanced in presence of oils
and fats; dermal absorption decreased in presence of soils with high
organic carbon content)
• Significant mucociliary clearance of inhaled particulate to gut
• Absorption from gut depends on presence of bile in intestinal lumen
Distribution:
• Widely distributed throughout the body
• Initial rapid uptake into well-perfused tissues (e.g., lung, kidney,
liver)
• Subsequent accumulation, retention, and slow release from fat
• High levels in gut (from any route) due to mucociliary clearance from
respiratory tract and hepatobiliary excretion of metabolites
• Limited placental transfer
Excretion:
• Excretion is rapid, with half-times of 22-30 h
• Primary route is biliary excretion to feces; urine is secondary route
• Excreted mainly as conjugated metabolites
• Small amounts excreted in milk
• Metabolism is rapid and occurs in many tissues
throughout the body
• Oxidized via CYP450; primary metabolites are
9,10-, 7,8-, 4,5-, and 2,3-dihydrodiols and
epoxides, as well as various phenols, quinones, and
derivatives.
• Oxidative metabolism can be induced by CYP450
inducers
• Oxidative metabolites conjugated with GSH,
glucuronic acid, and sulfate esters
ATSDR (1995);
I ARC (2010);
U.S. EPA (2017b);
U.S. EPA (2017c)
91
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-3. Summary of Toxicokinetic Data for BeP (CASRN 192-97-2) and Analogues
Compound
(CASRN)
Absorption, Distribution, Excretion
Metabolism
References
BbC
(214-17-5)
ND
ND
NA
BgC
(196-78-1)
ND
• Fjord region oxidation to form 11,12-diols
• Further metabolism via intermediary
ll,12-diol-13,14-oxides to form diastereomeric
tetrols
I ARC (2010)
DBacA
(215-58-7)
ND
• Oxidation to 10,11-dihydrodiol (major metabolite)
• Further oxidation to 10,ll-diol-12,13-epoxide
• Minor metabolites include 1,2- and 3,4-diols
I ARC (1983)
DBahA
(53-70-3)
ND
• Metabolic activation via the diol epoxide
mechanism to diols, oxides, catechols, phenols,
and hexols
I ARC (2010)
DBajA
(224-41-9)
ND
• Metabolic activation via the diol epoxide
mechanism to diols, bisdiols, and oxides
• Glucuronidation of diols
I ARC (2010)
Perylene
(198-55-0)
ND
ND
NA
Picene
(213-46-7)
ND
• Primary metabolites formed via oxidation include
dihydrodiols, phenols, and quinones
Piatt et al. (1988)
BaP = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene; CYP450 = cytochrome P450; DBacA = dibenz[a,c]anthracene;
DBahA = dibenz[a,h]anthracene; DBajA = dibenz[a,j]anthracene; GSH = glutathione; NA = not applicable; ND = no data.
92
Benzo[e]pyrene
-------
EPA/690/R-21/008F
STEP 5. CARCINOGENICITY OF BEP ANALOGUES AND MOA DISCUSSION
U.S. EPA cancer WOE descriptors for BeP and analogue compounds are shown in Tables C-4
(oral) and C-5 (inhalation). As noted in the PPRTV document, there is inadequate information to
assess the carcinogenic potential of BeP by oral or inhalation exposure. Only two of the potential
analogue compounds are characterized as having evidence of carcinogenic potential.
Additionally, several potential analogues have been identified as unmeasured components of
complex chemical mixtures that are known to be carcinogenic in humans. BaP was characterized
as "Carcinogenic to Humans" (U.S. EPA. 2017c) under the 2005 Guidelines for Carcinogen
Risk Assessment (U.S. EPA. 2005) and DBahA was considered a probable human carcinogen
(U.S. EPA. 1993) under the 1986 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 1986).
Reported oral slope factor (OSF) and inhalation unit risk (IUR) values were two- to threefold
higher for DBahA compared with BaP.
93
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-4. Comparison of Available Oral Carcinogenicity Toxicity Data for BeP (CASRN 192-97-2) and Analogues
Type of Data
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
Structure
cfi?
CO
ecu
9Vr9
/ V //
o
CASRN
192-97-2
50-32-8
214-17-5
196-78-1
215-58-7
53-70-3
224-41-9
198-55-0
213-46-7
U.S. EPA WOE
characterization
"Inadequate
Information to
Assess
Carcinogenic
Potential"
"Carcinogenic
to Humans "
ND
ND
ND
Probable human
carcinogen
(Group B2)
ND
"Inadequate
Information to
Assess
Carcinogenic
Potential"
ND
OSF (mg/kg-d) 1
NDr
1.4
NDr
NDr
NDr
4.12
NDr
NDr
NDr
Data set(s) used
for slope factor
derivation
ND
Alimentary tract
tumors
(multiple sites)
in female
B6C3F1 mice
ND
ND
ND
Pulmonary (alveolar
carcinomas)
ND
ND
ND
Other tumors
observed in animal
oral bioassays
ND
Auditory canal,
kidney, liver,
skin, mammary
gland
ND
ND
ND
Mammary,
forestomach,
hemangio-
endothelioma
ND
ND
ND
Study doses
(mg/kg-d)
ND
0,0.7,3.3, 16.5
ND
ND
ND
NS
ND
ND
ND
Route (method)
ND
Oral (diet)
ND
ND
ND
NS
ND
ND
ND
Duration
ND
2 yr
ND
ND
ND
NS
ND
ND
ND
94
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-4. Comparison of Available Oral Carcinogenicity Toxicity Data for BeP (CASRN 192-97-2) and Analogues
Type of Data
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
POD type
ND
BMDLio
0.071 mg/kg-d
ND
ND
ND
NS
ND
ND
ND
Source
NA
U.S. EPA
(2017b): U.S.
EPA (2017c)
NA
NA
NA
U.S. EPA (1990c);
CalEPA (2019);
CalEPA (1992)
NA
NA
NA
BaP = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene; BMDL = benchmark dose lower confidence limit;
DBacA = dibenz[a,c]anthracene; DBahA = dibenz[a,h]anthracene; DBajA = dibenz[a,j]anthracene; NA = not applicable; ND = no data; NDr = not determined; NS = not
specified; OSF = oral slope factor; POD = point of departure; WOE = weight of evidence.
95
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-5. Comparison of Available Inhalation Carcinogenicity Toxicity Data for BeP (CASRN 192-97-2) and
Analogues
Type of Data
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
Structure
co5°
^ <•'' \ }
... ,0
orCX/
CASRN
192-97-2
50-32-8
214-17-5
196-78-1
215-58-7
53-70-3
224-41-9
198-55-0
213-46-7
U.S. EPA WOE
characterization
"Inadequate
Information to
Assess
Carcinogenic
Potential"
"Carcinogenic to
Humans "
ND
ND
ND
Probable
human
carcinogen
(Group B2)
ND
"Inadequate
Information to
Assess
Carcinogenic
Potential"
ND
IUR (|ig/m3) 1
NDr
0.0006
NDr
NDr
NDr
0.00122
NDr
NDr
NDr
Data set(s) used for
unit risk derivation
ND
Squamous cell
neoplasia in upper
respiratory tract
(multiple sites),
esophagus, and
forestomach of male
Syrian golden hamster
ND
ND
ND
NS
ND
ND
ND
Other tumors
observed in animal
inhalation bioassays
ND
ND
ND
ND
ND
ND
ND
ND
ND
Study
concentrations
(mg/m3)
ND
0, 2.2, 9.5, 46.5
ND
ND
ND
NS
ND
ND
ND
Route (method)
ND
Inhalation (nose only)
ND
ND
ND
NS
ND
ND
ND
Duration
ND
Up to 130 wk
ND
ND
ND
NS
ND
ND
ND
96
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-5. Comparison of Available Inhalation Carcinogenicity Toxicity Data for BeP (CASRN 192-97-2) and
Analogues
Type of Data
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
POD type
ND
BMCLio 0.163 mg/m3
ND
ND
ND
NS
ND
ND
ND
Source
NA
U.S. EPA (2017b):
U.S. EPA (2017c)
NA
NA
NA
U.S. EPA
(1990c);
CalEPA (2019)
NA
NA
NA
BaP = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene; BMCL = benchmark concentration lower confidence limit;
DBacA = dibenz[a,c]anthracene; DBahA = dibenz[a,h]anthracene; DBajA = dibenz[a,j]anthracene; IUR = inhalation unit risk; NA = not applicable; ND = no data;
NDr = not determined; NS = not specified; POD = point of departure; WOE = weight of evidence.
97
Benzo[e]pyrene
-------
EPA/690/R-21/008F
As summarized in Tables C-4 (oral) and C-5 (inhalation), BaP is a multisite carcinogen in
laboratory animals following oral and inhalation exposure and DBahA is a multisite carcinogen
in laboratory animals following oral exposure. No oral or inhalation carcinogenicity data are
available for BeP or analogues other than BaP and DBahA. However, data from other routes
(e.g., dermal, injection, implantation) are available for all compounds except benzo[g]chrysene
(BgC) (see Table C-6). For BeP, evidence of carcinogenicity from other route studies is limited
to a positive finding for skin tumor initiation in one of six dermal initiation-promotion studies;
BeP was not carcinogenic in complete dermal, injection, or implantation studies. When
metabolites were tested directly, 9,10-dihydrobenzo[e]pyrene was a skin tumor initiator and the
9,10-dihydrodiol and a diol epoxide induced hepatic tumors in neonatal injection studies
(metabolite studies not included in Table C-6). For analogues, BaP and DBahA are the best
studied and demonstrate consistent carcinogenic potential across exposure routes. Most other
PAH analogues (benzo[b]chrysene, DBacA, DBajA, and picene) show evidence of
carcinogenicity in a limited number of studies; however, perylene shows negative or equivocal
findings in dermal and injection studies.
The U.S. EPA (2017c) proposed a mutagenic MO A for BaP carcinogenicity via all routes
of exposure. Proposed key events include (1) metabolism of BaP into DNA-reactive metabolites
(diol epoxides, radical cations, and o-quinone and reactive oxygen species [ROS]), (2) direct
DNA damage via DNA adducts and ROS-mediated damage, (3) DNA mutation, and (4) clonal
expansion of mutated cells. Alternative MO As that may contribute to tumor promotion include
aryl hydrocarbon receptor (AhR) affinity, immune suppression, cytotoxicity and inflammation,
and inhibition of gap junctional intercellular communication (U.S. EPA. 2017c). IARC (2010)
also proposed that the formation of DNA-reactive diol epoxides during metabolism of PAHs
underlies carcinogenic potential. A mutagenic MOA is plausible for all analogues, as well as
BeP, especially those that have demonstrated mutagenic potential and DNA adduct formation
(BeP, BgC, DBacA, DBahA, and DBajA) (see Table C-2).
98
Benzo[e]pyrene
-------
EPA 690 R-21 008F
Table C-6. Summary of Carcinogenicity Studies Using Other Exposure Routes for BeP (CASRN 192-97-2) Analogues
Exposure Route
(species)
BePa
192-97-2
BaPb
50-32-0
BbCb
214-17-5
BgCb
196-78-1
DBacAb
215-58-7
DBahAb
53-70-3
DBajAb
224-41-9
Peryleneb
198-55-0
Piceneb
213-46-7
Dermal (mouse)
Sk
Sk
Sk
Sk
Sk
Dermal initiation promotion (mouse)
Sk
Sk
Sk
Sk
Sk
Sk
(Sk)
Sk
i.v. Injection (mouse)
Lu
i.p. Injection (mouse)
Lu, Lv, St
(Lv)
Lu
i.p. Injection (rat)
Ms
s.c. Injection (mouse)
IS, Lu
IS, Lu
(IS)
IS, Lu
s.c. Injection (rat)
IS
IS
Intratracheal (hamster)
Lu, OR
OR
Intratracheal (rat or mouse)
Lu
Intrapulmonary (rat)
Lu
Lu
Intrapulmonary (mouse)
Lu
Buccal pouch application (hamster)
BP, St
99
Benzo[e]pyrene
-------
EPA 690 R-21 008F
Table C-6. Summary of Carcinogenicity Studies Using Other Exposure Routes for BeP (CASRN 192-97-2) Analogues
Exposure Route
(species)
BePa
192-97-2
BaPb
50-32-0
BbCb
214-17-5
BgCb
196-78-1
DBacAb
215-58-7
DBahAb
53-70-3
DBajAb
224-41-9
Peryleneb
198-55-0
Piceneb
213-46-7
Intramammary, intramamillary (rat)
Mm
Color Key:
Experiments in this test system 1 to 2 3 to 5
Experiments reporting statistically significant increase 1 to 2 3 to 5 >6
Experiments reporting equivocal result (+/-)
Tumor Site Key:
BP = buccal pouch; IS = injection site; Lu = lung; Lv = liver; Mm = mammary; Ms = mesothelioma, abdominal; OR = other respiratory tract; Sk = skin;
St = forestomach, stomach.
aSee Table 3B in main document for BeP references.
bData obtained from IARC (2010).
BaP = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene; DBacA =dibenz[a,c]anthracene ; DBaliA = dibenz[a,h]anthracene;
DBajA = dibenzo[a,j]anthracene; i.p. = intraperitoneal; s.c. = subcutaneous.
100
Benzo[e]pyrene
-------
EPA 690 R-21 008F
STEP 6. STRUCTURAL ALERTS AND SAR PREDICTIONS FOR BEP AND
ANALOGUES
Structural alerts and predictions for genotoxicity and carcinogenicity were identified
using computational tools as described in Appendix B. The model results for BeP and its
analogue compounds are shown in Table C-7. Concerns for carcinogenicity and/or mutagenicity
of BeP and its analogues were indicated by several models within each predictive tool (see
Table C-7). Table C-8 provides a list of the specific structural and mechanistic alerts that
underlie the findings of a concern for carcinogenicity or mutagenicity in Table C-7. These
include a structural alert for PAHs and mechanistic alerts for DNA alkylation and intercalation.
Table C-7. Heat Map Illustrating the Structural Alert and SAR Prediction
Results for BeP (CASRN 192-97-2) and Analogues3
Tool
Modelb
BeP
BaP
BbC
U
ex
ffl
DBacA
DBahA
DBajA
Perylene
Picene
Mutagenicity/genotoxicity alerts
OECD
QSAR
Toolbox
DNA alerts for Ames by OASIS
DNA alerts for CA and MNT by OASIS
In vitro mutagenicity (Ames test) alerts by ISS
In vivo mutagenicity (MN) alerts by ISS
Protein binding alerts for chromosomal aberration by OASIS
ToxRead
ToxRead (mutagenicity)
VEGA
Mutagenicity (Ames test) CONSENSUS model—assessment
Mutagenicity (Ames test) model (CAESAR)—assessment
Mutagenicity (Ames test) model (SARpy/IRFMN)—assessment
Mutagenicity (Ames test) model (ISS)—assessment
Mutagenicity (Ames test) model (A-NN/read-across)—assessment
Toxtree
Potential Salmonella tvphimurium TA100 mutagen based on QSAR
Unlikely to be a S. tvphimurium TA100 mutagen based on QSAR
Carcinogenicity alerts
OECD
QSAR
Toolbox
Carcinogenicity (genotoxicity and nongenotoxicity) alerts by ISS
OncoLogic
OncoLogic (prediction of the carcinogenic potential of the chemical)
VEGA
Carcinogenicity model (CAESAR)—assessment
Carcinogenicity model (ISS)—assessment
Carcinogenicity model (IRFMN/ANT ARES)—assessment
Carcinogenicity model (IRFMN/ISSCAN-CGX)—assessment
Carcinogenicity oral classification model (IRFMN)
Carcinogenicity inhalation classification model (IRFMN)
101
Benzo[e]pyrene
-------
EPA 690 R-21 008F
Table C-7. Heat Map Illustrating the Structural Alert and SAR Prediction
Results for BeP (CASRN 192-97-2) and Analogues3
Tool
Modelb
BeP
BaP
BbC
U
ex
ffl
DBacA
DBahA
DBajA
Perylene
Picene
ToxAlerts
Polycyclic aromatic hydrocarbons (for nongenotoxic carcinogenicity)
Toxtree
Potential carcinogen based on QSAR
Unlikely to be a carcinogen based on QSAR
Nongenotoxic carcinogenicity
Combined alerts
ToxAlerts
Polycyclic aromatic hydrocarbons (for genotoxic carcinogenicity)
Toxtree
Structural alert for genotoxic carcinogenicity
bModel results or alerts indicating no concern for carcinogenicity/mutagenicity.
bModel results outside the applicability domain for carcinogenicity/mutagenicity.
bModel results or alerts indicating concern for carcinogenicity/mutagenicity.
aAll tools and models described in Appendix B were used. Models with results are presented in the heat map
(models without results were omitted).
ANT ARES = Alternative Non-Testing Methods Assessed for REACH Substances; BaP = benzo[a]pyrene;
BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene; CA = chromosomal aberration;
CAESAR = Computer-Assisted Evaluation of industrial chemical Substances According to Regulations;
CONSENSUS = consensus assessment based on multiple models (CAESAR, SARpy, ISS, and A-NN);
DBacA = dibenz[a,c]anthracene; DBaliA = dibenz[a,h]anthracene; DBajA = dibenz[a,j]anthracene;
DNA = deoxyribonucleic acid; IRFMN = Istituto di Ricerche Fannacologiche Mario Negri; ISS = Istituto
Superiore di Sanita; ISSCAN-CGX = Istituto Superiore di Sanita Chemical Carcinogen; A-NN = A-nearest
neighbor; MN = micronucleus; MNT = micronucleus test; OECD = Organisation for Economic Co-operation and
Development; REACH = Registration Evaluation Authorisation and Restriction of Chemicals;
SAR = structure-activity relationship; QSAR = quantitative structure-analysis relationship; VEGA = Virtual
models for property Evaluation of chemicals within a Global Architecture.
102
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-8. Structural Alerts and Chemical Mechanisms for BeP
(CASRN 192-97-2) and Analogues
Structural Alert
Tools
Compounds
PAHs
OECD QSAR Toolbox
BeP, BaP, BbC, BgC, DBacA,
DBahA, DBajA, perylene,
picene
ToxAlerts
Toxtree
OncoLogic
BaP, BgC, DBajA, picene
Mechanistic Alert
Tools
Compounds
DNA intercalation
OECD QSAR Toolbox
BbC, BgC, DBacA, DBahA,
DBajA, picene
Alkylation after metabolically formed
carbenium ion species
Alkylation, direct acting epoxides and related
after CYP450-mediated metabolic activation
BaP = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene;
CYP450 = cytochrome P450; DBacA = dibenz[a,c]anthracene; DBahA = dibenz[a,h]anthracene
DBaj A = dibenz[a,j]anthracene; DNA = deoxyribonucleic acid; OECD = Organisation for Economic Co-operation
and Development; PAH = polycyclic aromatic hydrocarbon; QSAR = quantitative structure-activity relationship.
STEP 7. EVIDENCE INTEGRATION FOR SCREENING EVALUATION OF BEP
CARCINOGENICITY
Table C-9 presents the data for multiple lines of evidence pertinent to the screening
evaluation of the carcinogenic potential of BeP.
103
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-9. Integration of Evidence for BeP (CASRN 192-97-2) and Analogues
Evidence
Streams
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
Structure
r^i
CO$^
0^'
$3?
iy
r#'
CASRN
192-97-2
50-32-8
214-17-5
196-78-1
215-58-7
53-70-3
224-41-9
198-55-0
213-46-7
Analogue
selection and
evaluation
(see Steps 1
and 2)
Target
compound:
PAH, five
unsubstituted
benzene rings
PAH, five
unsubstituted
benzene rings
PAH, five
unsubstituted
benzene rings
PAH, five
unsubstituted
benzene rings
PAH, five
unsubstituted
benzene rings
PAH, live
unsubstituted
benzene rings
PAH, five
unsubstituted
benzene rings
PAH, five
unsubstituted
benzene rings
PAH, five
unsubstituted
benzene rings
Experimental
genotoxicity
data (see
Step 3)
Weak mutagen
with metabolic
activation;
predominately
negative for
clastogenicity
and DNA
damage/repair;
forms DNA
adducts; weak
inducer of cell
transformation
Mutagenic;
clastogenic;
DNA
damaging;
forms DNA
adducts;
induces cell
transformation
ND
Mutagenic;
DNA
damaging;
forms DNA
adducts
Mutagenic; not
clastogenic;
DNA
damaging;
forms DNA
adducts;
induces cell
transformation
Mutagenic;
clastogenic;
forms DNA
adducts;
induces cell
transformation
Mutagenic;
forms DNA
adducts
Mutagenic in
bacteria; not
mutagenic in
mammalian
cells;
clastogenic;
does not cause
DNA damage
in bacteria
Not mutagenic
104
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-9. Integration of Evidence for BeP (CASRN 192-97-2) and Analogues
Evidence
Streams
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
ADME
Common
Common
Presumed
Common
Common
Common
Common
Presumed
Common
evaluation (see
metabolic
metabolic
common
metabolic
metabolic
metabolic
metabolic
common
metabolic
Step 4)
pathways with
pathways with
metabolic
pathways with
pathways with
pathways with
pathways with
metabolic
pathways with
other PAHs
other PAHs
pathways with
other PAHs
other PAHs
other PAHs
other PAHs
pathways with
other PAHs
(oxidation and
(oxidation and
other PAHs
(oxidation and
(oxidation and
(oxidation and
(oxidation and
other PAHs
(oxidation and
glucuronide
glucuronide,
(oxidation and
glucuronide,
glucuronide,
glucuronide,
glucuronide
(oxidation and
glucuronide,
conjugation)
GSH, or sulfate
glucuronide,
GSH, or sulfate
GSH, or sulfate
GSH, or sulfate
conjugation)
glucuronide,
GSH, or sulfate
conjugation)
GSH, or sulfate
conjugation)
conjugation)
conjugation)
GSH, or sulfate
conjugation)
Metabolites:
conjugation)
Metabolites:
conjugation)
Major: k-region
Metabolites:
Metabolites:
Metabolites:
Metabolites:
diols, bisdiols,
Metabolites:
4,5-dihydrodiol,
9,10-, 7,8-,
Metabolites:
fjord region
Major:
diols, oxides,
oxides
Metabolites:
dihydrodiols,
phenols, and
4,5-, and
ND
11,12-diols,
10,11-di-
catechols,
ND
phenols, and
quinones
2,3-di-
11,12-diol-
hydrodiol,
phenols, and
quinones
hydrodiols and
12,14-oxides,
10,11-diol-
hexols
Minor:
epoxides, as
diastereomeric
12,13-epoxide
bay-region
well as various
tetrols
9,10-di-
phenols,
Minor: 1,2- and
hydrodiol,
quinones, and
3,4-diols
4,5,9,10-tetra-
derivatives
hydroxy-
4,5,9,10-tetra-
hydro-
benzo[e]pyrene
105
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-9. Integration of Evidence for BeP (CASRN 192-97-2) and Analogues
Evidence
Streams
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
Cancer data
No oral or
Multisite
No oral or
ND
No oral or
Multisite
No oral or
No oral or
No oral or
and MOA
inhalation data
carcinogen in
inhalation data
inhalation data
carcinogen in
inhalation data
inhalation data
inhalation data
(see Step 5)
rodents
rodents
Predominately
following oral
Skin tumor
Skin tumors in
following oral
Skin tumors in
Equivocal
Skin tumors in
negative in other
or inhalation
promotion in
dermal assays;
exposure
dermal assays;
evidence for
dermal assays;
route studies:
exposure
dermal assays
limited data
limited
skin tumors in
lung and
tumor initiator
evidence for
Carcinogenic in
evidence for
dermal assays;
injection site
in 1/6 studies,
Carcinogenic in
Inferred
liver tumors in
other route
injection site
negative in
tumors in
1/5 metabolites
other route
mutagenic
injection
studies
tumors
injection
injection
was a dermal
studies
MOA (based
studies
studies
studies
initiator,
on BaP)
Inferred
Inferred
2/7 metabolites
Proposed
Inferred
mutagenic
mutagenic
Inferred
Unknown
induced tumors
mutagenic
mutagenic
MOA (based
MOA (based
mutagenic
MOA (negative
in injection
MOA (U.S.
MOA (based
on BaP)
on BaP)
MOA (based
in mutation
studies
EPA, 2017c)
on BaP)
on BaP)
assay)
Inferred
mutagenic MOA
(based on BaP)
106
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Table C-9. Integration of Evidence for BeP (CASRN 192-97-2) and Analogues
Evidence
Streams
BeP
BaP
BbC
BgC
DBacA
DBahA
DBajA
Perylene
Picene
Common
Alerts:
Alerts:
Alerts:
Alerts:
Alerts:
Alerts:
Alerts:
Alerts:
Alerts:
structural alerts
• PAHs
• PAHs
• PAHs
• PAHs
• PAHs
• PAHs
• PAHs
• PAHs
• PAHs
and SAR
• DNA
• DNA
• DNA
• DNA
• DNA
• DNA
predictions
SAR
SAR
intercalation
intercalation
intercalation
intercalation
intercalation
SAR
intercalation
(see Step 6)
prediction:
prediction:
• Alkylation
• Alkylation
• Alkylation
• Alkylation
• Alkylation
prediction:
• Alkylation
Concerns for
Concerns for
(metabolites)
(metabolites)
(metabolites)
(metabolites)
(metabolites)
Concerns for
(metabolites)
mutagenicity
mutagenicity
mutagenicity
and carcino-
and carcino-
SAR
SAR
SAR
SAR
SAR
and carcino-
SAR
genicity in most
genicity in
prediction:
prediction:
prediction:
prediction:
prediction:
genicity in
prediction:
models
most models
Concerns for
Concerns for
Concerns for
Concerns for
Concerns for
most models
Concerns for
mutagenicity
mutagenicity
mutagenicity
mutagenicity
mutagenicity
mutagenicity
and carcino-
and carcino-
and carcino-
and carcino-
and carcino-
and carcino-
genicity in
genicity in
genicity in
genicity in
genicity in
genicity in
most models
most models
most models
most models
most models
most models
ADME = absorption, distribution, metabolism, and excretion; BaP. = benzo[a]pyrene; BbC = benzo[b]chrysene; BeP = benzo[e]pyrene; BgC = benzo[g]chrysene;
DBacA = dibenz[a,c]anthracene; DBahA = dibenz[a,h]anthracene; DBajA = dibenz[a,j]anthracene; DNA = deoxyribonucleic acid; GSH = glutathione; MOA = mode of
action; ND = no data; PAH = polycyclic aromatic hydrocarbon; SAR = structure-activity relationship.
107
Benzo[e]pyrene
-------
EPA/690/R-21/008F
STEP 8. QUALITATIVE LEVEL OF CONCERN FOR BEP POTENTIAL
CARCINOGENICITY
Table C-10 identifies the qualitative level of concern for potential carcinogenicity of BeP
based on the multiple lines of evidence described above. Because the proposed mutagenic MOA
for BaP is relevant for all exposure routes, a qualitative level of concern for BeP potential
carcinogenicity is applicable to both oral and inhalation exposures.
Table C-10. Qualitative Level of Concern for Carcinogenicity of
BeP (CASRN 192-97-2)
Level of Concern
Designation
Comments
Concern for potential
carcinogenicity
Selected
Concern is based on (1) limited evidence of skin tumor
initiation by BeP (at highest concentration tested), (2) limited
evidence of skin tumor initiation by BeP metabolite
9,10-dihydrobenzo[e]pyrene, (3) limited evidence of tumor
induction following neonatal i.p. exposure to specific BeP
dihydrodiol and expected epoxide metabolites, (4) evidence of
oral and/or inhalation carcinogenicity in animal studies of
analogues BaP and DBahA, and (5) plausibility of a
mutagenic MOA (potential, though limited, formation of
reactive dihydrodiols and epoxides; evidence of mutagenicity
with metabolic activation; formation of DNA adducts).
Inadequate information for
assigning qualitative level
of concern
NS
NA
BaP = benzo[a]pyrene; BeP = benzo[e]pyrene; DBahA = dibenz[a,h]anthracene; DNA = deoxyribonucleic acid;
i.p. = intraperitoneal; MOA = mode of action; NA = not applicable; NS = not selected.
108
Benzo[e]pyrene
-------
EPA/690/R-21/008F
APPENDIX D. REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). (2020). 2020 TLVs and
BEIs: Based on the documentation of the threshold limit values for chemical substances
and physical agents & biological exposure indices. Cincinnati, OH.
Ai-Daghri. NM; Alokail. MS; Abd-Airahman. SH; Draz. HM. (2014). Poly cyclic aromatic
hydrocarbon distribution in serum of Saudi children using HPLC-FLD: Marker elevations
in children with asthma. Environ Sci Pollut Res Int 21: 12085-12090.
http://dx.doi.org/10.1007/sll356-014-31Q8-0
Anderson. C; Hehr. A; Robbins. R; Hasan. R; Athar. M; Mukhtar. H; Elmets. CA. (1995).
Metabolic requirements for induction of contact hypersensitivity to immunotoxic
polyaromatic hydrocarbons. J Immunol 155: 3530-3537.
Andrews. AW; Thibautt LH; Liiinskv. W. (1978). The relationship between carcinogenicity and
mutagenicity of some polynuclear hydrocarbons. Mutat Res 51: 311-318.
http://dx.doi.org/10.1016/0027-5 107(78)90120-3
Archibong. AE; Invang. F; Ramesh. A; Greenwood. M; Navvar. T; Kopsombut. P; Hood. DB;
Nvanda. AM. (2002). Alteration of pregnancy related hormones and fetal survival in F-
344 rats exposed by inhalation to benzo(a)pyrene. Reprod Toxicol 16: 801-808.
http://dx.doi.org/10.1016/S0890-6238(02)00058-8
AT SDR (Agency for Toxic Substances and Disease Registry). (1995). Toxicological profile for
polycyclic aromatic hydrocarbons - Update [ATSDR Tox Profile], (CIS/97/00215).
Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service.
https://www. atsdr. cdc. gov/ substances/toxsub stance. asp?toxid=25
ATSDR (Agency for Toxic Substances and Disease Registry). (2005). Toxicological profile for
naphthalene, 1-methylnaphthalene, and 2-methylnaphthalene. (PB2006100004). Atlanta,
GA.
https://ntrl.ntis.gov/NTRI.dashboard/searchResults.xhtml'.'scareh0ucry PB2006100004
Ball. LM; Warren. SH: Sangaiah. R; Gold. A. (1991). Bacterial mutagenicity of two
cyclopentafused isomers of benzpyrene. Mutat Res 271: 271-279.
http://dx.doi.org/10.1016/0165-1218f9n90Q36-L
Blanton. RH; Lvte. M; Myers. MJ; Bick. PH. (1986). Immunomodulation by polyaromatic
hydrocarbons in mice and murine cells. Cancer Res 46: 2735-2739.
Brette. F; Shi els. HA; Gatti. GL; Cros. C; Incardona. JP; Scholz. ML; Block. BA. (2017). A
novel cardiotoxic mechanism for a pervasive global pollutant. Sci Rep 7: 41476.
http://dx.doi.org/10.1038/srep41476
Buening. MK; Levin. W; Wood. AW; Chang. RL; Lehr. RE; Taylor. CW; Yagi. H; Jerina. DM;
Connev. AH. (1980). Tumorigenie activity of benzo(e)pyrene derivatives on mouse skin
and in newborn mice. Cancer Res 40: 203-206.
CalEPA (California Environmental Protection Agency). (1992). Expedited cancer potency values
and proposed regulatory levels for certain Proposition 65 carcinogens. Berkeley, CA:
Office of Environmental Health Hazard Assessment, Reproductive and Cancer Hazard
Assessment Section, http://www.oehha.ca.gov/prop65/pdf/expcancer.pdf
https://oehha.ca.gov/air/crnr/technical-support-
109
¦potency-factors-2009
Benzo[e]pyrene
-------
EPA/690/R-21/008F
CalEPA (California Environmental Protection Agency). (2017). Prop 65: §69502.2(a)(1)(A).
Chemicals known to cause cancer and/or reproductive toxicity that are listed under
Health and Safety Code section 25249.8 of the California Safe Drinking Water and Toxic
Enforcement Act of 1986. Available online
CalEPA (California Environmental Protection Agency). (2019). Consolidated table of
OEHHA/ARB approved risk assessment health values (September 19, 2019 ed.).
Sacramento, CA: California Air Resources Board.
https://www.arb.ca.gov/toxics/healthval/contable.pdf
Casto. BC; Pieczvnski. WJ; Janosko. N; Dipaolo. JA. (1976). Significance of treatment interval
and DNA repair in the enhancement of viral transformation by chemical carcinogens and
mutagens. Chem Biol Interact 13: 105-125.
Chang. LH; Young. L. (1943). The metabolism of acenaphthene in the rat. J Biol Chem 151: 87-
91.
Chang. RL; Levin. W; Wood. AW; Lehr. RE; Kumar. S; Yagi. H; Jerina. DM; Connev. AH.
(1981). Tumorigenicity of the diastereomeric bay-region benzo(e)pyrene 9,10-diol-l 1,12-
epoxides in newborn mice. Cancer Res 41: 915-918.
ChemlDplus. (2021). Chem 1 Dpi us advanced. Available online at
https ://chem. nlm. nih. gov/chemidptus/
Chen. C; Tang. Y; Jiang. X; Qi. Y; Cheng. S; Qiu. C; Peng. B; Tu. B. (2012). Early postnatal
benzo(a)pyrene exposure in Sprague-Dawley rats causes persistent neurobehavioral
impairments that emerge postnatally and continue into adolescence and adulthood.
Toxicol Sci 125: 248-261. http://dx.doi.org/10.1093/toxsci/kfr265
Cherng. SH; Lin. ST; Lee. H. (1996). Modulatory effects of polycyclic aromatic hydrocarbons
on the mutagenicity of 1-nitropyrene: A structure-activity relationship study. Mutat Res
Genet Toxicol 367: 177-185. http://dx.doi.org/10.1016/S0165-1218(96)90075-0
Coulomb. H; Gu. Z; Audu. S; Chouroulinkov. I. (1981). The uptake and release of
benzo[a]pyrene and benzo[e]pyrene in vitro by Syrian hamster embryo cells as a function
of serum concentration. Carcinogenesis 2: 523-527.
http://dx.doi.Org/10.1093/carcin/2.6.523
CPDB (Carcinogenic Potency Database). (201 1). The carcinogenic potency project: The
carcinogenic potency database [Database]: Department of Energy; National Cancer
Institute; Environmental Protection Agency; National Institute of Environmental Health
Sciences; National Toxicology Program; University of California, Berkeley. Retrieved
from https://www.nlm.nih.gov/databases/download/cpdb.html
Curfs. DM; Knaapen. AM; Pachen. DM; Gijbels. MJ; Lutgens. E; Smook. ML; Kockx. MM;
Daemen. MJ; van Schooten. FJ. (2005). Polycyclic aromatic hydrocarbons induce an
inflammatory atherosclerotic plaque phenotype irrespective of their DNA binding
properties. FASEB J 19: 1290-1292. http://dx.doi.org/10.1096/fi.04-2269fie
Davila. PR; Lane. JL; Lauer. FT; Burchiel. SW. (1999). Protein tyrosine kinase activation by
polycyclic aromatic hydrocarbons in human HPB-ALL T cells. J Toxicol Environ Health
56: 249-261. http://dx.doi.org/10.1080/009841099158Q97
Davila. PR; Romero. PL; Burchiel. SW. (1996). Human T cells are highly sensitive to
suppression of mitogenesis by polycyclic aromatic hydrocarbons and this effect is
differentially reversed by alpha-naphthoflavone. Toxicol Appl Pharmacol 139: 333-341.
http://dx.doi.org/10.1006/taap.1996.0173
Pe Flora. S; Zanacchi. P; Camoirano. A; Bennicetti. C; Badolati. GS. (1984). Genotoxic activity
and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair
test [Review], Mutat Res 133: 161-198. http://dx.doi.org/10.1016/0165-l 110(84)90016-2
110
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Dean. JH; Luster. MI; Boorman. GA; I .aucr. LP; I .cubkc. RW; Lawson. L. (1983). Selective
immunosuppression resulting from exposure to the carcinogenic congener of
benzopyrene in B6C3F1 mice. Clin Exp Immunol 52: 199-206.
Deutsch-Wenzel. RP; Brune. H; Grimmer. G; Dettbarn. G; Misfeld. J. (1983). Experimental
studies in rat lungs on the carcinogenicity and dose-response relationships of eight
frequently occurring environmental polycyclic aromatic hydrocarbons. J Natl Cancer Inst
71: 539-544.
DiPaolo. JA; Donovan. P; Nelson. R. (1969). Quantitative studies of in vitro transformation by
chemical carcinogens. J Natl Cancer Inst 42: 867-874.
DSSTox (Distributed Structure-Searchable Toxicity). (2018). DSSTox database. Research
Triangle Park, NC: U.S. Environmental Protection Agency, National Center for
Computational Toxicology. Retrieved from https://www.epa.gov/chemical-
rcscarchdistributcd-structurc-scarchable-toxicitv-dsstox-database
Duncan. M; Brookes. P; Dipple. A. (1969). Metabolism and binding to cellular macromolecules
of a series of hydrocarbons by mouse embryo cells in culture. Int J Cancer 4: 813-819.
http://dx.doi.org/10.1002/ii c.2910040610
Dunkel. \ ('; Zeiger. E; Brusick. D; McCoy. E; McGregor. D; Mortelmans. K; Rosenkranz. HS;
Simmon. VF. (1984). Reproducibility of microbial mutagenicity assays: I. Tests with
Salmonella typhimurium and Escherichia coli using a standardized protocol. Environ
Mutagen 6: 1 -25 1. http://dx.doi.org/10.1002/em.28600607Q4
Durant. JL; Busbv. WF. Jr; Latlcur. A1Penman. BW; Crespi. CL. (1996). Human cell
mutagenicity of oxygenated, nitrated and unsubstituted polycyclic aromatic hydrocarbons
associated with urban aerosols. Mutat Res DNA Repair 371: 123-157.
http://dx.doi.org/10.1016/S0165-1218(96)90103-2
ECHA (European Chemicals Agency). (2019a). Substance infocard: Polycyclic-aromatic
hydrocarbons (PAH). https://echa.curopa.eu/substance-information/-
/substanceinfo/100.239.209
ECHA (European Chemicals Agency). (2019b). Summary of classification and labeling:
Benzo(e)pyrene (192-97-2). (601 -049-00-6). https://echa.europa.eu/information-on-
chemicals/cl-inventorv-database/-/discli/details/131125
Estrago-Franco. MF: Moustafa. MT: Riazi-Esfahani. M: Sapkal. AU: Piche-Lopez. R: Patil. AJ:
Sharma. A: Falatoonzadeh. P: Chwa. M: Luczv-Bachman. G: Kuppermann. BP: Kennev.
MC. (2016). Effects of benzo(e)pyrene on reactive oxygen/nitrogen species and
inflammatory cytokines induction in human RPE cells and attenuation by mitochondrial-
involved mechanism. Journal of Ophthalmic & Vision Research 11: 385-393.
http://dx.doi.org/10.4103/2008-322X.194Q91
Fujikawa. K: Fort FL: Samejima. K: Sakamoto. Y. (1993). Genotoxic potency in Drosophila
melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as
assayed in the DNA repair test. Mutat Res 290: 175-182. http://dx.doi.org/lQ.1016/QQ27-
5107(93)90157-B
Grantham. PH. (1963). Metabolism of fluorene-9-C 14 in rats, guinea pigs, and rabbits.
Biochemistry 2: 610-616. http://dx.doi.org/10.1021/bi0Q903aQ38
Gupta. P: Baneriee. DK: Bhargava. SK: Kaul. R: Shanker. VR. (1994). Abnormal pattern of lung
functions in rubber factory workers. J Indian Med Assoc 92: 260-263.
Gupta. P: Makhijani. SD: Meenakshi. C. (1998). Pulmonary function tests on rubber factory
workers exposed to polynuclear aromatic hydrocarbons. Indian J Physiol Allied Sci 52:
71-75.
Ill
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Gurtoo- HL; Parker. NB; Paigen. B; Havens. MB; Minowada. J; Freedman. HI. (1979).
Induction, inhibition and some enzymological properties of aryl hydrocarbon hydroxylase
in fresh mitogen-activated human lymphocytes. Cancer Res 39: 4620-4629.
Haas. BS; Brooks. EE; Schumann. KE; Dornfeld. SS. (1981). Synergistic, additive and
antagonistic mutagenic responses to binary mixtures of benzo(a)pyrene and
benzo(e)pyrene as detected by strains TA98 and TA100 in the Salmonella microsome
assay. Environ Mutagen 3: 159-166. http://dx.doi.org/10.1002/em.28600302Q7
Hardin. JA; Hinoshita. F; Sherr. DH. (1992). Mechanisms by which benzo[a]pyrene, an
environmental carcinogen, suppresses B cell lymphopoiesis. Toxicol Appl Pharmacol
117:155-164.
Huberman. E. (1977). Viral antigen induction and mutability of different genetic loci by
metabolically activated carcinogenic polycyclic hydrocarbons in cultured mammalian
cells. In Cold Spring Harbor conferences on cell proliferation (pp. 1521-1535).
(EMICBACK/28591). Cold Spring Harbor Laboratory: Cold Spring Harbor, NY.
IARC (International Agency for Research on Cancer). (1983). I ARC monographs on the
evaluation of the carcinogenic risk of chemicals to humans: Polynuclear aromatic
compounds, Part I. Chemical, environmental and experimental data [IARC Monograph],
Lyon, France: World Health Organization, International Agency for Research on Cancer.
http://monographs.iarc.fr/ENG/Monographs/voll-42/mono32.pdf
IARC (International Agency for Research on Cancer). (2010). Some non-heterocyclic polycyclic
aromatic hydrocarbons and some related exposures. IARC monographs on the evaluation
of carcinogenic risks to humans [IARC Monograph] (pp. 1-853). Lyon, France.
http://monographs.iarc.fr/ENG/Monographs/vol92/mono92.pdf
IARC (International Agency for Research on Cancer). (2018). IARC monographs on the
evaluation of carcinogenic risk to humans.
http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php
IPCS (International Programme on Chemical Safety). (2020). INCHEM: Chemical safety
information from intergovernmental organizations [Database], Geneva, Switzerland:
World Health Organization, Canadian Centre for Occupational Health and Safety. Inter-
Organization Programme for the Sound Management of Chemicals. Retrieved from
http://www.inchem.org/
Jacob. J; Schmotdt A; Grimmer. G. (1983). Benzo(e)pyrene metabolism in rat liver microsomes:
Dependence of the metabolite profile on the pretreatment of rats with various
monooxygenase inducers. Carcinogenesis 4: 905-910.
http://dx.doi.Org/10.1093/carcin/4.7.905
Jacob. J; Schmotdt A; Grimmer. G; Hamann. M; Raab. G. (1985). Benzo(e)pyrene metabolism:
Identification and quantitative analysis of metabolites formed by liver microsomes of
variously pretreated rats by means of capillary gas chromatography/mass spectrometry.
In J Cooke; AJ Dennis (Eds.), Polynuclear aromatic hydrocarbons: Mechanisms, methods
and metabolism (pp. 627-641). Columbus, OH: Battelle Memorial Institute.
Kaden. DA; Hites. RA; Thittv. WG. (1979). Mutagenicity of soot and associated polycyclic
aromatic hydrocarbons to Salmonella typhimurium. Cancer Res 39: 4152-4159.
Kamiva. T; Nagaoka. T; Omae. T; Ono. S; Otani. S; Yoshida. A. (2017). Benzo(e)pyrene inhibits
endothelium-dependent no-mediated dilation of retinal arterioles via superoxide
production and endoplasmic reticulum stress. Invest Ophthalmol Vis Sci 58: 5978-5984.
http://dx.doi.org/10.1167/iovs.17-21925
112
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Katz. M. (1979). Analysis of polycyclic aromatic hydrocarbons in atmospheric suspended
particulate matter. In H Egan; M Castegnaro; H Kunte; P Bogovski; EA Walker (Eds.),
Environmental carcinogens: Selected methods of analysis Analysis of polycyclic
aromatic hydrocarbons in environmental samples. Lyon, France: International Agency
For Research On Cancer. http://publications.iarc.fr/Book-And-Report-Series/Iarc-
Scientific-Publications/Environmental-Carcinogens-Selected-Methods-Of-Analvsis-
Volume-3-Analvsis-Of-Polvcvclic-Aromatic-Hvdrocarbons-In-Environmental-Samples-
1979
Kirkland. D; A ardent a. M; Henderson. L; Miiller. L. (2005). Evaluation of the ability of a battery
of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-
carcinogens: I. Sensitivity, specificity and relative predictivity. Mutat Res 584: 1-256.
http: //dx. doi. or g/10.1016/i. mr gentox .2005.02.004
Kitchin. KT; Brown. JL; Kulkarn. AP. (1992). Predictive assay for rodent carcinogenicity using
in vivo biochemical parameters: Operational characteristics and complementarity. Mutat
Res 266: 253-272. http://dx.doi.org/10.1016/0027-5107(92)90193-6
Kitchin. KT; Brown. JL; Kulkarni. AP. (1993). Predicting rodent carcinogenicity of Ames test
false positives by in vivo biochemical parameters. Mutat Res 290: 155-164.
http://dx.doi. org/10.1016/0027-5107(93)90155-9
Krieger. JA; Born. JL; Burchiel. SW. (1994). Persistence of calcium elevation in the HPB-all
human T-cell line correlates with immunosuppressive properties of polycyclic aromatic
hydrocarbons. Toxicol Appl Pharmacol 127: 268-274.
http://dx.doi.Org/10.1006/taap.1994.l 161
Krieger. JA; Davila. PR; Lvtton. J; Born. JL; Burchiel. SW. (1995). Inhibition of
sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCA) by polycyclic aromatic
hydrocarbons in HPB-ALL human T cells and other tissues. Toxicol Appl Pharmacol
133: 102-108. http://dx.doi.org/10.1006/taap. 1995.1131
Lavoie. EJ; Bedenko. EV; Hirota. N; Hecht. SS; Hoffmann. D. (1979). A comparison of the
mutagenicity, tumor-initiating activity and complete carcinogenicity of polynuclear
aromatic hydrocarbons. In PW Jones; P Leber (Eds.), Polynuclear aromatic hydrocarbons
(pp. 705-721). Ann Arbor, MI: Ann Arbor Science Publishers, Inc.
Lipniak. M; Brandvs. J. (1993). Toxicokinetics of fluoranthene, pyrene and benz(a)anthracene in
the rat. Polycycl Aromat Compd 3: 111-119.
http://dx.doi.org/10.1080/10406639308Q47863
Ma. X; Gibbons. JA; Babish. JG. (1991). Benzo(e)pyrene pretreatment of immature, female
C57BL/6J mice results in increased bioactivation of aflatoxin B1 in vitro. Toxicol Lett
59: 51-58. http://dx.doi.org/10.1016/0378-4274(9D90054-A
MacLeod. MC; Cohen. GM; Selkirk. JK. (1979). Metabolism and macromolecular binding of the
carcinogen benzo[a]pyrene and its relatively inert isomer benzo[e]pyrene by hamster
embryo cells. Cancer Res 39: 3463-3470.
MacLeod. MC; Mansfield. BK; Selkirk. JK. (1982). Time course of metabolism of
benzo(e)pyrene by hamster embryo cells and the effect of chemical modifiers. Chem Biol
Interact 40: 275-285. http://dx.doi.org/10.1016/0009-2797(82)90151-X
Mane. SS; Purnett. DM; Hsu. IC. (1990). Genotoxic effects of five polycyclic aromatic
hydrocarbons in human and rat mammary epithelial cells. Environ Mol Mutagen 15: 78-
82.
113
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Mansoor. S; Gupta. N; Patil. AJ; Estrago-Franco. MF; Ramirez. C; Migon. R; Sapkai A;
Kuppermann. BP; Kennev. MC. (2010). Inhibition of apoptosis in human retinal pigment
epithelial cells treated with benzo(e)pyrene, a toxic component of cigarette smoke. Invest
Ophthalmol Vis Sci 51: 2601-2607. http://dx.doi.Org/10.l 167/iovs.09-4121
McCann. J; Choi. E; Yamasaki. E; Ames. BN. (1975). Detection of carcinogens as mutagens in
the Salmonella/microsome test: Assay of 300 chemicals. Proc Natl Acad Sci USA 72:
5135-5139. http://dx.doi.org/10.1073/pnas.72.12.5135
Mersch-Sundermann. V; Mochavedi. S; Kevekordes. S. (1992). Genotoxicity of poly cyclic
aromatic hydrocarbons in Escherichia coli PQ37. Mutat Res 278: 1-9.
http://dx.doi.org/10.1016/0165-1218(92)90279-9
Mersch-Sundermann. V; Mochavedi. S; Kevekordes. S; Kern. S; Wintermann. F. (1993). The
genotoxicity of unsubstituted and nitrated polycyclic aromatic hydrocarbons. Anticancer
Res 13: 2037-2043.
Milo. GE; Blakestee. J; Yohn. DS; Dipaolo. JA. (1978). Biochemical activation of aryl
hydrocarbon hydroxylase activity, cellular distribution of polynuclear hydrocarbon
metabolites, and DNA damage by polynuclear hydrocarbon products in human cells in
vitro. Cancer Res 38: 1638-1644.
Motvkiewicz. G; Michalska. J; Pendzich. J; Mahisecka. E; Strozvk. M; Kalinowska. E;
Butkiewicz. D; Mietzvnska. D; Midro. A; San tell a. RM; Chorazv. M. (1998). A
molecular epidemiology study in women from Upper Silesia, Poland. Toxicol Lett 96-97:
195-202. http://dx.doi.org/10.1016/S0378-4274(98)00072-l
Munson. AE; White. KL. (1983). Fourth interim report on effect of sub chronic fourteen day
exposure to benzopyrene in B6C3F1 male and female mice on specific immunological
parameters with cover letter [TSCA Submission], (#FYI-AX-0382-0220.
TSCATS/031213). Washington, DC: Medical College of Virginia, American Petroleum
Institute.
Munson. AE: White. KL. (1986). Immunotoxicology of benzopyrenes in Fisher 344 rats (final
report) with cover letter dated 05-19-1986 [TSCA Submission], (OTS00003093. #FYI-
AX-0686-0309. TSCATS/200128). Washington, DC: Medical College of Virginia,
American Petroleum Institute.
Munson. AE: White. KL. (1992). Effects of sub chronic 14-day exposure to benzopyrene in
B6C3F1 male and female mice on specific immunological parameters [TSCA
Submission], (OTS0000901K4. #FYI-0892-0901. TSCATS/452123). Medical College of
Virginia, American Petroleum Institute.
Munson. AE: White. KL: Lvsv. HFL (1985). Effects of subchronic fourteen day exposure to
benzopyrene in B6C3F1 female mice on host resistance with cover letter 041585 [TSCA
Submission], (OTS00002202. #FYI-AX-0485-0220. TSCATS/035026). Washington,
DC: Medical College of Virginia, American Petroleum Institute.
Neish. WJ. (1948). Metabolism of fluorene in the rabbit. Biochem J 43: 533-537.
http://dx.doi.org/10.1042/bi0430533
NIOSH (National Institute for Occupational Safety and Health). (1986). Health hazard
evaluation report No. HETA-84-284-1701, Granite City Steel, Granite City, Illinois (pp.
84-284). (NIOSH/00163543).
NIOSH (National Institute for Occupational Safety and Health). (1987). Health hazard
evaluation report No. HETA-84-284-1701B, revised, Granite City Steel, Granite City,
Illinois. (HETA-84-284-1701B). Cincinnati, OH: U.S. Department of Health and Human
Services, http://www.cdc.gov/niosh/nioshtic-2/00173 104.html
114
Benzo[e]pyrene
-------
EPA/690/R-21/008F
NIOSH (National Institute for Occupational Safety and Health). (2018). NIOSH pocket guide to
chemical hazards. Index of chemical abstracts service registry numbers (CAS No.).
Atlanta, GA. http://www.cdc.gov/niosh/npg/npgdcas.html
NIST (National Institute of Standards and Technology). (2019). NIST chemistry webbook.
Benzo[e]pyrene (192-97-2). Standard reference database No. 69.
https://webbook.nist. gov/cgi/cbook.cgi?ID=192-97-2&Units=SI
NLM (National Library of Medicine). (2019a). HSDB annotation record: Benzo[e]pyrene.
Bethesda, MD: National Center for Biotechnology Information.
https://pubchem.ncbi.nlm.nih.gov/source/hsdb/4031
NLM (National Library of Medicine). (2019b). Pubchem compounds summary: Acenaphthene,
83-32-9. Available online at https://pubchem.ncbi.nlm .nih.gov/com pound/Acenaphthene
NLM (National Library of Medicine). (2019c). Pubchem compounds summary: Anthracene,
120-12-7. Available online at https://pubchem.ncbi.nlm.nih.gov/compound/8418
NLM (National Library of Medicine). (2019d). Pubchem compounds summary: Benzo[a]pyrene,
50-32-8. Available online at https://pubchem.ncbi.nlm. nih.gov/compound/2336
NLM (National Library of Medicine). (2019e). Pubchem compounds summary: Benzo[e]pyrene,
192-97-2. Available online at https://pubchem.ncbi.nlm.nih.gov/compound/9128
NLM (National Library of Medicine). (2019f). Pubchem compounds summary: Fluoranthene,
206-44-0. Available online at https://pubchem.ncbi.nlm.nih.gov/compound/9154
NLM (National Library of Medicine). (2019g). Pubchem compounds summary: Fluorene, 86-73-
7. Available online at https://pubchem.ncbi.nlm.nih.gov/compound/6853
NLM (National Library of Medicine). (2019h). Pubchem compounds summary: Pyrene, 129-00-
0. Available online at https://pubchem.ncbi.nlm.nih.gov/compound/31423
Novicki. PL; Rosenberg. MR; Michatopoutos. G. (1985). Inhibition of DNA synthesis by
chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes.
Cancer Res 45: 337-344.
NTP (National Toxicology Program). (2016). 14th Report on carcinogens. Research Triangle
Park, NC. https://ntp.niehs.nih.gov/pubhealth/roc/index-l.html
NTP (National Toxicology Program). (2017). NTP technical reports index. Available online at
https://ntp.niehs.nih.gov/results/summaries/chronicstudies/index.html
OECD (Organisation for Economic Co-operation and Development). (2019). The OECD QSAR
toolbox [version 4.3.1], Retrieved from http://www.oecd.org/chemicalsafety/risk-
assessment/occd-cisar-toolbox htm
OSHA (Occupational Safety & Health Administration). (2020). Air contaminants: Occupational
safety and health standards for shipyard employment, subpart Z, toxic and hazardous
substances. (OSHA Standard 1915.1000). Washington, DC.
https://www.osha. gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p
id10286
Patil. A J; Sharma. A: Seigel. GM: Gramaio. A: Chwa. M: Kuppermann. BP: Kennev. MC.
(2009). Effects of benzo(e)pyrene on the retinal neurosensory cells and human
microvascular endothelial cells in vitro. Curr Eye Res 34: 672-682.
http://dx.doi.org/10.1080/02713680903Q15892
Patton. WP: Rout ledge. MN; Jones. GD: Lewis. SE: Archer. DB: Davies. RJ: Chakravarthv. U.
(2002). Retinal pigment epithelial cell DNA is damaged by exposure to benzo[a]pyrene, a
constituent of cigarette smoke. Exp Eye Res 74: 513-522.
http://dx.doi.Org/10.1006/exer.2001.l 160
115
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Perm. A; Snyder. C. (1988). Arteriosclerotic plaque development is 'promoted" by polynuclear
aromatic hydrocarbons. Carcinogenesis 9: 2185-2189.
http://dx.doi.Org/10.1093/carcin/9.12.2185
Piatt KL; Aderhold. S; Kulpe. K; Fickler. M. (2008). Unexpected DNA damage caused by
polycyclic aromatic hydrocarbons under standard laboratory conditions. Mutat Res 650:
96-103. http://dx.doi.Org/10.1016/i.mrgentox.2007.09.011
Piatt. KL; Petrovic. P; Seidel. A; Becrmann. D; Ocsch. F. (1988). Microsomal metabolism of
picene. Chem Biol Interact 66: 157-175. http://dx.doi.org/10.1016/0009-2797(88)90069-
5
Probst. GS; Mcmahon. RE; Hill. LE; Thompson. CZ; Epp. JK; Neal. SB. (1981). Chemically-
induced unscheduled DNA synthesis in primary rat hepatocyte cultures: A comparison
with bacterial mutagenicity using 218 compounds. Environ Mutagen 3: 11-32.
http://dx.doi.org/10.1002/em.28600301Q3
Prodi. G; Rocchi. P; Gritti. S. (1975). The effect of polycyclic hydrocarbons on the synthesis of
DNA on lymphoid organs bone marrow and regenerating rat liver. Experientia 31: 358-
360.
Reddv. MY; Gupta. RC; Randerath. E; Randerath. K. (1984). 32 P-postlabeling test for covalent
DNA binding of chemicals in vivo: Application to a variety of aromatic carcinogens and
methylating agents. Carcinogenesis 5: 231-244. http://dx.doi.Org/10.1093/carcin/5.2.231
Rees. ED; Maiumdar. SK; Shuck. A. (1970). Changes in chromosomes of bone marrow after
intravenous injections of 7,12-dimethylbenz(a)anthracene and related compounds. Proc
Natl Acad Sci USA 66: 1228-1235. http://dx.doi.Org/10.1073/pnas.66.4.1228
Revis. NW; Bull. R; Laurie. D; Schiller. CA. (1984). The effectiveness of chemical carcinogens
to induce atherosclerosis in the white Carneau pigeon. Toxicology 32: 215-227.
http://dx.doi.org/10.1016/0300-483X(84)90075-l
Romero. PL; Mounho. BJ; Lauer. FT; Born. JL; Burchiel. SW. (1997). Depletion of glutathione
by benzo(a)pyrene metabolites, ionomycin, thapsigargin, and phorbol myristate in human
peripheral blood mononuclear cells. Toxicol Appl Pharmacol 144: 62-69.
http://dx.doi.org/10.1006/taap.1997.8113
Roszinskv-Kocher. G; Basler. A; Roehrborn. G. (1979). Mutagenicity of polycyclic
hydrocarbons. V. Induction of sister-chromatid exchanges in vivo. DNA Repair 66: 65-
67. http://dx.doi.org/10.1016/0165-1218(79)90008-9
Sakai. M; Yoshida. D; Mizusaki. S. (1985). Mutagenicity of polycyclic aromatic hydrocarbons
and quinones on Salmonella typhimurium TA97. Mutat Res 156: 61-67.
http://dx.doi.org/10.1016/0165-1218(85)90007-2
Salamone. MF; Heddle. JA; Katz. M. (1979). The mutagenic activity of thirty polycyclic
aromatic hydrocarbons (PAH) and oxides in urban airborne particulates. Environ Int 2:
37-43. http://dx.doi.org/10.1016/0160-4120(79)90092-8
Sato. S; Kitaiima. H; Konishi. S; Takizawa. H; Inui. N. (1987). Mouse strain differences in the
induction of micronuclei by polycyclic aromatic hydrocarbons. Mutat Res 192: 185-189.
http://dx.doi.org/10.1016/0165-7992(87)90054-6
Sawyer. TW; Chang. K; Harvey. RG; Digiovanni. J. (1987). Further investigations into the effect
of non-benzo ring bay-region methyl substituents on tumor-initiating activity of
polycyclic hydrocarbons. Cancer Lett 36: 317-324. http://dx.doi.org/10.lQ16/Q3Q4-
3835(87)90025-5
116
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Sctiiff. LJ; Elliott SF; Moore. SJ; Urcan. MS; Graham. JA. (1983). Unscheduled DNA synthesis
in hamster tracheal epithelium exposed in vitro to chemical carcinogens and
environmental pollutants. In MD Waters (Ed.), Short-term bioassays in the analysis of
complex environmental mixtures III (pp. 277-284). New York, NY: Springer.
http ://link. springer, com/chapter/10.1007%2F978 -1-4613-3611-2 19
Schmitt M; Kempf. C; Kempf. J. (1984). Endogenous activation by indirect muta-carcinogens
and DNA-repair synthesis in mouse-lung fibroblast cultures. Mutat Res 132: 197-201.
Scribner. JD. (1973). Tumor initiation by apparently noncarcinogenic polycyclic aromatic
hydrocarbons. J Natl Cancer Inst 50: 1717-1719. http://dx.doi.Org/10.1093/inci/50.6.1717
Segerback. D; Vodicka. P. (1993). Recoveries of DNA adducts of polycyclic aromatic
hydrocarbons in the 32P-postlabelling assay. Carcinogenesis 14: 2463-2469.
http://dx.doi.org/10.1093/carcin/14.12.2463
Selkirk. JK; MacLeod. MC. (1979). Metabolism and macromolecular binding of benzo(a)pyrene
and its noncarcinogenic isomer benzo(e)pyrene in cell culture. In PW Jones; P Leber
(Eds.), Polynuclear aromatic hydrocarbons: 3rd international symposium on chemistry
and biology - carcinogenesis and mutagenesis, Columbus, Oct 1978, proceedings (pp. 21-
35). Ann Arbor, Michigan: AASP.
Selkirk. JK; MacLeod. MC; Mansfield. BK; Nikbakht. A. (1982). Metabolic rationale for the
weak mutagenic and carcinogenic activity of benzo(e)pyrene. In T Sugimura; S Kondo;
H Takebe (Eds.), Environmental mutagens and carcinogens: Proceedings of the 3rd
international conference. New York, NY: Alan R. Liss.
Serdar. B; Brindlev. S; Doolev. G; Volckens. J; Juarez-Cotunga. E; Gan. R. (2016). Short-term
markers of DNA damage among roofers who work with hot asphalt. Environ Health 15:
99. http://dx.doi.org/10.1186/sl2940-016-0182-4
Sharma. A; Neekhra. A; Gramaio. AL; Patil. J; Chwa. M; Kuppermann. BP; Kennev. MC.
(2008). Effects of benzo(e)pyrene, a toxic component of cigarette smoke, on human
retinal pigment epithelial cells in vitro. Invest Ophthalmol Vis Sci 49: 5111-5117.
http://dx.doi.Org/10.l 167/iovs.08-2060
Simmon. VF. (1979). In vitro mutagenicity assays of chemical carcinogens and related
compounds with Salmonella typhimurium. J Natl Cancer Inst 62: 893-899.
Sims. P. (1964). Metabolism of polycyclic compounds. 25. The metabolism of anthracene and
some related compounds in rats. Biochem J 92: 621-631.
http ://dx. doi. org/10.1042/bi 0920621
Sims. P. (1970a). The metabolism of some aromatic hydrocarbons by mouse embryo cell
cultures. Biochem Pharmacol 19: 285-297. http://dx.doi.org/10.1016/00Q6-
2952(70)90348-5
Sims. P. (1970b). Qualitative and quantitative studies on the metabolism of a series of aromatic
hydrocarbons by rat-liver preparations. Biochem Pharmacol 19: 795-818.
http ://dx. doi. org/10.1016/0006-2952(70)90243 -1
Shiga. I'J: Gleason. GL; Mills. G: Ewald. L; Fu. PP; Lee. HM; Harvey. RG. (1980). Comparison
of the skin tumor-initiating activities of dihydrodiols and diol-epoxides of various
polycyclic aromatic hydrocarbons. Cancer Res 40: 1981-1984.
Slaga. TJ; Jecker. L; Bracken. WM; Weeks. CE. (1979). The effects of weak or non-carcinogenic
polycyclic hydrocarbons on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene skin
tumor-initiation. Cancer Lett 7: 51-59. http://dx.doi.org/10.1016/S0304-3835(79)80076-2
Teranishi. K; Hamada. K; Watanabe. H. (1975). Quantitative relationship between
carcinogenicity and mutagenicity of polyaromatic hydrocarbons in Salmonella
typhimurium mutants. DNA Repair 31: 97-102.
117
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Tokiwa. H; Nakanishi. Y; Sera. N; Kara. N; Inuzuka. S. (1998). Analysis of environmental
carcinogens associated with the incidence of lung cancer. Toxicol Lett 99: 33-41.
http://dx.doi.org/10.1016/S0378-4274f98)00136-2
Tom a. I; McCaffrey. TA. (2012). Transforming growth factor-beta and atherosclerosis:
interwoven atherogenic and atheroprotective aspects. Cell Tissue Res 347: 155-175.
http://dx.doi.org/10.1007/s00441-011-1189-3
U.S. EPA (U.S. Environmental Protection Agency). (1986). Guidelines for carcinogen risk
assessment [EPA Report] (pp. 33993-34003). (EPA/630/R-00/004). Washington, DC:
U.S. Environmental Protection Agency, Risk Assessment Forum.
https://www.epa.gov/iris/basic-information-about-integrated-risk-information-
svstem#risk
U.S. EPA (U.S. Environmental Protection Agency). (1990a). Integrated Risk Information System
(IRIS) chemical assessment summary for acenaphthene (CASRN 83-32-9). Washington,
DC: National Center for Environmental Assessment, Integrated Risk Information System.
https ://cfpub. epa. gov/ncea/iri s/iri s documents/documents/sub st/0442 summary. pdf
U.S. EPA (U.S. Environmental Protection Agency). (1990b). Integrated Risk Information
System (IRIS) chemical assessment summary for anthracene (CASRN 120-12-7).
Washington, DC: National Center for Environmental Assessment, Integrated Risk
Information System.
https://cfpub.epa.gov/ncea/iris/iris documents/documents/subst/0434 summarv.pdf
U.S. EPA (U.S. Environmental Protection Agency). (1990c). Integrated Risk Information System
(IRIS) chemical assessment summary for dibenz[a,h]anthracene (CASRN 53-70-3).
Washington, DC: National Center for Environmental Assessment, Integrated Risk
Information System.
https://cfpub.epa.gov/ncea/iris/iris documents/documents/subst/0456 summarv.pdf
U.S. EPA (U.S. Environmental Protection Agency). (1990d). Integrated Risk Information
System (IRIS) chemical assessment summary for fluoranthene (CASRN 206-44-0).
Washington, DC: National Center for Environmental Assessment, Integrated Risk
Information System.
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0444_summarv.pdf
U.S. EPA (U.S. Environmental Protection Agency). (1990e). Integrated Risk Information System
(IRIS) chemical assessment summary for fluorene (CASRN 86-73-7). Washington, DC:
National Center for Environmental Assessment, Integrated Risk Information System.
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0435_summarv.pdf
U.S. EPA (U.S. Environmental Protection Agency). (1990f). Integrated Risk Information System
(IRIS) chemical assessment summary for pyrene (CASRN 129-00-0). Washington, DC:
National Center for Environmental Assessment, Integrated Risk Information System.
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0445_summarv.pdf
U.S. EPA (U.S. Environmental Protection Agency). (1991). Guidelines for developmental
toxicity risk assessment. Fed Reg 56: 63798-63826.
U.S. EPA (U.S. Environmental Protection Agency). (1993). Integrated Risk Information System
(IRIS) chemical assessment summary for 1,2-dichloroethane (CASRN 107-06-2).
https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/0149_summarv.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2005). Guidelines for carcinogen risk
assessment [EPA Report], (EPA/630/P-03/001F). Washington, DC.
https://www. epa. gov/ sites/production/files/2013-
09 documents, cancer guidelines final 3-25-05.pdf
118
Benzo[e]pyrene
-------
EPA/690/R-21/008F
U.S. EPA (U.S. Environmental Protection Agency). (2007). Provisional peer-reviewed toxicity
values for perylene (CASRN 198-55-0) [EPA Report], Cincinnati, OH.
https://cfpub.epa.gov/ncea/pprtv/documents/Pervlene.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2009). Provisional peer-reviewed toxicity
values for anthracene (CASRN 120-12-7) [EPA Report], Cincinnati, OH.
https://cfpub.epa.gov/ncea/pprtv/documents/Anthracene.pdf
U.S. EPA (U.S. Environmental Protection Agency). (201 la). Chemical Assessment Clustering
Engine (ChemACE) [Database], Retrieved from https://www.epa.gov/tsca-screening-
tools/chemical-assessment-clustering-engine-chemace
U.S. EPA (U.S. Environmental Protection Agency). (201 lb). Health effects assessment summary
tables (HEAST) for superfund. Available online at https://epa-heast.ornl.gov/heast.php
U.S. EPA (U.S. Environmental Protection Agency). (201 lc). Provisional peer-reviewed toxicity
values for acenaphthene (CASRN 83-32-9) [EPA Report], Cincinnati, OH.
https://cfpub.epa.gov/ncea/pprtv/documents/Acenaphthene.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2012). Provisional peer-reviewed toxicity
values for fluoranthene (CASRN 206-44-0) [EPA Report], Cincinnati, OH.
https://cfpub.epa.gov/ncea/pprtv/documents/Fluoranthene.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2017a). IRIS carcinogens:
§69502.2(a)(1)(E). Chemicals that are identified as "carcinogenic to humans," "likely to
be carcinogenic to humans," or Group A, Bl, or B2 carcinogens in the United States
Environmental Protection Agency's Integrated Risk Information System. Available online
U.S. EPA (U.S. Environmental Protection Agency). (2017b). Toxicological review of
benzo[a]pyrene (CASRN 50-32-8): Supplemental information [EPA Report],
(EPA/635/R-17/003Fb). Washington, DC: U.S. Environmental Protection Agency,
Integrated Risk Information System.
https://cfpub.epa.gov/si/si public file download.cfm?p download id=535926&Lab=NC
EA
U.S. EPA (U.S. Environmental Protection Agency). (2017c). Toxicological review of
benzo[a]pyrene [CASRN 50-32-8] [EPA Report], (EPA/635/R-17/003Fa). Washington,
DC: U.S. Environmental Protection Agency, Integrated Risk Information System.
https://cfpub.epa. gov/ncea/iris/iris_documents/documents/toxreviews/013 6tr.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2018a). 2018 Edition of the drinking water
standards and health advisories [EPA Report], (EPA/822/F-18/001). Washington, DC:
U.S. Environmental Protection Agency, Office of Water.
https://www.epa.gOv/sites/production/files/2018-03/documents/dwtable2018.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2018b). How to access the TSCA
inventory. Download the non-confidential TSCA inventory [Database], Retrieved from
http://www2.epa.gov/tsca-inventorv/how-access-tsca-inventorv
U.S. EPA (U.S. Environmental Protection Agency). (2020a). Integrated risk information system.
IRIS assessments [Database], Washington, DC. Retrieved from http://www.epa.gov/iris/
U.S. EPA (U.S. Environmental Protection Agency). (2020b). Provisional peer-reviewed toxicity
values (PPRTVs) for superfund: Derivation support documents [Database], Washington,
DC. Retrieved from https://www.epa.gov/pprtv
Van Duuren. BL: Gotdschmidt BM. (1976). Cocarcinogenic and tumor-promoting agents in
tobacco carcinogenesis. J Natl Cancer Inst 56: 1237-1242.
Van Duuren. BL: Sivak. A: Langseth. L: Gotdschmidt BM: Segal. A. (1968). Initiators and
promoters in tobacco carcinogenesis. Natl Cane Inst Monogr 28: 173-180.
119
Benzo[e]pyrene
-------
EPA/690/R-21/008F
van Grevenvnghe. J; Rion. S; Le Ferrec. E; Le Yee. M; Amiot L; Fauchet R: Fardel. O. (2003).
Poly cyclic aromatic hydrocarbons inhibit differentiation of human monocytes into
macrophages. J Immunol 170: 2374-2381.
http://dx.doi.Org/10.4049/iimmunol.170.5.2374
Vandervort R; Lucas. JB. (1974). Health hazard evaluation determination report No. HHE-72-
77-109, Ashland Oil Incorporated, Catlettsburg, Kentucky (pp. 72-77). (NIOSH-TR-109-
74). Rockville, MD: National Institute for Occupational Safety and Health (NIOSH).
Ved Brat. S; Tong. C; Telang. S; Williams. GM. (1983). Comparison of sister chromatid
exchange and mammalian cell mutagenesis at the hypoxanthine guanine phosphoribosyl
transferase locus in adult rat liver epithelial cells [Abstract], Ann N Y Acad Sci 407: 474-
475. http://dx.doi.org/10.1111/i. 1749-6632.1983.tb47868.x
Vienneau. DS; Deboni. U; Wells. PG. (1995). Potential genoprotective role for UDP-
glucuronosyltransferases in chemical carcinogenesis: Initiation of micronuclei by
benzo(a)pyrene and benzo(e)pyrene in UDP-glucuronosyltransferase-deficient cultured
rat skin fibroblasts. Cancer Res 55: 1045-1051.
Walker. SA; Addai. AB; Mathis. M; Ramesh. A. (2007). Effect of dietary fat on metabolism and
DNA adduct formation after acute oral exposure of F-344 rats to fluoranthene. J Nutr
Biochem 18: 236-249. http://dx.doi.Org/10.1016/i.inutbio.2006.04.001
Wang. NC; Zhao. QJ; Wesselkamper. SC; Lambert. JC; Petersen. D; Hess-Wilson. JK. (2012).
Application of computational toxicological approaches in human health risk assessment.
I. A tiered surrogate approach. Regul Toxicol Pharmacol 63: 10-19.
http://dx.doi.Org/10.1016/i.vrtph.2012.02.006
White. GL; Fu. PP; Heflich. RH. (1985a). Effect of nitro substitution on the light-mediated
mutagenicity of polycyclic aromatic hydrocarbons in salmonella typhimurium TA98.
MutatRes 144: 1-7. http://dx.doi.org/10.1016/0165-7992(85)90115-0
White. KL; Holsapple. MP. (1984). Direct suppression of in vitro antibody production by mouse
spleen cells by the carcinogen benzo(a)pyrene but not by the noncarcinogenic congener
benzo(e)pyrene. Cancer Res 44: 3388-3393.
White. KL: Lvsv. HH; Holsapple. MP. (1985b). Immunosuppression by polycyclic aromatic
hydrocarbons: A structure-activity relationship in B6C3F1 and DBA/2 mice.
Immunopharmacology 9: 155-164. http://dx.doi.org/10.1016/0162-3109(85)90011-6
White. KL: McLoughlin. CE: Auttachoat. W: Smith. MJ. (2012). Validation of the Candida
albicans delayed-type hypersensitivity (DTH) model in the female Br.CsFi mouse for use
in immunotoxicological investigations. J Immunotoxicol 9: 141-147.
http://dx.doi.org/10.3109/1547691X.2011.636768
WHO (World Health Organization). (1998). Environmental Health Criteria 202: Selected non-
heterocyclic policyclic aromatic hydrocarbons [WHO EHC], Geneva, Switzerland.
http://www.inchem.Org/documents/ehc/ehc/ehc202.htm#SubSectionNumber:4.2.l
Winker. N: Tuschl. H: Kovac. R: Weber. E. (1997). Immunological investigations in a group of
workers exposed to various levels of polycyclic aromatic hydrocarbons. J Appl Toxicol
17: 23-29.
Woidani. A: Alfred. LJ. (1984). Alterations in cell-mediated immune functions induced in mouse
splenic lymphocytes by polycyclic aromatic hydrocarbons. Cancer Res 44: 942-945.
Woidani. A: Attarzadeh. M: Wolde-Tsadik. G: Alfred. LJ. (1984). Immunocytotoxicity effects of
polycyclic aromatic hydrocarbons on mouse lymphocytes. Toxicology 31: 181-189.
120
Benzo[e]pyrene
-------
EPA/690/R-21/008F
Wood. AW; Levin. W; Thakker. PR; Yagi. H; Chang. RL; Ryan. DE; Thomas. PE; Dansette.
PM; Whittaker. N; Turuiman. S; Lehar. RE; Kumar. S; Jerina. DM; Connev. AH. (1979).
Biological activity of benzo[e]pyrene. An assessment based on mutagenic activities and
metabolic profiles of the poly cyclic hydrocarbon and its derivatives. J Biol Chem 254:
4408-4415.
Wvnder. EL; Hoffmann. D. (1959). A study of tobacco carcinogenesis. VII. The role of higher
polycyclic hydrocarbons. Cancer 12: 1079-1086. http://dx.doi.org/10.1002/1097-
0142(19591 1 /12) 12:6< 1079: :aid-cncr2820120604>3,0.co;2-i
Zeiger. E; Anderson. B; Haworth. S; Lawlor. T; Mortelmans. K. (1992). Salmonella
mutagenicity tests: V. Results from the testing of 311 chemicals. Environ Mol Mutagen
19: 2-141. http://dx.doi.org/10.1002/em.28501906Q3
Zhao. W; Ramos. KS. (1995). Inhibition of DNA synthesis in primary cultures of adult rat
hepatocytes by benzo[a]pyrene and related aromatic hydrocarbons: Role of Ah receptor-
dependent events. Toxicology 99: 179-189. http://dx.doi.org/10.1016/030Q-
483 X(94)03028-Z
121
Benzo[e]pyrene
------- |